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Pigmentation in Anuran TestesAnatomical Pattern and Variation.

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THE ANATOMICAL RECORD 292:178–182 (2009)
Pigmentation in Anuran Testes:
Anatomical Pattern and Variation
Department of Biology, Sao Paulo State University (UNESP), IBILCE,
Sao Jose do Rio Preto, Sao Paulo, Brazil
In amphibians, pigmented cells are present in several organs, composing an extracutaneous pigmentary system. Seventeen species from
two families were studied to develop a protocol for pigmentary classification. The amount and distribution of these cells are variable, allowing the
establishment of anatomical patterns for visceral pigmentation in anuran
testes. Anat Rec, 292:178–182, 2009. Ó 2008 Wiley-Liss, Inc.
Key words: anurans; pigmentation testes; anatomical variation
Ectothermic vertebrates have a well-developed system
of melanin-containing cells, distributed among several
organs (liver, spleen, lung, heart, thymus, and gonads)
and tissues (meninges and connective tissues surrounding blood vessels) composing an extracutaneous pigmentary system (Gallone et al., 2002).
In amphibians, cells similar to melanocytes have been
described in the epidermis and in several organs (Agius
and Agbede, 1984; Zuasti et al., 1998; Oliveira and Zieri,
2005; Zieri et al., 2007) that produce and store melanin
in the interior of spherical or ovoid structures denominated melanosomes (Agius and Roberts, 2003; Zieri
et al., 2007). These cellular types are large and irregular, presenting a star-like or punctiform aspect (Oliveira
and Zieri, 2005).
In hematopoietic organs of lower vertebrates, pigmented cells with macrophagic activity, known as melanomacrophages, are found (Agius, 1980; Agius and
Agbede, 1984). These types of cells reside in the liver of
amphibians where they are also are known as Kupffer
cells (Sichel et al., 1997; Zuasti et al., 1998; Prelovisek
and Bulog, 2003), presenting different types of granules
in the cytoplasm that indicate chemically diversified
substances (Agius and Agbede, 1984; Herráez and
Zapata, 1991).
Because of the difficulty of detecting these pigmented
cells separately, it is necessary to establish a protocol to
determine their occurrence and distribution by dividing
them into categories according to the pigmentation intensity in the anuran testes.
This study aimed at characterizing anatomical patterns of pigmentation intensity in the testes of adult
anurans, with a minimum of five animals being analyzed from each species (17 species distributed into two
families): Leiuperidae (Physalaemus cuvieri (N 5 15), P.
olfersii (N 5 10), P. centralis (N 5 10), P. marmoratus
(N 5 20), Eupemphix nattereri (N 5 10), Pseudopaludicola cf. falcipes (N 5 5), and P. saltica (N 5 5)) and Leptodactylidae (Leptodactylus bokermanni (N 5 5), L. furnarius (N 5 11), L. fuscus (N 5 19), L. labyrinthicus (N
5 12), L. mystaceus (N 5 13), L. mystacinus (N 5 12),
L. ocellatus (N 5 17), L. podicipinus (N 5 14), L. chaquensis (N 5 5), and L. notoaktites (N 5 5)). The animals were collected in temporary ponds located in the
state of Sao Paulo during reproductive activity, between
September of 2006 and February of 2007.
The individuals were anesthetized, euthanized, and
submitted to morphological studies in the laboratory,
following the Guide for Care and Use of Laboratory
Animals (Protocol n8001/06-CEEA). All animals had the
organs of the abdominal cavity exposed by medial incision for macroscopic documentation under a stereomicroscope (Leica- MZ16), using the program Image Manager
50 (IM50) to capture the images. Only organ surfaces
Grant sponsor: State Research Foundation of Sao Paulo
(FAPESP); Grant numbers: 02/08016-9, 05/02919-5, 06/57990-9
and 08/52389-0.
*Correspondence to: Classius de Oliveira, Department of Biology, Sao Paulo State University (UNESP), IBILCE, Sao Jose do
Rio Preto, Sao Paulo, Brazil. Fax: 15-517-3221-2390.
Received 5 January 2008; Accepted 22 September 2008
DOI 10.1002/ar.20832
Published online 16 December 2008 in Wiley InterScience (www.
Visceral melanocytes are large and irregular in shape,
with intensely pigmented cytoplasm. Under a stereomicroscope, they may present star-like or punctiform morphology according to the presence or absence of cytoplasmic processes, respectively (Oliveira and Zieri, 2005).
They are typically distributed in organs and tissues,
with species-specific patterns of incidence and types
(Aoki et al., 1969; Trevisan et al., 1991; Christiansen
et al., 1996; Sichel et al., 1997; Zuasti et al., 1998; Barni
et al., 2002; Oliveira et al., 2002, 2003; Prelovisek and
Bulog, 2003; Oliveira and Zieri, 2005).
Pigmentary classification was accomplished by establishing testicular pigmentation intensity, ranging from
absent (Category 0) (Fig. 1) to entirely pigmented, when
an intense black coloration is observed (Category 3—
maximum intensity) (Fig. 2). Considering that the maximum pigmentation is 100%, a value of 50% for color intensity can be established to delimit two intermediate
categories, in which intensities >50% and <100% represent Category 2 while intensities <50% and >0% represent Category 1 (Fig. 3). The proposed classification is
an analog to a classic utilized classification for histochemical and cytochemical studies, in which the degree
of the reaction was standardized (between reagent-dye
and substrate-tissue) into a range of predetermined class
dependent from the perception of the observer: absence
of reaction (2), weak reaction (1), moderate (11), and
strong reaction (111). Grant et al. (2006) observed
three different states for pigmentation character in the
intestine and Dendrobatidae testes, not considering the
pigmentation intensity differences, but only the distribution of such pigmentation in the organ surface, adopting
state 0 for the absence of pigmentation; state 1 restricted to a small area of the organ and state 2 for completely pigmented organs. These changes were observed
during ontogeny, being interpreted as an evidence of
additivity. However, these variations were neither correlated to adult size, pigmentation of ventral surface nor
to individual maturity.
In this work, Category 0 expresses total absence of
pigmented cells on gonad surface, as observed in Leptodactylidae (Fig. 1), where milky-white coloration and
vascularization are evident. Category 1 consists of few
pigmented cells, constituting a discrete pigmentation.
Category 2 comprises a large quantity of pigmented cells
whose presence masks the whitish shade commonly
described for the vertebrate testicle. In Category 3, the
massive presence of pigmented cells renders an intense
pigmentation to the structure with evident alteration of
the usual color for this organ as described in vertebrates, obscuring the milky-white aspect and the superficial vascularization. Differences in pigmentation intensity were observed in some analyzed species of Leiuperidae. The representation of each category can be
observed in Fig. 3.
In Leptodactilydae family, a pattern of absent testicular pigmentation was found, occurring in 98% of all
specimens analyzed among the 10 species, although in
two, L. bokermanni and L. furnarius, only one individual manifested Category 1 (2%). In L. bokermanni, pigmentation occurred in the two gonads, whereas in L.
furnarius, only the right gonad was pigmented (Fig. 4).
By contrast, the family Leiuperidae displayed a testicular pigmentation pattern (Categories 1, 2, and 3) that
was present in 95.7% of all specimens analyzed in seven
different species, whereas one example each among
P. cuvieri, P. olfersii and P. marmoratus manifested Category 0 (4.3%).
All species analyzed from the family Leiuperidae displayed testicular pigmentation of various intensities.
Both species of the genus Pseudopaludicola demonstrated pigmentation in the gonads (Categories 2 and 3)
with no specimens showing an absence or lower degree
of pigmentation (Fig. 2). Physalaemus centralis was the
species with the greatest uniformity of pigmentation in
100% of individuals (Category 3). In Physalaemus
cuvieri, the testes also showed pigmentation in the testicular capsule (Category 2 in 27% and Category 3 in
67%); intra-individual variation occurred in one specimen analyzed (6%), whose left testicle was intensely pigmented (Category 3) and the right gonad showed absence of pigmentation. In Physalaemus olfersii, Category
2 was noted in only one individual (10%), with the
predominance of Category 3 in 80%; Category 0 was
observed only in one left gonad in one animal that also
presented intra-individual variation, with the right testicle also showing coloration of intensity 2. In Physalaemus marmoratus, testes range from being devoid of pigmentation (Category 0) to intensely pigmented (Category
3). This species was the one with the highest level of
intra-individual variation, occurring in six animals analyzed (30%), consisting of cases with one of the gonads
without pigment (Category 0), and the others pigmented
(categories 2 and 3), as well as a variation in coloration
intensity among the antimeres (Categories 1 and 2; Categories 2 and 3). This testicular pigmentation variation
among antimeres was also reported by Grant (2004), for
Colostethus panamensis (Dendrobatidae). In Eupemphix
nattereri, absence of testicular pigmentation was not
Fig. 1. Testes of species of the family Leptodactylidae demonstrating total absence of pigmentation, evidenced by milky-white coloration
of gonads as well as abundant vascularization. L f, Leptodactylus fuscus; L fr, Leptodactylus furnarius; L l, Leptodactylus labyrinthicus;
L mi, Leptodactylus mystacinus; L me, Leptodactylus mystaceus; L o,
Leptodactylus ocellatus; L p, Leptodactylus podicipiunus; L c, Leptodactylus chaquensis; L b, Leptodactylus bokermanni; L n, Leptodactylus notoaktites.
were analyzed for the distribution in categories according to the intensity and presence of melanocytes.
Fig. 2. Species of the family Leiuperidae with maximum intensity of testicular pigmentation: P cv,
Physalaemus cuvieri; P ct, Physalaemus centralis; Ps f, Pseudopaludicola falcipes; Ps s, Pseudopaludicola
Fig. 3. Differences of intensity in pigmentation represented by each category in the testes of Leiuperidae: Physalaemus marmoratus, Eupemphix nattereri, and Physalaemus olfersii.
Figure 1.
(Legend on page 179.)
Figure 2.
(Legend on page 179.)
Figure 3.
(Legend on page 179.)
Fig. 4.
Occurrence of pigmentation in testes of species of Leptodactylidae.
Fig. 5.
Occurrence of testicular pigmentation in Leiuperidae.
observed, Category 1 occurred in 20%, Category 2 in
70%, and Category 3 in 10% of the individual (Fig. 5).
Variation in color intensity also occurs in the surface
of other organs of the abdominal cavity, such as the rectum, pericardium, and vessels of the cardiac base, intestine, and heart. The simultaneous evaluation of diverse
structure may permit the establishment of patterns that
are species-specific. In this article, a categorization
protocol based on the coloration intensity conferred by
pigmented cells in anuran gonads has been proposed,
to allow a general morphological description of the
extracutaneous pigmentary system.
This pigmentation pattern can be used as additional
and complementary proposal to the classical methodologies based on morphometrics, external morphology, color
patterns, and osteological characters, used in taxonomic
revisions in Leptodactylidae (Nascimento et al., 2005,
2006). Hedges et al. (2008) estimated the relationships
between 344 species of the family Brachycephalidae
using DNA sequences from mitochondrial and nuclear
genes. DNA barcode has also been used in anurans as
an alternative to elucidate taxonomic questions (Smith
et al., 2008). Hawkins et al. (2007) used this technique
to diagnosis distinct populations of Dendropsophus minutus in the Guianas, which, although has made possible
the detection of genetic variations, is not suitable for
amphibians. All these methodologies are complementary
and, when used together, enable taxonomic inferences.
Morphofunctional aspects of this visceral pigmentation
present in ectothermic animals are still unknown (Corsaro et al., 2000), and may constitute characteristics
intrinsic to the species, or may be related to physiological responses and, therefore, present variations within
the category established for the species.
The authors thank Dr. Itamar Alves Martins and Silvio Cesar de Almeida for capturing and supplying the
animals, Dr. Francisco Langeani Neto and Dr. Reinaldo
José Fazzio Feres for reviewing the manuscript, Msc.
Paula Tavares Pinto Paiva for reviewing the language.
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patterns, variation, pigmentation, testesanatomical, anura
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