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Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): Occurrence and comparison
Zoologischer Anzeiger 250 (2011) 102–110
Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): Occurrence and comparison Lilian Franco-Belussia , Lia Raquel de Souza Santosb,1 , Rodrigo Zieria , Classius de Oliveirac,∗ a
Post-graduation Program in Animal Biology, IBILCE-UNESP/SJRP São Paulo State University (UNESP), campus São Jose do Rio Preto, Institute of Bioscience, Letters and Exact Sciences, Department of Biology, Brazil2 b Federal Institute of Goiás, IFG, campus Rio Verde, Goiás, Brazil c Department of Biology, IBILCE-UNESP/SJRP, Brazil Received 1 April 2009; received in revised form 13 July 2010; accepted 1 December 2010
Abstract Amphibians share with other ectothermic vertebrates an extracutaneous pigmentary system consisting of melanin-containing cells in various organs and tissues. This paper describes the interspecific variation in the visceral pigmentation and extracutaneous pigment system in four species of the genus Dendropsophus [i.e., D. elianeae, D. minutus, D. nanus, and D. sanborni (Anura: Hylidae)]. Fifteen adult males from each species were collected in the region of São José do Rio Preto (State of São Paulo, Brazil), and their visceral pigmentation was analyzed during the reproductive period. The individuals were weighed and measured, and the pigmented visceral cells were classified and documented in photographs. The shape and quantity of the pigment cells differed among the various structures of the same individual, as well as among the same structures of different species. Similarities and differences among these species were observed, and a visceral pigmentation pattern for anatomical structures was detected. In the digestive system of all species analyzed, there was an absence of pigment cells on the stomach and middle intestine. However, the pigmentation of the final portion of the intestine (i.e., the rectum) showed interspecific variation, with D. minutus presenting intense pigmentation, whereas the other species presented no pigmentation. Significant differences were detected also in the cardio-respiratory system, mesentery, and lumbosacral peritoneum. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. Keywords: Anurans; Extracutaneous pigmentary system; Melanocytes
1. Introduction Amphibians and other ectothermic vertebrates possess an extracutaneous pigmentary system in various tissues and
∗ Corresponding author at: Department of Biology, Institute of Bioscience, Letters and Exact Sciences, UNESP, São Paulo State University, CEP: 15040-000 São José do Rio Preto, São Paulo, Brazil. Fax: +55 17 3221 2390. E-mail address: [email protected] (C. de Oliveira). 1 Current address: Federal Institute of Goiás,IFG,campus Rio Verde GO,CEP: 75.908-000 Rio Verde, Goiás, Brazil. 2 www.ibilce.unesp.br.
organs, which comprises cells, whose cytoplasm contains melanin (Gallone et al., 2002). These melanin-containing cells are frequently found in the liver, spleen, kidneys, peritoneum, lung, heart, blood vessels, thymus, gonads, and meninges of fishes, amphibians, and reptiles (Agius, 1980; Agius and Agbede, 1984; Zuasti et al., 1990, 1998; Christiansen et al., 1996; Bagnara and Matsumoto, 2006; Gallone et al., 2002; Zieri et al., 2007; Pederzoli and Trevisan, 1990; Trevisan et al., 1991). These cells are similar to melanocytes (Zuasti et al., 1998; Agius and Agbede, 1984), which are derived from the neural crest (Bagnara and Matsumoto, 2006) and are able to produce and store melanin inside their cytoplasm (Agius and Roberts, 2003). In organs
0044-5231/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2010.12.001
L. Franco-Belussi et al. / Zoologischer Anzeiger 250 (2011) 102–110
with hematopoietic function, there are also cell types with phagocytic activity similar to that of macrophages (Agius, 1980). These cells are derived from hematopoietic stem cells (Sichel et al., 1997), which often conglomerate to form pigmented nodules called melanomacrophage centers (Agius, 1981). Current knowledge about visceral pigmentation, be its structural features or its diversity, is superficial, with the exception of a few morphological studies in anurans, such as Physalaemus marmoratus (Aoki et al., 1969; Oliveira and Zieri, 2005), Physalaemus cuvieri (Oliveira et al., 2002, 2003), Eupemphix nattereri (Oliveira and Zieri, 2005; Zieri et al., 2007), Rana esculenta (Cicero et al., 1989; Sichel et al., 1997; Barni et al., 1999; Gallone et al., 2002; Barni et al., 2002), Rana ridibunda (Akulenko, 1998) and, Xenopus laevis (Sichel et al., 1997; Zuasti et al., 1998). Our aim in this study was to characterize the visceral pigmentation in the cardiorespiratory, digestive and urogenital systems and their associated serosal membranes in four tree-frog species of the hylid genus Dendropsophus (i.e., D. elianeae, D. minutus, D. nanus, and D. sanborni) during their reproductive periods. We also evaluated the intra- and interspecific differences and searched for patterns of occurrence of pigmentation that could help to distinguish related species. Such an approach will increase our knowledge about the extracutaneous pigmentary system in neotropical anurans. The specimens were treated according to the ethical guidelines of São Paulo State University – UNESP/Botucatu, following the Guide for Care and Use of Laboratory Animals (Protocol No. 001/06-CEEA). In the laboratory, the specimens were anesthetized and euthanized by submersion in 20% ethanol. All specimens had the organs of the abdominal cavity exposed by medial incision for macroscopic documentation under a stereomicroscope (Leica-MZ16), using the program Image Manager 5.0 (Version 4.0.5, Media Cybernetics Inc., Bethesda, MD, USA) to capture the images.
2. Materials and methods Fifteen male specimens of each species (D. elianeae, D. minutus, D. nanus, and D. sanborni) were collected in the region of São José do Rio Preto (State of São Paulo, Brazil) during their reproductive period from October 2006 to March 2007. Some of the specimens used were from the Amphibian Collection of the Department of Botany and Zoology (DZSJRP-Amphibia) at the São Paulo State UniversityIBILCE/UNESP (D. elianeae: DZSJRP 6368, 7646, 7890, 7965, 7968–9, 8016, 8037, 8089, 8124, 8474–6, 8232, 8532). Visceral pigment cells were classified according to the protocol of Franco-Belussi et al. (2009), based on the differences in the intensity of pigmentation present on the gonads of anurans, and applied to other organs and tissues in this study. The pigmentary classification is based on the intensity of pigmentation, ranging from absence (category 0) to entirely pigmented, when an intense black coloration is observed (cat-
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egory 3 = maximal intensity), with the categories 1 and 2 representing a gradual increase in the intensity of pigmentation. For this study, the protocol of classification for the visceral pigmentation was applied to the following anatomical structures and regions of the specimens as follows: (1) pericardium and blood vessels at the basis of the heart; (2) heart; (3) lungs; (4) stomach; (5) middle intestine; (6) rectum; (7) intestinal mesentery, (8) kidney and renal blood vessels; (9) testes; (10) nerves of the lumbar plexus; (11) lumbosacral parietal peritoneum. To compare the differences between categories of pigmentation in each organ or region we used the G test for goodness of fit, with the corrections by Yates and Williams (Sokal and Rohlf, 1995). This test was implemented using the code provided by Prof. Peter Hurd available at http://www.psych.ualberta.ca/∼phurd/cruft/g.test.r. The test was run using the R software v. 2.11.1 (R Development Core Team, Informer Technology Inc. 2010).
3. Results Visceral pigment cells in the various organs and membranes of the abdominal cavity varied significantly in their occurrence and distribution among the species analyzed, allowing a classification of this pigmentation according to the intensity of pigmentation on an organ’s surface. This pigmentary classification is shown for D. elianeae, D. minutus, D. nanus, and D. sanborni in Fig. 1. There is no pigmentation (category 0) on the pericardium, heart, vertebral column, and kidneys of D. minutus, and on the lungs of D. sanborni. There is an intense black pigmentation (category 3) on the rectum of D. minutus. There are a few pigment cells creating only a little pigmentation (category 1) on the pericardium, heart, and vertebral column of D. nanus and on the lungs, kidneys, and rectum of D. elianeae. There is a large number of pigmented cells, which mask the color of the organ (category 2), on the kidneys of D. elianeae. The pigmentation on organs and structures of the abdominal cavity in the four species (D. elianeae, D. minutus, D. nanus and D. sanborni) are summarized in Figs. 2–5 and Table 1. The pigmentation of the pericardium revealed significant interspecific differences (G-test: G = 23.56; df = 9; p = 0.005), heart (G-test: G = 61.93; df = 9; p < 0.01), and lungs (G-test: G = 49.54; df = 9; p < 0.01) (Fig. 2). All 15 individuals of D. elianeae presented no visceral melanocytes (category 0) on the pericardium and cardiac blood vessels. In addition, 14 out of 15 individuals of D. minutus showed no visceral melanocytes, although one specimen presented only a little pigmentation (category 1). On the other hand, only 8 out of 15 specimens of D. nanus and 6 out of 15 specimens of D. sanborni presented no pigmentation. Only a little pigmentation (category 1) was found in 6 specimens of D. nanus and in 8 specimens of D. sanborni, and one specimen of each of those species presented a large number of pigmented cells
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Fig. 1. Photographs of organ surfaces and regions arranged according to the intensity of their pigmentation. Empty spaces represent absence of that pigmentation category. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation. Abbreviations: D nan, Dendropsophus nanus; D eli, D. elianeae; D min, D. minutus; D san, D. sanborni.
(category 2). All specimens of D. minutus, as well as 2 out of 15 specimens of D. elianeae, showed no visceral melanocytes on the heart. In contrast, 9 out of 15 specimens of D. elianeae presented only a little pigmentation (category 1) and 4 out of 15 specimens presented a large number of pigmented cells (category 2). In D. sanborni, 4 out of 15 specimens presented no pigmentation (category 0) in this region, and 11 out of 15 specimens presented only a little pigmentation (category 1). In contrast, the heart of all specimens of D. nanus was pigmented. The heart of 6 out of 15 specimens had a few visceral melanocytes (category 1), and the heart of 9 out of 15 specimens had a large number of pigment cells (category 2) (Table 1). On the lungs, 14 out of 15 specimens of D. minutus and D. sanborni showed no pigmentation (category 0) and 1 out of 15 specimens presented only a little pigmentation (category 1).
In D. nanus, 6 out of 15 specimens showed no pigmentation (category 0) in this region, whereas 9 out of 15 specimens presented only a little pigmentation (category 1). In contrast, the lungs of all specimens of D. elianeae were pigmented to some degree. The lungs of 13 out of 15 individuals had very little pigmentation (category 1), but 2 out of 15 specimens had a high degree of pigmentation (category 2) (Table 1). On the digestive system (Fig. 3), no pigment cells were observed on the stomach of all specimens of the four species analyzed. All specimens of D. nanus, D. sanborni and D. minutus presented no pigmentation on the middle intestine, whereas 1 out of 15 specimens of D. elianeae presented a few pigmented cells (category 1) in this region. A significant variation (G-test: G = 72.12; df = 9; p < 0.01) was observed on the terminal portion of the digestive system (i.e., the rectum). In this region, there was an intense pigmentation (category
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Fig. 2. Visceral pigmentation on the organs and associated structures of the cardio-respiratory system of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
3) in all specimens of D. minutus, in contrast to an absence of pigmentation in 12 out to 15 specimens of D. elianeae. In addition, 14 out of 15 individuals of D. nanus, and all specimens of D. sanborni, presented no pigmentation. In D. elianeae, 3 out of 15 specimens presented only a little pigmentation (category 1) and in D. nanus, only 1 out of 15 individuals presented this category (Table 1). On the urogenital system (Fig. 4), a complete absence of pigmentation was observed on the testes, urinary bladder, and fat bodies of all species. However, the kidneys presented a significant variation (G-test: G = 23.97; df = 9; p = 0.004). In D. minutus, 8 out of 15 specimens presented no pigmentation, 4 out of 15 specimens presented only a little pigmentation (category 1), and 3 out of 15 specimens presented a high degree of pigmentation (category 2). On the other hand, 5 out of 15 specimens of D. nanus presented no pigmentation, whereas 4 out of 15 specimens presented only a little pigmentation (category 1), and 6 out of 15 specimens presented a high degree of pigmentation (category 2). In D. sanborni, 5 out of 15 specimens presented no pigmentation in this region, however 10 out of 15 specimens presented only a little pigmentation (category 1). In D. elianeae, 2 out of 15 specimens presented no pigmentation on the kidneys, 3 out of 15 speci-
mens presented only a little pigmentation (category 1), and 10 out of 15 specimens presented a high degree of pigmentation (category 2) (Table 1). The occurrence of pigment cells is constant and does not vary significantly on the lumbar nerve plexus (G-test: G = 16.64; df = 9; p = 0.06). In D. elianeae, 14 out of 15 specimens showed no pigmentation in this region, and 1 out of 15 specimens presented only a little pigmentation (category 1). In D. nanus and D. minutus, 8 out of 15 specimens presented no pigmentation in this region, whereas 5 out of 15 specimens possessed only a little pigmentation (category 1), and 2 out of 15 specimens presented a high degree of pigmentation (category 2). In D. sanborni, 6 out of 15 specimens showed no pigmentation in this region and 9 out of 15 specimens presented only a little pigmentation (category 1) (Fig. 5). Interspecific variation is significant on the lumbosacral parietal peritoneum (G-test: G = 49.82; df = 9; p < 0.01) and intestinal mesentery (G-test: G = 25.63; df = 9; p = 0.002). In the lumbosacral parietal peritoneum, all specimens of D. elianeae presented no pigmentation. 13 out of 15 specimens of D. nanus showed no pigmentation, but 2 out of the 15 specimens presented only a little pigmentation (category 1). In D. minutus, an absence of pigmentation was observed in
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Fig. 3. Visceral pigmentation on the digestive system (i.e., stomach, middle intestine, and rectum) of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
Fig. 4. Visceral pigmentation on the urogenital system of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
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Fig. 5. Visceral pigmentation on the peritoneum, mesentery, and lumbar nerve plexus of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
10 out of 15 specimens, whereas 3 out of 15 specimens presented only a little pigmentation (category 1) in this region. In contrast, all individuals of D. sanborni presented no pigmentation, and 12 out of 15 specimens presented only a little pigmentation (category 1), and 3 out of 15 specimens presented a high degree of pigmentation. On the intestinal mesentery, 12 out of 15 specimens of D. nanus presented no pigmentation (category 0) in this region, whereas 2 out of 15 specimens had only a little pigmentation (category 1), and 1 out of 15 specimens presented a high degree of pigmentation (category 2). In D. sanborni, 6 out of 15 specimens presented an absence of pigmentation, and 9 out of 15 specimens presented only a little pigmentation. In D. minutus, 9 out of 15 specimens showed no pigmentation (category 0) in this region, but 6 out of 15 specimens presented a high degree of pigmentation (category 2). In D. elianeae, 10 out of 15 specimens presented an absence of pigmentation (category 0) on the intestinal mesentery, whereas 3 out of 15 specimens presented only a little pigmentation (category 1), and 2 out of 15 specimens presented a high degree of pigmentation (category 2) (Table 1). When comparing the four species (D. elianeae, D. minutus, D. nanus and D. sanborni) (Table 1), it is possible to observe that D. minutus presents the highest number of regions without any pigmentation (category 0), but this species was the only one that presented a region (i.e., rectum) with intense black coloration (category 3). In contrast, D. nanus is the species that possesses the highest number of regions with pigmentation mostly in the categories 1 and 2. The species
D. sanborni and D. elianeae have similar degrees of pigmentation.
4. Discussion Cells containing melanin are found not only in the skin, but also in other organs constituting the extracutaneous pigmentary system. In this study, pigment cells were observed in the cardio-respiratory system, as well as in the intestinal mesentery, lumbosacral peritoneum, lumbar nerve plexus, and kidneys. The visceral pigment cells are found in the connective tissue of the capsule and interstitium of organs or are associated with the adventitious tunics or serous membranes. They are large, approximately 150 m in diameter, and irregular in shape with an abundantly and intensely pigmented cytoplasm. They may have a dendritic shape due to cytoplasmic processes or a round shape in the absence of cytoplasmic processes (Oliveira and Zieri, 2005). The interspecific differences in the degree of pigmentation are statistically significant in most organs and structures, except in the lumbar nerve plexus and middle intestine. Even though our study comprises only a limited number of representatives of each species, it was still possible to discover peculiarities and variations that distinguish the species. Thus, by determining the occurrence and density of the visceral melanocytes in different organs and structures, significant differences could be found to facilitate the identification and differentiation of species. In contrast, Grant
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Table 1. Comparison of the number of individuals of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni, which exhibit particular categories of visceral pigmentation on the pericardium and blood vessels at the basis of the heart, heart, lungs, stomach, middle intestine, rectum, intestinal mesentery, kidney and renal blood vessels, testes, nerves of the lumbar plexus, and lumbosacral parietal peritoneum. Organs and regions
Pericardium and blood vessels
Heart
Lung
Stomach
Intestine
Rectum
Kidney and blood vessel
Testes
Lumbar nerve plexus
Lumoar Kanetai Kentonium Mesenleriurn
Vesicula Urinaria
Species
D. nanus D. sanbomi D. minutus D. elianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. elianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. elianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae
Categories Category 0
Category 1
Category 2
8 6 14 15
6 8 1
1 1
6 11
9
9 9 1 1 13
4
4 15 2 6 14 14 15 15 15 15 15 15 15 14 14 15
Category 3
2
1 1 15
12 5 5 8 2 15 15 15 15 8 6 8 14 13 10 15 12 6 9 10 15 15 15 15
et al. (2006) used only the pigmentation on the intestine as a character in a phylogenetic study. In their study, Grant et al. (2006) observed three different degrees of the pigmentation on the intestine and testes of Dendrobatidae species, depend-
3 4 10 4 3
6 3 10
5 9 5 1 2 12 3
2
2 9
1
3
2
3 2
6 2
ing on the distribution of pigments on the organ surface, but did not consider differences in the number of pigment cells. These differences were not correlated to body size, pigmentation of the ventral body surface, or sexual maturity and
L. Franco-Belussi et al. / Zoologischer Anzeiger 250 (2011) 102–110
were interpreted as evidence of additivity during ontogeny. This character invariably progresses from an absence of pigmentation to a presence confined to the anterior quarter of the intestine and later extends beyond the middle of the final portion of intestine (i.e., rectum). The absence of pigmentation (category 0) on the testes is a conspicuous feature of the Dendropsophus species analyzed, giving the gonads a white appearance, which was also observed in some individuals of the family Leptodactylidae [Leptodactylus bokermanni, Leptodactylus furnarius, Leptodactylus fuscus, Leptodactylus labyrinthicus, Leptodactylus mystaceus, Leptodactylus mystacinus, Leptodactylus latrans, Leptodactylus podicipinus, Leptodactylus chaquensis, and Leptodactylus notoaktites (Franco-Belussi et al., 2009)]. In contrast, in Leiuperidae species (P. cuvieri, Physalaemus olfersii, Physalaemus centralis, Physalaemus marmoratus, E. nattereri, Pseudopaludicola cf. falcipes and Physalaemus saltica), the testicular capsule is abundantly and intensely pigmented and appears intensely black (category 3) (FrancoBelussi et al., 2009). P. marmoratus (Aoki et al., 1969; Oliveira and Zieri, 2005), P. cuvieri (Oliveira et al., 2002, 2003), and E. nattereri (Oliveira and Zieri, 2005; Zieri et al., 2007) bear a large number of melanocyte-like pigment cells in the testicular interstitium. But a large number of anuran species do not possess any pigment cells in the gonads, although some authors (Oliveira and Zieri, 2005; Zieri et al., 2007; Franco-Belussi et al., 2009; Moresco and Oliveira, 2009) have described a close relationship of visceral melanocytes with testicular blood vessels. Melanocytes are associated also with blood vessels from other organs and connective tissue membranes (i.e., pericardium, intestinal mesentery, and lumbosacral parietal peritoneum). Especially in E. nattereri and P. marmoratus, the density of pigment cells in the interstitium and albugineous tunic of the gonads is intense, giving the testes a dark brown color (Oliveira and Zieri, 2005; Zieri et al., 2007). The species analyzed in this study did not present pigmentation on the testes. However, Moresco and Oliveira (2009) described a little pigmentation (category 1) in Rhinella schneideri, and an intense black coloration (category 3) in P. cuvieri during the breeding season. On the pericardium, an absence of pigmentation is a unique feature of D. minutus and D. elianeae, whereas there is a predominance of only a little pigmentation (category 1) in D. sanborni and D. nanus, similar to what was reported by Moresco and Oliveira (2009) for R. schneideri and P. cuvieri. Moresco and Oliveira (2009) also observed that there is no pigmentation on the heart of R. schneideri and P. cuvieri (category 0), similar to what was observed in this study for D. minutus. However, on the pericardium of D. elianeae and D. sanborni, only a little pigmentation (category 1) predominates, and on that of D. nanus, a high degree of pigmentation (category 2) predominates. On the lungs, there is no pigmentation (category 0) in D. minutus and D. sanborni, similar to what was described by
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Moresco and Oliveira (2009) for R. schneideri. However, D. elianeae and D. nanus present only little pigmentation, resembling P. cuvieri at the beginning of the breeding season as described by Moresco and Oliveira (2009). But in this case, the pigmentation varies during the breeding season, being predominantly absent at the end of the season (Moresco and Oliveira, 2009). On the stomach and middle intestine, the absence of pigmentation was observed in all individuals of the four species, a fact also observed by Moresco and Oliveira (2009) in R. schneideri. On the rectum, the absence of pigmentation is predominant in D. elianeae, D. sanborni, and D. nanus, as already described by Moresco and Oliveira (2009) for R. schneideri, but D. minutus presents an intense pigmentation (category 3) in all individuals analyzed. Only this species presented category 3 of pigmentation on the rectum. On the kidneys, the pigmentation is highly variable ranging from total absence (category 0) to a high degree of pigmentation (category 2) in all species analyzed. In D. minutus, the absence of pigmentation (category 0) is predominant, as was also reported for R. schneideri (Moresco and Oliveira, 2009). In D. elianeae, there is a high degree of pigmentation (category 2), whereas D. sanborni presents only a little pigmentation (category 1). In D. nanus, there is great variation in the degree of pigmentation from individuals without pigmentation (category 0) to individuals with only a little pigmentation (category 1), and individuals with a high degree of pigmentation (category 2). This variability may be related to the breeding season of these individuals, since in P. cuvieri the pigmentation in this region varies during the reproductive cycle (Moresco and Oliveira, 2009). Morphofunctional aspects of visceral melanocytes in ectothermic animals are still poorly understood. Although the pigmentation on an organ’s surface is similar among anuran species, the degree of pigmentation varies in certain regions and, therefore, can be used to distinguish similar species. These pigment cells may also vary according to physiological responses of the metabolism due to the presence of different substances within the melanocytes. Nevertheless, this visceral pigmentation may constitute intrinsic features of species.
Acknowledgments We would like to thank the São Paulo State Research Foundation (FAPESP) for funding this project (Proc. # 02/08016-9, 05/02919-5, 06/57990-9 and 08/52389-0) and also to Diogo B. Provete for critical reviewing the manuscript and helping with the English version.
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frogs and their relatives (Amphibia: Athesphatanura: Dendrobatidae). B. Am. Mus. Nat. Hist. 299, 1–262. Moresco, R.M., Oliveira, C., 2009. A comparative study of the extracutaneous pigmentary system in three anuran amphibian species evaluated during the breeding season. S. Am. J. Herpetol. 4, 1–8. Oliveira, C., Zanetoni, C., Zieri, R., 2002. Morphological observations on the testes of Physalaemus cuvieri (Amphibia, Anura). Rev. Chil. Anat. 20, 263–268. Oliveira, C., Santanna, A.C., Omena, P.M., Santos, L.R.S., Zieri, R., 2003. Morphological considerations on the seminiferous structures and testes of anuran amphibians: Bufo crucifer, Physalaemus cuvieri and Scinax fuscovarius. Biociências 11, 39–46. Oliveira, C., Zieri, R., 2005. Pigmentac¸ão testicular em Physalaemus nattereri (Steindachner) (Amphibia, Anura) com observac¸ões anatômicas sobre o sistema pigmentar extracutâneo. Rev. Bras. Zool. 22, 454–460. Pederzoli, A., Trevisan, P., 1990. Pigmentary system of the adult alpine Salamander atra aurorae. Pigm. Cell Res. 3, 80–89. R Development Core Team, 2010. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available from: http://www.R-project.org (accessed 20.06.10.). Sichel, G., Scalia, M., Mondio, F., Corsaro, C., 1997. The amphibian Kupffer cells build and demolish melanosomes: an ultrastructural point of view. Pigm. Cell Res. 10, 271–287. Sokal, R.R., Rohlf, F.J., 1995. Biometry: The Principles and Practice of Statistics in Biological Research, 3rd ed. W.H. Freeman and Company, New York. Trevisan, P., Pederzoli, A., Barozzi, G., 1991. Pigmentary system of the adult alpine salamander Salamandra atra atra. Pigm. Cell Res. 4, 151–157. Zieri, R., Taboga, S.R., Oliveira, C., 2007. Melanocytes in the testes of Eupemphix nattereri (Anura, Leiuperidae): histological, estereological and ultrastructural aspects. Anat. Rec. Part A 290, 795–800. Zuasti, A., Ferrer, C., Aroca, P., Solano, F., 1990. Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicentrarchus labrax (Pisces, Teleostei). Pigm. Cell Res. 3, 126–131. Zuasti, A., Jiménez-Cervantes, C., García-Borrón, J.C., Ferrer, C., 1998. The melanogenic system of Xenopus laevis. Arch. Histol. Cytol. 61, 305–316.
Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): Occurrence and comparison Lilian Franco-Belussia , Lia Raquel de Souza Santosb,1 , Rodrigo Zieria , Classius de Oliveirac,∗ a
Post-graduation Program in Animal Biology, IBILCE-UNESP/SJRP São Paulo State University (UNESP), campus São Jose do Rio Preto, Institute of Bioscience, Letters and Exact Sciences, Department of Biology, Brazil2 b Federal Institute of Goiás, IFG, campus Rio Verde, Goiás, Brazil c Department of Biology, IBILCE-UNESP/SJRP, Brazil Received 1 April 2009; received in revised form 13 July 2010; accepted 1 December 2010
Abstract Amphibians share with other ectothermic vertebrates an extracutaneous pigmentary system consisting of melanin-containing cells in various organs and tissues. This paper describes the interspecific variation in the visceral pigmentation and extracutaneous pigment system in four species of the genus Dendropsophus [i.e., D. elianeae, D. minutus, D. nanus, and D. sanborni (Anura: Hylidae)]. Fifteen adult males from each species were collected in the region of São José do Rio Preto (State of São Paulo, Brazil), and their visceral pigmentation was analyzed during the reproductive period. The individuals were weighed and measured, and the pigmented visceral cells were classified and documented in photographs. The shape and quantity of the pigment cells differed among the various structures of the same individual, as well as among the same structures of different species. Similarities and differences among these species were observed, and a visceral pigmentation pattern for anatomical structures was detected. In the digestive system of all species analyzed, there was an absence of pigment cells on the stomach and middle intestine. However, the pigmentation of the final portion of the intestine (i.e., the rectum) showed interspecific variation, with D. minutus presenting intense pigmentation, whereas the other species presented no pigmentation. Significant differences were detected also in the cardio-respiratory system, mesentery, and lumbosacral peritoneum. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. Keywords: Anurans; Extracutaneous pigmentary system; Melanocytes
1. Introduction Amphibians and other ectothermic vertebrates possess an extracutaneous pigmentary system in various tissues and
∗ Corresponding author at: Department of Biology, Institute of Bioscience, Letters and Exact Sciences, UNESP, São Paulo State University, CEP: 15040-000 São José do Rio Preto, São Paulo, Brazil. Fax: +55 17 3221 2390. E-mail address: [email protected] (C. de Oliveira). 1 Current address: Federal Institute of Goiás,IFG,campus Rio Verde GO,CEP: 75.908-000 Rio Verde, Goiás, Brazil. 2 www.ibilce.unesp.br.
organs, which comprises cells, whose cytoplasm contains melanin (Gallone et al., 2002). These melanin-containing cells are frequently found in the liver, spleen, kidneys, peritoneum, lung, heart, blood vessels, thymus, gonads, and meninges of fishes, amphibians, and reptiles (Agius, 1980; Agius and Agbede, 1984; Zuasti et al., 1990, 1998; Christiansen et al., 1996; Bagnara and Matsumoto, 2006; Gallone et al., 2002; Zieri et al., 2007; Pederzoli and Trevisan, 1990; Trevisan et al., 1991). These cells are similar to melanocytes (Zuasti et al., 1998; Agius and Agbede, 1984), which are derived from the neural crest (Bagnara and Matsumoto, 2006) and are able to produce and store melanin inside their cytoplasm (Agius and Roberts, 2003). In organs
0044-5231/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2010.12.001
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with hematopoietic function, there are also cell types with phagocytic activity similar to that of macrophages (Agius, 1980). These cells are derived from hematopoietic stem cells (Sichel et al., 1997), which often conglomerate to form pigmented nodules called melanomacrophage centers (Agius, 1981). Current knowledge about visceral pigmentation, be its structural features or its diversity, is superficial, with the exception of a few morphological studies in anurans, such as Physalaemus marmoratus (Aoki et al., 1969; Oliveira and Zieri, 2005), Physalaemus cuvieri (Oliveira et al., 2002, 2003), Eupemphix nattereri (Oliveira and Zieri, 2005; Zieri et al., 2007), Rana esculenta (Cicero et al., 1989; Sichel et al., 1997; Barni et al., 1999; Gallone et al., 2002; Barni et al., 2002), Rana ridibunda (Akulenko, 1998) and, Xenopus laevis (Sichel et al., 1997; Zuasti et al., 1998). Our aim in this study was to characterize the visceral pigmentation in the cardiorespiratory, digestive and urogenital systems and their associated serosal membranes in four tree-frog species of the hylid genus Dendropsophus (i.e., D. elianeae, D. minutus, D. nanus, and D. sanborni) during their reproductive periods. We also evaluated the intra- and interspecific differences and searched for patterns of occurrence of pigmentation that could help to distinguish related species. Such an approach will increase our knowledge about the extracutaneous pigmentary system in neotropical anurans. The specimens were treated according to the ethical guidelines of São Paulo State University – UNESP/Botucatu, following the Guide for Care and Use of Laboratory Animals (Protocol No. 001/06-CEEA). In the laboratory, the specimens were anesthetized and euthanized by submersion in 20% ethanol. All specimens had the organs of the abdominal cavity exposed by medial incision for macroscopic documentation under a stereomicroscope (Leica-MZ16), using the program Image Manager 5.0 (Version 4.0.5, Media Cybernetics Inc., Bethesda, MD, USA) to capture the images.
2. Materials and methods Fifteen male specimens of each species (D. elianeae, D. minutus, D. nanus, and D. sanborni) were collected in the region of São José do Rio Preto (State of São Paulo, Brazil) during their reproductive period from October 2006 to March 2007. Some of the specimens used were from the Amphibian Collection of the Department of Botany and Zoology (DZSJRP-Amphibia) at the São Paulo State UniversityIBILCE/UNESP (D. elianeae: DZSJRP 6368, 7646, 7890, 7965, 7968–9, 8016, 8037, 8089, 8124, 8474–6, 8232, 8532). Visceral pigment cells were classified according to the protocol of Franco-Belussi et al. (2009), based on the differences in the intensity of pigmentation present on the gonads of anurans, and applied to other organs and tissues in this study. The pigmentary classification is based on the intensity of pigmentation, ranging from absence (category 0) to entirely pigmented, when an intense black coloration is observed (cat-
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egory 3 = maximal intensity), with the categories 1 and 2 representing a gradual increase in the intensity of pigmentation. For this study, the protocol of classification for the visceral pigmentation was applied to the following anatomical structures and regions of the specimens as follows: (1) pericardium and blood vessels at the basis of the heart; (2) heart; (3) lungs; (4) stomach; (5) middle intestine; (6) rectum; (7) intestinal mesentery, (8) kidney and renal blood vessels; (9) testes; (10) nerves of the lumbar plexus; (11) lumbosacral parietal peritoneum. To compare the differences between categories of pigmentation in each organ or region we used the G test for goodness of fit, with the corrections by Yates and Williams (Sokal and Rohlf, 1995). This test was implemented using the code provided by Prof. Peter Hurd available at http://www.psych.ualberta.ca/∼phurd/cruft/g.test.r. The test was run using the R software v. 2.11.1 (R Development Core Team, Informer Technology Inc. 2010).
3. Results Visceral pigment cells in the various organs and membranes of the abdominal cavity varied significantly in their occurrence and distribution among the species analyzed, allowing a classification of this pigmentation according to the intensity of pigmentation on an organ’s surface. This pigmentary classification is shown for D. elianeae, D. minutus, D. nanus, and D. sanborni in Fig. 1. There is no pigmentation (category 0) on the pericardium, heart, vertebral column, and kidneys of D. minutus, and on the lungs of D. sanborni. There is an intense black pigmentation (category 3) on the rectum of D. minutus. There are a few pigment cells creating only a little pigmentation (category 1) on the pericardium, heart, and vertebral column of D. nanus and on the lungs, kidneys, and rectum of D. elianeae. There is a large number of pigmented cells, which mask the color of the organ (category 2), on the kidneys of D. elianeae. The pigmentation on organs and structures of the abdominal cavity in the four species (D. elianeae, D. minutus, D. nanus and D. sanborni) are summarized in Figs. 2–5 and Table 1. The pigmentation of the pericardium revealed significant interspecific differences (G-test: G = 23.56; df = 9; p = 0.005), heart (G-test: G = 61.93; df = 9; p < 0.01), and lungs (G-test: G = 49.54; df = 9; p < 0.01) (Fig. 2). All 15 individuals of D. elianeae presented no visceral melanocytes (category 0) on the pericardium and cardiac blood vessels. In addition, 14 out of 15 individuals of D. minutus showed no visceral melanocytes, although one specimen presented only a little pigmentation (category 1). On the other hand, only 8 out of 15 specimens of D. nanus and 6 out of 15 specimens of D. sanborni presented no pigmentation. Only a little pigmentation (category 1) was found in 6 specimens of D. nanus and in 8 specimens of D. sanborni, and one specimen of each of those species presented a large number of pigmented cells
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Fig. 1. Photographs of organ surfaces and regions arranged according to the intensity of their pigmentation. Empty spaces represent absence of that pigmentation category. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation. Abbreviations: D nan, Dendropsophus nanus; D eli, D. elianeae; D min, D. minutus; D san, D. sanborni.
(category 2). All specimens of D. minutus, as well as 2 out of 15 specimens of D. elianeae, showed no visceral melanocytes on the heart. In contrast, 9 out of 15 specimens of D. elianeae presented only a little pigmentation (category 1) and 4 out of 15 specimens presented a large number of pigmented cells (category 2). In D. sanborni, 4 out of 15 specimens presented no pigmentation (category 0) in this region, and 11 out of 15 specimens presented only a little pigmentation (category 1). In contrast, the heart of all specimens of D. nanus was pigmented. The heart of 6 out of 15 specimens had a few visceral melanocytes (category 1), and the heart of 9 out of 15 specimens had a large number of pigment cells (category 2) (Table 1). On the lungs, 14 out of 15 specimens of D. minutus and D. sanborni showed no pigmentation (category 0) and 1 out of 15 specimens presented only a little pigmentation (category 1).
In D. nanus, 6 out of 15 specimens showed no pigmentation (category 0) in this region, whereas 9 out of 15 specimens presented only a little pigmentation (category 1). In contrast, the lungs of all specimens of D. elianeae were pigmented to some degree. The lungs of 13 out of 15 individuals had very little pigmentation (category 1), but 2 out of 15 specimens had a high degree of pigmentation (category 2) (Table 1). On the digestive system (Fig. 3), no pigment cells were observed on the stomach of all specimens of the four species analyzed. All specimens of D. nanus, D. sanborni and D. minutus presented no pigmentation on the middle intestine, whereas 1 out of 15 specimens of D. elianeae presented a few pigmented cells (category 1) in this region. A significant variation (G-test: G = 72.12; df = 9; p < 0.01) was observed on the terminal portion of the digestive system (i.e., the rectum). In this region, there was an intense pigmentation (category
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Fig. 2. Visceral pigmentation on the organs and associated structures of the cardio-respiratory system of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
3) in all specimens of D. minutus, in contrast to an absence of pigmentation in 12 out to 15 specimens of D. elianeae. In addition, 14 out of 15 individuals of D. nanus, and all specimens of D. sanborni, presented no pigmentation. In D. elianeae, 3 out of 15 specimens presented only a little pigmentation (category 1) and in D. nanus, only 1 out of 15 individuals presented this category (Table 1). On the urogenital system (Fig. 4), a complete absence of pigmentation was observed on the testes, urinary bladder, and fat bodies of all species. However, the kidneys presented a significant variation (G-test: G = 23.97; df = 9; p = 0.004). In D. minutus, 8 out of 15 specimens presented no pigmentation, 4 out of 15 specimens presented only a little pigmentation (category 1), and 3 out of 15 specimens presented a high degree of pigmentation (category 2). On the other hand, 5 out of 15 specimens of D. nanus presented no pigmentation, whereas 4 out of 15 specimens presented only a little pigmentation (category 1), and 6 out of 15 specimens presented a high degree of pigmentation (category 2). In D. sanborni, 5 out of 15 specimens presented no pigmentation in this region, however 10 out of 15 specimens presented only a little pigmentation (category 1). In D. elianeae, 2 out of 15 specimens presented no pigmentation on the kidneys, 3 out of 15 speci-
mens presented only a little pigmentation (category 1), and 10 out of 15 specimens presented a high degree of pigmentation (category 2) (Table 1). The occurrence of pigment cells is constant and does not vary significantly on the lumbar nerve plexus (G-test: G = 16.64; df = 9; p = 0.06). In D. elianeae, 14 out of 15 specimens showed no pigmentation in this region, and 1 out of 15 specimens presented only a little pigmentation (category 1). In D. nanus and D. minutus, 8 out of 15 specimens presented no pigmentation in this region, whereas 5 out of 15 specimens possessed only a little pigmentation (category 1), and 2 out of 15 specimens presented a high degree of pigmentation (category 2). In D. sanborni, 6 out of 15 specimens showed no pigmentation in this region and 9 out of 15 specimens presented only a little pigmentation (category 1) (Fig. 5). Interspecific variation is significant on the lumbosacral parietal peritoneum (G-test: G = 49.82; df = 9; p < 0.01) and intestinal mesentery (G-test: G = 25.63; df = 9; p = 0.002). In the lumbosacral parietal peritoneum, all specimens of D. elianeae presented no pigmentation. 13 out of 15 specimens of D. nanus showed no pigmentation, but 2 out of the 15 specimens presented only a little pigmentation (category 1). In D. minutus, an absence of pigmentation was observed in
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Fig. 3. Visceral pigmentation on the digestive system (i.e., stomach, middle intestine, and rectum) of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
Fig. 4. Visceral pigmentation on the urogenital system of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
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Fig. 5. Visceral pigmentation on the peritoneum, mesentery, and lumbar nerve plexus of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni showing the number of individuals with particular categories of pigmentation. Category 0 = absence of pigmentation; category 1 = few pigmented cells; category 2 = a great number of pigmented cells; category 3 = intense pigmentation.
10 out of 15 specimens, whereas 3 out of 15 specimens presented only a little pigmentation (category 1) in this region. In contrast, all individuals of D. sanborni presented no pigmentation, and 12 out of 15 specimens presented only a little pigmentation (category 1), and 3 out of 15 specimens presented a high degree of pigmentation. On the intestinal mesentery, 12 out of 15 specimens of D. nanus presented no pigmentation (category 0) in this region, whereas 2 out of 15 specimens had only a little pigmentation (category 1), and 1 out of 15 specimens presented a high degree of pigmentation (category 2). In D. sanborni, 6 out of 15 specimens presented an absence of pigmentation, and 9 out of 15 specimens presented only a little pigmentation. In D. minutus, 9 out of 15 specimens showed no pigmentation (category 0) in this region, but 6 out of 15 specimens presented a high degree of pigmentation (category 2). In D. elianeae, 10 out of 15 specimens presented an absence of pigmentation (category 0) on the intestinal mesentery, whereas 3 out of 15 specimens presented only a little pigmentation (category 1), and 2 out of 15 specimens presented a high degree of pigmentation (category 2) (Table 1). When comparing the four species (D. elianeae, D. minutus, D. nanus and D. sanborni) (Table 1), it is possible to observe that D. minutus presents the highest number of regions without any pigmentation (category 0), but this species was the only one that presented a region (i.e., rectum) with intense black coloration (category 3). In contrast, D. nanus is the species that possesses the highest number of regions with pigmentation mostly in the categories 1 and 2. The species
D. sanborni and D. elianeae have similar degrees of pigmentation.
4. Discussion Cells containing melanin are found not only in the skin, but also in other organs constituting the extracutaneous pigmentary system. In this study, pigment cells were observed in the cardio-respiratory system, as well as in the intestinal mesentery, lumbosacral peritoneum, lumbar nerve plexus, and kidneys. The visceral pigment cells are found in the connective tissue of the capsule and interstitium of organs or are associated with the adventitious tunics or serous membranes. They are large, approximately 150 m in diameter, and irregular in shape with an abundantly and intensely pigmented cytoplasm. They may have a dendritic shape due to cytoplasmic processes or a round shape in the absence of cytoplasmic processes (Oliveira and Zieri, 2005). The interspecific differences in the degree of pigmentation are statistically significant in most organs and structures, except in the lumbar nerve plexus and middle intestine. Even though our study comprises only a limited number of representatives of each species, it was still possible to discover peculiarities and variations that distinguish the species. Thus, by determining the occurrence and density of the visceral melanocytes in different organs and structures, significant differences could be found to facilitate the identification and differentiation of species. In contrast, Grant
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Table 1. Comparison of the number of individuals of Dendropsophus nanus, D. elianeae, D. minutus and D. sanborni, which exhibit particular categories of visceral pigmentation on the pericardium and blood vessels at the basis of the heart, heart, lungs, stomach, middle intestine, rectum, intestinal mesentery, kidney and renal blood vessels, testes, nerves of the lumbar plexus, and lumbosacral parietal peritoneum. Organs and regions
Pericardium and blood vessels
Heart
Lung
Stomach
Intestine
Rectum
Kidney and blood vessel
Testes
Lumbar nerve plexus
Lumoar Kanetai Kentonium Mesenleriurn
Vesicula Urinaria
Species
D. nanus D. sanbomi D. minutus D. elianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. elianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. elianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae D. nanus D. sanbomi D. minutus D. etianeae
Categories Category 0
Category 1
Category 2
8 6 14 15
6 8 1
1 1
6 11
9
9 9 1 1 13
4
4 15 2 6 14 14 15 15 15 15 15 15 15 14 14 15
Category 3
2
1 1 15
12 5 5 8 2 15 15 15 15 8 6 8 14 13 10 15 12 6 9 10 15 15 15 15
et al. (2006) used only the pigmentation on the intestine as a character in a phylogenetic study. In their study, Grant et al. (2006) observed three different degrees of the pigmentation on the intestine and testes of Dendrobatidae species, depend-
3 4 10 4 3
6 3 10
5 9 5 1 2 12 3
2
2 9
1
3
2
3 2
6 2
ing on the distribution of pigments on the organ surface, but did not consider differences in the number of pigment cells. These differences were not correlated to body size, pigmentation of the ventral body surface, or sexual maturity and
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were interpreted as evidence of additivity during ontogeny. This character invariably progresses from an absence of pigmentation to a presence confined to the anterior quarter of the intestine and later extends beyond the middle of the final portion of intestine (i.e., rectum). The absence of pigmentation (category 0) on the testes is a conspicuous feature of the Dendropsophus species analyzed, giving the gonads a white appearance, which was also observed in some individuals of the family Leptodactylidae [Leptodactylus bokermanni, Leptodactylus furnarius, Leptodactylus fuscus, Leptodactylus labyrinthicus, Leptodactylus mystaceus, Leptodactylus mystacinus, Leptodactylus latrans, Leptodactylus podicipinus, Leptodactylus chaquensis, and Leptodactylus notoaktites (Franco-Belussi et al., 2009)]. In contrast, in Leiuperidae species (P. cuvieri, Physalaemus olfersii, Physalaemus centralis, Physalaemus marmoratus, E. nattereri, Pseudopaludicola cf. falcipes and Physalaemus saltica), the testicular capsule is abundantly and intensely pigmented and appears intensely black (category 3) (FrancoBelussi et al., 2009). P. marmoratus (Aoki et al., 1969; Oliveira and Zieri, 2005), P. cuvieri (Oliveira et al., 2002, 2003), and E. nattereri (Oliveira and Zieri, 2005; Zieri et al., 2007) bear a large number of melanocyte-like pigment cells in the testicular interstitium. But a large number of anuran species do not possess any pigment cells in the gonads, although some authors (Oliveira and Zieri, 2005; Zieri et al., 2007; Franco-Belussi et al., 2009; Moresco and Oliveira, 2009) have described a close relationship of visceral melanocytes with testicular blood vessels. Melanocytes are associated also with blood vessels from other organs and connective tissue membranes (i.e., pericardium, intestinal mesentery, and lumbosacral parietal peritoneum). Especially in E. nattereri and P. marmoratus, the density of pigment cells in the interstitium and albugineous tunic of the gonads is intense, giving the testes a dark brown color (Oliveira and Zieri, 2005; Zieri et al., 2007). The species analyzed in this study did not present pigmentation on the testes. However, Moresco and Oliveira (2009) described a little pigmentation (category 1) in Rhinella schneideri, and an intense black coloration (category 3) in P. cuvieri during the breeding season. On the pericardium, an absence of pigmentation is a unique feature of D. minutus and D. elianeae, whereas there is a predominance of only a little pigmentation (category 1) in D. sanborni and D. nanus, similar to what was reported by Moresco and Oliveira (2009) for R. schneideri and P. cuvieri. Moresco and Oliveira (2009) also observed that there is no pigmentation on the heart of R. schneideri and P. cuvieri (category 0), similar to what was observed in this study for D. minutus. However, on the pericardium of D. elianeae and D. sanborni, only a little pigmentation (category 1) predominates, and on that of D. nanus, a high degree of pigmentation (category 2) predominates. On the lungs, there is no pigmentation (category 0) in D. minutus and D. sanborni, similar to what was described by
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Moresco and Oliveira (2009) for R. schneideri. However, D. elianeae and D. nanus present only little pigmentation, resembling P. cuvieri at the beginning of the breeding season as described by Moresco and Oliveira (2009). But in this case, the pigmentation varies during the breeding season, being predominantly absent at the end of the season (Moresco and Oliveira, 2009). On the stomach and middle intestine, the absence of pigmentation was observed in all individuals of the four species, a fact also observed by Moresco and Oliveira (2009) in R. schneideri. On the rectum, the absence of pigmentation is predominant in D. elianeae, D. sanborni, and D. nanus, as already described by Moresco and Oliveira (2009) for R. schneideri, but D. minutus presents an intense pigmentation (category 3) in all individuals analyzed. Only this species presented category 3 of pigmentation on the rectum. On the kidneys, the pigmentation is highly variable ranging from total absence (category 0) to a high degree of pigmentation (category 2) in all species analyzed. In D. minutus, the absence of pigmentation (category 0) is predominant, as was also reported for R. schneideri (Moresco and Oliveira, 2009). In D. elianeae, there is a high degree of pigmentation (category 2), whereas D. sanborni presents only a little pigmentation (category 1). In D. nanus, there is great variation in the degree of pigmentation from individuals without pigmentation (category 0) to individuals with only a little pigmentation (category 1), and individuals with a high degree of pigmentation (category 2). This variability may be related to the breeding season of these individuals, since in P. cuvieri the pigmentation in this region varies during the reproductive cycle (Moresco and Oliveira, 2009). Morphofunctional aspects of visceral melanocytes in ectothermic animals are still poorly understood. Although the pigmentation on an organ’s surface is similar among anuran species, the degree of pigmentation varies in certain regions and, therefore, can be used to distinguish similar species. These pigment cells may also vary according to physiological responses of the metabolism due to the presence of different substances within the melanocytes. Nevertheless, this visceral pigmentation may constitute intrinsic features of species.
Acknowledgments We would like to thank the São Paulo State Research Foundation (FAPESP) for funding this project (Proc. # 02/08016-9, 05/02919-5, 06/57990-9 and 08/52389-0) and also to Diogo B. Provete for critical reviewing the manuscript and helping with the English version.
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