THE ANATOMICAL RECORD 292:178–182 (2009) Pigmentation in Anuran Testes: Anatomical Pattern and Variation LILIAN FRANCO-BELUSSI, RODRIGO ZIERI, LIA RAQUEL DE SOUZA SANTOS, RAFAELA MARIA MORESCO, AND CLASSIUS DE OLIVEIRA* Department of Biology, Sao Paulo State University (UNESP), IBILCE, Sao Jose do Rio Preto, Sao Paulo, Brazil ABSTRACT 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 Ó 2008 WILEY-LISS, INC. 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. E-mail: firstname.lastname@example.org Received 5 January 2008; Accepted 22 September 2008 DOI 10.1002/ar.20832 Published online 16 December 2008 in Wiley InterScience (www. interscience.wiley.com). PIGMENTATION IN ANURAN TESTES 179 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. RESULTS AND DISCUSSION 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 saltica. Fig. 3. Differences of intensity in pigmentation represented by each category in the testes of Leiuperidae: Physalaemus marmoratus, Eupemphix nattereri, and Physalaemus olfersii. 180 FRANCO-BELUSSI ET AL. Figure 1. (Legend on page 179.) Figure 2. (Legend on page 179.) Figure 3. (Legend on page 179.) PIGMENTATION IN ANURAN TESTES Fig. 4. Occurrence of pigmentation in testes of species of Leptodactylidae. Fig. 5. Occurrence of testicular pigmentation in Leiuperidae. 181 182 FRANCO-BELUSSI ET AL. 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. 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