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Wildlife Diseases JOURNAL OF WILDLIFE DISEASES INCIDENCE OF PIGMENTED SKIN TUMORS IN A POPULATION OF WILD MONTSENY BROOK NEWT (CALOTRITON ARNOLDI ) Albert Martı´nez-Silvestre,1,5 Fe`lix Amat,2 Ferran Bargallo´,3 and Salvador Carranza4 1 CRARC (Catalonia Reptile and Amphibian Rehabilitation Centre), Santa Clara s/n, 08783 Masquefa, Barcelona, Spain 2 A` rea d’Herpetologia, Museu de Granollers – Cie`ncies Naturals, Francesc Macia`51, 08400, Granollers, Barcelona, Spain 3 Hospital Zoolo`gic Badalona, 08912, Conquista, 74, Badalona, Barcelona, Spain 4 Institute of Evolutionary Biology (CSIC-UPF), Passeig Marı´tim de la Barceloneta, 37-49 08003 Barcelona, Spain 5 Corresponding author (email: [email protected]) Journal of Wildlife Diseases, 47(2), 2011, pp. 410–414 # Wildlife Disease Association 2011 INCIDENCE OF PIGMENTED SKIN TUMORS IN A POPULATION OF WILD MONTSENY BROOK NEWT (CALOTRITON ARNOLDI ) Albert Martı´nez-Silvestre,1,5 Fe`lix Amat,2 Ferran Bargallo´,3 and Salvador Carranza4 1 CRARC (Catalonia Reptile and Amphibian Rehabilitation Centre), Santa Clara s/n, 08783 Masquefa, Barcelona, Spain 2 A` rea d’Herpetologia, Museu de Granollers – Cie`ncies Naturals, Francesc Macia`51, 08400, Granollers, Barcelona, Spain 3 Hospital Zoolo`gic Badalona, 08912, Conquista, 74, Badalona, Barcelona, Spain 4 Institute of Evolutionary Biology (CSIC-UPF), Passeig Marı´tim de la Barceloneta, 37-49 08003 Barcelona, Spain 5 Corresponding author (email: [email protected]) ABSTRACT: We report the presence of pigmented skin tumors in three populations of the endangered amphibian Montseny brook newt, Calotriton arnoldi, one of the European amphibian species with the smallest distribution range (40 km2 in the Montseny Natural Park, Catalonia, Spain). Examination of one of the tumors by light microscopy was consistent with chromatophoroma and was most suggestive of a melanophoroma. Tumors were not found in juveniles. In adults, only two of three populations were affected. The proportions of males and females affected were not significantly different, but there was a positive correlation between body size and presence of tumors in both sexes. The etiology of chromatophoromas remains unknown but, in our study, they do not appear to have been caused by water quality or Ultraviolet B. Key words: Calotriton arnoldi, chromatophoroma, dermatology, emergent diseases, melanocytoma. INTRODUCTION Iberian Peninsula). Despite significant ef- forts over the last 5 yr to find more, Many factors negatively impact amphib- only seven populations are currently known. ians worldwide including habitat modifica- The total number of adults has been tion and loss, environmental contamina- estimated at less than 1,500 individuals. tion, climate change, infectious diseases, These newts are entirely aquatic and live commercial exploitation, and invasive spe- in fast-flowing steams in forested areas at cies (Crump, 2009). How these factors altitudes between 620 and 1,200 m (Amat present within the population may vary but, and Carranza, 2006). Based on its extre- in some cases (e.g., exposure to certain mely small range and population size, contaminants, changes in temperatures, Calotriton arnoldi is considered an endan- various pathogens), may result in skin gered species (Carranza, 2008). The demog- abnormalities including neoplasia. Skin raphy, activity, genetics, and morphology of neoplasms have been reported rarely in some populations are being investigated to amphibian field surveys (Khudoley and increase our knowledge of the species and to Mizgireuv, 1980) and are usually attributed plan appropriate conservation measures. to environmental degradation of streams, The spread of emergent diseases is becom- increase in temperature, or chromosomal ing a serious threat to amphibian conserva- alterations (Zavanella and Arias, 1982; tion worldwide, especially in extremely Zavanella, 1985; Wheeler et al., 2002; endangered species such as Calotriton Miller and Miller, 2005). Most spontaneous arnoldi. Part of the recovery plan (Carranza, skin neoplasms in newts have been de- 2008) is to investigate the existence of scribed from captive populations (Kous- diseases in this species and, if found, to soulakos et al., 1994). characterize them and analyze their popu- The Montseny brook newt (Calotriton lation incidence. arnoldi) was described by Carranza and Amat (2005). It is restricted to a geo- MATERIALS AND METHODS 2 graphic area of ,40 km within the Our study was performed with wild newts Montseny mountain massif (North Eastern examined from populations at three sites, 410 MARTI´NEZ-SILVESTRE ET AL.—MELANOCYTOMA IN THE NEWT CALOTRITON ARNOLDI 411 referred to herein as populations one, two, TABLE 1. Sample size of examined Montseny brook and three. Population one inhabits a small newts (Calotriton arnoldi) collected from Montseny stream in a beech forest at 1,200 m on a Natural Park, Catalonia, Spain, by population and south-facing mountain slope. Population two age class. is found in a tiny stream inside a mixed forest of oak and alder at 800 m in a north-facing Population Adults Juveniles Total valley that experiences severe summer drought. Population three is near the second One 98 9 107 population and is, therefore, at approximately Two 33 13 46 the same altitude and in the same habitat. Three 50 0 50 However, the stream where the newts live is Total 181 22 203 wider and deeper than the stream from population two. All of these streams are located at 41u459290N, 2u259460E. RESULTS Sampling was conducted at night, in spring and autumn, and included 21, five, and 19 We examined 203 newts (181 adults and visits to populations one, two, and three, 22 juveniles) from the three populations respectively, during 2006–2008. We caught (Table 1). Macroscopically, skin lesions newts by hand, determined their sex using secondary sexual characters (morphology of were intense, black, solitary, circum- the cloacal area and tail height), measured scribed, and irregularly defined nodules snout-vent length (SVL), and marked them with a diameter of 1–4 mm (Fig. 1). using Visible Implant Elastomers (VIE; North- Histologic examinations of samples from west Marine Technology, Inc., Seattle, Wash- the euthanized newt confirmed that the ington, USA). Newts were visually examined for body abnormalities (supranumery legs or skin formations were nonencapsulated digits, scoliosis, or double tail) and their chromatophoromas resulting from prolif- numbers and body positions were recorded. eration of cells containing black-brown One of the most-affected individuals was pigment. Although not confirmed by captured and euthanized with phenobarbital electron microscopy or special histo- (100 mg/kg intracoelomic) for a complete pathologic study. Samples of approximately chemic or immunohistochemic tech- 10 3 5 3 5 mm of skin and internal organs niques, this pigment was consistent with (esophagus, stomach, intestine, liver, gall melanin (Fig. 2). The tumor was restricted bladder, spleen, pancreas, adipose tissue, to the dermis and did not invade the striated muscle, brain, spinal cord, spinal underlying lymph sacs or internal organs, nerve, ganglia, kidney, ureter, testis (active), epididymis, deferent duct, bones [vertebrae, thus suggesting it was benign. Because skull, full posterior limb], heart, arteries, histopathologic results and biologic behav- cloaca, oropharynx, ear, nasal cavity, trachea, ior suggested a benign mass, these skin and lung) were removed. Tissues were fixed in nodules were considered melanocytomas. neutral-buffered 10% formalin, embedded in The nodes were restricted to the dermal paraffin, sectioned at 5 mm for light microsco- and epidermal area and were limited to py, and stained with hematoxylin and eosin for histopathologic examination. the dermis; invasion of underlying lymph Data were analyzed using conventional sacs was not observed and the cellular parametric statistics after log transformation proliferations did not affect internal or- of continuous variables. We used analysis of gans or body structures. In field surveys, variance (ANOVA) to evaluate differences in 27% (29 individuals) of the newts were body size among normal specimens and in newts with nodules. We investigated the affected by skin tumors in population one. relationship between body size and number In population two, only one individual, an of skin nodules using a Pearson parametric adult male, had skin tumors (2%) and no correlation. A chi-square test was used to test affected specimens were found in popula- differences in the incidence of body abnor- tion three. No skin tumors were found in malities between age classes (adults versus juveniles) and sexes. We also used this test to juvenile newts. infer the co-occurrence of different kinds of Adults in population one were signifi- body abnormalities. cantly more affected than juveniles (adult 412 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 2, APRIL 2011 FIGURE 1. Photographs of Montseny brook newts (Calotriton arnoldi) collected from Montseny Natural Park, Catalonia, Spain showing pigmented masses consistent with chromatophoromas (circled areas). Bar51 cm. prevalence531%). We did not find dif- ferences in the prevalence of skin tumors 5 5 between sexes (chi-square 0.018; df 1; FIGURE 2. Section of a pigmented skin mass P50.893). collected from a Montseny brook newt (Calotriton The mean (6SE) number of skin tumors arnoldi) collected from Montseny Natural Park, per affected newt was 2.360.3 (range 1– Catalonia, Spain. The pigment was most consistent 10). Tumors were present
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