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 )

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 differences 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 more frequently with melanin. The mass was well-demarcated, con- fined to the dermis, and most suggestive of a benign on the back and flanks than on other body mass. Hematoxylin and eosin stain. Bar51 mm. areas (Table 2). Body size was positively correlated with the number of skin tumors (r50.224; P50.026; n530). Mean SVL in mass. However, we do not know if all of the affected adult newts (61.360.4: range 56– pigmented skin tumors in the wild Mont- 67) was significantly larger than in unaf- seny brook newts we examined were fected individuals (59.360.3; range 53–65; melanophoromas, nor do we know if ANOVA, F1,96511.977; P,0.001). malignant pigmented tumors occur in this species. The absence of skin tumors in DISCUSSION immature newts, the correlation between SVL and the number of skin tumors, and We report the occurrence of pigmented the larger body size of affected newts skin tumors in three wild populations of the suggest a progressive development of pig- Montseny brook newt (Calotriton arnoldi). mented skin tumors throughout an individ- The tumor examined by light microscopy ual’s life. A similar pattern was observed in was consistent with a chromatophoroma Mexican axolotls (Ambystoma mexicanum), and most suggestive of a melanophoroma. which showed a high correlation between The morphology of the tumor was consis- age and the presence of melanophoromas tent with observations that it was a benign (Khudoley and Mizgireu, 1980). In that MARTI´NEZ-SILVESTRE ET AL.—MELANOCYTOMA IN THE NEWT CALOTRITON ARNOLDI 413

TABLE 2. Descriptive statistics (mean6standard error Another possible cause for the tumors is and range) of the numbers of chromatophoromas by body area and number of affected individuals (n530) of ultraviolet-B (UV-B) radiation. We found Montseny brook newts (Calotriton arnoldi) collected significantly more pigmented skin tumors from Montseny Natural Park, Catalonia, Spain. on the dorsum than on the ventrum of the newts. Both body areas have approximate- No. of Tumors per ly the same surface area, but only the affected individual Body area newts (mean6SE) Range dorsum is exposed to sunlight. Neverthe- less, the dorsum of this species is heavily Head 1 1 Back 16 1.960.17 1–3 pigmented and, hence, has a higher Flanks 16 1.360.19 1–4 density of chromophores than the trans- Venter 7 1.460.20 1–2 lucent ventrum, thereby favoring the Limbs 2 1.560.50 1–2 development of pigmented tumors in this Tail 4 3.260.75 2–5 area. Nevertheless, newts are aquatic and largely nocturnal stream dwellers, and the stream inhabited by this population is study, axolotls developed proliferation of protected in summer, when solar radiation melanophores as a nodular growth (as in is more intense, by a dense cover of beech our case) at 2.5 mo of age and developed trees. These conditions would make the additional masses 16 mo later. However, UV-B hypothesis seem less likely. the authors could not correlate the pres- As a consequence of climate change, the ence of chromatophoromas with body size, Montseny massif has experienced a rise in hereditary predisposition, or other physical environmental temperatures that has af- causes. fected the altitudinal succession of forest A thorough analysis of water quality for or butterfly communities (Pen˜ uelas and all seven streams where the Montseny Boada, 2003; Stefanescu et al., 2004). brook newt occurs was carried out to During the last century, its forests were check for any traces of metal and positive heavily exploited to make charcoal and to or negative ions (F. Amat and S. Carranza, make space for cultivation. As a result, the unpubl. data). The analyses showed no landscape of the Montseny mountain traces of water pollution (chemical or massif consisted of a mosaic of small biological), and water composition (dis- patches of forest, farms, and pastures solved solids, mineral contents, pH, etc.) (Boada, 1994). In the last 50 yr, however, did not differ between the stream inhab- the abandonment of traditional agricultur- ited by the significantly affected popula- al and forest exploitation has resulted in a tion and the other two, geographically vigorous expansion of forests, and water- close populations that were unaffected by courses have become drier as a result of chromatophoromas. Although some chem- the increased level of plant transpiration icals, particularly the fungicide manganese and the rise in temperature (Pen˜ uelas and ethylenebisdithiocarbamate (maneb), have Boada, 2003). All the streams where the been reported as a cause of pigmented Montseny brook newt populations occur skin tumors in captive European newts are affected by severe droughts in summer (Triturus cristatus; Zavanella et al., 1980), and winter. During these periods, the no data are available about this compound newts migrate into the rock crevices, in our study area. Population one from our where a subterranean cold watercourse study had a relatively high mitochondrial provides good conditions for survival. DNA variability of cytochrome b in However, adult newts are active in the comparison to the other populations stream when both climatic and hydrologic (Carranza and Amat, 2005), indicating conditions are favorable (spring and au- that endogamy was not a cause for the tumn), whereas juveniles are rarely ob- pigmented skin tumors in population one. served in the outer stream and pass most 414 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 2, APRIL 2011 of their life among the rocks. The lack of clearcut and in nearby forest in the southern pigmented skin tumors in juveniles sug- Blue Ridge Mountains. Journal of Herpetology 37: 445–452. gests that the agent responsible for these BOADA, M. 1994. Montseny, medi i home. Fundacio´ pathologies may be present in the upper Rectoria Vella. ICE. Barcelona, Fundacio´ La stream, where it progressively affects Caixa. pp. 56. adults during their lifetime. Skeletochro- CARRANZA, S. 2008. Calotriton arnoldi. In Interna- nologic studies have indicated that there is tional Union for the Conservation of Nature a correlation between body size and age in (IUCN) 2008. 2008 IUCN Red List of Threat- ened Species, 18 January 2008, Version 2010.4, salamanders (Ash et al., 2003). If this also www.iucnredlist.org. Accessed January 2011. holds true for Calotriton arnoldi,the ———, AND F. AMAT. 2005. Taxonomy, biogeography larger newts are also the oldest and, and evolution of Euproctus (Amphibia: Salaman- therefore, should be more affected by this dridae), with the resurrection of the genus external agent. This was corroborated by Calotriton and the description of a new endemic species from the Iberian Peninsula. Zoological the positive correlation between nodule Journal of the Linnean Society 145: 555–582. numbers and SVL in our study. CRUMP, M. L. 2009. Amphibian diversity and life history. In addition to UV-B radiation, other InAmphibianecologyandconservation.Ahandbook external causes may include the progres- of techniques, K. Dodd (ed.). Oxford University sive rise in temperature that has affected Press, New York, New York. pp. 3–19. KHUDOLEY, V. V., AND I. V. MIZGIREUV. 1980. On the stream surface as a result of environ- spontaneous skin tumours in Amphibia. Neo- mental changes in the Montseny mountain plasma 27: 289–293. massif. It is also possible that the inci- KOUSSOULAKOS, S., M. LELOUDA, AND H. J. ANTON. dence of age-associated pigmented skin 1994. Allografting of a nontransmissible, spon- tumors is due to cumulative sun exposure taneous dermal melanoma in the newt Triturus of adult newts over time. Indeed, studies cristatus. Tumor Biology 15: 141–146. MILLER, B. T., AND J. L. MILLER. 2005. Prevalence of of chromatophoromas of crested newts physical abnormalities in eastern hellbender (Triturus cristatus) have indicated a tem- (Cryptobranchus alleganiensis alleganiensis) perature-dependent growth rate for the populations of Middle Tennessee. Southeastern tumors (Zavanella, 1985). Long-term Naturalist 4: 513–520. monitoring of environmental and water PEN˜ UELAS, J., AND M. BOADA. 2003. A global change- induced biome shift in the Montseny mountains temperatures in the streams inhabited by (NE Spain). Global Change Biology 9: 131–140. this endangered species may further STEFANESCU,C.,S.HERRANDO, AND F. PA´ RAMO. 2004. elucidate the causes of this pathology. Butterfly species richness in the north-west Med- iterranean Basin: The role of natural and human- ACKNOWLEDGMENTS induced factors. Journal of Biogeography 31: 905–915. We are grateful to the Diputacio´ de WHEELER, V. A., M. I. MCCALLUM, AND S. E. TRAUTH. Barcelona (Natural Park of Montseny) and 2002. Abnormalities in the Ozark hellbender, the Generalitat de Catalunya (Departament de Cryptobranchus alleganiensis bishopi. Journal of Medi Ambient) for financial and logistic the Arkansas Academy of Science 56: 250–252. support to our investigation of the biology ZAVANELLA, T. 1985. Environmental temperature and and pathology of Calotriton arnoldi. We thank metastatic spread of melanoma in the crested Josep Roca and Joaquim Soler for their newt. Cancer Letters 27: 171–179. valuable comments on this manuscript. ———, AND E. ARIAS. 1982. Chromosome variation in two populations of the crested newt, Triturus LITERATURE CITED cristatus carnifex, with different melanoma AMAT, F., AND S. CARRANZA. 2006. Aproximacio´ a incidence. Cytobios 34: 75–82. l’evolucio´ i biologia del trito´ del Montseny. ———, N. P. ZAFFARONI, AND E. ARIAS. 1980. Testing Calotriton arnoldi (Amphibia, Salamandridae). of the fungicide maneb for carcinogenicity in Conservacio´ d’una raresa amagada i singular. two populations of the European crested newt. Monografies del Montseny 21: 165–191. Cancer Letters 10: 109–116. ASH, A. N., R. C. BRUCE,J.CASTANET, AND H. FRANCILLON-VIELLOT. 2003. Population parame- Submitted for publication 11 January 2010. ters of Plethodon metcalfi on a 10-year-old Accepted 20 December 2010.