Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes
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Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes Frank Pasmans1., Pascale Van Rooij1*., Mark Blooi1, Giulia Tessa2,3, Serge´ Bogaerts4, Giuseppe Sotgiu3, Trenton W. J. Garner5, Matthew C. Fisher6, Benedikt R. Schmidt7,8, Tonnie Woeltjes9, Wouter Beukema10, Stefano Bovero3, Connie Adriaensen1, Fabrizio Oneto11, Dario Ottonello11, An Martel1", Sebastiano Salvidio11" 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium, 2 Dipartimento di Scienze della Vita e Biologia dei Sistemi, Universita` degli Studi di Torino, Torino, Italy, 3 Zirichiltaggi – Sardinia Wildlife Conservation, Sassari, Italy, 4 Waalre, The Netherlands, 5 Institute of Zoology, Zoological Society of London, London, United Kingdom, 6 Department of Infectious Disease Epidemiology, Imperial College, School of Public Health, London, United Kingdom, 7 Institut fu¨r Evolutionsbiologie und Umweltwissenschaften, Universita¨tZu¨rich, Zu¨rich, Switzerland, 8 Koordinationsstelle fu¨r Amphibien- und Reptilienschutz in der Schweiz, Neuchaˆtel, Switzerland, 9 Stichting Reptielen, Amfibiee¨n en Vissen Onderzoek Nederland, Nijmegen, The Netherlands, 10 Faculty of Geo- Information Science and Earth Observation, University of Twente, Enschede, The Netherlands, 11 Dipartimento di Scienze della Terra, dell’Ambiente e della Vita, Universita` di Genova, Genova, Italia Abstract North America and the neotropics harbor nearly all species of plethodontid salamanders. In contrast, this family of caudate amphibians is represented in Europe and Asia by two genera, Speleomantes and Karsenia, which are confined to small geographic ranges. Compared to neotropical and North American plethodontids, mortality attributed to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has not been reported for European plethodontids, despite the established presence of Bd in their geographic distribution. We determined the extent to which Bd is present in populations of all eight species of European Speleomantes and show that Bd was undetectable in 921 skin swabs. We then compared the susceptibility of one of these species, Speleomantes strinatii, to experimental infection with a highly virulent isolate of Bd (BdGPL), and compared this to the susceptible species Alytes muletensis. Whereas the inoculated A. muletensis developed increasing Bd-loads over a 4-week period, none of five exposed S. strinatii were colonized by Bd beyond 2 weeks post inoculation. Finally, we determined the extent to which skin secretions of Speleomantes species are capable of killing Bd. Skin secretions of seven Speleomantes species showed pronounced killing activity against Bd over 24 hours. In conclusion, the absence of Bd in Speleomantes combined with resistance to experimental chytridiomycosis and highly efficient skin defenses indicate that the genus Speleomantes is a taxon unlikely to decline due to Bd. Citation: Pasmans F, Van Rooij P, Blooi M, Tessa G, Bogaerts S, et al. (2013) Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes. PLoS ONE 8(5): e63639. doi:10.1371/journal.pone.0063639 Editor: Ilse D. Jacobsen, Leibniz Institute for Natural Products Research and Infection Biology- Hans Knoell Institute, Germany Received October 25, 2012; Accepted April 4, 2013; Published May 20, 2013 Copyright: ß 2013 Pasmans et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this work was provided to PVR by research grant BOF08/24J/004 from Ghent University and to MB by a Dehousse grant from the Royal Zoological Society of Antwerp, Belgium. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors confirm hereby that co-authors Matthew C. Fisher and Benedikt R. Schmidt are PLOS ONE Editorial Board members. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected] . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction the two countries where the genus Speleomantes occurs, the aggressive lineage of the pathogen, the Bd global panzootic lineage With more than 430 species, the family Plethodontidae (BdGPL), also occurs [7]. Both infection and mortality due to Bd comprises the majority of extant urodelan species and has has been reported for both countries, including localities where experienced a marked evolutionary radiation in North, Central Speleomantes sp. are endemic [3,4,6,8]. Although habitat alteration and northern South America [1]. In the rest of the world, this has taken its toll on Speleomantes populations, enigmatic declines family is confined to the Maritime Alps, the central Apennine that would match chytridiomycosis driven declines witnessed mountains in continental Italy and Sardinia in Europe (the genus elsewhere have not been reported. Indeed, these salamanders are Speleomantes, containing 8 species), and to South Korea (1 species, among the most abundant vertebrates in suitable habitats [9]. The Karsenia koreana). The European plethodontids are closely related to skin is of vital importance to plethodontid salamanders, which rely the North American genus Hydromantes [1] and occupy an area exclusively on cutaneous respiration. Chytridiomycosis dramati- well known to be infected by the amphibian pathogenic fungus cally disturbs the skin function [10,11] and thus compromises Batrachochytrium dendrobatidis (Bd), one of the known drivers respiration. Therefore, chytrid infections in Speleomantes should underlying global amphibian declines [2–6]. In Italy and France, result in rapid killing of the plethodontid host, as has been PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63639 Resistance of Speleomantes to Chytridiomycosis hypothesized to be the case for several declining neotropical extracted in 100 ml PrepMan Ultra (Applied Biosystems, Foster plethodontids and demonstrated in some, but not all [12], North City, CA, USA), according to Hyatt et al. [26]. DNA samples American species [13–16]. Although different infection protocols were diluted 1:10 and quantitative PCR (qPCR) assays were have been used in these studies, they clearly demonstrate that performed in duplicate on a CFX96 Real Time System (BioRad some New World plethodontid species are easily colonized by the Laboratories, Hercules, CA, USA). Amplification conditions, fungus. primer and probe concentrations were according to Boyle et al. Hitherto no suspected chytridiomycosis associated declines in [27]. Within each assay, 1 positive control sample containing Bd Speleomantes have been observed, even in a region where DNA from a naturally infected and deceased Costa Rican chytridiomycosis occurs. This leads us to hypothesize first that Eleutherodactylus sp. as template and 3 negative control samples prevalence of lethal cutaneous infections, such as infections by Bd, with HPLC water as template were included. Samples were are low in European plethodontid salamanders. For this purpose, considered positive for Bd when a clear log-linear amplification we determined to what extent Bd is present in populations of all was observed, when the number of genomic equivalents (GE) of eight species of Speleomantes. Bd, defined as the measure of infection, was higher than the Susceptibility to clinical chytridiomycosis, however, varies detection limit of 0.1 GE and when amplification that met both of greatly among plethodontid species. In amphibian hosts, differ- the previous criteria was observed in both replicates. In case of ences in host susceptibility have been attributed to the presence of conflict between both replicates of the same sample, the sample fungicidal skin microbiota [17–19], antimicrobial peptides (re- was run again in duplicate. To control and estimate inhibition, a viewed in Rollins-Smith [20]), host genetics [21–23] and/or subset of samples negative for the presence of Bd (n = 84) was environmental factors (e.g. [8]), which may affect invasion of retested under the same conditions as described above, but with an TM amphibian skin by the pathogen [24]. This leads us to hypothesize exogenous internal positive control (VIC probe, Life technol- that resistance of European plethodontid salamanders is due to ogies, Austin, TX, USA) included as described by Hyatt et al. skin defenses that efficiently cope with Bd infection. Subsequently, [26]. The Bayesian 95% credible interval for prevalence was we then examined susceptibility of Speleomantes strinatii to experi- estimated as described by Lo¨tters et al. [28]. mental infection with a global panzootic lineage strain of Bd We assayed susceptibility to chytridiomycosis in five male (BdGPL isolate IA2011, [7]). Finally, we determined to what subadult S. strinatii by experimentally exposing them to a extent skin secretions of Speleomantes species are capable of killing controlled dose of Bd. Ten captive bred juvenile Alytes muletensis Bd. were used as susceptible, positive control animals [29]. The animals were housed individually in plastic boxes (20610610 cm) Materials and Methods lined with moist tissue, provided with PVC-tubes