Acta Herpetologica 18(1): 3-9, 2023 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-13279 Threats of the emerging pathogen Batrachochytrium salamandrivorans (Bsal) to Italian wild salamander populations Lorenzo Dondero1, Giorgia Allaria1, Giacomo Rosa1, Andrea Costa1, Gentile Francesco Ficetola2, Rob- erto Cogoni3, Elena Grasselli1, Sebastiano Salvidio1,* 1 Department of Earth, Environment and Life Sciences (DISTAV), University of Genova, Corso Europa 26, I-16132 Genova, Italy 2 Department of Environmental Science and Policy, University of Milan, Via Celoria 10, 20133 Milan, Italy 3 Unione Speleologica Cagliaritana, Via Scarlatti 11, I-09045 Quartu St Elena, Italy *Corresponding author. Email: sebastiano.salvidio@unige.it Submitted on: 2022, 23rd June; revised on: 2022, 13th November; accepted on: 2022, 23rd November Editor: Ilaria Bernabò Abstract. The salamander killing fungus Batrachochytrium salamandrivorans (Bsal), recently introduced from Asia, is threatening salamander populations in different parts of Europe. In fact, this pathogen is rapidly spreading in Central Europe and has been also introduced into NE Spain. Of special concern are those regions with an exceptionally high salamander diversity such as Italy, where 19 salamander species are present most of which are strictly endemic. In this study, we update the information on the presence of Bsal in Italian wild salamanders, by adding samples from two presumptive outbreak sites, one on the island of Sardinia and one on continental Italy (Liguria). In addition, we reviewed the potential susceptibility of all the Italian salamander species on the basis of laboratory experimental infec- tion trials, or from the tested susceptibility of the phylogenetically nearest species, according to the literature. Overall, 15 skin swabs from three species (Speleomantes sarrabusensis, Speleomantes strinatii and Salamandra salamandra) col- lected in the two presumptive Bsal outbreak sites were analysed by quantitative molecular methods, but none gave positive results. The majority of Italian salamander species and almost all of the endemic ones showed a high suscep- tibility to Bsal infection. Therefore, even if the presence of Bsal in Italian salamander populations has not been proven yet, the entire salamander fauna is highly threatened and preventive management actions should be taken. The need for strict biosecurity protocols on the international trade of captive or wild amphibians and for the implementation of preventive measures during field activities to limit the introduction and spread of the Bsal pathogen is again stressed. Keywords. Amphibians, biosecurity, chytridiomycosis, conservation, emerging infectious diseases, mitigation, real- time PCR. INTRODUCTION The chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are amphibian pathogens that were introduced in Europe from East Asia, probably through the international trade (O’Hanlon et al., 2018; Laking et al., 2019). The con- tinuous spreading of these emerging pathogens is now threatening wild amphibian populations in different parts of Europe (Scheele et al., 2019; Bosch et al., 2021). In particular, the salamander killing fungus Bsal is a real threat to European salamanders, because many species are extremely susceptible to this infection and, therefore, highly vulnerable (Martel et al., 2014). The pathogenic effect of Bsal is caused by its zoosporangia that pene- trate the skin cells of adult salamanders, breaking innate immunological defences, and producing tissue erosions and deep ulcerations that may disrupt the host infected 4 Lorenzo Dondero et alii skin respiratory and rehydration functions (Martel et al., 2013; Grogan et al., 2020). In this way, Bsal infections are capable of causing severe illness or even death of the infected amphibian hosts (Martel et al., 2013, 2014). In fact, Bsal is constantly expanding from the Netherlands, where it was first detected and where it caused a popu- lation collapse in the local fire salamander Salamandra salamandra population (Martel et al., 2013). Currently, Bsal has been confirmed in about 80 localities surround- ing Bunderbos, in the Netherlands, where it was first detected (Martel et al., 2013), and also in Belgium and Germany (e.g., Spitzen-van der Sluijs, 2016; Lötters et al, 2020; Schmeller et al., 2020; Thein et al., 2020). Bsal has also been detected in 2018 in Catalonia (Northeastern Spain), where it has infected the marbled newt Triturus marmoratus and caused a local mass mortality event of this species (Martel et al., 2020), while its presence in North Central Spain has not been yet confirmed (Bosch et al., 2021). Therefore, to date, the presence of Bsal in Europe has been confirmed in the wild in four coun- tries: The Netherlands, Belgium, Germany and Spain. The emergence and the diffusion of B. salamandrivorans through Europe, apparently facilitated by humans trans- ferring and introducing in the wild environment captive amphibians, is worrying and requests urgent prevention measures and monitoring actions to prevent further loss of amphibian diversity (Thomas et al., 2019). In particu- lar, the high salamander diversity of the southern Euro- pean peninsulas, such as Italy and Spain, appears at risk. Italy alone hosts 19 species of urodeles (Sindaco and Razzetti, 2021), many of which are endemic, such as the entire genus Salamandrina, the Italian newt Lissotriton italicus, the Sardinian brook newt Euproctus platycepha- lus, and seven species of cave salamanders belonging to the genus Speleomantes (Sindaco and Razzetti, 2021). However, to date, only two studies screened Bsal in Ital- ian salamanders (Grasselli et al., 2019, 2021). These stud- ies analysed by means of real-time PCR (qPCR) 136 skin swabs from 6 species of Italian wild salamanders and 53 from non-native individuals that were bred in pri- vate collections (Grasselli et al., 2019). The results from these studies were that none of the 189 Italian salaman- der swabs gave positive results. However, recent events raised concerns on the possibility of Bsal outbreaks in Italian populations. In the summer of 2021, a mass mor- tality of Speleomantes sarrabusensis was observed in Sar- dinia by the wildlife photographer Emanuele Biggi while, more recently (in spring 2022), a living fire salamander bearing skin lesions from Liguria was photographed by Michael Fahrbach and reported to the authors by Frank Pasmans from Ghent University. These observations were compatible with the suspected infection of Bsal on Italian amphibians but needed robust confirmation by molecular, and histological methods or by both. There- fore, the aim of this study was twofold: i) to expand the current knowledge about the presence of Bsal in wild salamander populations in Italy, adding the molecular data obtained from new skin samples collected from the two Bsal-suspected outbreaks and ii) to assess the sus- ceptibility to Bsal of all Italian salamanders, on the basis of published experimental studies or, in the absence of experimental evidence, from their phylogenetic affinities. This information will be needed to better plan Bsal miti- gation actions and also to guide strategic conservation and management efforts on the national territory of Italy. MATERIALS AND METHODS Origin of skin swab samples, DNA extraction and quanti- tative PCR We obtained skin swabs from two areas, one in Southern Sardinia and one in NW Italy. In Southern Sardinia (Monte Sette Fratelli), we sampled a protected underground site that hosts the largest known popula- tion of Speleomantes sarrabusensis. This site is closed by a gate and only an authorised person has access, how- ever, in this location several dead cave salamanders were observed from July to August 2021, by different herpetol- ogists. Skin swabs were collected a few days after the first mortality record (August 2021) from all the living S. sar- rabusensis individuals (two individuals), plus one recent- ly dead individual; a fourth swab was obtained from a Hyla sarda corpse found at the same site (Supplementary material, Fig. S1). In the province of Genova (Liguria, NW Italy), we collected twelve swabs in April and May 2022 along a small stream or in an adjacent artificial cave from liv- ing fire salamanders (Salamandra salamandra) and cave salamanders (Speleomantes strinatii) where an individual of Salamandra salamandra with suspect skin lesions was photographed (Supplementary material, Fig. S1). In both sites, all skin swabs were obtained with a standardised protocol used in previous Bd and Bsal studies on Ital- ian salamanders (Costa et al., 2021; Grasselli et al., 2019, 2021). Sterile cotton swabs were rubbed 30 times on the skin of different parts of the salamander’ body and were preserved in individual sterile plastic tubes at 4 °C until extraction (Spitzen-van der Sluijs et al., 2016). DNA was extracted in 200 μl of Prepman ULTRA (Thermo Fisher Scientific Technologies, Monza, Italy). Samples were then analysed for Bd and Bsal DNA using a duplex qPCR, tar- geting the ITS1 rRNA gene of Bd and 5.8S rRNA gene of Bsal, as described by Blooi et al. (2013). All samples 5Bsal threats to Italian salamanders were run in duplicate, together with standard curves obtained from suspensions of known numbers of Bd and Bsal zoospores (kindly provided by An Martel and Frank Pasmans) in the same plate, and results were expressed in genome equivalents (GEs) according to Thomas et al. (2018). Assessing Italian salamanders’ Bsal susceptibility The susceptibility of Italian salamanders to Bsal infection was estimated by collecting all the published data on laboratory experimental infection trials on the same salamander species. In absence of such experimen- tal evidence, the susceptibility was inferred from the phy- logenetically nearest species, adopting a precautionary approach and thus selecting the more threatening out- come. Species susceptibility was estimated as “High” if the experimental infection had a lethal outcome, “Mod- erate” if the gravity of infection was dose-dependent and “Low” if the infected salamander was able to clear infec- tion or remained asymptomatic. Moreover, we used the results of Beukema et al. (2018, Table 2) as an index of potential niche overlap between Bsal and Italian salamanders. Beukema et al. (2018) estimated both native and invasive ecological niches of Bsal and of all native European salamanders using three different ordination methods (see Material and Methods in Beukema et al., 2018). The statistical overlap between both Bsal native and invasive ecologi- cal niches and the current niche of Italian salamanders was then calculated (Beukema et al., 2018). In the pre- sent study, we counted all significant overlaps between Italian salamanders and Bsal native and invasive niche. Therefore, there were several possible outcomes per spe- cies, ranging from 0/6 if all overlaps were non-significant, to 6/6 if all the six niches overlapped significantly. This “overlap score” may be used as a gross indication of the environmental compatibility between each Italian species and the salamander killing fungus Bsal. In this study, we used species nomenclature following the most recent checklist of Italian Herpetofauna (Sinda- co and Razzetti, 2021), and we revised the available data on the 19 native Italian species. We also report informa- tion for one subspecies of alpine salamander (Salamandra atra aurorae), that is listed as a priority taxon in annex II of the European Union (EU) Directive “Habitats” 92/43/ CEE. According to Art. 1 letter (h) of this Directive, Italy has a particular responsibility for the conservation of this endemic taxon that shows an extremely small distribu- tional range limited to the Eastern Alps. RESULTS DNA testing We analysed one skin swab from Hyla sarda, three Speleomantes sarrabusensis, five Speleomantes strinatii and seven Salamandra salamandra. None of these swabs gave Bd or Bsal-positive results. Therefore, the possible Bsal infection in the Sardinian (prevalence 0; 95% con- fident limits 0.00-0.60), and Ligurian (prevalence 0; 95% confident limits 0.00-0.30) sites were not confirmed, but given the very small samples analysed, a high level of uncertainty concerning these results remains. To date, the number of Italian wild salamanders screened for Bsal is 151, obtained from nine species (Table 1). Bsal susceptibility Ten of the nineteen (53%) salamander species found in Italy were infected experimentally in laboratory trials with Bsal, thus providing direct evidence of their poten- tial susceptibility to the pathogen (Table 2). In the case of the cave salamanders (genus Speleomantes), three spe- cies were infected in laboratory experiments by Martel et al. (2014): S. genei, S. strinatii and S. imperialis. The two former species resulted highly susceptible (i.e., Bsal infection was lethal), while the latter was able to clear the infection, thus showing low susceptibility. In this study, following the precautionary approach, we considered all non-tested Speleomantes species to be highly susceptible, as already done by Gilbert et al. (2020). Overall, the majority of Italian salamander species, 15 out of 19 (79%), were shown or inferred to possess Table 1. Italian salamanders tested for Batrachochytrium sala- mandrivorans. a and b indicate presence of samples from the non- confirmed Bsal outbreaks in Liguria (2022) and Sardinia (2021), respectively. This table is an expanded version of Grasselli et al. (2019). Species or subspecies Sample (N) Bd positive Bsal positive Euproctus platycephalus 3 0 0 Ichthyosaura alpestris 76 1 0 Lissotriton italicus 22 0 0 Salamandra atra aurorae 3 0 0 Salamandra salamandraa 11 0 0 Salamandrina terdigitata 14 0 0 Speleomantes sarrabusensisb 3 0 0 Speleomantes strinatiia 5 0 0 Triturus carnifex 14 3 0 Total 151 4 0 6 Lorenzo Dondero et alii high susceptibility to Bsal infection, while two (11%) had moderate and two (11%) had low susceptibility (Table 2). When focusing on endemic salamanders, 10 out of 11 (91%) have, or were inferred to have, high sus- ceptibility, the only exception being S. imperialis (Table 2). Finally, only four species showed no niche overlap with Bsal pathogen. Three of them were salamanders found exclusively in the Mediterranean bioclimatic region (i.e., Lissotriton italicus, Speleomantes flavus and Speleomantes supramontis), while the fourth one (Sala- mandra lanzai) lives at high altitudes in the Alps. On the other hand, four species showed a relatively high overall niche overlap score with Bsal: Ichthyosaura alp- estris, Speleomantes ambrosii, Speleomantes strinatii and Triturus carnifex. The two latter also had a high experi- mental susceptibility towards the infection with the pathogen (Martel et al., 2014). DISCUSSION The diffusion of Bsal infection is a dramatic threat to salamander diversity in Central Europe and Spain (Martel et al., 2014; Bosch et al., 2021), and recently two potential outbreak sites have been recorded in Italy. How- ever, none of our qPCR samples from these sites pro- vided positive results. Clearly, these partial results can- not exclude the presence of Bsal-infected individuals in the studied sites or elsewhere, because of the very small sample size tested. The mass mortality event reported in Sardinia is particularly concerning, because the cave sala- mander population lives in a high-altitude protected site, where environmental stressors should be absent. There- fore, local Authorities in charge of the site management were immediately alerted and the water quality inside the site is monitored to report any further critical condi- tion and to report further changes as well as to mitigate a possible spread of any kind of pathogen, as indicated by Thomas et al. (2019). In the Ligurian site, no mass mortality was observed, and a monitoring programme is regularly taking place as a preventive mitigation measure (Salvidio, unpublished data). Our review of species susceptibility already con- ducted by Beukema et al. (2018) and Gilbert et al. (2020) shows that the great majority of Italian salamanders, and in particular many endemic taxa, are potentially highly vulnerable to Bsal infection. Indeed, the endemic genus Salamandrina and the many species belonging to the genus Speleomantes have been experimentally proven highly vulnerable to this infection. In addition, all Ital- Table 2. Susceptibility of Italian salamander species to Bsal infection, according to the literature or inferred from phylogenetic related spe- cies. Bsal susceptibility was updated from Gilbert et al. (2020). The overlap score was calculated from Table 2 of Beukema et al. (2018). * not reported in Beukema et al. (2018). Species Italian endemic IUCN Status Habitat Directive Bsal susceptibility Comments Reference Overlap score Euproctus platycephalus yes EN IV High Lethal, laboratory tested Martel et al., 2014 1/6 Ichthyosaura alpestris no LC -- Moderate Dose-dependent laboratory tested Martel et al., 2014 3/6 Lissotriton italicus yes LC IV High Lethal, laboratory tested Martel et al., 2014 0/6 Lissotriton vulgaris no LC -- Moderate Dose-dependent, laboratory tested Bates et al., 2019 1/6 Proteus anguinus no VU II/IV Low Asymptomatic, laboratory tested Li et al., 2020 2/6 Salamandra atra no LC IV High Inferred from congeneric species 2/6 Salamandra atra aurorae yes II*/IV High Inferred from congeneric species -* Salamandra lanzai no VU IV High Inferred from congeneric species 0/6 Salamandra salamandra no LC -- High Lethal, laboratory tested Martel et al., 2013, 2014 2/6 Salamandrina perspicillata yes LC II/IV High Lethal, laboratory tested Martel et al., 2014 1/6 Salamandrina terdigitata yes LC II/IV High Inferred from congeneric species 1/6 Speleomantes ambrosi yes NT II/IV High Inferred from congeneric species 3/6 Speleomantes flavus yes VU II/IV High Inferred from congeneric species 0/6 Speleomantes genei yes VU II/IV High Lethal, laboratory tested Martel et al., 2014 1/6 Speleomantes imperialis yes NT II/IV Low Clears infection, laboratory tested Martel et al., 2014 1/6 Speleomantes italicus yes NT II/IV High Inferred from congeneric species Martel et al., 2014 1/6 Speleomantes sarrabusensis yes VU II/IV High Inferred from congeneric species Martel et al., 2014 1/6 Speleomantes strinatii no NT II/IV High Lethal, laboratory tested Martel et al., 2014 3/6 Speleomantes supramontis yes EN II/IV High Inferred from congeneric species 0/6 Triturus carnifex no LC II/IV High Inferred from congeneric species 3/6 7Bsal threats to Italian salamanders ian species with few exceptions share a relatively high ecological niche overlap score with Bsal (Beukema et al., 2018 synthetized in Table 2 of this study), suggest- ing that, even in the Mediterranean region, salamanders inhabiting humid and cool microhabitats, such as Spe- leomantes strinatii and Triturus carnifex are exposed to a high risk. Concerning the EU priority micro-endemic subspecies of Alpine salamander, Salamandra atra auro- rae, Beukema et al. (2018) did not calculate niche over- lap, while a high susceptibility to Bsal was inferred from the experimental data on other Salamandra subspecies (Gilbert et al., 2010). Given the high conservation con- cern of this taxon possessing a restricted range of occur- rence (Romano et al., 2018), a demographic monitoring project has been implemented by the local province of Trento starting in 2017, while skin swabs have been col- lected from different sites in August 2022 (Romano, pers. com. September 2022). Active disease surveillance and large-scale monitor- ing of populations of the most threatened species should be the ideal preventive strategy, but the costs associated with these activities would be clearly prohibitive (Bosch et al., 2021). Therefore, a more cost-effective policy should integrate passive reporting and intervention in the cases of confirmed mass mortality events combined with strong preventive measures, as already suggested by Thomas et al. (2019). These measures should include test- ing for Bsal throughout the amphibian international com- merce to exclude the unintentional introduction of fungal pathogens from captive amphibians legally traded. More- over, controlling and eradicating invasive populations of alien amphibians that could act as Bsal intermediate hosts should be considered and realised, when possible. These preventive eradications should be undertaken especially for species that act as intermediate hosts and are associat- ed with the spread of amphibian chytrid pathogens, such as the African clawed frog Xenopus laevis and the North American bullfrog Lithobates catesbeianus (Pasmans et al., 2017). Moreover, it is pivotal that professional and amateur herpetologists always implement strict biosecuri- ty measures during field activities, to avoid the spread of the pathogen across distant study areas, and the existence of online information about biosecurity precautions, such as http://bsaleurope.com/, should be better advertised. Finally, herpetologists are not the only potential drivers of the spread of these pathogens, because unfor- tunately they can be spread by any kind of people per- forming outdoor activities. A broad dissemination of the issues of wildlife pathogens, and of the protocols to limit them, remains a fundamental preventive action (e.g., http://www-9.unipv.it/webshi/images/files/All.%20II%20 -%20CHITRIDIOMICOSI.pdf; http://bsaleurope.com/). ACKNOWLEDGMENTS We thank An Martel and Frank Pasmans for provid- ing DNA standards and continuous support to our labo- ratory, Emanuele Biggi and Alessandro Spiga for report- ing about the mass mortality in Sardinia. Permits to swab salamanders were provided by the Italian Ministry of Ecological Transition (0039130 of 15/04/2022). The com- ments and suggestions of Giulia Tessa and of an anony- mous Reviewer greatly improved this manuscript. SUPPLEMENTARY MATERIAL Supplementary material associated with this arti- cle can be found at manuscript number 13279 REFERENCES Bates, K., Shelton, J.M.G., Mercier, V.L., Hopkins, K.P., Harrison, X.A., Petrovan, S.O., Fisher, M.C. (2018): Captivity and Infection by the fungal pathogen Batra- chochytrium salamandrivorans perturb the amphibian skin microbiome. Front. Microbiol. 10: 1834. Beukema, W., Martel, A. Nguyen, T.T., Goka, K., Schmel- ler, D.S., Yuan, Z., Laking, A.E., Nguyen, T.Q., Lin, C.-F., Shelton, J., Loyau, A., Pasmans, F. (2018): Envi- ronmental context and differences between native and invasive observed niches of Batrachochytrium sala- mandrivorans affect invasion risk assessments in the Western Palaearctic. Divers. Distrib. 24: 1788-1801 Blooi, M., Pasmans, F., Longcore, J. E., Spitzen-van der Sluijs, A., Vercammen, F., Martel, A. (2013): Duplex real-time PCR (qPCR) for rapid simultaneous detec- tion of Batrachochytrium dendrobatidis and Batra- chochytrium salamandrivorans in amphibian samples. J. Clin. Microbiol. 51: 4173-4177. Blooi, M., Pasmans, F., Longcore, J.E., Spitzen-van der Sluijs, A., Vercammen, F., Martel, A. (2013): Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytri- um salamandrivorans in amphibian samples. J. Clin. Microbiol., 51: 4173-4177. Boyle, D.G., Boyle, D.B., Olsen, V., Morgan, J.A.T., Hyatt, A.D. (2004): Rapid quantitative detection of chytridi- omycosis. Dis. Aquat. Organ. 60: 141-148. Bosch, J., Martel, A., Sopniewski, J., Thumsová, B., Ayres, C., Scheele, B.C., Guillermo Velo-Antón, G., Pasmans, F. (2021): Batrachochytrium salamandrivorans threat to the Iberian urodele hotspot. J. Fungi 7: 644. 8 Lorenzo Dondero et alii Costa A., Dondero L., Allaria G., Morales Sanchez B.N., Salvidio S., Rosa G., Grasselli E. (2021). Modelling the amphibian chytrid fungus spread by connectivity analysis: towards a national monitoring network in Italy. Biodiv. Cons. 30: 2807-2825. Gilbert, M.J., Spitzen-van der Sluijs, A.M., Canessa, S., Bosch, J., Cunningham, A.A., Grasselli, E., Laudelout, A., Lötters, S., Miaud, C., Salvidio, S., Veith, A. Mar- tel, Pasmans, F. (2020). Mitigating Batrachochytrium salamandrivorans in Europe, Batrachochytrium sala- mandrivorans Action Plan for European Urodeles, European Commision: Nijmegen, The Netherlands, 2020. Grasselli, E., Bianchi, G., Salvidio, S. (2021): Batra- chochytrium salamandrivorans in Europe: the Italian contribution to the international mitigation project. In: Atti del XII Congresso Nazionale della Societas Herpetologica Italica (Rende, Cosenza), pp. 214-217. Tripepi, S., Ed, Ventura Edizioni, Senigallia. Grasselli, E., Bianchi, G. Dondero, L., Marchianò, V., Carafa, M., Perrone, M., Salvidio, S., (2019). First screening of Batracochytrium salamandrivorans (Bsal) in wild and captive salamanders from Italy. Salaman- dra 55: 124-126. Grogan. L.F., Humphries, J.E., Robert, J., Lanctôt, C.M., Nock, C.J., Newell, D.A., McCallum, H.I. (2020): Immunological aspects of chytridiomycosis. J. Fungi 6: 234. Laking, A.E., Ngo, H., Pasmans, F., Martel, A., Nguyen, T.T. (2019): Batrachochytrium salamandrivorans is the predominant chytrid fungus in Vietnamese salaman- ders. Sci. Rep. 7: 44443. Li, Z., Verbrugghe, E., Konstanjšek, R., Lukač, M., Pas- mans, F., Cizelj, I., Martel, A. (2020). Dampened virulence and limited proliferation of Batrachochytri- um salamandrivorans during subclinical infection of the troglobiont olm (Proteus anguinus). Sci. Rep. 10: 16480. Lötters, S., Wagner, N., Albaladejo, G., Böning, P., Dal- beck, L., Düssel, H., Feldmeier, S., Guschal, M., Kirst, K., Ohlhoff, D., Preissler, K., Reinhardt, T., Schlüp- mann, M., Schulte, U., Schulz, V., Steinfartz, S., Twi- etmeyer, S., Veith, M., Vences, M., Wegge, J. (2020): The amphibian pathogen Batrachochytrium salamand- rivorans in the hotspot of its European invasive range: past – present – future. Salamandra 56: 173-188. Martel, A., Blooi, M., Adriaensen, C., Van Rooij, P., Beu- kema, W., Fisher, M.C., Farrer, R.A., Schmidt, B.R., Tobler, U., Goka, K., Lips, K.R., Muletz, C., Zamudio, K., Bosch, J., Lötters, S., Wombwell, E., Garner, T.W.J., Cunningham, A.A., Spitzen-van der Sluijs, A., Sal- vidio, S., Ducatelle, R., Nishikawa, K., Nguyen, T.T., Kolby, J.E., Van Bocxlaer, I., Bossuyt, F., Pasmans, F. (2014): Recent introduction of a chytrid fungus endangers Western Palearctic salamanders. Science 346: 630-631. Martel, A., Spitzen-van der Sluijs, A., Blooi, M., Bert, W., Ducatelle, R., Fisher, M.C., Woeltjes, A., Bosman, W., Chiers, K., Bossuyt, F., (2013): Batrachochytrium sala- mandrivorans Sp Nov causes lethal chytridiomycosis in amphibians. Proc. Natl. Acad. Sci. USA 110: 15325- 15329. Martel, A., Vila-Escale, M., Fernandez-Giberteau, D., Martinez-Silvestre, A., Canessa, S., Van Praet, S., Pan- non, P., Chiers, K., Ferran, A., Kelly, M. (2020): Inte- gral chain management of wildlife diseases. Conserv. Lett. 13: e12707. O’Hanlon, S. J., Rieux, A., Farrer, R. A., Rosa, G. M., Waldman, B., Bataille, A., Kosch, T. A., Murray, K. A., Brankovics, B., Fumagalli, M., Martin, M. D., Wales, N., Alvarado-Rybak, M., Bates, K. A., Berger, L., Böll, S., Brookes, L., Clare, F., Courtois, E. A., Cunning- ham, A.A., Doherty-Bone, T.M., Ghosh, P., Gower, D.J., Hintz, W.E., Höglund, J., Jenkinson, T.S., Lin, C-F., Laurila, A., Loyau, A., Martel, A., Meurling, S., Miaud, C., Minting, P., Pasmans, F., Schmeller, D.S. Schmidt, B.R., Shelton, J.M.G., Skerratt, L.F., Smith, F., Soto-Azat, C., Spagnoletti, M., Tessa, G., Toledo, L.P., Valenzuela-Sánchez, A., Verster, R., Vörös, J., Webb, R.J. Wierzbicki, C., Wombwell, E., Zamudio, K.E., Aanensen, D.M., James, T.Y., Thomas, M., Gilbert, P., Weldon, C., Bosch, J., Balloux, F., Garner, T.M.J., Fish- er, M.C. (2018): Recent Asian origin of chytrid fungi causing global amphibian declines. Science 360: 621- 627. Pasmans, F., Bogaerts, S., Braeckman, J., Cunningham, A.A., Hellebuyck, T., Griffiths, R.A., Sparreboom, M., Schmidt, B.R., Martel, A. (2017): Future of keeping pet reptiles and amphibians: towards integrating ani- mal welfare, human health and environmental sus- tainability. Vet. Rec. 181: 450. Romano A., Costa A., Salvidio S., Menegon M., Garollo E., Tabarelli De Fatis K., Miserocchi D., Matteucci G., Pedrini P. (2018): Forest management and conser- vation of an elusive amphibian in the Alps: Habitat selection by the Golden Alpine Salamander reveals the importance of fine woody debris. For. Ecol. Man- ag. 424: 338-344. Scheele, B.C., Pasmans, F., Skerratt, L.F., Berger, L., Mar- tel, A., Beukema, W., Acevedo, A.A., Burrowes, P.A., Carvalho, T., Catenazzi, A., De la Riva, I., Fisher, M.C., Flechas, S.V., Foster, C.N., Frías-Álvarez, P., Garner, T.W.J., Gratwicke, B., Guayasamin, J.M., Hirschfeld, M., Kolby, J.E., Kosch, T.A., La Marca, E., 9Bsal threats to Italian salamanders Lindenmayer, D.B., Lips, K.R., Longo, A.V., Maneyro, R., McDonald, C.A., Mendelson, J.3rd, Palacios-Rod- riguez, P., Parra-Olea, G., Richards-Zawacki, C.L., Rödel, M.O., Rovito, S.M., Soto-Azat, C., Toledo, L.F., Voyles, J., Weldon, C., Whitfield, S.M., Wilkinson, M., Zamudio, K.R., Canessa, S. (2019): Amphibian fungal panzootic causes catastrophic and ongoing loss of bio- diversity. Science 363: 1459-1463. Schmeller, D.S., Utzel, R., Pasmans, F., Martel, A. (2020): Batrachochytrium salamandrivorans kills alpine newts (Ichthyosaura alpestris) in southernmost Germany. Salamandra 56: 230-232. Sindaco, R., Razzetti E. (2021): An updated check-list of Italian amphibians and reptiles. Nat. Hist. Sci. 8: 35-46. Spitzen-van der Sluijs, A., Martel, A., Asselberghs, J., Bales, E.K., Beukema,W., Bletz, M.C., Dalbeck, L., Goverse, E., Kerres, A., Kinet, T., (2016): Expand- ing distribution of lethal amphibian fungus Batra- chochytrium salamandrivorans in Europe. Emerg. Infect. Dis. 22: 1286-1288. Thein, J., Reck, U., Dittrich, G., Martel, A., Schulz, V., Hansbauer, G. (2020): Preliminary report on the occurrence of Batrachochytrium salamandrivorans in the Steigerwald, Bavaria, Germany. Salamandra 56: 227-229. Thomas, V., Wang, Y., van Rooij, P., Verbrugghe, E., Baláz, V., Bosch, J., Cunningham, A.A., Fischer, M.C., Gar ner, T.W.J., Gilbert, M.J., Grasselli, E., Kinet, T., Laudelout, A., Lötters, S., Loyau, A., Miaud, C., Sal- vidio, S., Schmeller, D.S., Schmidt, B.R., Spitzen- van der Sluijs, A., Steinfartz, S., Veith, M., Vences, M., Wagner, N., Canessa, S., Martel, A., Pasmans, F. (2019) Mitigating Batrachochytrium salamandrivorans in Europe. Amphibia-Reptilia 40: 265-290. Threats of the emerging pathogen Batrachochytrium salamandrivorans (Bsal) to Italian wild salamander populations Lorenzo Dondero1, Giorgia Allaria1, Giacomo Rosa1, Andrea Costa1, Gentile Francesco Ficetola2, Roberto Cogoni3, Elena Grasselli1, Sebastiano Salvidio1,* Age estimation and body size of the Parsley Frog, Pelodytes caucasicus Boulenger, 1896 from Lake Borçka Karagöl, Turkey Cantekin Dursun*, Serkan Gül, Nurhayat Özdemir Patterns of acoustic phenology in an anuran assemblage of the Yungas Andean forests of Argentina Martín Boullhesen1,2,*, Marcos Vaira1, Rubén Marcos Barquez2, Mauricio Sebastián Akmentins1 Diet and trophic niche overlap of four syntopic species of Physalaemus (Anura: Leptodactylidae) in southern Brazil Renata K. Farina1, Camila F. Moser2, Stefano Scali3, Mateus de Oliveira4, Patrícia Witt5, Alexandro Marques Tozetti1,* Screening of Ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of San Domenico e dei Serpari (Cocullo, AQ, Italy) Daniele Marini1,2, Ernesto Filippi3,*, Gianpaolo Montinaro4, Francesco C. Origgi5,6 Assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community Carter Dollen1,2, Tracy J. Coleman1,2, Travis R. Robbins1,2,* Revisiting the polyploidy in the genus Odontophrynus (Anura: Odontophrynidae) André Luis de Souza, Mayara Aparecida das Neves Micalichen, Roger Alves da Rocha, Rafael Bueno Noleto*