Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes

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.

Full text

Resistance to Chytridiomycosis in European
Plethodontid Salamanders of the Genus
Speleomantes
Frank Pasmans
1.
, Pascale Van Rooij
1
*
.
, Mark Blooi
1
, Giulia Tessa
2,3
, Serge
´Bogaerts
4
, Giuseppe Sotgiu
3
,
Trenton W. J. Garner
5
, Matthew C. Fisher
6
, Benedikt R. Schmidt
7,8
, Tonnie Woeltjes
9
, Wouter Beukema
10
,
Stefano Bovero
3
, Connie Adriaensen
1
, Fabrizio Oneto
11
, Dario Ottonello
11
, An Martel
1"
,
Sebastiano Salvidio
11"
1Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium, 2Dipartimento di Scienze della Vita e
Biologia dei Sistemi, Universita
`degli Studi di Torino, Torino, Italy, 3Zirichiltaggi – Sardinia Wildlife Conservation, Sassari, Italy, 4Waalre, The Netherlands, 5Institute of
Zoology, Zoological Society of London, London, United Kingdom, 6Department of Infectious Disease Epidemiology, Imperial College, School of Public Health, London,
United Kingdom, 7Institut fu
¨r Evolutionsbiologie und Umweltwissenschaften, Universita
¨tZu
¨rich, Zu
¨rich, Switzerland, 8Koordinationsstelle fu
¨r Amphibien- und
Reptilienschutz in der Schweiz, Neucha
ˆtel, Switzerland, 9Stichting 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, 11Dipartimento 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: ß2013 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: pascale.vanrooij@ugent.be
.These authors contributed equally to this work.
"These authors also contributed equally to this work.
Introduction
With more than 430 species, the family Plethodontidae
comprises the majority of extant urodelan species and has
experienced a marked evolutionary radiation in North, Central
and northern South America [1]. In the rest of the world, this
family is confined to the Maritime Alps, the central Apennine
mountains in continental Italy and Sardinia in Europe (the genus
Speleomantes, containing 8 species), and to South Korea (1 species,
Karsenia koreana). The European plethodontids are closely related to
the North American genus Hydromantes [1] and occupy an area
well known to be infected by the amphibian pathogenic fungus
Batrachochytrium dendrobatidis (Bd), one of the known drivers
underlying global amphibian declines [2–6]. In Italy and France,
the two countries where the genus Speleomantes occurs, the
aggressive lineage of the pathogen, the Bd global panzootic lineage
(BdGPL), also occurs [7]. Both infection and mortality due to Bd
has been reported for both countries, including localities where
Speleomantes sp. are endemic [3,4,6,8]. Although habitat alteration
has taken its toll on Speleomantes populations, enigmatic declines
that would match chytridiomycosis driven declines witnessed
elsewhere have not been reported. Indeed, these salamanders are
among the most abundant vertebrates in suitable habitats [9]. The
skin is of vital importance to plethodontid salamanders, which rely
exclusively on cutaneous respiration. Chytridiomycosis dramati-
cally disturbs the skin function [10,11] and thus compromises
respiration. Therefore, chytrid infections in Speleomantes should
result in rapid killing of the plethodontid host, as has been
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hypothesized to be the case for several declining neotropical
plethodontids and demonstrated in some, but not all [12], North
American species [13–16]. Although different infection protocols
have been used in these studies, they clearly demonstrate that
some New World plethodontid species are easily colonized by the
fungus.
Hitherto no suspected chytridiomycosis associated declines in
Speleomantes have been observed, even in a region where
chytridiomycosis occurs. This leads us to hypothesize first that
prevalence of lethal cutaneous infections, such as infections by Bd,
are low in European plethodontid salamanders. For this purpose,
we determined to what extent Bd is present in populations of all
eight species of Speleomantes.
Susceptibility to clinical chytridiomycosis, however, varies
greatly among plethodontid species. In amphibian hosts, differ-
ences in host susceptibility have been attributed to the presence of
fungicidal skin microbiota [17–19], antimicrobial peptides (re-
viewed in Rollins-Smith [20]), host genetics [21–23] and/or
environmental factors (e.g. [8]), which may affect invasion of
amphibian skin by the pathogen [24]. This leads us to hypothesize
that resistance of European plethodontid salamanders is due to
skin defenses that efficiently cope with Bd infection. Subsequently,
we then examined susceptibility of Speleomantes strinatii to experi-
mental infection with a global panzootic lineage strain of Bd
(BdGPL isolate IA2011, [7]). Finally, we determined to what
extent skin secretions of Speleomantes species are capable of killing
Bd.
Materials and Methods
All animal experiments were conducted according to biosecurity
and ethical guidelines and approved by the ethical committee of
the Faculty of Veterinary Medicine, Ghent University (EC2011-
073). All species involved in this study are protected as defined in
Annex IV of the EU Habitats Directive (Council Directive 92/43/
EEC on the Conservation of natural habitats and of wild fauna
and flora). The entire experiment was submitted and approved by
the Italian Ministry of Environment that issued permits to SS (issue
numbers: DPN-2010-0010807 and PNM-2012-0007331). In Italy,
state permits are valid over the entire country, since wildlife is a
public property (national law 157/92). In addition however, when
salamanders were sampled inside Protected Areas, local permits
were also obtained from the ‘‘Parco Regionale Frasassi and Gola
della Rossa’’ (permit number 3774/2012) the ‘‘Parco Regionale
delle Alpi Apuane’’ (permit number DD.5/2012) and ‘‘Parco
Regionale Alpi Marittime’’ (permit DD.165a/2011 issued to DO
and FO). Permit PNM-2012-0007331 was also valid to capture
animals (outside Protected Areas) to be used in experimental
infections at Ghent University (EC2011-073). Since the study did
not involve work on living animals in Italian laboratories,
authorisation from the Italian Ministry of Health was not required.
Between December 2004 and September 2012, we sampled 921
specimens including examples of all 8 recognized species of
Speleomantes (Speleomantes ambrosii,S. flavus,S. genei,S. imperialis,S.
italicus,S. sarrabusensis,S. strinatii,S. supramontis) at 65 localities in
mainland Italy and southern France (351 samples) and Sardinia
(570 samples) (Fig. 1, Table S1). Samples were collected by
rubbing the abdomen, feet and the ventral side of the tail at least
10 times as has been described by Van Rooij et al. [25] using a
rayon tipped swab (160 C, Copan Italia S.p.A., Brescia, Italy).
The sex of the adults was recorded for the three continental species
(S. italicus,S. ambrosii and S. strinatii) by checking for the presence of
the typical male mental gland [9] and the ratio females: males:
juveniles was approximately 1:1:1. DNA from the swabs was
extracted in 100 ml PrepMan Ultra (Applied Biosystems, Foster
City, CA, USA), according to Hyatt et al. [26]. DNA samples
were diluted 1:10 and quantitative PCR (qPCR) assays were
performed in duplicate on a CFX96 Real Time System (BioRad
Laboratories, Hercules, CA, USA). Amplification conditions,
primer and probe concentrations were according to Boyle et al.
[27]. Within each assay, 1 positive control sample containing Bd
DNA from a naturally infected and deceased Costa Rican
Eleutherodactylus sp. as template and 3 negative control samples
with HPLC water as template were included. Samples were
considered positive for Bd when a clear log-linear amplification
was observed, when the number of genomic equivalents (GE) of
Bd, defined as the measure of infection, was higher than the
detection limit of 0.1 GE and when amplification that met both of
the previous criteria was observed in both replicates. In case of
conflict between both replicates of the same sample, the sample
was run again in duplicate. To control and estimate inhibition, a
subset of samples negative for the presence of Bd (n = 84) was
retested under the same conditions as described above, but with an
exogenous internal positive control (VIC
TM
probe, Life technol-
ogies, Austin, TX, USA) included as described by Hyatt et al.
[26]. The Bayesian 95% credible interval for prevalence was
estimated as described by Lo¨tters et al. [28].
We assayed susceptibility to chytridiomycosis in five male
subadult S. strinatii by experimentally exposing them to a
controlled dose of Bd. Ten captive bred juvenile Alytes muletensis
were used as susceptible, positive control animals [29]. The
animals were housed individually in plastic boxes (20610610 cm)
lined with moist tissue, provided with PVC-tubes as shelter and
kept at 18uC. Crickets were provided as food items ad libitum. All
animals were sampled for the presence of Bd before inoculation
using the method described above. A virulent isolate of the global
panzootic lineage of Bd (BdGPL IA2011) [7], isolated in 2011 in
the Spanish Pyrenees and capable of causing severe chytridiomy-
cosis in urodelans was used in this study. All animals were exposed
to a single dose of 1 ml of distilled water containing 10
5
zoospores/ml. Skin swabs were collected weekly for 4 weeks and
processed as described above to determine the infection load for
Bd each animal exhibited over the course of the experiment. After
termination of the experiment, all animals were treated with
voriconazole [30]. The S. strinatii specimens are still kept following
strict biosecurity guidelines at the clinic for Exotic Animals and
Avian Diseases (Faculty of Veterinary Medicine, Ghent University)
for further follow-up.
To determine the extent to which skin secretions of Speleomantes
are capable of killing Bd zoospores, skin secretions were collected
non-invasively from wild individuals of 7 of the 8 Speleomantes
species (Table S2) and processed within 1 h (S. strinatii), 48 h (S.
ambrosii, S. italicus, S. flavus, S. supramontis, S. genei)or72h(S.
sarrabusensis). For this purpose, a microbiological inoculating loop
was gently rubbed over the dorsal tail until white skin secretions
accumulated on the loop. Prior to sampling sterile loops were cut
off, stored individually in sterile vials and the total weight of each
vial was determined. Collected skin secretions were weighed to the
nearest 0.1 mg by subtracting the weight of the vial and the
inoculation loop from the total weight. Varying amounts of skin
secretions were collected per Speleomantes specimen, ranging from
3.1 to 32.7 mg. Collected skin secretions were not further diluted
prior exposure to Bd zoospores. Loops with secretions were
incubated in a zoospore suspension. To keep the ratio between the
amount of skin secretions and Bd zoospores added constant, 10 ml
of zoospore suspension containing 10
6
zoospores/ml distilled
water was added per mg skin secretion. Samples were incubated
for 24 h at 20uC. At 0 and 24 h of incubation, the number of
Resistance of Speleomantes to Chytridiomycosis
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viable zoospores was assessed using qPCR on the zoospores that
were pretreated with ethidium monoazide (EMA, Sigma-Aldrich
Inc., Bornem, Belgium) as described by and validated in Blooi
et al. [31]. Viable/death differentiation is obtained by covalent
binding of EMA to DNA in dead Bd by photoactivation. EMA
penetrates only dead Bd with compromised membranes and DNA
covalently bound to EMA cannot be PCR amplified [32]. In brief,
at 0 and 24 h of incubation a 5 ml aliquot was taken from each
sample, transferred into a 24-well plate and 195 ml TGhL broth
(tryptone, gelatin hydrolysate, lactose) was added to each well for
its protective effect on viable Bd zoospores during EMA treatment.
Negative controls for skin secretion activity consisted of zoospore
suspensions not exposed to skin secretions in order to quantify the
‘natural’ loss of viability in Bd zoospores, while positive controls
were heat-killed zoospores. Five ml of a 1 mg/ml stock solution of
EMA in dimethyl formamide was added to 200 ml zoospore
suspension in TGhL broth to obtain a final concentration of
25 mg/ml EMA, incubated for 10 minutes, protected from light
and exposed to a 500 W halogen light at 20 cm distance for 5
minutes. Then, samples were washed by centrifugation (5000 rpm,
5 min, 20uC), the supernatant was discarded and the pellet was
suspended in HPLC water. In parallel, the total amount of Bd
zoospores in all samples, including controls, was enumerated using
exactly the same procedure as described above, only 5 ml HPLC
water was added to each sample instead of EMA. DNA extraction
and qPCR were then done as described above. Killing activity was
expressed as log(10) reduction of viable spores in a given sample
compared to the negative controls.
Results
None of the 921 skin swabs collected from any Speleomantes sp.
tested positive for Bd. The Bayesian 95% credible interval for the
observed prevalence of 0% is (0.0000, 0.0040). However, in 12 out
of 84 samples tested (14%) PCR-inhibition occurred that could not
be abolished by diluting the samples 1/100. Extrapolated to the
total number of individual salamanders tested, this reduces the
reliable number of Bd-negative salamanders to 789 with a
corresponding 95% credible interval of (0.0000, 0.0047).
None of the experimental animals tested positive for Bd prior to
the exposure. Over the four week infection period, 9/10 Alytes
muletensis developed marked infection with increasing Bd loads. Of
the 5 inoculated Speleomantes strinatii, 3 animals exhibited weak
infections (small GE value) at 7 days post infection (dpi) with an
average of 5.561.8 GE per swab. At 14 dpi, one salamander was
borderline positive (0.2 GE per swab) but at 21 and 28 dpi all
salamanders tested negative for Bd. In contrast, A. muletensis
exhibited median GE counts of 91 at 21 dpi and 3920 at 28 dpi
(Fig. 2). No clinical signs were noticed in the infected
salamanders. For animal welfare reasons, A. muletensis were treated
at 4 weeks pi using voriconazole to clear infection, as described by
Martel et al. [30].
Skin secretions of all Speleomantes species were capable of
efficiently killing Bd zoospores (Fig. 3). Exposure to skin secretions
resulted in a 200 to 20000 fold reduction of the number of viable
spores within 24 h post exposure.
Figure 1. Sampling locations for
Bd
in Europe. The boxed areas in the larger map of Europe (A) show the geographic locations. Expanded maps
show the collection sites in southeastern France, mainland Italy (B) and Sardinia (C). For map (B), the colours represent the species Speleomantes
strinatii (yellow), S. ambrosii (bright green) and S. italicus (red). For map (C), the colours represent the species S. genei (blue), S. sarrabusensis (purple),
S. imperialis (dark green), S. supramontis (pink) and S. flavus (orange). Localities are indicated by symbols proportional to sample size.
doi:10.1371/journal.pone.0063639.g001
Resistance of Speleomantes to Chytridiomycosis
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Discussion
Bd infections appear to be highly uncommon, if not absent, in
adult and juvenile Speleomantes of all species and throughout their
range. This observation is strengthened by the recent publication
of Chiari et al. [33], reporting absence of Bd in Sardanian S. flavus,
S. genei,S. imperialis,S. sarrabusensis and S. supramontis (n = 143). The
genus Speleomantes occupies an ecological niche highly suitable to
Bd colonization, persistence and spread due to relatively low
preferred body temperatures (,18.5uC, reviewed by Lanza et al.
[9]) and higher humidity environments. Contacts that would
facilitate interspecific transmission are also common because at
some locations salamanders occur outside of caves and are found
under retreat sites with other species that are capable of carrying
infections (FP personal observations, see also [3,4,6]). Intraspecific
transmission would also be highly likely, as courtship involves
intimate contact, salamanders crowd together in summer retreats,
and juveniles also exhibit highly aggregated distribution patterns at
certain times of the year [34]. Thus, substantial opportunity exists
for Bd for introduction into Speleomantes populations and to amplify
rapidly once introduced, but neither of these seems to have
occurred to any significant degree. Field studies of other
amphibian species have reported low prevalence or absence of
detectable infection in species that show seasonal fluctuations in
prevalence [35–37]. We find this an unlikely explanation for the
observed absence of infection. Moreover, for the present study
samples were taken from December to September and over
multiple years. Seasonally mediated fluctuations are associated
with strong variation in environmental metrics that influence Bd
growth and reproduction [35,36,38] suggesting that more stable
environments should result in more consistent patterns of infection
(see [39]). However, recent evidence suggests that variable
temperatures may be most favorable for Bd driven declines,
probably due to temperature drop induced zoospore release [40].
Variation of prevalence should be minimized in the cave-dwelling
Speleomantes sp., where relatively stable cave temperatures and
moisture regimes should buffer against environmentally mediated
changes in prevalence. Further, our experimental results indicate
the infection is unlikely in at least one species and the majority of
Speleomantes species are equipped with the tools to resist infection.
Exposing S. strinatii to a highly virulent Bd strain and in a
manner that resulted in potentially lethal infection in a susceptible
host did not result in persistent infection of the salamanders. It is
possible that the low GE values detected until two weeks post
inoculation in S. strinatii represent dead Bd cells or some form of Bd
DNA contamination rather than active infection. Thus it is
possible that S. strinatii is extremely efficient at blocking epidermal
colonization by Bd even when exposed to a highly concentrated
and strong dose of Bd zoospores, but our experimental design
Figure 2. Experimental infection of
Speleomantes strinatii
with
Bd
.Infection loads are represented as log (10) genomic equivalents (GE)
of Bd in skin swabs from Speleomantes strinatii (left panel) and
compared with Alytes muletensis (right panel) serving as positive control
animals, up to four weeks post experimental inoculation with Bd. Each
symbol represents an individual animal.
doi:10.1371/journal.pone.0063639.g002
Figure 3. Killing activity of skin secretions of
Speleomantes
species against
Bd
.Killing activity of Speleomantes skin secretions at
physiological concentrations is expressed as log(10) viable spores of Bd added to the skin secretions –log(10) viable spores recovered 24 h later.
Results are presented as mean genomic equivalents of Bd (GE) 6standard error (SEM); n = sample size.
doi:10.1371/journal.pone.0063639.g003
Resistance of Speleomantes to Chytridiomycosis
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prevents us from distinguishing between this and rapid clearing of
infection. Since successful epidermal colonization by Bd requires
keratinocyte invasion [22,41,42], we hypothesized that Speleomantes
skin contains highly effective fungicidal properties that prevent
skin invasion. Indeed, we showed that Speleomantes skin secretions
were very efficient in killing Bd zoospores as assessed using a
recently developed and highly reproducible assay [31]. Factors
present in the skin secretions that account for the observed Bd
killing need further identification but probably include antimicro-
bial peptides (AMP) [43–45] and/or bacterially produced metab-
olites [17,18,46]. AMPs that play a defensive role against invasion
by pathogenic microorganisms have been described for other
Ambystomidae and plethodontid species [47–49] but not charac-
terized. Hitherto, only the antifungal metabolites 2,4-diacetylph-
loroglucinol, indol-3-carboxaldehyde and violacein have been
identified that are secreted by symbiotic bacteria residing on the
skin of plethodontid species Plethodon cinereus and Hemidactylium
scutatum [17,46]. Moreover, these metabolites may work synergis-
tically with AMPs to inhibit colonization of the skin by Bd [50].
Further characterization of such AMPs and the composition of
microbial skin communities, combined with the study of their
assessment in plethodontid species and/or populations can open
new perspectives for further understanding factors mediating
resistance towards chytridiomycosis, its control and mitigation. In
addition, a study of microbial skin communities has the potential
to direct probiotic conservation strategies for susceptible species in
the area.
The apparent absence of Bd and chytridiomycosis driven
declines in Speleomantes throughout their range, lack of colonization
or sustained infection in experimentally infected animals and
pronounced Bd killing capacity of Speleomantes skin secretions
together suggest the genus Speleomantes to be refractory to Bd
infection and thus resistant to chytridiomycosis. Resistance to
chytridiomycosis would at least in part explain the localized
persistence of the genus Speleomantes in the presence of highly
virulent BdGPL strains in Europe. This situation differs markedly
from some of the plethodontids in North America that are
susceptible to infection and those in the Neotropics that underwent
recent and sharp chytridiomycosis driven declines upon the arrival
of Bd [15,16,51–55]. While our results are preliminary evidence
that the genus Speleomantes is a low-risk taxon for decline due to
chytridiomycosis, we recommend additional studies that further
investigate the risk Bd may pose to this unique amphibian taxon.
Supporting Information
Table S1 Overview of the sampled Speleomantes spe-
cies, sampling localities, sample size and sampling
dates. Seconds have been removed from coordinates to prevent
illegal collection.
(DOCX)
Table S2 Overview of the sampled Speleomantes spe-
cies for collection of skin secretions and respective
sampling localities. Seconds have been removed from
coordinates to prevent illegal collection
(DOCX)
Acknowledgments
We thank in particular Paolo Casale, Giacomo Bruni, Sandro Casali,
David Fiacchini, Carlo Torricelli and Olivier Gerriet for field assistance.
Author Contributions
Conceived and designed the experiments: FP TWJG MCF SS. Performed
the experiments: FP PVR MB GT SB GS TW SB CA FO DO AM SS.
Analyzed the data: FP PVR MB GT WB BRS AM SS. Wrote the paper:
FP PVR TWJG MCF WB BRS AM SS.
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