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Biological Invasions
ISSN 1387-3547
Biol Invasions
DOI 10.1007/s10530-016-1161-y
Invasive North American bullfrogs
transmit lethal fungus Batrachochytrium
dendrobatidis infections to native
amphibian host species
Claude Miaud, Tony Dejean, Karine
Savard, Annie Millery-Vigues, Alice
Valentini, Nadine Curt Grand Gaudin &
Trenton W.J.Garner
1 23
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ORIGINAL PAPER
Invasive North American bullfrogs transmit lethal fungus
Batrachochytrium dendrobatidis infections to native
amphibian host species
Claude Miaud .Tony Dejean .Karine Savard .Annie Millery-Vigues .
Alice Valentini .Nadine Curt Grand Gaudin .Trenton W. J. Garner
Received: 13 March 2015 / Accepted: 10 May 2016
ÓSpringer International Publishing Switzerland 2016
Abstract Invasive species can be a threat to native
species in several ways, including transmitting lethal
infections caused by the parasites they carry. How-
ever, invasive species may also be plagued by novel
and lethal infections they acquire when invading,
making inferences regarding the ability of an invasive
host to vector disease difficult from field observations
of infection and disease. This is the case for the
pathogenic fungus Batrachochytrium dendrobatidis
(Bd) in Europe and one invasive host species, the
North American bullfrog Lithobates catesbeianus,
hypothesized to be responsible for vectoring lethal
infection to European native amphibians. We tested
this hypothesis experimentally using the alpine newt
Ichthyosaura alpestris as our model native host. Our
results show that infected bullfrog tadpoles are
effective vectors of Bd. Native adult newts co-housed
with experimentally infected bullfrog tadpoles
became Bd infected (molecular and histological tests).
Moreover, the exposed adult newts suffered mortality
while the majority of infected bullfrog tadpoles
survived until metamorphosis. These results cannot
resolve the historical role of alien species in estab-
lishing the distribution of Bd across Europe or other
regions in the world where this species was intro-
duced, but they show its potential role as a Bd
reservoir capable of transmitting lethal infections to
native amphibians. Finally, our results also suggest
that the removal of infected bullfrogs from aquatic
environments may serve to reduce the availability of
Bd in European amphibian communities, offering
another justification for bullfrog eradication
C. Miaud (&)T. Dejean K. Savard
A. Millery-Vigues A. Valentini N. Curt Grand Gaudin
UMR CNRS 5553, Laboratoire d’E
´cologie Alpine,
Universite
´Savoie-Mont-Blanc,
73376 Le-Bourget-Du-Lac, France
e-mail: claude.miaud@cefe.cnrs.fr
C. Miaud
PSL University, Ecole Pratique des Hautes Etudes,
Bioge
´ographie et Ecologie des Verte
´bre
´s, Centre
d’Ecologie Fonctionnelle et E
´volutive (UMR 5175),
Campus CNRS, 34293 Montpellier, France
T. Dejean
Parc naturel re
´gional Pe
´rigord-Limousin,
24450 La Coquille, France
K. Savard
Agriculture and Agri-Food Canada, Ottawa,
ON K1A 0C6, Canada
T. W. J. Garner
Institute of Zoology, Zoological Society of London,
Regents Park, London NW1 4RY, UK
T. W. J. Garner
Environmental Sciences and Development, Northwest
University, Private Bag X6001, Potchefstroom 2531,
South Africa
123
Biol Invasions
DOI 10.1007/s10530-016-1161-y
Author's personal copy
programmes that are currently underway or may be
considered.
Keywords Introduced amphibian Disease Cross-
contamination Fungus Alpine newt
Introduction
Invasive, non-native species are considered to be one
of the greatest threats to biodiversity and threaten
native species through a variety of mechanisms. The
co-introduction of parasites capable of eliciting sig-
nificant pathogenesis in naı
¨ve native hosts is thought
to be one of the major mechanisms behind biodiversity
loss attributable to invasive species (Daszak et al.
2000; Prenter et al. 2004; Crowl et al. 2008). Indeed,
parasites that are transported with invasive species
tend to reach equivalent prevalence in native species
(Torchin et al. 2003), sometimes with devastating
consequences (Martel et al. 2014; Doddington et al.
2013; Bosch et al. 2013). However, invasive species
may carry significantly reduced parasite diversity
when invading (Torchin et al. 2003) and commonly
become infected with parasites that occur in endemic
residents (Colautti et al. 2004;Bu
¨rgi and Mills 2014).
Invasive species that are infected with resident para-
sites can suffer costs exceeding those experienced by
the native host species (Wolfe et al. 2004)or
equivalent to those experienced by native species
infected with newly introduced parasites (Heger and
Jeschke 2014). The unpredictability of these relation-
ships means that patterns of parasite infection and
disease in native and invasive hosts do not always
indicate which host may be serving as a vector for the
parasite.
Batrachochytrium dendrobatidis (Bd), a global
fungal pathogen of amphibians, is presumed to be an
invasive parasite in many parts of its range (Farrer
et al. 2011). Bd invasion is commonly attributed to the
release of infected, asymptomatic species that have
been displaced as a result of trade (Hanselmann et al.
2004). A prime example is that of the North American
bullfrog Lithobates catesbeianus (Hanselmann et al.
2004). Due to their ubiquity as a traded species
infected with Bd (Fisher and Garner 2007; Bai et al.
2010; Schloegel et al. 2009), their distribution, and a
consistent pattern of infection with Bd (Garner et al.
2006), they have been proposed to be important
vectors of Bd into native amphibians. Bullfrogs may
contribute to maintaining Bd in native amphibian
community (Peterson and McKenzie 2014). The
distribution of invasive bullfrogs appears as a poor
predictor of Bd distributions (Richardson et al. 2014;
Bataille et al. 2013). Native bullfrogs do have the
ability to transmit infection to species that occur
within their natural range (Greenspan et al. 2012), and
invasive bullfrogs tend to produce a higher number of
Bd zoospores relative to native species (Peterson and
McKenzie 2014) but do not appear to sustain infec-
tions for prolonged periods of time and can die from
heavy infections (Gervasi et al. 2013). The evidence
that invasive bullfrogs can act as significant vectors of
chytridiomycosis to native hosts is relatively weak.
Bullfrogs have been widely introduced in Europe
in an uncoordinated, multinational effort to establish
viable populations for the trade in frog legs (Ficetola
et al. 2007). Invasive bullfrog populations were
consistently founded by a small number of adults
directly transported from their native range, and
much of the current distribution in Europe probably
arose through translocation from these founder
populations (Ficetola et al. 2008). This small
number of potential transport vectors is incompat-
ible with the widespread, pervasive distribution of
Bd across Europe (Olson et al. 2013) and the
patterns of Bd invasion in areas of Europe where
bullfrogs are absent (Bielby et al. 2013; Bosch et al.
2013; Walker et al. 2008,2010). Introduced bull-
frogs can potentially transmit Bd to native amphib-
ians, but spill-over can be from native hosts to
invasive bullfrogs. If this were the case, invasive
bullfrogs would be accruing infection from native
hosts, and we would predict that native species
commonly infected with Bd would be relatively
tolerant of infection while bullfrogs would exhibit
costs such as the post-metamorphic mortality of
bullfrogs experiencing strong infections in their
native range, as described by Gervasi et al. (2013).
The possibility does remain that bullfrogs act as
vectors of infection and disease in Europe and can
act as significant reservoir hosts. Indeed, Bd has
been documented infecting bullfrog populations
across Europe (Garner et al. 2006). In this instance,
we predict that native European amphibians would
be susceptible to lethal chytridiomycosis caused by
transmission from infected bullfrogs, bullfrogs
C. Miaud et al.
123
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would not exhibit significant costs associated with
exposure to and infection with Bd.
In this paper, we experimentally determine if
invasive bullfrogs significant vectors of Bd to Euro-
pean amphibians. We cohoused experimentally
infected bullfrog tadpoles with adult native amphib-
ians, in this case, the alpine newt. We selected the
alpine newt because it is known to be infected with Bd
across Europe (Zampiglia et al. 2013; Sztatecsny and
Glaser 2011) but little is known about its susceptibility
to lethal chytridiomycosis. We considered bullfrog
tadpoles as the appropriate life history stage for
assessing reservoir status of the species because
tadpoles with prolonged larval periods are commonly
cited as significant reservoirs of infection (Briggs et al.
2010; Walker et al. 2010). We recorded infection
status, determined through molecular diagnostics and
histology, burden of infection, and survival in both
bullfrog tadpoles and adult alpine newts. For bullfrogs,
we measured mortality rates until the onset of
metamorphosis, as significant costs associated with
larval infection initially manifest when metamorpho-
sis is near to completion (Gervasi et al. 2013; Walker
et al. 2010; Garner et al. 2009).
Materials and methods
One clutch of American bullfrog (Lithobates cates-
beianus) spawn was collected in June 2009 at an
artificial pond in Ambare
`s in southwestern France
(44°5602200N, 0°3100400 E; 20 m a.s.l.). Eggs were
hatched and larvae reared in the laboratory in three
plastic containers (400 9600 9200 mm), each con-
taining approximately 35 L of aged tap water. Larvae
were fed flaked goldfish food provided ad libitum
during this and the subsequent exposure periods (see
below). In May 2010, 30 of the 250 available tadpoles
(Gosner stages 26–30; Gosner 1960) were selected
randomly and examined for evidence of infection by
swab-sampling their mouthparts (swab ref. M01-
MW100, Kitvia Co.) and testing DNA extracted from
these swabs using the TaqMan Assay described by
Boyle et al. (2004). Because the extraction reagent is a
PCR inhibitor, samples were diluted by a ratio of 1:10
prior to attempted PCR amplification. For all molec-
ular assessments of infection, amplifications yielding
quantitative scores of 0.1 genomic equivalents (GE;
untransformed value) or greater were considered Bd-
positive, allowing us to assign individuals as either
‘infected’ or ‘not infected’.
Twenty tadpoles were transferred to plastic con-
tainers (240 9160 9144 mm) filled with approxi-
mately 2 L of aged tap water and maintained as such
until the end of the experiment as negative controls for
infection with Bd. Over the course of the next 20 days,
we individually exposed another 120 of the remaining
tadpoles five times to 30,000 zoospores using a Bd
culture isolated from a dead, recently metamorphosed
Alytes obstetricans. The dead Alytes was collected at a
recurrent A. obstetricans mass-mortality site located in
the French Pyrenees where only the global pandemic
lineage (Bd-GPL) is known to occur and was geno-
typed as such (Farrer et al. 2011,2013). Before each
exposure, 40 of the 160 mL of water in each tadpole
container were replaced and all visible tadpole faeces
removed using a disposable sterile pipette. Seven days
after the fifth exposure, all tadpoles were again swab-
sampled and tested for evidence of infection using the
qPCR molecular diagnostic.
At the same time, we collected 40 male alpine
newts (Ichthysaura alpestris) from artificial ponds
located on the Bourget-du-Lac campus of the Univer-
sity of Savoie-Mont-Blanc (45°3803000N, 5°5200200 E;
240 m a.s.l.). Newts (mean mass ±SD =1.8 ±
0.23 g) were also housed individually, swab sampled
over the fore- and hindlimbs, abdomen and cloaca and
swabs tested for evidence of infection using the qPCR
molecular diagnostic. Individual newts were then
housed in 40 plastic containers (240 9160 9
144 mm) containing 1.5 L of aged tap water. Ten of
these experimental units were left as is, containing
only a single newt. We added three bullfrog tadpoles
to all of the other 30 replicates; 10 with unexposed and
presumably uninfected tadpoles, and 20 with exposed
and presumably infected tadpoles. Tadpoles and newts
were cohoused for 15 days at 20.1 ±1.0 °C and on a
16 h/8 h artificial day/night schedule. Water levels
were assessed daily and topped up when needed with
aged tap water. On day 15, tadpoles were removed,
water levels reduced by 500 mL and containers tilted
to allow newts to have access to a terrestrial area (a
plastic box, 140 9140 mm, placed within the exper-
imental unit). While cohoused with tadpoles and
during the post-exposure period, newts were fed
chironomid larvae every 48 h. Newt containers were
cleaned every day with a disposable, sterile plastic
pipette to remove feces and food remains. For 29 days
Transmit lethal fungus Batrachochytrium dendrobatidis infections
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after tadpoles were removed we recorded newt
mortality and all newts were again swab sampled
after death or as survivors at the end of the
experiment. Tadpoles that were cohoused with newts
(1 per replicate involving exposed tadpoles, n =30,
and 2 per unexposed replicates, n =20) were
rehoused individually in plastic containers as per the
negative control tadpoles. Tadpoles were maintained
as such until the onset of metamorphosis (Gosner
stage 42; Gosner 1960) and then swab-sampled across
the epidermis for evidence of infection. We switched
swab sampling to skin at this stage because tadpoles
have shed keratinized mouthparts, which are the
target of infection earlier in development, and
because keratinization of the stratum corneum that
occurs at this time becomes the new target for Bd
infection.
Dead newts were stored in 70°alcohol. The four
newts exposed to Bd-infected bullfrog tadpoles alive
at the end of the experiment were sacrificed with an
overdose of 10 mL/L of phenoxyethanol. Ten cross
sections (4 lm) were taken from skin sampled from
the interior proximal part of the hind foot of each newt.
The skin was embedded in tissue-teck (Sakura
Fineteck, USA) and frozen at -18 °C. Cross sections
were cut using a LEICA CM3050 S freezing
microtome, stained with Ehrlich’s haematoxylin, and
examined for evidence of infection with Bd using light
microscopy.
Statistical analyses were performed with the Pro-
gram R (R Development Core Team, 2010). We used
log rank tests to test for differences amongst treatment
groups for both bullfrog tadpoles and alpine newts. We
also assessed the differences in alpine newt mortality
between the 3 treatments using survival analysis
(Kaplan–Meier estimate), with ‘time until death’ as
the response variable. Individuals without a corre-
sponding time until death (i.e., survived to the end of
the experiment, n =23) were removed from the
analysis.
Results
Bullfrog tadpole Bd status and survival
The 30 tadpoles from which unexposed tadpoles were
selected for cohousing with newts (n =10) tested
negative for Bd DNA (Table 1). The 60 tadpoles
experimentally exposed to Bd zoospores were com-
prehensively infected with Bd (mean GE ±1 SD:
58.6 ±32.8) at the start of the cohousing period with
Table 1 Design and results of the cross contamination experiment, the co-housing of alpine newt Ichthyosaura alpestris with
American bullfrog tadpoles Lithobates catesbeianus infected by the fungus Batrachochytrium dendrobatidis
Before the experiment qPCR test
Bd?Bd-
Alpine newts alone (n =40) 0 40
Bullfrog tadpoles alone (n =20) 0 20
Bullfrog tadpoles Bd?(n =60) 60 0
Bullfrog tadpoles Bd-(n =30) 0 30
During the experiment qPCR test Histological test
Bd?Bd-Bd?Bd-
Alpine newt alone (n =10) 0 10 – –
Alpine newt with Bd-tadpoles (n =10) 0 10 – –
Alpine newts with Bd?tadpoles (n =20) overall 14 6 17 3
Alpine newts with Bd?tadpoles which died (n =16) 11 5 14 2
Alpine newts with Bd?tadpoles which survived (n =4) 3 1 1 2
Bullfrog tadpoles Bd?=tadpole experimentally infected with Bd and co-housed with alpine newts. Bullfrog tadpoles
Bd-=tadpole Bd-and co-housed with alpine newts. Alpine newt with Bd-or Bd?tadpoles =1 alpine newt adult is co-
housed with three American Bullfrog tadpoles
C. Miaud et al.
123
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newts (n =20, Table 1). Exposed tadpoles that were
removed from experimental replicates all tested
positive for infection on day 15 (n =60, mean
GE ±1 SD: 39.6 ±23.3), those from unexposed
replicates did not test positive. Twenty of the 26
tadpoles from the exposed replicates surviving to the
end of the experiment also tested positive (n =20,
mean GE ±1 SD: 49.7 ±29.3), whereas no unex-
posed tadpoles tested positive on day 44.
Tadpoles started to metamorphose (Gosner stage
42) on day 70. Only seven tadpoles did not survive to
this date, among them 4 exposed to Bd and cohoused
with alpine newt, 2 unexposed to Bd and cohoused with
alpine newt, and 1 control (unexposed to Bd and alone).
Survival of bullfrog tadpoles did not differ across
tadpole treatment groups (20 tadpoles housed alone
and the two newt experiment treatments: Fig. 1a; Log
rank test, Chi square test =1.3, df =2, p=0.526).
Alpine newt Bd status and survival
All 40 male alpine newts tested negative for Bd before
cohousing (Table 1). Newts housed alone (n =10) or
with unexposed bullfrog tadpoles (n =10) for
15 days tested negative for Bd at the end of the
experiment. Alternatively, 14 newts cohoused with
infected bullfrog tadpoles tested positive for infection
either at time of death or at the end of the experiment
(mean GE ±1 SD: 7.6 ±6.2). The remaining 6
newts cohoused with infected bullfrog tadpoles tested
negative for Bd DNA during this experiment. Among
the 16 newts cohoused with infected bullfrog tadpoles
died during the experiment, 11 tested PCR positive for
infection. Of the 4 newts cohoused with infected
bullfrog tadpoles that survived, 3 were positive for Bd
(respectively 3.15, 4.4 and 46.4 GE).
Histological examinations were performed on the
skin of all the newts that were exposed to infected
bullfrog tadpoles (16 dead newts and the 4 newts alive
at the end of the experiments, Table 1). Intracellular
thalli and zoosporangia at various stages of maturation
were observed in the 11 newts that died and tested PCR
positive for Bd, in 3 of the 5 dead newts that tested PCR
negative for Bd, and in the 3 newts PCR positive for Bd
which survived to the end of the experiment. One newt
that survived and PCR tested negative for Bd also had
no observable thalli and zoosporangia.
Significant variation of mortality occurred among
newt treatments: 16 of 20 newts exposed to Bd-infected
bullfrog tadpoles were dead by the end of the exper-
iment, only one of the 20 newts that were not exposed to
Bd or tadpoles died. Newts began dying on day 26,
9 days after bullfrog tadpoles were removed (Fig. 1b).
Cohousing newts with infected tadpoles significantly
affected the mortality rate (Log rank test, Chi
square =21.7, df =2, p=1.91 910
-05
). At day
30, survival of newts cohoused with infected tadpoles
0 10203040506070
0.0 0.2 0.4 0.6 0.8 1.0
Days surviving
Proportion alive
010203040
0.0 0.2 0.4 0.6 0.8 1.0
Days surviving
Proportion alive
(a)
(b)
Fig. 1 Survival curves for American bullfrog tadpoles Litho-
bates catesbeianus tadpoles (a) and alpine newt Ichthyosaura
alpestris (b). For both figures, animals housed singly and not
exposed to the fungus Batrachochytrium dendrobatidis (Bd) are
represented by the dotted line, animals cohoused with unin-
fected animals are represented by the solid line and cohoused
animals where tadpoles were infected with Bd are represented
by broken line
Transmit lethal fungus Batrachochytrium dendrobatidis infections
123
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was reduced by 25 % when compared to newts
cohoused with uninfected tadpoles or newts reared
alone (Table 2). At day 38, survival of newts cohoused
with infected tadpoles was reduced by 80 % compared
to newts cohoused with uninfected tadpoles and 70 %
compared to those reared alone (Table 2).
Discussion
Genetic and genomic data have been used to describe
geographically widespread Bd and endemic Bd lin-
eages (Farrer et al. 2011,2013). The contact between
allopatric populations of Bd could allow recombina-
tion, genera of virulent lineages, and lead to contem-
porary amphibian disease emergence (Farrer et al.
2011). Increased sampling and analysis confirmed that
Bd is composed of multiple divergent lineages, but
which appear endemic in some parts of its range and
novel (i.e. emerging) in others (Rosenblum et al.
2013). Perhaps more relevant to this study, patterns of
mutation, recombination and aneuploidy make resolv-
ing historical relationships of isolates, even within
lineages, problematic (Farrer et al. 2013). Because of
this, it is questionable if the relationship between
invasive amphibian hosts and history of Bd invasion
can ever be clearly elucidated.
Nevertheless, introducing infected hosts of any kind
to naı
¨ve amphibian communities increases host den-
sity, elevating transmission rates (Rachowicz and
Vredenburg 2004), and prevalence of infection, which
may be vectored into susceptible species. The Amer-
ican bullfrog is a good candidate to fulfil this role: it has
been globally introduced (review in Ficetola et al.
2007) and carries Bd in native (Ouellet et al. 2005) and
introduced populations in Asia (Bai et al. 2010),
Europe (Garner et al. 2006), North America (Peterson
and McKenzie 2014) and South America (Hanselmann
et al. 2004; Schloegel et al. 2010). Direct evidence of
the role of bullfrog as a reservoir of local Bd lineages
and/or introduction of allopatric lineages to native
amphibian communities lacking, but in Colorado,
amphibian communities invaded by non-native bull-
frogs were more likely to support Bd infected individ-
uals (Peterson and McKenzie 2014). The transmission
of Bd from native American bullfrog juveniles to
syntopic wood frog tadpoles (Lithobates sylvaticus)
was shown experimentally by Greenspan et al. (2012).
Extending on their work, our experiment shows that
infected and non-native bullfrog tadpoles can transmit
Table 2 Survival of alpine newts Ichthyosaura alpestris co-housed with American bullfrog tadpoles Lithobates catesbeianus
infected by the fungus Batrachochytrium dendrobatidis
Time n n death Survival SE Lower 95 % CI Upper 95 % CI
Treatment =newts with Bd positive tadpoles
a
26 20 1 0.95 0.0487 0.8591 1.000
28 19 3 0.80 0.0894 0.6426 0.996
30 16 1 0.75 0.0968 0.5823 0.966
32 15 1 0.70 0.1025 0.5254 0.933
33 14 1 0.65 0.1067 0.4712 0.897
36 13 1 0.60 0.1095 0.4195 0.858
38 12 8 0.20 0.0894 0.0832 0.481
Treatment =newt as control (newt alone)
b
30 10 0 1.0 – – –
33 10 1 0.90 0.0949 0.7320 1.0000
38 10 1 0.9 0.0949 0.7320 1.0000
Time =days since the beginning of the experiment, n =number of alive alpine newts, n death =number of dead alpine newt along
the last 24 h
a
12 dead in this treatment
b
1 dead in the control at day 33
C. Miaud et al.
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Bd to adult alpine newts under experimental condi-
tions. Newts exposed to infected bullfrog tadpoles in
our study readily developed infections in a matter of
days and sustained these infections for weeks after
exposure without any need for re-exposure beyond the
15 days of cohousing. In the wild, bullfrog populations
are well-established in France and geographically
overlap with native alpine newt populations in one
region (Ficetola et al. 2007). Temporally the potential
for spill-over exists, as breeding by adult newts
coincides with the presence of bullfrog tadpoles for a
period of months (Michelin et al. 2014), and tadpoles
we experimentally exposed were still infected 70 days
after initial exposure, with no significant decrease in
infection burden. This was strong enough to transmit
infection to at least 70 % of the cohoused newts. Newly
metamorphosed bullfrogs are not always an efficient
reservoir species for Bd and may experience heavy
mortality (Gervasi et al. 2013), but bullfrog meta-
morphs in our study did not suffer mortality from this
virulent Bd-GPL lineage. Range overlap, persistent,
and strong burdens of infection and high prevalence, in
this case across life history stages, are all key traits of a
competent vector, as transmission is more likely when
infectious particles are available for transmission over
a longer time span (Murray et al. 2009).
In nature newts commonly leave water and stay on
land for significant periods of time. Behavioural
avoidance of aquatic zoospores has been described
in another species (e.g. McMahon et al. 2014), and our
experiment offered no opportunity for newts to escape
from the water. The aquatic environment is important
for transmission of zoospores, and the heavy infec-
tions consistently generated in our study by cohousing
with bullfrog tadpoles were of similar strength to
burdens estimated from newts captured from aquatic
environments in the wild at sites where newts occur at
high densities (Garner et al. 2005). Bullfrogs have not
been detected at the newt study sites sampled by
Garner et al. (2005), and mortality of alpine newts
attributable to chytridiomycosis has never been
reported. The impact of host community structure on
probability of infection and strength of infection with
Bd is a common theme in amphibian host/chytrid
systems, where increased density of hosts harbouring
the heaviest infections is expected to elicit greater
prevalence and heavier infections (Searle et al. 2011;
but see Bielby et al. 2015). Infections of tadpoles were
far stronger than newts in our study, which may go
some way towards explaining why experimental newts
experienced significant mortality, newts occupying
ponds lacking a heterospecific reservoir exhibiting
stronger infections appear not to. Further study of the
relationships between habitat choice, host community
composition, and susceptibility of alpine newts to
infection and chytridiomycosis is certainly warranted.
Some newts that died did not exhibit detectable in-
fection using either diagnostic method. Studies have
reported increased risk of mortality during prolonged
exposure to Bd even with no evidence of infection at
time of death (Luquet et al. 2012; Garner et al. 2009).
Resisting infection with Bd is probably costly, poten-
tially increasing the mortality risk of these individuals.
But not in all cases: of the four survivors, at least 3
exhibited significant levels of infection. One hypoth-
esis for this could be inter-individual variation in
immune defence. Innate immunity in the form of skin
antimicrobial peptides secretions can act as a first line
of defence against Bd, and has been shown to allow
tolerance of infection (Woodhams et al. 2007; Rollins-
Smith 2009; Ramsey et al. 2010). Whatever compo-
nent of immunity may be responsible for tolerance or
resistance, repeated exposure to Bd has been shown to
immunize against subsequent costs (McMahon et al.
2014; but see Cashins et al. 2013). This seems unlikely
in our case as we have never detected infection in the
source population for the newts we used (102 adults
tested with qPCR, C. Miaud, unpublished data), and
none of our experimental animals tested positive
before exposure. These are strong indications that the
alpine newts used in the experiment were Bd naı
¨ve.
These experimental findings are the first evidence
of death attributable to exposure to an infection with
Bd in alpine newts, adding to an ever-growing list of
European amphibian species that may be deleteriously
affected through interactions with this fungus (Bala
´z
ˇ
et al. 2014; Bosch et al. 2013; Garner et al. 2013;
Luquet et al. 2012; Bielby et al. 2009; Garner et al.
2009; Bosch et al. 2001). Additional surveillance for
Bd-related newt mortality in the wild is called for to
investigate potential disease-associated decline under
natural conditions.
Conclusion
We conclude from our experiment that invasive
bullfrogs are effective reservoirs of Bd, capable of
Transmit lethal fungus Batrachochytrium dendrobatidis infections
123
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transmitting infections to native hosts. Infections with
the Bd-GPL lineage transmitted by invasive bullfrogs
can be sustained for weeks after initial exposure and
have the capacity to cause significant mortality in
native species. Although we cannot resolve the debate
regarding the role invasive hosts have played in
introducing Bd to Europe, we do conclude that
infected, invasive bullfrog tadpoles will increase the
likelihood that infection with Bd will be maintained in
a European amphibian community (Spitzen-van der
Sluijs et al. 2014). Experimental results (this study) do
not always reflect field conditions, but spill-over from
bullfrog tadpoles to native European amphibians has
the potential to drive mortality in native species.
Removal of invasive bullfrogs as a conservation
strategy has been adopted in several European coun-
tries based on the conclusion that bullfrogs can cause
native-species declines due to competition and preda-
tion (Kupferberg 1997; Lawler et al. 1999). Our study
further justifies these efforts. Even if removal may not
eliminate infections in native hosts, any reduction in
density of infected hosts capable of transmitting to
susceptible hosts should reduce the likelihood of
infections reaching potentially lethal thresholds
(Peterson and McKenzie 2014). Exposure duration,
zoospore load, and virulence can dictate the severity of
the costs associated with exposure and infection (e.g.
Briggs et al. 2010), and the removal of infected
bullfrogs has a strong likelihood of reducing the
impact of Bd on other, native susceptible host species.
Acknowledgments All experimental work done here was
ethically reviewed at Universite
´de Savoie-Mont-Blanc.
Authorization to catch Alpine newts was provided by the
regional authorities (DREAL Rho
ˆne-Alpes, permit No.
2009–2010). CM, TD, AM, NCGG and AV were supported
by ANR through the EU BiodivERsA-funded project R.A.C.E.
(Risk Assessment of Chytridiomycosis to European amphibian
biodiversity, M. Fisher coordinator), TWJG was supported by a
NERC (Grant NE/G002193/1) through the EU BiodivERsA-
funded project R.A.C.E., but is currently supported for research
on chytridiomycosis by NERC standard grant NE/K012509/1.
KS was hosted in France thanks to the Office Franco-
Que
´be
´quois pour la Jeunesse, programme Formation & Emploi.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest: none of the authors of this paper has a
financial or personal relationship with other people or organi-
zations that could inappropriately influence or bias the content
of the paper.
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