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Infection prevalence in three lowland species of Harlequin toads from the threatened genus Atelopus

Herpetological Review 46(4), 2015
Herpetological Review, 2015, 46(4), 528–532.
© 2015 by Society for the Study of Amphibians and Reptiles
Infection Prevalence in Three Lowland Species of
Harlequin Toads from the Threatened Genus Atelopus
Atelopus, a species-rich Neotropical genus within the
family Bufonidae (Lötters 1996), is distributed from Costa Rica
south to Bolivia and eastward into the Guyanas (La Marca et al.
2005). Despite declines and disappearances of Atelopus species
occurring before Batrachochytrium dendrobatidis (hereafter, Bd)
was described (Longcore et al. 1999), Bd has been implicated in
losses (La Marca et al. 2005; Lampo et al. 2006). The majority of
these species occur in the Andes Range above 1500 m, and are
considered one of the most threatened genera of amphibians
worldwide. At least 71% (69 of 97) of Atelopus species are listed
as Critically Endangered or Extinct (IUCN 2015). Still, many
species have uncertain conservation status because they remain
undescribed (Coloma et al. 2010) or because they require
systematic revision (Lötters et al. 2011). The most affected
species within the genus occur along mid-to-high elevation
streams with nearly 75% of those species facing serious declines
(La Marca et al. 2005). Colombia harbors the highest number
of Atelopus species (Lötters 1996) and, similar to other parts of
its distribution, nearly all species that occur in the mid-to-high
elevations have declined sharply (Rueda-Almonacid et al. 2005).
In the Colombian lowlands, however, species still persist at
altitudes ranging from 0–600 m and there are no reported declines
in this group (Rueda-Almonacid et al. 2005). This difference
in survival is usually attributed to Bd not being able to survive
well at lower elevations, which in the tropics usually means
higher and less variable environmental temperatures. In order
to evaluate whether Bd can be considered as a potential threat
for Atelopus species, we assessed Bd prevalence and infection
intensity on three lowland species that occurring between 5–115
m elevation, examining live animals during contemporary
field surveys in 2007 to 2012 and archived museum specimens
collected between 1968 and 2000.
We studied three Atelopus species occurring in three different
localities in the coastal forests of Colombia (Fig. 1). We visited
each site 3–5 times between 2007 and 2012 (Table 1). Atelopus aff.
elegans, from Gorgona Island (2.966667°N, 78.166667°W; 6–115
m elev.) was sampled three times (July 2007, July 2010, and June
2012). Atelopus spurrelli, in the municipality of Arusí (5.566667°N,
77.5°W; 90 m elev.) on the Pacific coast was sampled five times
(May and November 2009, June and September 2011, and June
2012). Atelopus aff. limosus, an undescribed species occurring
near the municipality of Capurganá (8.6°N, 773333°W; 150 m),
close to the border between Panama and Colombia, was sampled
three times (September 2008, October 2009, and July 2012).
Survey teams composed of 2–5 people conducted field
trips to each locality for 2–8 days per trip. To maximize capture
success, we surveyed along streams and along forest trails using
visual encounter surveys (Heyer et al. 1994). For each animal
we collected the following information to look for potential
Department of Biological Sciences, Universidad de los Andes,
Bogotá, AA 4976, Colombia
Department of Biology, San Francisco State University,
San Francisco, California 94132-1722, USA
Department of Biological Sciences, Universidad de los Andes,
Bogotá, AA 4976, Colombia
*Corresponding author; e-mail: vicky
Fig. 1. Colombia locations of the Atelopus field sites: (¡) Atelopus aff.
elegans; () Atelopus aff. limosus; and (æ) Atelopus spurrelli. The dis-
tribution of A. aff. elegans is restricted to Gorgona Island near the
Pacific Coast of Colombia, the distribution of A. aff. limosus is un-
known, and the stippled area is the distribution of A. spurrelli (IUCN
Herpetological Review 46(4), 2015
patterns in Bd occurrence: sex and life-stage (male, female; adult,
juvenile), snout–vent length (mm), and weight (g). To examine
the hypothesis that lowland conditions may be unsuitable for Bd
occurrence, body temperature was measured using an infrared
thermometer (OAKTON ® InfraPro) with a resolution of 0.2°C.
From a distance of 50 cm, we pointed the laser to the frog’s dorsal
surface or the exact place where the individual was perched before
To determine infection status and to avoid cross contam-
ination, animals were collected using clean, decontaminated
equipment, individually handled with fresh disposable gloves,
and placed in individual bags prior to obtaining the skin swab
samples. Each animal was sampled by running a synthetic cot-
ton swab (skin swabs) over the ventral surface, the inner thigh
area and the plantar surface of the hind-feet webbing for a total
of 50 strokes. Skin swabs were preserved either in 96% ethanol
or were air dried and stored at -20°C until processed. To diag-
nose Bd infection we used two techniques: end-point PCR and
Real-time TaqMan PCR – qPCR. When we started the surveys our
main goal was simply to determine Bd presence and thus we ran
end-point PCR and qPCR assay to determine presence/absence
of the pathogen. Starting in 2012 we performed qPCR assay, to
test not only for the presence of Bd but also for the infection in-
tensity (pathogen load) and report it as the relative number of
zoospores (Zoospore Equivalents, ZE) on each animal.
For end-point PCR, DNA was extracted using GeneReleas-
er® (Bioventures Inc., Carlsbad, California, USA). We used the
primers developed by Annis et al. (2004) to amplify the ITS1-ITS2
region specifically in B. dendrobatidis: Bd1a (5’- CAGTGTGCCAT-
Amplifications were performed in an MJ Research Peltier Ther-
mal Cycler (PTC-200), as follows: an initial 2 min denaturation at
95°C, followed by 35 cycles of DNA amplification (45 sec at 95°C,
45 sec at 55°C, and 1 min at 72°C), and a final extension at 72°C
for 10 min completed the amplifications. Each reaction con-
sisted of 0.5 mL of each primer (1 mM), 3.0 mL of doubly distilled
DNA-free water, 6 mL of GoTaq® Green Master Mix (1X; Prome-
ga) and 2 mL of the DNA extract. The amplified fragments were
separated by electrophoresis in 1% agarose gels. The qPCR assay
was performed using a BIO-RAD PCR machine (to diagnose) and
an Applied BioSystems 7300 Real Time PCR System (to quantify
zoospore loads). For the qPCR, DNA was extracted using Prep-
Man Ultra. Extractions and qPCR’s were performed following the
methods described by Hyatt et al. (2007) and Boyle et al. (2004).
Each reaction consisted of 0.45 mL of each primer (50 mM), 5.975
ml of water, 12.5 ml of TaqMan, 0.625 mL of MGB Probe and 5 ml of
the DNA template. To quantify infection intensity we used stan-
dards of known concentrations and negative controls.
To determine the historical presence of Bd, we swabbed
museum specimens of A. spurrelli and A. aff. elegans. Specimens
for A. aff. limosus were not available (Table 2). We used the
molecular methods and protocols described by Cheng et al.
(2011). In order to decrease the chances of contamination we
rinsed each frog with 70% EtOH and we used fresh gloves for
each frog handled. We ran a synthetic cotton swab (MWE 113)
over the ventral surface, inner thighs, abdomen, and between
toes for a total of 30 strokes. DNA was extracted using Prepman
Ultra. Each sample was run in triplicate by using qPCR assay
following Boyle et al. (2004) using an Applied BioSystems 7300
Real Time PCR System.
table 1. Batrachochytrium dendrobatidis prevalence among three lowland Atelopus species in Colombia. The number in parenthesis repre-
sents the number of Bd-positive individuals. p/h = person/hours. Prevalence CI 95% = Prevalence of infection, in parenthesis are the Bayesian
Credible Intervals based on 95% confidence. Bold numbers indicate summaries per species.
Species Date Sex and Life Stage Search Total Prevalence Technique
Female Male Juvenile Efforts (p/h) CI (95%)
Atelopus aff. elegans Jul 2007 15 (4) 57 (10) 6 (1) 144 78 (15) 19 (11.6–29) PCR
Jul 2010 12 (2) 25 (5) 46 (2) 150 83 (9) 10.7 (5.4–18.6) PCR
Jun 2012 5 (0) 60 (2) 82 (1) 120 147 (3) 2 (0.5–5.3) qPCR
308 (27) 8.7 (5.9–12.2)
Atelopus spurrelli May 2009 1 (0) 67 (1) 6 (0) 96 74 (1) 1 (0.1–6.1) PCR
Nov 2009 3 (0) 40 (1) 2 (1) 60 45 (2) 4 (0.9–13.5) PCR
Jun 2011 0 (0) 9 (1) 0 (0) 72 9 (1) 11 (1.2–41.4) PCR
Sep 2011 1 (0) 4 (1) 7 (0) 96 12 (1) 8 (0.9–32.8) PCR
Jun 2012 3 (0) 9 (1) 20 (0) 144 32 (1) 3 (0.3–13.6) qPCR
172 (6) 3.4 (1.4–7.0)
Atelopus aff. limosus Sep 2008 1 (0) 8 (0) 2 (0) 48 11 (0) 0 (0–15.6) PCR
Oct 2009 0 (0) 61 (0) 11 (0) 48 72 (0) 0 (0–2.6) PCR
Jul 2012 8 (0) 87 (0) 0 (0) 72 95 (0) 0 (0–1.9) qPCR
178 (0) 0 (0–1.0)
table 2. Museum specimens swabbed. The asterisk (*) indicates the
year when Batrachochytrium dendrobatidis (Bd) was first detected.
Species Year No. swabbed No.
Atelopus aff. elegans 1968 54 0
1977 1 0
1978 12 0
1985 24 0
1986 20 0
1987 1 0
2000* 25 3
Atelopus spurrelli 1968 6 0
1969 91 0
1983 31 0
Herpetological Review 46(4), 2015
We analyzed skin swabs from a total of 658 individuals
collected during the field surveys, including 308 A. aff. elegans,
178 A. aff. limosus, and 172 A. spurrelli (Table 1). We analyzed for
Bd presence with 384 samples (end-point PCR and qPCR), and
Bd zoospore equivalents or infection intensity with 274 swabs
(qPCR assay). In total we detected Bd in 6 (3.5%) A. spurrelli and
27 (8.7%) A. aff. elegans. We have infection intensity data for four
samples taken in 2012: one male A. spurrelli (16.9 ZE), two males
(63 and 65490 ZE) and one juvenile (726 ZE) A. aff. elegans. Per
sample date, the prevalence of the infection (i.e., the number of
infected animals out of the total number of individuals sampled)
was very low, ranging from 1–11% in A. spurrelli and from 2–19%
in A. aff. elegans (no Bd-positive A. aff. limosus). From 2007 to 2012,
the prevalence of Bd in A. aff. elegans dropped steadily (Table 1)
(G-test test of independence with Williams’ correction, c
19.792, P = 5.037 × 10
); conversely, there was not a discernible
pattern in the prevalence of Bd in A. spurrelli during that same
time frame (G-test, c
= 2.325, P = 0.6762) (Fig. 2). Visual checks
of animals that were released after capture did not reveal any signs
of chytridiomycosis in any species. We found that Bd prevalence
was not influenced by body size (N = 658, Z = 1.067, P = 0.286).
However, in A. aff. elegans the prevalence was higher for males (c
11.412, df = 2, P = 0.003). Frog body temperatures ranged between
23.2–28.2°C (
= 25.1°C) in A. aff. elegans, 22.6–29.3°C (
= 25.2°C)
in A. aff. limosus, and 22.5–28.5°C (
= 25.4°C) in A. spurrelli. We
detected differences in body temperature among surveys only for
A. aff. limosus (F = 136.5, P < 0.01). To provide a retrospective view
of Bd emergence, we swabbed available museum specimens: 128
formalin-preserved specimens of A. spurrelli collected between
1968 and 1983 and 137 A. aff. elegans collected between 1968 and
2000 (Table 2). We detected Bd presence in three (12%) A. aff.
elegans collected in 2000.
Visual encounter surveys showed that from 2009 to 2012,
A. spurrelli were encountered at a much lower rate. We spent
approximately 414 h (person hours) surveying for individuals
of A. aff. elegans, and 168 h and 468 h for individuals of A. aff.
limosus and A. spurrelli, respectively. By the end of the study, A.
spurrelli were difficult to find despite intense effort (Table 1). Our
findings suggest a decrease in abundance of A. spurrelli during
the course of our study. We first detected Bd in one of 74 frogs
sampled in 2009 in the wild, then in the following surveys (2009–
2012), A. spurrelli became increasingly harder to find. Although
we acknowledge that we did not conduct systematic population
abundance monitoring, we did not detect chytridiomycosis and
Bd prevalence from our samples was low, we hypothesize that
Bd may have contributed to the apparent decline. We cannot
rule out the possibility that this drop in abundance over the time
frame sampled might be due to other factors, including natural
population variation (Pechmann et al. 1991). The apparent
decline of A. spurrelli is puzzling due to the presence of cutaneous
bacteria with antifungal properties (Flechas et al. 2012), the
fact that these frogs occur in an environment with relatively
high temperatures (22.6–29.3°C) which may be unsuitable for
optimal Bd growth (Johnson et al. 2003; Piotrowski et al. 2004),
and no observed habitat degradation or other stressors. Based
on our retrospective study of museum specimens, we found no
evidence of Bd presence in this species before the first positive
record in 2009. Unfortunately, the lack of systematic population
surveys makes it hard to compare abundances between
years, which would allow for detecting gradual versus sudden
population collapse. Other species of Atelopus have declined
due to habitat destruction and not Bd (Tarvin et al. 2014), but
we found no indication of habitat alteration or disturbance in
our study area. Therefore we suggest that systematic surveys
(population monitoring and disease diagnostics) be conducted
to assess the current status of A. spurrelli from Arusí in order to
more accurately estimate future trajectories for this population.
A recent study conducted on the same three Atelopus
species that are the focus here revealed that A. aff. elegans, the
species that we found to be most common despite Bd infection,
harbors the highest proportion of bacterial isolates with anti-
Bd activity; these bacteria also exhibited the strongest anti-
Bd effect (Flechas et al. 2012). An alternative explanation for
species survival when exposed to lethal strains of Bd could
be an environmental effect that limits Bd growth (Rowley and
Alford 2007; Bustamante et al. 2010; Zumbado-Ulate et al. 2014).
Various studies have proposed the existence of climatic refugia
from chytridiomycosis-driven amphibian declines (Puschendorf
et al. 2011; 2013) as a mechanism to allow susceptible species
to persist despite infection (Puschendorf et al. 2005). Our three
study species occur in tropical coastal forests that exhibit high
temperatures, above the optimal physiological range of the
pathogen (Johnson et al. 2003; Piotrowski et al. 2004). This
temperature regime might limit Bd growth causing a decrease in
zoospores production, thereby slowing transmission rates and
allowing individuals the time to mount an immune response or
simply tolerate the pathogen. However, we suggest that when
the pathogen first arrives it could cause high mortality in the
naïve populations.
Knowing the relative time frame of the emergence of a
pathogen in host populations is important for understanding
present-day disease dynamics (Briggs et al. 2010; Vredenburg et
al. 2010). For example, host pathogen dynamics can move from
unstable dynamics soon after pathogens emerge in naïve host
populations (e.g., epizootic state) to more stable dynamics long
after emergence events (e.g., enzootic state). As part of our study,
we provide a retrospective view of Bd emergence in two of our
Fig. 2. Prevalence of Batrachochytrium dendrobatidis infection over
time for the three Atelopus species from Colombia. Lines indicate the
Bayesian Credible Intervals.
Herpetological Review 46(4), 2015
three study species (specimens were not available for the third
species). We sampled museum specimens using a molecular
technique that has been shown to accurately reflect Bd presence
or absence in specimens collected up to a century ago (Cheng et
al. 2011; Rodriguez et al. 2014). We first detected Bd in three of
25 individuals of A. aff. elegans collected in 2000. Since there is a
lack of sampling between 1987 and 2000, we cannot discard the
possibility that Bd arrived to the island earlier. The elapsed time
could fit an enzootic pattern of host/pathogen dynamic, where
A. aff. elegans populations remain stable despite the presence of
the pathogen.
Atelopus aff. limosus, our third study species, occurs near the
border between Colombia and Panamá. This species is believed
to have a wider distribution in the Darién region of Panamá (R.
Ibáñez, pers. comm.), and this area was considered until recently
one of the last Bd-free areas in Central America (Rebollar et al.
2014). Our data from 2008 to 2012 did not find any evidence of
Bd, supporting the idea that the pathogen has not arrived to
Capurganá. However, the situation is critical because Bd was
detected in 2012 in the Darién Province in Panamá (Rebollar et
al. 2014) very close (~50 km) to our study site in Colombia. We
predict that Bd will spread across the Colombian border very
soon. The fact that this is a Bd-naïve area and the high infection
susceptibility reported for the genus, we believe that Bd may have
a considerable impact on the Atelopus aff. limosus population.
However, we also expect that the suboptimal environmental
conditions (i.e., high temperatures) will limit Bd growth and
colonization rate, which may allow for host tolerance of the
infection or evolution of host resistance allowing for coexistence
between host and pathogen.
Acknowledgments.For assistance in the field we thank A. Batis-
ta, L. Barrientos, G. Corredor, A. J. Crawford, T. Escovar, C. Guarnizo,
M. González, J. A. Hernández, J. Méndez, A. Muñoz, A. Paz, A. Ro-
dríguez, P. Rodríguez, C. Sarmiento, C. Silva, F. Vargas-Salinas, and
B. Velásquez. We would like to thank J. D. Lynch at the Instituto de
Ciencias Naturales – Universidad Nacional de Colombia for allow-
ing us to swab museum specimens under his care, and A. J. Craw-
ford, V. Ramírez, and C. Sarmiento for their assistance in swabbing.
We thank C. Sarmiento and S. Ellison for assistance in the labora-
tory and A. Paz for creating the distribution map. We would like to
thank the personnel at Parque Nacional Natural Gorgona for logistic
support. This study was completed with financial support from US
Fish & Wildlife Service – Wildlife without Borders (96200-0-G228), As-
sociation of Zoos and Aquariums – Conservation Endowment Fund
(08-836), Conservation International Critically Endangered Species
Fund, and National Science Foundation (IOS-1258133). Procedures
for capture and handling of live animals in the field were approved
by the Colombian National Parks Authority and the Ministry of the
Environment, under permits DTSO 019-09, DTSO 001-09 and Nº 10
March 7, 2012. We would like to thank D. Olson, S. Lötters, and an
anonymous reviewer for helpful comments and suggestions that
greatly improved this manuscript.
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© 2015 by Society for the Study of Amphibians and Reptiles
Ranavirus Detected in Lithobates clamitans and
L. catesbeianus in Indiana
Ranaviruses are an emerging group of pathogens that threaten
populations of all three classes of ectothermic vertebrates,
especially amphibians (Gray and Miller 2013; Duffus et al. 2015).
Infections have now been documented on six continents and in
more than 175 species, and are very likely under documented
(Duffus et al. 2015). The pathogenicity and broad host range of
ranaviruses make them serious threats to both wild and captive
populations of ectothermic vertebrate species worldwide.
Despite an increased awareness and study of ranaviral infection,
much remains unknown about many aspects of their ecology,
including geographic distribution.
To date, the distribution of ranavirus in Indiana, USA, is
poorly known. Only two studies have tested wild amphibian and
reptile populations for the presence of ranavirus (Currylow et al.
2014; Winzeler et al. 2015). In south-central Indiana, Currylow et
al. (2014) found PCR evidence of ranavirus in Eastern Box Turtles
(Terrapene carolina carolina) and larval amphibians (Ambystoma
sp and unidentified anuran larvae). Overall, ranavirus infections
were detected in 2.2% of larval amphibians and 3.0% of Eastern
Box Turtles sampled. However, Winzeler et al. (2015) found no
SYBR quantitative realtime PCR (qPCR) evidence of ranavirus
in Green Frog (Lithobates clamitans) tadpoles at five sites
distributed across Indiana. In a third study, Kimble et al. (2014)
report the presence of ranavirus in a wild-caught, newly captive
population of Eastern Box Turtles that was likely initially infected
in the wild. Although these studies provide some evidence
that ranaviruses are present within Indiana, there is a need for
broader surveillance to improve our understanding of ranavirus
distribution. Our objective was to test for ranavirus infections in
larval Green Frogs and American Bullfrogs (L. catesbeianus) from
15 sites distributed across Indiana.
In 2013 and 2014, the Indiana Department of Natural
Resources collected live larvae in an ad hoc manner using dipnets
or seines from 15 public areas distributed across the state (Table 1,
Fig. 1). Field equipment was disinfected between sites with 0.75%
Nolvasan (2% chlorhexidine diacetate; Fort Dodge Animal Health,
Fort Dodge, Iowa; Bryan et al. 2009). Individuals were euthanized
with a 30-minute soak in a 3% solution of neutral-buffered tricaine
Department of Forestry and Natural Resources,
Purdue University, West Lafayette, Indiana 47907, USA
*Corresponding author; e-mail:
Fig. 1. Map of Indiana showing sample locations for the current study
(stars), that of Currylow et al. (2014; circles), Winzeler et al. (2015;
triangles), and Kimble et al. (2014; diamonds). Filled symbols indi-
cate sites where ranavirus was detected; empty symbols indicate it
was not. These symbols indicate the county, not the exact sampling
... Harlequin toads from the genus Atelopus are considered one of the most threatened groups of amphibians worldwide, with at least 71% of the species listed as critically endangered according to the International Union for Conservation of Nature (2016). Although many species have been declining, some species from the lowlands still persist in their natural habitats (Flechas, Vredenburg & Amézquita, 2015). Other species, such as A. elegans from Ecuador, has only been found in one of its historical collection localities. ...
... The genus Atelopus is one of the most threatened groups of amphibians worldwide. Although more than 70% of the species in the genus are listed as critically endangered according to the International Union for Conservation ofNature (2016), some species have persisted in the wild (Flechas, Vredenburg & Amézquita, 2015). In other cases, such as A. elegans from Ecuador, this species has been recently rediscovered but only in one of the historical localities. ...
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Many amphibian species are at risk of extinction in their natural habitats due to the presence of the fungal pathogen Batrachochytrium dendrobatidis ( Bd ). For the most highly endangered species , captive assurance colonies have been established as an emergency measure to avoid extinction. Experimental research has suggested that symbiotic microorganisms in the skin of amphibians play a key role against Bd . While previous studies have addressed the effects of captivity on the cutaneous bacterial community, it remains poorly studied whether and how captive conditions affect the proportion of beneficial bacteria or their anti- Bd performance on amphibian hosts. In this study we sampled three amphibian species of the highly threatened genus, Atelopus , that remain in the wild but are also part of ex situ breeding programs in Colombia and Ecuador. Our goals were to (1) estimate the diversity of culturable bacterial assemblages in these three species of Atelopus , (2) describe the effect of captivity on the composition of skin microbiota, and (3) examine how captivity affects the bacterial ability to inhibit Bd growth. Using challenge assays we tested each bacterial isolate against Bd , and through sequencing of the 16S rRNA gene, we identified species from thirteen genera of bacteria that inhibited Bd growth. Surprisingly, we did not detect a reduction in skin bacteria diversity in captive frogs. Moreover, we found that frogs in captivity still harbor bacteria with anti- Bd activity. Although the scope of our study is limited to a few species and to the culturable portion of the bacterial community, our results indicate that captive programs do not necessarily change bacterial communities of the toad skins in a way that impedes the control of Bd in case of an eventual reintroduction.
... Most field research and surveys of Bd have been conducted at middle and high elevations, where declines were initially observed ( ), but recent reports of Bd in the lowlands suggest we need to survey more widely ( Brem & Lips 2008, Kriger & Hero 2008, McCracken et al. 2009, Kilburn et al. 2010, Flechas et al. 2012, Whitfield et al. 2012, Zumbado-Ulate et al. 2014). Bd should be considered a threat to species occurring at low elevations as well, although population declines have been rarely reported ( Kilburn et al. 2010, Flechas et al. 2015). In this study, 19 of 32 surveyed localities in the lowlands below 1000 m, and 13 of 25 below 500 m tested positive for Bd (59% and 52% of sites, respectively). ...
... Despite its limited geographic scope, our study provides a dense sampling that allowed us to recognize heretofore-unappreciated variation in observed and predicted Bd prevalence among lowland sites. For example, the Choco lowlands of the Pacific coast, previously thought to have a very low probability of Bd occurrence, may in fact be facing a major threat with many positive localities ( Flechas et al. 2015). Given that the Choco region hosts high amphibian biodiversity, we highlight the urgent need to develop a monitoring program for the Pacific lowlands of Colombia. ...
Global amphibian declines have been attributed to several factors including the chytrid fungal pathogen, Batrachochytrium dendrobatidis (Bd), that infects hosts’ skin and causes death by inhibiting immune response and impairing osmoregulatory function. Here, we integrate extensive new field data with previously published locality records of Bd in Colombia, a megadiverse and environmentally heterogeneous country in northwestern South America, to determine the relative importance of environmental variables and reproductive mode for predicting the risk of Bd infection in amphibians. We surveyed 81 localities across Colombia and sampled 2876 individual amphibians belonging to 14 taxonomic families. Through a combination of end-point PCR and real-time PCR analyses, Bd was detected in 338 individuals (12%) representing 43 localities (53%) distributed from sea level to 3200 m. We found that annual mean temperature and variables related with seasonality in precipitation and temperature appeared to define the most suitable areas for the establishment of the pathogen. In addition, prevalence of infection appeared to be higher in species with a terrestrial reproductive mode. Our study provides the first large-scale study of the current and potential distribution of Bd in the biodiversity hotspot centered on Colombia. We hope the newly provided information on the extent of the distribution of the pathogen and the potential areas where Bd may impact the amphibian fauna will inform decision making by environmental authorities and future conservation action.
... Few populations of harlequin toads have persisted for decades with endemic infection with Bd [17,[48][49][50][51], despite the devastating effect this pathogen had in the past on most species in this genus [8,52]. A lower Bd virulence in warm habitats, where it tends grow slowly, has been proposed to explain the higher survival of lowland populations of some frog species (i.e. ...
... Other studies have used the term thermal refuge to refer to habitats with lower prevalence of infection, although the underlying mechanisms leading to lower prevalence have not been explicitly defined in most cases [48,67]. Classic epidemiological models have demonstrated that pathogen transmission rates and disease-induced mortality may both affect the prevalence of infection [41]. ...
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Harlequin toads (Atelopus) are among the most severely impacted amphibians by the emergenceof chytridiomycosis, a fungal disease caused by the pathogen Batrachochytrium dendrobatidis (Bd). Many species disappeared while others suffered drastic contractions oftheir geographic distribution to lower altitudes. A diminished virulence of Bd in warm habitats was proposed to explain the survival of lowland populations of harlequin toads (i.e. thermal refuge hypothesis). To understand the mechanisms that allow some populations to reach an endemic equilibrium with this pathogen, we estimated demographic and epidemiological parameters at one remnant population of Atelopus cruciger in Venezuela using mark recapture data from 2007–2013. We demonstrated that Bd is highly virulent for A. cruciger,increasing the odds of dying of infected adults four times in relation to uninfected ones and-reducing the life expectancy of reproductive toads to a few weeks. Despite an estimated annual loss of 18% of the reproductive population due to Bd-induced mortality, this population has persisted in an endemic equilibrium for the last decade through the large recruitment of healthy adults every year. Given the high vulnerability of harlequin toads to Bd in lowland populations, thermal refuges need to be redefined as habitats of reduced transmission rather than attenuated virulence.
... The conservation status of at least 30 species is uncertain because they remain undescribed (Coloma et al. 2010) or because a comprehensive systematic revision is required La Marca and Lötters. 2008;Lötters et al. 2011;Flechas et al. 2015). Peru contains 19 nominal species of Atelopus and three confirmed candidate new species from the Andes and Amazon regions (Frost 2016;Rueda-Almonacid et al. 2005). ...
... In this report, we provide new distributional data for A. seminiferus and recommend an update to the map of its known geographic distribution (Fig. 1). We also tested for the prevalence of the chytrid fungus Batrachochytrium dendrobatidis (Bd), a pathogen that has been linked with population declines of harlequin frogs throughout tropical America (Lampo et al. 2006;Venegas et al. 2008;Flechas et al. 2015). Additionally, given that other factors such as habitat loss may have caused population declines in many other amphibian species (Catenazzi and von May 2014;Tarvin et al. 2014), we noted the type of habitat used by A. seminiferus in the region. ...
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We provide information of the distribution, habitat, and conservation status of the harlequin frog Atelopus seminiferus, a poorly known species from northern Peru. Multiple individuals of A. seminiferus were detected inside the Alto Mayo Protected Forest, San Martin region, 87‒98 km northwest from the type locality. Additionally, we used skin swab samples to test for the prevalence of the chytrid fungus Batrachochytrium dendrobatidis (Bd), a pathogen that has been linked with population declines of harlequin frogs throughout tropical America. Our findings represent the first record of A. seminiferus inside a natural protected area, and we recommend an update of the IUCN Red List geographic range map of this species. Though we did not detect individuals infected by Bd, additional surveys are required to further assess the elevational distribution and potential for chytrid fungus infection of this Critically Endangered species.
... Some species of harlequin toads have been rediscovered in recent years (A. varius [13], A. nepiozomus [14], A. palmatus [14], A. bomolochos [15], A. ignescens [16], A. nanay [17], A. longirostris [18] and A. carrikeri [19]), and few populations appear to be recovering despite the presence of Bd in some of its individuals [20][21][22][23]. The mechanisms that allow these populations to coexist with the fungus are not fully understood. ...
Full-text available
Chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis ( Bd ), has been linked with the disappearance of amphibian populations worldwide. Harlequin toads ( Atelopus ) are among the most severely impacted genera. Two species are already considered extinct and most of the others are at high risk of extinction. The recent rediscovery of harlequin toad populations coexisting with Bd suggest that the pathogen can maintain enzootic cycles at some locations. The mechanisms promoting coexistence, however, are not well understood. We explore the dynamics of Bd infection in harlequin toads by modeling a two-stage host population with transmission through environmental reservoirs. Simulations showed that variations in the recruitment of adults and the persistence of zoospores in the environment were more likely to drive shifts between extinction and coexistence than changes in the vulnerability of toads to infection with Bd . These findings highlight the need to identify mechanisms for assuring adult recruitment or minimizing transmission from potential reservoirs, biotic or abiotic, in recovering populations.
... Despite the focus on highlands for most Bd-related studies, the few studies conducted in lowlands of Central America have found new locations where this pathogen occurs, suggesting that Bd is more widely distributed than previously thought (Flechas, Vredenburg, & Amézquita, 2015;Kilburn et al., 2010;von May, Catenazzi, Santa-Cruz, & Vredenburg, 2018;Whitfield et al., 2013;Whitfield, Kerby, Gentry, & Donnelly, 2012;Woodhams et al., 2008;Zumbado-Ulate et al., 2014). Predictive models and abiotic suitability for Bd across heterogenous landscapes (Brannelly, Martin, Llewelyn, Skerratt, & Berger, 2018 2000. ...
Full-text available
Numerous species of amphibians declined in Central America during the 1980s and 1990s. These declines mostly affected highland stream amphibians and have been primarily linked to chytridiomycosis, a deadly disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Since then, the majority of field studies on Bd in the Tropics have been conducted in midland and highland environments (>800 m) mainly because the environmental conditions of mountain ranges match the range of ideal abiotic conditions for Bd in the laboratory. This unbalanced sampling has led researchers to largely overlook host–pathogen dynamics in lowlands, where other amphibian species declined during the same period. We conducted a survey testing for Bd in 47 species (n = 348) in four lowland sites in Costa Rica to identify local host–pathogen dynamics and to describe the abiotic environment of these sites. We de‐tected Bd in three sampling sites and 70% of the surveyed species. We found evidence that lowland study sites exhibit enzootic dynamics with low infection inten‐sity and moderate to high prevalence (55% overall prevalence). Additionally, we found evidence that every study site represents an independent climatic zone, where local climatic differences may explain variations in Bd disease dynamics. We recommend more detection surveys across lowlands and other sites that have been historically considered unsuitable for Bd occurrence. These data can be used to identify sites for potential disease outbreaks and amphibian rediscoveries.
... However, several studies have documented Bd infection in lowland rainforest amphibians over the past decade (Flechas et al., 2012;Kosch, Morales, & Summers, 2012;McCracken, Gaertner, Forstner, & Hahn, 2009;Whitfield, Kerby, Gentry, & Donnelly, 2012). These findings lent support to the idea that some lowland tropical forest habitats offer suitable environmental conditions for Bd growth, even though most lowland areas may contain suboptimal conditions limiting the pathogen spread and growth (Flechas, Vredenburg, & Ame´zquita, 2015). A recent study focusing on museum-stored specimens of species in the genus Leptodactylus (Anura: Leptodactylidae) showed that Bd has been present in the lowland Amazon for more than 80 years (Becker, Rodriguez, Lambertini, Toledo, & Haddad, 2016). ...
Full-text available
Until recently, it was assumed that the pathogenic fungus Batrachochytrium dendrobatidis (Bd) was not widely distributed in warm ecosystems such as lowland tropical rainforests because high environmental temperatures limit its growth. However, several studies have documented Bd infection in lowland rainforest amphibians over the past decade. In addition, a recent study focusing on museum-stored specimens showed that Bd has been present in the lowland Amazon for more than 80 years. These findings lent support to the idea that some lowland rainforest habitats offer suitable environmental conditions for Bd growth, even though most lowland areas may contain suboptimal conditions limiting the pathogen spread and growth. Here, we surveyed four sites in southeast Peru to examine the prevalence and the intensity of infection of Bd in lowland Amazonian amphibians and to fill a gap between two areas where Bd has been present for more than a decade. In one of these “hotspots” of Bd infection, the upper slopes of Manu National Park, several species experienced population declines attributed to Bd epizootics over the past 15 years. We also examined the thermal profile of the main microhabitats used by lowland Amazonian frogs to infer whether these microhabitats offer suitable thermal conditions for Bd growth. We detected Bd in nine lowland frog species and variation in prevalence of infection across years. Our findings suggest that the temperatures in the leaf litter and understory vegetation of some habitats offer suitable conditions for Bd growth.
... Hace tiempo se viene alertando sobre la declinación de algunos taxones y poblaciones de anuros ( Cadavid et al., 2005) en las cordilleras norandinas (e.g., La Marca, 2005;Valera-Leal et al., 2011), lo cual ha sido asociado, principalmente, al cambio climático, la degradación o disminución del hábitat y a la relación de estos organismos con patógenos, factores que podrían estar actuando sinérgicamente. En los últimos años se ha venido revisando y evaluando la presencia del hongo Batrachochytrium dendrobatidis y sus posibles efectos sobre las poblaciones Papéis Avulsos de Zoologia, 57(39), 2017 de anfibios andinas (ver Sánchez et al., 2008;Velásquez et al., 2008;Urbina & Galeano, 2011;Vásquez-Ochoa et al., 2012;Flechas et al., 2015;Acevedo et al., 2016;Acevedo et al., en prensa). Por ejemplo, especies del género Atelopus han mostrado un gran declive, situación que ha sido bastante asociada con la infección de Bd detectada en varias de poblaciones de estos anfibios (ver Lampo et al., 2007); sin embargo, no hay evidencia clara o directa de que las desapariciones hayan sido causadas por el quítrido (ver Tarvin et al., 2014). ...
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Knowledge of Andean anurans is constantly increasing; for this reason, it needs an update. We reviewed scientific literature and museum records to determine the species richness of an-urans in the northern Andes and their conservation status based on the IUCN categories. The northern Andes harbor 740 species of anurans, belonging to 13 families and 63 genera. The family with the highest species number was Craugastoridae (334 spp.), followed by Bufonidae (92 spp.), and Centrolenidae (81 spp.). The Cordillera Central of Colombia harbors the highest species richness (199 spp.), while the Cordillera de la Costa Oriental de Venezuela contains the lowest species number (17 spp.). Most species (74%) is endemic to the northern Andes, but no family is endemic to the northern Andes. About 50% species is threatened and 20% is included in the category of deficient data. The species richness patterns in northern Andes might be caused, among other reasons, by historical factors, but also result of sampling lack in some areas.
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Technical Report
The Harlequin Toad (Atelopus) Conservation Action Plan (HarleCAP), created by the Atelopus Survival Initiative, promotes strategies to synchronize efforts and exchange resources, knowledge, and capacities among stakeholders through regional coordination and inter- and multi-disciplinary approaches to: 1) Produce baseline knowledge, 2) Ensure viable populations in natural habitats, 3) Maintain and manage captive populations for future reintroduction, 4) Increase visibility of Atelopus, and 5) Foster multi-stakeholder collaboration and participation. By incorporating a monitoring and evaluation framework, the HarleCAP will advance priorities and strategies to ensure Atelopus conservation, updating objectives and actions as threats and conservation successes evolve.
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The demise of amphibians? Rapid spread of disease is a hazard in our interconnected world. The chytrid fungus Batrachochytrium dendrobatidis was identified in amphibian populations about 20 years ago and has caused death and species extinction at a global scale. Scheele et al. found that the fungus has caused declines in amphibian populations everywhere except at its origin in Asia (see the Perspective by Greenberg and Palen). A majority of species and populations are still experiencing decline, but there is evidence of limited recovery in some species. The analysis also suggests some conditions that predict resilience. Science , this issue p. 1459 ; see also p. 1386
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We studied populations of frogs of the genus Atelopus from the Pasto Massif of the Andes in southern Colombia and northern Ecuador, and from the Huancabamba depression in southern Ecuador and northern Perú and conclude that they belong to six species, five of which are described as new to science. Atelopus angelito is recorded for the first time from Ecuador and its range is extended 183 km (airline) southwest of its type locality in Departamento del Cauca, Colombia. We distinguish the five new species from similar ones using features of coloration, skin texture, and morphometrics. We also include osteological data for four of the new species. A putative hybrid zone at Provincia Imbabura, Ecuador, is proposed to exist between the non-sister taxa A. ignescens and one of the new species. Because recent records of four of the new species and A. angelito are lacking despite search efforts, we hypothesize that they are possibly extinct, as are many other Andean Atelopus. Thus, we categorize these species, applying IUCN Red List criteria, as Critically Endangered (Possibly Extinct). No search efforts have been carried out for one new species (from La Victoria, Colombia); thus, it is included under the Data Deficient category. The conservation of Atelopus is briefly discussed.
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Captive and wild frogs from North and Central America and Australia recently have died with epidermal infections by chytridiomycete fungi. We isolated a chytridiomycete into pure culture from a captive, blue poison dart frog that died at the National Zoological Park in Washington, D.C. Using this isolate, we photographed developmental stages on nutrient agar, examined zoospores with transmission electron microscopy, and inoculated test frogs. This inoperculate chytrid develops either monocentrically or colonially and has thread-like rhizoids that arise from single or multiple areas on the developing zoosporangium. The taxonomically important features of the kinetosomal region of the zoospore indicate that this chytrid is a member of the Chytridiales but differs from other chytrids studied with transmission electron microscopy. Its microtubule root, which begins at kinetosome triplets 9-1 and extends parallel to the kinetosome into the aggregation of ribosomes, is distinctive. Histologic examination of test frogs revealed that the pure culture infected the skin of test frogs, whereas the skin of control frogs remained free of infection. The fungus is described as Batrachochytrium dendrobatidis gen. et sp. nov.
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Approximately one-third of amphibian species are experiencing population declines. Among the most affected groups are the Harlequin frogs (Bufonidae: Atelopus), nearly all of which are threatened with extinction. We monitored one of the last known Ecuadorian populations of Atelopus spurnarius (Pebas Stubfoot Toad) with a mark-recapture survey between February 2009 and December 2010. We aimed to determine the effect of the fungal pathogen Batrachochytrium dendrobatidis (Bd), previously recorded at the site, on population size and survival. We also analyzed the effect of selective logging which occurred along the transect in April 2010. We tested every individual captured or recaptured for Bd presence using end-point PCR. All 679 Bd swabs (from 356 individuals) were negative, suggesting that Bd did not influence population size and survival. Population size increased during the first 9 months of the study (August 2009-April 2010), from an estimated 47 to 92 individuals, but then decreased by November 2010 to an estimated 48 individuals. Probability of survival was 0.13 lower in months following selective logging compared to previous months; emigration decreased by 0.37 after the logging event. Pairs in amplexus were found between April and December, suggesting a long, if not year-round, reproductive period. Although most Atelopus spp. declines have been attributed to Bd, we present a case in which Bd has been detected in the area yet changes in population size cannot be attributed to chytridiomycosis. Analyses of survival and male body condition index suggest that the decline at the end of the study was the result of habitat destruction.
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Population declines and extinctions of numerous species of amphibians, especially stream-breeding frogs, have been linked to the emerging infectious disease chytridiomycosis, caused by the chytrid fungus, Batrachochytrium dendrobatidis. In Central America, most of the 34 species of the Craugastor punctariolus species group have disappeared in recent years in high- and low-elevation rainforests. Distribution models for B. dendrobatidis and the continuous presence of the extirpated stream-dwelling species, Craugastor ranoides, in the driest site of Costa Rica (Santa Elena Peninsula), suggest that environmental conditions might restrict the growth and development of B. dendrobatidis, existing as a refuge from chytridiomycosis-driven extinction. We conducted field surveys to detect and quantify the pathogen using Real-time PCR in samples from 15 species of frogs in two locations of tropical dry forest. In Santa Elena Peninsula, we swabbed 310 frogs, and only one sample of the species, C. ranoides, tested positive for B. dendrobatidis (prevalence <0.1%). In Santa Rosa Station, we swabbed 100 frogs, and nine samples from three species tested positive (prevalence = 9.0%). We failed to detect signs of chytridiomycosis in any of the 410 sampled frogs, and low quantities of genetic equivalents (between 0 and 1073) were obtained from the ten positive samples. The difference in the prevalence between locations might be due not only to the hotter and drier conditions of Santa Elena Peninsula but also to the different compositions of species in both locations. Our results suggest that B. dendrobatidis is at the edge of its distribution in these dry and hot environments of tropical dry forest. This study supports the existence of climatic refuges from chytridiomycosis and highlights the importance of tropical dry forest conservation for amphibians in the face of epidemic disease.
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The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is one of the main causes of amphibian population declines and extinctions all over the world. In the Neotropics, this fungal disease has caused catastrophic declines in the highlands as it has spread throughout Central America down to Panamá. In this study, we determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 15-34%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11-24 zoospore equivalents per site. Our work indicates that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen.
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In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus) have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd) at temperatures above 25°C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation) or not (exaptation) have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.
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We surveyed the population status of the Neotropical toad genus Atelopus, and document recent catastrophic declines that are more severe than previously reported for any amphibian genus. Of 113 species that have been described or are candidates for description, data indicate that in 42 species, population sizes have been reduced by at least half and only ten species have stable populations. The status of the remaining taxa is unknown. At least 30 species have been missing from all known localities for at least 8 yr and are feared extinct. Most of these species were last seen between 1984 and 1996. All species restricted to elevations of above 1000 m have declined and 75 percent have disappeared, while 58 percent of lowland species have declined and 38 percent have disappeared. Habitat loss was not related to declines once we controlled for the effects of elevation. In fact, 22 species that occur in protected areas have disappeared. The fungal disease Batrachochytrium dendrobatidis has been documented from nine species that have declined, and may explain declines in higher elevation species that occur in undisturbed habitats. Climate change may also play a role, but other potential factors such as environmental contamination, trade, and introduced species are unlikely to have affected more than a handful of species. Widespread declines and extinctions in Atelopus may reflect population changes in other Neotropical amphibians that are more difficult to survey, and the loss of this trophic group may have cascading effects on other species in tropical ecosystems.
The recent global spread of the amphibian killing fungus (Batrachochytrium dendrobatidis [Bd]) has been closely tied to anthropogenic activities, however regional patterns of spread are not completely understood. Using historical samples, we can test whether Bd was a spreading or endemic pathogen in a region within a particular time frame, because those two disease states provide different predictions for the regional demographic dynamics and population genetics of Bd. Testing historical patterns of pathogen prevalence and population genetics under these predictions is key to understanding the evolution and origin of Bd. Focusing on the Atlantic Forest of Brazil, we used qPCR assays to determine the presence or absence of Bd on 2799 preserved post-metamorphic anurans collected between 1894 and 2010 and used semi-nested PCRs to determine the frequency of rRNA ITS1 haplotypes from 52 samples. Our earliest date of detection was 1894. A mean prevalence of 23.9% over time and spatiotemporal patterns of Bd clusters indicate that Bd has been enzootic in the Brazilian Atlantic Forest with no evidence of regional spread within the last 116 years. ITS1 haplotypes confirm the long-term presence of two divergent strains of Bd (BdGPL and Bd-Brazil) and three spatiotemporally broad genetic demes within BdGPL, indicating that Bd was not introduced into southeast Brazil by the bullfrog trade. Our data show that the evolutionary history and pathogen dynamics of Bd in Brazil is better explained by the endemic pathogen hypothesis.