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Detection of Batrachochytrium dendrobatidis in Mexican Bolitoglossine Salamanders Using an Optimal Sampling Protocol

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Detection of Batrachochytrium dendrobatidis in Mexican Bolitoglossine Salamanders Using an Optimal Sampling Protocol

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The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further studies would be required to draw a causal link.
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Detection of Batrachochytrium dendrobatidis in Mexican
Bolitoglossine Salamanders Using an Optimal Sampling
Protocol
Pascale Van Rooij,
1
An Martel,
1
Joachim Nerz,
2
Sebastian Voitel,
3
Filip Van Immerseel,
1
Freddy Haesebrouck,
1
and Frank Pasmans
1
1
Laboratory of Bacteriology and Mycology, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,
Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2
Ja
¨gerstraße 50, 71032 Bo
¨blingen, Germany
3
Spangenbergstraße 81, 06295 Eisleben, Germany
Abstract: The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of
chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we
establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this
protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the
presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of
the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to
detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal
hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of
the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence
of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild
salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further
studies would be required to draw a causal link.
Keywords: Batrachochytrium dendrobatidis, bolitoglossine, salamander, swabbing protocol, captive, wild
Since the 1960s, significant declines and extinctions of
amphibians have been observed worldwide (Stuart et al.
2004) and potential causes include overexploitation, habitat
loss, climate change, and infectious diseases (Stuart et al.
2004; Rohr et al. 2008;Lo
¨tters et al. 2009; Rohr and Raffel
2010). Several population declines have been linked to the
presence of a pathogenic chytrid fungus, Batrachochytrium
dendrobatidis (Bd; La Marca et al. 2005; Lips et al. 2006),
which invades keratinized epithelial cells and can cause
increased tissue growth (hyperplasia) and thickening of the
cornified skin layers (hyperkeratosis) (Berger et al. 1998).
Salamanders of the family Plethodontidae, commonly
called ‘‘lungless salamanders’’, contribute significantly to the
biological diversity of Central America (Larson et al. 2006;
Wake and Vredenburg 2008), but are experiencing signifi-
cant declines that might be caused by chytridiomycosis
(Parra-Olea et al. 1999; Lips et al. 2006; Rovito et al. 2009).
For instance, Bd was recently detected in plethodontid
Correspondence to: Pascale Van Rooij, e-mail: pascale.vanrooij@ugent.be
EcoHealth
DOI: 10.1007/s10393-011-0704-z
Short Communication
Ó2011 International Association for Ecology and Health
salamander populations that are declining in Mexico and
Guatemala (Cheng et al. 2011). Here we establish a swabbing
protocol to maximize the detection probability of Bd in
salamanders and then used this protocol to examine captive
and wild Mexican bolitoglossine salamanders of 14 different
species for the presence of Bd.
Thirty-three dead captive adult bolitoglossine sala-
manders (Bolitoglossinae, Plethodontidae, Caudata), each
preserved in 70% ethanol in a separate container, were used
to establish an efficient swabbing protocol for Bd. The
specimens had not been fixed in formalin prior to storage
in ethanol. To determine whether these animals were
infected with Bd, a pelvic tissue sample was taken from
each specimen. To prevent possible cross-contamination,
disposable gloves and dissection material were changed
between containers. For each sample, DNA was extracted
from the tissue using proteinase K digestion, following the
protocol of Bandi et al. (1994), with 1:10 dilutions stored at
-20°C. Quantitative PCR (qPCR) assays were performed
on a CFX96 Real Time System (BioRad Laboratories,
Hercules, CA, USA), with amplification conditions and
primer and probe concentrations according to Boyle et al.
(2004). For each sample, qPCR assays were performed in
duplicate. Amplification standards of 1000, 100, 10, 1, and
0.1 zoospore genomic equivalents (GEs) were included
within each assay, as well as 3 negative and 1 positive
control sample. A result was considered positive when
values higher than 0.1 GEs were obtained twice.
Animals with high Bd loads were selected for the
sampling of seven selected body sites: chin, plantar side of
forelimb and hindlimb, dorsum, dorsal and ventral sides of
the tail, and the abdomen (Fig. 1; Table 1). Each of these
sites was rubbed 5 times with separate sterile synthetic
swabs (160 C, Copan Italia S.p.A., Brescia, Italy). DNA was
extracted according to Hyatt et al. (2007) and Bd was
quantified as described above. To standardize the surface
area sampled, surface area morphometry of each body part
was performed using Optimas 6.5 image analysis software
(Media Cybernetics Inc., Bethesda, MD, USA) so that GEs
of Bd could be quantified per cm
2
of skin. To validate the
qPCR results, histological slides were examined for the
presence of Bd. To do so, for each salamander, two sam-
pling sites, one with the minimum and the other with the
maximum GEs, were excised, fixed in 10% neutral buffered
formalin, embedded in paraffin and stained with hema-
toxylin and eosin (HE) and with an immunoperoxidase
(IPX) stain for Bd,as described by Berger et al. (2002).
Thirty-three dead adult and 28 healthy adult captive
Mexican plethodontid salamanders from two private collec-
tions were screened for the presence ofBd. Then, the swabbing
protocol was used on 17 healthy (no clinical signs of chy-
tridiomycosis) adult wild bolitoglossine salamanders during
an opportunistic field survey conducted in the Ve
´racruz
and Me
´xico regions of Mexico in August 2010 (Fig. 2). These
regions were selected because they represent the natural
habitat of the examined captive species. All of the animals
were sampled with a sterile synthetic swab over the seven
aforementioned body regions as described above. An overview
of all the animals sampled, together with their species desig-
nation and sampling localities is given in Table 2.
Data were log transformed to correct for non-nor-
mality. A one-way ANOVA was used to determine whether
the abundance of Bd (GE/cm
2
) differed according to the
body part sampled. A post hoc Tukey HSD test was used to
Figure 1. Assessing the effect of the body part sampled on the detection of Bd in Caudata. Schematic presentation of the body sites that were
swab sampled: 1chin, 2plantar side forelimb, 3ventral side abdomen, 4plantar side hindlimb, 5ventral side tail, 6dorsum, 7dorsal side tail.
P. Van Rooij et al.
identify which body parts were significantly different from
one another. All tests were performed in SPSS (Version 17;
SPSS Inc., Chicago, IL, USA).
Based on the analysis of pelvic tissue, evidence of Bd
infection was found in sixteen out of 33 (48%) dead
captive bolitoglossine salamanders, comprising 6 species:
Bolitoglossa platydactyla,Bolitoglossa rufescens,Pseudoeu-
rycea belli,Pseudoeurycea cephalica,Pseudoeurycea leprosa,
and Pseudoeurycea longicauda. Tail loss or tail autotomy
was observed in 8 out of the 16 infected salamanders
belonging to the species Bolitoglossa platydactyla,
B. rufescens,P. cephalica, and P. leprosa, suggesting that
infections might induce tail autotomy. Animals with GEs
of Bd in pelvic skin samples equal to or higher than 20
were used to determine the effect of the body part sampled
on the detection of Bd (Table 1). Eleven animals belong-
ing to the species B. platydactyla (n= 2), B. rufescens
(n= 6), P. cephalica (n= 1), and P. leprosa (n= 2) were
included in this study. All seven sampling sites
tested positive in the infected salamanders, except for 2
Table 1. Density of Batrachochytrium dendrobatidis in different locations on the bodies of infected bolitoglossine salamanders
Sampling site Sample
type
Mean GE ±SE/cm
2
(log 10)
Min. GE/cm
2
(log 10)
Max. GE/cm
2
(log 10)
No. of
positives
No. of
samples
Prevalence
(%)
Significance
level (P)
Chin Swab 2.31 ±3.70 Neg. 4.23 9 11 82 0.046*
Plantar side forelimb Swab 3.45 ±3.58 1.76 4.04 11 11 100 0.920
Dorsum Swab 2.55 ±2.77 Neg. 3.28 10 11 91 0.002*
Dorsal side tail Swab 3.06 ±3.30 Neg. 3.76 11 11 100 0.034*
Ventral side abdomen Swab 2.92 ±3.36 Neg. 3.89 9 11 82 0.002*
Plantar side hindlimb Swab 3.89 ±4.14 Neg. 4.68 10 11 91 0.818
Ventral side tail Swab 3.06 ±3.38 Neg. 3.92 11 11 100 0.038*
Pelvic region Tissue 4.43 ±4.49 2.45 5.01 11 11 100 N/A
Neg. samples where Bd was not detected, N/A not applicable.
* Significant difference between a swabbed site compared to the pelvic tissue samples.
Figure 2. Localities in Mexico
surveyed for the presence of
Batrachochytrium dendrobatidis.
The stars indicate localities where
Bd was detected; the circles indi-
cate localities where Bd was not
detected. The numbers correspond
with the species mentioned in
Table 2;thenumbers indicated
with an asterisk represent the
species found to be positive for
Bd.
Detection of Bd in Bolitoglossine Salamanders
Table 2. Overview of all bolitoglossine species examined for the presence of Batrachochytrium dendrobatidis using the swab protocol for
Caudata
Species Site Latitude
(°N)
Longitude
(°W)
IUCN Red
List Category
Wild/
captive
No. infected/
No. examined
[prevalence %
(95% CI)]
GE
Bolitoglossa
rufescens
1
Cordoba, Veracruz 18 52.200 97 00.200 LC Wild 1/1 8
Bolitoglossa
rufescens
2
Orizaba, Veracruz 18 50.600 97 01.800 LC Wild 1/1 5
Chiropterotriton
chiropterus
3
Zempoala, Veracruz 19 03.892 99 18.814 CR Wild 0/1
Chiropterotriton
lavae
4
La Joya, Me
´xico 19 37.426 97 01.862 CR Wild 0/1
Chiropterotriton
orculus
5
Rio Frio, Me
´xico 19 04.400 98 41.300 VU Wild 0/1
Chiropterotriton
orculus
6
Popocatepetl, Me
´xico 19 02.800 99 52.500 VU Wild 0/1
Pseudoeurycea
cephalica
7,8
Zempoala, Veracruz 19 03.900 99 18.800 NT Wild 2/24,19
Pseudoeurycea
cephalica
9
Rio Frio, Me
´xico 19 22.400 98 39.400 NT Wild 1/16
Pseudoeurycea
cephalica
10
Hidalgo, Me
´xico 20 11.300 98 44.800 NT Wild 1/1 6
Pseudoeurycea
firscheini
11
Orizaba, Veracruz 18 42.900 97 20.500 EN Wild 1/118
Pseudoeurycea
leprosa
12
Rio Frio, Me
´xico 19 21.873 98 40.651 VU Wild 0/1
Pseudoeurycea
leprosa
13
Zempoala, Veracruz 19 04.219 99 20.430 VU Wild 0/1
Pseudoeurycea
leprosa
14
Rio Frio, Me
´xico 19 22.400 98 39.400 VU Wild 1/11
Pseudoeurycea
leprosa
15
Popocatepetl, Me
´xico 19 04.400 98 41.300 VU Wild 1/112
Pseudoeurycea
longicauda
16
N/A 19 26.254 100 10.683 EN Wild 0/1
Pseudoeurycea
robertsi
17
Nevado de Toluca,
Me
´xico
19 02.800 99 52.500 CR Wild 0/1
Wild total 9/17 [52.9%
(28.5–77.4)]
Bolitoglossa
platydactyla
NT Captive 0/2
Bolitoglossa
platydactyla
NT Captive 2/2 379, 3191
Bolitoglossa
rufescens
LC Captive 6/7 1, 5, 68, 152,
386, 589
Chiropterotriton
chiropterus
CR Captive 0/2
P. Van Rooij et al.
B. rufescens specimens (3/7 and 6/7 sites positive) and 1
Pseudoeurycea leprosa specimen (6/7 sites positive) (data
not shown).
Based on the qPCR results, the plantar side of the
forelimbs and hindlimbs and the ventral side of the tail
consistently had the highest Bd densities, and the dorsum
and chin had the lowest Bd densities. In 9 out of the 11
samples with high GE values of Bd, the histological slides
showed few to abundant sporangia embedded in the upper
keratinized layers of the epidermis (Fig. 3), coinciding with
hyperkeratosis but not epidermal hyperplasia. In 10 out of
the 11 samples with low GEs, no histological lesions could
be observed.
We detected a significant main effect of body part on
Bd density (P= 0.025). However, Tukey’s post hoc mul-
tiple comparison test did not detect significant differences
Table 2. continued
Species Site Latitude
(°N)
Longitude
(°W)
IUCN Red
List Category
Wild/
captive
No. infected/
No. examined
[prevalence %
(95% CI)]
GE
Chiropterotriton
chiropterus
CR Captive 0/6
Chiropterotriton
chiropterus
CR Captive 0/5
Chiropterotriton
multidentatus
EN Captive 0/1
Pseudoeurycea
belli
VU Captive 2/4 1, 9
Pseudoeurycea
belli
VU Captive 0/2
Pseudoeurycea
cephalica
NT Captive 0/1
Pseudoeurycea
cephalica
NT Captive 2/3 18, 117
Pseudoeurycea
leprosa
VU Captive 0/14
Pseudoerycea
leprosa
VU Captive 3/3 41, 151, 1331
Pseudoeurycea
longicauda
EN Captive 1/4 2
Pseudoeurycea
nigromaculata
CR Captive 0/1
Pseudoeurycea
robertsi
CR Captive 0/2
Pseudoeurycea
sp. nov.*
N/A Captive 0/1
Thorius
troglodytes
EN Captive 0/1
Captive total 16/61 [26.2%
(15.1–37.4)]
Species and numbers indicated in bold are qPCR positive for Bd and are presented with the corresponding genomic equivalents (GEs); the indexed numbers
refer to the localities were the wild species were sampled (Fig. 2).
LC least concern, NT near threatened, VU vulnerable, EN endangered, CR critically endangered, N/A no data available.
Specimens from the species are dead.
* Species are under description by Parra-Olea & Wake.
Detection of Bd in Bolitoglossine Salamanders
among the body parts. To determine whether swabbing
might be a viable alternative to destructive tissue sampling,
we compared Bd densities in skin samples from the pelvic
region to Bd densities from swab samples of the seven body
parts. For every body part, the mean Bd density was lower
on the swab sample than it was in the tissue sample, sug-
gesting that swabbing underestimates Bd densities. How-
ever, some caution should be used interpreting these results
given that there is variation in Bd densities among body
parts and all swab samples were compared to a single tissue
sample from the pelvic region. Although all the swab
samples had less Bd than the tissue sample, a multiple
comparison test revealed that Bd density in the tissue
sample was not significantly different from Bd densities in
the swab samples of the forelimbs (P= 0.920) or hindlimbs
(P= 0.818). Hence, in salamanders, swab sampling of the
limbs might provide a reliable, nondestructive alternative
to tissue sampling for Bd quantification.
Bd was not detected in any of the 28 healthy, living
Mexican bolitoglossine salamanders, whereas 48% (16/33)
of the dead captive animals carried high zoospore loads of
up to 3191 GEs (Table 2). Fifty-three percent of the wild
animals (9/17) sampled were positive for Bd,and Bd was
detected in seven localities (Fig. 2). None of the wild ani-
mals showed obvious clinical signs of chytridiomycosis, and
the positive wild animals carried rather low zoospore loads.
A high incidence of Bd infections was found in the
sampled bolitoglossine salamanders with the pelvic region,
the forelimbs and hindlimbs and the ventral side of the tail
having the highest Bd densities. To maximize the chances
of detecting Bd in Caudata, we recommend sampling these
body parts. In contrast, in Anura (frogs and toads) the
ventral skin and the toes seem more predisposed to high Bd
densities (Berger et al. 2005; Puschendorf and Bolan
˜os
2006).
There is a paucity of data demonstrating susceptibility
of plethodontid salamanders to Bd infection. We revealed
that clinically healthy captive salamanders were free of Bd
infection, but that in half of the dead captive salamanders
Bd was present, combined with hyperkeratosis, a hallmark
of clinical Bd infections. This result is consistent with
recent infection trials revealing high virulence of Bd to
B. rufescens and P. leprosa (Cheng et al. 2011) and the
report of Pasmans et al. (2004) demonstrating lethal Bd
infection in captive Bolitoglossa dofleini. Bd was detected in
half of the wild Mexican bolitoglossine salamanders sam-
pled but was not associated with any obvious clinical signs
of disease. Although the high prevalence of Bd in dead
captive salamanders, its absence in clinically healthy living
ones, and its presence in wild salamanders is consistent
with Bd being involved in recent bolitoglossine population
declines, further studies will be necessary before Bd is
conclusively demonstrated to be a causal factor in the
decline of Mexican bolitoglossine salamanders.
ACKNOWLEDGMENTS
This study was funded by a research grant from Ghent
University to Pascale Van Rooij (BOF08/24J/004). Poly-
clonal antibodies against B. dendrobatidis were kindly
provided by Dr. Alex D. Hyatt (Australian Animal Health
Laboratory, CSIRO, Victoria, Australia). We are grateful to
Arnaud Jamin and Eike Amthauer for kindly providing
Plethodontid specimens, David Van Rooij (RCMG, Ghent
University, Belgium) for providing help in mapping of the
sampling localities and two anonymous referees for
providing helpful suggestions that greatly improved the
manuscript.
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... We here assessed the regulation of virulence mechanisms of B. salamandrivorans and its ability to attach and invade the skin of olms versus fire salamanders as a proxy for pathogenicity and colonization capacity 7 using an ex vivo protocol modified from Van Rooij et al. 22 . Three 5 mm and three 8 mm diameter, full thickness ventral skin biopsies were collected from each of the six olms of the infection trial after euthanasia, and from six B. salamandrivorans negative fire salamanders. ...
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jats:title>Abstract Emerging infections add to existing threats to the survival of amphibians worldwide. The olm ( Proteus anguinus ) is a vulnerable, troglobiont urodele species with a small European range and restricted to underground karstic systems. Population declines to emerging threats like the chytrid fungus Batrachochytrium salamandrivorans , are likely to go unnoticed due to inaccessibility of the species’ habitat. We here studied the interaction between olms and B. salamandrivorans . Experimental inoculation of olms resulted in low-level, asymptomatic but persistent infections, with limbs as predilection sites. The lack of exponential fungal growth in the olms’ epidermis correlated with limited fungal proliferation and dampened virulence gene expression after exposure to olm skin compounds. The olm is one of few western Palearctic urodeles that is tolerant to B. salamandrivorans infection and may act as a subterranean disease reservoir, yet costs of subclinical infection may compromise olm fitness on the long term.</jats:p
... Appropriate permit numbers and information may be obtained from first author upon request. We rubbed rayon-tipped sterile swabs (MW-113, Medical Wire & Equipment, Corsham, England) over the plantar side of one front and one hind limb, the ventral tail surface of caudates, the dorsal side of the body, and the ventral surface of the body 5 times each 28 . We placed the swabs into sterile plastic vials with 20 μl of sterile deionized water. ...
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The salamander chytrid fungus (Batrachochytrium salamandrivorans [Bsal]) is causing massive mortality of salamanders in Europe. The potential for spread via international trade into North America and the high diversity of salamanders has catalyzed concern about Bsal in the U.S. Surveillance programs for invading pathogens must initially meet challenges that include low rates of occurrence on the landscape, low prevalence at a site, and imperfect detection of the diagnostic tests. We implemented a large-scale survey to determine if Bsal was present in North America designed to target taxa and localities where Bsal was determined highest risk to be present based on species susceptibility and geography. Our analysis included a Bayesian model to estimate the probability of occurrence of Bsal given our prior knowledge of the occurrence and prevalence of the pathogen. We failed to detect Bsal in any of 11,189 samples from 594 sites in 223 counties within 35 U.S. states and one site in Mexico. Our modeling indicates that Bsal is highly unlikely to occur within wild amphibians in the U.S. and suggests that the best proactive response is to continue mitigation efforts against the introduction and establishment of the disease and to develop plans to reduce impacts should Bsal establish.
... Samples were also collected at 6 additional sites in Germany and 1 in the Netherlands, which were located >100 km from the nearest known outbreak (Technical Appendix Table 2). Sampling was conducted by swabbing skin (Hyatt et al., 2007;Van Rooij et al., 2011) of live animals and collecting skin samples from dead animals. All samples were kept frozen at -20°C until they were analyzed for the presence of Bsal DNA via real-time PCR, as described . ...
... Studies reporting the presence of Bd in Mexico exist Bolom-Huet et al., 2019;Hernández-Martínez et al., 2019;Hale et al., 2005;Frías-Alvarez et al., 2008;Muñoz-Alonso, 2010;Cheng et al., 2011;Van Rooij et al., 2011;Luja et al., 2012;Luría-Manzano et al., 2011;Murrieta-Galindo et al., 2014;Mendoza-Almeralla et al., 2016b;Cabrera-Hernández, 2012;García-Feria et al., 2017;Peralta-García et al., 2018;Familiar-López, 2010;Gómez, 2013;Cortes, 2014;López-Velázquez, 2014;Mendoza-Almeralla, 2016a;Ortiz-Millán, 2016;Solís-Sotelo, 2017). ...
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Amphibian populations are globally declining at an alarming rate, and infectious diseases are among the main causes of their decline. Two micro-parasites, the fungus Batrachochytrium dendrobatidis (BD) and the virus Ranavirus (RV) have caused mass mortality of amphibians and population declines. Other less understood epizootics are caused by macro-parasites, such as Trombiculoidea chiggers. Infection with chiggers can affect frog behavior and survival. Furthermore, synergistic effects of co-infection with both macro and micro-parasites may lead to higher morbidity. To better understand these potential synergies, we investigated the presence and co-infection by chiggers, BD and RV in the endemic frog Tlalocohyla smithii (T. smithii). Co-infection of BD, RV, and/or chiggers is expected in suitable habitats, with high canopies (which generate more humid environments necessary for survival), sites with less amphibian diversity (dilution effect), and lower water quality (habitat preferences). On one hand co-infection of BD, RV, and/or chiggers is expected in habitats that are suitable for their co-occurrence; and if infection with one parasite facilitates infection with the others. On the other hand, co-infection could decrease if these parasites were to differ in their micro-environmental requirements (i.e. niche apportionment). A total of 116 frogs of T. smithii were studied during 2014 and 2016 in three streams within the Chamela-Cuixmala Biosphere Reserve in Jalisco, Mexico. Our results show that 31% of the frogs were infected with Trombiculoidea chiggers (Hannemania sp. and Eutrombicula alfreddugesi); Hannemania prevalence increased with air temperature and decreased in sites with high canopies and with water pH values above 8.5 and below 6.7. BD prevalence was 2.6%, RV prevalence was 0%, and none of the frogs infected with chiggers were coinfected with BD. Together, this study suggests that chiggers do not facilitate infection with BD, as these may be apportioned in different micro-habitats. Nevertheless, the statistical power to assure this is low as BD prevalence is 2%. We recommend further epidemiological monitoring of multiple parasites in different geographical locations in order to provide insight on the true hazards, risks and conservation options for amphibian populations.
... Sequences for other species of Thorius were obtained from GenBank and sequences were aligned using Muscle v.3.8 (Edgar 2004). The pairwise GTR distance between sequences from the two populations was calculated using PAUP v4.165 (Swofford 2003). Sequences are deposited in GenBank (Table 1). ...
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Infection records of Batrachochytrium dendrobatidis (Bd), a pathogen that has devastated amphibian populations worldwide, have rapidly increased since the pathogen’s discovery. Dealing with so many records makes it difficult to (a) know where, when and in which species infections have been detected, (b) understand how widespread and pervasive Bd is and (c) prioritize study and management areas. We conducted a systematic review of papers and compiled a database with Bd infection records. Our dataset covers 71 amphibian families and 119 countries. The data revealed how widespread and adaptable Bd is, being able to infect over 50% of all tested amphibian species, with over 1000 confirmed host species and being present in 86 countries. The distribution of infected species is uneven among and within countries. Areas where the distributions of many infected species overlap are readily visible; these are regions where Bd likely develops well. Conversely, areas where the distributions of species that tested negative overlap, such as the Atlantic Coast in the USA, suggest the presence of Bd refuges. Finally, we report how the number of tested and infected species has changed through time, and provide a list of oldest detection records per country.
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Rapid, early, and reliable detection of invasive pathogenic microorganisms is essential in order to either predict or delineate an outbreak, and monitor appropriate mitigation measures. The chytrid fungus Batrachochytrium salamandrivorans is expanding in Europe, and infection with this fungus may cause massive mortality in urodelans (salamanders and newts). In this study, we designed and validated species-specific primers and a probe for detection of B. salamandrivorans in water. In a garden pond in close proximity to the B. salamandrivorans index site in the Netherlands, B. salamandrivorans-infected newts had been detected in 2015 and have been monitored since. In 2016 and 2017, no B. salamandrivorans was detected at this site, but in 2018 B. salamandrivorans flared up in this isolated pond which allowed validation of the technique in situ. We here present the development of an environmental DNA technique that successfully detects B. salamandrivorans DNA in natural waterbodies even at low concentrations. This technique may be further validated to play a role in B. salamandrivorans range delineation and surveillance in both natural waterbodies and in captive collections.
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Batrachochytrium dendrobatidis is a fungal pathogen that has been implicated in amphibian declines worldwide. Histopathologic techniques have been used to diagnose the disease, but their sensitivity bas not been determined. it is also unclear whether the probability of detection varies between skin samples derived from different body parts. We examined 24 Fitzinger's rainfrogs; (Eleutherodactylus fitzingeri) with chytridiomycosis. This is a common frog species with a broad range and high abundance throughout most of Costa Rica. We sampled 12 different body, parts from each animal, and alternated the staining between a routinely used stain (hematoxylin and eosin [H&E]), and a more fungus-specific stain (periodic acid-Schiff [PAS]). The pelvic patch and the innermost finger of the hand were consistently the best places to detect the disease, although significant differences were found only with the gular area, the abdomen, and toes four and five. We found more positive samples using PAS than using H&E in all body parts, although significant differences were detected only in samples derived from the pelvic patch. Using the best combination of factors (stain and body part) and animals with the lightest infections (to test the sensitivity of the technique), we calculated that at least 17 sections are needed in order to reach 95% confidence that a frog is or is not infected. We conclude that the choice of stain and body part can significantly alter estimates of prevalence of B. dendrobatidis.
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Populations of Mexican plethodontid salamanders have been surveyed non-systematically over the last 25 years. In light of many reports of disappearance of amphibians around the world, we checked for persistence of reported species at ten of these sites. All of the commoner species persist (we observed individuals representing a total of 30 species). While observed densities of many species of Mexican plethodontids are lower to much lower than was the case 20 to 25 years ago, evidence for recent extinctions, such as has been reported for amphibian taxa elsewhere, is equivocal or lacking. Habitat modification has contributed to difficulties in finding certain species.
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Epidermal changes caused by a chytridiomycete fungus (Chytridiomycota; Chytridiales) were found in sick and dead adult anurans collected from montane rain forests in Queensland (Australia) and Panama during mass mortality events associated with significant population declines. We also have found this new disease associated with morbidity and mortality in wild and captive anurans from additional locations in Australia and Central America. This is the first report of parasitism of a vertebrate by a member of the phylum Chytridiomycota. Experimental data support the conclusion that cutaneous chytridiomycosis is a fatal disease of anurans, and we hypothesize that it is the proximate cause of these recent amphibian declines.
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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.
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Amphibians highlight the global biodiversity crisis because ∼40% of all amphibian species are currently in decline. Species have disappeared even in protected habitats (e.g., the enigmatic extinction of the golden toad, Bufo periglenes, from Costa Rica). The emergence of a fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been implicated in a number of declines that have occurred in the last decade, but few studies have been able to test retroactively whether Bd emergence was linked to earlier declines and extinctions. We describe a noninvasive PCR sampling technique that detects Bd in formalin-preserved museum specimens. We detected Bd by PCR in 83-90% (n = 38) of samples that were identified as positive by histology. We examined specimens collected before, during, and after major amphibian decline events at established study sites in southern Mexico, Guatemala, and Costa Rica. A pattern of Bd emergence coincident with decline at these localities is revealed-the absence of Bd over multiple years at all localities followed by the concurrent emergence of Bd in various species at each locality during a period of population decline. The geographical and chronological emergence of Bd at these localities also indicates a southbound spread from southern Mexico in the early 1970s to western Guatemala in the 1980s/1990s and to Monteverde, Costa Rica by 1987. We find evidence of a historical "Bd epidemic wave" that began in Mexico and subsequently spread to Central America. We describe a technique that can be used to screen museum specimens from other amphibian decline sites around the world.
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Amphibians are globally declining and approximately one-third of all species are threatened with extinction. Some of the most severe declines have occurred suddenly and for unknown reasons in apparently pristine habitats. It has been hypothesized that these "rapid enigmatic declines" are the result of a panzootic of the disease chytridiomycosis caused by globally emerging amphibian chytrid fungus. In a Species Distribution Model, we identified the potential distribution of this pathogen. Areas and species from which rapid enigmatic decline are known significantly overlap with those of highest environmental suitability to the chytrid fungus. We confirm the plausibility of a link between rapid enigmatic decline in worldwide amphibian species and epizootic chytridiomycosis.
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We document major declines of many species of salamanders at several sites in Central America and Mexico, with emphasis on the San Marcos region of Guatemala, one of the best studied and most diverse salamander communities in the Neotropics. Profound declines of several formerly abundant species, including 2 apparent extinctions, are revealed. Terrestrial microhabitat specialists at mid- to high elevations have declined more than microhabitat generalists. These terrestrial microhabitat specialists have largely disappeared from multiple sites in western Guatemala, including in well-protected areas, suggesting that the phenomenon cannot be explained solely by localized habitat destruction. Major declines in southern Mexican plethodontid salamanders occurred in the late 1970s to early 1980s, concurrent with or preceding many reported frog declines. The species in decline comprise several major evolutionary lineages of tropical salamanders, underscoring that significant portions of the phylogenetic diversity of Neotropical salamanders are at risk. Our results highlight the urgent need to document and understand Neotropical salamander declines as part of the larger effort to conserve global amphibian diversity.
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Human alteration of the environment has arguably propelled the Earth into its sixth mass extinction event and amphibians, the most threatened of all vertebrate taxa, are at the forefront. Many of the worldwide amphibian declines have been caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), and two contrasting hypotheses have been proposed to explain these declines. Positive correlations between global warming and Bd-related declines sparked the chytrid-thermal-optimum hypothesis, which proposes that global warming increased cloud cover in warm years that drove the convergence of daytime and nighttime temperatures toward the thermal optimum for Bd growth. In contrast, the spatiotemporal-spread hypothesis states that Bd-related declines are caused by the introduction and spread of Bd, independent of climate change. We provide a rigorous test of these hypotheses by evaluating (i) whether cloud cover, temperature convergence, and predicted temperature-dependent Bd growth are significant positive predictors of amphibian extinctions in the genus Atelopus and (ii) whether spatial structure in the timing of these extinctions can be detected without making assumptions about the location, timing, or number of Bd emergences. We show that there is spatial structure to the timing of Atelopus spp. extinctions but that the cause of this structure remains equivocal, emphasizing the need for further molecular characterization of Bd. We also show that the reported positive multi-decade correlation between Atelopus spp. extinctions and mean tropical air temperature in the previous year is indeed robust, but the evidence that it is causal is weak because numerous other variables, including regional banana and beer production, were better predictors of these extinctions. Finally, almost all of our findings were opposite to the predictions of the chytrid-thermal-optimum hypothesis. Although climate change is likely to play an important role in worldwide amphibian declines, more convincing evidence is needed of a causal link. • chytridiomycosis • climate change • emerging infectious disease • extinction • global warming
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Many scientists argue that we are either entering or in the midst of the sixth great mass extinction. Intense human pressure, both direct and indirect, is having profound effects on natural environments. The amphibians--frogs, salamanders, and caecilians--may be the only major group currently at risk globally. A detailed worldwide assessment and subsequent updates show that one-third or more of the 6,300 species are threatened with extinction. This trend is likely to accelerate because most amphibians occur in the tropics and have small geographic ranges that make them susceptible to extinction. The increasing pressure from habitat destruction and climate change is likely to have major impacts on narrowly adapted and distributed species. We show that salamanders on tropical mountains are particularly at risk. A new and significant threat to amphibians is a virulent, emerging infectious disease, chytridiomycosis, which appears to be globally distributed, and its effects may be exacerbated by global warming. This disease, which is caused by a fungal pathogen and implicated in serious declines and extinctions of >200 species of amphibians, poses the greatest threat to biodiversity of any known disease. Our data for frogs in the Sierra Nevada of California show that the fungus is having a devastating impact on native species, already weakened by the effects of pollution and introduced predators. A general message from amphibians is that we may have little time to stave off a potential mass extinction.
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The role of global climate change in the decline of biodiversity and the emergence of infectious diseases remains controversial, and the effect of climatic variability, in particular, has largely been ignored. For instance, it was recently revealed that the proposed link between climate change and widespread amphibian declines, putatively caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), was tenuous because it was based on a temporally confounded correlation. Here we provide temporally unconfounded evidence that global El Niño climatic events drive widespread amphibian losses in genus Atelopus via increased regional temperature variability, which can reduce amphibian defenses against pathogens. Of 26 climate variables tested, only factors associated with temperature variability could account for the spatiotemporal patterns of declines thought to be associated with Bd. Climatic predictors of declines became significant only after controlling for a pattern consistent with epidemic spread (by temporally detrending the data). This presumed spread accounted for 59% of the temporal variation in amphibian losses, whereas El Niño accounted for 59% of the remaining variation. Hence, we could account for 83% of the variation in declines with these two variables alone. Given that global climate change seems to increase temperature variability, extreme climatic events, and the strength of Central Pacific El Niño episodes, climate change might exacerbate worldwide enigmatic declines of amphibians, presumably by increasing susceptibility to disease. These results suggest that changes to temperature variability associated with climate change might be as significant to biodiversity losses and disease emergence as changes to mean temperature.