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No detection of chytrid in first systematic screening of Bombina variegata pachypus (Anura: Bombinatoridae) in Liguria, northern Italy

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Abstract

The Apennine Yellow-bellied toad Bombina variegata pachypus, a small anuran endemic to peninsular Italy, has been declining throughout its range over the last 30 years. Although mortality by chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, was first reported for the species in 2004, its role in the decline has not yet been assessed. Between 2011 and 2012 we sampled eight populations of B. v. pachypus in Liguria, northern Italy, swabbing 86 and 143 individuals respectively, corresponding to between 24 and 80% of the estimated individuals within each population. We did not detect chytrid in any the samples collected. For the three largest populations in the region, we can rule out infections of prevalence greater than 10% with at least 98% confidence. Research at a larger scale is urgently needed to clarify the role of B. dendrobatidis in the decline of this and other amphibians in Italy.
ISSN 1827-9635 (print) © Firenze University Press
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Acta Herpetologica 8(1): 59-63, 2013
No detection of chytrid in rst systematic screening of Bombina variegata
pachypus (Anura: Bombinatoridae) in Liguria, northern Italy
S C,*, A M, F P
1 ARC Centre of Excellence for Environmental Decisions, School of Botany, University of Melbourne, 3010 VIC, Australia. *Correspond-
ing author. E-mail: canessas@unimelb.edu.au
2 Faculty of Veterinary Medicine, Ghent University, Merelbeke 9820, Belgium
Submitted on: 2012, 7th Decembre; revised on: 2013, 10th April; accepted on: 2013, 19th April.
Abstract. e Apennine Yellow-bellied toad Bombina variegata pachypus, a small anuran endemic to peninsular Italy,
has been declining throughout its range over the last 30 years. Although mortality by chytridiomycosis, caused by
the fungus Batrachochytrium dendrobatidis, was rst reported for the species in 2004, its role in the decline has not
yet been assessed. Between 2011 and 2012 we sampled eight populations of B. v. pachypus in Liguria, northern Ita-
ly, swabbing 86 and 143 individuals respectively, corresponding to between 24 and 80% of the estimated individuals
within each population. We did not detect chytrid in any the samples collected. For the three largest populations in
the region, we can rule out infections of prevalence greater than 10% with at least 98% condence. Research at a larg-
er scale is urgently needed to clarify the role of B. dendrobatidis in the decline of this and other amphibians in Italy.
Keywords. Bombina, chytrid, condence, Liguria, monitoring, population size, prevalence.
e amphibian chytrid fungus Batrachochytrium dend-
robatidis is the main pathogen driver of the global amphib-
ian decline (Fisher et al., 2009). Die-os and extinctions
have occurred in all continents (Berger et al., 1998; Con-
radie et al., 2011; Swei et al., 2011) including several cas-
es in Europe (Garner et al., 2005). In Italy, infection by
chytridiomycosis has been documented for several species
in the last decade (reviewed in Tessa et al., 2013): further
information on the distribution of B. dendrobatidis in Italy
is needed to understand the causes of local and regional
declines and inform conservation eorts.
e Apennine yellow-bellied toad Bombina variegata
pachypus (Anura: Bombinatoridae) was the rst amphib-
ian species in Italy with conrmed mortality by chytridi-
omycosis, reported by Stagni et al. (2004) on captive
individuals caught from three populations in the north-
eastern Apennines. Recent evidence suggests chytrid is
present in populations of B. v. pachypus along the Ital-
ian peninsula, with infections dating back as early as the
late 1970s (Canestrelli et al., 2013). B. v. pachypus has
declined steeply over the last 30 years, with widespread
local disappearances: although habitat loss is believed to
represent the main driver of the decline (Mirabile et al.,
2009; Canessa et al., 2013), chytridiomycosis is accepted
as a potential threat to the species (see for example its
Red List assessment in IUCN, 2011). However, to our
knowledge, no systematic assessment of the spread of
chytrid among populations of B. v. pachypus in Italy has
been undertaken to date.
In order to clarify this issue at least in part, we con-
ducted surveys throughout the northern Italian region of
Liguria in 2011 and 2012. A recent assessment of the sta-
tus of B. v. pachypus in this area showed that over 50%
of the known populations disappeared between 2001 and
2010: habitat loss relating to the abandonment of tradi-
tional farming practices has been suggested as the main
cause of decline (Canessa et al., 2013). A small-scale sur-
vey (ve individuals tested) carried out in 2005 at a single
site in the region failed to detect any presence of B. dend-
robatidis (Adams et al., 2008).
60 Stefano Canessa et al.
In 2011, between 31 July and 4 August we visited all
seven known breeding sites of B. v. pachypus in Liguria,
located within the catchments of the Lavagna and Vara
rivers in the eastern part of the region, encompassing an
area of approximately 800 km2. In 2012, we repeated our
visits at ve of these seven populations: the three sites
believed to host the largest populations were sampled
three times during the 2012 season in order to maxi-
mize the overall proportion of the population that could
be tested (22 June, 24 July and 26 July). We also tested
all the animals present at a small captive breeding centre
within the study area: these individuals had been caught
in the wild and kept in a semi-natural enclosure with no
contact with other amphibians during the study period.
We captured all detected individuals by hand and
photographed them to record their unique ventral pat-
tern: this allowed us to calculate the number of unique
individuals tested in addition to the total number of
swabs collected at sites that were visited multiple times.
To minimize the chance of spreading pathogens, we fol-
lowed recommendations by Speare et al. (2004) when
handling individuals, and sites within dierent catch-
ments were never sampled during the same day. We col-
lected samples as described by Hyatt et al. (2007) using
cotton-tip swabs and then released individuals at the
point of capture. Samples were stored at –4 °C until fur-
ther analysis. We only tested post-metamorphic indi-
viduals: although pathogen prevalence can be higher in
tadpoles, facilitating detection, we chose to avoid any
impact to the few remaining populations of the species in
the region that could result from applying lethal or non-
lethal sampling to larvae. Swabbing is less sensitive than
other methods, and can still damage tadpole mouthparts
and facilitate infections in addition to stress resulting
from handling (Retallick et al., 2006).
B. dendrobatidis DNA in the samples, real-time
Taqman PCR assays were conducted on a CFX96 Real
Time System (BioRad). Amplication conditions, primer
and probe concentrations were according to Boyle et al.
(2004). Due to economic constraints, samples were run in
duplicate. Consistent results between the two runs were
classied as correct: we considered a test result as posi-
tive where both qPCR runs gave results above 0.1 GE,
with standard deviation smaller than the average of both
repeats and suitable amplication curves.
After obtaining the test results, we estimated the
mean prevalence (with 95% credible intervals) of the
pathogen in the population using a Bayesian equal-tailed
Jerey prior (Brown et al., 2001). To assess the sensitivity
of our sampling program, we then calculated the proba-
bility of detecting 5, 10 and 30% infections with the num-
ber of individuals sampled at each site, by solving
C = 1 – (1 – p)n
where C is the probability of detecting at least one infect-
ed individual from n tested in a population with p preva-
lence of the disease (DiGiacomo and Koepsell, 1986).
Additionally, for the three largest populations in the
study we were able to use an existing multi-year mark-
recapture database to estimate population size at the
time of sampling (Canessa, unpubl. data). To account
for open-population dynamics such as temporary emi-
gration, oen observed for B. variegata (Hartel, 2008),
we used an open-population Jolly-Seber model (Wagner
et al., 2011). We t the model in a Bayesian framework
using date- and population-dependent recapture rates
(code in Kéry and Schaub, 2011). We used JAGS (Plum-
mer, 2005) to obtain samples from three Markov chains
over 100,000 iterations, aer discarding the rst 10,000 as
a burn-in.
In 2011, we collected 81 swabs from 81 individuals in
seven wild populations, corresponding to between 24 and
42% of the estimated sizes of respective populations (as
estimated from 95% condence intervals from the Jolly-
Seber model): at two sites we captured only one and four
individuals respectively (Table 1). In 2012, we captured
and swabbed a total of 143 individuals at four of the
wild locations on four separate occasions, correspond-
ing to between 66 and 80% of the estimated sizes of the
tested populations. Several individuals were re-captured
and swabbed in two or three occasions in 2012, yielding
a total of 224 swabs for that season. All individuals at the
captive breeding centre were also caught and tested (four
in 2011 and three in 2012): we did not calculate con-
dence intervals or sampling power for this site.
All samples tested negative for B. dendrobatidis. We
captured more than 10 individuals only at four sites: in
2011, this resulted in upper 95% condence intervals (CI)
of prevalence between 0.09 and 0.13 (Table 1). For three
of these populations, in 2012 we were able to reduce the
upper CIs to 0.05 or less; at the fourth site, only seven
individuals were captured in 2012, resulting in an upper
CI of 0.23. For the three most intensively sampled pop-
ulations, we can be at least 97% condent that chytrid
would have been detected at these populations for a
prevalence of at least 10%, and more than 83% condent
for a prevalence of at least 5% (Table 1). For the smaller
populations sampled (less than 10 individuals), we could
make no reliable inference regarding the prevalence of
infection, although in sites with more than one capture,
the probability of having missed large infections (> 30%
prevalence) was still lower than 50%.
Although reliable estimates of abundance could not
be obtained for the smaller populations in the study, we
61
No detection of chytrid in Ligurian B. variegata
observed relatively high recapture rates (> 40%) for the
larger populations, suggesting the small sample sizes at
some sites might reect actual abundances. Nevertheless,
the estimates we present for populations with less than 10
sampled individuals can only be considered as prelimi-
nary and do not allow reliable inference. For the largest
populations in the region the large sample sizes, particu-
larly for 2012, allows us to rule out with sucient con-
dence infections of prevalence greater than 10%.
Additional information about the expected preva-
lence of chytrid infection in B. v. pachypus is needed to
make more precise inference: for example. Canestrelli et
al. (2013) recently found that prevalence of B. dendroba-
tidis in present and historical specimens of B. v. pachy-
pus across the Italian peninsula we never below 12%.
Amongst populations of B. variegata in Hungary, the
infection rate has been estimated between 12% and 29%
(Vörös, pers. comm.). In the Netherlands and Flanders
(Belgium), Spitzen-van der Sluijs et al. (2010) reported a
prevalence of infection of 5.9% for B. variegata out of 255
individuals sampled. Holding this relatively high base rate
as a guideline, the failure to detect chytrid in the Ligurian
populations may indicate lower susceptibility due to local
conditions, bias related to the date of sampling (patterns
of chytrid infection are highly seasonal: see Kriger and
Hero, 2007), or possibly actual absence of the pathogen
from the region at present. In addition, breeding sites of
B. v. pachypus in Liguria have been regularly monitored
throughout the breeding season since 2008 and no obvi-
ous sign of chytrid infection (e.g. individual mortality or
abnormal behaviour) has ever been reported (Arillo et al.,
2009; Arillo et al., 2011).
It is dicult to infer whether chytridiomycosis might
have had a role in past local extinctions in the region.
Most disappearances in the region in recent years have
occurred at sites in agricultural areas (Canessa et al.,
2013). However, the existing literature suggests such sites
might be less likely to host permanent foci of chytrid
infection, due to their low elevation and open vegeta-
tion types, resulting in higher water temperatures (> 30
°C in August) and regular desiccation, unsuitable for the
pathogen (Johnson et al., 2003). For example, Hyne et al.
Table 1. Results for all screened populations of B. v. pachypus in 2011 and 2012.
Population
Fav Lo1 Lo2 LoP Pav Per Pin Te v
2011
Total swabs14 17 21 8 17 1 13 1
Individuals swabbed24 17 21 8 17 1 13 1
Population size3-68 ± 2 67 ± 2 -35 ± 4 -47 ± 3 -
% tested4- 25.0 31.3 - 48.6 - 27.7 -
Prevalence (95% CI)50.36 0.11 0.09 0.21 0.11 0.77 0.13 0.77
Detection p = 0.0560.19 0.58 0.66 0.34 0.58 0.05 0.49 0.05
p = 0.170.34 0.83 0.89 0.57 0.83 0.10 0.75 0.10
p = 0.380.76 1.00 1.00 0.94 1.00 0.30 0.99 0.30
2012
Total swabs1- 106 60 - 7 - 51 -
Individuals swabbed2- 55 43 - 7 - 35 -
Population size3-69 ± 2 69 ± 2 -35 ± 4 -48 ± 2 -
% tested4- 79.7 62.3 - 20.0 - 72.9 -
Prevalence (95% CI)5- 0.04 0.04 - 0.23 - 0.05 -
Detection p = 0.056- 0.94 0.89 - 0.30 - 0.83 -
p = 0.17- 1.00 0.99 - 0.52 - 0.97 -
p = 0.38- 1.00 1.00 - 0.92 - 1.00 -
1 Total number of swabs collected
2 Total numbers of unique individuals swabbed, where repeated visits were carried out (Lo1, Lo2 and Pin in 2012)
3 Population size at the time of sampling (± standard deviation), estimated using the Jolly-Seber open population model – a dash indicates
no estimate could be made for that population/year
4 Proportion of the estimated population size (mean) that was tested
5 Upper 95% equal-tailed Jerey prior condence interval (lower interval is 0 in all cases).
6, 7, 8 Probability of detecting at least one infected individual if the pathogen were present in the population with a prevalence of 0.05, 0.1
and 0.3 respectively.
62 Stefano Canessa et al.
(2009) found lowland species persisting without notice-
able population declines in the presence of chytrid in an
agricultural landscape with relatively warm climate. Con-
versely, particularly high impacts of chytrid worldwide
have occurred in forest areas with permanent hydrop-
eriods and cooler mesoclimates (Berger et al., 1998): all
remaining large populations of B. v. pachypus in Liguria
occur in such areas and therefore require special atten-
tion. At the moment, monitoring must continue across
all known sites to ensure prompt detection of possible
outbreaks. Quantifying the eciency of monitoring (e.g.
capture rates of individuals and test sensitivities) can help
inform screening programs to achieve the best results for
the eort invested (Lachish et al., 2012).
Although B. v. pachypus is recognized as an endan-
gered species in Italy, this is, to our knowledge, the rst
systematic screening of any of its populations for B. den-
drobatidis. Conservation plans for the species include
habitat restoration and ex-situ initiatives (Di Cerbo and
Ferri, 2002; Gentilli et al., 2002; Arillo et al., 2011). Addi-
tional information about the role of chytridiomycosis
in the decline of B. v. pachypus is urgently needed, both
to inform actions (for example, habitat restoration may
not be sucient where disease is the main threat) and
to raise awareness of the need to implement safety pro-
tocols (Phillott et al., 2010). In general, further research
about the distribution and prevalence of chytrid amongst
amphibian species in Italy should be seen as a prior-
ity, particularly in areas of known and ongoing declines.
Surveys at the local and regional scale, whilst relatively
inexpensive, can provide valuable information about the
spread and impact of the pathogen and help optimize
conservation strategies.
ACKNOWLEDGEMENTS
We thank A. Morán Ordoñez, F. Oneto, D. Ottonello, P.
Piana and S. Salvidio for help with surveys. Comments from
two anonymous referees helped improve an earlier version of
the manuscript. Capture and handling permits granted by the
Ministero dell’Ambiente e della Tutela del Territorio (DPN-
2010-370, 12-01-297 2010). Field work was supported by fund-
ing from the Societas Europaea Herpetologica and the Society
for the Study of Amphibians and Reptiles. Manuscript prepa-
ration was supported by the University of Melbourne and the
ARC Centre of Excellence for Environmental Decisions.
REFERENCES
Adams, M.J., Galvan, S., Scalera, R., Grieco, C., Sindaco,
R. (2008): Batrachochytrium dendrobatidis in amphib-
ian populations in Italy. Herpetol. Rev. 39: 324-326.
Arillo, A., Braida, L., Canessa, S., Ferravante, C., Oneto,
F., Ottonello, D., Salvidio, S. (2011): Azioni di sal-
vaguardia di Bombina pachypus (Bonaparte, 1838) in
Liguria. Pianura 27: 171-173.
Arillo, A., Braida, L., Canessa, S., Oneto, F., Ottonello, D.,
Raineri, A. (2009): Problematiche di conservazione delle
popolazioni di Bombina pachypus (Bonaparte, 1838) in
Liguria. Boll. Mus. Ist. Biol. Univ. Genova 71: 89.
Berger, L., Speare, R., Daszak, P., Green, D.E., Cunning-
ham, A.A., Goggin, C.L., Slocombe, R., Ragan, M.A.,
Hyatt, A.D., McDonald, K.R., Hines, H.B., Lips, K.R.,
Marantelli, G., Parkes, H. (1998): Chytridiomycosis
causes amphibian mortality associated with population
declines in the rain forests of Australia and Central
America. Proc. Natl. Acad. Sci. U. S. A. 95: 9031-9036.
Boyle, D.G., Boyle, D.B., Olsen, V., Morgan, J.A.T., Hyatt,
A.D. (2004): Rapid quantitative detection of chytridio-
mycosis (Batrachochytrium dendrobatidis) in amphib-
ian samples using real-time Taqman PCR assay. Dis.
Aquat. Org. 60: 141-148.
Brown, L.D., Cai, T.T., DasGupta, A. (2001): Interval
estimation for a binomial proportion. Stat. Sc. 16:
101-117.
Canessa, S., Oneto, F., Ottonello, D., Arillo, A., Salvidio,
S. (2013): Land abandonment may reduce distur-
bance and aect the breeding sites of an endangered
amphibian in northern Italy. Oryx 47: 280-287.
Canestrelli, D., Zampiglia, M., Nascetti, G. (2013): Wide-
spread occurrence of Batrachochytrium dendrobatidis
in contemporary and historical samples of the Endan-
gered Bombina pachypus along the Italian peninsula.
PloS One: e63349.
Conradie, W., Weldon, C., Smith, K.G., Du Preez, L.H.
(2011): Seasonal pattern of chytridiomycosis in com-
mon river frog (Amietia angolensis) tadpoles in the
South African Grassland Biome. Afr. Zool. 46: 95-102.
Di Cerbo, A.R., Ferri, V. (2002): Guidelines for the
improvement of aquatic habitats of Bombina variegata
and Bombina pachypus. Riv. Idrobiol. 40: 73-84.
DiGiacomo, R.F., Koepsell, T.D. (1986): Sampling for
detection of infection or disease in animal popula-
tions. J. Am. Vet. Med. Assoc. 189: 22-23.
Fisher, M.C., Garner, T.W.J., Walker, S.F. (2009): Global
emergence of Batrachochytrium dendrobatidis and
Amphibian chytridiomycosis in space, time, and host.
Annu. Rev. Microbiol. 63: 291-310.
Garner, T.W.J., Walker, S., Bosch, J., Hyatt, A.D., Cun-
ningham, A.A., Fisher, M.C. (2005): Chytrid fungus
in Europe. Emerg. Infect. Dis. 11: 1639-1641.
Gentilli, A., Scali, S., Barbieri, F., Bernini, F. (2002): A
three-year project for the management and the con-
servation of amphibians in Northern Italy. Biota 3:
27-33.
63
No detection of chytrid in Ligurian B. variegata
Hartel, T. (2008): Movement activity in a Bombina varie-
gata population from a deciduous forested landscape.
North-Western J. Zool. 4: 79-90.
Hyatt, A.D., Boyle, D.G., Olsen, V., Boyle, D.B., Berger,
L., Obendorf, D., Dalton, A., Kriger, K.M., Hero, J.M.,
Hines, H.B. (2007): Diagnostic assays and sampling
protocols for the detection of Batrachochytrium dend-
robatidis. Dis. Aquat. Org. 73: 175-192.
Hyne, R.V., Spolyarich, N., Wilson, S.P., Patra, R.W.,
Byrne, M., Gordon, G., SánchezBayo, F., Palmer,
C.G. (2009): Distribution of frogs in rice bays within
an irrigated agricultural area: links to pesticide usage
and farm practices. Environ. Toxicol. Chem. 28:
1255-1265.
IUCN. (2011): Bombina pachypus. In: IUCN Red List of
reatened Species, version 2011.2. 2011.2. Retrieved
27 January 2012.
Johnson, M.L., Berger, L., Phillips, L., Speare, R. (2003):
Fungicidal effects of chemical disinfectants, UV
light, desiccation and heat on the amphibian chytrid,
Batrachochytrium dendrobatidis. Dis. Aquat. Org. 57:
255-260.
Kéry, M., Schaub, M. (2011): Bayesian population analy-
sis using WinBUGS: a hierarchical perspective. Aca-
demic Press, Burlington.
Kriger, K.M., Hero, J.M. (2007): Large-scale seasonal vari-
ation in the prevalence and severity of chytridiomyco-
sis. J. Zool. 271: 352-359.
Lachish, S., Gopalaswamy, A.M., Knowles, S.C.L., Shel-
don, B.C. (2012): Site-occupancy modelling as a novel
framework for assessing test sensitivity and estimating
wildlife disease prevalence from imperfect diagnostic
tests. Methods Ecol. Evol. 3: 339-348.
Mirabile, M., Melletti, M., Venchi, A., Bologna, M.A.
(2009): e reproduction of the Apennine yellow-
bellied toad (Bombina pachypus) in central Italy.
Amphibia-Reptilia 30: 303-312.
Phillott, A.D., Speare, R., Hines, H.B., Skerratt, L.F., Mey-
er, E., McDonald, K.R., Cashins, S.D., Mendez, D.,
Berger, L. (2010): Minimising exposure of amphibians
to pathogens during eld studies. Dis. Aquat. Org. 92:
175-185.
Plummer, M. (2005): JAGS: just another Gibbs sampler.
Proceedings of the 3rd International Workshop on
Distributed Statistical Computing (DSC 2003), Vien-
na, Austria.
Retallick, R.W., Miera, V., Richards, K.L., Field, K.J., Col-
lins, J.P. (2006): A non-lethal technique for detecting
the chytrid fungus Batrachochytrium dendrobatidis on
tadpoles. Dis. Aquat. Org. 72: 77.
Speare, R., Berger, L., Skerratt, L.F., Alford, R., Mendez,
D., Cashins, S., Kenyon, N., Hauselberger, K., Rowley,
J. (2004): Hygiene protocol for handling amphibians
in eld studies. Unpublished report by the Amphibian
Diseases Group, James Cook University, Townsville.
Spitzen-van der Sluijs, A., Zollinger, R., Bosman, W., Pas-
mans, F., Martel, A., Van Rooij, P., Clare, F. (2010):
Short report: Batrachochytrium dendrobatidis in
amphibians in the Netherlands and Flanders (Bel-
gium). Stichting RAVON. Nijmegen, the Netherlands.
Stagni, G., Dall’olio, R., Fusini, U., Mazzotti, S., Scoccian-
ti, C., Serra, A. (2004): Declining populations of apen-
nine yellow-bellied toad Bombina pachypus in the
northern Apennines (Italy): is Batrachochytrium dend-
robatidis the main cause? Ital. J. Zool. 71: 151-154.
Swei, A., Rowley, J.J.L., Rodder, D., Diesmos, M.L.L.,
Diesmos, A.C., Briggs, C.J., Brown, R., Trung Tien,
C., Cheng, T.L., Chong, R.A., Han, B., Hero, J.M., Huy
Duc, H., Kusrini, M.D., Duong i uy, L., McGuire,
J.A., Meegaskumbura, M., Min, M., Mulcahy, D.G.,
Neang, T., Phimmachak, S., Rao, D., Reeder, N.M.,
Schoville, S.D., Sivongxay, N., Srei, N., Min, M.S., Rao,
D.Q. (2011): Is chytridiomycosis an emerging infec-
tious disease in Asia? PLoS ONE: e23179.
Tessa, G., Angelini, C., Bielby, J., Bovero, S., Giacoma, C.,
Sotgiu, G., Garner, T.W.J. (2013): e pandemic path-
ogen of amphibians, Batrachochytrium dendrobatidis
(Phylum Chytridiomicota), in Italy. Ital. J. Zool. 80:
1-11.
Wagner, N., Pellet, J., Lotters, S., Schmidt, B.R., Schmitt,
T. (2011): The superpopulation approach for esti-
mating the population size of’prolonged’breeding
amphibians: Examples from Europe. Amphibia-Rep-
tilia 32: 323-332.
... This programme aimed to locally improve the conservation status of Apennine Yellow-bellied toad populations and at the same time to promote the traditional farming activities that, in the rural landscape of Liguria, play an important role in maintaining the diversity of amphibian populations breeding sites (Canessa et al., 2013b;Romano et al., 2013). The preliminary results concerning population size estimation and Bd analyses have been published by Canessa et al. (2013a). Therefore, in this study we will report only about the results concerning the Bd screening for 2013, the colonisation of the newly-created artificial or semi-natural habitats and the restocking activities. ...
... Chytridiomycosis detection. All the main populations known in Liguria (Canessa et al., 2013a) have been screened for the pathogen Batracochytrium dendrobatidis (Bd) by means of PCR assay. Yellow bellied toads were swabbed in the field at elast 30 times with cottone sterile swabs that were conserved at 4° C until DAN esxtraction. ...
... During the 2013 survey, 100 swabs of B. v. pachypus were analysed for Bd by qPCR analysis and all of them were negative, confirming the previous results obtained by Canessa et al. (2013a) that sampled the same populations in 2011 and 212 (Table 1). Table 2), where in the spring 2014 they began to breed successfully. ...
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The Apennine Yellow-bellied toad, Bombina variegata pachypus is endemic to the Italian peninsula. Land abandonment, chytrid infection and climate change are negatively impacting Yellow-bellied toad populations in many parts of its range. In Liguria, a northwestern administrative Italian region, a regional conservation programme has been implemented to contrast the decline. Breeding sites were restored and created, the size and the sanitary status of populations was monitored and restocking was carried out. In the period 2011-2014, nine artificial breeding sites were created, four semi-natural water bodies and a small breeding facility was also created from which tadpoles were introduced in a newly-created site. In Liguria, Apennine Yellow-bellied toad populations are relatively small, but appear stable and non-infected by Batracochytrium dendrobatidis. Many of the new or restored breeding sites were colonised by the Apennine Yellow-bellied toad or by other amphibians, and a new reproductive population of Bombina variegata pachypus became established after restocking. Overall, these results confirm that the creation of small artificial breeding sites is a very successful strategy for conserving amphibian populations. This strategy is also useful for maintaining traditional rural activities in the Mediterranean landscape.
... A screening program for known populations in the region was carried out in 2011 and 2012 to clarify the extent and magnitude of possible infections: during this program, a total of 86 and 143 individuals were caught and swabbed at eight populations in the region in 2011 and 2012, respectively (full description of sampling and diagnostic methods in Canessa et al. 2013a). In 2011, swabs were collected during a single visit to each site in late July. ...
... As the first step of our analysis, we sought to obtain estimates of prevalence from the 2011 data described in Canessa et al. (2013a). We implemented the post hoc binomial model (Eq. ...
Article
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The amphibian chytrid fungus, Batrachochytrium dendrobatidis, is one of the main factors in global amphibian decline. Accurate knowledge of its presence and prevalence in an area is needed to trigger conservation actions. However, imperfect capture rates determine the number of individuals caught and tested during field surveys, and contribute to the uncertainty surrounding estimates of prevalence. Screening programs should be planned with the objective of minimizing such uncertainty. We show how this can be achieved by using predictive models that incorporate information about population size and capture rates. Using as a case study an existing screening program for three populations of the yellow-bellied toad (Bombina variegata pachypus) in northern Italy, we sought to quantify the effect of seasonal variation in individual capture rates on the uncertainty surrounding estimates of chytrid prevalence. We obtained estimates of population size and capture rates from mark–recapture data, and found wide seasonal variation in the individual recapture rates. We then incorporated this information in a binomial model to predict the estimates of prevalence that would be obtained by sampling at different times in the season, assuming no infected individuals were found. Sampling during the period of maximum capture probability was predicted to decrease upper 95% credible intervals by a maximum of 36%, compared with least suitable periods, with greater gains when using uninformative priors. We evaluated model predictions by comparing them with the results of screening surveys in 2012. The observed results closely matched the predicted figures for all populations, suggesting that this method can be reliably used to maximize the sampling size of surveillance programs, thus improving their efficiency.
... Besides, in some areas along the Apennine mountain range the observed decline of the Apennine yellow-bellied toad, Bombina pachypus, was attributed to Bd infection (Stagni et al. 2004;Canestrelli et al. 2013). However, declines are also observed in other areas where Bd has been screened for, but is apparently absent (Canessa et al. 2013a(Canessa et al. , 2019. In these latter areas, the Apennine yellow-bellied toads' declines were attributed to major habitat changes rather than pathogens (Canessa et al. 2013b). ...
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The emerging amphibian disease, Batrachochytrium dendrobatidis (Bd), is driving population declines worldwide and even species extinctions in Australia, South and Central America. In order to mitigate effects of Bd on amphibian populations, high-exposed areas should be identified at the local scale and effective conservation measures should be planned at the national level. This assessment is actually lacking in the Mediterranean basin, and in particular in Italy, one of the most relevant amphibian diversity hotspots in the entire region. In this study, we reviewed the available information on Bd in Italy, and conducted a 5-year molecular screening on 1274 individual skin swabs belonging to 18 species. Overall, we found presence of Bd in 13 species and in a total of 56 known occurrence locations for peninsular Italy and Sardinia. We used these occurrence locations and climate data to model habitat suitability of Bd for current and future climatic scenarios. We then employed electric circuit theory to model landscape permeability to the diffusion of Bd, using a resistance map. With this procedure, we were able to model, for the first time, the diffusion pathways of Bd at the landscape scale, characterising the main future pathways towards areas with a high probability of Bd occurrence. Thus, we identified six national protected areas that will become pivotal for a nationally-based strategic plan in order to monitor, mitigate and possibly contrast Bd diffusion in Italy.
... where C is the probability of detecting at least one Bd positive individual from a representative sample (n) and the plausible prevalence of the disease (p) expressed as a fraction ranging from 0 to 1 (e.g., 0.05, 0.10, 0.30; Canessa et al. 2013). ...
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Since 2004, over 750 students have participated in Team Salamander, the longest-running group-studies experience at the University of Tennessee at Chattanooga (UTC). Like most curricula, Team Salamander is based on predetermined benchmarks that have to be met. However, rather than following the traditional syllabus-based lecture/laboratory model, the program is a hands-on cooperative experience that encourages students to become independent and forward-thinking. Ultimately, we strive for participants to become natural-science practitioners and informed citizens, and several program participants have continued on to graduate or professional school in natural-science disciplines. The Team Salamander program has contributed to UTC by teaching students to think creatively and connecting them with community partners. Specifically, Team Salamander has constructed a framework that provides students with job skills through hosting workshops on wetland conservation, participating in regional bioblitzes, and training visiting scientists. Team Salamander equips and empowers its members to facilitate change by encouraging them to become practicing scientists and community leaders that positively impact others.
... The Iberian region became the centre of European chytridiomycosis research due to the declining populations of three species in the genus Alytes (Bosch et al. 2001, Bosch & Rincón 2008, Rosa et al. 2013. The Apennine Peninsula has been the subject of several local Bd mapping projects (e.g., Simoncelli et al. 2005, Canessa et al. 2013, Tessa et al. 2013) that also included northern Italy (Stagni et al. 2004) and the island of Sardinia (Bielby et al. 2009). In the Balkans region, on the other hand, only few samples have been analysed and published to date, of which none (Garner et al. 2005, Vörös & Jelić 2011 or only small numbers proved Bd-positive (Vörös et al. 2013). ...
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The Mediterranean region, consisting of, amongst other areas, three main southern European peninsulas (the Iberian, Apennine, and Balkan) is known as one of the world's biodiversity hotspots. The Iberian Peninsula was the first place in Europe where amphibians were confirmed to be infected by the amphibian chytrid fungus, Batrachochytrium dendro batidis (Bd). Whereas the Iberian and Apennine peninsulas have been subject to intensive chytridiomycosis research , this study represents the first large-scale Bd survey in the Balkans. Over a two-year period, 454 skin swab samples of amphibians were collected in Montenegro, Albania, and the Republic of Macedonia of which 65 samples (14.3%) were Bd-positive. Bd was detected in 5 out of 11 sampled species (Bombina variegata, Hyla arborea, Lissotriton vulgaris, Pelo­ phylax sp., and Triturus macedonicus) at 13 out of 38 localities. Infection rates did not differ between countries but varied greatly between species with a maximum in Pelophylax sp. (> 30%). Within positive Pelophylax samples, infection loads were constant across areas and age groups. Considering the Balkans' biodiversity and the potential threat to local endem-ics and genetic richness, future monitoring is vital for assessing Bd presence, prevalence and infection trends in the region.
... Many Swiss populations of amphibians are missing from many historical localities, although causes are complex and none have definitively been attributed to disease. The situation is similar to that in Italy, where Bd has been present for several decades and populations of some species are declining, but no causative link has been established [79,80]. Spitzen-Van Der Sluijs et al. [81] showed that in the Netherlands Bd was present in many species but at low prevalence and low intensity. ...
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Chytridiomycosis is an emerging infectious disease of amphibians that affects over 700 species on all continents where amphibians occur. The amphibian–chytridiomycosis system is complex, and the response of any amphibian species to chytrid depends on many aspects of the ecology and evolutionary history of the amphibian, the genotype and phenotype of the fungus, and how the biological and physical environment can mediate that interaction. Impacts of chytridiomycosis on amphibians are varied; some species have been driven extinct, populations of others have declined severely, whereas still others have not obviously declined. Understanding patterns and mechanisms of amphibian responses to chytrids is critical for conservation and management. Robust estimates of population numbers are needed to identify species at risk, prioritize taxa for conservation actions, design management strategies for managing populations and species, and to develop effective measures to reduce impacts of chytrids on amphibians. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.
... where C is the probability of detecting at least one Bd positive individual from a representative sample (n) and the plausible prevalence of the disease (p) expressed as a fraction ranging from 0 to 1 (e.g., 0.05, 0.10, 0.30; Canessa et al. 2013). ...
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Although human-related disturbance is usually detrimental for biodiversity, in some instances it can simulate natural processes and benefit certain species. Changes in the disturbance regime, both natural and human-driven, can affect species that rely on it. The Apennine yellow-bellied toad Bombina variegata pachypus, an amphibian endemic to peninsular Italy, has declined throughout its range in the last 3 decades. We sought to identify the drivers of the decline in the region of Liguria, at the north-western limit of its distribution. In 2009 and 2010 we surveyed sites where the species occurred until 2005 and related the persistence of breeding activity to the characteristics of sites. Populations had disappeared from 50% of the sites between 2005 and 2009. Current breeding sites have less aquatic and bank vegetation, fewer predators and better insolation. Frequent disturbance events (desiccation and floods) were related to reduced vegetation, which in turn may decrease predator densities and increase insolation. In this region disturbance is provided by natural factors or, in the case of artificial water bodies, by regular maintenance carried out by landowners. The widespread land abandonment in Liguria can disrupt disturbance regimes, interrupting the removal of vegetation, and thus rapidly reduce the suitability of artificial sites. This was confirmed in our study, with most abandoned breeding sites occurring in formerly cultivated areas. Possible short-term conservation actions include creating new ponds, maintaining artificial water bodies and clearing vegetation. However, long-term conservation may be more problematic as the land abandonment process is unlikely to be reversed.
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Batrachochytrium dendrobatidis is considered a main driver of the worldwide declines and extinctions of amphibian populations. Nonetheless, fundamental questions about its epidemiology, including whether it acts mainly as a "lone killer" or in conjunction with other factors, remain largely open. In this paper we analysed contemporary and historical samples of the endangered Apennine yellow-bellied toad (Bombina pachypus) along the Italian peninsula, in order to assess the presence of the pathogen and its spreading dynamics. Once common throughout its range, B. pachypus started to decline after the mid-1990s in the northern and central regions, whereas no declines have been observed so far in the southern region. We show that Batrachochytrium dendrobatidis is currently widespread along the entire peninsula, and that this was already so at least as early as the late 1970s, that is, well before the beginning of the observed declines. This temporal mismatch between pathogen occurrence and host decline, as well as the spatial pattern of the declines, suggests that the pathogen has not acted as a "lone killer", but in conjunction with other factors. Among the potentially interacting factors, we identified two as the most probable, genetic diversity of host populations and recent climate changes. We discuss the plausibility of this scenario and its implications on the conservation of B. pachypus populations.
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Bayesian statistics has exploded into biology and its sub-disciplines, such as ecology, over the past decade. The free software program WinBUGS and its open-source sister OpenBugs is currently the only flexible and general-purpose program available with which the average ecologist can conduct standard and non-standard Bayesian statistics. Bayesian Population Analysis Using WinBUGS goes right to the heart of the matter by providing ecologists with a comprehensive, yet concise, guide to applying WinBUGS to the types of models that they use most often: linear (LM), generalized linear (GLM), linear mixed (LMM) and generalized linear mixed models (GLMM). Comprehensive and richly-commented examples illustrate a wide range of models that are most relevant to the research of a modern population ecologist. All WinBUGS/OpenBUGS analyses are completely integrated in software R. Includes complete documentation of all R and WinBUGS code required to conduct analyses and shows all the necessary steps from having the data in a text file out of Excel to interpreting and processing the output from WinBUGS in R.
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Worldwide amphibian declines and species losses are global problems and emerging infectious diseases have been identified as one of the major threats. The worst of these is chytridiomycosis, an amphibian disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Here we review what is known of the distribution of Bd and chytridiomycosis in Italy. We critically summarize the evidence in support of the hypothesis that Bd is an invasive pathogen in Italy. Last we provide recommendations for immediate research needs, both for basic science and applied conservation.