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Herpetological Review 46(1), 2015
AMPHIBIAN DISEASES 41
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© 2015 by Society for the Study of Amphibians and Reptiles
Chytrid Fungus (Batrachochytrium dendrobatidis)
Undetected in the Two Orders of Seychelles Amphibians
Infection by the fungal pathogen Batrachochytrium
dendrobatidis (Bd) is a major driver in global amphibian
declines (Berger et al. 1998; Skerratt et al. 2007) and can occur
in all three orders of Amphibia, having been most recently
documented in the Gymnophiona (Doherty-Bone et al. 2013;
Gower et al. 2013). Bd has been detected in 71 countries and
695 amphibian species (Olson and Ronnenberg 2014) but to our
knowledge no screening for its presence or anecdotal evidence
of chytridiomycosis has been reported from the Seychelles
Archipelago, a biodiversity hotspot (Myers et al. 2000) with
a very high proportion (86%) of endemic amphibian genera
(Poynton 1999) (Fig. 1).
Eleven of the 12 species of Seychelles amphibians (Table
1) are globally significant. The four species of sooglossid
frog are unique in being the only amphibian family endemic
to an island group, and the three genera of caecilians (all
endemic, comprising a radiation of six species) are the
only gymnophionan genera confined to islands. The single
hyperoliid frog species occurring here is endemic to the
Seychelles, leaving the single ptychadenid frog as the only
non-endemic amphibian (Nussbaum 1984). Being primarily
fossorial, caecilians are considered difficult study organisms
JIM LABISKO*
Durrell Institute of Conservation and Ecology,
School of Anthropology and Conservation,
University of Kent, Canterbury CT2 7NR, UK
SIMON T. MADDOCK
Department of Life Sciences, The Natural History Museum,
Cromwell Road, London SW7 5BD, UK; Department of Genetics,
Evolution and Environment, University College London,
Gower Street, London WC1E 6BT, UK
MICHELLE L. TAYLOR
Durrell Institute of Conservation and Ecology,
School of Anthropology and Conservation,
University of Kent, Canterbury CT2 7NR, UK
LINDSAY CHONG-SENG
Plant Conservation Action group,
P O Box 392, Victoria, Mahé, Seychelles
DAVID J. GOWER
Department of Life Sciences, The Natural History Museum,
Cromwell Road, London SW7 5BD, UK
FELICITY J. WYNNE
Institute of Zoology, Zoological Society of London,
Regents Park, London NW1 4RY, UK
EMMA WOMBWELL
Durrell Institute of Conservation and Ecology,
School of Anthropology and Conservation, University of Kent,
Canterbury CT2 7NR, UK; Institute of Zoology,
Zoological Society of London, Regents Park, London NW1 4RY, UK
CHARLES MOREL
Natural History Museum, Victoria, Mahé, Seychelles
GEORGIA C. A. FRENCH
Amphibian and Reptile Conservation Trust,
655a Christchurch Road, Boscombe, BH1 4AP, UK
NANCY BUNBURY
Seychelles Islands Foundation, PO BOX 853, Victoria, Mahé, Seychelles
KAY S. BRADFIELD
Perth Zoo, South Perth, WA 6151, Australia
*Corresponding author; e-mail: jl412@kent.ac.uk
Fig. 1. Location map showing the position of the Seychelles Archi-
pelago in relation to the African coast and Madagascar. Zoomed
panel shows the main inner islands. Map generated using ArcMap
10.1 (ESRI 2012).
Herpetological Review 46(1), 2015
42 AMPHIBIAN DISEASES
and collection usually requires dedicated digging (Gower and
Wilkinson 2005), such that there have been few caecilian Bd
field surveys (Gower et al. 2013). Similarly, sooglossid frogs
are cryptic in their habits, undergoing direct development as
part of a completely terrestrial life cycle in often inaccessible,
elevated areas of moist forest (Nussbaum 1984; Nussbaum and
Wu 2007). In contrast, the Seychelles Treefrog (Tachycnemis
seychellensis: see Maddock et al. 2014), and the likely introduced
Mascarene Ridged Frog (Ptychadena mascareniensis: see
Vences et al. 2004) can be easier to encounter, forming breeding
aggregations around marshes, streams, and temporary pools,
especially in the evening (T. seychellensis) and/or after rain
events (P. mascareniensis) (Nussbaum 1984). Bd is known to
infect P. mascareniensis in mainland Africa (www.bd-maps.net/
surveillance/s_species.asp; accessed 3 March 2014; Goldberg
2007), while Olson et al. (2013) identified a 50% prevalence of
Bd in African Hyperoliidae, the family to which T. seychellensis
belongs.
Between May 2010 and March 2013, skin swabs were taken
from wild-caught caecilians and metamorphosed anurans at
multiple locations across six of the Seychelles’ inner islands
(Fig. 2). All sampled specimens were swabbed alive and within
24 hours of capture, using rayon-tipped MW100 fine-tip swabs
(Medical Wire and Equipment, Corsham, Wiltshire, England).
Swabbing protocol generally followed best-practice methods
available at the time (e.g., Smith 2011). However, our sampling
was undertaken by three separate research groups operating
over the period, with differing primary research aims. Therefore,
some deviation from standard biosecurity protocol did occur,
including capture and initial handling of amphibians without
gloves, and (although infrequent) housing more than one animal
(but always of the same species) in the same plastic bag upon
capture. The principle aim was to gather a representative sample
from sites within broadly separate locations, and not to swab
every individual encountered or captured. A large proportion
of sooglossid frog swabbing was undertaken in the field by a
single person (JL). These mostly very small anurans (except for
individuals of Sooglossus thomasseti measuring greater than
25 mm snout–vent length) were cleaned of debris and soil by
transferring them to a small plastic zip-lock bag and rinsing with
fresh water sourced at the sampling locality. Although this rinsing
may have removed some zoospores, it was performed to reduce
the increased likelihood of PCR inhibition. The water was then
drained and the animal gently restrained and swabbed within the
tABle 1. Distribution of Seychelles amphibians (adapted from Nussbaum 1984), including the 2009 discovery of Sooglossidae on Praslin ( Tay-
lor et al. 2012) and their IUCN Red List status (IUCN 2013): LC – Least Concern; EN – Endangered; CR – Critically Endangered. Caecilian
taxonomy follows Wilkinson et al. (2011).
Order Family Species Islands present Conservation status
Anura Hyperolidae Tachycnemis seychellensis La Digue, Mahé, Praslin, Silhouette LC
Anura Ptychadenidae Ptychadena mascareniensis Cerf, Curieuse, Frégate, Grand Soeur, LC
La Digue, Mahé, Northa, Praslin, Silhouette
Anura Sooglossidae Sooglossus sechellensis Mahé, Praslin, Silhouette EN
Anura Sooglossidae Sooglossus thomasseti Mahé, Silhouette CR
Anura Sooglossidae Sechellophryne gardineri Mahé, Silhouette EN
Anura Sooglossidae Sechellophryne pipilodryas Silhouette CR
Gymnophiona Indotyphlidae Grandisonia alternans Félicité, Frégate, La Digue, Mahé, LC
St. Anne, Silhouette
Gymnophiona Indotyphlidae Grandisonia larvata Félicité, La Digue, Mahé, Praslin, LC
St. Anne, Silhouette
Gymnophiona Indotyphlidae Grandisonia sechellensis Mahé, Praslin, Silhouette LC
Gymnophiona Indotyphlidae Hypogeophis brevis Mahé EN
Gymnophiona Indotyphlidae Hypogeophis rostratus Cerf, Curieuse, Félicité, Frégate, Grand Soeur, LC
La Digue, Mahé, Praslin, St. Anne, Silhouette
Gymnophiona Indotyphlidae Praslinia cooperi Mahé, Praslin EN
a New locality record
Herpetological Review 46(1), 2015
AMPHIBIAN DISEASES 43
bag, removing the need for direct handling and minimizing risk
of cross-contamination. Larger anurans, and sooglossid frogs
processed when field assistance was available, were sampled
using the standard technique (Smith 2011). Caecilians were
swabbed dorsally, laterally, and ventrally along the length of the
body, and also around the vent and head. Gloves were changed
between each individual except where two or more specimens
had been housed in the same plastic bag. All amphibians not
retained as vouchers as part of broader research aims were
released at their place of capture. Swabs were kept in the dark
and transferred to cool storage, mostly refrigerated within 12 h
and frozen within one week of completed fieldwork.
DNA extractions and quantitative real time Taqman®
polymerase chain reaction (qPCR) assays were performed at the
Institute of Zoology (London, UK), using methods adapted from
Boyle et al. (2004). DNA was extracted from swabs using bead
beating with 0.5 mm silica beads and 60 µl PrepMan Ultra (Hyatt
et al. 2007). The qPCR amplifications were performed in 25 µl
reactions using Bd primers ITS-1 (forward): 5’-CCT TGA TAT AAT
ACA GTG TGC CAT ATG TC-3’ and 5.8S (reverse): 5’-AGC CAA
GAG ATC CGT TGT CAA A-3’, specific to the ITS-1/5.8S region of
rDNA (Boyle et al. 2004). Standards of 100, 10, 1 and 0.1 Bd DNA
genomic equivalents and negative controls were used in each
run. Bovine serum albumin (BSA) was included in the TaqMan®
master mix, to reduce inhibition of the PCR (Garland et al. 2010).
Each sample was run in duplicate and no amplification in either
replicate indicated a negative result.
A total of 291 skin swabs were obtained from Seychelles
amphibians, representing 10 of the 12 species known to occur
across the archipelago. All 213 anurans and 78 caecilians tested
negative for Bd (comprising 66 Sooglossus sechellensis [Fig. 3];
13 S. thomasseti, 14 Sechellophryne gardineri, 99 Tachycnemis
seychellensis, 21 Ptychadena mascareniensis, 18 Grandisonia
alternans [Fig. 4], 7 G. larvata, 10 G. sechellensis, 6 Hypogeophis
brevis, 23 H. rostratus, 14 unidentified caecilians) (Table 2). No
macroscopic presentation of chytridiomycosis-like symptoms
or associated mortality was observed in any sampled individual,
or in any amphibian encountered during the fieldwork. Our
results suggest a widespread absence of Bd between 2010 and
2013 across six of the eleven amphibian-inhabited islands of
the Seychelles archipelago. Some caution should be exercised in
interpreting our results as indicating that the Seychelles are free
of Bd. Many of the locations sampled were in close proximity to
one another and/or linked by contiguous habitat, and so could
be described as a single location for the purposes of achieving
the recommended minimum sample size of 30 amphibians per
site (Smith 2011), but the temporal differences between site
surveys and overall limited sampling invariably resulted in this
not being achieved (Table 2), leading to the possibility of type II
errors. Also, the recommendation for sampling >59 individuals
to detect Bd when infection rate is low (Skerratt et al. 2008)
was achieved for only two species sampled (T. seychellensis, S.
sechellensis), two islands sampled (Mahé and Praslin), and for the
two pooled samples of all anurans and all caecilians. Prevalence
of Bd infection in anurans indicates seasonal peaks in the cooler
months (Berger et al. 2004; Retallick et al. 2004; Kriger and Hero
2007) even with little seasonal temperature variation (Whitfield at
al. 2012). Sampling of Seychelles amphibians was not undertaken
during the two (historically) coolest months of July and August.
However, housing more than one animal in the same capture bag,
although limited in occurrence, increased the opportunity for
cross-contamination from chytrid zoospores, making detection of
Bd more likely by fostering false-positive results for infection.
Fig. 2. Sampling localities for Batrachochytrium dendrobatidis across
the Seychelles inner islands. Map generated using ArcMap 10.1 (ESRI
2012).
Fig. 3. Sooglossus sechellensis from Mahé, Seychelles Archipelago.
IMAGE BY JIM LABISKO
Fig. 4. Grandisonia alternans from Silhouette, Seychelles Archipelago.
IMAGE BY DAVID GO WER
Herpetological Review 46(1), 2015
44 AMPHIBIAN DISEASES
Direct development—a reproductive mode adopted by
the Sooglossidae and at least one Seychelles caecilian (H.
rostratus; Nussbaum 1984)—may provide a limiting factor to
the transmission of Bd between and among amphibians (Todd
2007; but see Longo and Burrowes 2010). The probably more
vagile T. seychellensis and P. mascareniensis require water bodies
for aquatic larval development, and potentially present more
suitable hosts for the dispersal and spread of Bd. Due to previous
evidence of human-mediated trans-oceanic dispersal (Vences
et al. 2004), P. mascareniensis in particular may lend itself to
continuing introduction and transportation by way of tourist and/
or domestic traffic, especially among the main islands of Mahé,
Praslin, and La Digue (Fig. 1). This species’ propensity for dispersal
was evidenced first-hand during fieldwork following the discovery
of a novel, reproducing population on North Island. Similarly, and
despite no currently recorded infection or capacity as a host for
Bd (www.bd-maps.net/surveillance/s_species.asp; accessed 21
September 2014), the recent discovery of Asian Common Toads
(Duttaphrynus melanostictus) on the east coast of Madagascar,
having likely arrived in shipping containers from Asia (Kolby
2014a), highlights a further risk to Seychelles endemic fauna as
a potential disease vector. The significantly shorter shipping
distances among Madagascar, the Mascarenes, and Seychelles
islands undoubtedly elevates the risk of further human-mediated
dispersal of this potentially invasive species.
Links between climate, temperature, and Bd have been
documented (see Pounds et al. 2006; Bosch et al. 2007; Lips et al.
2008; Rohr and Raffel 2010; Olson et al. 2013). In tropical regions,
elevated, moist, and riparian habitats are home to amphibian
species considered most likely to be severely threatened by Bd
(Wake and Vredenburg 2008). The endemic Seychelles amphibians
fall into at least two, and often all three of these categories which,
combined with their restricted range, highlights the requirement
for targeted conservation measures (Sodhi et al. 2008). In light of
the recent discovery of Bd in Malagasy anurans (Kolby 2014b),
and given the potential susceptibility of Seychelles amphibians,
continued disease monitoring warrants consideration as part of
ongoing conservation work for this globally significant amphibian
community. Effective implementation of Seychelles’ recently
approved Biosecurity Act (Animal and Plant Biosecurity Act, 2014)
is consistent with maintaining such vigilance.
Acknowledgments.─—This research was supported by the Durrell
Institute of Conservation and Ecology; The Natural History Museum,
London; University College London; Seychelles Islands Foundation;
Institute of Zoology; Seychelles National Parks Authority; The System-
atics Association; BBSRC’s SynTax scheme; and the Darwin Initiative
(grant 19-002). We thank T. Garner for facilitating laboratory work at
the Institute of Zoology, London; Seychelles Bureau of Standards for
permission to carry out fieldwork; Seychelles Department of Environ-
ment for permission to collect and export samples; Islands Develop-
ment Company for permissions and hosting on Silhouette; Island
Conservation Society for field assistance on Silhouette; M. La Bus-
chagne for access to Coco de Mer Hotel land on Praslin; North Island
Seychelles for permissions and hosting on North Island; R. Bristol, R.
Griffiths, and J. Groombridge for organisational and field assistance; K.
Beaver, D. Birch, P. Haupt, M. Jean-Baptiste, C. Kaiser-Bunbur y, J. Mou-
gal, M. Pierre, N. Pierre, D. Quatre, A. Reuleaux, H. Richards, A. Rob-
erts, and many other NGO staff, researchers, and Seychellois for their
in- and ex-situ support. We also thank D. Olson and an anonymous
reviewer for helpful suggestions on a previous draft of this manuscript.
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