Download First Record of Batrachochytrium dendrobatidis Infecting Four Frog

EcoHealth
DOI: 10.1007/s10393-011-0685-y
Ó 2011 International Association for Ecology and Health
Short Communication
First Record of Batrachochytrium dendrobatidis Infecting Four
Frog Families from Peninsular Malaysia
Anna E. Savage,1 L. Lee Grismer,2 Shahrul Anuar,3 Chan Kin Onn,2,4 Jesse L. Grismer,5 Evan Quah,3
Mohd Abdul Muin,6 Norhayati Ahmad,7 Melissa Lenker,1 and Kelly R. Zamudio1
1
Department of Ecology and Evolutionary Biology, Cornell University, E145 Corson Hall, Ithaca, NY 14853
Department of Biology, La Sierra University, 4500 Riverwalk Parkway, Riverside, CA 92515-8247
3
School of Biological Sciences, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia
4
Institute for Environment and Development (LESTARI), Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor Darul Ehsan, Malaysia
5
Department of Ecology and Evolutionary Biology and Natural History Museum & Biodiversity Research Center, University of Kansas, Dyche Hall, 1345
Jayhawk Blvd., Lawrence, KS 66045-7561
6
Centre for Drug Research, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia
7
School of Environment and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor
Darul Ehsan, Malaysia
2
Abstract: The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects amphibians on every continent
where they occur and is linked to the decline of over 200 amphibian species worldwide. At present, only three
published Bd surveys exist for mainland Asia, and Bd has been detected in South Korea alone. In this article, we
report the first survey for Bd in Peninsular Malaysia. We swabbed 127 individuals from the six amphibian
families that occur on Peninsular Malaysia, including two orders, 27 genera, and 47 species. We detected Bd on
10 out of 127 individuals from four of five states and five of 11 localities, placing the 95% confidence interval
for overall prevalence at 4–14%. We detected no variation in Bd prevalence among regions, elevations, or
taxonomic groups. The infection intensity ranged from 1 to 157,000 genome equivalents. The presence of Bd
infections in native species without clinical signs of disease suggests that Bd may be endemic to the region.
Alternately, Bd may have been introduced from non-native amphibians because of the substantial amphibian
food trade in Peninsular Malaysia. Under both scenarios, management efforts should be implemented to limit
the spread of non-native Bd and protect the tremendous amphibian diversity in Peninsular Malaysia.
Keywords: chytridiomycosis, Batrachochytrium dendrobatidis, amphibian pathogen, Peninsular Malaysia
The fungus Batrachochytrium dendrobatidis (Bd) causes the
amphibian disease chytridiomycosis and has been linked to
the decline or disappearance of over 200 amphibian species
worldwide (Skerratt et al., 2007). Initially, Bd infections
were investigated, and thus documented exclusively in the
Correspondence to: Anna E. Savage, e-mail: [email protected]
amphibian populations experiencing conspicuous declines
(Berger et al., 1998). However, the rapid expansion of Bd
surveying in recent years led to the discovery that Bd infects
numerous amphibian species without causing fatal chytridiomycosis. Sub-lethal Bd infections have now been
documented on every continent where the amphibians
occur, including the United States (Green and Dodd,
2007; Longcore et al., 2007; Pearl et al., 2007), Mexico
Anna E. Savage et al.
(Frias-Alvarez et al., 2008), Brazil (Carnaval et al., 2006),
Uganda (Goldberg et al., 2007), Australia (Van Sluys and
Hero, 2010), and Asia (Kusrini et al., 2008; Yang et al.,
2009; Goka et al., 2009).
The discovery of sub-lethal Bd infections in numerous
species worldwide begs the question of whether Bd is an
emerging infectious disease because it was recently spread
across the globe (NPH, the ‘‘novel pathogen hypothesis’’;
Rachowicz et al., 2005), or if Bd is a globally distributed
fungus that has become pathogenic because of the environmental alterations such as the climate change (EPH, the
‘‘endemic pathogen hypothesis’’; Pounds et al., 2006).
Support for either hypothesis would be greatly enhanced by
a critical data set, comprehensive Bd surveys from every
global region in which the amphibians occur, unbiased
toward locations of conspicuous chytridiomycosis. At
present, there are only six published surveys of Bd in Asia,
which were conducted in South Korea (Yang et al., 2009),
Japan (Une et al., 2008; Goka et al., 2009), Indonesia
(Kusrini et al., 2008), Hong Kong (Rowley et al., 2007), and
Thailand (McLeod et al., 2008). In contrast, over 750 species have been surveyed for Bd in Europe, Africa, Australia,
and the Americas (Fisher et al., 2009). This sampling bias
understandably arose from heightened study of regions
with documented chytridiomycosis, but our current
inability to resolve the origins of Bd demands more
amphibian surveys in the regions where Bd has not yet been
detected. Indeed, to distinguish between the NPH and the
EPH in explaining global chytridiomycosis, the ability to
exclude Bd from a region of the world is equally important
in discovering a novel Bd infection.
To this end, we present the first survey of Bd in Peninsular Malaysia, a region of high amphibian biodiversity
with no published records of Bd presence or absence. In
other regions of the world, Bd infection and concomitant
amphibian declines can vary by species (Rovito et al.,
2009), latitude (Kriger et al., 2007), and elevation (Laurance et al., 1996). Survey locations were, therefore, selected
based on the following criteria: (1) to maximize the number of amphibian species encountered, (2) to encompass a
latitudinal range, and (3) to cover the elevational range that
occurs in Peninsular Malaysia.
From March 14 to March 24, 2010, we conducted the
amphibian surveys in five states of Peninsular Malaysia;
Kedah, Perak, and Perlis, three of the four states that border
Thailand; Pahang, the largest state in Peninsular Malaysia;
and Penang, a state that includes Penang Island and
the adjacent continental region of Seberang Perai. Mean
temperatures during this month ranged from 23.0°C to
36.0°C (mean: 27.1°C) in lowland areas and 13.5°C to
25.7°C (mean: 18.5°C) in upland areas (Malaysian Meterological Department 2010). Amphibians were captured by
hand and stored individually in sterile plastic bags until they
were swabbed (within 3-h of capture). All swabbed individuals were euthanized, preserved, and deposited in the La
Sierra University Herpetological Collection (LSUHC), La
Sierra University, Riverside, California, USA (LSUHC
numbers 9578-89, 9596, 9606-12, 9636-61, 9674-81, 9691-2
9696-9, 9700-1, 9705-20, 9729-36, and 9740-71), with the
exception of a newly discovered rhacophorid of the genus
Chiromantis (Chan et al., unpublished), of which the
holotype has been deposited at the Zoological Reference
Collection at the Raffles Museum of Biodiversity Research,
National University of Singapore (ZRC).
Using the standardized sampling protocol detailed in
Hyatt et al. (2007), we swabbed the epidermis of all the
encountered amphibians with sterile fine-tip swabs (Medical Wire & Equipment Co. MW113). We swabbed each
frog while wearing a fresh pair of disposable gloves to
prevent cross-contamination among individuals. Swabs
were stored individually in 100% ethanol, kept cool and
away from sunlight starting immediately after collection,
and processed 1–3 weeks after collection. DNA was
extracted from each swab using 50 ll of PrepMan Ultra
(Applied Biosystems) according to the modified manufacturer’s protocol of Hyatt et al. (2007). The infection
intensity of all the samples was determined using quantitative real-time PCR (Boyle et al., 2004; Hyatt et al., 2007).
Infection intensity was measured as the number of genome
equivalents (GE) per swab, calculated by multiplying the
genome equivalent values generated during the real-time
PCR by the dilution factor of the template DNA. Swabs
were categorized as Bd-positive at 1 GE and as Bd-negative at <1 GE. We calculated 95% Clopper–Pearson
binomial confidence intervals for Bd prevalence from the
observed proportions of infection among our samples.
We swabbed 127 individuals from the six amphibian
families that occur on Peninsular Malaysia, including two
orders (Anura and Gymnophiona), 27 genera, and 47
species (Table 1) accounting for nearly 44% of the known
amphibian fauna. To our knowledge, this is the first
published record of a Bd survey of the amphibians from
Peninsular Malaysia, as well as the first survey that includes any caecilian species. Ten out of 127 individuals
from four of five states and five of 11 localities were Bd
infected (Fig. 1), corresponding to a 95% confidence
Bd in Peninsular Malaysia
Table 1. Native amphibians sampled for Batrachochytrium dendrobatidis in Peninsular Malaysia
Species
Gymnophiona: Ichthyophiidae
Ichthyophis sp.
Caudacaecilia larutensis
Anura: Bufonidae
Ansonia jeetsukumarani
Ansonia malayana
Ansonia penangensis
Duttaphrynus melanostictus
Ingerophrynus parvus
Pedostibes hosii
Phrynoidis aspera
Anura: Megophryidae
Leptobrachium hendricksoni
Leptolalax heteropus
Xenophrys aceras
Xenophrys longipes
Anura: Microhylidae
Kaloula pulchra
Metaphrynella pollicaris
Microhyla annectens
Microhyla butleri
Microhyla heymonsi
Microhyla mantheyi
Micryletta inornata
Anura: Ranidae
Amolops larutensis
Fejervarya cancrivora
Fejervarya limnocharis
Hylarana banjarana
Hylarana erythraea
Hylarana glandulosa
Hylarana labialis
State
Site
Elevation
Bd positive / sampled
Penang
Perak
Penang Botanical Gardens
Bukit Larut
85 m
1161 m
0/1
0/1
Pahang
Perak
Penang
Penang
Penang
Penang
Kedah
Kedah
Kedah
Pahang
Penang
Perak
Bukit Fraser
Bukit Larut
Teluk Bahang Rec.
Pulau Jerejak
Teluk Bahang Rec.
Teluk Bahang Rec.
Sungai Sedim
Sungai Sedim
Sungai Tupah
Bukit Fraser
Teluk Bahang Rec.
Bukit Larut
1291 m
1161 m
67 m
21 m
67 m
67 m
126 m
126 m
88 m
1291 m
67 m
1161 m
0/2
0/2
0/1
0/1
0/1
0/4
0/2
0/1
0/1
0/1
0/2
0/1
Kedah
Perak
Pahang
Pahang
Pahang
Perak
Sungai Sedim
Bukit Larut
Bukit Fraser
Bukit Fraser
Cameron Highlands
Bukit Larut
126 m
1161 m
1291 m
1291 m
1418 m
1161 m
1/2
0/2
0/1
0/1
0/2
0/2
Perlis
Penang
Perak
Pahang
Pahang
Pahang
Penang
Kedah
Penang
Kedah
Perlis
Gua Kelam
Pulau Jerejak
Bukit Larut
Bukit Fraser
Bukit Fraser
Cameron Highlands
Teluk Bahang Rec. Forest
Sungai Sedim
Teluk Bahang Rec. Forest
Sungai Sedim
Kampung Chabang
37 m
21 m
1161 m
1291 m
1291 m
1418 m
67 m
126 m
67 m
126 m
41 m
0/1
1/2
1/2
0/2
1/1
0/3
0/1
0/2
1/1
0/1
0/1
Pahang
Kedah
Penang
Penang
Perlis
Pahang
Pahang
Penang
Penang
Penang
Kedah
Bukit Fraser
Sungai Sedim
Pulau Jerejak
Teluk Bahang Rec. Forest
Kampung Chabang
Bukit Fraser
Cameron Highlands
Pulau Jerejak
Teluk Bahang Rec. Forest
Teluk Bahang Rec. Forest
Sungai Sedim
1291 m
126 m
21 m
67 m
41 m
1291 m
1418 m
21 m
67 m
126 m
126 m
0/2
0/3
0/2
0/2
0/1
0/2
0/1
0/1
0/2
1/3
0/3
Forest
Forest
Forest
Forest
Anna E. Savage et al.
Table 1. Native amphibians sampled for Batrachochytrium dendrobatidis in Peninsular Malaysia
Species
Hylarana luctuosa
Hylarana picturata
Limnonectes blythii
Limnonectes kuhlii
Limnonectes laticeps
Limnonectes plicatellus
Occidozyga laevis
Odorrana hosii
Taylorana hascheana
Anura: Rhacophoridae
Chiromantis sp.
Nyctixalus pictus
Philautus petersi
Philautus vermiculatus
Polypedates leucomystax
Polypedates macrotis
Rhacophorus bipunctatus
Rhacophorus nigropalmatus
Rhacophorus prominanus
Rhacophorus prominanus
Theloderma asperum
Total
State
Site
Elevation
Bd positive / sampled
Kedah
Penang
Kedah
Kedah
Kedah
Kedah
Penang
Perak
Kedah
Kedah
Kedah
Pahang
Perak
Kedah
Kedah
Kedah
Kedah
Penang
Sungai Tupah
Teluk Bahang Rec. Forest
Sungai Sedim
Sungai Sedim
Sungai Sedim
Sungai Tupah
Teluk Bahang Rec. Forest
Bukit Larut
Gunung Jerai
Sungai Sedim
Sungai Tupah
Bukit Fraser
Bukit Larut
Gunung Jerai
Sungai Sedim
Sungai Sedim
Sungai Sedim
Penang Botanical Gardens
88 m
67 m
126 m
126 m
126 m
88 m
67 m
1611 m
618 m
126 m
88 m
1291 m
1161 m
618 m
126 m
126 m
126 m
85 m
0/1
0/1
0/1
1/3
0/2
0/1
0/2
0/2
0/1
1/1
0/1
0/4
0/2
0/1
0/1
0/1
0/2
0/1
Perlis
Penang
Pahang
Pahang
Perak
Pahang
Pahang
Perak
Kedah
Kedah
Kedah
Penang
Penang
Kedah
Pahang
Perak
Kedah
Pahang
Kedah
Pahang
Perlis State Park
Penang Botanical Gardens
Bukit Fraser
Cameron Highlands
Bukit Larut
Bukit Fraser
Cameron Highlands
Bukit Larut
Gunung Jerai
Sungai Sedim
Sungai Tupah
Pulau Jerejak
Teluk Bahang Rec. Forest
Sungai Sedim
Bukit Fraser
Bukit Larut
Sungai Sedim
Bukit Fraser
Sungai Sedim
Cameron Highlands
37 m
85 m
1291 m
1418 m
1161 m
1291 m
1418 m
1161 m
618 m
126 m
88 m
21 m
67 m
126 m
1291 m
1161 m
126 m
1291 m
126 m
1418 m
0/1
0/2
0/1
0/3
1/2
0/1
0/2
1/2
0/1
0/1
0/1
0/1
0/2
0/1
0/1
0/1
0/2
0/1
0/2
0/1
10/127
interval for overall Bd prevalence of 4–14%. However, Bd
prevalence and susceptibility can vary among species
(Pearl et al., 2007, Frias-Alvarez et al., 2008), geographic
regions, or elevations (Lips et al., 2006; Laurance et al.,
1996). Consequently, we calculated 95% confidence
intervals for Bd prevalence within regions, elevations, and
taxonomic groups (Table 2), each of which may experience unique Bd dynamics. All confidence intervals were
overlapping. Thus, we detected no significant regional,
elevational, or taxonomic differences in Bd prevalence
Bd in Peninsular Malaysia
Figure 1. Map of Peninsular
Malaysia showing Batrachochytrium dendrobatidis survey localities.
Bd negative sites are shown with
open symbols and Bd positive
sites are shown with filled symbols. Within Bd positive sites, pie
charts indicate the proportion of
infected individuals in black and
uninfected individuals in white.
Numbers refer to localities as
follows: 1, Teluk Bahang Forest
Reserve; 2, Pulau Jerejak; 3, Sungai Sedim; 4, Penang Botanic
Gardens; 5, Bukit Larut; 6, Perlis
State Park; 7, Gua Kelam and
Kampung Chabang; 8, Sungai
Tupah; 9, Gunung Jerai; 10,
Cameron Highlands; 11, Fraser’s
Hill
given our sample sizes. However, we predict that additional sampling would reveal higher Bd prevalence at
higher elevations, as the cooler temperatures in high elevation regions of Peninsular Malaysia are optimal for Bd
growth (Piotrowski et al., 2004). Among the 10 Bd infected
individuals, infection intensity ranged from 1 to 157,000
GE (Fig. 2). Infection intensities were <181 GE in eight
individuals and >13,200 GE in two individuals. None of
these individuals showed signs of disease in the field.
Before our study, Bd was only known to infect mainland Asian amphibians from South Korea (Une et al.,
2008), where infection prevalence was 38.8% across the
seven species examined and 61.9% across the three species
that were infected (Yang et al., 2009). The other two Bd
surveys from mainland Asia were conducted in Thailand
(McLeod et al., 2008) and Hong Kong (Rowley et al., 2007)
and found no evidence of Bd infections. Our finding of
Bd in four states and 10 frog species of Peninsular Malaysia
is consistent with the hypothesis that Bd is endemic to the
region (the EPH). However, a reasonable alternate
hypothesis is that Bd currently infects at least two regions of
mainland Asia because of the introduction (the NPH) via
non-native amphibians (Johnson and Speare, 2003).
Genetic analysis of South Korean and Peninsular Malaysian
Bd isolates will be necessary to provide support for either
hypothesis. The only Asian Bd isolates that have undergone
genetic analysis are from Japan, and these isolates showed
sufficient genetic differentiation to implicate Japan as a
possible origin of worldwide Bd (Goka et al., 2009). This
surprising result underscores that any prediction of Bd
strain origins based on the presence of infections alone
should be interpreted with caution.
Anna E. Savage et al.
Detection of Bd in 10 species and four frog families
from Peninsular Malaysia has important implications for
the amphibian conservation. Although we did not observe
signs of morbidity or mortality, two individuals were
infected with extremely high pathogen loads, suggesting
Table 2. Bd prevalence with 95% Clopper-Pearson binomial
confidence intervals for individuals grouped by amphibian family,
Malaysian state, and elevation
Group
Family
Ichthyophiidae
Bufonidae
Megophryidae
Microhylidae
Ranidae
Rhacophoridae
State
Kedah
Pahang
Penang
Perak
Perlis
Elevation
Low (21–126 m)
Mid (618 m)
High (1161–1611 m)
All individuals
Sample
size
Bd
prevalence (%)
95% CI
2
19
10
17
50
29
0
0
10
24
6
7
0–84%
0–19%
0.3–45%
7–50%
1–17%
1–23%
39
32
33
19
4
8
3
9
16
0
2–21%
0.01–16%
2–24%
3–40%
0–60%
73
3
51
127
8
0
8
8
3–17%
0–71%
2–19%
4–14%
full-blown chytridiomycosis rather than the presence of a
native skin microbe. In contrast, the other eight infected
frogs had low pathogen loads, and Bd prevalence was low
across our sampling of the amphibians and localities.
However, we did not sample across seasons, and temperature and humidity have strong effects on the Bd growth
(Piotrowski et al., 2004). The origins and implications of
Bd in Peninsular Malaysia thus remain enigmatic. Bd may
indeed be a native microbe in this region, but due to the
deadly effects of this pathogen on other continents, our
discovery of Bd in frogs of Peninsular Malaysia should be
considered a conservation threat unless the NPH can be
excluded. In fact, one potential scenario that is consistent
with the NPH is that the infections we detected were
acquired from encounters with the feral imported
amphibians harboring Bd. Peninsular Malaysia has a large
frog farming industry that includes the non-native species,
Lithobates catesbeianus and Holoplobatrachus rugulosus
(John, 2005). Lithobates catesbeianus is a worldwide carrier
of Bd (Garner et al., 2006), and naturalized and breeding
populations of L. catesbeianus have been detected in
Peninsular Malaysia (Chan et al., 2008). If non-native Bd
strains are introduced, Malaysian amphibians are at high
risk of chytridiomycosis epidemics and catastrophic
declines, as seen when Bd first appeared in Central America
and Australia (Lips et al., 2006; Laurance et al., 1996).
Management efforts should, therefore, focus on preventing
the escape of imported amphibians, testing feral populations of L. catesbeianus for Bd infections, and restricting
further importation of the non-native amphibians.
Figure 2. Bd infection intensity (measured in genome equivalents) among the
10 Bd infected native frog species from
Peninsular Malaysia
Bd in Peninsular Malaysia
ACKNOWLEDGMENTS
A research pass (40/200/19 SJ.1105) was issued to LLG by
the Economic Planning Unit, Prime Minister’s Department. This research was supported partially by a grant from
the College of Arts and Sciences, La Sierra University to
L.L.G., a USM Grant to S.A., and a travel grant to A.E.S.
from the National Science Foundation Graduate Research
Fellowship Program.
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