Download Detecting the Distribution of the Chytrid Fungus in the Philippines

Detecting the Distribution of the Chytrid Fungus in
the Philippines
By 1*Mae Lowe L. Diesmos, 2Arvin C. Diesmos, 3Cameron D. Siler, 4*Vance T. Vredenburg, 3Rafe M. Brown
G
lobally over 30% of the 6,000+ amphibians species are
currently threatened with extinction (1). Amphibian biologists have recorded over 150 species of frogs that have vanished from many parts of the world particularly in North, Central,
and South America, Europe and in Australia. Chytridiomycosis, an
emerging infectious disease specific to amphibians and is caused
by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), has
been directly associated with these extinction events (2–4). Mass
amphibian mortalities were recorded in areas where evidence of
high Bd spore counts are known, most especially in closed fresh
water ecosystems. Chytridiomycosis is also known to interact with
other environmental factors such as habitat destruction and climate change, triggering massive declines of amphibian populations
in countries where the pathogenic disease is known (5, 6).
In 2008, we detected the first case of Bd infection in an assemblage
of frogs at two localities on the island of Luzon: on Mts. PalaypalayMataas Na Gulod in Cavite Province and on Mt. Labo in Camarines
Norte Province (7). We subsequently found chytrid-positive frog
populations from our surveys of other areas across the country (8,
9). Here we summarize information on the status and distribution
of this emerging infectious disease in the Philippines covering the
period from 2004 to 2011.
Our sampling sites include natural forested areas (primary and
secondary forest from near sea level to over 1,000 m elevation)
and man-modified environments (second-growth, agricultural
areas, and gardens). During the course of the study, we sampled
over 3,000 frogs belonging to at least 30 species via standardized
swabbing protocol (five strokes each on inner thighs of hind legs,
on webbing of each foot, and on abdomen). A drop of 95% ethyl
alcohol was added to each swab, air-dried, and the swabs were
placed inside individually labeled micro-centrifuge tubes. Samples
were analyzed using Real-Time PCR (Polymerase Chain Reaction)
assay (9, 10). To estimate prevalence and infection intensity, we
calculated a measure of the number of Bd zoospores found on each
swab that we refer to as zoospore equivalents. To calculate for prevalence, samples were categorized as Bd-positive when zoospore
equivalents were ≥ 1 and Bd-negative when zoospore equivalents
were < 1 (9).
Chytrid fungus in Philippine frogs
From an initial two localities, we detected Bd-infected frog assemblages in 15 other sites on the major islands of Luzon, Negros and
Mindanao. We further expect to find an increasing trend in the
number of chytrid-positive localities with the completion of our
1
Department of Biological Sciences, College of Science of the University
of Santo Tomas, Manila, Philippines. 2 Herpetology Section, Zoology
Division, National Museum of the Philippines, Manila, Philippines. 3
Natural History Museum and Department of Ecology and Evolutionary
Biology, University of Kansas, Lawrence, Kansas, USA. 4 Department of
Biology, San Francisco State University, San Francisco, California, USA.
(Corresponding authors: MLLD = [email protected]; VTV =
[email protected])
48 | FrogLog 20 (5), Number 104 (October 2012)
Luzon
Mindanao
Fig. 1: The chytrid fungus Batrachochytrium dendrobatidis (Bd) is now found in at
least 17 localities on three major islands across the Philippines. Thus far, highest
prevalence of Bd-infection (with over 60% of samples) were detected from two
localities.
analyses of additional materials from multiple sampling sites
across the archipelago.
At least seven species of frogs were infected with Bd, these are
Limnonectes macrocephalus (Inger, 1954), L. magnus (Stejneger,
1910), L. woodworthi (Taylor, 1923), Occidozyga laevis (Günther,
1858), Hylarana grandocula (Taylor, 1920), H. similis (Günther,
1873) and Sanguirana luzonensis (Boulenger, 1896). These species
are associated with aquatic environments and are typically found
in clear, fast-flowing mountain streams and rivers (11). Except
for O. laevis, all of these species are endemic to the Philippines.
None of our samples of alien invasive frog species known from the
Philippines (12), such as Rhinella marina (Linnaeus, 1758), Hoplobatrachus rugulosus (Wiegmann, 1834) and Hylarana erythraea
(Schlegel, 1837), were positive for chytrid fungus.
Infection levels were found to be generally low and ranged from
3–10% of our samples. However, we detected high levels of infection (> 10%) from two localities, Mts. Palaypalay-Mataas Na Gulod
on Luzon Island and Cotabato Cordillera in South Cotabato Province on Mindanao Island. This level of infection, based on studies
from several regions in Central and South America, is known to
result in amphibian declines or the extinction of affected populations (9, 10).
Research Priorities
Results of our ongoing field surveys demonstrate that Bd is widespread in the Philippines. Thus far, evidence of mass die-offs or
local extinction of amphibian populations is yet to be detected. This
B
A
C
E
D
F
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Fig. 2: Among the species that we found to be infected with Bd include Limnonectes macrocephalus (Fig. 2A), L. magnus (Fig. 2B), L. woodworthi (Fig. 2C), Occidozyga laevis
(Fig. 2D) (Dicroglossidae), Hylarana grandocula (Fig. 2E), H. similis, (Fig. 2F) and Sanguirana luzonensis (Fig. 2G) (Ranidae). Nearly all of these species are endemic to the
Philippines. Photos: A. C. Diesmos.
is an issue that must be considered as top research priority in the
region. Bd has the potential to infect numerous Philippine amphibian species and may cause large-scale species extinctions, given the
high levels of richness and species endemicity among Philippine
amphibians and the extent to which numerous critical habitats are
already being degraded (2, 8, 12).
Based on initial results of our studies, we recommend that: (1) a
comprehensive and sustained field surveys be undertaken to cover
as many islands and localities as possible and to sample various
habitats; (2) there is a need to perform ecological experiments that
will examine the effects of Bd on both infected and unexposed species and assemblages, and (3) a long-term monitoring and research
program need to be established, which may prove to be more effective through partnerships among government agencies, research
and academic institutions and conservation groups.
Acknowledgements
Funding and logistical support were generously provided by the US
National Science Foundation, National Geographic, University of
Kansas, National Museum of the Philippines, University of Santo
Tomas and San Francisco State University. The Protected Areas
and Wildlife Bureau (Philippine Department of Environment and
Natural Resources) and the National Museum of the Philippines
furnished research and collecting permits. We are indebted to our
many colleagues and students from the Philippines and the US for
their invaluable assistance both in the field and in the laboratory.
References
1. IUCN Red List of Threatened Species. Version 2012.1 (IUCN, 2012; www.
iucnredlist.org).
2. S. N. Stuart, M. Hoffmann, J. S. Chanson, N. A. Cox, R. Berridge, P. Ramani,
B. E. Young, Eds., Threatened Amphibians of the World (Lynx Ediciones,
Barcelona, Spain, 2008).
3. K. R. Lips et al., Biol. Conserv. 119, 4 (2004).
4. L. F. Skerratt et al., EcoHealth 4, 2 (2007).
5. K. R. Lips et al., PLoS Biology 6, e72 (2008).
6. J. A. Pounds et al., Nature 439, doi: 10.1038 (2006).
7. A. C. Diesmos, M. L. L. Diesmos, R. M. Brown, Philippine Daily Inquirer 14
February (2010).
8. A. C. Diesmos, R. M. Brown, in: Proceedings of the Conference “Biology of the
Amphibians in the Sunda Region, Southe-east Asia,” (Univ. of Malaysia Sarawak,
Kota Samarahan, 2011), pp. 26–49.
9. A. Swei et al., PLoS ONE 6, e23179 (2011).
10.A. D. Hyatt et al., Diseases of Aquatic Organisms 73, 3 (2007).
11. R. F. Inger, Fieldiana: Zoology 33 (1954).
12.A. C. Diesmos, M. L. Diesmos, R. M. Brown, J. Env. Sci. Management 9, 2
(2006).
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