Conservation Ecology Program School of Bioresources and Technology

1. Survey of the Amphibian Chytrid Fungus, Batrachochytriumdendrobatidis from Relatively Undisturbed Evergreen Forest in Western Thailand Presented by: PichaniSaengtharatip Conservation Ecology Program School of Bioresources and Technology King Mongkut’s University of Technology Thonburi

2. Chytrid fungus (Bd): Batrachochytrium dendrobatidis - Significant threat to amphibian populations globally - > 200 spp. lead to population declines & extinctions around the world infected adult (Berger et al., 1998; IUCN, 2008; Swei et al., 2011)

3. EX In Costa Rica Harlequin frog (Atelopus spp.) Climate change + Bd has had 67% of the 110 species disappeared within the last two decades

4. Bd motile zoospore; aquatic & has flagellate swim in water & penetrate frog skin sporangium zoospore sporangium (Berger, 2000 ; Longcore, 1999)

5. The presence ofBd in … 1. South Africa 2. North & Central America 3. South America 4. Australia 5. Europe 6. Asia

6. Bd has emerged in 11 countries; Japan, Indonesia, South Korea, China, Malaysia, Laos, Philippines, Vietnam, Cambodia, Singapore and Thailand Asia detected non-detected

7. Infection prevalence - South America (in Chile) (SolÍs et al., 2010) overall 24% - Mass mortality in Australia (Berger et al., 1998) - Asia (Swei et al., 2011) overall 2.3% (6 of 15 countries) Asia Low infection rates at most undisturbed sites No reports about mass die-offs or enigmatic population declines in SE Asia Bd may or may not be widespread (Rowley et al., 2010)

8. Increasing the movement and/or susceptibility to Bd - Global climate change - Commercial trade Frog farming Wild frogs for food Pet trade (Blaustein, 2006)

9. Thailand In 2012, sampled from Songkla province using real-time PCR techniques (Vörös et al., 2012) 1 out of 6 sampled individuals tested positive for Bd, but with low infection Lesser toad (Ingerophrynus parvus)

10. Thailand may face increased risk Importation of amphibians for … Commercial trade Consumption Frog farming Pets Cultural belief of making merit releasing live animals bought from markets Driving the emergence of Bd

11. Why Bd is not currently linked to significant amphibian declines in Asia ? 1. Bd has not fully emerged in Asia 2. Bd is endemic to Asia & shared evolutionary history with native species who are resistant 3. Bd emergence is inhibited by abiotic or biotic condition that unique in Asia (Goka et al., 2009; Swei et al, 2011)

12. Objective To investigate whether there was any evidence for the presence of Bd in a relatively undisturbed evergreen forest

13. Study Area Huai Kha Khaeng Wildlife Sanctuary where Bd was first detected

14. Methods • - Sep - Oct 2013 • - Huai Chang Tai stream • - 550 - 1150 msl • - Randomly collected 5-6 encountered skin swabs at each elevation • - n = 5-6 individuals x 4 zones x 2 months = 50 • - Samples were fixed in 70% ethanol & stored at room temp • - Wildlife Forensic Science Unit laboratory, DNP

15. - Extraction of DNA from Swab Samples Protocol from the ArchivePure DNA Kit for genomic DNA purification from buccal cells (5 PRIME, Germany) With some modification described byConservation Research and Education Division of Thailand’s Zoological Park Organization (ZPO, 2010) - Conventional PCR Assay • ITS-1/5.8S DNA region of Bd • Two specific primers for Bd(Annis et al., 2004; Garner et al., 2005; Goka et al., 2009) • Bd1a & Bd2a • - PCR products were separated on 1.5% agarose gels • - Bands of DNA fragments were viewed under UV light

16. Skin swabs from 10 species (50 adult frogs)example results from 5 samples band approx. 300 bp M = 1 kb DNA ladder Gene Ruler N = negative control P = positive control

17. Discussion In 2010 (Kamolnorranart et al.) ZPO found Bd, but only in non-native specimens from a zoo In 2012 (Vörös et al., 2012) Bd was detected in the wild BUT, Had a low infection intensity & Did not display the most common clinical signs of infection 2399 observations of frogs did not detect any symptomatic frogs, dead or alive

18. - Genetic evidence of ancestral Bd haplotype that might be endemic to Asia (Goka et al., 2009) - It is possibly present in natural areas at very low levels, but yet undetected - Real-time PCR more sensitive than conventional PCR (Kriger et al., 2006) - Some of our results reported here may be false negatives Next mission … We will work on re-examining these samples using real-time PCR to determine whether they contain trace amounts of naturally occurring Bd

19. Conclusion • Further comprehensive follow-up studies on distribution of Bd in SE Asia • To monitor status of areas where positives are detected to see if prevalence & infection intensity rise • Conduct surveillance of Bd so that it will not cause the same levels of infection seen in other biogeographic regions • More data will help us obtain a better understanding of this threat to native amphibians within the region • Help authorities & concerned citizens mitigate this hazard

21. Acknowledgements S. Duangchantrasiri and KNR research assistants & staffs, Khao Nang Rum Wildlife Research Station T. Vichitsoonthonkul, King Mongkut’s University of Technology Thonburi R. Songchan & members of “Survey and monitoring of newly emerging ‘Chytridiomycosis’ disease in Thai amphibians” Project, Wildlife Research Division, DNP Wildlife Forensic Science Unit laboratory, DNP

22. THANK YOU!!!

24. Taxonomy chytrid fungus (Bd) phylum Chytridiomycota class Chytridiomycetes order Chytridiales genus Batrachochytrium dendrobatidis (Longcore, 1999)

25. Physiology range of temperatures for fungus growth in vitro temperature 4–25 °C optimum for growth temperature 17-25 °C pH 6-7 at 4 °C, it has the ability to persist & grow slowly at 30 °C kill 50% of pure cultures (Piotrowski, 2004)

26. Clinical signs infects keratinized tissues - mouthparts in tadpole loss of the jaw sheath - skin in adult cause thickened skin resulting in interruption of gas exchange & osmoregulation normal tadpole infected tadpole infected adult (Berger et al., 1998; Kusrini et al., 2008)

27. Histological examination (Haemotoxylin and eosin stained section of infected frog skin) sporangium with discharge papillae containing zoospores Epidermis (Berger, 2000)

28. Scanning electron microscopical examination sporangium 10 µm (Berger, 1998)