Amphibian Surveys Reveal No Instances of Batrachochytrium Dendrobatidis and Suggest Low Prevalence of Chytrid Fungus in Thailand
Total Page：16
File Type：pdf, Size：1020Kb
290 AMPHIBIAN AND REPTILE DISEASES Herpetological Review, 2019, 50(2), 290–298. © 2019 by Society for the Study of Amphibians and Reptiles Amphibian Surveys Reveal No Instances of Batrachochytrium dendrobatidis and Suggest Low Prevalence of Chytrid Fungus in Thailand Chytridiomycosis, the amphibian infectious disease caused by the fungal organism Batrachochytrium dendrobatidis (Bd), occurs globally with high prevalence and has been implicated in the population declines of amphibians in Africa (Hopkins and Channing 2003), Australia (Berger et al. 1998), Central America (Berger et al. 1998), Europe (Bosch et al. 2001), North America (Vredenburg et al. 2010), and South America (de Queiroz Carnaval et al. 2006). However, studies on the prevalence of Bd in East Asian amphibians present a different story—surveys conducted in parts of China, Japan, and the Korean peninsula have found low prevalence of Bd and have observed few negative effects on individuals that have tested positive for Bd (e.g., Goka et al. 2009; Swei et al. 2011; Bai et al. 2012; Bataille et al. 2013). Additionally, when sequencing the Bd strains found in the region, high diversity of Bd haplotypes were detected across sampling localities, suggesting that Bd has been evolving in the region for an extended period (Bai et al. 2012; Bataille et al. 2013). Recently, whole-genome sequencing of 234 isolates of Bd revealed a hyperdiverse East Asian Bd lineage with the highest SNP diversity found in wild South Korean frogs (O’Hanlon et al. 2018). Based on subsequent demographic and diversification analyses, it is hypothesized that Bd originated in the Korean Peninsula and spread globally throughout the 20th century (O’Hanlon et al. 2018). Nevertheless, there remain several regions of the globe that have not been well tested for Bd presence; this lack of data hinders our understanding of the origin, direction, and timing of the pathogen’s global spread. Additionally, without Bd prevalence data, it is difficult to develop strategies for conservation and disease management of sensitive species or habitats. One of ELYSE S. FREITAS* Sam Noble Oklahoma Museum of Natural History, University of Oklahoma, 2401 Chautauqua Ave, Norman, Oklahoma 73072, USA; Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, Oklahoma 73019, USA ATTAPOL RUJIRAWAN NATEE AMPAI PIYAWAN PUANPRAPAI SIRIPORN YODTHONG KORKHWAN TERMPRAYOON Department of Zoology, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan Rd, Chatuchak, Bangkok, 10900 Thailand CAMERON D. SILER Sam Noble Oklahoma Museum of Natural History, University of Oklahoma, 2401 Chautauqua Ave, Norman, Oklahoma 73072, USA; Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, Oklahoma 73019, USA ANCHALEE AOWPHOL Department of Zoology, Faculty of Science, Kasetsart University, FIG. 1. Map of Thailand showing locations where amphibians were 50 Ngam Wong Wan Rd, Chatuchak, Bangkok, 10900 Thailand sampled for Batrachochytrium dendrobatidis (black circles show specific localities, orange regions show the sampled provinces). The *Corresponding author; e-mail: [email protected] inset map shows Southeast Asia with Thailand highlighted in pink. Herpetological Review 50(2), 2019 AMPHIBIAN AND REPTILE DISEASES 291 (241 species) in mainland Southeast Asia (AmphibiaWeb 2018). Therefore, understanding the prevalence of Bd in Thailand is critical for higher-level analyses of the pathogen’s prevalence and distribution across Southeast Asia. There have been three studies to date on the occurrence of Bd in Thailand: 1) McLeod et al. (2008) examined histological samples from 123 fluid-preserved historical museum specimens housed at the Natural History Museum of Chulalongkorn University in Bangkok, representing 28 species from localities across the country, and found no instances of Bd infection; 2) Vörös et al. (2012) tested skin swabs of six individuals of the toad Ingerophrynus parvus at the Prince of Songkla University Protected Area in southern Thailand and found one individual that was positive for Bd, suggesting an overall high prevalence of disease in that region, albeit with limited sampling; 3) Techangamsuwan et al. (2017) tested skin swabs of 259 wild- caught amphibians and 233 captive amphibians at zoos and parks across Thailand, including a zoo near Prince of Songkla University, and found Bd presence only in captive non-native species. Here, we present the first study investigating the prevalence of chytrid fungus on wild amphibians in non- protected areas in northern and southern Thailand using the most comprehensive spatial sampling effort to date. We conducted amphibian surveys in five provinces in northern Thailand in March 2017 and in three provinces in southern Thailand in June 2017 (Fig. 1). In general, sampling was opportunistic and occurred as part of biodiversity surveys targeting northern and southern Thailand amphibian species for vouchered phylogenetic analyses. All surveys were carried out after dark between 1900–2130 h, and temperature and humidity measurements were taken at the start of each sampling period with a Kestrel 2000 pocket weather meter (Kestrel Instruments, FIG. 2. Map of Thailand showing gradients in: A) annual mean Boothwyn, Pennsylvania, USA). For samples collected in temperature; B) annual temperature differential (difference between northern Thailand, the temperature and relative humidity for the maximum temperature and the minimum temperature; C) annual mean precipitation; and D) the mean precipitation of the each sampling locality ranged from 21.3–32.2°C and 27.4–70.1%, warmest month. Northern Thailand has an overall higher temperature respectively, and in southern Thailand measured 20.3–30.0°C differential and lower precipitation than southern Thailand. Data and 68.7–100%, respectively. Amphibians were captured by from WorldClim (2017). hand, placed into individual plastic bags to avoid potential cross-contamination of individuals, and euthanized the same these understudied regions is Southeast Asia. Despite comprising night of capture with an injection of MS-222 (Conroy et al. four major biodiversity hotspots (Myers et al. 2000), there is a 2009). After euthanization, each individual was rinsed briefly paucity of studies on Bd prevalence across the region (but see with tap water to remove any loose debris and mud on the body Diesmos et al. 2012 [Philippines]; Rowley et al. 2013 [Cambodia that may have interfered with our ability to detect Bd (i.e., PCR and Vietnam]; and LeBlanc et al. 2014 [Peninsular Malaysia], inhibitors). We acknowledge that this may have decreased our which suggested extremely low Bd prevalence; and Chong et al. ability to detect Bd in an individual with low loads of infection 2018 [Singapore], which suggested moderate Bd prevalence). (Brem et al. 2007). Individuals were swabbed with a sterile, The most thorough survey of Southeast Asia to date, conducted rayon-tipped swab (Medical Wire; Corsham, Wiltshire, England) by Swei et al. (2011), found that only 1.4% of more than 2,800 ten times each on the dorsum, venter, medial surface of the individuals tested positive for Bd, with positive samples found in left thigh, left foot webbing, throat, and mouth (juveniles and Indonesia (0.25% infection rate), Laos (0.73%), Malaysia (0.90%), adults) or ten times on the oral disc (tadpoles) (modified from and the Philippines (8.01%). Hyatt et al. 2007). A control swab was dipped and swirled in Despite the high numbers of samples collected throughout the tap water used for rinsing and tested for chytrid to ensure Southeast Asia by Swei et al. (2011), sampling was opportunistic that there was no contamination of individuals from the water and large areas of the region were missed, including Thailand. source—all control swabs tested negative for chytrid. Each swab The general paucity of countrywide data regarding Bd prevalence was then placed into individual 1.5-ml Eppendorf tubes and in Thailand is surprising given its unique and complex allowed to dry unsealed for an hour, before being stored after biogeography. Within Southeast Asia, Thailand represents a the completion of field surveys in a -20°C freezer until extracted. major biogeographic transition zone between Indochinese and We sexed each specimen via gonadal inspection and vouchered South Asian species in the north and Sunda Shelf species in the individuals at the Zoological Museum of Kasetsart University, south (Woodruff 2003; Parnell 2013), making it an important Bangkok, Thailand (ZMKU). Extractions of Bd DNA were done region for biodiversity. Currently, there are 145 species of at the Sam Noble Museum Genomics Core Facility (University amphibians recognized in the country, second only to Vietnam of Oklahoma, Norman, Oklahoma, USA) with PrepMan Ultra Herpetological Review 50(2), 2019 292 AMPHIBIAN AND REPTILE DISEASES TABLE 1. The number of amphibian individuals for each age class (tadpole, juvenile, adult) and sex (male, female) sampled for Batrachochy- trium dendrobatidis (Bd) at each locality in Thailand (coordinates; elevation). The localities are listed in the chronological order that they were sampled. The right column shows the calculated 95% confidence interval for the maximum potential Bd prevalence in each species for each locality, given the sample size, and for each locality, given the total number of individuals sampled. Locality Species Tadpole Juvenile Adult Adult Max. chytrid male female prevalence in population (%) 19 March 2017: Tak Province: Mae Sot Duttaphrynus melanostictus