Acta Herpetologica 11(1): 81-84, 2016 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-17406 Yeasts in amphibians are common: isolation and the first molecular characterization from Thailand Srisupaph Poonlaphdecha, Alexis Ribas* Biodiversity Research Group, Faculty of Science, Udon Thani Rajabhat University, Udon Thani 41000, Thailand. *Corresponding author. E-mail: alexisribas@hotmail.com Submitted on: 2015, 6th November; revised on: 2016, 21th March; accepted on: 2016, 25th March Editor: Fabio Maria Guarino Abstract. A survey of the presence of yeasts in frogs and toads in Thailand was conducted using standard mycologi- cal examination techniques. The results, which were confirmed with molecular techniques, revealed the presence of five yeast species – Cryptococcus liquefaciens, C. heveanensis, Pseudozyma hubeiensis, Rhodotorula mucilaginosa and R. minuta – in the bile of these amphibians. Although previous works have isolated yeasts from amphibian gastrointes- tinal tracts and skin, it is questionable whether these yeasts were acquired by ingestion or were commensals on adult individuals. Frog farms, an urban area and protected natural areas were surveyed and all tested positive for yeasts, which shows their ubiquity in both wild and farm-reared frogs. Additionally, the finding of yeasts in five different spe- cies of frogs and toads shows that there is a wide spectrum of hosts in this vertebrate group. Our results thus suggest that yeasts are likely to be widespread among amphibians in different habitat types and in a wide range of host species. Keywords. Amphibian, yeast, Rhodotorula, Pseudozyma, Cryptococcus Frogs are parasitized by several taxa, the most-stud- ied of which include helminths, bacteria, viruses and the chytrid fungus Batrachochytrium dendrobatidis. Parasites cause high mortality rates in wild amphibian populations (Densmore and Green, 2007), while infectious diseases are a concern in amphibians kept in captivity as pets or reared on farms as a food source. Although not strictly pathogenic, symbiont organisms have been found on frog skins, where they are reported to act as a barrier against chytridiomicosis, and have also been isolated in the guts of these vertebrates (Walke et al., 2014). Anecdotal reports of yeasts – opportunistic pathogens that are ubiq- uitous in nature – exist for tadpoles and adult amphib- ians. Steinwascher (1979) found that Candida humicola was abundant in the faeces of some large tadpoles of Rana clamitans in Australia, where it acts as a mutualis- tic symbiont. Rhodotorula glutinis was isolated from the tadpoles of marsh frogs (Limnodynastes peronii) in Aus- tralia, where it plays a role in suppressing the growth of the mosquito larvae that inhabit the same pond (Mokany and Shine, 2003). In the study by Sammon et al. (2010), yeast were isolated from the faeces and the skin of adult Australian green tree frogs (Litoria caeruela), although no subsequent molecular confirmation was undertaken. As all studies of yeast from the gastrointestinal tracts of amphibians (tadpoles and adults) to date have only focused on faeces, it is still unclear whether or not yeasts form part of the natural microflora of amphibians or whether they are acquired from the environment through the ingestion of water and thus appear in faeces. The yeast Candida humicola can modify the behavior of tad- poles (Kiesecker et al., 1999) and so should be considered pathogenic rather than symbiotic, as has been previously reported (Steinwascher, 1979). Consequently, the distri- bution of yeasts in both wild and farm-reared popula- tions of amphibians is of interest. In the course of a par- asitological survey of adult frogs and toads in Thailand, the observation under the microscope of yeast-like cells 82 Srisupaph Poonlaphdecha, Alexis Ribas in bile led us to suspect that yeasts were present in these amphibians. We thus conducted culture and molecular characterization to confirm the validity of these micro- scopic observations. Fieldwork was carried out in March–June 2014. Frogs were collected by hand in three types of habitats: frog farms, urban areas and protected natural areas. On Farm 1, where chicken and fish are also bred, frogs are raised in walled concrete enclosures at high densities. On Farm 2, frogs are raised in shallow ponds which were covered by plastic on the floor and net as a wall, it also has chickens in the very close area. On Farm 3, frogs are raised in ponds and coexist with fish and ducks, although they are physically separated by netting. All farms are located in Udon Thani Province. The second habitat surveyed was an urban area on the Udon Thani Rajabhat University campus, where there are many buildings and green spaces. The third environ- ment surveyed were two protected forest areas, Na Yung- Nam Som Natural Park (344 km2, Udon Thani Province) and Phu Wua Wildlife Sanctuary (186.5 km2, Bueng Kan Province), that both harbour great floral and faunal bio- diversity. Once collected, all samples were immediately transported alive to a laboratory at Udon Thani Rajabhat University and identified following Taylor (1962) and the nomenclature used in an online database of the world’s amphibians (Frost, 2015). Amphibians were anesthetized, killed using MS222 (ethyl-4-aminobenzoate) and then dissected. Their gall bladders were removed and placed on sterile Petri dishes. Syringes were used to suck bile from the gall bladders, which was then transferred onto Yeast Malt agar (HiMe- dia Laboratories) and cultured using a spread-plate tech- nique. The YM culture plates were incubated at 25°C for 5–7 days. Single colonies were selected, streaked onto YM agar plates and then incubated at 25°C for 5–7 days. The purified colonies were examined microscopically and those with yeast-like shapes were subjected to further molecular studies. Isolation of DNA was carried out by boiling of cells with lysis buffer according to the methods of Maniatis et al., (1982) with slight modification. A loopful of yeast cells was transferred to 1.5 ml Eppendorf tube. The 100 µl of lysis buffer was added. Cell suspensions were boiled in water bath or metal block bath for 15 min. After boiling, 100 µl of 2.5 M potassium acetate (pH 7.5) was added and placed on ice for 1 hour, and centrifugated at 14,000 rpm for 5 min. Supernatant was extracted twice with 100 µl of chloroform: isoamyl alcohol (24:1 v/v). DNA was precipitated with isopropanol, placed at 20°C for 10 min and centrifugated at 15,000 rpm for 15 min. DNA pellet was rinsed with 70% and 90% ethanol and then dried up (15-30 min at room temperature). The dried DNA was dissolved in 30 µl milli Q water. The amplification and sequencing of 13 isolates were performed at the National Center for Genetic Engineer- ing and Biotechnology (BIOTEC), Bangkok (Thailand). The divergent D1/D2 domain of 26S rDNA was ampli- fied with primers NL-1 (5’-GCA TAT CAA TAA GCG GAG GAA AAG-3’) and NL4 (5’-GGT CCG TGT TTC AAG ACG G-3’) (Kurtzman and Robnett, 1998). Ampli- fication was carried out in a 100 μl reaction mixture con- taining 100 ng of genomic DNA, 2.5 U of Taq polymer- ase (Thermo Scientific), 20 mM of each dNTP, 40 mM of each primer, 10 mM Tris-HCl and 1.5 mM MgCl2. The reaction was pre-denatured at 94°C for 5 min, then repeated for 30 PCR cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2.5 min and then final extension at 72°C for 10 min. The amplified DNA was purified using a QIAquick PCR Puri- fication Kit according to the manufacturer’s instructions. The nucleotide sequences of D1/D2 domain of 26S rDNA were directly determined using PCR products following Kurtzman and Robnett (1998) with slight modifications. Cyclic sequencing of the D1/D2 domain was conducted with an NL1 forward primer (59-GCA TAT CAA TAA GCG GAG GAA AAG-39) and an NL4 reverse primer (59-GGT CCG TGT TTC AAG ACG G-39) using an ABI PrismTM BigDyeTM Terminator Cycle Sequence Ready Reaction Kit (Applied Biosystems, Stafford, USA) follow- ing the manufacturer’s instructions. The thirteen obtained sequences (576 to 612 base pairs) of D1/D2 domain of 26S rDNA were compared using a BLAST Homology Search (http://www.ncbi. nlm.nih.gov/blast): AF070432, AF181515, AF189948, DQ008953 and FJ534905. Our obtained sequences were deposited in Genbank, accession numbers KU893128 to KU893140. Growth of yeast colonies on YM agar (pink and white) was observed in samples from 12 amphibians from 3 of the 90 farms (3.33%), 2 of the 7 amphibians from the urban area (28.57%) and 7 of the 31 amphibians (21.87%) from the wild (total=129 specimens). One spec- imen of Hoplobatrachus rugulosus from a farm harboured two gross colony morphologies (Table 1). Molecular characterization confirmed the presence of Cryptococcus heveanensis, C. liquefaciens, Pseudozyma hubeiensis, Rho- dotorula minuta and R. mucilaginosa (see Table 1). The yeasts isolated from the amphibians are common in the environment and have been reported as oppor- tunistic pathogens in vertebrates. Cryptococcus liquefa- ciens is a common yeast that is able to survive in extreme habitats such as the deep sea (Buzzini and Margesin, 2013) and can be found in the microbiota of human 83Yeasts in amphibians skin (Zhang et al., 2011). It has also been reported as an opportunistic pathogen (Takemura et al., 2015) and is able to cause fatalities in humans (Conde-Pereira et al., 2015), but has never before been reported in amphibians. Cryptococcus cuniculus has previously been reported from rabbits (Shin et al., 2006). Pseudozyma hubeiensis, a basidiomycetous yeast, was recently isolated in China from the leaves of a number of plant species (Wang et al., 2006), while Liou et al. (2009) have described a new species of this genus in Thailand from the region in which the current study was carried out. To our knowledge, there are no previous reports of this genus of yeast in vertebrates. The genus Rhodotorula is a common yeast found in air, soil, lakes, ocean water, milk and fruit juice (Wirth and Goldani, 2012), and is able to survive in adverse conditions. Rhodotorula spp. including the two spe- cies found in amphibians in the present study have been observed as opportunistic pathogens in humans (Wirth and Goldani, 2012). Recent studies have reported the presence of Rhodotorula yeasts in vertebrates (Chitko- Mckown et al., 2013) including a female lamb (but with no associated pathology), in skin lesions in sea lions in an aquarium (Alvarez-Perez et al., 2010) and as a nor- mal component of the microbiota of salmonids (Raggi et al., 2014). Our results suggest that the role of yeasts in wild and farmed amphibians as pathogens has to date been underestimated and it is likely that extrinsic and intrinsic factors induce their pathogenicity. Our results show a high diversity of yeast species (5) in amphibians compared to the number of species found in vertebrates. However, only a few studies of yeasts in vertebrates have ever been conducted. It is possible that the aquatic habi- tat of adult amphibians (partially dependent on water and varying according to the species) may contribute to the life cycle of these yeasts. Our examination of bile samples revealed that yeasts colonize the internal organs of frogs. Although the pres- ence of Candida spp. in human gall bladders has previ- ously been reported (Diebel et al., 1996), we believe that this is the first report of yeast from the gall bladders of non-human vertebrates. The study by Sammon et al. (2010) found a minimum of six species of yeasts (Rho- dotorula sp., Trichosporon spp., Candida sp., C. famata and C. glabrata) in faeces from five frogs and our find- ings confirm this high diversity. We isolated five species of yeasts, which suggests that the biodiversity of yeasts in frogs has been insufficiently studied. Additionally, our study confirms the ubiquity of yeasts in these amphibians since they were found in two wild protected areas, in an urban area and on frog farms. Our study also shows that yeasts are widespread in a number of species of amphibi- ans, including both those associated with aquatic habitats and those with more terrestrial habitats (toads). ACKNOWLEDGEMENTS This study was supported by a grant from the Research and Development Institute, Udon Thani Rajab- hat University. We would like to thank Renu Sirimong- konthaworn and Tiwarat Thalernkietleela from Udon Thani Inland Fisheries Research and Development Center, Taweep Kampaengmuang, Director of Phu Wua Wildlife Sanctuary, and Rungsun Loupha, Director of Na Yung-Nam Som protected area. The research in Phu Wua Wildlife Sanctuary and Na Yung-Nam Som pro- tected area was approved by the Department of National Table 1. Distribution of the yeast species confirmed by molecular analysis according to habitat and host. Sampling Point Habitat Anuran Species Species of Yeasts % Similarity Farm 2 Farm Hoplobatrachus rugulosus Rhodotorula mucilaginosa 100.00% Farm 2 Farm Hoplobatrachus rugulosus Rhodotorula mucilaginosa 100.00% UDRU Urban Area Kaloula pulchra Rhodotorula mucilaginosa 100.00% Phu Wua Wildlife Sanctuary Wild Sylvirana faber Rhodotorula mucilaginosa 99.80% UDRU Urban Area Duttaphrynus melanostictus Rhodotorula mucilaginosa 99.50% Phu Wua Wildlife Sanctuary Wild Kaloula pulchra Rhodotorula minuta 99.70% Farm 2 Farm Hoplobatrachus rugulosus Cryptococcus liquefaciens 100.00% Phu Wua Wildlife Sanctuary Wild Sylvirana faber Cryptococcus liquefaciens 100.00% Na Yung-Nam Som protected area Wild Duttaphrynus melanostictus Cryptococcus liquefaciens 100.00% Na Yung-Nam Som protected area Wild Kaloula pulchra Cryptococcus liquefaciens 100.00% UDRU Urban Area Duttaphrynus melanostictus Cryptococcus heveanensis 99.80% Na Yung-Nam Som protected area Wild Fejervarya limnocharis Psudozyma hubeiensis 99.80% Na Yung-Nam Som protected area Wild Duttaphrynus melanostictus Psudozyma hubeiensis 99.80% 84 Srisupaph Poonlaphdecha, Alexis Ribas Parks, Wildlife and Plant Conservation and the National Research Council of Thailand (Number 0002/7387). 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