Field Surveys for the Detection of Chytridiomycosis in North Georgia Amphibian Populations S. Cruz and J. Nations; Advisors: J. M. Morgan and N. L. Hyslop Department of Biology, The University of North Georgia, Gainesville We conducted amphibian surveys in the northeast Georgia Piedmont using active night searches on 52 occasions between spring 2013 and fall 2015 (Fig. 1) at three study sites across the region. In addition to active surveys, we also conducted passive sampling of tree frogs in multiple areas of one study site during 35 sampling occasions from September 2013 through August 2015 (excluding winter). For these passive surveys, we used 4 different diameters of 1m tall poly-vinyl chloride (PVC) pipes (4cm, 3cm, 2cm and 1.5cm) (6) installed vertically into the ground in 90m long transects (Fig. 2). We moved the pipe transects periodically to capture new individuals at the site. In addition to the vertically installed pipes, we also installed 6 additional 60 cm long pipes that were in a “T” form and suspended from trees. Species found during surveys were captured while wearing gloves and placed in a new plastic bag. Frogs were swabbed with a sterile polyester swab (Fig. 3) using procedures in Pessier and Mendelson (7) and returned back to the pipe or wetland location where it was found within 15 minutes of capture. The procedures included swabbing the ventral skin surfaces times including the front and hind feet, thighs and abdomen where infection tends to be reported on species (7). We placed swabs in individual sterile tubes then returned them to the laboratory where they were analyzed using PCR (8) to detect for the presence of Bd. In addition to skin swabs, we also recorded the location and time of capture, weather conditions, snout to urostyle length (SUL), life stage, microhabitat, and collection method. ( 1) Beebee, T. J., R. A. Griffiths The amphibian decline crisis: a watershed for conservation biology. Biological Conservation 125: 271–285. (2) Daszak, P., L. Berger, A.A. Cunningham, A.D. Hyatt, D.E. Green, and R. Speare Emerging infectious diseases and amphibian population decline. Emerging Infectious Diseases 86: 735–748. (3) Daszak, P., D.E. Scott, A.M. Kilpatrick, C. Faggioni, J. W. Gibbons, & D. Porter Amphibian population declines at Savannah River site are linked to climate, not chytridiomycosis. Ecology issue 86: 3232–3237. (4) Rothermel, B.B., S.C. Walls, J.C. Mitchell, C.K. Dodd Jr, L.K. Irwin, D.E. Green, V.M. Vasquez, J.W. Petranka, and D.J. Stevenson Widespread occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in the southeastern USA. Diseases of Aquatic Organisms 82.1: 3–18. (6)E. O'Neill, and B. Robin PVC Pipe Refugia: A sampling method for studying treefrogs. Patuxent Wildlife Research Center USGS. (7) Pessier, A.P., and J.R. Mendelson A Manual for Control of Infectious Diseases in Amphibian Survival Assurance Colonies and Reintroduction Programs. IUCN/SSC Conservation Breeding Specialist Group: Apple Valley, MN. (8) Goka, K., J. Yokoyama, Y. Une, T. Kuroki, K. Suzuki, M. Nakahara, A. Kobayashi, S. Inaba, T. Mizutani and A.D. Hyatt Amphibian chytridiomycosis in Japan: distribution, haplotypes and possible route of entry into Japan. Molecular Ecology, 18: 4757–4774. (9) Young, S., L. Berger, & R. Speare Amphibian chytridiomycosis: strategies for captive management and conservation. Amphibians & Chytridiomycosis: Strategies for Captive Management 41: 85–95. (10) Forzan, M.J., H. Gunn, & P. Scott Chytridiomycosis in an Aquarium Collection of Frogs: Diagnosis, Treament, and Control. Journal of Zoo and Wildlife Medicine 39: 406–411. Amphibians are one of most threatened classes of vertebrates due to multiple factors including habitat destruction, climate change, and disease (1). Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is a potential, significant contributing factor to worldwide amphibian population declines (2). Studies, however, suggest that chytrid is not always causative of declines (3), demonstrating the importance of long term monitoring of both environmental data and chytrid presence to elucidate Bd’s effect on populations. Past surveys of the fungus have confirmed its occurrence in multiple amphibian species in the southeastern USA (4); however, little research has been completed in the northern Piedmont region of Georgia to investigate the distribution of the fungus. Our data can serve as a baseline for Bd distribution in the region. Chytridiomycosis is a fungal disease caused by the pathogen Batrachochytrium dendrobatidis (Bd), which is a contributing factor to global amphibian population declines. Although Bd is distributed globally, little research has been conducted on north Georgia’s amphibian populations. We surveyed for the presence of Bd in amphibian populations in the northeast Georgia Piedmont region 3 different locations using active night searches and passive sampling techniques from spring 2013 through fall During night searches, amphibians were located in wetlands and captured by hand. We used poly-vinyl chloride (PVC) pipe traps for passive sampling during the day. Following captures, we collected environmental and physical data from each individual, swabbed the skin for Bd detection using sterile polyester tipped swabs, and released individuals at their capture site. We changed gloves and disinfected equipment between each capture. Collected skin swabs were analyzed using polymerase chain reaction (PCR) testing to determine the presence of Bd. To date, PCR techniques have detected a positive Bd sample at one of the sites. Sampling, along with capture-mark-recapture methods will continue throughout 2016 to contribute to conservation efforts and knowledge of Bd in the region. Figure 2: Photo of a PVC pipe trap plot at site 1. This plot consists of two 2cm pipes and one 3cm pipe. The positive Bd sample from a green tree frog (Hyla cinerea) found in a t-pipe suspended in a tree in a wetland is potentially noteworthy because Bd is a primarily water-borne pathogen (9). The high abundance of the species at the site is also noteworthy because it is the only species with a positive result but is also the species with the highest presence recorded so far. Bd could be present on the other species as well, but more PCR testing needs to be done before any conclusions can be made. Surveillance and rapid detection of Bd is important to effective management of Chytridiomycosis (9,10). Future research efforts include continuing sampling at the three existing study sites in the region with the addition of new sites. In addition, in summer 2015 we started a capture-mark-recapture study on the tree frog population where the positive Bd result was obtained to monitor the population size in relation to Bd infection. With this research we hope to expand our knowledge of the occurrence of Chytridiomycosis in the region. Figure 3: Ventral and thigh swabbing of a bullfrog, using a polyester swab. Research was funded by The University of North Georgia Faculty Scholar Grant and the UNG Department of Biology. To date, we have collected 320 individual samples from 3 study sites using active surveys and 90 individual samples from the PVC passive sampling. We sampled from spring through fall of each year in temperatures ranging from 10.1°C to 31.5°C and a humidity ranging from 35% to 95% (Fig.4). Passive sampling has yielded 2.57 frogs per sampling event while active sampling yielded 5.89 per event. The species accumulation curve for site 1 is nearing asymptote while the one for site 2, which has fewer sampling occasions is still rising (Fig. 5). Species abundance (Fig. 6) at all 3 sites indicates greater encounters with green tree frogs (Hyla cinerea), green frogs (Rana clamitans), and leopard frogs (Rana pipiens). PCR testing has been completed on 97 samples to date and has yielded one positive result, a green tree frog (Hyla cinerea) (Fig. 7) found in a hanging t-pipe at site 1 on October 18, The frog was caught in 28.7°C air temperature with 83% humidity and was in good condition. The frog had a SUL of 18.5mm indicating that it was a small adult. Figure 6: Amphibian species accumulation curves for sites 1 & 2. Figure 4: Average humidity at sampling locations. Figure 5: Relative abundance of species at sites 1 & 2. Figure 1: Night sampling at site 3, spring Figure 7: Green tree frog being swabbed.