2013 White-nose Syndrome Workshop

Oral Presentation Abstracts Summer surveillance for Geomyces destructans on bats using contaminated hibernacula: implications for timing of transmission A. Ballmann1*, M. Torkelson1, D. Blehert1, E. Bohuski1, M. Verant1, C. Meteyer1, and R. Russell1

1US Geological Survey – National Wildlife Health Center Persistence of viable Geomyces destructans (Gd), the pathogenic agent responsible for white-nose syndrome (WNS), in hibernacula [Lorch et al. 2012 Mycologia] and the continuous or periodic use of some contaminated hibernacula by bats throughout the summer pose a potential risk for exposure from an environmental reservoir. Bats exposed to viable Gd in late summer could transport the fungus to other bats via direct contact [Lorch et al. 2011 Nature] or to other locations well-suited for the growth and maintenance of the fungus. In order to explore transmission risks outside of the winter season, this cross-sectional study examined late summer occupants at select hibernacula confirmed to be contaminated with Gd as potential sources for Gd transmission in the fall. Wing swabs from 618 bats, representing 8 species, were collected between July 18 – August 22, 2012 from 8 hibernacula (6 Gd contaminated, 2 presumably “clean” sites) located in the Ohio River Valley region. Feces were collected opportunistically from individual bats. In addition, environmental samples (cave sediment and wall swabs) were collected from multiple areas within each site to assess for Gd presence and viability. Finally, swabs collected from research equipment and personnel at the conclusion of each trap night will be assessed for the presence of Gd to evaluate the risk of cross-contamination among bats related to late summer/fall trapping activities conducted at hibernacula within the WNS endemic region. Sample analyses are on-going. Thus far, molecular testing has detected Gd on a small number of bats at several hibernacula and viable fungus on a bat has been demonstrated during late summer. The low recapture rate of marked bats at all locations on subsequent trap nights suggests a high degree of movement among bats using these sites in late summer and indicates that fall swarm activity could play an important role in pathogen dissemination on the landscape. Pseudogymnoascus [=Geomyces] destructans in the Environment Hazel A. Barton1* and Hannah T. Reynolds1 1University of Akron Since its initial description, questions have surrounded the origin of Pseudogymnoascus destructans. While phylogenetic evidence points to an origin in Europe, a question remains as to how the fungus first entered the European bat population. With its close relationship to other soil-dwelling fungi, we examined whether P. destructans expressed the necessary phenotype for an environmental lifestyle. Our results demonstrate that P. destructans possesses many of the enzymes necessary for saprophytic growth, as well as lipases and hemolysins that could aid in pathogenesis. Our work also demonstrates that a saprophytic lifestyle is possible for P. destructans, albeit at a reduced rate when compared to environmental Pseudogymnoasci andGeomyces. This saprophytic growth allows P. destructans to be amplified in the environment and has significant implications for epidemiology. The reduced capacity of P. destructans for saprotrophy suggests that the pathogen has adapted to its host, suggesting that P. destructans has had a long coevolutionary history with its host.

What Proportion of the Bats that occur at the Entrance of Caves and Abandoned Mines Hibernate there? Hugh Broders1* and Lynne Burns2 1Saint Mary’s University, 2Dalhousie University In the late summer and early fall in temperate areas, large numbers of bats congregate at the entrances of caves and abandoned mines which are used during the winter as hibernacula. There are several hypotheses to explain these swarms including courtship and mating, and information transfer about potential hibernating sites or migration routes. It is known that some bats that visit a site during swarming do not necessarily hibernate at that same site, although some certainly do. Characterization of the proportion of bats that occur at the entrance of underground sites during the spring and also during the fall swarming period that eventually hibernate there is not well known and could provide insight into the movement dynamics, site fidelity and population structure of temperate hibernating species. Thus, our objective was to compare the proportion of bats (males and females) that were tagged during spring emergence and fall swarming that actually hibernate inside the mine. At an abandoned gold mine at Rawdon, NS, Canada, we trapped and PIT-tagged 338 M. lucifugus and 224 M. septentrionalis during emergence in the spring or swarming in the fall. We have actively searched for PIT- tagged bats during each of 1-3 winter counts inside the mine from 2008-2012 where we expect that we were able to count and scan >90% of the ≈1200 bats present (pre-WNS). We have 660 individual relocation records of tagged bats during hibernation. Generally, M. lucifugus were more likely to be recaptured during hibernation than M. septentrionalis and, for both species, males were more likely to be recaptured than females.

Identification of a promising Trichoderma polysproum strain for biocontrol of Geomyces Destructans Tao Zhang1, Holland DeFiglio1, Vishnu Chaturvedi1, and Sudha Chaturvedi1* 1Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, USA. Geomyces destructans (Gd) is a cold-loving (‘psychrophile’) fungus that causes white nose syndrome (WNS) in hibernating bats. The WNS caused mass mortality of affected bats in the caves and mines of US Northeast, Midwest, South and some provinces in Canada. It is imperative that the disease cycle is interrupted in natural habitats of bats by reducing the fungal burden available for new infections. We have previously reported that Gd is highly susceptible to several antifungal drugs. However, spraying such drugs in caves and mines is not feasible, as it will be an extremely expensive preposition and these drugs will also kill other fungi, which can have undesirable consequences on the ecosystem of caves and mines. Biocontrol of plant pathogenic fungi by other soil fungi is a viable, biocompatible, and inexpensive approach for disease management in agriculture. A US FWS funded project allowed us to carry out a systematic survey of Gd for cataloging fungi found in caves and mines. To our surprise, we have isolated Trichoderma polysporum, which belongs to a class of well-known biocontrol fungi. Our preliminary observations indicated that the indigenous T. polysporum impedes the growth of Gd in the laboratory and it is able to grow between 6˚-15˚ C, a property so far not recorded in literature. Of particular interest, metabolites produced by T. polysporum were antagonistic to Gd, but not against closely related fungus G. pannorum. We also found that T.

polysporum secretes inhibitory metabolites in the medium when grown at 6˚-15o

C. These

metabolites appears to be fungicidal in nature, as 1,000 Gd spores inoculated in medium containing 10% of the T. polysporum extract neither germinated nor produced a single colony-forming unit upon culture on solid medium. Next, we determined if active metabolites in the T. polysporum extract

are proteins or a mix of proteins, lipids and carbohydrates. The extract exposed to heat at 45o C or treated with proteinase K had similar inhibitory activity against Gd as control extract indicating that the inhibitory metabolites are a mixture of compounds. Further experimental data based on high performance liquid chromatograpy (HPLC) combined with Gd inhibition activity detection indicated that there are presumably two mixture of compounds in T. polysporum extracts, i.e. high polarity compound(s) with higher inhibition activity to Gd; and low polarity compound(s) with lower inhibition activity to Gd. Further work is underway to determine the precise nature of these active compounds in T. polysporum extract by HPLC, Mass Spectrometry and NMR analyses. We are also conducting genome mining for relevant gene(s) or gene cluster. The ultimate aim of this investigation is to engineer a T. polysporum strain for use in field condition to control the spread of Gd and break the WNS disease cycle. Bacterially mediated contact-independent antagonism of Geomyces destructans Christopher T Cornelison1* 1Applied and Environmental Microbiology, Georgia State University The recently identified causative agent of White Nose Syndrome (WNS), Geomyces destructans, has been responsible for the mortality of 5.7 million North American bats since its emergence in 2006. A primary focus of the National Response Plan established by the US Fish and Wildlife Service in 2011 was the identification of biological and chemical control agents. In an effort to identify potential biological and chemical control agents for WNS 6 previously described bacterially produced volatile organic compounds (VOCs) and multiply induced Rhodococcus rhodochrous DAP96253 were screened for anti-Geomyces destructans activity. Geomyces destructans spores and mycelial plugs were exposed to the VOCs and induced Rhodococcus in a closed air space at 15°C and 4°C and evaluated for inhibition of spore germination and radial growth. Subsequently, in situ application methods for induced Rhodococcus such as fixed cell catalyst and fermentation cell paste in non-growth conditions were screened with positive results. Additionally, Rhodococcus was assayed for ex vivo activity via exposure to bat tissue ex-plants inoculated with G. destructans spores. All VOCs inhibited radial growth of mycelial plugs and growth from spores at both temperatures at concentrations as low as 4µM. The greatest observed inhibitory effect of the VOCs was at low temperature (4°C). Induced Rhodococcus completely inhibited growth from spores at 15°C and had a strong fungistatic effect at 4°C. Additionally, induced Rhodococcus inhibited Geomyces destructans growth from spores when cultured in a shared air space with bat tissue explants inoculated with Geomyces destructans spores at 7°C. The identification of bacterially produced VOCs and inducible biological agents with anti-Geomyces destructans activity expands the pool of potential biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Geomyces destructans Infection across North America Winifred F. Frick1*, Tina Cheng1, Kate Langwig1, Kevin Drees2, Amanda Janicki3, Gary McCracken3, Jeff Foster2, and A. Marm Kilpatrick1 1University of California, Santa Cruz, 2Arizona University, 3University of Tennessee, Knoxville Transmission dynamics of Geomyces destructans in wild bats remain poorly understood. Determining prevalence, intensity of infection, and transmissibility among individuals in wild bat populations aids development of effective management strategies for controlling spread and mitigating impacts of WNS. We assessed prevalence of Geomyces destructans infection by swab sampling bats in 20 states across enzootic, epizootic, and leading edge regions in North America during the 2011-2012 and 2012-2103 winter seasons. Prevalence varies by species in highly impacted regions even within the same hibernacula and by region depending on time since WNS first detected. We also compare infection prevalence and intensity on bats to hibernacula wall substrates. By non-invasive swab sampling of multiple individuals at sites, we are able to provide early detection methods of presence of G.destructans before disease symptoms of mortality and visible infection are apparent. Our results are useful to track spread of G. destructans at a continental scale and determining factors associated with risk of arrival of G. destructans, disease progression, and impacts to populations. Understanding the critical recovery phase of white nose syndrome Nathan W. Fuller1*, Liam P. McGuire2, Heather W. Mayberry2, Paul M. Cryan3, and Craig K.R. Willis2 1Boston University, 2University of Winnipeg, 3US Geological Survey Most research on effects of white nose syndrome (WNS) has focused on the pathophysiology of winter mortality and corresponding effects of WNS on hibernaculum populations. However, a fundamentally important time for survivors of the disease, that has received little attention, is the period immediately following emergence from hibernation. During this critical time, survivors face a significant physical and physiological challenge as they migrate to summer habitats, potentially begin gestation in the case of reproductive females, and begin to recover from wing damage. To improve understanding of the healing process following infection, we collected bats from WNS-positive sites in eastern Canada and transported them to a captive facility where they were monitored during recovery. Both UV and white fluorescent light photographs were taken at regular intervals and 6 mm wing punches were collected at days 0, 3, 5, 7, 14, 25 and 40 post-hibernation to document healing on both macro- and microscopic scales. We also used a standardized protocol to swab the forearms of bats for qPCR analysis of fungal load, and obtained wing surface lipid samples at each sampling time. Our study confirms that WNS-related wing damage gets more severe in early spring, immediately after bats emerge and healing begins but is dramatically less apparent by 40 days post-emergence. This suggests that many, though not all, bats which survive to emerge are able to quickly recover from the effects of WNS. We also report new data on the relationship between healing and Gd prevalence during the healing phase. Our results have implications for WNS management and surveillance strategies and suggest that the early spring healing phase is critical to individual survival and population dynamics. Environmental conditions inside winter hibernation sites predict white-nose syndrome mortality in bats David T. S. Hayman1, 2*, Paul M. Cryan3, Juliet R. C. Pulliam2, 4, 5, Colleen T. Webb1 1Department of Biology, Colorado State University, 2Department of Biology, University of Florida,

3U.S. Geological Survey, Fort Collins Science Center, 4Emerging Pathogens Institute, University of Florida, 5Fogarty International Center, National Institutes of Health White-nose syndrome (WNS) is a fungal disease of bats that is causing a devastating epizootic across eastern North America. The population declines for several bat species are unprecedented and a few once-numerous species are thought to be at risk of extinction. Geomyces destructans, the fungus that causes the disease, grows well in cool conditions, such as those characteristic of bat hibernacula. Bats hibernating in sites with higher humidity may also decline more rapidly after the arrival of G. destructans. Thus, incorporating temperature and humidity-dependent fungal growth into models to predict bat mortality is critical if these key features of fungal ecology are predictors of bat mortality. We use mathematical models of the energy requirements of over-wintering bats at different hibernacula temperatures, and fungal growth rates at different temperatures and levels of ambient humidity, to predict bat mortality from WNS. Although reasons for observed differences in mortality of hibernating bats due to WNS have been unclear, our results reveal that WNS mortality may be closely associated with hibernation sites that are both warmer and wetter, the type used by the most affected species. Our findings underscore the importance of environmental monitoring during WNS surveillance, and may allow for the prediction of which sites and species might be most affected by disease. Band Retention And Band-related Mortality in the Little Brown Bat, Myotis lucifugus Alan Hicks1*,Allen Kurta2, Tom Ingersoll3, John Paul White4, Bill Scullon5, Joseph Kath6, Jonathan Reichard7 1Vesper Environmental LLC, 2Eastern Michigan University, 3NIMBioS, University of Tennessee, Knoxville, 4Wisconsin Department of Natural Resources, 5Michigan Department of Natural Resources, 6Illinois Department of Natural Resources, 7U.S. Fish and Wildlife Service Accurately assessing the status of bat species in regions depleted by WNS requires a clear understanding of survival, recruitment, and disease-related behavioral changes. All these estimates require that we be able to uniquely mark and subsequently recapture individual bats; the two options for marking are split ring bands or passive integrated transponders (PIT). For either of these methods, former standards for acceptable risk to bats that were applied when species were common are no longer valid today. We must clearly understand the risks to bats associated with the markers and their application, and from subsequent efforts to gather recapture data. In late March and early April 2012 we applied 2.9 mm lipped aluminum bands (Porzana LTD, Sussex, England) to 1,469 bats within 7 WNS- free hibernacula in Illinois, Wisconsin and Michigan, to determine band retention and band- related morbidity and mortality rates. Half of the bats were banded on one wing and half on both wings. Bands were applied by multiple individuals using banding pliers to improve the consistency of application. Banding sites and nearby hibernacula were revisited in early December 2012 and 568 banded bats were identified. Banding sites were again visited in late March 2013 and crews once more recaptured all accessible banded bats. We discuss the results of these recapture efforts and the role of banding in monitoring the survivors of WNS. Beneficial bacteria on bats inhibit the growth of Geomyces destructans Joseph R. Hoyt1, Tina L. Cheng1, Kate E. Langwig1, Winifred F. Frick1, A. Marm Kilpatrick1

1University of California, Santa Cruz

White-nose syndrome threatens several hibernating bat species with extinction, and at present, there are no known effective treatments available. Our study shows that bacteria found naturally occurring on bats can inhibit the growth of Geomyces destructans and have promise for development as a biocontrol. Bacteria were cultured from swabs collected from multiple bat species in the field during hibernation and then challenged against Geomyces destructans in the lab. There were nine bacteria cultured from the swabs that showed inhibition of Geomyces destructans growth. We conducted two experiments to help determine a candidate bacterium. The first experiment looked at the strength and duration of inhibition produced by each bacterium, and the second experiment looked at effectiveness of the bacteria at different concentrations. This work shows that bacteria naturally found on bat skin can inhibit the growth of Geomyces destructans and could potentially be used as a biocontrol on live bats to reduce the mortality associated with white-nose syndrome. The Effect of Temperature and Infectious Dose on White-nose Syndrome in Captive Myotis lucifugus Joseph S. Johnson1*, Ken Field1, Jim McMichael1, Melissa Meierhofer1, Daniel Stern1, and DeeAnn Reeder1 1Bucknell University, Lewisburg, PA. Since the discovery of white-nose syndrome (WNS) in North America in 2006, much has been learned about how infection with the fungus Geomyces destructans (Gd) leads to large-scale mortality among cave-hibernating bats. Much remains unresolved; however, including how variation in environmental conditions and level of exposure to the fungal pathogen promote differences in mortality in infected bats. To better understand WNS mortality rates, we collected 71 male and 77 female little brown bats (Myotis lucifugus) from the wild and placed them into artificial hibernacula. Captive bats were randomly placed into control and experimental groups artificially hibernated at 4° and 10° C. At each temperature, 14-15 bats were inoculated with a control solution or a solution containing 500, 5000, 50000, or 500000 fungal spores. Bats were left in hibernation for 21 weeks to assess the influence of temperature and infectious dose on the progression of WNS and resultant mortality. Data presented will include analyses of mortality, decline in body condition, rate of periodic arousals from hibernation, and wing damage as influenced by sex, body condition entering hibernation, hibernacula temperature, and infectious dose. These data will provide important insights into patterns of mortality among wild populations of little brown bats in eastern North America, and help to understand future population declines as WNS continues to spread.

Seasonal Transmission Dynamics of Geomyces destructans Kate E. Langwig1*, Winifred F. Frick1, Kevin P. Drees2, Rick Reynolds3, Thomas H. Kunz4, Jeff T. Foster2, A. Marm Kilpatrick1 1University of California Santa Cruz, 2Northern Arizona University, 3Virginia Department of Game and Inland Fisheries, 4Boston University Understanding seasonal changes in Geomyces destructans transmission on multiple host species will allow for targeted disease management that may reduce the consequences of the recently emerged disease, white-nose syndrome. Differences in transmission among species may drive differences in impacts, and ultimately determine which species will persist or be driven extinct from disease. In addition, faster spatial spread of the pathogen will be observed if transmission occurs from mother to offspring during the summer, or during fall swarm when bats are highly mobile. We investigated seasonal transmission of G. destructans. Swabs from exposed wing and muzzle tissue of bats were

collected and analyzed using real-time quantitative PCR. Bats became infected when returning to hibernacula during the fall, but infectious loads were very low until bats entered hibernation. We found strong evidence of intense transmission during the winter, and by late winter nearly all bats were infected. For some species, pathogen loads increased significantly throughout hibernation, and females were more likely to be infected than males, possibly due to earlier entry into torpor. Pathogen prevalence and loads on little brown myotis decrease rapidly upon emergence from hibernation, suggesting that hibernacula serve as the source of the yearly epidemic. We found that infected bats in the summer were more likely to have a high wing damage scores (Reichard and Kunz) than uninfected bats. There was, however, limited support to suggest that summer is an important period for G. destructans transmission. Changes in pathogen loads coupled with early timing of infection may be important factors in WNS mortality, and drive local transmission. The Canadian Response to White-nose Syndrome Frederick Leighton1* 1Canadian Cooperative Wildlife Health Centre As is the case elsewhere, responding to White-nose Syndrome (WNS) has challenged Canada’s capacity and resources. Only about 60 people across the country are actively engaged with this issue and, for many of these, it is a secondary or tertiary level of engagement. Management of bat populations is the responsibility of provincial (10) and territorial (3) governments; there is no federal government mandate except on federally-owned lands. In 2010, a small core of government, university and NGO biologists initiated periodic teleconferences to share information about WNS and to seek advice and collaborations. This ad hoc group has evolved into Canada’s Inter-agency WNS Committee, an unofficial but functional group-for-common-purpose which works under the general authority and approval of the inter-government Canadian Wildlife Directors Committee. The Inter-agency Committee developed a Canadian WNS management plan in 2011-12, and then, at a 4-day workshop in November 2012, a set of comprehensive action plans to achieve the goals of the management plan. All such planning has been done in close consultation with the American response program. In January 2013, a set of priority actions was extracted from the seven Action Plans and a loose administrative structure to foster collaborative implementation of these priority actions was established. This structure includes three small scientific technical working groups to coordinate and advise on 1) surveillance and diagnostics, 2) population monitoring and 3) mitigation and two other working groups on 4) communication and outreach and 5) data management to support the work of the scientific committees and interact with other stakeholders. To achieve harmonized continental response to WNS, participants on these working groups include at least one member of each of the seven US Action Plan groups. The Canadian Cooperative Wildlife Health Centre serves as secretariat and coordinator for the Inter-agency Committee and its working groups. This system of a national committee and 5 working groups is designed to be sustainable among the small number of active WNS workers in Canada and to facilitate collaboration and best uses of resources. In 2012-13, WNS was detected in several new locations in eastern Canada as were substantial declines in previously-affected populations. No westward extension of the range of WNS or of Geomyces destructans in Canada was detected as of 1 June 2013. Little Brown, Northern Long-eared and Tri-colored bats have been given endangered status in Ontario and this is anticipated soon in Nova Scotia. National status as endangered species by Environment Canada has been recommended by COSEWIC, but, as yet, there has been no response to this recommendation by the Minister.

Understanding Genetic Mechanisms of Pathogenicity: Why can one fungus cause so much trouble when its close relatives do not? Daniel L. Lindner1*, Andrew Minnis1, Jeff Lorch2, Jonathan Palmer2, David Blehert3

1US Forest Service, 2University of Wisconsin, Madison, 3US Geological Survey-National Wildlife Health Center Controlling disease is almost impossible without a basic understanding of the mechanisms a pathogen uses to cause disease in the host. In the case of white-nose syndrome (WNS), a basic understanding of disease mechanisms is beginning to emerge, especially in relation to host physiology, but little is known about the fungal side of the equation. In order to better understand the mechanisms of fungal pathogenicity, we initiated a search for closely related but non-pathogenic relatives of Pseudogymnoascus destructans, the causal agent of WNS, to use for genomic comparisons. To date, the nearest relatives of P. destructans have been identified in North America, and comparisons are being made between populations of these fungi in North America, Europe and the southern hemisphere. In addition to elucidating mechanisms of pathogenicity, these genomic comparisons should also help to shed light on the potential mechanisms this fungus may use to sexually reproduce, information that will be critical to determining whether new and potentially more pathogenic versions of the fungus may arise over time.

Tracking White-Nose Syndrome and Other Threats: A Population Monitoring Program for North American Bats Susan Loeb1*, Jeremy Coleman2, Laura Ellison3, Thomas Rodhouse4 and Thomas Ingersoll5 1USFS Southern Research Station, 2U.S. Fish and Wildlife Service, 3U.S. Geological Survey, 4National Park Service, 5Department of Defense Bats in North America are facing unprecedented threats including White-Nose Syndrome, wind energy development, habitat loss and fragmentation, and climate change. Yet, there is currently no coordinated monitoring program to track changes in their populations in response to these threats. A group of scientists and statisticians from the U.S., Canada, and Mexico is developing the North American Bat Monitoring Program (NABat), a framework for bat monitoring across North America. NABat will provide the statistical, biological and administrative architecture for coordinated bat population monitoring that will promote effective decision-making and long-term viability of bat populations across the continent by providing robust data on changes in bat distributions and abundance. The sampling framework will likely be comprised of a nested grid consisting of 50 km cells as primary units and 10 km cells as secondary units. A spatially balanced design will be used to select grid cells so that unequal and changing survey efforts can be accounted for in design-based and model-based population estimates of status and trend. The primary data sources for the monitoring program will be maternity and hibernacula counts and acoustic data collected along driving transects or at stationary points across the landscape. Data will be housed and managed by the Bat Population Data (BPD) Project, an initiative of the USGS Fort Collins Science Center to foster data sharing and collaboration through a web-based bat population data management application. A newly described non-lethal skin infection of hibernating bats can be mistaken for white-nose syndrome

Jeffrey Lorch1*, Andrew Minnis2, Carol Meteyer2, David Redell3, Jennifer Redell3, J. Paul White3, Heather Kaarakka3, Laura Muller1, Michelle Verant2, Daniel Lindner4, and David Blehert2 1University of Wisconsin, Madison, 2U.S. Geological Survey-National Wildlife Health Center, 3Wisconsin Department of Natural Resources, 4US Forest Service White-nose syndrome (WNS) is a disease of hibernating bats caused by the fungus Geomyces destructans. Prior to the discovery of WNS in 2007, we are unaware of reports of bats with fungal skin infections (dermatitis) during hibernation. However, individuals of Myotis lucifugus (little brown bat) and Perimyotis subflavus (eastern pipistrelle) with grossly visible white material on their wings (and occasionally on the ears and/or muzzle) that superficially resembles clinical signs of WNS were recently reported from Wisconsin, a state in which G. destructans has not yet been identified. Tape impressions, culture, direct DNA amplification and sequencing, and histologic examination were conducted on wing skin from these bats, and fungi were consistently associated with the observed dermatitis. We determined that these fungi represent at least two undescribed species within the genus Trichophyton, a group that contains known dermatophytes. An additional twelve bats submitted to the U.S. Geological Survey National Wildlife Health Center for diagnostic testing due to observation of suspect fungal skin infection, but that tested negative for G. destructans, were re-examined to determine whether Trichophyton spp. may have been associated with these skin infections. Amplification and sequencing of fungal DNA from three of these bats confirmed presence of an identical Trichophyton spp. on animals from Indiana and Texas, suggesting the fungus may be widely distributed in the central U.S. Furthermore, bats from hibernacula in Wisconsin known to harbor infected animals have maintained stable populations without reported mortality since the dermatitis was first noticed in 2010, indicating that these Trichophyton spp. do not pose a lethal threat to North American bats. Rather, the fungi may be endemic to the region, but had been largely overlooked prior to emergence of WNS. Although Trichophyton dermatitis may superficially resemble WNS, laboratory and histopathologic analyses can easily distinguish between Trichophyton spp. infections and those caused by G. destructans. Intraspecific Lipid Profiling of Bats and Changes during White-Nose Syndrome Progression

Evan Pannkuk1*, Nathan Fuller2, Hannah Blair3, David Gilmore3, Brett Savary3, and Thomas Risch3 1Arkansas State University, Jonesboro, 2Boston University, 3Arkansas Biosciences Institute

The microbial community colonizing skin obtains nutrients from the host integumentary matrix. Mechanisms responsible for microbial integumentary molecule acquisition may provide targets for disease prophylaxis. Lipid profiling provides evidence at a molecular level of initial nutrient levels in a host/pathogen system. Furthermore, differences in lipid profiles contribute to understanding of taxonomy, life history, and clinical signs of disease. We obtained lipid profiles for 13 species of bats and compared necrotic vs. healthy Myotis lucifugus wing tissue. Bat sebaceous lipids are a complex mixture of glycerolipids, sterols, wax/sterol esters, squalene, and free fatty acyls (FFAs). Bat fatty acid methyl ester profiles are species specific. Lipid localization was observed on wing tissue by imaging mass spectrometry. M. lucifugus wing tissue damaged from Pseudogymnoascus destructans infection resulted in lower total lipid amount but increases in lysophospholipid proportion. These results indicate that cellular membranes are degraded during white-nose syndrome (WNS) progression resulting in altered chemical properties of the protein/lipid matrix comprising wing integument. Further experimentation should highlight differences in the physical properties of damaged cellular membranes including polarity, enzyme diffusion, and fungal response to these compounds.

Modelling Geomyces destructans Distribution in North America and Eurasia Using Ecological Niche Modelling: What Can we Learn? Sabastien Puechmaille1* and Hugo Rebelo2 1University College Dublin, 2Center in Biodiversity and Genetic Resources White-nose syndrome (WNS), an emerging infectious disease caused by the fungus Geomyces destructans (Gd), has been expanding year after year in North America, suggesting a recent introduction of the fungus (probably from Europe). Given the massive mortalities associated with WNS in North America, it is of prime importance to predict areas suitable for its causative agent, Gd. By modelling the occupied niche by Gd in Europe, we predicted its distribution in North America, and which ecological factors could be limiting its distribution. To achieve this, we used a species distribution modelling technique maximum entropy modelling - that has been proven to accurately predict current species distributions. To reduce uncertainties in models projections we used the reciprocal modelling approach. This way the full niche of the species is considered (including data from both Europe and North America). We also calculated the similarities/differences in niche occupied by Gd between North America and Europe and highlight potential adaptations of Gd in North America. Finally, we determined potential dispersal routes that Gd could use to expand its current distribution. Results indicated that Gd distribution was limited by temperature variables. Model predictions comprised the currently known distribution of Gd in Europe but also areas where Gd has not yet been detected. Results showed a mismatch between realized niche in Europe and North America. This indicates that Gd is probably not occupying its entire potential ecological niche in Europe or that it recently adapted to North American conditions. Anti-Geomyces destructans Drug Candidates: Comparative In Vitro assays for Antifungal Activity Alison Robbins MS, DVM1*, Brittany Pereira1, Travis Beeler1, Donna Akiyoshi1 1Tufts Cummings School of Veterinary Medicine Hibernating bat populations continue to severely decline across North America as Geomyces destructans (Gd) infection spreads to new geographic locations and new bat species. To date there have been no successful interventions that increase survival in hibernating bats. Treatment trials in hibernating bats using the antifungal medication terbinafine applied through topical and systemic drug applications modes have not increased winter survival. Terbinafine had been reported to have significant fungistatic and fungicidal activity against Gd grown in culture. In this study we conducted in vitro trials to compare the antifungal activity of terbinafine to several other compounds as possible candidates for direct treatment of Gd infected bats or the hibernation environment. The anti-Gd drug activity of terbinafine and several other compounds was measured by using both liquid culture and direct plating techniques. Gd was grown in YNBG liquid culture medium incubated with different concentrations of the test drugs in 96 well plates at 12-13 C° for 10 -14 days. Levels of drug inhibition were evaluated by measuring the OD530 of each well using a Synergy H4 Multi-Detection Plate Reader (BioTek). Gd viability and quantitative drug inhibition results were also assessed by plating aliquots from each well containing various concentrations of each drug on to Sabouraud agar plates. After two weeks of growth, the colonies were counted and recorded. The results of in Vitro drug inhibition activity of terbinafine and other compounds will be compared and reported in this presentation.

Investigation of a terbinafine impregnated subcutaneous implant for the prevention or treatment of white nose syndrome in little brown bats (Myotis lucifugus) Marcy J. Souza1*, DeeAnn Reeder2, Brooke Hines3, Rachel Dutkosky1, Edward Ramsay1, Tim Cairns4, Kimberly Newkirk1, Sherry Cox1 1University of Tennessee College of Veterinary Medicine, Knoxville, 2Bucknell University, 3Kentucky Department of Fish and Wildlife Resources, and 4Melatek LLC No successful treatment of bats infected with Geomyces destructans (Gd) has been determined, but terbinafine, an antifungal medication, has been shown to inhibit growth of Gd in vitro. A terbinafine-impregnated subcutaneous implant that slowly releases terbinafine over the course of months has been designed and could potentially be used to treat bats infected with Gd. During the winter of 2011-2012, 40 bats were collected from the wild, while hibernating, and a terbinafine-impregnated implant was placed subcutaneously over the dorsum. Each bat received one of four different implants: 0 mg, 2 mg, 4 mg, or 8 mg of terbinafine. The bats were then kept in an artificial hibernaculum for approximately 3 months. At the completion of the trial, each bat was euthanized and the whole body was placed in formalin. Tissue samples were evaluated for histologic evidence of toxicity, and severity of fungal lesions (number of lesions per mm2 of wing and muzzle tissue) by histology, and wing tissue samples were collected and analyzed by HPLC for terbinafine concentrations. The same procedures were performed on bats that died during the trial. No evidence of toxicity was observed in any of the bats, and there were no significant differences in the number of lesions amongst treatment groups. No terbinafine was present in tissue of control bats and skin terbinafine concentrations ranged from 5 to 11,262 ng/g in the treatment groups. Further field and laboratory trials of the implant were conducted during the winter of 2012-2013 and results are pending. Does Gd suppress fungal diversity on hibernating bats? A comparison of pre-and post WNS fungal associations on bats in eastern Canada K.J. Vanderwolf1*, D.F. McAlpine2, and D. Malloch2 1Canadian Wildlife Federation, 2New Brunswick Museum Data documenting fungi present on hibernating bats are still exceedingly rare, making it difficult to predict what if any impact the introduction of Geomyces destructans (Gd) might have on these assemblages. It has been suggested that fungal diversity on hibernating bats may be suppressed by the presence of Gd. However, we hypothesized no change, since with the exception of Gd, fungi are not known to use live bats as hosts or substrates. Here we compare and contrast fungal assemblages from hibernating Myotis sp. based on a unique dataset collected immediately pre- and post-WNS infection in eastern Canada, while also seeking to identify any fungi that may interact with Gd in culture. Preliminary data indicate that over-all fungal diversity on hibernating bats did not change with the introduction of Gd. Most notably, several fungal isolates examined post-WNS appear to be antagonistic to G. destructans in vitro, although the nature of these interactions is still under study. Cave walls were also sampled for fungi. The fungal diversity on cave walls post-WNS was similar to that found on bats; Gd was frequently cultured from both substrates. Gd was also cultured from arthropods in caves post-WNS, although their role as vectors is likely minor. Assessment of physiological effects of white-nose syndrome in bats using doubly labeled water and clinical chemistry

M. Verant1*, J.R. Speakman1, C.U. Meteyer1, P.M. Cryan1, D.S. Blehert1 1US Geological Survey – National Wildlife Health Center The physiological effects of white-nose syndrome (WNS) and mechanisms of mortality due to infection with Geomyces destructans in hibernating bats are not fully understood. Associated wing damage is hypothesized to disrupt vital physiological functions, and preliminary evidence indicates increased arousal frequency, water loss, and electrolyte imbalances occur. We used the doubly labeled water (DLW) method in hibernating bats infected with G. destructans in the laboratory to evaluate the role of water balance and energy use in WNS pathogenesis. Blood chemistries were analyzed to assess physiologic disturbances. Preliminary analyses indicate bats with WNS increased total body water content over the course of the experiment compared to sham-treated bats, supporting the observation that bats utilize fat reserves at a faster rate when infected with G. destructans. Additionally, bats with WNS demonstrated a primary respiratory acidosis characterized by elevated levels of carbon dioxide (PCO2) and lower blood pH than controls. Renal compensation, evidenced by elevated bicarbonate (HCO3-), was not sufficient to normalize blood pH. Potassium concentrations were also higher in infected bats. There were no differences in sodium concentrations, but chloride was marginally lower in infected bats likely due to secondary renal excretion. Blood glucose levels were also lower in infected bats, but there was no evidence of lactic- or keto-acidosis. Although normal blood chemistry reference values for microchiropterans do not exist, significant differences observed between infected and control bats in this study demonstrate increased energy use among bats with WNS together with life-threatening respiratory acidosis, providing further insight into a mechanistic explanation for mortality during hibernation. Does chitosan combat growth of Geomyces destructans and prevent White Nose Syndrome? An experimental test Maarten J. Vonhof1*, Timothy C. Carter2, M. Kevin Keel3, Joseph S. Johnson4, and DeeAnn M. Reeder4 1Western Michigan University, 2Ball State University, 3National Park Service, 4Bucknell University As white-nose syndrome (WNS) continues to increase in prevalence and expand across North America from the original locus, there is an urgent need to develop mechanisms and strategies to reduce mortality rates and limit transmission to new regions. We tested the efficacy of the non-chemical, naturally occurring biopolymer chitosan for the prevention and treatment of WNS. Chitosan is a powerful antimicrobial (antibacterial and antifungal) agent, has extensive wound-healing properties, and is fully biocompatible, biodegradable, and nontoxic. Our preliminary tests of serial dilutions of two different chitosan preparations showed that one preparation exhibited strong antifungal properties against Gd (84% inhibition) at the highest concentration we tested in standard fungal plating assays. We then tested this preparation on experimentally-infected little brown bats in vivo to determine its influence on fungal growth, bat arousal behavior, and the histopathology of WNS. Little brown bats were collected at a hibernaculum in Illinois and brought into the lab where we set up three experimental treatments with Gd-infected bats that received topical application of chitosan either one or two times (single treatment of chitosan early in the hibernation season, single treatment late, and double treatment early + late) and three control groups (infected + untreated, infected + solution control, uninfected + chitosan). Bats were outfitted with modified iButton temperature data loggers and were monitored continuously with video cameras. At the end of the experiment we quantified the reduction in fungal growth and tissue damage, fat loss, and arousal frequency and duration relative to

infected but untreated controls. We will present the results of these experiments and discuss the utility of chitosan as a potential treatment for WNS. Understanding hibernation using PIT tags and temperature telemetry: Update from Central Canada K.J.O. Norquay1, Z. Czenze1, M.A.Collis1 and *C.K.R. Willis1* 1Department of Biology, University of Winnipeg White-nose syndrome (WNS) disrupts torpor/arousal cycles and, therefore, energy balance during hibernation. As a result, one way of thinking about WNS is as a disease of winter endurance; Bats with the largest fat reserves, the greatest ability to endure deep, long torpor bouts and the ability to hibernate for the longest duration could be the most likely to survive. Despite the importance of understanding these critical aspects of hibernation, both before and after WNS, little is known about relationships between body condition, torpor/arousal patterns and hibernation phenology (i.e., the timing of immergence into and emergence from hibernation). To address these knowledge gaps, and in preparation for the arrival of WNS, to date we have marked nearly 5,000 little brown bats with passive transponders (PIT tags) and are remotely recording the arrival and departure of individuals at hibernacula throughout a 30,000+ km2 study area in central Canada. To address specific questions about hibernation energetics and clustering we have also recorded body temperatures of bats using temperature telemetry and clustering behavior using infrared camera traps. Here we report new data testing the hypotheses that body condition, demography and hibernaculum characteristics (e.g., temperature and humidity) influence: 1) torpor/arousal cycles during hibernation; 2) clustering behavior during hibernation and 3) hibernation phenology. We also report new data illustrating the potential benefits and limitations of PIT tags as a method to monitor bat populations.

2013 White-nose Syndrome Workshop Poster Abstracts

Acoustic Bat Inventory on National Wildlife Refuges in Eastern Oregon, Eastern Washington, and Idaho Jenny Barnett1 1US Fish and Wildlife Service, Inventory and Monitoring Initiative In 2012, the Region 1 Inventory and Monitoring Program, U. S. Fish and Wildlife Service initiated an acoustic inventory of bats. The work focused on National Wildlife Refuges in eastern Oregon, eastern Washington, and Idaho. Project objectives included developing protocols for acoustic bat detection on National Wildlife Refuges (NWR) ; documenting occurrence, distribution, and activity rates of bat species on NWRs; and providing baseline data for monitoring future impact of White-nose Syndrome. Sampling protocol was aligned with the Oregon/Washington Bat Grid to facilitate use of locally collected data in regional analysis. Pettersson D500x full-spectrum bat detectors and SonoBat software were used to record and analyze bat calls. Bat detectors were deployed on 13 Refuges in 2012. Sampling continues in 2013, with an additional 4 refuge participating. Thirteen species of bats were detected in 2013. Project methodology and preliminary results will be presented. Changes in Winter Activity of Bats in the Great Smoky Mountains National Park, Tennessee, Due to White-nose Syndrome Riley Bernard1, James Carr2, and Gary McCracken1 1University of Tennessee Knoxville; 2Great Smoky Mountains National Park, USA The White-nose syndrome (WNS) epizootic, characterized by the psychrophilic fungus Geomyces destructans (Gd), has caused unprecedented mortality of hibernating bats throughout the Northeastern United States. Gd was first recorded in the Great Smoky Mountains National Park (GSMNP) during winter 2009/2010 and bats were confirmed WNS positive in winter 2011/2012. By January 2013, Park rangers began receiving numerous reports regarding daytime activity of bats within the Park, including contact with a Park visitor. The goal of this research is to evaluate possible behavioral changes of bats during winter hibernation following progression of WNS. We hypothesize that WNS affected caves in southern latitudes may experience delayed mortality due to warmer ambient temperatures during hibernation. Using ultrasonic detectors and temperature data we have established that bats in GSMNP are active throughout the winter. Bat calls were recorded during each acoustic survey night (140 sample nights) regardless of low nightly temperatures. By year three of WNS confirmation, we see a significant increase in activity levels during two of the coldest months of the year (January: t-ratio -4.36, p < 0.000 and March: t-ratio 2.7, p < 0.011), suggesting a time lag in mortality due to WNS when compared to affected regions in the Northeast. Although we have documented this trend in GSMNP, we have yet to see a precipitous change in winter activity levels of bats at other caves in Tennessee. Analysis of pigment produced by Geomyces destructans in response to control agent exposure

Bacterially mediated contact-independent antagonism of Geomyces destructans Christopher T Cornelison1 and Kyle Gabriel1 1Georgia State University Fungi commonly produce pigments in response to diverse cellular and environmental stimuli. The pigment melanin is produced by a number of fungi, providing protection from photo- radioactive- and chemically-induced oxidative damage and increasing pathogenicity as a virulence factor in select vertebrate pathogens. Geomyces destructans has been observed exuding diffusible pigment into its growth media and producing pigmented conidia after exposures to several bacterially-produced antifungal VOCs, and the bacterial control agent Rhodococcus rhodochrous DAP 96253. Growth on media supplemented with L-DOPA, a melanin precursor, has induced drastic mycelial, conidial, and media-diffused pigmentation confirming G. destructans melanogenic potential. Hyaline G. destructans and purified synthetic melanin were compared to control agent-exposed G. destructans and extracts with the use copper sulfide-silver staining with light microscopy and ultraviolet-visible (UV-VIS) spectroscopy. These analyses suggest melanin to be the main constituent of the pigment extracts. Purification by thin layer chromatography (TLC) and analysis by Infrared Spectroscopy and nuclear magnetic resonance (NMR) spectroscopy are currently underway to identify the particular chemical composition of the pigment. The role of melanin as a virulence factor in other vertebrate-pathogenic fungi such as Cryptococcus neoformans warrants additional investigation to determine the role of this secondary metabolite in the manifestation of White-Nose Syndrome in bats. Winter Activity of Four Bat Species at Four Desert Hibernacula in Idaho Bill Doering1 and Jericho Whiting2 1Power Engineers, 2Gonzales-Stoller Surveillance Documenting activity patterns of hibernating bats in western North America is important for understanding the behavioral ecology of these mammals, especially before white-nose syndrome potentially affects some of these species. Activity patterns of bats during winter in western North America, however, are poorly understood. We acoustically monitored 4 caves (Middle Butte, Rattlesnake, East Boundary and Aviator caves) that are important hibernacula (up to 705 hibernating bats) in southeastern Idaho during winter (November to March) from 2011 to 2013. At those caves, AnaBat detectors were set for 234 sampling nights during 2011 to 2012 and for 320 sampling nights during 2012 to 2013. We used filters in AnaLookW to quantify bat passes (sequences ≥ 2 calls separated by ≥ 1 second), and documented western small-footed myotis (Myotis cilolabrium), Townsend’s big-eared bat (Corynorhinus townsendii townsendii), big brown bat (Eptesicus fuscus), and the little brown myotis (Myotis lucifugus) flying outside of caves during mid-winter. Activity was highly sporadic and differed among species, with western small-footed myotis being most active during winter (bat passes = 2,303), followed by Townsend’s big-eared bat (bat passes = 694). We recorded the highest diversity (4 species) and the most activity (3,051 bat passes) at Middle Butte Cave. Our study documents normal activity patterns outside of caves for mutiple species of bats during hibernation. These results improve our understanding of the behavioral ecology of these species in western North America prior to the potential arrival white-nose syndrome, and the possibility of this disease altering winter behavior of these mammals.

Quality Control for qPCR Detection of Geomyces spp. in Hibernaculum Sediment and Bat Feces Kevin Drees1*, Colin Sobek1, Jeffrey Foster1 1Center for Microbial Genetics and Genomics, Northern Arizona University Fungi of the genus Geomyces are common members of soil microbial communities in cold climates. Geomyces destructans has been cultured and detected with molecular methods in the sediments of hibernacula affected by White-Nose Syndrome (WNS). Also, bats groom themselves after hibernation, and may pass G. destructans in their feces, a potential vehicle for introducing G. destructans into new hibernacula, and cave sediment may serve as a reservoir for the organism. It may be possible to detect G. destructans in these materials in advance of an outbreak and take steps to prevent it. DNA extractions from both soil and feces are complicated by the presence of a large quantity of matrix material and PCR-inhibitory molecules, reducing DNA yields and assay sensitivity. Furthermore, target DNA may be at a low concentration in samples. In order to control for these phenomena, we developed quality control procedures for use with a qPCR assay including optimizing a bead-based DNA extraction method to maximize DNA yield from Geomyces fungi in sediment and feces, a quantified Geomyces spp. conidia preparation with extractions to determine percent yield of target DNA, optimization of bovine serum albumin to minimize PCR inhibition, use of an endogenous qPCR control to detect PCR inhibition, and development of a synthetic positive control to detect cross-contamination of control DNA on a qPCR plate. All of these methods should lead to more accurate, sensitive, and reliable detection of G. destructans from environmental sources. Tracking the Arrival and Spread of Pseudogymnoascus destructans and White-nose Syndrome in Missouri Anthony G. Elliott1, Shelly N. Colatskie1, Dr. Winifred F. Frick2, and Amanda Janicki3 1Missouri Department of Conservation, 2University of California Santa Cruz, 3University of Tennessee Missouri is home to the six bat species having suffered mortality from White-nose Syndrome (WNS) plus the endangered gray bat, known to contract WNS, and the southeastern myotis which has tested positive for presence of Pseudogymnoascus destructans (Pd). Missouri provides a good opportunity to track the arrival of Pd and subsequent development of WNS. We have conducted extensive WNS surveillance since the winter of 2009/2010. In April 2010 we found the first sign of Pd in Pike County and in May Pd was found in Shannon County. Winter WNS surveillance was conducted in over 30 sites during the winter of 2010/2011 including the private cave in Pike County. We found no outbreaks of WNS and bats appeared healthy, but swab sampling in March 2011 detected Pd at an additional Shannon County Cave. During the winter of 2011/2012 MDC WNS we visited over 40 sites. Bats from 2 caves in Lincoln County were submitted for laboratory testing based on visible fungal growth. These cases were confirmed with WNS and were the first documented cases west of the Mississippi River. Swab samples from 2 additional counties tested positive for the presence of Pd. During the winter of 2012/2013 we conducted surveillance in over 40 sites. Nine additional caves were confirmed as WNS positive making a total of 11 caves in 4 counties in Missouri that are WNS confirmed though we have not documented any mortality. Swab samples from 2013 indicate the presence of Pd in 2 additional counties.

National Speleological Society Supported WNS Research Summary Jennifer Foote1 1National Speleological Society The National Speleological Society (NSS) is the nation’s oldest and largest organization dedicated to the study, exploration, and conservation of caves and their natural resources. The NSS has been involved at the forefront of the investigation into white-nose syndrome since its discovery in New York in 2007. Our individual members have assisted in bat field surveys, both underground during hibernation season, and above ground conducting emergence surveys and summer acoustical monitoring. They have also actively participated in state and federal WNS planning. Some of our best scientists are involved directly in lab and field research, and several of the state and federal agency personnel working on WNS are NSS members, as well. The NSS has participated in virtually all the national WNS conferences and symposia, helping underwrite the first national convening on WNS in Albany, New York in 2009. At that conference, the NSS was asked to take the lead in raising and granting money for critical WNS research, as state and federal funding streams could not react quickly enough through their lengthy budget processes. Our members stepped up to the plate, and hundreds of them have made individual contributions, totaling over $120,000, supporting 20 Rapid Response Fund grants for critical and timely white-nose syndrome research. Cryptic Geomyces destructans Infections in the Southeastern United States Amanda Janicki1, Winifred Frick2, Jeff Foster3, and Gary McCracken1 1University of Tennessee; 2University of California at Santa Cruz; 3Northern Arizona University White-Nose Syndrome is an epizootic in hibernating bats and the causal agent has been identified as the fungus, Geomyces destructans. Detection of G. destructans in the field is limited by the ability of researchers to visually document fungal growth on affected bats and submit these bats for laboratory testing. Swabbing bats for fungal DNA provides non-invasive sampling for G. destructans, and the opportunity to document the accuracy of visual observations for detecting the fungus. To determine the presence of G. destructans, bats were swabbed on their muzzle and forearm, and it was noted whether any fungus was visible. qPCR was used to detect G. destructans DNA, if present. As part of a continent-wide study, 665 bats of 7 species were swabbed at 18 hibernacula in 3 states (TN, MO, AL) in the winter of 2011-2012, and 624 bats of 10 species were swabbed at 19 hibernacula in 4 states (TN, MO, AL, KY) in the winter of 2012-2013. G. destructans DNA was detected by swabbing as early as mid-October and as late as the end of May on bats mist-netted near cave entrances. Results suggest the occurrence of “cryptic” infections; 109 bats of 6 species from the first winter of swabbing tested positive for G. destructans DNA without showing any signs of visible fungus. Only one little brown bat (Myotis lucifugus) tested negative for G. destructans DNA while showing signs of visible fungus. These data are useful for management purposes in documenting the presence of G. destructans in hibernacula. A spatio-temporal assessment of WNS effects on summer and winter bat populations in West Virginia Catherine Johnson1, Craig Stihler2, and Frederica Wood3 1US Forest Service-Monongahela National Forest, 2West Virginia Division of Natural Resources, 3US Forest Service – Northern Research Station

The West Virginia Division of Natural Resources (WVDNR) and the Monongahela National Forest (MNF) have been monitoring both winter and summer bat populations in the same area of the state for 30 and 15 years, respectively. Since the first observation of WNS at caves in Pendleton County, WV in January 2009, wintering populations of several bat species have declined precipitously across much of the state. The impact of WNS on summer bat populations has also been significant, both in terms of numbers of individuals captured at long-term mist-netting sites and demographic parameters of local populations. Information from these two sources is complementary – some species (e.g., Myotis lucifugus) are well-represented in both summer and winter surveys, some species are under-represented in counts of hibernacula that are regularly surveyed but are commonly captured during summer mist-net surveys (e.g., M. septentrionalis), and others are better-accounted for in hibernacula counts than in summer surveys (e.g., Perimyotis subflavus). We review the results of pre- and post-WNS winter hibernacula counts and summer bat surveys conducted on the MNF as WNS spread across the state of WV and compare the decline of individual bat species seen in hibernacula with that of summer populations in the same spatial areas. Post-WNS changes in demographic data that are evident from mist-net survey data, as well as the ability to attach transmitters to individuals to track movements and identify roost locations, underlines the importance of incorporating such survey efforts into a comprehensive bat population monitoring program. Potential Spread of WNS in the Pacific Northwest: Use of an Individual-based Simulation Model to Explore Epizootic Dynamics in a Bat Metapopulation. Guy R. Knudsen1, Sybill K. Amelon2, and Rita D. Dixon3 1University of Idaho, 2USDA Forest Service, 3Idaho Department of Fish and Game Framing WNS epidemiological knowledge in a predictive context helps to understand potential for disease spread in western bat populations. We use an individual-based modeling (“IBM”) approach: attributes of individual bats at discrete geographic locations are tracked over time, allowing analysis of processes where events among individuals have emergent metapopulation consequences. Knudsen and Schotzko (1991, 1999) used an IBM to simulate aphid dynamics and epizootics caused by a fungal entomopathogen. Hallam and McCracken (2011) used an IBM to show that culling symptomatic bats is an ineffective WNS control measure. IBM advantages include: the spatial structure of a system can be explicitly constructed, and there is flexibility to accommodate habitats that are spatially heterogeneous with respect to biotic and abiotic conditions, along with phenotypic heterogeneity among hosts. Also, for several eastern bat species, large winter populations converge in few hibernacula, whereas some western species occur in small colonies or colony assemblages. It is possible that smaller, more dispersed host colonies may limit ability of the pathogen to spread. Our model simulates epizootic dynamics in metapopulations of up to 107 bats, in up to 50 hibernacula. Pathogen transmission occurs by bat-to-bat contact (or from inoculum on abiotic surfaces). Healthy and latently-infected bats occasionally move between hibernacula, but infectious bats (exhibiting sporulation) do not. Simulations suggest an interplay between local pathogen transmission efficiency and inter-hibernaculum movement, which may result in rapid geographic spread of the epizootic, or, conversely, localized extinctions of both host and pathogen populations which then serve to limit epizootic spread.

The Misuse of Relative Humidity in Ecological Studies Allen Kurta1

1Eastern Michigan University Biologists often are interested in whether ambient moisture levels affect the activity of bats or their patterns of roost selection, presumably because of the decreased or increased rates of evaporative water loss. Almost invariably, field ecologists measure relative humidity and use this parameter in subsequent univariate or multivariate analyses. Relative humidity is defined as the ratio of the amount of water vapor in the air divided by the maximum amount of water vapor that could physically exist in the air (saturation vapor pressure), which is not a constant but varies with temperature. Evaporation from an animal depends on the absolute amount of moisture in the air (not the relative amount), as well as other factors, especially the animal’s surface temperature. Consequently, it is easy to demonstrate that expected evaporation from the body surface of a bat hibernating at, for example, 95% relative humidity and 2 C will be less than the evaporation expected for a bat hibernating at 95% relative humidity and 10 C. If the bat is active, with a surface temperature of 35 C, the reverse is true—greater water loss occurs at an ambient temperature of 2 C than at 10 C, even though relative humidity remains the same. Relative humidity is not a reliable predictor of evaporative water loss in variable field environments. Bats Using Caves In Southeast Alaska May Be Susceptible To White Nose Syndrome

Ellen W. Lance1 Stephen W. Lewis2 1US Fish and Wildlife Service, Anchorage, AK, 2Tenakee Springs, AK Five species of bats occur regularly in southeast Alaska: little brown bats (Myotis lucifugus), long-legged bats (M. volans), California bats (M. californicus), Keen’s Myotis (M. keenii), and silver-haired bats (Lasionycteris noctivagans); little brown bats are the most common and widely distributed species and are known to hibernate in caves in southeast Alaska. White-nose syndrome has devastated bat populations on the east coast of the United States, and the disease is spreading west and north, affecting bats west of the Mississippi River and into Ontario, Canada. It seems only a matter of time before white-nose syndrome spreads to Alaska’s borders. Geomyces destructans, the fungus that causes white-nose syndrome in hibernating bats, requires specific environmental conditions to grow. To determine the potential viability of G. destructans, we examined climate variables measured in five caves in southeast Alaska with year-round bat activity. Temperature and relative humidity was continuously measured from 1999 through 2003 in the caves located on Prince of Wales Island. Our data revealed that cave temperature and relative humidity are within the range of the thermal and moisture conditions required for G. destructans to thrive. Should white-nose syndrome migrate further west and north, Alaskan bats may be at risk. An Improved Method for Processing Bat Patagia for Histopathology Lisa A. Last1 and M. Kevin Keel2

1Southeastern Cooperative Wildlife Disease Study, University of Georgia, 2University of California-Davis, Davis, CA Histopathological examination remains the preferred method for confirmation of White-Nose Syndrome (WNS) infection. However, bats pose unique problems in processing tissues due to their small size and

the elasticity of the patagia. We present a novel method for improved processing of tissues utilizing adhesive to coat the tissues prior to rolling on a wax dowel and fixation. This method results in a tightly curled spiral that holds its shape during trimming into cross-sections and embedding, easing handling of tissues. Resultant sections are of equal or improved quality to those obtained without adhesive and there is no visible residue. The method is simple to employ and teach to others and uses inexpensive, readily available materials. It can be effectively used on tissues of varying postmortem quality from all bat species currently studied. Montana’s Bat and White-Nose Syndrome Surveillance Efforts Bryce Maxell1, Lauri Hanauska-Brown2, Amie Shovlain3, Jake Chaffin4 1Montana Natural Heritage Program, 2Montana Fish, Wildlife, and Parks, 3US Forest Service - Beaverhead-Deerlodge National Forest, 4Bureau of Land Management - Montana/Dakotas State BLM Office Montana’s bat populations face a wide array of conservation issues, including loss of roosting sites, collision and drowning hazards at sites where they forage and drink, barotrauma and collisions with wind turbines, and the potential arrival of Geomyces destructans, the cold-adapted soil fungus that causes White-Nose Syndrome and has decimated bat populations in eastern North America. These conservation issues, and the low reproductive output of bats, highlight the need to gather baseline information that can be used to mitigate impacts to populations. Beginning in the fall of 2011, a collaborative effort was initiated to document roost habitat characteristics and year-round spatial and temporal activity patterns of Montana’s bats. To-date, collaborators have deployed over 30 temperature and relative humidity data loggers near known winter bat roosts; most known bat hibernacula in Montana are now being monitored. Collaborators have also established a nearly statewide array of 48 passive ultrasonic detector/recorder stations that are deployed year-round and powered by solar panels and deep cycle batteries. Through June 2013, these recording stations have resulted in more than 1.3 million sound files containing nearly 5 terabytes of information. Highlights to-date include numerous first records of species in regions with previously limited bat survey effort, numerous first records of bat activity during the fall, winter, and spring months, documentation of temperatures at which bats are active year-round, documentation of winter bat roost temperatures, documentation of nightly activity patterns throughout the year, and the potential year-round presence of species previously considered migratory. Long-term monitoring of Eptesicus fuscus maternity colonies in Delaware; the first 5years Erin Adams1, Sarah Bouboulis1, Holly Niederriter1, Sarah Baker1 1Delaware Division of Fish and Wildlife, Species Research and Conservation Program. Delaware Division of Fish and Wildlife monitored big brown bat populations from 2009 to 2013 using volunteer emergence count data at maternity colonies throughout Delaware and by conducting catches at select benchmark sites. These projects were designed to monitor Delaware’s most abundant species in an effort to detect indirect impacts of White-nose Syndrome and to better understand and protect bats in Delaware. In 2013, counts at select sites were conducted every 2 weeks to assess the best time to conduct pre-and post-volant counts. Documented movements among sites are also discussed. Overall, counts at the large maternity colonies have been relatively stable, recapture rates remain high and bat health (as measured by body mass index) has remained steady. Although likely dependent on weather conditions from year to year, the post-volant count increase at benchmark sites in 2013

occurred throughout the month of July, indicating that single post volant counts may not capture peak numbers. Slight dips in numbers before peaks were documented for four of the five sites. Collection of data at weekly or bi-monthly intervals in future years is needed to better evaluate maternity colony trends. Bats in the US National Park System: conservation challenges and opportunities Tom Rodhouse1, Kevin Castle1, Tom Philippi1, Alison Loar1, Paul Cryan2

1National Park Service, 2US Geologic Survey The emergence of white-nose syndrome (WNS) and its devastating impacts on bat populations in North America have heightened the need for National Park Service (NPS) and other land management agencies to improve our understanding of the population status of bats. Other stressors, including fatalities at wind turbines, climate change, and habitat loss underscore the growing body of evidence that many bat populations within NPS lands may be in peril. In order for NPS to contribute most effectively to a coordinated national bat monitoring program and other bat conservation efforts in the face of WNS and other stressors, the agency needs a better assessment of bats occurring in NPS units. We have compiled information on bat species in NPS units across the country using of the NPSpecies database, then prepared basic biogeographic summaries and identified knowledge gaps and opportunities for enhanced inventory, monitoring, and research. Of the 270 NPS units that were included in the biological inventory effort funded by the Natural Resource Challege that began 13 years ago, 37 (13%) NPS units report as present ≥90% of the bat species expected based on NatureServe range maps. Another 20% of NPS units report ≥70%-90% of bat species present, relative to expected. We consider the remaining 67% of NPS units in need of additional inventory effort. Based on these data, there are 49 bat species reported in at least one NPS unit, out of 57 total species of bats whose ranges overlap with the NPS footprint, including islands in the Caribbean and tropical Pacific. Species diversity across NPS covaries positively with mean elevation of units, but less so with the size (area) of units. All 7 species of bats currently known to be affected by WNS are well-represented in the NPS system, although Perimyotis subflavus is reported present only in 5 units. The species currently hardest-hit by WNS, Myotis lucifugus, is reported present in 121 units, underscoring the challenge that NPS faces with respect to WNS. These and other patterns will be used as a baseline of information for NPS and will help guide the agency’s ongoing efforts to preserve and protect bat populations for future generations. Introduction of Geomyces destructans-infected bats to an abandoned military bunker at Aroostook National Wildlife Refuge, Maine Steve Agius1, Alyssa Bennett2, Scott Darling2, Carl Herzog3, Jon Reichard1, Susanna von Oettingen1, Kate Langwig4, Jeff Foster 4, Marm Kilpatrick5, Winnifred Frick 5, and Tina Cheng 5

1US Fish and Wildlife Service, 2Vermont Fish and Wildlife Department, 3New York Department of Environmental Conservation, 4Northern Arizona University, 5University of California, Santa Cruz Artificial hibernacula offer potential winter refugia at which environmental factors conducive to the growth and persistence of the bat-killing fungus, Geomyces destructans (Gd), can be managed or eliminated through chemical, biological or other means. Former military bunkers may closely approximate environmental conditions in natural hibernacula and could be used as small bat hibernacula for experimental or conservation purposes. The primary objective of our experimental translocation of Gd-infected bats was to determine if cave bats can hibernate in military bunkers that exhibit appropriate temperature and humidity regimes.

After relatively minor environmental manipulations, a former military bunker at Aroostook National Wildlife Refuge (ANOWR) in Limestone, Maine was selected for a bat translocation experiment. Fifteen male little brown bats (Myotis lucifugus) infected with Gd were moved from each of two hibernacula, one in Vermont and one in New York, in December 2012 to the bunker at ANOWR. Environmental samples were taken from the bunker for PCR anaylsis of Gd presence before bats were introduced and after bats were removed. Gd was not detected in the pre-hibernation samples from the bunker but the majority of samples at the end of the hibernation season (42/80) contained Gd. Of the thirty translocated bats, nine survived and were returned at the end of March, 2013 to their respective hibernacula. We conclude that Northeastern cave bats can successfully hibernate in artificial hibernacula created out of ex-military bunkers if suitable conditions exist, although the logistical challenges to applying this management approach are significant. We are still investigating potential avenues of decontamination and future experimental uses of the bunker. Crucial Hibernacula for Bats in Southern Idaho: Implications for Conservation and Management Jericho Whiting1, Joe Lowe2, Scott Earl2, April Earl2, Bill Doering3, Devin Englestead2, Justin Frye2, Rob Cavallaro4, Todd Stefanic5 and Bill Bosworth4 1Gonzales-Stoller Surveillance, 2U. S. Bureau of Land Management, 3Power Engineers, 4Idaho Department of Fish and Game, 5National Park Service Bat populations are being impacted by human disturbance and modification of hibernacula. Identifying important hibernacula and counting hibernating bats are effective ways to conserve these mammals. We compiled periodic counts of hibernating bats during winter (November to March) in 36 caves from 1984 to 2013 to document the number of caves used by bats, as well as to investigate if the number of bats hibernating varied by colony size. Researchers counted 24,919 bats representing 6 species. Townsend’s big-eared bats (Corynorhinus townsendii townsendii) comprised 95.8% (23,874 individuals) of those bats and used 35 caves, and western small-footed myotis (Myotis ciliolabrum) comprised 4.1% (1,014) of those bats and used 19 caves. Twenty caves were substantial hibernacula (≥ 20 individuals) for Townsend’s big-eared bats, and five caves were substantial hibernacula (≥ 5 individuals) for western small-footed myotis. The largest hibernating colony of C. t. townsendii occupied Kid’s Cave ( X = 1,446, SD = 516.3, range = 619 to 1,994 individuals). The largest hibernating colony of M. ciliolabrum occupied Fool’s Wading Pool ( X = 87, SD = 51.4, range = 32 to 146 individuals). Smaller hibernating colonies varied more in the number of bats counted than did larger colonies. We document one of the largest densities of caves used by hibernating bats in the western USA, as well as possibly the largest reported hibernacula for Townsend’s big-eared bats and western small-footed myotis in their distributions. This information provides important context regarding hibernating bats prior to major threats (i.e., white-nose syndrome) occurring in southern Idaho.

Mycobiome of the White Nose Syndrome (WNS) afflicted Caves and Mines in the Northeastern United States Tao Zhang1, Tanya Victor1, Xiaojiang Li1, Joe C. Okoniewski2, Al C. Hicks2, Carl J. Herzog2, Sudha Chaturvedi1, and Vishnu Chaturvedi1 1New York State Department of Health, 2New York State Department of Environmental Conservation The current investigations of the bat White Nose Syndrome (WNS) have yet to provide answers as to how the causative fungus Geomyces destructans first appeared in the Northeast and how a single clone

has spread rapidly in the US and Canada. Finally, urgent steps are still needed for the mitigation or control of G. destructans in the affected environment to augment the ongoing efforts to save bats from extinction. We hypothesized that a focus on the fungal ecology of the affected sites would yield important clues about the disease cycle and possible steps that can be used for its disruption in the wild. The present study was conducted in 2010/2011 in NY and VT in twelve caves and mines worst affected by WNS. We aimed to catalogue G. destructans and all other fungi (mycobiome) in these sites by the simultaneous use of culture-dependent and culture-independent methods. A combination of specialized media previously standardized in our laboratory, were used for the fungal recovery, and morphology and molecular approaches were used for final identifications. For the culture-independent study, environmental DNA samples were amplified with several universal fungal primers and the resulting amplicons were cloned into bacterial vectors for nucleotide sequencing. A total of 656 fungal isolates were obtained from twelve caves and mines including the live recovery of G. destructans from 11 of the 12 samples. The most abundant fungi were cosmopolitan inhabitants of temperate environments - Penicillium species, Geomyces species other than G. destructans, Oidiodendron species, Kernia species, Mortierella species and Trichosporon species. A total of 703 nucleotide sequences that met the definition of operational taxonomic units (OTUs) were recovered from 24 sediment or swab samples collected from six different WNS-affected sites. Most OTUs belonged to unidentified, uncultured clones deposited in the databases as environmental sequences. Among OTUs with excellent nucleotide matches were Penicillium species, Geomyces species, Debaromyces species, Trichosporon species, and Helicostylum species. As expected, the results were not congruent for different fungi recovered from culture-dependent and independent methods. Further statistical analyses revealed that the fungal community structure was significantly different at the sample sites. The near universal presence of G. destructans in this milieu indicated that the fungus persists in the environment in the absence of bats and it competes successfully with the dominant fungal genera in these habitats. Further analyses are currently underway to define ecology, abundance and physiology of fungal communities in WNS affected sites.