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White-nose syndrome (WNS) is caused by the fungus Pseudogymnoascus destructans and has led to the deaths of millions of North American bats since it was first documented in New York in 2006. Since the first cases were recorded, WNS has spread rapidly across North America and is now confirmed or suspected in 40 US states and seven Canadian provinces. Often, the presence of P. destructans is detected in a cave or hibernaculum before signs of WNS manifest in the resident bat population, making presence of the fungus a more reliable assessment of potential epidemic spread than expansion of manifested WNS. An analysis of 43 cave internal climates across the state of Texas revealed a pattern of thermal suitability for P. destructans that correlated significantly with landscape (elevation, lithology) and external climate (mean surface temperature and precipitation). We generated a predictive model to assess the potential spread of P. destructans through Texas karst systems based on external features that correlate with suitable internal climates for fungal growth. Applications of this model to external climatic variables from 2019 showed seasonally varying patterns of suitability for fungal growth in select regions of Texas karst systems. Results from these surveys and models showed that internal climates of Texas caves are likely able to sustain the growth of P. destructans and could cause disease and resulting declines in Texas bats, and act as stepping-stones for the fungus, allowing it to travel southward into Mexican and Central American cave systems. The resulting work will inform researchers and natural resource managers of areas of significant concern to monitor for the spread of WNS.

The identification of bat colonies is essential to conserve and manage these globally threatened mammals. Caves offer potential roosting locations (hibernacula) to hibernating bat species; however, identifying regions where bat-occupied caves exist can be time-consuming. In Texas, caves are often on privately owned land, creating difficulties for accessing and managing potential hibernacula. The tricolored bat (Perimyotis subflavus), a species susceptible to white-nose syndrome, hibernates in caves in the winter in Texas. We sought to identify and quantify site-specific structural and environmental features that influence the presence and abundance of overwintering tricolored bats. We surveyed caves for bats and recorded environmental and structural features of 116 caves January–February 2016, December–February 2016–2017, January–February 2018, and December–February 2018–2019. We used a zero-inflated Poisson regression to identify which features best explained the presence and abundance of tricolored bats. We found that bat presence increased as cave length decreased, and as Normalized Difference Vegetation Index (NDVI) and external vapor pressure deficit increased. Bat abundance increased as number of portals, cave length, NDVI, and external temperature increased. Combining surface data with subsurface features can assist with identifying specific karst regions and known caves within those regions for survey and management efforts.

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Subterranean ecosystems are among the most widespread environments on Earth, yet we still have poor knowledge of their biodiversity. To raise awareness of subterranean ecosystems, the essential services they provide, and their unique conservation challenges, 2021 and 2022 were designated International Years of Caves and Karst. As these ecosystems have traditionally been overlooked in global conservation agendas and multilateral agreements, a quantitative assessment of solution-based approaches to safeguard subterranean biota and associated habitats is timely. This assessment allows researchers and practitioners to understand the progress made and research needs in subterranean ecology and management. We conducted a systematic review of peer-reviewed and grey literature focused on subterranean ecosystems globally (terrestrial, freshwater, and saltwater systems), to quantify the available evidence-base for the effectiveness of conservation interventions. We selected 708 publications from the years 1964 to 2021 that discussed, recommended, or implemented 1,954 conservation interventions in subterranean ecosystems. We noted a steep increase in the number of studies from the 2000s while, surprisingly, the proportion of studies quantifying the impact of conservation interventions has steadily and significantly decreased in recent years. The effectiveness of 31% of conservation interventions has been tested statistically. We further highlight that 64% of the reported research occurred in the Palearctic and Nearctic biogeographic regions. Assessments of the effectiveness of conservation interventions were heavily biased towards indirect measures (monitoring and risk assessment), a limited sample of organisms (mostly arthropods and bats), and more accessible systems (terrestrial caves). Our results indicate that most conservation science in the field of subterranean biology does not apply a rigorous quantitative approach, resulting in sparse evidence for the effectiveness of interventions. This raises the important question of how to make conservation efforts more feasible to implement, cost-effective, and long-lasting. Although there is no single remedy, we propose a suite of potential solutions to focus our efforts better towards increasing statistical testing and stress the importance of standardising study reporting to facilitate metaanalytical exercises. We also provide a database summarising the available literature, which will help to build quantitative knowledge about interventions likely to yield the greatest impacts depending upon the subterranean species and habitats of interest. We view this as a starting point to shift away from the widespread tendency of recommending conservation interventions based on anecdotal and expert-based information rather than scientific evidence, without quantitatively testing their effectiveness., This study is funded by the European Commission via the Marie Sklodowska-Curie Individual Fellowships program (H2020-MSCA-IF-2019; project number 882221), awarded to S.M. Additional support is provided by the PRIN SHOWCAVE “A multidisciplinary research project to study, classify and mitigate the environmental impact in tourist caves” (project number 2017HTXT2R; funded by the Italian Ministry of Education, University and Research). M.B.M. acknowledges support from the Kone Foundation (project number 202007611). T.D., C.F., V.Z., and M.Z. were supported by the Slovenian Research Agency, through core programme P1-0184 and P6-0119. O.T.M. was supported by a grant of the Romanian Ministry of Research, Innovation and Digitization, CNCS/CCCDI— UEFISCDI, project number 2/2019 (DARKFOOD), within PNCDI III. S.I.S. acknowledges funding by MEMOBIC (EU Operational Programme Research, Development and Education No. CZ.02.2.69/0.0/ 0.0/16_027/0008357), and by the Ministry of Education, Youth and Sports of the Czech Republic (grant number CZ.02.1.01/0.0/0.0/16 025/0007417). E.P. is supported by the PON “Research and Innovation” Programme (Axis IV “Education and Research for recovery” – Action IV.6 “Research contracts on Green themes”).

Many North American bat species hibernate in both natural and artificial roosts. Although hibernacula can have high internal climate stability, they still retain spatial variability in their thermal regimes, resulting in various “microclimates” throughout the roost that differ in their characteristics (e.g., temperature and air moisture). These microclimate components can be influenced by factors such as the number of entrances, the depth of the roost, and distance to the nearest entrance of the roost. Tri‐colored bats are commonly found roosting in caves in winter, but they can also be found roosting in large numbers in culverts, providing the unique opportunity to investigate factors influencing microclimates of bats in both natural and artificial roost sites. As tri‐colored bats are currently under consideration for federal listing, information of this type could be useful in aiding in the conservation and management of this species through a better understanding of what factors affect the microclimate near roosting bats. We collected data on microclimate temperature and microclimate actual water vapor pressure (AWVP) from a total of 760 overwintering tri‐colored bats at 18 caves and 44 culverts. Using linear mixed models analysis, we found that variation in bat microclimate temperatures was best explained by external temperature and distance from nearest entrance in both caves and culverts. External temperature had a greater influence on microclimate temperatures in culverts than caves. We found that variation in microclimate AWVP was best explained by external temperature, distance from nearest entrance, and proportion from entrance (proportion of the total length of the roost from the nearest entrance) in culvert‐roosting bats. Variation in microclimate AWVP was best explained by external temperature and proportion from entrance in cave‐roosting bats. Our results suggest that bat microclimate temperature and AWVP are influenced by similar factors in both artificial and natural roosts, although the relative contribution of these factors differs between roost types., Tri‐colored bats are commonly found winter roosting in caves, but they can also be found roosting in culverts, providing the unique opportunity to investigate factors influencing bat microclimate temperatures in both natural and artificial roost sites. We collected data on microclimate temperatures and actual water vapor pressure near roosting bats in 18 caves and 44 culverts. Our results suggest that bat microclimate components are influenced by the same factors in both artificial and natural roosts, although the relative contribution of these factors differs between roost types.

Time and budgetary resources are often a limiting factor in the collection of large-scale ecological data. If data collected by citizen scientists were comparable to data collected by researchers, it would allow for more efficient data collection over a broad geographic area. Here, we compare the quality of data on bat activity collected by citizens (high school students and teachers) and researchers. Both researchers and citizen scientists used the same comprehensive instructions when choosing study sites. We found no differences in total bat activity minutes recorded by citizens and researchers. Instead, citizen scientists collected data from a wider variety of habitats than researchers. Involvement of citizens also increased the geographical coverage of data collection, resulting in the northernmost documentation of the Nathusius’s pipistrelle so far in Finland. Therefore, bat research can benefit from the use of citizen science when participants are given precise instructions and calibrated data collection equipment. Citizen science projects also have other far-reaching benefits, increasing, for example, the scientific literacy and interest in natural sciences of citizens. Involving citizens in science projects also has the potential to enhance their willingness to conserve nature.Open Research StatementData are not yet provided, but will uploaded Dryad upon publication.

Predicting the emergence and spread of infectious diseases is critical for the effective conservation of biodiversity. White-nose syndrome (WNS), an emerging infectious disease of bats, has resulted in high mortality in eastern North America. Because the fungal causative agent Pseudogymnoascus destructans is constrained by temperature and humidity, spread dynamics may vary by geography. Environmental conditions in the southern part of the continent are different than the northeast, where disease dynamics are typically studied, making it difficult to predict how the disease will manifest. Herein, we modelled WNS pathogen spread in Texas based on cave densities and average dispersal distances of hosts, projecting these results out to 10 years. We parameterized a predictive model of WNS epidemiology and its effects on bat populations with observed cave environmental data. Our model suggests that bat populations in northern Texas will be more affected by WNS mortality than southern Texas. As such, we recommend prioritizing the preservation of large overwintering colonies of bats in north Texas through management actions. Our model illustrates that infectious disease spread and infectious disease severity can become uncoupled over a gradient of environmental variation and highlight the importance of understanding host, pathogen and environmental conditions across a breadth of environments.

The tri-colored bat (Perimyotis subflavus) is being reviewed for listing under the Endangered Species Act by the United States Fish and Wildlife Service. It is also listed as a species of greatest conservation need by the Texas Parks and Wildlife Department due to its susceptibility to white-nose syndrome (WNS) in other states. Several colonies of hibernating tri-colored bats have been documented roosting in culverts. Culverts are widespread in Texas as part of roadway infrastructure; thus, our objective was to understand and quantify which structural and environmental factors best explain culvert use and abundance of hibernating tri-colored bats in Texas. We selected and surveyed 207 culverts for presence of tri-colored bats using the Generalized Random Tessellation Stratified (GRTS) design and opportunistic sampling across 10 of 12 Texas level III ecoregions during the winters of 2016–2017 and 2017–2018. We recorded environmental and structural features of culverts at each site. We used a zero-inflated Poisson regression to identify which culvert features best explained presence and abundance of hibernating tri-colored bats. We found that number of culvert sections predicted presence of tri-colored bats. We also found that abundance of tri-colored bats was influenced by length of culvert, elevation, number of sections, portal height, portal obstruction, aspect, external VPD, external temperature, and NDVI. With the current threats to tri-colored bat populations, there is a need to consider management of culvert roosts. In addition, it is imperative to further investigate the potential susceptibility to WNS of culvert-roosting bats at more southern latitudes for local and regional planning efforts.

Predicting the emergence and spread of infectious diseases is critical for effective conservation of biodiversity. White-nose syndrome (WNS), an emerging infectious disease of bats, has resulted in high mortality in eastern North America. Because the fungal causative agentPseudogymnoascus destructansis constrained by temperature and humidity, spread dynamics may vary greatly by geography. Environmental conditions in the southern part of the continent, where disease dynamics are typically studied, making it difficult to predict how the disease will manifest. Herein, we modeled the spread of WNS in Texas based on available cave densities and average dispersal distances of species occupying these sites, and projected these results out to 10 years. We parameterized a predictive model of WNS epidemiology and its effects on hibernatory bat populations with observed environmental data from bat hibernation sites in Texas. Our model suggests that bat populations in northern Texas will be more affected by WNS mortality than southern Texas. As such, we recommend prioritizing the preservation of large overwintering colonies of bats in north Texas through management actions. Our model further illustrates that infectious disease spread and infectious disease severity can become uncoupled over a gradient of environmental variation. Finally, our results highlight the importance of understanding host, pathogen and environmental conditions in various settings to elucidate what may happen across a breadth of environments.

Natural hibernation sites used by bats in areas that lack cave features have long remained unresolved. To investigate hibernation site selection and winter activity of boreal bats, we recorded bat calls using passive acoustic monitoring at 16 sites in South-Western Finland. These sites included four rock outcrops with crevices and cave features, three glacial erratics or boulder fields, three ancient shores, three root cellars and three control sites where we did not expect bats to be overwintering. Our results revealed echolocation calls of Eptesicus nilssonii, Plecotus auritus and Myotis sp. We recorded significantly more activity near rock outcrops compared to other habitats, excluding root cellars. We also found that ambient temperature had a positive effect on bat activity and found evidence that P. auritus may be using low barometric pressure as a proxy for suitable foraging conditions during the winter. Our results suggest that rock outcrops may be more important to bats than previously acknowledged, highlighting the need to take these sites in account in planning of conservation measures. Furthermore, our findings underline the suitability of using acoustic monitoring in homing on hibernation sites that are not otherwise accessible.

There are few studies of day-roosting ecology of bats inhabiting the southernmost forests of South America, where cool summer temperatures and land management practices pose several challenges. The goal of the present study was to describe day-roosting habitats and patterns of thermoregulation in two bat species occurring on Tierra del Fuego, Myotis chiloensis (Chilean myotis) and Histiotus magellanicus (southern big-eared brown bat), during late spring. To do so, we tagged 17 bats with temperature-sensitive radio-transmitters, located 17 day-roosts, and collected 81 days of skin temperature data. We concurrently recorded ambient air temperature to determine its effect on torpor use. Both species were found roosting in large diameter (77.8 ± 6 cm), typically live, Nothofagus pumilio trees (lenga) located on the edges of forest gaps or within stands primarily composed of smaller, younger trees. Bats of both species frequently used torpor, with skin temperatures dropping below a torpor threshold on 89% of days (n = 72) and daily minimum skin temperatures averaging 16.5 °C over the course of our study. Average daily air temperature was a significant predictor of torpor use, with lower skin temperatures and more time spent in torpor observed on colder days. Minimum skin temperature and time spent torpid did not vary between bat species, nor did the characteristics of day-roosts. These data show that spring temperatures in Tierra del Fuego pose an energetic challenge that bats meet through frequent use of torpor, and likely, habitat selection. We recommend local conservation efforts keep these thermal challenges in mind by retaining large trees, which may provide warmer microclimates or room for social groups.

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field. S.M acknowledge support by the CAWEB project “Testing macroecological theory using simplified systems”, funded by the European Commission through Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) individual fellowships (Grant no. 882221). I.R.A. was supported by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/ CP1375/CT0003. D.E. was supported by IFREMER and by the CERES “Climate change and European Aquatic Resources” project funded by European Commission through Horizon 2020 research and innovation programme (Grant no. 678193). C.F., Z.F., and M.Z. were supported by the Slovenian Research Agency (program P1-0184, project N1-0069). J.E.K. was funded by NSF awards DEB1754231 and IOS1933428, and EDGE award 1923372. F.M. was supported by the French National Research Agency projects CONVERGENOMICS (ANR-15-CE32-0005) and EUR H2O’Lyon (ANR-17-EURE-0018). A.M. was supported by the ANCAVE project “Anchialine caves to understand evolutionary processes”, funded by the European Commission through Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) individual fellowships (Grant no. 745530). P.A.V.B. was supported by the project AZORESBIOPORTAL – PORBIOTA (ACORES-01-0145-FEDER-000072), financed by FEDER in 85% and by Azorean Public funds by 15% through Operational Program Azores 2020. A.S.P.S.R. was supported by VILLUM FONDEN (Grant no. 15471) and by a Carlsbergfondet grant (CF19-0609). T.G.P. was supported by Vale S.A. and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for individual fellowship (RDP 00092-18). T.P. was supported by the Karst Research Programme P6-0119, LifeWatch ERIC, RI-SI LifeWatch, and EU H2020 project eLTER. info:eu-repo/semantics/publishedVersion

The devastating bat fungal disease, white-nose syndrome (WNS), does not appear to affect all species equally. To experimentally determine susceptibility differences between species, we exposed hibernating naïve little brown myotis (Myotis lucifugus) and big brown bats (Eptesicus fuscus) to the fungus that causes WNS, Pseudogymnoascus destructans (Pd). After hibernating under identical conditions, Pd lesions were significantly more prevalent and more severe in little brown myotis. This species difference in pathology correlates with susceptibility to WNS in the wild and suggests that survival is related to different host physiological responses. We observed another fungal infection, associated with neutrophilic inflammation, that was equally present in all bats. This suggests that both species are capable of generating a response to cold tolerant fungi and that Pd may have evolved mechanisms for evading host responses that are effective in at least some bat species. These host-pathogen interactions are likely mediated not just by host physiological responses, but also by host behavior. Pd-exposed big brown bats, the less affected species, spent more time in torpor than did control animals, while little brown myotis did not exhibit this change. This differential thermoregulatory response to Pd infection by big brown bat hosts may allow for a more effective (or less pathological) immune response to tissue invasion.

Few studies have described winter microclimate selection by bats in the southern United States. This is of particular importance as the cold-adapted fungus, Pseudogymnoascus destructans, which causes the fatal bat disease white-nose syndrome (WNS), continues to spread into southern United States. To better understand the suitability of winter bat habitats for the growth of P. destructans in this region, we collected roost temperature and vapor pressure deficit from 97 hibernacula in six ecoregions in Texas during winter 2016-17 and 2017-18. We also measured skin temperature of Rafinesque's big-eared bats (Corynorhinus townsendii), Townsend's big-eared bats (C. townsendii), big-brown bats (Eptesicus fuscus), southeastern myotis (Myotis austroriparius), cave myotis (M. velifer), tri-colored bats (Perimyotis subflavus), and Mexican free-tailed bats (Tadarida brasiliensis) during hibernation to study their use of torpor in these habitats. We found that temperatures within hibernacula were strongly correlated with external air temperatures and were often within the optimal range of temperatures for P. destructans growth. Hibernacula and skin temperatures differed among species, with Rafinesque's big-eared bats, southeastern myotis, and Mexican free-tailed bats occupying warmer microclimates and having higher torpid skin temperatures. For species that were broadly distributed throughout Texas, hibernacula and skin temperatures differed within species by ecoregion; Tri-colored bats and cave myotis in colder, northern regions occupied colder microclimates within hibernacula and exhibited colder skin temperatures, than individuals of the same species in warmer, southern regions. These data illustrate the variability in microclimates used as hibernacula by bats in Texas and suggest similar variation in susceptibility to WNS in the state. Thus, monitoring microclimates at winter roosts may help predict where WNS may develop, and where management efforts would be most effective.

[This corrects the article DOI: 10.1371/journal.pone.0220839.].

Although aberrant coloration is known to occur in mammals, few records of the phenomenon exist in the literature for North American bats. Here we report on 2 leucistic Perimyotis subflavus (Tri-colored Bats) in Leon County, TX, that had a distinct white patch on their bodies. Our observation is the third documentation of leucism in Tri-colored Bats.

Host responses to infection with novel pathogens are costly and require trade-offs among physiologic systems. One such pathogen is the fungus Pseudogymnoascus destructans (Pd) that causes white-nose syndrome (WNS) and has led to mass mortality of hibernating bats in eastern North America. Although infection with Pd does not always result in death, we hypothesized that bats that survive infection suffer significant consequences that negatively impact the ability of females to reproduce. To understand the physiologic consequences of surviving infection with Pd, we assessed differences in wing damage, mass-specific resting metabolic rate, and reproductive rate between little brown myotis ( Myotis lucifugus) that survived a winter in captivity after inoculation with Pd (WNS survivors) and comparable, uninfected bats. Survivors of WNS had significantly more damaged wing tissue and displayed elevated mass-specific metabolic rates compared with Pd-uninfected bats after emergence from hibernation. The WNS survivors and Pd-uninfected bats did not significantly differ in their reproductive capacity, at least in captivity. However, our metabolic data demonstrated greater energetic costs during spring in WNS survivors compared with uninfected bats, which may have led to other consequences for postpartum fitness. We suggest that, after surviving the energetic constraints of winter, temperate hibernating bats infected with Pd faced a second energetic bottleneck after emerging from hibernation.

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans (Pd) that affects bats during hibernation. Although millions of bats have died from WNS in North America, mass mortality has not been observed among European bats infected by the fungus, leading to the suggestion that bats in Europe are immune. We tested the hypothesis that an antibody-mediated immune response can provide protection against WNS by quantifying antibodies reactive to Pd in blood samples from seven species of free-ranging bats in North America and two free-ranging species in Europe. We also quantified antibodies in blood samples from little brown myotis (Myotis lucifugus) that were part of a captive colony that we injected with live Pd spores mixed with adjuvant, as well as individuals surviving a captive Pd infection trial. Seroprevalence of antibodies against Pd, as well as antibody titers, was greater among little brown myotis than among four other species of cave-hibernating bats in North America, including species with markedly lower WNS mortality rates. Among little brown myotis, the greatest titers occurred in populations occupying regions with longer histories of WNS, where bats lacked secondary symptoms of WNS. We detected antibodies cross-reactive with Pd among little brown myotis naïve to the fungus. We observed high titers among captive little brown myotis injected with Pd. We did not detect antibodies against Pd in Pd-infected European bats during winter, and titers during the active season were lower than among little brown myotis. These results show that antibody-mediated immunity cannot explain survival of European bats infected with Pd and that little brown myotis respond differently to Pd than species with higher WNS survival rates. Although it appears that some species of bats in North America may be developing resistance to WNS, an antibody-mediated immune response does not provide an explanation for these remnant populations.

Bridges provide roost structures for bats in temperate regions of the US, including Texas, where Tadarida brasiliensis (Mexican Free-tailed Bats) are common occupants. In March 2018, we documented 1 Mexican Free-tailed Bat with Pseudogymnoascus destructans, the fungal causative agent of white-nose syndrome (WNS), in an artificial structure in Texas, thus making the ability to quantify their movements and occupancy critical for understanding WNS ecology. To determine which attributes influenced day-roosting activity by Mexican-Free-tailed Bats, we surveyed for roosting bats 70 box-beam bridges in 21 Texas counties and collected structural, weather, and landscape-characteristic data. We analyzed the data with a stepwise multiple logistic regression model to isolate variables significantly correlated with presence of day-roosting Mexican Freetailed Bats. Of 70 bridges sampled, 14 (20%) contained day-roosting Mexican Free-tailed Bats and 17 (24%) bridges had signs indicating bat use. In the best-fitting logistic regression model, bridge width, number of spans, and elevation had a positive influence on bat occupancy, whereas average temperature for the month of July 2016 negatively influenced bat occupancy. Bridge age also had a positive influence on bat occupancy, but the effect lessens in older bridges. These data show that structural and environmental characteristics are significant predictors of bridge use by Mexican Free-tailed Bats.

An estimated 5.7 million or more bats died in North America between 2006 and 2012 due to infection with the fungus Pseudogymnoascus destructans (Pd) that causes white-nose syndrome (WNS) during hibernation. The behavioral and physiological changes associated with hibernation leave bats vulnerable to WNS, but the persistence of bats within the contaminated regions of North America suggests that survival might vary predictably among individuals or in relation to environmental conditions. To investigate variables influencing WNS mortality, we conducted a captive study of 147 little brown myotis (Myotis lucifugus) inoculated with 0, 500, 5 000, 50 000, or 500 000 Pd conidia and hibernated for five months at either 4 or 10°C. We found that female bats were significantly more likely to survive hibernation, as were bats hibernated at 4°C, and bats with greater body condition at the start of hibernation. Although all bats inoculated with Pd exhibited shorter torpor bouts compared to controls, a characteristic of WNS, only bats inoculated with 500 conidia had significantly lower survival odds compared to controls. These data show that host and environmental characteristics are significant predictors of WNS mortality, and that exposure to up to 500 conidia is sufficient to cause a fatal infection. These results also illustrate a need to quantify dynamics of Pd exposure in free-ranging bats, as dynamics of WNS produced in captive studies inoculating bats with several hundred thousand conidia may differ from those in the wild.