Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white‐nose syndrome

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.     

Body mass and hibernation microclimate may predict bat susceptibility to white‐nose syndrome

In multihost disease systems, differences in mortality between species may reflect variation in host physiology, morphology, and behavior. In systems where the pathogen can persist in the environment, microclimate conditions, and the adaptation of the host to these conditions, may also impact mortality. White‐nose syndrome (WNS) is an emerging disease of hibernating bats caused by an environmentally persistent fungus, Pseudogymnoascus destructans. We assessed the effects of body mass, torpid metabolic rate, evaporative water loss, and hibernaculum temperature and water vapor deficit on predicted overwinter survival of bats infected by P. destructans. We used a hibernation energetics model in an individual‐based model framework to predict the probability of survival of nine bat species at eight sampling sites across North America. The model predicts time until fat exhaustion as a function of species‐specific host characteristics, hibernaculum microclimate, and fungal growth. We fit a linear model to determine relationships with each variable and predicted survival and semipartial correlation coefficients to determine the major drivers in variation in bat survival. We found host body mass and hibernaculum water vapor deficit explained over half of the variation in survival with WNS across species. As previous work on the interplay between host and pathogen physiology and the environment has focused on species with narrow microclimate preferences, our view on this relationship is limited. Our results highlight some key predictors of interspecific survival among western bat species and provide a framework to assess impacts of WNS as the fungus continues to spread into western North America.     

Major histocompatibility complex variation is similar in little brown bats before and after white‐nose syndrome outbreak

White‐nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans (Pd), has driven alarming declines in North American hibernating bats, such as little brown bat (Myotis lucifugus). During hibernation, infected little brown bats are able to initiate anti‐Pd immune responses, indicating pathogen‐mediated selection on the major histocompatibility complex (MHC) genes. However, such immune responses may not be protective as they interrupt torpor, elevate energy costs, and potentially lead to higher mortality rates. To assess whether WNS drives selection on MHC genes, we compared the MHC DRB gene in little brown bats pre‐ (Wisconsin) and post‐ (Michigan, New York, Vermont, and Pennsylvania) WNS (detection spanning 2014–2015). We genotyped 131 individuals and found 45 nucleotide alleles (27 amino acid alleles) indicating a maximum of 3 loci (1–5 alleles per individual). We observed high allelic admixture and a lack of genetic differentiation both among sampling sites and between pre‐ and post‐WNS populations, indicating no signal of selection on MHC genes. However, post‐WNS populations exhibited decreased allelic richness, reflecting effects from bottleneck and drift following rapid population declines. We propose that mechanisms other than adaptive immunity are more likely driving current persistence of little brown bats in affected regions.     

Temperature-Dependent Growth of Geomyces destructans,the Fungus That Causes Bat White-Nose Syndrome

White-nose syndrome (WNS) is an emergent disease estimated to have killed over five million North American bats. Caused by the psychrophilic fungus Geomyces destructans, WNS specifically affects bats during hibernation. We describe temperature-dependent growth performance and morphology for six independent isolates of G. destructans from North America and Europe. Thermal performance curves for all isolates displayed an intermediate peak with rapid decline in performance above the peak. Optimal temperatures for growth were between 12.5 and 15.8°C, and the upper critical temperature for growth was between 19.0 and 19.8°C. Growth rates varied across isolates, irrespective of geographic origin, and above 12°C all isolates displayed atypical morphology that may have implications for proliferation of the fungus. This study demonstrates that small variations in temperature, consistent with those inherent of bat hibernacula, affect growth performance and physiology of G. destructans, which may influence temperature-dependent progression and severity of WNS in wild bats.     

Host,Pathogen, and Environmental Characteristics Predict White-Nose Syndrome Mortality in Captive Little Brown Myotis (Myotis lucifugus)

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.     

Sebaceous Lipid Profiling of Bat Integumentary Tissues: Quantitative Analysis of Free Fatty Acids,Monoacylglycerides, Squalene,and Sterols

White‐nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans and is devastating North American bat populations. Sebaceous lipids secreted from host integumentary tissues are implicated in the initial attachment and recognition of host tissues by pathogenic fungi. We are interested in determining if ratios of lipid classes in sebum can be used as biomarkers to diagnose severity of fungal infection in bats. To first establish lipid compositions in bats, we isolated secreted and integral lipid fractions from the hair and wing tissues of three species: big brown bats (Eptesicus fuscus), Eastern red bats (Lasiurus borealis), and evening bats (Nycticeius humeralis). Sterols, FFAs, MAGs, and squalene were derivatized as trimethylsilyl esters, separated by gas chromatography, and identified by mass spectrometry. Ratios of sterol to squalene in different tissues were determined, and cholesterol as a disease biomarker was assessed. Free sterol was the dominant lipid class of bat integument. Squalene/sterol ratio is highest in wing sebum. Secreted wing lipid contained higher proportions of saturated FFAs and MAGs than integral wing or secreted hair lipid. These compounds are targets for investigating responses of P. destructans to specific host lipid compounds and as biomarkers to diagnose WNS.     

Will reduced host connectivity curb the spread of a devastating epidemic?

The white‐nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans, is threatening the cave‐dwelling bat fauna of North America by killing individuals by the thousands in hibernacula each winter since its appearance in New York State less than ten years ago. Epidemiological models predict that WNS will reach the western coast of the USA by 2035, potentially eliminating most populations of susceptible bat species in its path (Frick et al. 2015; O'Regan et al. 2015). These models were built and validated using distributional data from the early years of the epidemic, which spread throughout eastern North America following a route driven by cave density and winter severity (Maher et al. 2012). In this issue of Molecular Ecology, Wilder et al. (2015) refine these findings by showing that connectivity among host populations, as assessed by population genetic markers, is crucial in determining the spread of the pathogen. Because host connectivity is much reduced in the hitherto disease free western half of North America, Wilder et al. make the reassuring prediction that the disease will spread more slowly west of the Great Plains.     

Rapid Real-Time PCR Assay for Culture and Tissue Identification of <Emphasis Type="Italic">Geomyces destructans</Emphasis>: the Etiologic Agent of Bat Geomycosis (White Nose Syndrome)

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS–PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10⁻¹–5 × 10⁷), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)–PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.     

Wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology

White-nose syndrome (WNS) is causing unprecedented declines in several species of North American bats. The characteristic lesions of WNS are caused by the fungus Geomyces destructans, which erodes and replaces the living skin of bats while they hibernate. It is unknown how this infection kills the bats. We review here the unique physiological importance of wings to hibernating bats in relation to the damage caused by G. destructans and propose that mortality is caused by catastrophic disruption of wing-dependent physiological functions. Mechanisms of disease associated with G. destructans seem specific to hibernating bats and are most analogous to disease caused by chytrid fungus in amphibians.     

Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality

Background

The dramatic mass mortalities amongst hibernating bats in Northeastern America caused by “white nose-syndrome” (WNS) continue to threaten populations of different bat species. The cold-loving fungus, Geomyces destructans, is the most likely causative agent leading to extensive destruction of the skin, particularly the wing membranes. Recent investigations in Europe confirmed the presence of the fungus G. destructans without associated mass mortality in hibernating bats in six countries but its distribution remains poorly known.

Methodology/Principal Findings

We collected data on the presence of bats with white fungal growth in 12 countries in Europe between 2003 and 2010 and conducted morphological and genetic analysis to confirm the identity of the fungus as Geomyces destructans. Our results demonstrate the presence of the fungus in eight countries spanning over 2000 km from West to East and provide compelling photographic evidence for its presence in another four countries including Romania, and Turkey. Furthermore, matching prevalence data of a hibernaculum monitored over two consecutive years with data from across Europe show that the temporal occurrence of the fungus, which first becomes visible around February, peaks in March but can still be seen in some torpid bats in May or June, is strikingly similar throughout Europe. Finally, we isolated and cultured G. destructans from a cave wall adjacent to a bat with fungal growth.

Conclusions/Significance

G. destructans is widely found over large areas of the European continent without associated mass mortalities in bats, suggesting that the fungus is native to Europe. The characterisation of the temporal variation in G. destructans growth on bats provides reference data for studying the spatio-temporal dynamic of the fungus. Finally, the presence of G. destructans spores on cave walls suggests that hibernacula could act as passive vectors and/or reservoirs for G. destructans and therefore, might play an important role in the transmission process.     

Use of predictive distribution models to describe habitat selection by bats in Colorado,USA

Numerous processes operating at landscape scales threaten bats (e.g., habitat loss, disease). Temperate bat species are rarely examined at commensurate scales because of logistical and modeling constraints. Recent modeling approaches now allow for presence-only datasets, like those often available for bats, to assist with the development of predictive distribution models. We describe the use of presence-only data and rigorous predictive distribution models to examine habitat selection by bats across Colorado, USA. We applied hierarchical Bayesian models to bat locations from 1906–2018 to examine relationships of 13 species with landscape covariates. We considered differences in type of activity (foraging, roosting, hibernation), seasonality (summer vs. winter), and scale (1, 5, 10, and 15-km buffers). These findings generated statewide probability of use models to guide management of bat species in response to threats (e.g., white-nose syndrome [WNS]). Analysis of buffers suggest selection of land cover and environmental covariates occurs at different scales depending on the species and activity. Pinyon (Pinus spp.)-juniper (Juniperus spp.) appeared as a positive association in the highest number of models, followed by montane woodland, supporting the importance of these forest types to bats in Colorado. Other covariates commonly associated with bats in Colorado include westerly longitudes, and negative associations with montane shrubland. Mechanical treatments within pinyon-juniper and montane woodlands should be conducted with caution to avoid harming bat communities. We developed hibernation models for only 2 species, making apparent the lack of winter records for bat species in the state. We also provide a composite predictive surface of small-bodied bats in Colorado that delineates where these species, vulnerable to WNS, converge. This tool provides managers with focal points to apply surveillance and response strategies for the impending arrival of the disease.     

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography–mass spectrometry (GC–MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.     

Changes in body condition of hibernating bats support the thrifty female hypothesis and predict consequences for populations with white-nose syndrome

White-nose syndrome (WNS) is a new disease of bats that has devastated populations in eastern North America. Infection with the fungus, Geomyces destructans, is thought to increase the time bats spend out of torpor during hibernation, leading to starvation. Little is known about hibernation in healthy, free-ranging bats and more data are needed to help predict consequences of WNS. Trade-offs presumably exist between the energetic benefits and physiological/ecological costs of torpor, leading to the prediction that the relative importance of spring energy reserves should affect an individual''s use of torpor and depletion of energy reserves during winter. Myotis lucifugus mate during fall and winter but females do not become pregnant until after spring emergence. Thus, female reproductive success depends on spring fat reserves while male reproductive success does not. Consequently, females should be “thrifty” in their use of fat compared to males. We measured body condition index (BCI; mass/forearm length) of 432 M. lucifugus in Manitoba, Canada during the winter of 2009/2010. Bats were captured during the fall mating period (n = 200), early hibernation (n = 125), and late hibernation (n = 128). Adult females entered hibernation with greater fat reserves and consumed those reserves more slowly than adult males and young of the year. Consequently, adult females may be more likely than males or young of the year to survive the disruption of energy balance associated with WNS, although surviving females may not have sufficient reserves to support reproduction.     

Plasma proteomic profiles differ between European and North American myotid bats colonized by Pseudogymnoascus destructans

Emerging fungal diseases have become challenges for wildlife health and conservation. North American hibernating bat species are threatened by the psychrophilic fungus Pseudogymnoascus destructans (Pd) causing the disease called white‐nose syndrome (WNS) with unprecedented mortality rates. The fungus is widespread in North America and Europe, however, disease is not manifested in European bats. Differences in epidemiology and pathology indicate an evolution of resistance or tolerance mechanisms towards Pd in European bats. We compared the proteomic profile of blood plasma in healthy and Pd‐colonized European Myotis myotis and North American Myotis lucifugus in order to identify pathophysiological changes associated with Pd colonization, which might also explain the differences in bat survival. Expression analyses of plasma proteins revealed differences in healthy and Pd‐colonized M. lucifugus, but not in M. myotis. We identified differentially expressed proteins for acute phase response, constitutive and adaptive immunity, oxidative stress defence, metabolism and structural proteins of exosomes and desmosomes, suggesting a systemic response against Pd in North American M. lucifugus but not European M. myotis. The differences in plasma proteomic profiles between European and North American bat species colonized by Pd suggest European bats have evolved tolerance mechanisms towards Pd infection.     

Psychrophilic and Psychrotolerant Fungi on Bats and the Presence of Geomyces spp. on Bat Wings Prior to the Arrival of White Nose Syndrome

Since 2006, Geomyces destructans, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus Geomyces and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera Cladosporium, Fusarium, Geomyces, Mortierella, Penicillium, and Trichosporon. Eleven Geomyces isolates were obtained and placed in at least seven distinct Geomyces clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all Geomyces strains isolated are psychrotolerant, unlike G. destructans, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several Geomyces isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant Geomyces strains isolated from bats.     

Long‐term changes in bat activity in Quebec suggest climatic responses and summer niche partitioning associated with white‐nose syndrome

In North America, the greatest and most sudden threat to hibernating bats is white‐nose syndrome (WNS), which has caused massive declines in populations since 2006. Other determinants of bat dynamics, such as the climate, and the effect of reduction in the number of individuals sharing foraging space and summer roosting habitat may have an effect on population dynamics. We analyzed transect acoustic bat surveys conducted with ultrasonic detectors in 16 regions in Quebec, Canada, between 2000 and 2015. We used piecewise regression to describe changes in activity over time for each species and a meta‐analytic approach to measure its association with the North Atlantic Oscillation (NAO). As expected, mouse‐eared bat (Myotis spp.) activity sharply declined after the onset of WNS, down by 79% after 3 years. In contrast, big brown/silver‐haired bat activity increased over the same period, possibly due to a release of competition. Hoary bats and red bats remained present, although their activity did not increase. Myotis activity was positively correlated with a one‐year lag to the NAO index, associated with cold conditions in winter, but warm autumns. Big brown/silver‐haired and hoary bats were also more active during NAO‐positive years but without a lag. We conclude that combinations of threats may create rapid shifts in community compositions and that a more balanced research agenda that integrates a wider range of threats would help better understand and manage those changes.     

Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry

White-nose syndrome (WNS) caused by the pathogenic fungus Pseudogymnoascus destructans is decimating the populations of several hibernating North American bat species. Little is known about the molecular interplay between pathogen and host in this disease. Fluorescence microscopy ambient ionization mass spectrometry was used to generate metabolic profiles from the wings of both healthy and diseased bats of the genus Myotis. Fungal siderophores, molecules that scavenge iron from the environment, were detected on the wings of bats with WNS, but not on healthy bats. This work is among the first examples in which microbial molecules are directly detected from an infected host and highlights the ability of atmospheric ionization methodologies to provide direct molecular insight into infection.     

Evaporative water loss is a plausible explanation for mortality of bats from white-nose syndrome

White-nose syndrome (WNS) has caused alarming declines of North American bat populations in the 5 years since its discovery. Affected bats appear to starve during hibernation, possibly because of disruption of normal cycles of torpor and arousal. The importance of hydration state and evaporative water loss (EWL) for influencing the duration of torpor bouts in hibernating mammals recently led to "the dehydration hypothesis," that cutaneous infection of the wing membranes of bats with the fungus Geomyces destructans causes dehydration which in turn, increases arousal frequency during hibernation. This hypothesis predicts that uninfected individuals of species most susceptible to WNS, like little brown bats (Myotis lucifugus), exhibit high rates of EWL compared to less susceptible species. We tested the feasibility of this prediction using data from the literature and new data quantifying EWL in Natterer's bats (Myotis nattereri), a species that is, like other European bats, sympatric with G. destructans but does not appear to suffer significant mortality from WNS. We found that little brown bats exhibited significantly higher rates of normothermic EWL than did other bat species for which comparable EWL data are available. We also found that Natterer's bats exhibited significantly lower rates of EWL, in both wet and dry air, compared with values predicted for little brown bats exposed to identical relative humidity (RH). We used a population model to show that the increase in EWL required to cause the pattern of mortality observed for WNS-affected little brown bats was small, equivalent to a solitary bat hibernating exposed to RH of ～95%, or clusters hibernating in ～87% RH, as opposed to typical near-saturation conditions. Both of these results suggest the dehydration hypothesis is plausible and worth pursuing as a possible explanation for mortality of bats from WNS.