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White-nose syndrome (WNS) is an emerging fungal disease of bats caused by Pseudogymnoascus destructans. Since it was first detected near Albany, NY, in 2006, the fungus has spread across eastern North America, killing unprecedented numbers of hibernating bats. The devastating impacts of WNS on Nearctic bat species are attributed to the likely introduction of P.destructans from Eurasia to naive host populations in eastern North America. Since 2006, the disease has spread in a gradual wavelike pattern consistent with introduction of the pathogen at a single location. Here, we describe the first detection of P.destructans in western North America in a little brown bat (Myotis lucifugus) from near Seattle, WA, far from the previously recognized geographic distribution of the fungus. Whole-genome sequencing and phylogenetic analyses indicated that the isolate of P.destructans from Washington grouped with other isolates of a presumed clonal lineage from the eastern United States. Thus, the occurrence of P.destructans in Washington does not likely represent a novel introduction of the fungus from Eurasia, and the lack of intensive surveillance in the western United States makes it difficult to interpret whether the occurrence of P.destructans in the Pacific Northwest is disjunct from that in eastern North America. Although there is uncertainty surrounding the impacts of WNS in the Pacific Northwest, the presence of the pathogen in western North America could have major consequences for bat conservation. IMPORTANCE White-nose syndrome (WNS) represents one of the most consequential wildlife diseases of modern times. Since it was first documented in New York in 2006, the disease has killed millions of bats and threatens several formerly abundant species with extirpation or extinction. The spread of WNS in eastern North America has been relatively gradual, inducing optimism that disease mitigation strategies could be established in time to conserve bats susceptible to WNS in western North America. The recent detection of the fungus that causes WNS in the Pacific Northwest, far from its previous known distribution, increases the urgency for understanding the long-term impacts of this disease and for developing strategies to conserve imperiled bat species.

Bats play a crucial role in the ecosystem. However, North American bat populations have experienced a dramatic decline since 2006 due to white-nose syndrome, a disease caused by Pseudogymnoascus destructans (Pd). This fungus can invade and damage the skin on bat wings and muzzles during hibernation. Since 2021, Pd has been reported at selected sites in western Canada, the region with the highest bat diversity in Canada, eliciting urgent calls for action among diverse stakeholders. Here we analyze nine metagenomes of bat guanos and wing swabs and the genomes of five Pd strains from western Canada to investigate the distribution and diversity of Pd in this region. Pd was found in all nine metagenomic samples and the metagenome sequences enabled us to identify the associated bat species. Divergence time estimates of Pd based on whole-genome sequences suggest that Pd likely entered Alberta two to five years before its first official report. Furthermore, we found evidence of abundant gene copy number variations in this species. Together, our metagenomic and genomic analyses indicate that Pd is more prevalent than currently recognized and is evolving and diversifying. Continued surveillance with more comprehensive methods is needed to accurately track its spread and facilitate timely management of white-nose syndrome in North America.

Chapter 136 - White-nose syndrome: A fungal disease of North American hibernating bats

The skin microbiome is an essential line of host defense against pathogens, yet our understanding of microbial communities and how they change when hosts become infected is limited. We investigated skin microbial composition in three North American bat species (Myotis lucifugus, Eptesicus fuscus, and Perimyotis subflavus) that have been impacted by the infectious disease, white-nose syndrome, caused by an invasive fungal pathogen, Pseudogymnoascus destructans. We compared bacterial and fungal composition from 154 skin swab samples and 70 environmental samples using a targeted 16S rRNA and internal transcribed spacer amplicon approach. We found that for M. lucifugus, a species that experiences high mortality from white-nose syndrome, bacterial microbiome diversity was dramatically lower when P. destructans was present. Key bacterial families-including those potentially involved in pathogen defense-significantly differed in abundance in bats infected with P. destructans compared to uninfected bats. However, skin bacterial diversity was not lower in E. fuscus or P. subflavus when P. destructans was present despite populations of the latter species declining sharply from white-nose syndrome. The fungal species present on bats substantially overlapped with the fungal taxa present in the environment at the site where the bat was sampled, but fungal community composition was unaffected by the presence of P. destructans for any of the three bat species. This species-specific alteration in bat skin bacterial microbiomes after pathogen invasion may suggest a mechanism for the severity of white-nose syndrome in M. lucifugus but not for other bat species impacted by the disease.IMPORTANCEInherent complexities in the composition of microbiomes can often preclude investigations of microbe-associated diseases. Instead of single organisms being associated with disease, community characteristics may be more relevant. Longitudinal microbiome studies of the same individual bats as pathogens arrive and infect a population are the ideal experiment but remain logistically challenging; therefore, investigations like our approach that are able to correlate invasive pathogens to alterations within a microbiome may be the next best alternative. The results of this study potentially suggest that microbiome-host interactions may determine the likelihood of infection. However, the contrasting relationship between Pd and the bacterial microbiomes of Myotis lucifugus and Perimyotis subflavus indicate that we are just beginning to understand how the bat microbiome interacts with a fungal invader such as Pd.

Populations of some cave-hibernating bats have undergone declines in recent years due to an introduced fungus (Pseudogymnoascus destructans) that causes the disease white-nose syndrome (WNS), which is often fatal to bats during hibernation. Unprecedented declines in cave-hibernating species have the potential to change community composition via numerous mechanisms, including competitive release. We trapped bats for 6 years in the Ouachita Mountains of Arkansas prior to the arrival of WNS in the region and compared capture rates of 7 species to capture rates collected in 2020–2021 after WNS establishment. We found a 98% decline in Myotis septentrionalis (Northern Long-eared Bat) and a 77% decline in Perimyotis subflavus (Tricolored Bat) after WNS became prevalent. Nycticeius humeralis (Evening Bat) captures increased by 220% after WNS. Capture rates of Eptesicus fuscus (Big Brown Bat) increased by 100%, but this increase was not significant. We also found no significant differences in captures rates for Lasiurus borealis (Eastern Red Bat), Lasiurus seminolus (Seminole Bat), and Lasiurus cinereus (Hoary Bat) after the arrival of WNS. Our results indicate that the forest bat community of the Ouachita Mountains has been altered since the arrival of WNS in 2012, but it is unknown if these changes are permanent or if species will decline further or recover via adaptive or genetic changes in their populations in the future.

White-nose syndrome (WNS) is an emerging fungal epizootic disease that has caused large-scale mortality in several species of North American bats. The fungus that causes WNS, Pseudogymnoascus destructans (Pd), has also been detected in bat species without diagnostic signs of WNS. Although these species could play a role in WNS spread, understanding of the spatial and temporal extents of Pd occurrence on WNS-resistant species is limited. This study evaluated the presence of Pd on 272 individuals of three species of migratory tree-roosting bats: hoary (Lasiurus cinereus), eastern red (Lasiurus borealis), and silver-haired (Lasionycteris noctivagans) bats, obtained opportunistically during summer and autumn from throughout much of their ranges in North America. We also compared tissue sampling protocols (i.e., tissue swabbing, fur swabbing, and DNA extraction of excised wing tissue). We detected Pd on three eastern red bats from Illinois and Ohio, US, one silver-haired bat from West Virginia, US, and one hoary bat from New York, US, all via DNA extracted from wing tissue of carcasses. These results document the first publicly reported detections of Pd on a hoary bat and on migratory bats during the autumn migratory period, and demonstrate the potential for using carcasses salvaged at wind-energy facilities to monitor for Pd.

Temperature is a critically important factor in many infectious disease systems, because it can regulate responses in both the host and the pathogen. White-nose syndrome (WNS) in bats is a severe infectious disease caused by the temperature-sensitive fungus, Pseudogymnoascus destructans (Pd). One feature of WNS is an increase in the frequency of arousal bouts (i.e. when bat body temperatures are elevated) in Pd-infected bats during hibernation. While several studies have proposed that increased frequency of arousals may play a role in the pathophysiology of WNS, it is unknown if the temperature fluctuations might mediate Pd growth. We hypothesized that exposure to a high frequency of elevated temperatures would reduce Pd growth due to thermal constraints on the pathogen. We simulated the thermal conditions for arousal bouts of uninfected and infected bats during hibernation (fluctuating from 8 to 25°C at two different rates) and quantified Pd growth in vitro. We found that increased exposure to high temperatures significantly reduced Pd growth. Because temperature is one of the most critical abiotic factors mediating host-pathogen interactions, resolving how Pd responds to fluctuating temperatures will provide insights for understanding WNS in bats and other fungal diseases.

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

The causative agent of White-Nose Syndrome (WNS) in bats is the psychrophilic fungus, Pseudogymnoascus destructans. Since its discovery in the winter of 2006-2007 there has been a flurry of research to better understand this pathogenic fungus as well as find a treatment to save the bats. There is promise in finding a potential treatment through the use of safe Volatile Organic Compounds (VOC). There was potential for the use of 1-octen-3-ol, mushroom alcohol as a treatment for WNS. The first chapter shows the effectiveness of the R and S enantiomers and the racemic form of 1-octen-3-ol (mushroom alcohol) as well as trans-2-hexenal (leaf aldehyde), on mycelial plugs and conidiospores at temperatures of 5, 10 and 15˚C. 1-octen-3-ol was able to inhibit mycelial and conidiospore growth of P. destructans at 0.4 and 0.8 μmol/mL and the R enantiomer of this compound was more effective than the S enantiomer, supporting the finding that biological systems can be sensitive to stereochemistry. trans-2-hexenal was more effective than 1-octen-3-ol and showed fungicidal activity at 0.05 μmol/mL to both conidiospores and mycelia of P. destructans. The second chapter is a transcriptomics study to understand a potential pathway trans-2-hexenal takes in inhibiting P. destructans growth. P. destructans was treated with trans-2-hexenal at sublethal concentrations, and RNA was extracted for Illumina sequencing. It was found that trans-2-hexenal was able to inhibit growth, possibly through downregulating the production of essential genes, like Superoxide Dismutase. Also discovered was the downregulation of known endopeptidase enzyme, Destrucin-2, indicating trans-2-hexenal may also able to reduce virulence while inhibiting growth.

To the Editor: Bat geomycosis (white nose syndrome) is caused by the psychrophilic fungus Geomyces destructans, which has rapidly spread in the United States and Canada since it was first reported from Albany, New York (1,2). In 2011, a single genotype of G. destructans was found in bats with geomycosis in different parts of New York (3). The findings raised the possibility of clonal spread of a new pathogen with serious implications for the survival of the affected bat populations (4). To provide information for devising conservation measures, we explored whether this emerging infectious disease is caused by a novel pathogen (5). To do so, we genotyped G. destructans isolates from the midwestern and southern United States. During 2010 and 2011, a total of 11 cultures of G. destructans were isolated and identified: 1 each from Pennsylvania and Ohio, 3 from North Carolina, and 6 from West Virginia (Figure). The cultures came from 8 little brown bats (Myotis lucifugus) and 3 tri-colored bats (Perimyotis subflavus). Two recent G. destructans isolates from New York and 1 G. pannorum isolate were included as controls. Genomic DNA was prepared from fungal growth by the conventional glass bead treatment, phenol–chloroform extraction, and ethanol precipitation. PCR amplifications of 8 G. destructans gene fragments (ALR, Bpntase, DHC1, GPHN, PCS, POB3, SRP72, and VPS13) were performed as described (3). The amplicons were sequenced and nucleotides were aligned by Sequencher 4.8 (www.genecodes.com); phylogenetic analyses were done using PAUP*4.0 software (www.sinauer.com). Figure A) Consensus maximum-parsimony tree of 8 concatenated gene fragments of Geomyces destructans. Data were derived from 13 G. destructans test isolates. G. destructans M1379 and G. pannorum M1378 were used as controls in this study; they were described in ... A total of 4,722 nt sequences were obtained from 8 gene fragments of 13 G. destructans isolates (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:JQ029780-JQ029883,start_term:JQ029780,end_term:JQ029883,start_term_id:380040511,end_term_id:380040717}}JQ029780-JQ029883) and 1 G. pannorum isolate (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:HQ834330,term_id:386776025,term_text:HQ834330}}HQ834330, {type:entrez-nucleotide,attrs:{text:HQ834347,term_id:386776059,term_text:HQ834347}}HQ834347, {type:entrez-nucleotide,attrs:{text:HQ834364,term_id:386776093,term_text:HQ834364}}HQ834364, {type:entrez-nucleotide,attrs:{text:HQ834381,term_id:386776127,term_text:HQ834381}}HQ834381, {type:entrez-nucleotide,attrs:{text:HQ834398,term_id:386776161,term_text:HQ834398}}HQ834398, {type:entrez-nucleotide,attrs:{text:HQ834415,term_id:386776195,term_text:HQ834415}}HQ834415, {type:entrez-nucleotide,attrs:{text:HQ834432,term_id:386776229,term_text:HQ834432}}HQ834432, and {type:entrez-nucleotide,attrs:{text:HQ834449,term_id:386776263,term_text:HQ834449}}HQ834449). Multiple alignments of these sequences showed 100% identity, and the aligned nucleotides matched perfectly with those of earlier G. destructans sequences for the same gene fragments analyzed from New York isolates (3). The nucleotide alignments of 8 sequences showed differences from those obtained from the closely related fungus, G. pannorum. Maximum-parsimony trees were generated by using sequences from each gene fragment. These trees showed a single clade of G. destructans strains distinct from G. pannorum; similar topologies were obtained when different phylogenetics methods were used for analysis (details not shown). A consensus maximum-parsimony tree derived from the 8 concatenated gene fragments also showed a single clade of G. destructans isolates from New York and the midwestern and southern United States (Figure). The data obtained in this study strongly indicate further clonal spread of G. destructans from its origin near Albany, New York. The locations in which G. destructans was detected in the current study were spread across 5 states, which were >800 miles from Albany. The test isolates were compared with a New York isolate from 2008, which provided a 4-year temporal variation in our sampling. Bats of 2 species were positive for G. desctructans in the current samples, and they yielded the same G. destructans genotype. Thus, there is evidence for host-independent spread of a single clone of G. destructans. These data would support the novel-pathogen hypothesis for the origin of bat geomycosis (5). However, these conclusions are based on limited sampling because isolations of G. destructans from affected bats are uncommon. The demonstration of pure fungal culture in the affected animals is still not the standard for geomycosis diagnostics, and most geomycosis is confirmed by bat morphologic appearance or histopathologic examination. Additionally, our phylogenetics analyses were limited to ≈5 kbp of fungal genomes, which could lead to sampling bias (3). Ideally, a large number of G. destructans isolates, including isolates from Europe, and additional polymorphic markers would be needed to determine the novel or local origin of this pathogen (6,7). The environmental factors that led to introduction or reemergence of G. destructans in mines and caves remain unknown, and their contribution in the spread of the fungus through air, water, and soil is yet to be determined (8). Although no direct evidence has emerged, a role for anthropomorphic activities (occupational or recreational) in this spread is a distinct possibility (9). We provide genetic evidence for further spread of a single genotype of G. destructans from Albany, New York, to locations in the midwestern and southern United States. Experimental transmission of geomycosis from infected bats to healthy bats by direct contact has recently been confirmed (10). Therefore, G. destructans might be rapidly spreading along summer and winter migration routes of bats, which present ample opportunities for mixing of healthy and diseased animals.

Assessing the scope and severity of threats is necessary for evaluating impacts on populations to inform conservation planning. Quantitative threat assessment often requires monitoring programs that provide reliable data over relevant spatial and temporal scales, yet such programs can be difficult to justify until there is an apparent stressor. Leveraging efforts of wildlife management agencies to record winter counts of hibernating bats, we collated data for 5 species from over 200 sites across 27 U.S. states and 2 Canadian provinces from 1995 to 2018 to determine the impact of white‐nose syndrome (WNS), a deadly disease of hibernating bats. We estimated declines of winter counts of bat colonies at sites where the invasive fungus that causes WNS (Pseudogymnoascus destructans) had been detected to assess the threat impact of WNS. Three species undergoing species status assessment by the U.S. Fish and Wildlife Service (Myotis septentrionalis, Myotis lucifugus, and Perimyotis subflavus) declined by more than 90%, which warrants classifying the severity of the WNS threat as extreme based on criteria used by NatureServe. The scope of the WNS threat as defined by NatureServe criteria was large (36% of Myotis lucifugus range) to pervasive (79% of Myotis septentrionalis range) for these species. Declines for 2 other species (Myotis sodalis and Eptesicus fuscus) were less severe but still qualified as moderate to serious based on NatureServe criteria. Data‐sharing across jurisdictions provided a comprehensive evaluation of scope and severity of the threat of WNS and indicated regional differences that can inform response efforts at international, national, and state or provincial jurisdictions. We assessed the threat impact of an emerging infectious disease by uniting monitoring efforts across jurisdictional boundaries and demonstrated the importance of coordinated monitoring programs, such as the North American Bat Monitoring Program (NABat), for data‐driven conservation assessments and planning.

Pseudogymnoascus destructans is the causative fungal agent of white-nose syndrome (WNS), an emerging fungal-borne epizootic. WNS is responsible for a catastrophic decline of hibernating bats in North America, yet we have limited understanding of the physiological interactions between pathogen and host. Pseudogymnoascus destructans severely damages wings and tail membranes, by causing dryness that leads to whole sections crumbling off. Four possible mechanisms have been proposed by which infection could lead to dehydration; in this study, we tested one: P. destructans infection could cause disruption to passive gas-exchange pathways across the wing membranes, thereby causing a compensatory increase in water-intensive pulmonary respiration. We hypothesized that total evaporative water loss would be greater when passive gas exchange was inhibited. We found that bats did not lose more water when passive pathways were blocked. This study provides evidence against the proposed proximal mechanism that disruption to passive gas exchange causes dehydration and death to WNS-infected bats.

The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer-based qPCR test for P. destructans to refine quantification capabilities of this assay.

There is a serious concern that white‐nose syndrome (WNS), a fungal disease causing severe population declines in North American bats, could soon threaten bats on the Australian continent. Despite an ‘almost certain' risk of incursion within the next ten years, and high virulence in naïve bat populations, we remain uncertain about the vulnerability of Australian bats toWNS. In this study, we intersected occurrences for the 27 cave roosting bat species in Australia with interpolated data on mean annual surface temperature, which provides a proxy for thermal conditions within a cave and hence its suitability for growth by the fungal pathogenPseudogymnoascus destructans. Our analysis identifies favourable roost thermal conditions within 30–100% of the ranges of eight bat species across south‐eastern Australia, including for seven species already listed as threatened with extinction. These results demonstrate the potential for widespread exposure toP. destructansand suggest thatWNScould pose a serious risk to the conservation of Australia's bat fauna. The impacts of exposure toP. destructanswill depend, however, on the sensitivity of bats to developingWNS, and a more comprehensive vulnerability assessment is currently prevented by a lack of information on the hibernation biology of Australian bats. Thus, given the clear potential for widespread exposure of Australia's bats toP. destructansdemonstrated by our study, two specific policy actions seem justified: (i) urgent implementation of border controls that identify and decontaminate cave‐associated fomites and (ii) dedicated funding to enable research on key aspects of bat winter behaviour and hibernation physiology. Further, as accidental translocation of this fungus could also pose a risk to other naïve bat faunas in cooler regions of southern Africa and South America, we argue that a proactive, globally coordinated approach is required to understand and mitigate the potential impacts ofWNSspreading to Southern Hemisphere bats.

Pseudogymnoascus destructans, the fungus that causes white-nose syndrome in hibernating bats, has spread across eastern North America over the past decade and decimated bat populations. The saprotrophic growth of P. destructans may help to perpetuate the white-nose syndrome epidemic, and recent model predictions suggest that sufficiently reducing the environmental growth of P. destructans could help mitigate or prevent white-nose syndrome-associated bat colony collapse. In this study, we screened 301 microbes from diverse environmental samples for their ability to inhibit the growth of P. destructans. We identified 145 antagonistic isolates, 53 of which completely or nearly completely inhibited the growth of P. destructans in co-culture. Further analysis of our best antagonists indicated that these microbes have different modes of action and may have some specificity in inhibiting P. destructans. The results suggest that naturally-occurring microbes and/or their metabolites may be considered further as candidates to ameliorate bat colony collapse due to P. destructans.