Causative Agent Of White-nose Syndrome Research Articles

Impact of putatively beneficial genomic loci on gene expression in little brown bats (Myotis lucifugus, Le Conte, 1831) affected by white-nose syndrome.

Genome-wide scans for selection have become a popular tool for investigating evolutionary responses in wildlife to emerging diseases. However, genome scans are susceptible to false positives and do little to demonstrate specific mechanisms by which loci impact survival. Linking putatively resistant genotypes to observable phenotypes increases confidence in genome scan results and provides evidence of survival mechanisms that can guide conservation and management efforts. Here we used an expression quantitative trait loci (eQTL) analysis to uncover relationships between gene expression and alleles associated with the survival of little brown bats (Myotis lucifugus) despite infection with the causative agent of white-nose syndrome. We found that 25 of the 63 single-nucleotide polymorphisms (SNPs) associated with survival were related to gene expression in wing tissue. The differentially expressed genes have functional annotations associated with the innate immune system, metabolism, circadian rhythms, and the cellular response to stress. In addition, we observed differential expression of multiple genes with survival implications related to loci in linkage disequilibrium with focal SNPs. Together, these findings support the selective function of these loci and suggest that part of the mechanism driving survival may be the alteration of immune and other responses in epithelial tissue.

The skin I live in: Pathogenesis of white-nose syndrome of bats.

The emergence of white-nose syndrome (WNS) in North America has resulted in mass mortalities of hibernating bats and total extirpation of local populations. The need to mitigate this disease has stirred a significant body of research to understand its pathogenesis. Pseudogymnoascus destructans, the causative agent of WNS, is a psychrophilic (cold-loving) fungus that resides within the class Leotiomycetes, which contains mainly plant pathogens and is unrelated to other consequential pathogens of animals. In this review, we revisit the unique biology of hibernating bats and P. destructans and provide an updated analysis of the stages and mechanisms of WNS progression. The extreme life history of hibernating bats, the psychrophilic nature of P. destructans, and its evolutionary distance from other well-characterized animal-infecting fungi translate into unique host-pathogen interactions, many of them yet to be discovered.

Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

Fungal Diversity in Korean Caves and Cave-Inhabiting Bats with Attention to Pseudogymnoascus Species

Pseudogymnoascus is a psychrophilic fungus, which is a genus widely distributed in cold regions around the world. Recently, the presence of Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome (WNS) belonging to Pseudogymnoascus, has been reported in neighboring countries of Korea. However, no investigation on Pd has been reported in Korea. In this study, cave-inhabiting bats and their habitats were investigated in terms of the diversity of cave fungi, and we tried to confirm the presence of Pd. Three caves suspected of hosting Pd were selected, and 83 environmental and 53 bat samples were collected. A total of 154 fungal strains belonging to 31 different genera were isolated, and 20 of 154 were confirmed to belong to Pseudogymnoascus. Pd-diagnostic PCR was performed to check whether Pd was present in the isolated Pseudogymnoascus, and seven positives were confirmed. However, phylogenetic analyses revealed that no isolates belonged or were closely related to the clade with Pd. Although samples were collected from limited areas, undescribed Pseudogymnoascus species were isolated, and it was confirmed that Korean isolates were distributed in various clades. In conclusion, it is hypothesized that Korean Pseudogymnoascus presents high diversity.

First Isolation of Pseudogymnoascus destructans, the Fungal Causative Agent of White-Nose Syndrome, in Korean Bats (Myotis petax)

White-nose syndrome (WNS), caused by Pseudogymnoascus destructans (Pd), is a lethal fungal disease that affects hibernating bats in North America. Recently, the presence of Pd was reported in countries neighboring Korea. However, Pd has not been investigated in Korea. Therefore, this study aimed to identify the presence of Pd in Korean bats. Altogether, wings from 241 bats were collected from 13 cities and cultured. A total of 79 fungal colonies were isolated, and two isolates were identified as Pd using polymerase chain reaction. Of the nine bat species captured in 13 cities, Pd was isolated only from Myotis petax in Goryeong. Atypical, curved conidia were observed in two isolated fungal colonies. Although histological lesions were not observed by hematoxylin and eosin or periodic acid–Schiff staining, fungal invasion was observed in the tissue sections. Taken together, these results confirmed the presence of Pd in Korean bats and suggest the possibility of WNS outbreaks in Korean bats. This is the first report of the isolation and molecular analysis of Pd from Korean bats.

Microbial isolates with Anti-Pseudogymnoascus destructans activities from Western Canadian bat wings

Pseudogymnoascus destructans (Pd) is the causative agent of white-nose syndrome, which has resulted in the death of millions of bats in North America (NA) since 2006. Based on mortalities in eastern NA, the westward spread of infections likely poses a significant threat to western NA bats. To help prevent/reduce Pd infections in bats in western NA, we isolated bacteria from the wings of wild bats and screened for inhibitory activity against Pd. In total, we obtained 1,362 bacterial isolates from 265 wild bats of 13 species in western Canada. Among the 1,362 isolates, 96 showed inhibitory activity against Pd based on a coculture assay. The inhibitory activities varied widely among these isolates, ranging from slowing fungal growth to complete inhibition. Interestingly, host bats containing isolates with anti-Pd activities were widely distributed, with no apparent geographic or species-specific pattern. However, characteristics of roosting sites and host demography showed significant associations with the isolation of anti-Pd bacteria. Specifically, anthropogenic roosts and swabs from young males had higher frequencies of anti-Pd bacteria than those from natural roosts and those from other sex and age-groups, respectively. These anti-Pd bacteria could be potentially used to help mitigate the impact of WNS. Field trials using these as well as additional microbes from future screenings are needed in order to determine their effectiveness for the prevention and treatment against WNS.

Bacterial community dynamics on bats and the implications for pathogen resistance.

The bats skin microbiota plays an important role in reducing pathogen infection, including the deadly fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose syndrome. However, the dynamic of skin bacterial communities response to environmental perturbations remains poorly described. We characterized skin bacterial community over time and space in Rhinolophus ferrumequinum, a species with high resistance to the infection with P. destructans. We collected environmental covariate data to determine what factors influenced changes in community structure. We observed significant temporal and spatial shifts in the skin bacterial community, which was mainly associated with variation in operational taxonomic units. The skin bacterial community differed by the environmental microbial reservoirs and was most influenced by host body condition, bat roosting temperature and geographic distance between sites, but was not influenced by pathogen infection. Furthermore, the skin microbiota was enriched in particular taxa with antifungal abilities, such as Enterococcus, Burkholderia, Flavobacterium, Pseudomonas, Corynebacterium and Rhodococcus. And specific strains of Pseudomonas, Corynebacterium and Rhodococcus even inhibited P. destructans growth. Our findings provide new insights in characterizing the variation in bacterial communities can inform us about the processes of driving community assembly and predict the host's ability to resist or survive pathogen infection.

Trans-2-hexenal downregulates several pathogenicity genes of Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats.

White-nose syndrome is an emergent wildlife disease that has killed millions of North American bats. It is caused by Pseudogymnoascus destructans, a cold-loving, invasive fungal pathogen that grows on bat tissues and disrupts normal hibernation patterns. Previous work identified trans-2-hexenal as a fungistatic volatile compound that potentially could be used as a fumigant against P. destructans in bat hibernacula. To determine the physiological responses of the fungus to trans-2-hexenal exposure, we characterized the P. destructans transcriptome in the presence and absence of trans-2-hexenal. Specifically, we analyzed the effects of sublethal concentrations (5 μmol/L, 10 μmol/L, and 20 μmol/L) of gas-phase trans-2-hexenal of the fungus grown in liquid culture. Among the three treatments, a total of 407 unique differentially expressed genes (DEGs) were identified, of which 74 were commonly affected across all three treatments, with 44 upregulated and 30 downregulated. Downregulated DEGs included several probable virulence genes including those coding for a high-affinity iron permease, a superoxide dismutase, and two protein-degrading enzymes. There was an accompanying upregulation of an ion homeostasis gene, as well as several genes involved in transcription, translation, and other essential cellular processes. These data provide insights into the mechanisms of action of trans-2-hexenal as an anti-fungal fumigant that is active at cold temperatures and will guide future studies on the molecular mechanisms by which six carbon volatiles inhibit growth of P. destructans and other pathogenic fungi.

Determinants of defence strategies of a hibernating European bat species towards the fungal pathogen Pseudogymnoascus destructans

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in North America, has decimated bat populations within a decade. The fungus impacts bats during hibernation when physiological functions, including immune responses, are down-regulated. Studies have shown that Pd is native to Europe, where it is not associated with mass mortalities. Moreover, genomic and proteomic studies indicated that European bats may have evolved an effective immune defence, which is lacking in North American bats. However, it is still unclear which defence strategy enables European bats to cope with the pathogen. Here, we analyzed selected physiological and immunological parameters in torpid, Pd infected European greater mouse-eared bats (Myotis myotis) showing three different levels of infection (asymptomatic, mild and severe symptoms). From a subset of the studied bats we tracked skin temperatures during one month of hibernation. Contrasting North American bats, arousal patterns remained unaffected by Pd infections in M. myotis. In general, heavier M. myotis aroused more often from hibernation and showed less severe disease symptoms than lean individuals; most likely because heavy bats were capable of reducing the Pd load more effectively than lean individuals. In the blood of severely infected bats, we found higher gene expression levels of an inflammatory cytokine (IL-1β), but lower levels of an acute phase protein (haptoglobin), reactive oxygen metabolites (ROMs) and plasma non-enzymatic antioxidant capacity (OXY) compared to conspecifics with lower levels of infection. We conclude that M. myotis, and possibly also other European bat species, tolerate Pd infections during torpor by using selected acute phase response parameters at baseline levels, yet without arousing from torpor and without synthesizing additional immune molecules.

Streptomyces buecherae sp. nov., an actinomycete isolated from multiple bat species.

A putative novel clade within the genus Streptomyces was discovered following antifungal screening against Pseudogymnoascus destructans, the causative agent of white-nose syndrome, and described using multi-locus sequencing analysis. Swabs from both the cave myotisbat (Myotis velifer) and the Brazilian free-tailed bat (Tadarida brasiliensis) in southern New Mexico bore isolates AC536, AC541T and AC563, which were characterised using phylogenetic, morphological, and phenotypic analyses. Multi-locus sequence analysis positions AC541T with neighbors Streptomyces rubidus (NRRL B-24619T), Streptomyces guanduensis (NRRL B-24617T), and Streptomyces yeochonensis (NRRL B-24245T). A complete genome of the type strain was assembled to determine its taxonomy and secondary metabolite potential. ANI comparisons between all closely related types strains are shown to be well below the 95-96% species delineation. DNA-DNA relatedness between AC541T and its nearest neighbors ranged between 23.7 and 24.1% confirming novelty. Approximately 1.49Mb or 17.76% of the whole genome is devoted to natural product biosynthesis. The DNA G + C content of the genomic DNA of the type strain is 73.13mol%. Micromorphology depicts ovoid spores with smooth surfaces in flexuous chains. Strains presented an ivory to yellow hue on most ISP media except inorganic salts-starch agar (ISP4) and can grow on D-glucose, mannitol, and D-fructose, but exhibited little to no growth on L-arabinose, sucrose, D-xylose, inositol, L-rhamnose, D-raffinose, and cellulose. This clade possesses the capability to grow from 10 to 45°C and 12.5% (w/v) NaCl. There was strain growth variation in pH, but all isolates thrive at alkaline levels. Based on our polyphasic study of AC541T, the strain warrants the assignment to a novel species, for which the name Streptomyces buecherae sp. nov. is proposed. The type strain is AC541T (= JCM 34263T, = ATCC TSD201T).

Antifungal Potential of the Skin Microbiota of Hibernating Big Brown Bats (Eptesicus fuscus) Infected With the Causal Agent of White-Nose Syndrome.

Little is known about skin microbiota in the context of the disease white-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Pd), that has caused enormous declines of hibernating North American bats over the past decade. Interestingly, some hibernating species, such as the big brown bat (Eptesicus fuscus), appear resistant to the disease and their skin microbiota could play a role. However, a comprehensive analysis of the skin microbiota of E. fuscus in the context of Pd has not been done. In January 2017, we captured hibernating E. fuscus, sampled their skin microbiota, and inoculated them with Pd or sham inoculum. We allowed the bats to hibernate in the lab under controlled conditions for 11 weeks and then sampled their skin microbiota to test the following hypotheses: (1) Pd infection would not disrupt the skin microbiota of Pd-resistant E. fuscus; and (2) microbial taxa with antifungal properties would be abundant both before and after inoculation with Pd. Using high-throughput 16S rRNA gene sequencing, we discovered that beta diversity of Pd-inoculated bats changed more over time than that of sham-inoculated bats. Still, the most abundant taxa in the community were stable throughout the experiment. Among the most abundant taxa, Pseudomonas and Rhodococcus are known for antifungal potential against Pd and other fungi. Thus, in contrast to hypothesis 1, Pd infection destabilized the skin microbiota but consistent with hypothesis 2, bacteria with known antifungal properties remained abundant and stable on the skin. This study is the first to provide a comprehensive survey of skin microbiota of E. fuscus, suggesting potential associations between the bat skin microbiota and resistance to the Pd infection and WNS. These results set the stage for future studies to characterize microbiota gene expression, better understand mechanisms of resistance to WNS, and help develop conservation strategies.

Experimental evaluation of genomic DNA degradation rates for the pathogen Pseudogymnoascus destructans (Pd) in bat guano.

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in bats (WNS), has led to dramatic declines of bat populations in eastern North America. In the spring of 2016, WNS was first detected at several locations in Washington State, USA, which has prompted the need for large scale surveillance efforts to monitor the spread of Pd. Pd is typically detected in bats using invasive methods requiring capturing and swabbing individual bats. However, Pd can also be detected in guano, which may provide an efficient, affordable, and noninvasive means to monitor Pd in bats across North America. The widespread implementation of Pd surveillance in guano is hindered by substantial uncertainty about the probability of detecting Pd when present, and how this probability is influenced by the time since defecation, local environmental conditions, the amount of guano sampled, and the original concentration of DNA shed in the guano. In addition, the expected degradation rate of Pd DNA depends on whether the Pd DNA found in guano represents extracellular DNA fragments, intracellular DNA from dead Pd fungal cells, or from intracellular and viable Pd cells. While this is currently unknown, it has been posited that most environmental DNA, such as Pd found in guano long after defecation, is fragmented extracellular DNA. Using non-viable isolated DNA at precise quantities, we experimentally characterized the degradation rates of Pd DNA in guano samples. We spiked 450 guano samples with Pd gDNA in a 10-fold dilution series from 1 million to 1,000 fg and placed them in variable environmental conditions at five sites at Mount Rainier National Park in Washington State, which is a priority location for Pd surveillance. We evaluated DNA degradation over 70 days by quantifying the amount of DNA in samples collected every 14 days using real-time quantitative PCR (qPCR). Our sampling period was from July 10th to September 17th 2018 which overlaps with bat movement between summer roosts as well as movement from maternity colonies fall swarms. We detected Pd DNA in guano 56 and 70 days after inoculation with 1 million and 100,000 fg respectively, while the lowest quantity (1,000 fg) was detected until 42 days. Detection probability was variable among sites and lower where samples were left exposed without overhead cover. If Pd is shed as extracellular DNA in guano at quantities above 1,000 fg, then guano collection is likely to provide an effective tool for environmental screening of Pd that can be employed in an early detection and rapid response framework throughout Washington and other regions where this disease is rapidly emerging.

Streptomyces corynorhini sp. nov., isolated from Townsend's big-eared bats (Corynorhinus townsendii).

Four bacterial strains, with the capability of inhibiting Pseudogymnoascus destructans, the causative agent of white-nose syndrome, were isolated from male Townsend's big-eared bats (Corynorhinus townsendii, Family: Vespertilionidae) in New Mexico. Isolates AC161, AC162, AC208, and AC230T were characterised as a novel clade using morphological, phenotypic and phylogenetic analysis. A draft genome of the type strain was completed to determine its taxonomy and secondary metabolite biosynthetic potential. Multi-locus sequence analysis nests AC230T with neighbours Streptomyces scopuliridis (NRRL B-24574T), Streptomyces lushanensis (NRRL B-24994T), Streptomyces odonnellii (NRRL B-24891T) and Streptomyces niveus (NRRL 2466T). Further phylogenetic analysis showed the MLSA distances between AC230T and its near neighbours are much greater than the generally accepted threshold (> 0.007) for bacterial species delineation. DNA-DNA relatedness between AC230T and its near neighbours ranged between 25.7 ± 2.1 and 29.9 ± 2.4%. The DNA G+C content of the genomic DNA of the type strain is 71.7mol%. Isolate AC230T presents a white to ivory hue on most ISP media and its micromorphology exhibits ovoid spores with smooth surfaces in flexuous chains. Based on our study of AC230T, the strain warrants the assignment to a novel species, for which the name Streptomyces corynorhini sp. nov. is proposed. The type strain is AC230T (= JCM 33171T, = ATCC TSD155T).

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.

Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

Unsuspected retreats: autumn transitional roosts and presumed winter hibernacula of little brown myotis in Colorado

Documentation of autumn and winter roosts of many species of hibernating bats are lacking from western North America. However, recent evidence suggests that rather than using caves and mines, many individuals and some species of bats may roost in inconspicuous rock crevices at these times of year. I investigated autumn use of rock crevices and other roosts by the little brown myotis (Myotis lucifugus) in the Rocky Mountains of western Colorado through radiotelemetry (n = 38). Objectives were to determine the types and characteristics of roosts, describe patterns of movements to these roosts from summer colonies, and contrast findings with results of surveys of bats in caves and abandoned mines in Colorado during autumn and winter. Forty-four autumn transitional roosts and presumed hibernacula were located in buildings, trees, and rock crevices. Bats used short-distance movements changing in elevation to autumn transitional roosts and presumed hibernacula rather than major latitudinal migrations. Roost type and distance from capture site to roosts had the highest variable importance at the landscape scale. Microclimate comparisons showed that buildings provided warmer minimum average temperatures, which may benefit juvenile bats early in the transition season. Tree roost temperatures during autumn would allow bats to conserve energy by using daily torpor and passive rewarming to assist with afternoon arousals. Rock crevice roosts in talus were found to be suitable for hibernation by exhibiting the coolest average temperatures and maintaining the highest relative humidity levels. Autumn access and spring egress to high-elevation talus sites used by these bats were not obstructed by winter snow pack. These rock crevices also provided temperature and humidity levels that would support the persistence and growth of Pseudogymnoascus destructans (Pd), the causal agent of white-nose syndrome. However, bats in this study appeared to roost alone, which could inhibit the bat-to-bat spread of Pd. Surveys of caves and mines within and surrounding the study area revealed few hibernating little brown myotis, suggesting that most individuals in Colorado may instead utilize rock crevices as roosts during winter.

Summer activity patterns of four resident south Texas bat species

Although the activity patterns of bats are recognized to vary widely, both, temporally and spatially, the underlying drivers for those patterns remain poorly understood. Presently, studies focusing on foraging bat ecology have only generalized activity and influence of the environment (across sampling periods, sampling nights, or seasons). The influence of environmental features on foraging bat activity on a finer temporal scale (e.g., throughout a single sampling night) is markedly less studied. The recent emergence of Pseudogymnoascus destructans, a cold-adapted fungus and the causative agent of white-nose syndrome (WNS), has led to the precipitous declines in North American bat populations since its arrival in 2006. P. destructans has been recently documented in 10 Texas counties and, although WNS primarily affects hibernation cycles, it is recognized to have significant detrimental impacts on summer bat activity. It is imperative we understand and provide a baseline of summer bat activity patterns prior to the arrival of WNS to better manage and protect these, relatively understudied, populations of bat species in south Texas. We used a mixed model approach and acoustic monitoring to quantify how shrub density, canopy height, distance to water, roads, and bare ground coverage influenced nightly foraging activities of four summer resident bat species in south Texas: Eastern Red Bat (Lasirius borealis), Cave Myotis (Myotis velifer), Evening Bat (Nycticeius humeralis), and Brazilian Free-tailed Bat (Tadarida brasiliensis). Total bat activity peaked at 2:00-a for all species included in our study. A secondary, smaller peak was notable between 5:00-a and 6:00-a and activity decreased sharply immediately following 6:00-a. However, the trend for total bat activity differed substantially from trends exhibited by the individual species. Eastern red-bats and Evening bats exhibited bimodal nightly activity patterns and showed variable associations with water proximity whereas Cave bats and Brazilian free-tailed bats exhibited unimodal activity patterns and strong associations with canopy height and roads. Our results support the concept that insectivorous bats partition resources, temporally and spatially, in the presence of other sympatric species.

Phenotypic Divergence along Geographic Gradients Reveals Potential for Rapid Adaptation of the White-Nose Syndrome Pathogen, Pseudogymnoascus destructans, in North America.

White-nose syndrome (WNS) is an ongoing epizootic affecting multiple species of North American bats, caused by epidermal infections of the psychrophilic filamentous fungus Pseudogymnoascus destructans Since its introduction from Europe, WNS has spread rapidly across eastern North America and resulted in high mortality rates in bats. At present, the mechanisms behind its spread and the extent of its adaptation to different geographic and ecological niches remain unknown. The objective of this study was to examine the geographic patterns of phenotypic variation and the potential evidence for adaptation among strains representing broad geographic locations in eastern North America. The morphological features of these strains were evaluated on artificial medium, and the viability of asexual arthroconidia of representative strains was investigated after storage at high (23°C), moderate (14°C), and low (4°C) temperatures at different lengths of time. Our analyses identified evidence for a geographic pattern of colony morphology changes among the clonal descendants of the fungus, with trait values correlated with increased distance from the epicenter of WNS. Our genomic comparisons of three representative isolates revealed novel genetic polymorphisms and suggested potential candidate mutations that might be related to some of the phenotypic changes. These results show that even though this pathogen arrived in North America only recently and reproduces asexually, there has been substantial evolution and phenotypic diversification during its rapid clonal expansion.IMPORTANCE The causal agent of white-nose syndrome in bats is Pseudogymnoascus destructans, a filamentous fungus recently introduced from its native range in Europe. Infections caused by P. destructans have progressed across the eastern parts of Canada and the United States over the last 10 years. It is not clear how the disease is spread, as the pathogen is unable to grow above 23°C and ambient temperature can act as a barrier when hosts disperse. Here, we explore the patterns of phenotypic diversity and the germination of the fungal asexual spores, arthroconidia, from strains across a sizeable area of the epizootic range. Our analyses revealed evidence of adaptation along geographic gradients during its expansion. The results have implications for understanding the diversification of P. destructans and the limits of WNS spread in North America. Given the rapidly expanding distribution of WNS, a detailed understanding of the genetic bases for phenotypic variations in growth, reproduction, and dispersal of P. destructans is urgently needed to help control this disease.

Pseudogymnoascus destructans: Causative Agent of White-Nose Syndrome in Bats Is Inhibited by Safe Volatile Organic Compounds.

White-nose syndrome (WNS) is caused by Pseudogymnoascus destructans, a psychrophilic fungus that infects hibernating bats and has caused a serious decline in some species. Natural aroma compounds have been used to control growth of fungal food storage pathogens, so we hypothesized that a similar strategy could work for control of P. destructans. The effectiveness of exposure to low concentrations of the vapor phase of four of these compounds was tested on mycelial plugs and conidiospores at temperatures of 5, 10 and 15 °C. Here we report the efficacy of vapor phase mushroom alcohol (1-octen-3-ol) for inhibiting mycelial and conidiospore growth of P. destructans at 0.4 and 0.8 µmol/mL and demonstrate that the R enantiomer of this compound is more effective than the S enantiomer, supporting the finding that biological systems can be sensitive to stereochemistry. Further, we report that vapor phase leaf aldehyde (trans-2-hexenal), a common aroma compound associated with cut grass odors and also the major volatile compound in extra virgin olive oil, is more effective than mushroom alcohol. At 0.05 µmol/mL, trans-2-hexenal is fungicidal to both conidiospores and mycelia of P. destructans.

Use of Box-Beam Bridges as Day Roosts by Mexican Free-tailed Bats (Tadarida brasiliensis) in Texas

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.

DISPERSAL HAZARDS OF PSEUDOGYMNOASCUS DESTRUCTANS BY BATS AND HUMAN ACTIVITY AT HIBERNACULA IN SUMMER.

Bats occupying hibernacula during summer are exposed to Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome (WNS), and may contribute to its dispersal. Furthermore, equipment and clothing exposed to cave environments are a potential source for human-assisted spread of Pd. To explore dispersal hazards for Pd during the nonhibernal season, we tested samples that were collected from bats, the environment, and equipment at hibernacula in the eastern US between 18 July-22 August 2012. Study sites included six hibernacula known to harbor bats with Pd with varying winter-count impacts from WNS and two hibernacula (control sites) without prior history of WNS. Nucleic acid from Pd was detected from wing-skin swabs or guano from 40 of 617 bats (7% prevalence), including males and females of five species at five sites where WNS had previously been confirmed as well as from one control site. Analysis of guano collected during summer demonstrated a higher apparent prevalence of Pd among bats (17%, 37/223) than did analysis of wing-skin swabs (1%, 4/617). Viable Pd cultured from wing skin (2%, 1/56) and low recapture rates at all sites suggested bats harboring Pd during summer could contribute to pathogen dispersal. Additionally, Pd DNA was detected on clothing and trapping equipment used inside and near hibernacula, and Pd was detected in sediment more readily than in swabs of hibernaculum walls. Statistically significant differences in environmental abundance of Pd were not detected among sites, but prevalence of Pd differed between sites and among bat species. Overall, bats using hibernacula in summer can harbor Pd on their skin and in their guano, and demonstration of Pd on clothing, traps, and other equipment used at hibernacula during summertime within the WNS-affected region indicates risk for pathogen dispersal during the nonhibernal season.