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White-Nose Syndrome is an ongoing fungal epizootic caused by epidermal infections of the fungus, Pseudogymnoascus destructans (P. destructans), affecting hibernating bat species in North America. Emerging early in 2006 in New York State, infections of P. destructans have spread to 38 US States and seven Canadian Provinces. Since then, clonal isolates of P. destructans have accumulated genotypic and phenotypic variations in North America. Using microsatellite and single nucleotide polymorphism markers, we investigated the population structure and genetic relationships among P. destructans isolates from diverse regions in North America to understand its pattern of spread, and to test hypotheses about factors that contribute to transmission. We found limited support for genetic isolation of P. destructans populations by geographic distance, and instead identified evidence for gene flow among geographic regions. Interestingly, allelic association tests revealed evidence for recombination in the North American P. destructans population. Our landscape genetic analyses revealed that the population structure of P. destructans in North America was significantly influenced by anthropogenic impacts on the landscape. Our results have important implications for understanding the mechanism(s) of P. destructans spread. [ABSTRACT FROM AUTHOR]

We document white‐nose syndrome (WNS), a lethal disease of bats caused by the fungus Pseudogymnoascus destructans (Pd), and hibernacula microclimate in New Brunswick, Canada. Our study area represents a more northern region than is common for hibernacula microclimate investigations, providing insight as to how WNS may impact bats at higher latitudes. To determine the impact of the March 2011 arrival of Pd in New Brunswick and the role of hibernacula microclimate on overwintering bat mortality, we surveyed bat numbers at hibernacula twice a year from 2009 to 2015. We also collected data from iButton temperature loggers deployed at all sites and data from HOBO temperature and humidity loggers at three sites. Bat species found in New Brunswick hibernacula include Myotis lucifugus (Little Brown Bat) and M. septentrionalis (Northern Long‐eared Bat), with small numbers of Perimyotis subflavus (Tricolored Bat). All known hibernacula in the province were Pd‐positive with WNS‐positive bats by winter 2013. A 99% decrease in the overwintering bat population in New Brunswick was observed between 2011 and 2015. We did not observe P. subflavus during surveys 2013–2015 and the species appears to be extirpated from these sites. Bats did not appear to choose hibernacula based on winter temperatures, but dark zone (zone where no light penetrates) winter temperatures did not differ among our study sites. Winter dark zone temperatures were warmer and less variable than entrance or above ground temperatures. We observed visible Pd growth on hibernating bats in New Brunswick during early winter surveys (November), even though hibernacula temperatures were colder than optimum for in vitro Pd growth. This suggests that cold hibernacula temperatures encountered near the apparent northern range limit for Pd do not sufficiently slow fungal growth to prevent the onset of WNS and associated bat mortality over the winter. [ABSTRACT FROM AUTHOR]

Drinking water is a critical resource for hibernating bats and its importance may be further increased when disease affects water balance. White-nose syndrome (WNS), a fungal disease that causes dehydration and electrolyte imbalance in bats, is associated with high mortality rates of several hibernating bat species in North America. Aside from restoring water balance, hibernaculum water sources may also provide minerals to bats, which could contribute to restoring electrolyte balance and reducing the impacts of WNS. However, hibernacula water sources may also be a source of toxic elements, such as heavy metals. We collected water samples from 12 hibernacula in New Brunswick, Canada, and determined the concentrations of 18 elements in each water sample (n = 103 samples). Aluminum, barium, calcium, chloride, magnesium, manganese, potassium, and sodium were the most common elements detected, with concentrations of aluminum, lead, and manganese above drinking water recommendations (developed for human consumption) in some samples. The concentrations of electrolytes in cave water were orders of magnitude below therapeutic concentrations. Sampling period (early hibernation, late hibernation) did not affect results, but water profiles differed among sites and sample types within a site (running water, standing water, ceiling drip, and ice). The water profiles we recorded in our study suggest little potential for secondary consequences of drinking water, whether positive (i.e., electrolyte or mineral supplementation) or negative (i.e., heavy metal contamination). [ABSTRACT FROM AUTHOR]

The biogeography of cave fungi and factors that influence community composition are poorly known. The movement of animals into caves from the outside environment is thought to be one factor that affects cave mycota. Islands often have different faunal assemblages from the mainland, and this may affect the fungal diversity of island caves. In 2014 we swabbed walls in three natural solution caves on Anticosti Island, Quebec, to determine the composition of cave fungal assemblages present relative to well-studied nearby mainland sites. At least one of these caves, Grotte à la Patate in Anticosti National Park, appears to support overwintering bats. Culture-dependent methods were used to establish pure cultures, and fungi were identified by a combination of morphology and genetic sequencing. A total of 54 fungal taxa were identified, with a mean of 7.4 ± 3.9 taxa per swab. The most common taxa isolated were Penicillium spp., Pseudogymnoascus pannorum sensu lato, P. roseus, Trichoderma sp., Cladosporium spp., Thysanophora spp., Mucor sp., and Trichosporon dulcitum. Pseudogymnoascus destructans (Pd), the causative agent of the fungal disease white-nose syndrome in bats, was not detected, and we conclude that Pd was not present in the three sampled caves as of summer 2014. Two of the caves did not appear to be suitable bat hibernacula based on microclimate, although diverse fungal assemblages were detected on the walls. Several other fungal taxa common to bat hibernacula on the mainland, in addition to Pd, were lacking from Anticosti Island caves. We suggest that fungal assemblages on Anticosti Island are influenced by the absence of non-volant cave-visiting mammals on the island, particularly porcupines (Erethizon dorsatum) and raccoons (Procyon lotor), both frequent cave associates elsewhere in Maritime Canada. [ABSTRACT FROM AUTHOR]

Pseudogymnoascus destructans (Pd) causes the fungal disease white-nose syndrome (WNS), which has led to high mortality in some hibernating bat species in eastern North America. The ability to detect viable Pd in hibernacula is important for understanding the role the environment plays as a reservoir for infectious Pd. Previous studies have generally used the high-sugar medium Sabouraud-dextrose (SAB) and have had low yields of viable Pd from environmental samples of Pd-positive hibernacula. While cultureindependent methods (i.e., molecular genetics) have previously shown much better success in detecting Pd, these methods cannot determine viability. In 2012 and 2015, we swabbed walls in four hibernacula with WNS-positive bats in New Brunswick, Canada, and cultured the samples using dextrose-peptone-yeast extract agar (DPYA), SAB, and Malt extract (MEA) media. Samples cultured on DPYA produced viable Pd 43.7 to 50.0% more frequently than SAB, with a maximum overall return for DPYA among sites of 62.5% Pd-positive samples over both years. During the initial outbreak of WNS in our study region, Pd-positive swabs were produced from 40.0 to 83.3% of samples on DPYA, whereas SAB produced a maximum of 40.0%. At one site we detected Pd from 83.3% of swabs cultured on DPYA and 0% on SAB. MEA produced no viable Pd. Our figures for Pd detection are as high as or higher than previously published culture-independent methods, while also confirming the viability of the Pd present. We found that the yield of viable Pd from hibernacula walls decreased from 2012 to 2015 as the hibernating bat population decreased due to WNS mortality, but patterns varied amongst hibernacula, and overall, were not statistically different. It is possible that environmental growth of Pd contributes to its persistence within hibernacula. We suggest that future studies on the environmental persistence of viable Pd discontinue the use of high-sugar media that lack inhibitory fungal growth ingredients, such as SAB and MEA, as they favor fast-growing fungal species that overgrow and mask slowergrowing fungi such as Pd. [ABSTRACT FROM AUTHOR]

The introduction of Pseudogymnoascus destructans (Pd) to North America, agent of white-nose syndrome in hibernating bats, has increased interest in fungi from underground habitats. While bats are assumed to be the main vector transmitting Pd cave-to-cave, the role of other fauna is unexplored. We documented the fungi associated with over-wintering arthropods in Pd-positive hibernacula, including sites where bats had been recently extirpated or near-extirpated, to determine if arthropods carried Pd, and to compare fungal assemblages on arthropods to bats. We isolated 87 fungal taxa in 64 genera from arthropods. Viable Pd was cultured from 15.3% of arthropods, most frequently from harvestmen (Nelima elegans). Fungal assemblages on arthropods were similar to those on bats. The different fungal assemblages documented among arthropods may be due to divergent patterns of movement, aggregation, feeding, or other factors. While it is unlikely that arthropods play a major role in the transmission dynamics of Pd, we demonstrate that arthropods may carry viable Pd spores and therefore have the potential to transport Pd, either naturally or anthropogenically, within or among hibernacula. This underlines the need for those entering hibernacula to observe decontamination procedures and for such procedures to evolve as our understanding of potential mechanisms of Pd dispersal improve. [ABSTRACT FROM AUTHOR]

The introduction of the fungal pathogen Pseudogymnoascus destructans (Pd) to North America has stimulated research on the poorly known mycology of caves. It is possible that the introduction of Pd reduces the diversity of fungi associated with bats hibernating in caves. To test this hypothesis we examined the fungal assemblages associated with hibernating bats (Myotis spp.) pre- and post- white-nose syndrome (WNS) infection in eastern Canada using culture-dependent methods. We found the mean number of fungal taxa isolated from bats/ hibernaculum was not significantly different between pre-infection (29.6 ± 6.1SD) and postinfection with WNS (32.4 ± 4.3). Although the number of fungal taxa per bat was significantly higher on Myotis lucifugus vs. M. septentrionalis, evidence suggests that this is a reflection of environmental features of individual hibernacula, rather than any biological difference between bat species. The composition and number of the most common and widespread fungal taxa on hibernating Myotis spp. did not change with the introduction of Pd to hibernacula. We found no evidence to suggest that Pd interacts with other fungi on the external surface of bats in hibernacula, even among fungal species of the same genus. However, our data do suggest that environmental characteristics of individual caves can have a significant influence on the fungal assemblages cultured from hibernating bats at specific hibernacula. Following the mortality of thousands of WNS-infected Myotis spp. in one hibernacula, we found that those fungal taxa growing on dead bats were cultured with increased frequency from live bats. This suggests that fungal assemblages on live bats may be sensitive to sporadic introductions of new fungal substrates to hibernacula. [ABSTRACT FROM AUTHOR]

The tricolored bat (Perimyotis subflavus) is threatened by white-nose syndrome (WNS), a fungal disease caused by Pseudogymnoascus destructans (Pd) and was recently ranked as endangered under the Canadian Species-at-Risk Act. There have been few prior studies on the fungi associated with over-wintering bats. Such information is important in assessing overall fungal diversity within the cave habitat, in determining the ecological role that bats may play as dispersers of fungi, and in the identification of fungal species potentially antagonistic to Pd. We swabbed twenty-two P. subflavus overwintering in caves and mines in New Brunswick, Canada, in 2012 and 2013. This produced 408 isolates comprising 60 taxa in 49 fungal genera with an average of 10.2 ± 3.9SD fungal taxa recorded per bat. We found fungal assemblages on P. subflavus (post- WNS) very similar to those we cultured previously from Myotis spp. (pre-WNS) at the same sites. We suggest that the variation in fungal assemblages observed from site-to-site on hibernating P. subflavus is largely due to environmental and ecological characteristics of individual caves, rather than the presence of Pd or roosting habits. [ABSTRACT FROM AUTHOR]

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition - dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America. [ABSTRACT FROM AUTHOR]

The emergence of the fungal disease white-nose syndrome (WNS) among hibernating bats in North America and its causative pathogen, Geomyces destructans, underscores how little is known about fungi associated with bats and their subterranean environments. Investigating 8 caves and mines in New Brunswick, Canada, we cultured a diverse array of fungi from the fur and skin of apparently healthy, hibernating Myotis lucifugus (Little Brown Bat) and M. septentrionalis (Northern Long-eared Bat) in the year prior to the emergence of WNS in the province. Among the 117 isolated fungal taxa, we found an array of psychrophilic, psychrotrophic, keratinolytic, coprophilous, and saprobic fungi. The most common taxa were Geomycespannorum sensu lato, Penicillium spp., Mortierella spp., Mucor spp., Cephalotrichum stemonitis, Leuconeurospora spp., Penicillium solitum, Cladosporium spp., and Trichosporon dulcitum. Each bat hosted 6.9 ± 3 (SD) fungal taxa, and 30.8 ± 5 taxa were isolated per hibernaculum. Number bftaxa isolated per bat was positively correlated with mean and minimum winter temperatures in the dark zones of hibernacula. Forty-seven of the taxa have never been reported in caves, and an additional 31 taxa are new records for North American caves. The presence of Geomyces pannorum sensu lato on 70% of hibernating bats may complicate results of diagnostic techniques used for identifying G. destructans. Bats hibernating in eastern Canada harbor a rich reservoir of fungal species and probably play a role in moving fungal spores into and between hibernacula, as well as onto the landscape. [ABSTRACT FROM AUTHOR]

Studies of fungi in caves have become increasingly important with the advent of white-nose syndrome (WNS), a disease caused by the invasive fungus Pseudogymnoascus destructans (Pd) that has killed an estimated 6.5 million North American bats. We swabbed cave walls in New Brunswick, Canada, in 2012 and 2015 to determine whether the culturable fungal assemblage on cave walls changed after the introduction of Pd and subsequent decrease in hibernating bat populations. We also compared fungal assemblages on cave walls to previous studies on the fungal assemblages of arthropods and hibernating bats in the same sites. The fungal diversity of bats and cave walls was more similar than on arthropods. The diversity and composition of fungal assemblages on cave walls was significantly different among media types and sites but did not differ over time. Therefore, no change in the culturable fungal assemblage present on cave walls was detected with the introduction of Pd and subsequent disappearance of the hibernating bat population over a 3-year period. This suggests that fungi documented in caves in the region prior to the outbreak of Pd do not require regular transmission of spores by bats to maintain fungal diversity at these sites. [ABSTRACT FROM AUTHOR]

Pseudogymnoascus destructans is the causal agent of bat white-nose syndrome (WNS), which is devastating some North American bat populations. Previous transcriptome studies provided insight regarding the molecular mechanisms involved in WNS; however, it is unclear how different environmental parameters could influence pathogenicity. This information could be useful in developing management strategies to mitigate the negative impacts of P. destructans on bats. We cultured three P. destructans isolates from Atlantic Canada on two growth media (potato dextrose agar and Sabouraud dextrose agar) that differ in their nitrogen source, and at two separate incubation temperatures (4 C and 15 C) that approximate the temperature range of bat hibernacula during the winter and a temperature within its optimal mycelial growth range. We conducted RNA sequencing to determine transcript levels in each sample and performed differential gene expression (DGE) analyses to test the influence of growth medium and incubation temperature on gene expression. We also compared our in vitro results with previous RNA-sequencing data sets generated from P. destructans growing on the wings of a susceptible host, Myotis lucifugus. Our findings point to a critical role for substrate and incubation temperature in influencing the P. destructans transcriptome. DGE analyses suggested that growth medium plays a larger role than temperature in determining P. destructans gene expression and that although the psychrophilic fungus responds to different nitrogen sources, it may have evolved for continued growth at a broad range of low temperatures. Further, our data suggest that down-regulation of the RNA-interference pathway and increased fatty acid metabolism are involved in the P. destructans-bat interaction. Finally, we speculate that to reduce the activation of host defense responses, P. destructans minimizes changes in the expression of genes encoding secreted proteins during bat colonization. [ABSTRACT FROM AUTHOR]

Sorex maritimensis (Maritime Shrew) is endemic to New Brunswick and Nova Scotia. We suggest that post-glacial dispersal northward by this species from a coastal plain refugium located in the mid-Atlantic, rather than dispersal from a refugium off Nova Scotia as previously proposed, is more parsimonious with current understanding of the glacial history of the region. A range extension into northeastern New Brunswick shows that the species is distributed north-south in New Brunswick and suggests that wetlands in heavily forested regions meet the ecological requirements of the Maritime Shrew. Such wetlands may provide avenues for dispersal and ensure connectivity among populations. Habitat descriptions for the Maritime Shrew indicate a wetland-associated species adaptable enough to utilize disturbance-dominated and anthropogenic wetlands. Further investigations of the biology of the Maritime Shrew are required to inform future habitat and wetland protection policies that should consider the requirements of this poorly known Canadian mammal. [ABSTRACT FROM AUTHOR]

Wildlife are exposed to neurotoxic mercury at locations distant from anthropogenic emission sources because of long-range atmospheric transport of this metal. In this study, mercury bioaccumulation in insectivorous bat species (Mammalia: Chiroptera) was investigated on a broad geographic scale in Canada. Fur was analyzed (n = 1178) for total mercury from 43 locations spanning 20° latitude and 77° longitude. Total mercury and methylmercury concentrations in fur were positively correlated with concentrations in internal tissues (brain, liver, kidney) for a small subset (n = 21) of little brown bats ( Myotis lucifugus ) and big brown bats ( Eptesicus fuscus ), validating the use of fur to indicate internal mercury exposure. Brain methylmercury concentrations were approximately 10% of total mercury concentrations in fur. Three bat species were mainly collected (little brown bats, big brown bats, and northern long-eared bats [ M. septentrionalis ]), with little brown bats having lower total mercury concentrations in their fur than the other two species at sites where both species were sampled. On average, juvenile bats had lower total mercury concentrations than adults but no differences were found between males and females of a species. Combining our dataset with previously published data for eastern Canada, median total mercury concentrations in fur of little brown bats ranged from 0.88–12.78 μg/g among 11 provinces and territories. Highest concentrations were found in eastern Canada where bats are most endangered from introduced disease. Model estimates of atmospheric mercury deposition indicated that eastern Canada was exposed to greater mercury deposition than central and western sites. Further, mean total mercury concentrations in fur of adult little brown bats were positively correlated with site-specific estimates of atmospheric mercury deposition. This study provides the largest geographic coverage of mercury measurements in bats to date and indicates that atmospheric mercury deposition is important in determining spatial patterns of mercury accumulation in a mammalian species. [ABSTRACT FROM AUTHOR]

White-nose syndrome is one of the most lethal wildlife diseases, killing over 5 million North American bats since it was first reported in 2006. The causal agent of the disease is a psychrophilic filamentous fungus, Pseudogymnoascus destructans. The fungus is widely distributed in North America and Europe and has recently been found in some parts of Asia, but interestingly, no mass mortality is observed in European or Asian bats. Here we report a novel double-stranded RNA virus found in North American isolates of the fungus and show that the virus can be used as a tool to study the epidemiology of White-nose syndrome. The virus, termed Pseudogymnoascus destructans partitivirus-pa, contains 2 genomic segments, dsRNA 1 and dsRNA 2 of 1.76 kbp and 1.59 kbp respectively, each possessing a single open reading frame, and forms isometric particles approximately 30 nm in diameter, characteristic of the genus Gammapartitivirus in the family Partitiviridae. Phylogenetic analysis revealed that the virus is closely related to Penicillium stoloniferum virus S. We were able to cure P. destructans of the virus by treating fungal cultures with polyethylene glycol. Examination of 62 isolates of P. destructans including 35 from United States, 10 from Canada and 17 from Europe showed virus infection only in North American isolates of the fungus. Bayesian phylogenetic analysis using nucleotide sequences of the viral coat protein geographically clustered North American isolates indicating fungal spread followed by local adaptation of P. destructans in different regions of the United States and Canada. This is the first demonstration that a mycovirus potentially can be used to study fungal disease epidemiology. [ABSTRACT FROM AUTHOR]

Two species of Leuconeurospora, Leuconeurospora capsici (J.F.H. Beyma) Malloch, Sigler & Hambleton and Leuconeurospora polypaeciloides Malloch, Sigler & Hambleton (Ascomycota: Pseudeurotiaceae), are reported from the fur and skin of hibernating bats and from other substrata in caves in New Brunswick, Canada. Separate analyses using ITS, RPB1, and RPB2 DNA sequence data are in agreement and show these two species and the type species, Leuconeurospora pulcherrima (G. Winter) Malloch & Cain, form discrete clades within a distinct Leuconeurospora clade. The three species are distinguishable morphologically by their anamorphs, having dark conidia in L. capsici, hyaline conidia in L. polypaeciloides, and no conidia in L. pulcherrima. Ascomata and ascospores are produced in L. pulcherrima and in mated isolates of L. polypaeciloides, but have not been observed in L. capsici. Leuconeurospora species are psychrotolerant, with faster growth and heavier conidial development at 7 °C than at 22 °C. Of 151 bats sampled from 10 caves, 51 yielded isolates of L. polypaeciloides and 15 yielded L. capsici. The results were not uniform: neither species was isolated from bats in three caves, while three caves yielded isolates of both species and two yielded L. polypaeciloides only. These species were also isolated from cave walls and arthropods in the cave. [ABSTRACT FROM AUTHOR]