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.

White-nose syndrome (WNS) is an epizootic disease caused by the fungus Pseudogymnoascus destructans (Pd) that has led to severe declines in the populations of several North American bat species (Hoyt et al. 2021). Although Pd loads peak in affected species during the winter, Pd may persist on some WNS-affected species into summer (Carpenter et al. 2016; Huebschman et al. 2019). Besides the 12 bat species affected by WNS, six species without diagnostic signs of WNS have tested positive for Pd, including two migratory tree-roosting species that are infrequently found in winter hibernation sites: eastern red (Lasiurus borealis) and silver-haired (Lasionycteris noctivagans) bats (Bernard et al. 2015; Huebschman et al. 2019). However, previous studies indicating that tree-roosting species can harbor Pd were not specifically focused on such species and thus had small sample sizes (the former detected Pd on 1/3 silver-haired bats and 2/6 eastern red bats, and the latter detected Pd on 1/14 eastern red bats). Thus, understanding of Pd pervasiveness on tree-roosting species remains limited.

We aimed to test for the presence of Pd on three migratory tree-roosting bat species: eastern red, silver-haired, and hoary (Lasiurus cinereus) bats from throughout North America (Table 1). If sufficient numbers of Pdpositive individuals existed in our data set, a secondary aim was to provide an initial comparison of detection rates tissue sampling protocols: fungal DNA extracted from wing tissue, swabbing muzzle and forearm, and swabbing feet and tail membranes (uropatagia). We predicted that a small proportion of tree-roosting bats sampled would test positive for Pd, and that sampling of salvaged carcasses might allow for detection of Pd. Samples were opportunistically obtained during the course of collections for other projects (Pylant et al. 2016; Sovic et al. 2016; Nelson et al. 2018; Campbell et al. 2020) during 2003–18. We conducted DNA extraction from swabs following Verant et al. (2016), with modifications described in the Supplementary Material.

Traditional muzzle-and-forearm swabbing was performed on 38 live bats captured by mist net and harp trap during foraging (n=36), swarm (n=1), and emergence surveys (n=1) near hibernacula entrances as part of ongoing monitoring efforts (for example, Nagel and Gates 2017). For a few individuals, feet and uropatagium were also swabbed, because those body parts may be the most likely to encounter and retain Pd from cave substrate (Fig. 1). Carcasses of 234 individuals were collected during postconstruction monitoring surveys at 25 wind-energy facilities throughout the continental US between May and September (Table 1), with one additional carcass obtained via wild salvage in a residential area. We used only individuals that were identified to species and thus in relatively fresh, rather than highly degraded, condition. Because these samples were collected opportunistically, they were not necessarily obtained using a standardized or uniform protocol. However, Pd spores are highly resilient (Hoyt et al. 2015) and probably persist in a viable state on carcasses well beyond when they are collected in the field. Before extraction of DNA from tissue, whole carcasses were kept in individual bags and already-excised tissue in 95% ethanol; all tissue was stored at –80 C. Approximately 35 mm2 of wing tissue was excised from the lower plagiopatagium of carcasses. We extracted DNA from tissue using DNeasy Blood & Tissue Kits (Qiagen Inc., Valencia, California, USA). We followed the kit protocol, with the addition of a 6-min centrifuge step following tissue digestion to remove fur and pigments. When whole carcasses were available, we also swabbed the muzzle and forearm.

Samples were tested for Pd DNA using a quantitative PCR assay (Verant et al. 2016; Supplementary Material) at the University of Maryland Center for Environmental Science. Consistent with previous studies, we considered a sample positive for Pd DNA if exponential amplification of fluorescent intensity above background occurred within 40 quantification cycles (Cq; Bernard et al. 2015; Carpenter et al. 2016; Huebschman et al. 2019). Samples with one or more positive results were rerun in duplicate on a second plate for a total of four runs. We considered an individual Pd positive when a Cq value was determined for at least one of the four replicates. Detections on one of multiple replicates per sample are common when Pd quantities are near the assay's detection limit (Bernard et al. 2015; Carpenter et al. 2016; Huebschman et al. 2019).

We performed quantitative PCR pathogen detection on a total of 322 samples representing 272 individuals (119 samples from 95 individual eastern red bats, 76 samples from 75 individual hoary bats, and 127 samples from 102 individual silver-haired bats; Table 2). Five bats had Cq values indicating exponential amplification (Cq range = 31.7–37.0) and thus were considered positive for Pd: one hoary (from New York, US), three eastern red (from Illinois and Ohio, US), and one silver-haired bat (from West Virginia, US, Table 3). These positive numbers represent approximately 1%, 3%, and 1%, respectively, of individuals of these species that we tested. All positive samples were from DNA extracted from wing tissue from turbine-killed individuals; none of the Pd-positive individuals had skin or fur swab samples available. Each positive sample was collected after Pd had been documented in the state where they were collected (White-nose Syndrome Response Team 2019).

This study represents the first publicly available report of detection of Pd on a hoary bat and additional detections of Pd on eastern red and silver-haired bats. Additionally, it represents the first publicly reported detection of Pd on a silver-haired bat outside of the winter, and of Pd on tree-roosting bats collected in Illinois, Ohio, New York, and West Virginia. Previous studies documented Pd in eastern red and silver-haired bats in Tennessee and Wisconsin (Bernard et al. 2015; Huebschman et al. 2019), bringing the number of states observed to four for eastern red, two for silver-haired, and one for hoary bats. We detected Pd on bats sampled during the early summer (June) and late summer and autumn (August and September), suggesting that these WNS-resistant species might have detectable fungal loads before and during their migratory period (Cryan 2003). However, in conjunction with previous results (Bernard et al. 2015; Carpenter et al. 2016; Huebschman et al. 2019), our results suggest that the rates at which these tree-roosting species carry Pd is relatively low.

Because of the opportunistic nature of some sampling, we cannot completely rule out the possibility of cross-contamination by Pd of carcasses processed before shipment to our laboratory. However, to the best of our knowledge, carcasses were widely distributed on the landscape, and handled and stored individually. Furthermore, potential cross-contamination of bat tissue by Pd via handling and processing is unlikely to be dramatically higher than that of bats captured in the same mist net or harp trap, protocols widely used as part of WNS monitoring efforts (Ballmann et al. 2017; USGS-NWHC 2020).

Sampling protocol may play an important role in the detectability of Pd, especially in WNS-resistant species with presumably low Pd levels. Although all Pd-positive samples in our study were obtained from carcasses sampled with DNA extraction only (Table 2), the small number of positive detections in our data set and the lack of swab samples from the individuals that tested positive limit our ability to assess whether DNA extraction of the relatively large sections of bat tissue we used has a differential Pd detection rate than other methods. Extracting all DNA from a relatively large section of tissue probably maximizes the chances of detecting Pd beyond skin swabbing and wing biopsy (Janicki et al. 2015). Given the general availability of salvaged bat carcasses of the three species evaluated here (Arnett and Baerwald 2013), our results suggest that such carcasses might be useful for Pd monitoring.

The mechanisms by which hoary, eastern red, and silver-haired bats might be occasionally exposed to Pd outside of winter remain unclear. Although silver-haired bats sometimes hibernate in Pd-positive hibernacula, they are rarely found roosting there during the summer or during their autumn migrations (Kunz 1982; Barclay et al. 1988). Hoary and eastern red bats are almost exclusively tree-roosting species and are thought to enter hibernacula very rarely (Shump and Shump 1982a, b). One possible exposure mechanism is interspecific interactions, such as multispecies swarming and aggression behaviors (Myers 1960; Brokaw et al. 2016; Neubaum and Siemers 2021). Resistance to WNS and propensity for long-distance dispersal and seasonal migration (Cryan et al. 2014) suggest that WNS-resistant migratory bats could be capable of acting as vectors to spread WNS across large geographic scales (Maher et al. 2012; see also Escobar et al. 2014). Future studies could explore potential mechanisms of Pd exposure, persistence, and possible transmission by these species.

We thank the scientists who contributed to sample collection and procurement, including Meghan Lout, Carlyle Meekins, and Brad Romano. Samples were collected in the course of projects with ethical board approval and following state and federal guidelines and permitting. Thanks to Ana V. Longo and two anonymous reviewers for valuable feedback. Support was provided by the Michael L. May Graduate Fellowship in Biology and Graduate Student Funding Award Fellowship from the Department of Biology at the University of Florida, as well as grants from the National Park Service (P14AC01762 and P11AC30805).

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-00160.

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Supplementary data