Southeastern Naturalist 127 K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 22001166 SOUTHEASTERN NATURALIST 1V5o(1l.) :1152,7 N–1o3. 71 Monitoring the Status of Gray Bats (Myotis grisescens) in Virginia, 2009–2014, and Potential Impacts of White-nose Syndrome Karen E. Powers1,*, Richard J. Reynolds2 , Wil Orndorff3, Brenna A. Hyzy1,4, Christopher S. Hobson5, and W. Mark Ford6 Abstract - Myotis grisescens (Gray Bat) is a federally endangered species distributed over the mid-South with a summer range that extends across the upper Tennessee River Basin, including southwest Virginia. Given the onset of White-nose Syndrome (WNS) in the Commonwealth in the winter of 2009, we initiated yearly surveys in late summer 2009 to monitor the status of known summer populations. Our objectives were to examine the relative health of these bats using body mass index (BMI), and determine any changes in juvenile recruitment across sites and years. We did not find any marked changes in BMI across years after WNS for Gray Bats. This finding suggests that surviving bats are either not negatively impacted by WNS or have recovered sufficiently by late summer as to not document obvious differences across years. After limiting our analyses of juvenile recruitment to only the individuals that we had definitively aged via backlit photos (2010–2014), we found a non-significant declining trend in juvenile recruitment; a trend that merits continued monitoring in the years to come. As Gray Bats have only recently shown to be susceptible to WNS infection, it is possible that observable population declines are forthcoming. Introduction The summer range of Myotis grisescens A.H. Howell (Gray Bat) extends across the upper Tennessee River Basin (Virginia, Tennessee, North Carolina, Georgia; Chapman 2007). In southwest Virginia, published literature suggests that summer colonies are believed to be mostly bachelor colonies (5 caves in Lee and Scott counties), with only 1 known maternity colony (Washington County, VA, and Sullivan County, TN; Fig. 1; Timpone et al. 2011). This maternity colony’s exit counts have ranged from 1500 to 9300 in the last two decades, with counts of 2500–3000 in 2013 and 2014 (Virginia Department of Game and Inland Fisheries [VDGIF], Verona, VA, unpubl. data). However, exit counts from this site and others have been inconsistent (i.e., time of year, observer expertise, and equipment) and have not been replicated in a statistically sound effort. Summer captures away from the immediate vicinity of these locations are believed to be rare, with published reports of just 4 captures of solitary males in 1Biology Department, Radford University, Radford, VA 24142. 2Virginia Department of Game and Inland Fisheries, Verona, VA 24482. 3Virginia Department of Conservation and Recreation, Natural Heritage Program, Christiansburg, VA 24073. 4Current address - University of Wisconsin-Stevens Point, Stevens Point, WI 54481. 5Virginia Department of Conservation and Recreation, Natural Heritage Program, Richmond, VA 23219. 6US Geological Survey, Virginia Cooperative Fish and Wildlife Research Unit, Blacksburg, VA 24061. *Corresponding author - kpowers4@radford.edu. Manuscript Editor: Roger Applegate Southeastern Naturalist K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 128 Russell County in the last decade (Timpone et al. 2011). Captures from the 1990s until present-day suggest that small colonies exist throughout southwestern Virginia (Fig. 1, Table 1; VDGIF, unpubl. data). Many of these sites are caves, but Gray Bats have been found roosting in bridges too. They also have been captured along the types of riparian habitats where they forage, similar to other parts of the species’ range (Johnson et al. 2010). Based on band recaptures and returns, records indicate most Gray Bats that over-summer in southwest Virginia migrate in the fall to a winter hibernaculum at Pearson’s Cave in Hawkins County, eastern Tennessee (Fig. 1; R. Reynolds, VDGIF, Verona, VA, unpubl. data). However, in 2013, a single male from Scott County was documented in Fern Cave in northeastern Alabama (Jackson County; E. Spadgenske, US Fish and Wildlife Service, Birmingham, AL, pers. comm.). In Tennessee, the largest Gray Bat populations are documented in Hubbard’s Cave (Warren County; >520,000 Gray Bats reported in winter 2006 declining to <250,000 by 2014), Pearson’s Cave (>278,000 Gray Bats reported in winter 2007 declining to <200,000 by 2014), and Bellamy Cave (Montgomery County; >139,000 Gray Bats reported in winter 2006 rising to >340,000 by 2013 before declining to >240,000 in 2014) (Flock 2013, 2014; Martin 2007). Additional hibernacula in Tennessee intermittently support up to several hundred Gray Bats (Flock 2013, 2014; Holliday 2012). Although summer-resident Gray Bats from Virginia have only thus far been tracked to Pearson’s and Fern caves, it is possible that some may be associated with these additional, or other undocumented, hibernacula in Tennessee, Alabama, or Georgia. There are no significant Gray Bat hibernacula documented in Virginia, but a few (1–10) individuals have been observed hibernating at cave sites frequented in the summer (Table 1). Figure 1. Documented summer locations of Gray Bats in Virginia, broken down by habitat (cave, bridge, stream) and type (bachelor or maternity colony, mistnet capture). Nearly all winter band returns on Virginia Gray Bats are from Pearson’s Cave, TN (labelled “hibernaculum”). Southeastern Naturalist 129 K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 The Gray Bat has been listed as federally endangered since 1976 (Chapman 2007). Endangerment primarily has been a result of anthropogenic disturbance to their hibernacula, and secondarily a result of organochlorine pesticide bioaccumulation (Tuttle 1979). Efforts to secure the hibernacula (e.g., gates and laws restricting public access) have positively impacted this species with population increases since the time of listing (Martin et al. 2003). The potential impact to Gray Bats from the invasive fungus Pseudogymnoascus destructans (Blehert & Gargas) Minnis & D.L. Lindner that causes White-nose Syndrome (WNS) is largely unknown. Detected in the United States in upstate New York in 2006 (Frick et al. 2010), the fungus was first confirmed in Gray Bat hibernacula in winter 2009–2010 in Missouri. However, the first confirmed case via histopathology of active infection on a Gray Bat was first reported in 2 major Gray Bat winter hibernacula in 2012 in Tennessee (Bellamy Cave: visited 27 February, and Pearson’s Cave: visited 27 March; Holliday 2012). Because accurate counts are often difficult to obtain, even in the main 3 caves, it’s difficult to document mass mortalities for Gray Bats. Table 1. Documented summer locations of Gray Bats in southwestern Virginia, ca. 1990–2014. Reported are county and watershed, type of occurrence (if known), and number of bats observed or estimated. * indicates that small numbers (less than 10 individuals) of Gray Bats have been found during winter months. With the exception of mist-netting captures, site names have been omitted to protect the exact location of the federally endangered bats on private lands. No. observed/ estimated Site Type of occurrence in summer County Watershed Copper Creek, Mist net on stream 15 Scott Clinch/Copper Creek Nickelsville Dry Creek Mist net on stream 1 + 1 Lee Powell/Dry Creek Dungannon Bridges Unknown ≤1500A Scott Clinch Kents Ridge Mist net on stream 1 Russell Clinch Lee Cave 1* Bachelor ≥2000 Lee Powell Lee Cave 2 Bachelor ≤200 Lee Powell Lee Cave 3* Bachelor ≤200 Lee Powell Lee Cave 4 Bachelor 100 Lee Powell Lee Cave 5 Bachelor 40 Lee Powell Lee Cave 6 Bachelor 275 Lee Powell Lee Cave 7 Historic Undetermined Lee Powell/Hardy Creek Lee Cave 8 Historic Undetermined Lee Powell Lee Cave 9 Bachelor ≤200 Lee Powell Reed Creek, Kent Mist net on stream 1 Wythe New Russell Cave 1* Bachelor 1000 Russell Clinch/Little River Scott Cave 1* Bachelor ≤4000 Scott Clinch/Copper Creek Scott Cave 2 Bachelor 250 Scott Clinch/Copper Creek Scott Cave 3* Bachelor 600–1200 Scott Clinch Scott Cave 4 Unknown Undetermined Scott Clinch Smyth Cave 1* Bachelor 1000 Smyth Holston Tazewell Cave 1* Incidental hiber 1 Tazewell Clinch/Little River Washington 1 Maternity roost 2500–9000 Washington South Holston A May be mixed with other Myotis spp. Southeastern Naturalist K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 130 However, reported counts in Flock (2013, 2014) suggest that bats are moving among caves from year-to-year and that WNS impacts on the species have so far been negligible. Within known hibernacula, individuals have been noted to move to warmer, more humid microhabitats to hibernate (C. Holliday, The Nature Conservancy, Gainesboro, TN, pers. comm.). Admittedly, it is difficult to access such locales (e.g., the stream channel in Bellamy Cave) and accurately count individuals (Flock 2014). In other Myotid bats, mortality is partly due to the fungus penetrating the dermis of exposed tissue and causing an irritating sensation that results in frequent arousal of the infected bats. Verant et al. (2014) suggested that the fungus alters fat-energy utilization by Myotis lucifugus Le Conte (Little Brown Bat) to nearly twice that of uninfected individuals. The direct effects of the fungus on Gray Bats, however, are unknown. To determine if and how WNS is impacting summer colonies of Gray Bats in southwestern Virginia, our objectives were: (1) to examine the relative health of these bats, and (2) to determine any changes in juvenile recruitment across sites and years. Indirect measures of health, such as body mass (Boyles et al. 2007) and body mass index (BMI, calculated as weight[g]/ forearm length [mm]; Chappell and Titman 1983) have shown mixed results in linking WNS effects to bats (e.g., Powers et al. 2015, Reeder et al. 2012). To add complexity, differences in weight gain are documented between sexes (females are generally heavier than males of comparable size, due to reproductive demands; Gerell and Lundberg 1990) and between age categories (juveniles [young of the year; YOY] gain proportionally less weight than adults; Kunz et al. 1998). Therefore, year-to-year comparisons must take into account the interactions of date, sex, age, and species. Unfortunately, no indices other than BMI have been developed as more suitable indicators of bat health during summer months and it remains the best, albeit imperfect, monitoring tool available. Additionally, WNS has been tied to markedly lower juvenile recruitment in other species of Myotis (e.g., Francl et al. 2012) because physiologically stressed individuals likely are less capable of successfully raising young to volancy stage (USFWS 2015), so we used this measure as a complement to BMI measures to assess Gray Bat status. Documenting significant declines in juveniles in the region could be indicative of negative impacts of WNS on Gray Bats. Given past findings on both metrics (e.g., Francl et al. 2012, Powers et al. 2015), we predicted that BMI in Gray Bats would not have significantly declined since the onset of WNS, but that juvenile recruitment would be lower in the years since WNS has been confirmed in this species (2012–2014). Methods Field methods In late August and early September, 2009–2014, we conducted yearly Gray Bat surveys at 1 maternity colony (long box culvert in Washington County, VA, and Sullivan County, TN) and up to 4 bachelor colonies (caves in Lee, Scott, and Russell counties, VA). To maximize capture success, we employed one or more capture techniques—harp traps, single-high mistnets, and hand captures—depending on the Southeastern Naturalist 131 K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 physical characteristics of the site. We opened traps at sunset, and captured bats for 1–3 hours after sunset on nights with air temperature of ≥10 °C and no precipitation or strong winds (Francl 2008). For all Gray Bats captured, we recorded sex, right forearm length (± 0.1 mm), and weight (± 0.1 g). All Gray Bats were banded with 2.9-mm bands (Porzana Ltd., East Sussex, UK) to document recaptures. Netting procedures included standard WNS decontamination protocol, as required by the US Fish and Wildlife Service (e.g., https://www.whitenosesyndrome.org/sites/ default/files/resource/national_wns_revise_final_6.25.12.pdf). Analytical methods and statistics In 2009, we determined age of bats (adult or young-of-the-year juvenile) in the field by visual examination of the wings (degree of epiphyseal fusion; Anthony 1988, Haarsma 2008). However, due to the time of year, we acknowledged that delineation between juvenile and adult was difficult to assess ocularly, and assessments were inconsistent among observers. Accordingly, we excluded 2009 age data in our analysis that were based solely on ocular scoring. However, from 2010 to 2014, we determined age by examining backlit photographs of the wings for each individual bat captured. This allowed for a more accurate, consistent determination of age, which was completed by one viewer (B.A. Hyzy). We calculated the BMI (Chappell and Titman 1983) for each individual to serve as a proxy for bat health. Given the acknowledged differences in BMI between sexes (Gerell and Lundberg 1990) and ages (Kunz et al. 1998), all analyses involving this metric were completed separately for males and females and for adults and juvenile age classes. We examined potential changes in BMI across years and sites using a generalized linear mixed model (PROC GLIMIX, SAS 9.4, SAS Institute, Cary NC), with all sex and age differences acknowledged. The generalized linear mixed model is best suited for analyzing both fixed and random effects from input and response data from non-normal distributions (Bolker et al. 2009). To determine if the percent of juveniles in the populations were declining over time, we performed a robust regression using the MM estimator (PROC ROBUSTREG, SAS 9.4, SAS Institute, Cary, NC; Yohai 1987) weighted by cave population size to compare the percent of juveniles captured across years (2010–2014). Results Across 6 years of surveys, we captured 2949 Gray Bats. Males comprised 69.2% of all captures, and captures per year ranged from 366 to 591 individuals (Table 2). For years in which reliable adult–juvenile distinctions were available (2010–2014), average BMI followed expected biases (adult BMI > juvenile BMI and female BMI > male BMI; Table 2). These data were highly variable, and the generalized linear mixed models failed to detect any significant changes in BMI across sites or years (Table 3, P > 0.9 for all models) for any age/sex combination. In examining changes in age ratios across years via linear regression, we found a decreasing trend in the percentage of juveniles in the population (r2 = 0.123, P = 0.067; Fig. 2). Southeastern Naturalist K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 132 Table 3. Results of general linear mixed models, examining trends in body mass index values for age/ sex combinations of Gray Bats in southwestern Virginia. Models examined differences across years (2010–2014), capture sites, and the year-site interaction. Model Adult ♀ Adult ♂ Juvenile ♀ Juvenile ♂ Year df 4, 601 4, 1342 4, 150 4, 260 F 0.01 0.01 0.02 0.01 P 0.999 0.999 0.999 0.999 Site df 3, 601 4, 1342 3, 150 4, 260 F 0.04 0.36 0.01 0.02 P 1.000 0.834 1.000 0.999 Year*site df 10, 601 11, 1342 3, 150 9, 260 F 0.04 0.03 0.01 0.01 P 1.000 1.000 0.999 1.000 Table 2. Mean body mass index (BMI), standard deviation (StDev) of BMI, and sample size (n) for each age/sex combination of Gray Bats in southwestern Virginia, 2010–2014. Adult ♀ Adult ♂ Juvenile ♀ Juvenile ♂ Year n BMI StDev n BMI StDev n BMI StDev n BMI StDev 2010 124 0.236 0.004 345 0.236 0.011 34 0.238 0.010 88 0.219 0.003 2011 94 0.242 0.004 249 0.229 0.002 63 0.228 0.004 91 0.211 0.004 2012 120 0.234 0.003 298 0.226 0.002 15 0.214 0.006 32 0.220 0.004 2013 101 0.232 0.003 213 0.223 0.003 22 0.206 0.003 30 0.206 0.002 2014 180 0.232 0.005 258 0.215 0.002 27 0.189 0.014 37 0.210 0.003 Figure 2. Age ratios of Gray Bats in southwestern Virginia for each site plotted across years of study (2010–2014) with best-fit trend line and 95% confidence intervals (dashed lines) from a weighted robust regression (r2 = 0.123, P = 0.067). Southeastern Naturalist 133 K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 Discussion As predicted, we did not find any marked changes in BMI across years for Gray Bats in southwest Virginia following the advent of WNS. This is a positive, albeit not surprising, discovery, given non-significant relationships between BMI and presumed WNS impacts findings in previous studies for other more-affected bat species (e.g., Powers et al. 2015, Reeder et al. 2012). One explanation is that affected individuals have recovered sufficiently by August–September as to not document obvious differences across years. Affected Little Brown Bats have exemplified this seasonal healing trend (Fuller et al. 2011), and seasonal differences in fungal loadings have been documented (Langwig et al. 2014). A second explanation for our non-significant BMI–WNS relationships is that Gray Bats at these sites are not being negatively impacted by WNS. Given the lack of significant mortalities in Gray Bat hibernacula, we support this second hypothesis. As a year-round cave-obligate species, Gray Bats join other cave-obligate species in the eastern United States (and Europe) as seemingly unaffected by this fungal pathogen (Bernard et al. 2015, Martinkova et al. 2010, Whibbelt et al. 2010). The physiological pathway in which Gray Bats and others persist despite the presence of P. destructans is yet unknown. A naturally occurring bacteria on the wings and pelage of Little Brown Bats has shown promise in individual recovery (Hoyt et al. 2015); however, this avenue has not yet been investigated in Gray Bats. With recent discoveries regarding this bacterium and a better understanding of enzymatic pathways of the fungus (O’Donoghue et al. 2015), bats face a highly complex physiological disease that managers are just now beginning to document. In an examination of the juvenile recruitment analysis from 2010–2014, we found slight declines as time progressed, though not statistically significant. As Gray Bats have only recently shown to be prone to WNS infection (2012), it is possible that our observations have occurred too early in the disease process to record population declines. Such a lag has been suspected in Myotis septentrionalis Trouessart (Northern Long-eared Bat), in Virginia (Powers et al. 2014) and Kentucky (A. Silvis, Virginia Tech, Blacksburg, VA, unpubl. data) although this has been difficult to document (Thogmartin et al. 2012). We suggest continued monitoring of these summer populations in Virginia to determine potential increased rates of infection as WNS continues to spread. In summary, we acknowledge that Gray Bats are still in the early years of documented infection, and that infection rates are well below those documented in other co-occurring species of Myotis. Researchers have not documented mass mortalities in hibernating Gray Bats due to WNS infection, and we have not observed any marked declines in summer colony health or population counts in Virginia. The physiological explanation for Gray Bat persistence despite the presence of P. destructans in hibernacula is yet unknown. If Gray Bats continue to be mostly unaffected by P. destructans, there may be opportunity for population growth and range expansion to the north due to drastic (>95%) reductions in populations of the other dominant riparian-feeding bat species such as the Little Brown Bat (Powers et al. 2015). We suggest increased Southeastern Naturalist K.E. Powers, R.J. Reynolds , W. Orndorff, B.A. Hyzy, C.S. Hobson, and W.M. Ford 2016 Vol. 15, No. 1 134 surveillance of Gray Bat behavior along the northeastern boundary of the species’ range to determine if the decimation in populations of the Little Brown Bat has created an opportunity for the Gray Bat to exploit vacated niches and distributional space as has been observed elsewhere for other WNS-impacted species (Jachowski et al. 2014). Documentation of potential foraging habitat and water-quality monitoring of those riparian habitats also would build upon our knowledge of Gray Bats in Virginia. Further, because the capture efforts did not estimate total population size at our study sites, we suggest that future efforts at these locales focus on exit counts. Standard methods, using trained observers with night-vision goggles at the culvert or cave opening (e.g., Kunz 2003, Sabol and Hudson 1995), should be repeated for multiple nights and at the same time each year. This non-invasive effort would not harm these bats, already known to be sensitive to disturbance and cave alteration (Tuttle 1979), and would help managers track populations in a post- WNS environment. Acknowledgments This study was completed with funds provided by the Virginia Department of Game and Inland Fisheries through a White-nose Syndrome Program Grant and Wildlife Restoration Program Grant from the US Fish and Wildlife Service. We thank M. Krager and Natural Tunnel State Park for providing housing for the authors and volunteers. We thank numerous volunteers for assistance in the field: T. (Canniff) Adler, B. Balfour, J. Bentley, R.C. Bland, J. Bower, J. Castle, D. Crowder, E. Crowder, A. Futrell, K. Hamed and his students from Virginia Highlands Community College, J. 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