Northeastern Naturalist Vol. 23, No. 2 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 195 2016 NORTHEASTERN NATURALIST 23(2):195–204 Changes in Rates of Capture and Demographics of Myotis septentrionalis (Northern Long-eared Bat) in Western Virginia before and after Onset of White-nose Syndrome Richard J. Reynolds1,*, Karen E. Powers2, Wil Orndorff 3, W. Mark Ford4, and Christopher S. Hobson5 Abstract - Documenting the impacts of white-nose syndrome (WNS) on demographic patterns, such as annual survivorship and recruitment, is important to understanding the extirpation or possible stabilization and recovery of species over time. To document demographic impacts of WNS on Myotis septentrionalis (Northern Long-eared Bat), we mistnetted at sites in western Virginia where Northern Long-eared Bats were captured in summer before (1990–2009) and after (2011–2013) the onset of WNS. Our mean capture rates per hour, adjusted for area of net and sampling duration, declined significantly from 0.102 bats/ m2/h before WNS to 0.005 bats/m2/h (-95.1%) by 2013. We noted a time lag in the rate of decline between published data based on bats captured during the swarming season and our summer mist-netting captures from the same geographic area. Although proportions of pregnant or lactating females did not vary statistically in samples obtained before and after the onset of WNS, the proportion of juvenile bats declined significantly (-76.7%), indicating that the viability of Northern Long-eared Bats in western Virginia is tenuous. Introduction With the onset and spread of white-nose syndrome (WNS), the number of Myotis septentrionalis (Trouessart) (Northern Long-eared Bats) has declined precipitously, prompting the US Fish and Wildlife Service (USFWS 2015a) to list the species as threatened in April 2015. The listing is based primarily on decreasing counts at hibernacula and to a lesser extent on declines in summer mist-netting captures and secondary impacts (survivorship, fecundity, recruitment). Although the USFWS (2015a) has identified more than 1100 hibernacula throughout the range of the species, many contain only a few (1–3) individuals. However, detection of hibernating Northern Long-eared Bats may be difficult because this species often roosts in cracks and crevices (Caceres and Barclay 2000), and if detectability is low, counts during hibernation may not be the best indicator of population trends (Turner et al. 2011). Tree-roosting bats, including Northern Long-eared Bats, often exhibit fidelity to summer habitat and roosts (Barclay and Kurta 2007). For example, Perry (2011) conducted an extensive 8-year mist-netting effort in the Ouachita 1Virginia Department of Game and Inland Fisheries, Verona, VA 24482. 2Biology Department, Radford University, Radford, VA 24142. 3Virginia Department of Conservation and Recreation, Natural Heritage Program, Christiansburg, VA 24073. 4US Geological Survey, Virginia Cooperative Fish and Wildlife Research Unit, Blacksburg, VA 24061. 5Virginia Department of Conservation and Recreation, Natural Heritage Program, Richmond, VA 23219. *Corresponding author - rick.reynolds@dgif.virginia.gov. Manuscript Editor: Alan Kurta Northeastern Naturalist 196 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 Vol. 23, No. 2 Mountains, AR, and demonstrated site fidelity for Northern Long-eared Bats; Silvis et al. (2015) showed repeated interannual use of day-roosting areas in Kentucky. This apparent site fidelity suggests that annual mist-netting near important habitat features potentially provides a suitable method for monitoring bats (population indices and demographics) outside the hibernation period (Francl et al. 2012, Moosman et al. 2013). In Virginia, Northern Long-eared Bats traditionally have been difficult to detect during winter surveys, with only 2 hibernacula having records of >10 individuals (Virginia Department of Game and Inland Fisheries [VDGIF], Verona, VA, unpubl. data). Nevertheless, Northern Long-eared Bats were commonly captured in mountainous regions of western Virginia during summer mist-netting surveys before the onset of WNS (Hobson 1993, Timpone et al. 2011). WNS was first detected in Virginia in February 2009, and the disease had spread throughout the mountainous areas of the state by post-hibernation 2013 (Turner et al. 2011; VGDIF, unpubl. data). Declines in the number of Northern Long-eared Bats were first reported in western Virginia by Powers et al. (2015) based on harp-trapping surveys at caves and mines during fall swarming (mid-September through late October). Powers et al. (2015) reported a decline of >50% by 2010, >70% by 2011, and >90% by 2012–2013. Because of these reduced captures during swarming, the cryptic nature of Northern Long-eared Bats during hibernation, and their historic detectability during summer, we chose to focus on summer surveys to assess demographic changes before and after the onset of WNS and substantiate the declines observed during fall swarming. Methods Site description Our study area consisted of 26 sites in western Virginia within the Blue Ridge, Ridge and Valley, and Appalachian Plateau physiographic provinces, extending from Highland County in the north to Lee County in the southwest (McNab and Avers 1995; Fig. 1). Most sites were located in the George Washington and Jefferson National Forest. We selected these sites for contemporary surveys based on successful capture of Northern Long-eared Bats before the onset of WNS. The pre-WNS studies were completed between 1990 and 2009 as part of surveys for the endangered Myotis sodalis Miller and Allen (Indiana Bat; Ford and Chapman 2007). We conducted netting between 15 May and 15 August. Sites included closed-canopy upland water holes created for wildlife (n = 44 survey nights), 1st and 2nd order woodland streams (22), forested road ruts (7), and cave entrances (14). Although we initially chose these sites based on their suitability for mist-netting Indiana Bats, these locations were within a forest matrix also suitable for the Northern Long-eared Bat (Ford et al. 2005). The predominant forest in this area was mature Appalachian Quercus (oak)–Carya (hickory) or mixed oak–montane Pinus (pine) (Simon et al. 2005). Forest-management activities (prescribed fire and timber harvest) occurred close to some locations, but canopy cover at these sites remained intact throughout our study. Northeastern Naturalist Vol. 23, No. 2 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 197 Field surveys We conducted surveys for Northern Long-eared Bats during periods when pregnant females (mid-May–early July), lactating females (mid-June–late July), and volant juveniles (early July–August) were found in Virginia. We defined these periods by the condition of Northern Long-eared Bats that were captured in Virginia between 1990 and 2013. Before WNS, 11, 16, and 13 surveys, and after WNS, 26, 33, and 29 surveys were conducted during periods in which pregnant females, lactating females, and volant juveniles occurred, respectively. Before WNS (1990–2009), netting occurred at most sites only once per season, but 1 site was surveyed twice (15 May and 15 August 1996). After the onset of WNS, we visited 13 sites (14 survey nights) in 2011, 18 sites in 2012 (18 survey nights), and 25 sites in 2013 (28 survey nights). We surveyed a single location in 2011 and 3 sites in 2013 a second time due to poor weather conditions (rain or low temperatures) on the first night. Weather or logistical reasons prevented us from visiting all 26 sites each year. All efforts included in our analyses were conducted on nights with temperatures above 10 ºC, winds less than 4 m/sec, and intermittent or no rain (Francl 2008, USFWS 2015b). We captured bats using mist nets (38-mm mesh, reduced bag, Avinet, Dryden, NY) and replicated the original sampling effort at each site, including the number of nets (1–6), height of nets (2.6–9 m), and duration of sampling (1.2–5 h). We standardized sampling effort to a unit of effort (1 unit of effort = 1 m2 of net in place for 1 h), as defined by the Pennsylvania Game Commission (2011). We identified bats to species and fitted individuals with a unique numbered band (2.9-mm diameter, Porzana, Birmingham, UK) on the forearm. We recorded the sex of each animal. We determined age (adult vs. juvenile) by degree of ossification of epiphyseal plates Figure 1. Locations of areas and number of sites in which sampling occurred for Northern Long-eared Bats in western Virginia between 1990 and 2013. Northeastern Naturalist 198 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 Vol. 23, No. 2 in the phalanges (Anthony 1988) and assessed reproductive condition of females by palpating the abdomen for evidence of embryos and examining the nipples for signs of lactation (Haarsma 2008, Racey 1988). We omitted recaptures of banded individuals within the same night from our analyses. Data analysis To examine changes in capture rate (standardized by area of nets and mist-netting duration) and the actual and predicted proportion of Northern Long-eared Bats that were pregnant, lactating, or juvenile, we fit generalized linear mixed models (PROC GLIMMIX; SAS 9.4, SAS Inc., Cary, NC) to capture data. We examined changes in capture rate across years and with a post-hoc comparison, using least-squares means of the fixed effect. We assigned year relative to infection (i.e., before versus after WNS) and day of year as fixed effects to proportions of pregnant females, lactating adults, and juveniles to examine if reproductive measures differed because of WNS and to account for variation as a result of natural phenology through the summer. We tested proportion of females lactating against day + (day × day) to reflect the quadratic relationship of a seasonal rise to a maximum and subsequent decline during summer. We used chi-square procedures to test for differences in the proportion of sites with captures of Northern long-eared Bats across years. We set alpha = 0.1 for all tests, due to the exploratory nature of our analysis and to guard against a Type II error of rejecting the null hypothesis that WNS impacts were not observed. Results Capture rates Capture rates of Northern Long-eared Bats, adjusted for area of net and mistnetting duration, differed between surveys conducted before and after the onset of WNS (F = 3.75, df = 3, 87; P = 0.014). Prior to WNS, 175 Northern Long-eared Bats were captured at a mean adjusted capture rate of 0.102 bats/m2/h, during 27 survey-nights at 26 sites. In 2011, capture rate declined (-23.5%), but not significantly; we caught 115 Northern Long-eared Bats at a rate of 0.078 bats/m2/h during 14 survey nights at 13 sites. Our capture rate declined significantly (P = 0.005) in 2012 (-73.5%) compared to surveys before WNS; we netted 61 Northern Long-eared Bats at a rate of 0.027 bats/m2/h, during 18 survey-nights at 18 sites. The significant (P = 0.005) decline continued in 2013 (-95.1%) compared to those before WNS and 2011; in 2013, we captured only 7 Northern Long-eared Bats at a rate of 0.005 bats/m2/h, during 28 survey nights at 25 sites. The proportion of sites with captures of Northern Long-eared Bats also declined significantly (χ2 = 22.87, df = 2; P < 0.001), with captures at 12 of 13 sites in 2011 (-7.7%), 12 of 18 sites in 2012 (-33.3%), and 4 of 25 sites in 2013 (-84.0%). Reproductive trends Although proportions of pregnant or lactating female Northern Long-eared Bats numerically declined from before and after the onset of WNS, these changes were not significant (Table 1, Fig. 2). However, the proportion of juvenile bats did Northeastern Naturalist Vol. 23, No. 2 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 199 Figure 2. Generalized linear mixed-model comparisons of before (1990– 2009; black lines) and after infection (2010–2013; gray lines) by WNS, for proportion of female Myotis septentrionalis that were pregnant (a) or lactating (b), and proportion of juveniles captured (c). Black (before WNS) and grey (after WNS) dots represent actual proportion of juveniles captured for a particular day. Northeastern Naturalist 200 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 Vol. 23, No. 2 decline significantly, decreasing from 40% of captures before arrival of WNS to less than 10% after WNS (Table 1, Fig. 2). As expected, the proportion of pregnant females and proportion of juveniles declined and increased, respectively, over the summer, whereas the proportion of lactating females rose and fell through the summer, concomitant with the period following birth through weaning (Fig. 2). Discussion The results of our summer mist-netting surveys provide further evidence of the dramatic decrease in number of Northern Long-eared Bats in Virginia, which was indicated by pre-hibernation and hibernation surveys (Powers et al. 2015), and are similar to declines described in other states (Francl et al. 2012, Moosman et al. 2013, Turner et al. 2011). In addition, our data indicate an apparent lag between declines observed during swarming (Powers et al. 2015) and our summer mist-netting in the same mountainous region of western Virginia. Powers et al. (2015) first noted a decrease in Northern Long-eared Bats starting in 2010 and reaching >90% in 2012–2013. Although declines were noticeable during swarming surveys in 2011 (>70%), our capture rates in the summer of the same year showed a smaller, statistically insignificant decline (less than 25%) compared to capture rates before infection with WNS. Our decrease in summer capture rates in 2012 (>70%) was closer to that observed during fall swarming (>90%) in 2012, and our summer capture rates in 2013 mirrored those observed during fall swarming of that year (>90%). Physical impacts (wing damage) from WNS heal over summer (Fuller et al. 2011), when fungal-spore loads are low or nonexistent (Langwig et al. 2014), so we expected similar percent decreases in capture rates between summer and swarming surveys conducted in the same year in the same geographic area. One explanation for this time lag may be the presence of geographically different populations during these periods. It is possible that a WNS-impacted northern population may be Table 1. Parameter estimates for reproductive trends before versus after the onset of white-nose syndrome (WNS) for Northern Long-eared Bats (Myotis septentrionalis) in western Virginia, 1990–2013. Effect Estimate SE df F P Proportion Pregnant Intercept 16.38 5.48 1, 172 WNS -0.74 0.52 1, 172 2.01 0.16 Day -0.11 0.03 1, 172 10.18 less than 0.01 Proportion Lactating Intercept -288.93 76.57 1, 172 WNS -0.54 0.36 1, 172 2.25 0.14 Day 3.26 0.83 1, 172 15.42 less than 0.001 Day + (Day × Day) -0.01 0.002 1, 172 15.6 less than 0.001 Proportion Juveniles Intercept -9.12 1.89 1, 173 WNS -1.97 1.78 1, 173 2.81 0.09 Day 0.04 0.01 1, 173 16.17 less than 0.001 Northeastern Naturalist Vol. 23, No. 2 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 201 present during swarming with an unaffected or recently affected southern population present during summer. However, the limited literature on migration of the Northern Long-eared Bat indicates that it is a short-distance migrant, moving at most 50–90 km (Caire et al. 1979, Griffin 1940, Nagorsen and Brigham 1993) and suggesting that populations (i.e., capture rates) are not strongly influenced by long migratory movements. The direct distance between the most northern and southern mist-netting and swarming sites is ~390 km, about 4 times the longest documented migratory movement for the Northern Long-eared Bat. An alternative explanation may be the number and locations of the sample sites during these periods. Walsh et al. (1996) showed a significant gradient in bat abundance along a south–north geographical axis between London and Aberdeen in the UK, and Larsen et al. (2001) demonstrated that increasing sampling effort improved precision in evaluating regional population trends. During swarming in 2011, Powers et al. (2015) conducted 14 surveys at 9 sites, and in that summer we conducted 14 surveys at 13 sites. Although monitoring additional locations would be preferred, both surveys included sites from both the most northern and southern region of the project area, guarding against geographic bias. Additional ongoing efforts to document migratory patterns and distances of Northern Long-eared Bats may help clarify the time lag we observed. To our knowledge, this is the first case of a time lag in declines of relative abundance represented by 2 survey periods in the same year in the same geographic area. Secondary impacts (e.g., reproductive patterns) of WNS are important in understanding the ability and potential for populations to stabilize or recover. Cryan et al. (2010) postulated that serious wing damage caused by WNS could compromise reproductive success, and Francl et al. (2012) noted pregnancy rates for Northern Long-eared Bats that were half the historic levels late in the season in West Virginia. Francl et al. (2012) also postulated that reproduction would be delayed due to the added energetic demands caused by infection with WNS. Although Francl et al. (2012) found that rates of lactation were similar before and after WNS, the peak occurred 17 days earlier than before infection, which was opposite their prediction. This shift may be a response to improve survivorship of young, as suggested by the results of Frick et al. (2010) who reported that Myotis lucifugus (LeConte) (Little Brown Bat) born early in the summer were more likely to survive their first year than those born later. Although we did not observe a significant change in the proportion of pregnant or lactating females or a shift in the dates of occurrence for pregnant or lactating females, the proportion of juveniles declined significantly after WNS (Fig. 2). In 2013, most mist-netting events yielded no juveniles from late June through August, and functionally, recruitment fell to 0 for western Virginia. Francl et al. (2012) noted a similar, albeit less severe, trend in West Virginia, where the proportion of juveniles declined by 60% in 2010, 1 year after WNS was detected. Maslo et al. (2015) conducted a vital sensitivity analysis for a hibernating colony of Little Brown Bats and determined that adult and juvenile survival, as opposed to fecundity, are the demographic parameters most important to maximize recovery. If these parameters are equally critical to the Northern Long-eared Bat, Northeastern Naturalist 202 R.J. Reynolds, K.E. Powers, W. Orndorff , W.M. Ford, and C.S. Hobson 2016 Vol. 23, No. 2 then the lack of recruitment is alarming and calls into question the viability of the Northern Long-eared Bat over the long term. Although documenting population changes through counts at hibernacula and catch per unit effort provides insight into the degree of decline, additional sampling is needed to understand the secondary impacts of this disease on Northern Long-eared Bats. Our study highlights the need for research focused on maternity colonies throughout the range of the species to better understand the demographic impacts of WNS and the potential for this species to stabilize or recover over time. Acknowledgments This study was funded by a White-nose Syndrome Program Grant provided by the Virginia Department of Game and Inland Fisheries, and a Wildlife Restoration Program Grant from the US Fish and Wildlife Service. We thank numerous volunteers for assistance in the field: T. (Canniff) Adler, B. Balfour, J. Beeler, J. Bentley, M. Blanchard, L. Boggs, J. Bower, N. Brewer, L. Coleman, A. Futrell, J. Hallacher, J. Huth, B. Hyzy, J. Kiser, E. Koertge, D. Landgren, B. Meyer, N. Miller, Z. Orndorff, D. 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