Frequent Arousal from Hibernation Linked to Severity of Infection and Mortality in Bats with White-Nose Syndrome

White-nose syndrome (WNS), an emerging infectious disease that has killed over 5.5 million hibernating bats, is named for the causative agent, a white fungus (Geomyces destructans (Gd)) that invades the skin of torpid bats. During hibernation, arousals to warm (euthermic) body temperatures are normal but deplete fat stores. Temperature-sensitive dataloggers were attached to the backs of 504 free-ranging little brown bats (Myotis lucifugus) in hibernacula located throughout the northeastern USA. Dataloggers were retrieved at the end of the hibernation season and complete profiles of skin temperature data were available from 83 bats, which were categorized as: (1) unaffected, (2) WNS-affected but alive at time of datalogger removal, or (3) WNS-affected but found dead at time of datalogger removal. Histological confirmation of WNS severity (as indexed by degree of fungal infection) as well as confirmation of presence/absence of DNA from Gd by PCR was determined for 26 animals. We demonstrated that WNS-affected bats aroused to euthermic body temperatures more frequently than unaffected bats, likely contributing to subsequent mortality. Within the subset of WNS-affected bats that were found dead at the time of datalogger removal, the number of arousal bouts since datalogger attachment significantly predicted date of death. Additionally, the severity of cutaneous Gd infection correlated with the number of arousal episodes from torpor during hibernation. Thus, increased frequency of arousal from torpor likely contributes to WNS-associated mortality, but the question of how Gd infection induces increased arousals remains unanswered

Intraspecific Comparison of Population Structure, Genetic Diversity, and Dispersal Among Three Subspecies of Townsend’s Big-Eared Bats, \u3ci\u3eCorynorhinus townsendii townsendii, C. t. pallescens\u3c/i\u3e, and the Endangered \u3ci\u3eC. t. birginianus\u3c/i\u3e

Townsend’s big-eared bat, Corynorhinus townsendii, is distributed broadly across western North America and in two isolated, endangered populations in central and eastern United States. There are five subspecies of C. townsendii; C. t. pallescens, C. t. australis, C. t. townsendii, C. t. ingens, and C. t. virginianus with varying degrees of concern over the conservation status of each. The aim of this study was to use mitochondrial and microsatellite DNA data to examine genetic diversity, population differentiation, and dispersal of three C. townsendii subspecies. C. t. virginianus is found in isolated populations in the eastern United States and was listed as endangered under the Endangered Species Act in 1979. Concern also exists about declining populations of two western subspecies, C. t. pallescens and C. t. townsendii. Using a comparative approach, estimates of the genetic diversity within populations of the endangered subspecies, C. t. virginianus, were found to be significantly lower than within populations of the two western subspecies. Further, both classes of molecular markers revealed significant differentiation among regional populations of C. t. virginianus with most genetic diversity distributed among populations. Genetic diversity was not significantly different between C. t. townsendii and C. t. pallescens. Some populations of C. t. townsendii are not genetically differentiated from populations of C. t. pallescens in areas of sympatry. For the western subspecies gene flow appears to occur primarily through male dispersal. Finally, geographic regions representing significantly differentiated and genetically unique populations of C. townsendii virginianus are recognized as distinct evolutionary significant units

Distribution and spread of WNS throughout North America.

<p>Spread of WNS by hibernation season through the winter of 2010–2011 is shown along with locations of study sites, indicated by stars (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038920#pone-0038920-t001" target="_blank">Table 1</a>). Confirmed sites have been officially reported by each state or province based upon histological confirmation of infection with the fungal pathogen <i>Geomyces destructans</i> (Gd); bats from suspect sites have clinical signs of WNS but lack laboratory confirmation. The inset shows a little brown bat infected with Gd from site #1 in Vermont. This site was WNS confirmed in 2008–2009, when bats were studied. Bats from site # 2 in Pennsylvania were studied in 2008–2009 (for 8 weeks only in the spring), when no signs of WNS were present, in 2009–2010, when a single bat from this site showed infection with Gd without mass mortality and in 2010–2011, when bats in this site were heavily infected. Bats from site #3 in Pennsylvania were studied in 2008–2009 (no WNS), 2009–2010 (when Gd was noted but without mass mortality) and in 2010–2011, when bats in this site were heavily infected. Bats from site #4 in Pennsylvania were studied in 2009–2010 (for 8 weeks only in the spring), when bats were heavily infected. Bats from site #5 in West Virginia were studied in 2008–2009, when there was no evidence of Gd presence – which was also the case for bats from site #6 in Michigan, which were studied all three years.</p

Torpor bout length (TBL) as a function of date of death and BMI.

<p>For the 12 bats that died from WNS, BMI at the beginning of hibernation was not related to TBL (3A), nor was BMI predictive of the date of death (3B). However, TBL significantly predicted date of death in WNS-affected bats that were found dead at the time of datalogger retrieval (3C) (r² = 0.58). Bats that died sooner were arousing to euthermic temperatures much more frequently than those that lived longer.</p

Torpor bout length (TBL) in days by WNS status.

<p>WNS was associated with decreased TBL: bats that were affected by WNS but still alive at the collection of dataloggers (March) had shorter TBLs than unaffected bats (but this difference was not significant). Significantly shorter TBLs were seen in WNS-affected bats that were found dead at the time of datalogger collection compared to affected but alive bats (2A). Bats were categorized as: unaffected, WNS-affected and alive at time of datalogger removal (‘WNS-alive’), and WNS-affected and dead when loggers were removed in the spring (‘WNS-dead’). Numbers in brackets indicate sample size and boxes sharing the same letter are not significantly different from each other. Boxes depict the 25th and 75th percentiles, lines within boxes mark the median, and whiskers represent 95th and the 5th percentiles. Outliers are indicated with open circles. Additional panels illustrate sample temperature profile of an unaffected (B) and an affected (C) bat, during the winter of 2009. The bat illustrated in C displayed daily arousals at the end of its life, which was seen in several of these animals. Each of the ‘WNS-dead’ bats died at the end of their last arousal.</p

Temperature-sensitive datalogger deployment and retrieval (bat recapture) data, by study year, hibernacula site number (see Fig. 1), and sex for the 504 loggers deployed.

<p>Whether data were successfully downloaded from the logger and ultimately used in the analyses of this paper, are also described.</p>*<p># of males/# of females and date of deployment or retrieval of loggers.</p>**<p>bats were occasionally excluded from analyses due to incomplete data (e.g., BMI not recorded) or problems with downloaded data.</p>+<p>loggers only deployed mid-winter (January-March) as opposed to the full-hibernation season (November-March).</p