**1. Introduction**

White-nose Syndrome (WNS) is an emergent mycosis that affects some bat species in North America and is caused by an extensive cutaneous infection with the fungus *Pseudogymnoascus destructans* (*Pd*) during hibernation. It was first observed at a single cave in New York State during the winter of 2006–2007, and then spread to 5 more caves/mines in New York State during the winter of 2007–2008 [1]. *Pseudogymnoascus destructans* (*Pd*) has since spread to 39 U.S. States and 7 Canadian provinces, and it was introduced to North America from Europe [2]. This fungus grows on the muzzle, wings, and ears of afflicted bats during hibernation, with hyphae penetrating both the epidermis and dermis, consuming hair follicles, sebaceous and sweat glands [3–5]. *Pseudogymnoascus destructans* grows at ambient temperatures ranging from 1.9 to 15°C, although the hyphal morphology of this fungus exhibits heat stress at an ambient temperature (Ta) > 12°C, and growth ceases altogether at Ta > 19°C [6]. The arrival of *Pd* has led to severe population declines for 4 of the 6 bat species that hibernate in the Northeastern United States and Canada. Within 1–2 years after the arrival of *Pd* at a hibernation site, the number of little brown (*Myotis lucifugus*), northern long-eared (*Myotis septentrionalis*), Indiana

(*Myotis sodalis*), and tricolored (*Perimyotis subflavus*) bats decreases by 75–95% due to high over-winter mortality during the hibernation period [7]. Extensive cutaneous infections with *Pd* have been shown to be the cause of WNS in laboratory inoculation/hibernation experiments with captive *M. lucifugus* [8]. Bats suffering from WNS have numerous skin lesions caused by *Pd* infections on their wings, face, and ears. These *Pd* lesions display an orange-yellow fluorescence when illuminated by long-wave (365–385 nm) UV light [9]. The mechanism by which an extensive cutaneous infection with *Pd* leads to WNS and subsequent death during hibernation in some North American bat species is due to the effects of these infections on the hibernation energetics of small bats.

## **2. Hibernation physiology and white-nose syndrome**

Mammals and birds are unique among animals in that they are homeothermic endotherms, maintaining a constant core body temperature (Tb) over a wide range of ambient temperatures (Ta) through a high metabolic rate [10]. Prolonged periods of high metabolic heat production by mammals and birds requires high rates of energy intake. Food availability in the wild often fluctuates, and consequently the energetic costs of maintaining a high Tb (32–42°C) via endothermy becomes prohibitively expensive during certain environmental conditions. Not all mammals and birds are permanently homeothermic, but instead enter periods of torpor [11]. Torpor is a period when metabolic rate and Tb are greatly reduced. It involves the regulation of Tb at a new and substantially lower level, with a new minimum Tb being maintained. Torpor is a controlled reduction in metabolic rate, Tb, and a suite of physiological processes in endotherms [12]. Mammalian and avian species that employ torpor are therefore classified as heterothermic endotherms [11]. Metabolic rates during torpor can be less than 5% of basal metabolic rate with a corresponding Tb of just 0.5–1.0°C above ambient temperature (Ta) in most instances [13]. Mammals and birds generally employ one of two common patterns of torpor, depending upon species: prolonged torpor during hibernation, and daily torpor. Daily torpor occurs when a heterotherm has torpor bouts that are 3–12 h in duration. Hibernation is seasonal, usually from late summer/autumn to the following spring. Hibernators do not remain torpid continuously throughout the hibernation season; instead, bouts of torpor last from days to weeks, interrupted by brief periods of high metabolic rates and high Tb called arousal episodes. Hibernation is the most common pattern of torpor found in mammals. Numerous studies have revealed that daily torpor occurs in at least 42 bird species, and 78 mammalian species as well. At present, only 1 bird species (the Common Poorwill, *Phalaenoptilus nuttallii*) is known to hibernate, whereas hibernation has been document in about 100 mammalian species [14].

Stored triacylglycerols mobilized from white adipose tissue (WAT) are the primary energy source utilized during mammalian hibernation [15]. The periods of high metabolic rates and Tb known as arousal episodes normally account for 80–90% of stored lipids (energy) utilized during hibernation [16, 17], but their physiological function is poorly understood. A number of physiological/biochemical alterations that occur during torpor are reversed during arousal episodes. These alterations include dendritic retraction, leukocyte sequestration in secondary lymphatic organs, endocytosis, and protein degradation [18]. Periodic arousals from torpor thus serve to rectify physiological/biochemical imbalances that occur during torpor. Heterothermic mammals typically undergo an extensive period of feeding and fattening for several weeks prior to the onset of hibernation, during

which body fat levels increase by 4 to 7-fold. The body fat content of *M. lucifugus* increases from 7 to 27% body mass during the 2 months prior to hibernation [19, 20], for example.

The energetic constraints that normal arousal episodes place on the physiological ecology of hibernating bats are illustrated by examining the winter physiological ecology of one bat species that is now severely impacted by WNS, *M. lucifugus*. Although the normal arousal episodes of hibernating *M. lucifugus* are usually less than 1 h in duration, they nonetheless account for 80–90% of all energy utilized during hibernation by this species [21]. Each of these arousal episodes requires the utilization of about 110 mg of stored depot lipids [22]. The body mass of *M. lucifugus* at the onset hibernation averages 8.5 g, indicating that about 2.0 g of depot lipids (triacylglycerols) are stored by each bat to support the entire 190-d hibernation period [23]. If arousal episodes consume a total of at least 80% of the lipids utilized during the entire hibernation period, then about 1.6 g of the 2.0 g stored by *M. lucifugus* are required to fuel them. Hibernating *M. lucifugus* thus have enough stored energy to support only about 14–15 arousal episodes during the entire period. Free-ranging *M. lucifugus* hibernating at ambient temperatures of 5.5 to 12.0°C consequently have normal torpor bouts averaging 12.4 to 19.7 d in length [22, 24] which enables them to survive a 190-d hibernation period without depleting their energy reserves prior to the spring.

Studies on free-ranging *M. lucifugus* revealed that bats with extensive *Pd* infections arouse more frequently from torpor during hibernation, and consequently their torpor bouts were much shorter than the normal range of 12.4 to 19.7 d previously reported for this species. Individuals with extensive cutaneous *Pd* infections (lesions) had a mean (± SE) torpor bout duration of 7.93 ± 2.49 d between arousal episodes, whereas those with no *Pd* lesions had a mean torpor duration of 16.32 ± 6.65 d which was significantly longer [25]. This 51% reduction in torpor bout duration produced by extensive cutaneous *Pd* infections made arousal episodes more frequent, which increased the rate of energy expenditure during the entire hibernation period. This increased rate of energy expenditure during hibernation is WNS, which leads to the premature depletion of body fat reserves prior to the normal spring emergence from hibernation when food (arthropods) first becomes available, which in turn causes of death [26]. The mechanism by which a severe cutaneous infection with *Pd* increases the frequently of arousal episodes during hibernation is related to the degree of evaporative water loss from the skin surface. The normal rate of evaporative water loss (EWL) of bats is considerable during torpor, and it is thought that they periodically arouse from torpor to drink in order to restore their water balance [27]. Hibernating bats have been observed drinking during arousal episodes [28]. It thus has been proposed that the numerous skin lesions caused by severe *Pd* infections may increase the EWL of affected bats, which in turn would cause them to arouse from torpor more frequently to drink. Analyses of blood samples collected from both *Pd* infected and uninfected *M. lucifugus* during hibernation support this hypothesis [29, 30]. Laboratory inoculation/hibernation experiments with *M. lucifugus* revealed that the mean EWL rate of individuals with numerous cutaneous *Pd* lesions was 1.6-fold greater than that for bats with no *Pd* lesions [31]. Interpreting these findings together reveals that when cutaneous *Pd* infections result in numerous skin lesions, WNS is caused by a corresponding increase in the rate of cutaneous EWL, which in turns leads to both reduced torpor bout lengths and more frequent arousal episodes. This subsequently results in a greater rate of energy expenditure during hibernation that produces a premature depletion of body fat reserves during hibernation, before feeding can occur in the spring.
