Challenge or inoculation studies (e.g. Lorch et al. 2011; Warnecke et al. 2012; Wilcox et al. 2014) and comparative studies of bats from affected versus unaffected hibernacula (Moore et al. 2011; Storm and Boyles 2011; Reeder et al. 2012; Brownlee-Bouboulis and Reeder 2013) have led to progress in our understanding of mechanisms underlying WNS. The wings of bats are physiological active tissue involved in gas exchange and fluid balance. In general, results of physiological studies are converging on a consensus that cutaneous infection of the wings accounts for the physiological and behavioural effects of WNS (Cryan et al. 2010).
Lorch et al. (2011) experimentally inoculated the wings of healthy M. lucifugus with P. destructans for comparison to sham-inoculated controls. They housed bats in temperatureand humidity-controlled incubators that maintained environmental conditions approaching natural hibernacula [82 % relative humidity (RH) at 6.5 °C]. This experiment resolved a critical question by demonstrating that experimental infection with P. destructans caused the defining characteristics of WNS (e.g. cupping erosions in the epidermis associated with fungal growth, Meteyer et al. 2009). They also found that P. destructans spread from infected to un-infected bats housed in the same cages but did not spread between cages in the same incubator confirming contact but not airborne transmission of the causal pathogen under laboratory conditions. Lorch et al. (2011) did not detect differences in survival between infected and un-infected bats possibly because the experimental duration was shorter than a typical hibernation season and/ or because humidity in this experiment was lower than that of hibernacula used by M. lucifugus in the wild, potentially influencing hibernation patterns of both control and infected bats. Warnecke et al. (2012) repeated aspects of Lorch et al.'s (2011) experiment but increased ambient humidity to >97 % RH at 7 °C and ran the experiments for 120 days (vs. 102 days in Lorch et al. 2011). In Warnecke et al.'s (2012) experiment, all sham-inoculated bats survived four months of hibernation, while infected bats exhibited a significant increase in the frequency of periodic arousals, reduced fat reserves and reduced survival, thus confirming that infection with P. destructans alone causes the pathology that defines WNS, altered torpor behaviour and mortality. A field study comparing arousal frequency of bats in affected versus unaffected caves (Reeder et al. 2012) also reported a difference in arousal frequency similar to that observed by Warnecke et al. (2012). Together these findings suggest a strong role for increased arousal frequency and altered energy balance in WNS pathophysiology.
Comparisons of control and infected bats have also provided insight into immune responses (or lack of responses) of bats during and after hibernation. Hibernators generally exhibit down-regulated immune function during winter and bat species affected by WNS appear to be no exception (Meteyer et al. 2009, 2012; Moore et al. 2011). During hibernation, there is little evidence of initiation of an inflammatory response or recruitment of immune cells in bats infected by
P. destructans based on histopathology (Meteyer et al. 2009, 2012). Despite the absence of an inflammatory response, however, variation in other aspects of cellular immunity may have a role to play. Moore et al. (2013) found differences in immunological responses of M. lucifugus in affected versus unaffected hibernacula, specifically higher leukocyte counts, reduced antioxidant activity and lower levels of interleukin-4 (an important precursor for differentiation of T-cells) in bats from WNS-affected caves. Although comparisons between populations of bats in different hibernacula are challenging to interpret because of the potential for underlying differences between bats independent of infection, these findings suggest that even the hardest-hit bat species attempt some, albeit weak, immune response to P. destructans infection. This also raises the possibility that some bats may be better equipped to resist infection than others (Puechmaille et al. 2011c) with the potential for directional selection on immune function if these differences are heritable and provide a survival advantage.
Immune responses of bats to WNS could be as much a disadvantage as an advantage. Meteyer et al. (2012) recently reported the disheartening paradox that some survivors of WNS exhibit characteristic signs of immune reconstitution inflammatory syndrome (IRIS). When infected bats emerge from hibernation and their immune function resumes, they exhibit a massive neutrophilic inflammatory response to the fungal infection. This response appears to dramatically increase tissue damage and may reflect an over-reaction to infection because euthermic body temperatures in spring would likely be sufficient to combat the fungal infection (Chaturvedi et al. 2010; Puechmaille et al. 2011b; Verant et al. 2012). The response is likely energetically expensive and the resulting wing damage could compromise flight ability and, therefore, spring energy balance by increasing healing and immunity costs, while reducing potential foraging efficiency at a time when energy balance is critical to support reproduction. Further studies of the role of IRIS in the ecology of WNS are essential for understanding the potential for populations to recover from WNS.
A down-regulated immune response in hibernating bats generally, combined with increased arousal frequency (Boyles and Willis 2010; Reeder et al. 2012; Warnecke et al. 2012) and possibly increased metabolic rate and body temperature during torpor following infection (Storm and Boyles 2011; Verant et al. 2014), appears to result in premature fat depletion and starvation. However, why fungal infection would increase arousal frequency is still not fully understood. Cryan et al. (2010) proposed the hypothesis that fungal damage to the wings of bats could lead to increased evaporative water loss (EWL) across damaged epidermis. Rates of EWL during torpor are a strong predictor of arousal frequency in hibernators (Ben-Hamo et al. 2013; Thomas and Cloutier 1992; Thomas and Geiser 1997) so an increase in EWL or fluid loss due to skin damage from infection by
P. destructans could lead to the observed effects on arousals. Willis et al. (2011) used data on water loss and arousal frequency in healthy bats, combined with an individual-based model quantifying survival of hypothetical populations of bats, to demonstrate that even a small increase in EWL resulting from infection could cause the same patterns of arousal and mortality observed for infected bats, thus highlighting the plausibility of the dehydration hypothesis.
Two independent datasets from both captive and free-ranging bats also support a role for dehydration and fluid loss in WNS pathophysiology (Cryan et al. 2013; Warnecke et al. 2013). In addition to high hematocrit levels consistent with dramatic fluid loss, Cryan et al. (2013) and Warnecke et al. (2013) both found evidence of electrolyte depletion (with no evidence of renal pathology), consistent with hypotonic dehydration due to fluid loss across damaged wings. Presumably infected bats lose fluid containing both water and electrolytes across injured wing tissue but can only replenish or partially replenish water stores by drinking, because electrolytes are not available in hibernacula. Warnecke et al. (2013) also found preliminary evidence of a respiratory response to metabolic acidosis in infected bats which they hypothesized reflect reduced perfusion of infected tissues, localized anaerobic metabolism and acidosis, and increased respiratory rate to increase CO2 excretion and counter acidosis. In addition to increased arousal frequency, these physiological responses also predict increased metabolic costs and elevated body temperature during torpor. To date, measurements of torpid body temperature with enough precision to test this hypothesis are unavailable but these would be valuable, especially alongside measurements of metabolism during torpor and arousal in infected versus un-infected bats.
Other physiological mechanisms could also be at play. Willis and Wilcox (2014) reviewed three (of many potential) hormone systems that could be influenced by WNS, both within individuals and via selection on traits which could favour survival. For example, the lipostat hormone leptin is strongly associated with winter energy balance and pre-hibernation fattening. Bats must enter a state of leptin resistance during fall to accumulate adequate fat stores to survive the winter. If, as the evidence suggests, WNS represents a challenge for hibernation endurance, bats with the greatest leptin resistance (and therefore potential fat stores) in autumn may be best equipped to survive increased arousals associated with WNS (Willis and Wilcox 2014). Interactions between WNS and other hormone systems important for seasonal energetics, body temperature regulation and energy and fluid balance (e.g. glucocorticoids, melatonin, thyroid hormone, vasopressin, androgens) could also play important roles in disease dynamics and evolution of remnant populations and are worth further study.
In addition to physiological research, recent studies have also examined behavioural mechanisms associated with WNS that could reflect either adaptive responses to disease or maladaptive pathological responses. Langwig et al. (2012) reported that a much greater proportion of the M. lucifugus surveyed in WNS-affected caves after the emergence of the disease were hibernating solitarily (i.e. without clustering) compared to bats surveyed before WNS. This could reflect a behavioural change by individuals following infection or selection by WNS for bats which tend to roost individually (Langwig et al. 2012). Wilcox et al. (2014) reported behavioural observations of bats inoculated with P. destructans and found evidence supporting the former hypothesis. Infected bats gradually reduced their clustering behaviour as hibernation progressed. Wilcox et al. (2014) also observed a reduction in behavioural activity during arousals, in general, for affected bats. Taken together, reduced clustering and reduced activity by infected bats could reflect general patterns known as “sickness behaviour”, a coordinated response to infection characterized in part by lethargy presumably to save energy for immune responses (Adelman and Martin 2009). These behaviours could also reduce the potential for transmission among individuals in a social group within a hibernaculum. Even bats that have already been infected with P. destructans could benefit by reduced subsequent exposure to other infected individuals because new contacts could lead to additional areas of infection in the wings, exacerbating disease severity. On the other hand, reduced clustering behaviour could increase energy expenditure and EWL leading to negative consequences for survival. More work is needed to understand the survival consequences of a range of physiological and behavioural responses to WNS.