BACKGROUND
since emergence of white-nose syndrome  in  <dig>  bat populations of eastern north america have declined precipitously due to disease-related mortality .
the causative agent of wns is the fungus pseudogymnoascus  destructans , which erodes unfurred skin comprising wing membranes, muzzles, and ears of hibernating bats, inducing physiological perturbations, altered behavior, and death  <cit> .
although underlying causes for mortality from this invasive cutaneous mycosis remain unclear, proposed mechanisms include disruptions to vital homeostatic functions such as thermoregulation and water balance  <cit> .
for example, water and electrolyte losses across the ulcerated wing epithelium have been proposed to cause hypotonic dehydration  <cit>  and acid–base disturbances  <cit> .
consequently, alterations in behavior have been observed in infected bats, including increased frequency of arousal from torpor during hibernation  <cit>  and unusual day flights during winter  <cit> .
high metabolic demands of such activities  likely also contribute to mortality of bats prior to spring emergence by accelerating depletion of fat reserves.
however, physiological data linking altered behavior to increased energy demands in bats with wns are lacking.
the doubly labeled water  method is widely applicable to the study of energetics in relation to homeostasis, behavioral adaptations, and resource allocation in both animals and humans  <cit> .
this method is based on dynamic flux of hydrogen and oxygen through the body and ability to measure these flux rates over a period of time using labeled isotopes, 2h and 18o  <cit> .
following administration of these exogenous isotopes, they equilibrate throughout the body water pool.
the total body water volume  can then be estimated from the dilution spaces of the isotopes when introduced at known concentrations and serves as a valuable indicator of body composition   <cit> .
notably the dlw method has been used in temperate-zone insectivorous bats in the wild , but there are no published reports of this method being used in bats hibernating over a protracted time period .
to evaluate proposed causes of mortality from wns, we used the dlw method to quantify energy expenditure and changes in body composition of hibernating little brown bats  experimentally infected with p. destructans to test the hypothesis that wns increases metabolic demands during hibernation.
we predicted that infected bats would exhibit greater changes in body composition, specifically decreased proportion of fat mass, over the course of the experiment compared to negative control bats as a result of higher daily energy expenditures and fat utilization.
to further characterize previously reported physiologic outcomes associated with wns, we analyzed blood chemistries of all bats at the end of the experiment to assess acid–base balance, electrolytes, and hydration status.
RESULTS
infection status and torpor patterns
of the  <dig> bats treated with conidia from p. destructans,  <dig> bats  developed epidermal wing lesions characteristic of wns by the end of the 98-d experiment.
the majority of these bats  had mild to moderate wns , while the remaining two had moderate to severe wns .
sex had no effect on the probability that a bat developed wns .
all infected bats, including animals that did not develop detectable wns by histology , were pcr-positive for p. destructans; all bats in the control  group were pcr-negative for the fungal pathogen.
four infected bats and five control bats died prior to the end of the experiment.
average torpor bout duration for infected bats following dlw injection was  <dig>   d with average arousal duration of  <dig>  min.
average torpor bout duration for control bats following dlw injection was  <dig>   d with average arousal duration of  <dig>  min.
differences in torpor-arousal patterns of bats between treatment groups were not significant .
blood chemistry
blood chemistry parameters were analyzed for  <dig> infected bats  and  <dig> control bats  from which sufficient blood sample volumes were collected.
one infected bat did not have skin lesions characteristic of wns on histopathology, but there were no discernible differences across parameters when compared to infected bats with confirmed wns.
additionally, we were unable to collect sufficient blood volume for analysis from the two bats with most advanced pathology .
thus, blood chemistry values presented herein for infected bats represent bats with mild wns pathology .
infected bats had significantly lower blood ph than controls .
this acidemia was associated with a significant elevation of pco <dig> in infected bats compared to controls , indicating that bats had respiratory acidosis.
bicarbonate levels of infected bats were also significantly higher than those of non-infected controls , evident of a compensatory renal response to a chronic acidosis.
the accumulation of bicarbonate in blood of infected bats was also reflected in an elevated base excess compared to controls .
there were no differences in sodium or chloride concentrations between treatment groups, but potassium concentration was significantly higher in the infected bats than it was in the controls .
glucose concentrations were lower in infected bats but not significantly , and anion gap values were not different from controls nor elevated as would be expected if acidemia resulted from metabolic lactic or keto-acidosis.
other measures of hydration status  were equivalent between treatment groups .
overall, there were no effects of sex  =  <dig> , p =  <dig> ) or the interaction of sex and treatment  =  <dig>  , p =  <dig> ) on measured blood parameters.
see additional file  <dig> for a complete table of blood chemistry parameter estimations compared to available reference values.table  <dig>
blood chemistry comparisons
parameter
k
df
unadjusted p-value α =  <dig>
holm-bonferroni corrected p-value
pco2
hco3
−
cl−
na+
abbreviations: pco
<dig> dissolved carbon dioxide, k potassium, hco
3
− bicarbonate, be base excess, ag anion gap, cl
− chloride, bun blood urea nitrogen, tp total protein, na
+ sodium, hct hematocrit.
results of t-tests used to compare mean blood chemistry parameters for little brown bats  either experimentally infected with pseudogymnoascus destructans or negative  controls.
significant differences between treatment groups  were determined by p-values < holm-bonferroni p-values corrected by the index of comparison .
blood chemistry parameters.
box-and-whisker plots of blood chemistry values for hibernating little brown bats  experimentally infected with pseudogymnoascus destructans and negative  controls.
parameters for acid-base balance , electrolytes  and hydration status  are shown.
the median , upper and lower quartiles , and maximum and minimum values  are shown.
potential outliers  shown were not confirmed by bonferonni outlier tests.
significant differences  were determined at α =  <dig>  corrected for multiple comparisons by the holm-bonferonni method.
measurements of daily energy expenditure and total body water
upon collection of final blood samples,  <dig> d after injection with dlw, labeled isotope concentrations had decreased to levels statistically indistinguishable from background levels.
consequently, isotope turnover rates and daily energy expenditure  could not be determined for bats euthanized at the end of the experiment.
however, one bat in the control group that died  <dig> d after initial injection of dlw had detectable isotope concentrations at time of death.
for this bat, kd and ko were  <dig> , calculated respiratory co <dig> production  was  <dig>  ml/min, and resultant dee was  <dig>  kj/day.
measurements of total body water  were available for  <dig> bats  following initial injection of dlw and for  <dig> bats  after final injection .
the reduction in sample size is attributed to mortality of bats during the experiment or inability to collect a sufficient amount of blood for isotope analyses.
at the time of initial dlw injection, mean tbw as percent of body mass  was significantly lower for infected bats than controls , but mean body mass was not different between groups .
after  <dig> d hibernation, changes in tbw and body mass were compared for infected and control bats for which paired measurements were available.
body mass decreased significantly in both treatment groups , but the loss in body mass was equal between groups.
changes in tbw % bm were marginally significant between infected and control bats, but not when corrected for multiple comparisons .
within groups, infected bats, which all developed mild wns  exhibited a significant increase in tbw % bm ; increase in tbw % bm among control bats  was not significant .table  <dig>
body composition measurements
parameter
pd
infected bats
control bats
all data
n = 22
n = 10
paired data
n = 14
n = 4
values of body mass  and total body water as percentage of body mass  were used to estimate net fat energy use over  <dig> d for individual little brown bats  experimentally infected with pseudogymnoascus destructans and negative control  bats.
values in the table are mean  and n = sample size.
initial tbw % bm was significantly different between treatment groups.
over the course of the experiment, infected bats demonstrated a significant increase in tbw % bm and used significantly more fat energy than non-infected bats.
body mass decreased significantly over the experiment in infected and control bats but there was no difference between groups.
doubly labeled water comparisons
measurement
k
df
unadjusted p-value α =  <dig>
holm-bonferroni corrected p-value
results of t-tests used to compare doubly labeled water measurements for little brown bats  either experimentally infected with pseudogymnoascus destructans  or non-infected controls .
total body water is represented as percentage of body mass .
significant differences between treatment groups  were determined by p-values < holm-bonferroni p-values corrected by the index of comparison .
as trends in tbw, namely tbw % bm, reflect changes in body composition, net fat energy utilization was calculated from the change in tbw  and body mass measurements for all bats with paired data assuming constant 73% water content of lean mass .
based upon these calculations, mean total fat energy utilization for the  <dig> d after which bats were administered dlw was significantly higher for bats with wns compared to negative controls .
discussion
results of this study support the hypothesis that infection with p. destructans and subsequent development of wns increases energy  use in hibernating bats and provide key information for understanding the progression of physiologic disturbances that ultimately lead to mortality from this disease.
specifically, isotope-based estimates of changes in body composition provided evidence that hibernating little brown bats with wns utilized twice as much energy as non-infected control bats housed under equivalent experimental conditions.
however, the greater energy use by infected bats was not associated with an increased rate or duration of arousals from torpor.
this implies that bats, even with mild wns lesions, have an elevated metabolism prior to the onset of altered arousal patterns characteristic of late-stage infections  <cit> .
additionally, bats with early-stage wns developed severe, chronic respiratory acidosis and hyperkalemia .
integrating these results with those reported by others , we propose a mechanistic multi-stage disease progression model for wns that encompasses our current knowledge of disease pathology and physiologic sequelae, including death, that result following infection by p. destructans .figure  <dig>
disease progression model for bat white-nose syndrome .
we propose a mechanistic multi-stage disease model for wns in a hibernating bat that encompasses current knowledge on the progression of fungal-induced wing pathology and physiologic sequelae leading to mortality from disease.
initial colonization and invasion of the wing epidermis by pseudogymnoascus destructans  results in increased energy expenditure, chronic respiratory acidosis , and hyperkalemia .
erosion and ulceration of the epidermis stimulate increased frequencies of arousal from torpor, which remove excess co <dig> and normalize blood ph, but contribute to dehydration and depletion of fat reserves.
as wing pathology becomes more extensive and severe, these effects are exacerbated by water and electrolyte loss across the epidermis , which stimulate more frequent arousals and create a positive feedback loop that ultimately leads to mortality when energy reserves and compensatory mechanisms become exhausted.
as shown in this study, early stages of wns involving fungal colonization of the wing membrane with progression to erosion and ulceration of the epidermis, are characterized by increased co <dig> levels in blood, resultant acidemia, and hyperkalemia.
the accumulation of co <dig> may stem from either increased co <dig> production from an elevated metabolic rate associated with infection, decreased co <dig> expiration, possibly due to inhibition of diffusion across the damaged wing epithelium  <cit> , and/or a compensatory response by the host to attempt to further lower torpor metabolic rates  by inducing a hypercapnic  acidosis .
this acidosis may then contribute to the observed hyperkalemia by an acidosis-induced extracellular shift of potassium.
intracellular potassium ions may also leak into the blood through damaged and necrotic cell membranes caused by hyphal invasion of the epidermis.
overall, these physiologic effects result in a chronic respiratory acidosis, hyperkalemia, and reduction of fat reserves among bats during early stages of wns.
consistent with results observed in bats at later and more severe stages of wns  <cit> , we propose that once pco <dig> elevates beyond a tolerance threshold, chemoreceptors stimulate hyperventilation, and resulting increased arousals from torpor serve to remove excess co <dig>  returning blood ph to normal .
the high energy demand of these arousals then likely further contributes to accelerated depletion of fat reserves.
additionally, increased ventilatory rates and greater vapor pressure difference with increased body temperatures during arousals would contribute to greater evaporative water loss  <cit>  and dehydration.
together, these outcomes are consistent with data previously published for little brown bats with more severe wns pathology that exhibited increased frequency of arousal from torpor, decreased pco <dig>  normal blood ph, and dehydration  <cit> .
as wns progresses towards more extensive and severe wing lesions, dehydration may be further exacerbated by water and electrolyte loss across the damaged epidermis of the wing  <cit> , further stimulating arousal from hibernation to drink  <cit> .
positive feedback loops are then established that link worsening disease-associated wing pathology to further increases in arousal frequency, water loss, and energy use resulting in additional observed acute physiologic changes, including hypocapnia, hypoglycemia, hyponatremia, hypochloremia, and emaciation .
once compensatory mechanisms such as cellular buffering, respiratory and metabolic regulation, and/or behavioral adaptations are exhausted, this suite of disturbances ultimately leads to mortality unless the bat has sufficient energy reserves to persist until spring emergence and clear the infection following a return to a metabolically active state  <cit> .
although normal reference ranges for blood chemistry values in microchiropteran species are generally lacking  <cit> , deviations of measured parameters in infected bats from the negative controls in this study, together with published information for apparently healthy hibernating little brown bats , suggest pathologic and potentially life-threatening physiological disturbances associated with early-stage wns infections .
bats with wns in this study had almost 40% higher mean pco <dig> than negative control bats , and pco <dig> values above 90 mmhg are generally considered to be lethal in other non-hibernating animals and humans.
elevated pco <dig> levels and associated acidemia are known to interfere with enzymatic functions, reduce metabolic activity, and cause central depression of respiration; in severe cases, such elevated levels can lead to coma and death.
direct calculations of energy expenditure could not be determined for most bats in this study because final isotope concentrations were indistinguishable from background levels.
however, the increase in tbw as a percent of body mass observed in infected bats indicated that bats with wns had higher proportions of lean tissue mass to fat tissue mass at the end of the study.
this finding implies that bats with wns used significantly more fat energy reserves compared to negative controls despite hibernating under equivalent conditions.
from the estimated changes in fat content over the 67-d measurement period, infected bats utilized  <dig>  kj/d, while control bats utilized  <dig>  kj/d.
these results indicate that bats with wns expended approximately twice as much energy during hibernation as non-infected control bats.
some caution must be taken in interpreting these results due to the small number of control bats for which paired tbw measurements were available to calculate estimates of fat use.
however, the range of expected daily energy use of  <dig>  to  <dig>  kj/d predicted for healthy hibernating m. lucifugus at the mean temperature of our study  <cit>  is consistent with the daily rates of energy use observed in control bats in this experiment, and lower than rates observed in infected bats.
there were no differences in torpor-arousal patterns between treatment groups in this study suggesting that wns causes an increase in metabolism that is not directly associated with arousal from torpor and occurs at early stages in disease progression.
additionally, there were no differences between infected and control bats in tskin maintained during torpor or arousal bouts.
alternatively, metabolic costs associated with infection and development of wing pathology may be linked to increased costs of thermoregulation caused by inhibition of peripheral vasoconstriction during torpor and arousal  <cit> , catabolism of fat to generate metabolic water in response to increased water loss, or additional energetic costs associated with the host-pathogen interaction.
rising concentrations of pco <dig> in a mammal would normally stimulate increased respiration to release excess co <dig>  however, the unique physiology of mammalian hibernators allows for active suppression of respiration during torpor, and under these conditions, blood pco <dig> increases to levels higher than those observed in metabolically active mammals  <cit> .
this elevation of pco <dig> is thought to be an integral part of hibernation physiology as induction of an acidotic state serves to reduce metabolic rate and thermogenesis  <cit> .
additionally, the resultant high pco <dig> gradient improves ventilation efficiency, thereby minimizing energy costs of respiration during torpor  <cit> .
despite this tolerance for a respiratory acidosis  <cit> , a hibernating mammal must still be able to regulate pco <dig> for proper physiologic function.
if co <dig> elimination routes, such as passive diffusion of co <dig> across the wing epithelium  <cit>  are compromised by disease, as hypothesized for bats with wns  <cit> , persistently rising blood pco <dig> levels would cause the severe chronic respiratory acidosis we have observed.
thus, underlying causes for the high pco <dig> levels in bats with wns are likely a combination of the uniquely adapted physiology of a hibernating mammal compounded by pathologically induced insult to these physiological mechanisms.
bat wns presents a new paradigm for the study of infectious disease.
never before has a fungal skin pathogen been known to specifically infect a hibernating mammal, causing severe physiologic disturbances and mortality.
although substantial efforts have been devoted to understanding chytridiomycosis, a lethal fungal skin disease of amphibians, prior to wns there had been no in-depth study of disease processes in a metabolically repressed animal caused by a psychrophilic and metabolically active pathogen.
the poorly characterized capacity of bats to compensate for and respond to infection during hibernation demonstrates the difficulty of understanding how host-pathogen interactions influence disease manifestation and mortality.
CONCLUSIONS
this study demonstrated that infection with p. destructans and subsequent development of wns increased energy  use in hibernating bats prior to the onset of altered arousal patterns associated with later stages of wns.
severe, chronic respiratory acidosis and hyperkalemia were also apparent in bats that developed mild wns.
with these results, we present a multi-stage disease progression model for wns as a framework for understanding the pathogenesis and underlying causes of mortality due to wns .
this model integrates a range of published work  with data from this study into the first attempt to mechanistically define a comprehensive conceptual model of what may occur during development of wns in a hibernating bat – from initial colonization of wing skin by p. destructans until the death of the animal.
this model identifies key testable hypotheses necessary to develop a comprehensive understanding of the physiologic effects of wns on hibernating bats.
ultimately, this knowledge will be critical for guiding effective and properly timed management actions to moderate physiologic effects of wns and minimize morbidity and mortality from this devastating disease.

 since emergence of white-nose syndrome in bat populations of eastern north america have declined precipitously due to disease-related mortality. the causative agent of wns is the fungus pseudogymnoascus destructans, which erodes unfurred skin comprising wing membranes, muzzles, and ears of hibernating bats, inducing physiological perturbations, altered behavior, and death. although underlying causes for mortality from this invasive cutaneous mycosis remain unclear, proposed mechanisms include disruptions to vital homeostatic functions such as thermoregulation and water balance. for example, water and electrolyte losses across the ulcerated wing epithelium have been proposed to cause hypotonic dehydration and acid–base disturbances. consequently, alterations in behavior have been observed in infected bats, including increased frequency of arousal from torpor during hibernation and unusual day flights during winter. high metabolic demands of such activities likely also contribute to mortality of bats prior to spring emergence by accelerating depletion of fat reserves. however, physiological data linking altered behavior to increased energy demands in bats with wns are lacking. the doubly labeled water method is widely applicable to the study of energetics in relation to homeostasis, behavioral adaptations, and resource allocation in both animals and humans. this method is based on dynamic flux of hydrogen and oxygen through the body and ability to measure these flux rates over a period of time using labeled isotopes, 2h and 18o. following administration of these exogenous isotopes, they equilibrate throughout the body water pool. the total body water volume can then be estimated from the dilution spaces of the isotopes when introduced at known concentrations and serves as a valuable indicator of body composition. notably the dlw method has been used in temperate-zone insectivorous bats in the wild, but there are no published reports of this method being used in bats hibernating over a protracted time period. to evaluate proposed causes of mortality from wns, we used the dlw method to quantify energy expenditure and changes in body composition of hibernating little brown bats experimentally infected with p. destructans to test the hypothesis that wns increases metabolic demands during hibernation. we predicted that infected bats would exhibit greater changes in body composition, specifically decreased proportion of fat mass, over the course of the experiment compared to negative control bats as a result of higher daily energy expenditures and fat utilization. to further characterize previously reported physiologic outcomes associated with wns, we analyzed blood chemistries of all bats at the end of the experiment to assess acid–base balance, electrolytes, and hydration status.  infection status and torpor patterns of the bats treated with conidia from p. destructans, bats developed epidermal wing lesions characteristic of wns by the end of the 98-d experiment. the majority of these bats had mild to moderate wns, while the remaining two had moderate to severe wns. sex had no effect on the probability that a bat developed wns. all infected bats, including animals that did not develop detectable wns by histology, were pcr-positive for p. destructans; all bats in the control group were pcr-negative for the fungal pathogen. four infected bats and five control bats died prior to the end of the experiment. average torpor bout duration for infected bats following dlw injection was d with average arousal duration of min. average torpor bout duration for control bats following dlw injection was d with average arousal duration of min. differences in torpor-arousal patterns of bats between treatment groups were not significant. blood chemistry blood chemistry parameters were analyzed for infected bats and control bats from which sufficient blood sample volumes were collected. one infected bat did not have skin lesions characteristic of wns on histopathology, but there were no discernible differences across parameters when compared to infected bats with confirmed wns. additionally, we were unable to collect sufficient blood volume for analysis from the two bats with most advanced pathology. thus, blood chemistry values presented herein for infected bats represent bats with mild wns pathology. infected bats had significantly lower blood ph than controls. this acidemia was associated with a significant elevation of pco in infected bats compared to controls, indicating that bats had respiratory acidosis. bicarbonate levels of infected bats were also significantly higher than those of non-infected controls, evident of a compensatory renal response to a chronic acidosis. the accumulation of bicarbonate in blood of infected bats was also reflected in an elevated base excess compared to controls. there were no differences in sodium or chloride concentrations between treatment groups, but potassium concentration was significantly higher in the infected bats than it was in the controls. glucose concentrations were lower in infected bats but not significantly, and anion gap values were not different from controls nor elevated as would be expected if acidemia resulted from metabolic lactic or keto-acidosis. other measures of hydration status were equivalent between treatment groups. overall, there were no effects of sex =, p = ) or the interaction of sex and treatment =, p = ) on measured blood parameters. see additional file for a complete table of blood chemistry parameter estimations compared to available reference values.table blood chemistry comparisons parameter k df unadjusted p-value α =  holm-bonferroni corrected p-value pco2 hco3 − cl− na+ abbreviations: pco dissolved carbon dioxide, k potassium, hco 3 − bicarbonate, be base excess, ag anion gap, cl − chloride, bun blood urea nitrogen, tp total protein, na + sodium, hct hematocrit. results of t-tests used to compare mean blood chemistry parameters for little brown bats either experimentally infected with pseudogymnoascus destructans or negative controls. significant differences between treatment groups were determined by p-values < holm-bonferroni p-values corrected by the index of comparison. blood chemistry parameters. box-and-whisker plots of blood chemistry values for hibernating little brown bats experimentally infected with pseudogymnoascus destructans and negative controls. parameters for acid-base balance, electrolytes and hydration status are shown. the median, upper and lower quartiles, and maximum and minimum values are shown. potential outliers shown were not confirmed by bonferonni outlier tests. significant differences were determined at α = corrected for multiple comparisons by the holm-bonferonni method. measurements of daily energy expenditure and total body water upon collection of final blood samples, d after injection with dlw, labeled isotope concentrations had decreased to levels statistically indistinguishable from background levels. consequently, isotope turnover rates and daily energy expenditure could not be determined for bats euthanized at the end of the experiment. however, one bat in the control group that died d after initial injection of dlw had detectable isotope concentrations at time of death. for this bat, kd and ko were, calculated respiratory co production was  ml/min, and resultant dee was  kj/day. measurements of total body water were available for bats following initial injection of dlw and for bats after final injection. the reduction in sample size is attributed to mortality of bats during the experiment or inability to collect a sufficient amount of blood for isotope analyses. at the time of initial dlw injection, mean tbw as percent of body mass was significantly lower for infected bats than controls, but mean body mass was not different between groups. after d hibernation, changes in tbw and body mass were compared for infected and control bats for which paired measurements were available. body mass decreased significantly in both treatment groups, but the loss in body mass was equal between groups. changes in tbw % bm were marginally significant between infected and control bats, but not when corrected for multiple comparisons. within groups, infected bats, which all developed mild wns exhibited a significant increase in tbw % bm; increase in tbw % bm among control bats was not significant.table body composition measurements parameter pd infected bats control bats all data n = 22 n = 10 paired data n = 14 n = 4 values of body mass and total body water as percentage of body mass were used to estimate net fat energy use over d for individual little brown bats experimentally infected with pseudogymnoascus destructans and negative control bats. values in the table are mean and n = sample size. initial tbw % bm was significantly different between treatment groups. over the course of the experiment, infected bats demonstrated a significant increase in tbw % bm and used significantly more fat energy than non-infected bats. body mass decreased significantly over the experiment in infected and control bats but there was no difference between groups. doubly labeled water comparisons measurement k df unadjusted p-value α =  holm-bonferroni corrected p-value results of t-tests used to compare doubly labeled water measurements for little brown bats either experimentally infected with pseudogymnoascus destructans or non-infected controls. total body water is represented as percentage of body mass. significant differences between treatment groups were determined by p-values < holm-bonferroni p-values corrected by the index of comparison. as trends in tbw, namely tbw % bm, reflect changes in body composition, net fat energy utilization was calculated from the change in tbw and body mass measurements for all bats with paired data assuming constant 73% water content of lean mass. based upon these calculations, mean total fat energy utilization for the d after which bats were administered dlw was significantly higher for bats with wns compared to negative controls. discussion results of this study support the hypothesis that infection with p. destructans and subsequent development of wns increases energy use in hibernating bats and provide key information for understanding the progression of physiologic disturbances that ultimately lead to mortality from this disease. specifically, isotope-based estimates of changes in body composition provided evidence that hibernating little brown bats with wns utilized twice as much energy as non-infected control bats housed under equivalent experimental conditions. however, the greater energy use by infected bats was not associated with an increased rate or duration of arousals from torpor. this implies that bats, even with mild wns lesions, have an elevated metabolism prior to the onset of altered arousal patterns characteristic of late-stage infections. additionally, bats with early-stage wns developed severe, chronic respiratory acidosis and hyperkalemia. integrating these results with those reported by others, we propose a mechanistic multi-stage disease progression model for wns that encompasses our current knowledge of disease pathology and physiologic sequelae, including death, that result following infection by p. destructans.figure disease progression model for bat white-nose syndrome. we propose a mechanistic multi-stage disease model for wns in a hibernating bat that encompasses current knowledge on the progression of fungal-induced wing pathology and physiologic sequelae leading to mortality from disease. initial colonization and invasion of the wing epidermis by pseudogymnoascus destructans results in increased energy expenditure, chronic respiratory acidosis, and hyperkalemia. erosion and ulceration of the epidermis stimulate increased frequencies of arousal from torpor, which remove excess co and normalize blood ph, but contribute to dehydration and depletion of fat reserves. as wing pathology becomes more extensive and severe, these effects are exacerbated by water and electrolyte loss across the epidermis, which stimulate more frequent arousals and create a positive feedback loop that ultimately leads to mortality when energy reserves and compensatory mechanisms become exhausted. as shown in this study, early stages of wns involving fungal colonization of the wing membrane with progression to erosion and ulceration of the epidermis, are characterized by increased co levels in blood, resultant acidemia, and hyperkalemia. the accumulation of co may stem from either increased co production from an elevated metabolic rate associated with infection, decreased co expiration, possibly due to inhibition of diffusion across the damaged wing epithelium, and/or a compensatory response by the host to attempt to further lower torpor metabolic rates by inducing a hypercapnic acidosis. this acidosis may then contribute to the observed hyperkalemia by an acidosis-induced extracellular shift of potassium. intracellular potassium ions may also leak into the blood through damaged and necrotic cell membranes caused by hyphal invasion of the epidermis. overall, these physiologic effects result in a chronic respiratory acidosis, hyperkalemia, and reduction of fat reserves among bats during early stages of wns. consistent with results observed in bats at later and more severe stages of wns, we propose that once pco elevates beyond a tolerance threshold, chemoreceptors stimulate hyperventilation, and resulting increased arousals from torpor serve to remove excess co returning blood ph to normal. the high energy demand of these arousals then likely further contributes to accelerated depletion of fat reserves. additionally, increased ventilatory rates and greater vapor pressure difference with increased body temperatures during arousals would contribute to greater evaporative water loss and dehydration. together, these outcomes are consistent with data previously published for little brown bats with more severe wns pathology that exhibited increased frequency of arousal from torpor, decreased pco normal blood ph, and dehydration. as wns progresses towards more extensive and severe wing lesions, dehydration may be further exacerbated by water and electrolyte loss across the damaged epidermis of the wing, further stimulating arousal from hibernation to drink. positive feedback loops are then established that link worsening disease-associated wing pathology to further increases in arousal frequency, water loss, and energy use resulting in additional observed acute physiologic changes, including hypocapnia, hypoglycemia, hyponatremia, hypochloremia, and emaciation. once compensatory mechanisms such as cellular buffering, respiratory and metabolic regulation, and/or behavioral adaptations are exhausted, this suite of disturbances ultimately leads to mortality unless the bat has sufficient energy reserves to persist until spring emergence and clear the infection following a return to a metabolically active state. although normal reference ranges for blood chemistry values in microchiropteran species are generally lacking, deviations of measured parameters in infected bats from the negative controls in this study, together with published information for apparently healthy hibernating little brown bats, suggest pathologic and potentially life-threatening physiological disturbances associated with early-stage wns infections. bats with wns in this study had almost 40% higher mean pco than negative control bats, and pco values above 90 mmhg are generally considered to be lethal in other non-hibernating animals and humans. elevated pco levels and associated acidemia are known to interfere with enzymatic functions, reduce metabolic activity, and cause central depression of respiration; in severe cases, such elevated levels can lead to coma and death. direct calculations of energy expenditure could not be determined for most bats in this study because final isotope concentrations were indistinguishable from background levels. however, the increase in tbw as a percent of body mass observed in infected bats indicated that bats with wns had higher proportions of lean tissue mass to fat tissue mass at the end of the study. this finding implies that bats with wns used significantly more fat energy reserves compared to negative controls despite hibernating under equivalent conditions. from the estimated changes in fat content over the 67-d measurement period, infected bats utilized  kj/d, while control bats utilized  kj/d. these results indicate that bats with wns expended approximately twice as much energy during hibernation as non-infected control bats. some caution must be taken in interpreting these results due to the small number of control bats for which paired tbw measurements were available to calculate estimates of fat use. however, the range of expected daily energy use of to  kj/d predicted for healthy hibernating m. lucifugus at the mean temperature of our study is consistent with the daily rates of energy use observed in control bats in this experiment, and lower than rates observed in infected bats. there were no differences in torpor-arousal patterns between treatment groups in this study suggesting that wns causes an increase in metabolism that is not directly associated with arousal from torpor and occurs at early stages in disease progression. additionally, there were no differences between infected and control bats in tskin maintained during torpor or arousal bouts. alternatively, metabolic costs associated with infection and development of wing pathology may be linked to increased costs of thermoregulation caused by inhibition of peripheral vasoconstriction during torpor and arousal, catabolism of fat to generate metabolic water in response to increased water loss, or additional energetic costs associated with the host-pathogen interaction. rising concentrations of pco in a mammal would normally stimulate increased respiration to release excess co however, the unique physiology of mammalian hibernators allows for active suppression of respiration during torpor, and under these conditions, blood pco increases to levels higher than those observed in metabolically active mammals. this elevation of pco is thought to be an integral part of hibernation physiology as induction of an acidotic state serves to reduce metabolic rate and thermogenesis. additionally, the resultant high pco gradient improves ventilation efficiency, thereby minimizing energy costs of respiration during torpor. despite this tolerance for a respiratory acidosis, a hibernating mammal must still be able to regulate pco for proper physiologic function. if co elimination routes, such as passive diffusion of co across the wing epithelium are compromised by disease, as hypothesized for bats with wns, persistently rising blood pco levels would cause the severe chronic respiratory acidosis we have observed. thus, underlying causes for the high pco levels in bats with wns are likely a combination of the uniquely adapted physiology of a hibernating mammal compounded by pathologically induced insult to these physiological mechanisms. bat wns presents a new paradigm for the study of infectious disease. never before has a fungal skin pathogen been known to specifically infect a hibernating mammal, causing severe physiologic disturbances and mortality. although substantial efforts have been devoted to understanding chytridiomycosis, a lethal fungal skin disease of amphibians, prior to wns there had been no in-depth study of disease processes in a metabolically repressed animal caused by a psychrophilic and metabolically active pathogen. the poorly characterized capacity of bats to compensate for and respond to infection during hibernation demonstrates the difficulty of understanding how host-pathogen interactions influence disease manifestation and mortality.  this study demonstrated that infection with p. destructans and subsequent development of wns increased energy use in hibernating bats prior to the onset of altered arousal patterns associated with later stages of wns. severe, chronic respiratory acidosis and hyperkalemia were also apparent in bats that developed mild wns. with these results, we present a multi-stage disease progression model for wns as a framework for understanding the pathogenesis and underlying causes of mortality due to wns. this model integrates a range of published work with data from this study into the first attempt to mechanistically define a comprehensive conceptual model of what may occur during development of wns in a hibernating bat – from initial colonization of wing skin by p. destructans until the death of the animal. this model identifies key testable hypotheses necessary to develop a comprehensive understanding of the physiologic effects of wns on hibernating bats. ultimately, this knowledge will be critical for guiding effective and properly timed management actions to moderate physiologic effects of wns and minimize morbidity and mortality from this devastating disease. 
these differences were apparent in early stages of infection when torpor-arousal patterns were equivalent between infected and non-infected animals, suggesting that p. destructans has complex physiological impacts on its host prior to onset of clinical signs indicative of late-stage infections. integrating these novel findings on the physiological changes that occur in early-stage wns with those previously documented in late-stage infections, we propose a multi-stage disease progression model that mechanistically describes the pathologic and physiologic effects underlying mortality of wns in hibernating bats. this model identifies testable hypotheses for better understanding this disease, knowledge that will be critical for defining effective disease mitigation strategies aimed at reducing morbidity and mortality that results from wns. additionally, bats with mild to moderate skin lesions associated with early-stage wns demonstrated a chronic respiratory acidosis characterized by significantly elevated dissolved carbon dioxide, acidemia, and elevated bicarbonate. we used the doubly labeled water method and clinical chemistry to evaluate energy use, body composition changes, and blood chemistry perturbations in hibernating little brown bats experimentally infected with p. destructans to better understand the physiological processes that underlie mortality from wns. these data indicated that fat energy utilization, as demonstrated by changes in body composition, was two-fold higher for bats with wns compared to negative controls. the physiological effects of white-nose syndrome in hibernating bats and ultimate causes of mortality from infection with pseudogymnoascus destructans are not fully understood. white-nose syndromebatsdoubly labeled waterissue-copyright-statement© the author 2014 increased frequency of arousal from torpor described among hibernating bats with late-stage wns is thought to accelerate depletion of fat reserves, but the physiological mechanisms that lead to these alterations in hibernation behavior have not been elucidated.
 the physiological effects of white-nose syndrome in hibernating bats and ultimate causes of mortality from infection with pseudogymnoascus destructans are not fully understood. increased frequency of arousal from torpor described among hibernating bats with late-stage wns is thought to accelerate depletion of fat reserves, but the physiological mechanisms that lead to these alterations in hibernation behavior have not been elucidated. we used the doubly labeled water method and clinical chemistry to evaluate energy use, body composition changes, and blood chemistry perturbations in hibernating little brown bats experimentally infected with p. destructans to better understand the physiological processes that underlie mortality from wns.  these data indicated that fat energy utilization, as demonstrated by changes in body composition, was two-fold higher for bats with wns compared to negative controls. these differences were apparent in early stages of infection when torpor-arousal patterns were equivalent between infected and non-infected animals, suggesting that p. destructans has complex physiological impacts on its host prior to onset of clinical signs indicative of late-stage infections. additionally, bats with mild to moderate skin lesions associated with early-stage wns demonstrated a chronic respiratory acidosis characterized by significantly elevated dissolved carbon dioxide, acidemia, and elevated bicarbonate. potassium concentrations were also significantly higher among infected bats, but sodium, chloride, and other hydration parameters were equivalent to controls.  integrating these novel findings on the physiological changes that occur in early-stage wns with those previously documented in late-stage infections, we propose a multi-stage disease progression model that mechanistically describes the pathologic and physiologic effects underlying mortality of wns in hibernating bats. this model identifies testable hypotheses for better understanding this disease, knowledge that will be critical for defining effective disease mitigation strategies aimed at reducing morbidity and mortality that results from wns. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords white-nose syndromebatsdoubly labeled waterissue-copyright-statement© the author 2014 