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Eukaryon

Regional Extinction of Myotis lucifugus Due to Psychrophilic Geomyces destructans and Persisting White-Nose Syndrome

Lauren Leeber
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045
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Abstract

Over the past seven years bat populations in North America have been rapidly declining. In some cases this was due to the psychrophilic fungus Geomyces destructans, the etiologic agent of White-nose syndrome (WNS). The underlying mechanisms and reasoning behind why WNS has such deleterious effects were not known. It was thought that separate biological, behavioral, and environmental factors contributed to the severity of WNS independent of one another. All of the individual factors thought to cause mortality from WNS were compiled and evaluated based on current research. The origins of WNS were also unknown and evidence of Geomyces destructans had been found in low levels on European bats, however it did not cause mass mortality as in the North American bats. Therefore in addition to the mechanisms of WNS, the origins of Geomyces destructans and WNS were assessed. It was determined, due to the synergistic effects of these factors and the rapid spread of WNS, that many bat species would be regionally extinct in North America within the next decade. 

Introduction

White-nose syndrome (WNS) is an emerging infectious disease causing catastrophic declines among bat populations in North America. Geomyces destructans, a psychrophilic fungus, has been shown to be the etiologic agent of WNS. The disease’s name is derived from the hallmark white powdery growth on cutaneous tissue of the muzzle, ears, and wings. While fungal colonization and cutaneous lesions are a consistent pathological finding amongst WNS affected bats, it is not yet clear whether the fungus is the primary cause of mortality. Recently, it was discovered that G. destructans also colonizes the cutaneous tissue of European bat populations with no unusual levels of bat mortality (Wibbelt et al., 2010; Martínková et al., 2010; Puechmaille et al., 2011) which suggests there may be underlying factors which promote WNS (Turner et al., 2009; Puechmaille et al., 2011). The presence of G. destructans in both European and North American bat populations suggests that it is either native to both continents and has become increasingly pathogenic in North American populations, either by mutation or environment, or that the fungus arrived as an invader from Europe to North America (Warnecke et al., 2012). A causal link between G. destructans and WNS has yet to be found and therefore reasons for increased bat mortality and high pathogenicity in North American bat populations are not known. Given that little is known about G. destructans and the population dynamics of many bat species it is difficult to study this newly emerging infectious disease (Moore et al., 2013).

Bats are a widespread and diverse group of animals; they comprise roughly one- fourth of all mammals, amounting to nearly 1,000 species (Lorch et al., 2011). They are an integral part of their ecosystem as well as our agricultural production. Specifically bats are the key predator of night-flying insects and help suppress insect populations, particularly in wet environments. One individual may eat more than 600 insects per hour and roughly 6,000 to 8,000 per night. Bats also pollinate crops and disperse seeds (Lorch et al., 2011). Specifically the little brown bat (Myotis lucifugus) is a secondary consumer and both lower and higher trophic levels would be affected by the extinction of the Myotis (Lorch et al., 2011). 

Since its documented arrival in 2006 WNS has killed more than 6.7 million bats across the U.S. and within four providences in Canada (Turner et al., 2011). Thus far, nine bat species from three genera have been shown to carry G. destructans and mortality has been shown in six of the nine infected species (Turner et al., 2011). While the infection has not been studied extensively in many species, when looking at the decline in bat levels it is clear that WNS takes a devastating toll. Prior to the arrival of WNS one species in particular, the little brown bat (Myotis lucifugus) was the most common and widespread bat species in North America (Warnecke et al., 2012). However, population models have predicted that Myotis lucifugus will be regionally extinct by 2026 (Frick et al., 2010). Due to synergistic effects of multiple behavioral, biological, and environmental factors many bat species will be regionally extinct in North America within the next decade. 

Characteristics of Geomyces destructans
In order to more appropriately understand the underlying cause of mortality in WNS-affect bats G. destructans and other Geomyces isolates in both North America and Europe need to be elucidated. Geomyces species are fairly common and have been found in cave deposits, Antarctic soils, Arctic cryopegs, and submarine soils (Lorch et al., 2013; Marshall, 1998; Slinkina et al., 2010; Rice & Currah, 2006; Kochkina et al., 2007; Linder et al., 2011; Vanderwolf et al., 2013). Geomyces destructans is a know psychrophile, meaning that it is cold tolerant. It has a growth temperature ranging from 3 to 20°C and suspended growth at 24°C and higher (Gargas et al., 2009; Chaturvedi et al., 2010; Verant et al., 2012). With this knowledge it is not surprising that all of the affected bat species hibernate in caves or mines, as they provide a humid environment at which the fungus can maintain suspended growth (Turner et al., 2011).

It has been shown that there are no differences between growth patterns in fungal isolates from North America and Europe (Verant et al., 2012). However, it has previously been determined that isolates of G. destructans from both North America and Europe cause mortality in North American bats (Warnecke et al., 2012). This suggests that the disparity between WNS manifestation does not result from variation in the continent- specific isolate of the pathogen (Verant et al., 2012).

Symptoms of White-Nose Syndrome

The symptoms of WNS are consistent in all instances of occurrence, though it progress in stages, therefore mortality can occur any time. Geomyces destructans colonizes the skin of bats during hibernation, when bats are in a state of torpor, which leads to the powdery, white, filamentous fugal presence, shown in Figure 1 (Appendix A). Often the notable white fungus revealing WNS can be groomed by the bats and therefore will not be present. However, wing damage is a characteristic, which researchers use to identify WNS-affected bats, which can be seen in Figure 2 (Appendix A) (Blehert et al., 2009). Other characteristics associated with WNS include low fat reserves possibly leading to emaciation (Blehert et al., 2009; Courtin et al., 2010; Meteyer et al., 2009; Warnecke et al., 2012), increased frequency in arousal from torpor (Warnecke et al., 2012; Reeder et al., 2012) and atypical activity such as sightings outside the hibernacula in mid winter (Moore et al., 2013). As stated previously, G. destructans causes extensive invasion of fungal hyphae permeating the subcutaneous tissue, which causes ulcerative necrotic areas and wing membrane destruction (Meteyer et al., 2009). The epidermal basal membrane of the wing is destroyed by the hyphae, which affects the full span and thickness of the wing (Wibbelt et al., 2013). On the muzzle, fungal hyphae pack hair follicles and invade sebaceous and apocrine glands (Wibbelt et al., 2013). Both the sebaceous and apocrine glands deal with lubrication of the skin, either through sebum or sweat, respectively (Wibbelt et al., 2013).

Origins of White-Nose Syndrome

There are two current hypotheses about the origins of WNS in North America; these are the endemic pathogen hypothesis and the novel pathogen hypothesis (Rachowicz et al., 2005). The endemic pathogen hypothesis suggests that a pathogen was present but becomes increasingly pathogenic via genetic mutation or a change in environmental conditions. The novel pathogen hypothesis establishes that the pathogen has arrived in a new geographic location and has encountered a naïve host population (Rachowicz et al., 2005). It is known that G. destructans occurs in lower levels on eight Myotis species but there has yet to be any evidence of mortality (Puechmaille et al., 2011; Wibbelt et al., 2010). In fact G. destructans was not studied until WNS and its effects were seen in North America (Blehert et al., 2009). There are lower levels of WNS occurrence in Europe and a key way of determining which hypothesis is more probable by comparing European G. destructans with North American G. destructans and determining whether the European isolate thrives off a naïve host (Blehert et al., 2009). Warnecke et al. (2012) conducted a study to determine whether a G. destructans isolate from Europe (EUGd) would have the same effect as one from North America (NAGd) on North American bats (Blehert et al., 2009). They also studied whether or not inoculation with G. destructans was enough to cause mortality in North American bats. It was found that North American bats were susceptible to both NAGd and EUGd, providing support for the novel pathogen hypothesis (Blehert et al., 2009). It was also found that the lack of mortality in European bats most likely can be explained based on differences in physiologic and behavioral responses rather than a higher pathogenicity of NAGd (Wibbelt et al., 2010). Wibbelt et al., (2013) suggested that European bats may have coevolved resistance either through immunological response or have developed tolerance through behavioral adaptations with G. destructans. These researchers have recently determined that, unlike North American bats, the hyphae of G. destructans does not extend past the epidermis and adnexae and did not show deep invasion into the underlying connective tissue and does not extend past the superficial corneal stratum (Wibbelt et al., 2013). Knowing the origins of WNS can help in determining a course of action for conservation and recovery of affected bat species. 

Transmission

Importantly, Lorch et al. (2011) evaluated whether WNS was transmissible between bats by co-housing hibernating bats with WNS and unaffected bats. Of the originally unaffected bats eighty-nine percent developed WNS lesions by day 102 (Lorch et al., 2011). This reveals how rapidly the infection can spread due to the nature of how bats hibernate. The bat species that are most affected by WNS behaviorally form tight, mixed- species clusters, which facilitates transfer of G. destructans (Lorch et al., 2011). Fungal pathogens also have a unique capacity to drive host populations to extinction because of their ability to survive in host-free environments (Casadevall, 2005). 

Reasons for Mortality in White-Nose Syndrome-affected Bats
Torpor and Arousal Frequency

When bats hibernate they enter a state of torpor during which they experience decreased physiological activity as well as decreased body temperature and metabolic rate usually due to a decrease in food availability (Blehert et al., 2009). Torpor can be an advantageous way to avoid starvation during the winter months when food is scarce (Blehert et al., 2009). However, increased torpor length may increase susceptibility to mortality from G. destructans (Blehert et al., 2009). Torpor can be interrupted by periods of brief arousal during which bats become normothermic (Geiser et al., 2004). These arousals are generally no longer than 24 hours but account for most of the bat’s over-winter energy expenditure due to the high metabolic cost of thermoregulation during times of normothermia (Geiser et al., 2004; Thomas, Dorais, & Bergeron, 1990). Because food is scarce hibernating bats rely heavily on stored fat (Thomas, Dorais, & Bergeron, 1990). It is thought that one reason for mortality in WNS-affected bats is premature fat depletion due to increased frequency of arousal caused by the infection, which can lead to eventual starvation (WNS Strategy Group, 2008).

Warnecke et al. (2012) also assessed whether G. destructans-infected bats exhibited increased frequency or duration of arousals during hibernation. Arousal frequency was shown to be significantly greater in bats affected with G. destructans. Bats affected with NAGd had three times greater arousal frequency than non-affected bats and EUGd bats had four times greater arousal frequency than that of the control bats (Warnecke et al., 2012). Therefore providing evidence for the hypothesis that G. destructans causes increased frequency arousal in bats and that it is most likely one of the main mechanisms underlying mortality (Warnecke et al., 2012). This is also consistent with the energetic model predicted by Boyles and Willis (2009) during which premature depletion of fat stores would lead to starvation and dehydration. Warnecke et al., (2012) also determined that each additional arousal shortens the time that a bat is able to hibernate by 9 days. Because of premature fat depletion one of the characteristics of bats affected with WNS is observed flying outside the hibernacula during the daytime in winter, it is thought that this is to search for food as it is also characteristic to see emaciated WNS-affected carcasses (Warnecke et al., 2012). Another idea is that they leave the hibernacula to try and elevate body temperature in order to produce an immunological response and fight off the infection (Prendergast et al., 2002). One study, conducted under drier conditions than Warnecke et al., (2012) found reductions in mortality as well as severity of WNS suggesting that G. destructans does not proliferate as easily in drier conditions (Lorch et al., 2011). 

Dehydration and Electrolyte Depletion

Yet another possible explanation for mortality is that evaporative water loss during hibernation leads to dehydration (Thomas & Geiser, 1997). As previously mentioned G. destructans causes serious wing damage; bat wings are specialized because they are comprised of two thin membranes with two layers of skin surrounding a layer of connective tissue, blood vessels, and nerves. Due to their increased surface area, wings play a key role in maintaining homeostasis (Cryan et al., 2010). Damage to the wings can therefore elevate cutaneous water loss (Willis et al., 2011) therefore flying bats could be searching for water (Cryan et al, 2010). Cryan et al. (2013) conducted a study in which they looked at electrolyte levels in WNS-affected bats. They found that bats with more severe wing damage due to G. destructans became clinically hyponatremic (low Na+) and hypochloremic (low Cl-) (Cryan et al., 2013). Normally when dehydrated you would see increased leaves of both Na+ and Cl- however it is thought that bats may be experiencing hypotonic dehydration, during which the amount of stored water is sufficient but Na+ and Cl- are low as seen in Table 1 (Appendix B). Hypotonic dehydration can occur due to depletion of electrolytes and is separate from total body water (Cryan et al., 2013). Electrolytes can be depleted due to a number of reasons, lack of available food, electrolyte-rich water, or they could be leaking out of the damaged wing lesions (Cryan et al., 2013). This suggests that bat wings are crucial, especially when the bat is in torpor. 

Metabolic Rate and Temperature

While we have previously determined that differences do not exist between continental isolates of G. destructans, the severity of the infection is mediated by environmental conditions such as temperature and humidity. During hibernation the body temperature of Myotis lucifugus drops to roughly 7.2 °C from temperatures of up to 54 °C when not hibernating (Verant et al., 2012). This basal temperature falls within the optimal growth range of G. destructans and therefore could serve as a potential reason for colonization. Temperature also changes morphological characteristics of G. destructans grown in culture at temperatures of 12 °C and higher (Verant et al., 2012). At this higher temperature the fungus was characterized by increased septation and thickened hyphae as well as production of arthrospores and chlamydospore-like structures. This is important because arthrospores act as the primary means of propagation when under adverse conditions or if under stress (Rashid et al., 2001; Yazdanparast et al., 2006). While chlamydospores act as desiccation-resistant structures (Lin, X. & Heitman, J., 2005), all of these combined factors are possibly giving rise to the rapid spread of G. destructans in bat populations (Verant et al., 2012).

Basal temperature during torpor decreases and as a result lowers the bat’s metabolic rate, allowing the bat to go an extended period of time without food (Verant et al., 2012). As shown in Figure 3 (Appendix A) for a relatively small hibernator (7 g) as body temperature decreases, metabolic rate (ml O2 g-1 h-1) decreases (Geiser, 2004). However, this allows G. destructans to spread due to its psychrophilic nature (Wibbelt et al., 2013). Figure 4 (Appendix A) is derived from data collected by Warnecke et al. (2012) comparing NAGd and EUGd inoculates to a sham-inoculate control. The difference between the two G. destructans inoculated groups and the control group is very clear. The increased temperature to euthermia are arousals from torpor and then as it decreases the bats re-enter torpor. Both G. destructans inoculated groups had significantly more (p < .05) arousals from topor. Increased arousal leads to depleted fat stores resulting in flights out of the hibernacula in mid-winter and hypotonic dehydration. Therefore implying that none of these factors are acting independently and instead coalesce to cause greater damage. 

Immunological Response

Little is known about bat immunology and even less is known about immunological responses during hibernation and torpor. Moore et al. (2013) looked at immunoglobulin, leukocyte, cytokine, antioxidant, and interleukin-4 levels in both WNS-affected and WNS-unaffected bats. Because the immunological response to fungal infection in bats has not been studied previously the researchers compiled a list of what should happen when there is an invasion of the skin (Moore et al., 2013). These mechanisms included: phagocytosis by resident and recruited innate immune cells, such as marcrophages and neutrophils, respiratory burst, edema, vascular reaction and an increase in acute phase proteins (Kupper & Fuhlbrigge, 2004). It should also be characterized by activation of complement proteins in the stratum corneum (Kupper & Fuhlbrigge, 2004) as well as activation of both dendritic cells and mast cells (Takeda, Kaisho, & Akira, 2013). In order to build resistance to G. destructans effector functions mediated by T-lymphocytes and the development of immunological memory specific to G. destructans must occur (Romani, 2004). Also, antibody- dependent cellular cytotoxicity may play a role in establishing immunological memory (Shoham, & Levitz, 2005). Moore et al. (2013) found that total circulating leukocyte counts (WBC) were significantly higher in WNS-affected bats, as shown in Figure 5 (Appendix A). Furthermore, WBC was elevated in times of rewarming and euthermia (elevated body temperature) (Moore et al., 2013). This is compared to unaffected bats that showed no significant change in WBC due to change in body temperature (Moore et al., 2013). Bats cannot fight off G. destructans in a state of torpor but elevated WBC during euthermia suggests that infected bats are attempting to fight the infection. White- nose syndrome-affected bats also showed decreased levels of antioxidants and interleukin-4 (IL-4), which is a cytokine that induces T-cell differentiation (Moore et al., 2013). This provides support for the idea that changes in thermoregulatory behavior such as increased frequency arousal are linked to an attempt to fight infection (Moore et al., 2013). Whether or not this is an effective strategy for combating the effects of G. destructans is unknown.

During hibernation, bats do not have inflammatory cell recruitment at the site of G. destructans infection; this is consistent with temperature-induced inhibition of immune cell trafficking (Meteyer et al., 2012). However, as previously discussed, during euthermia WNS-affected bats experience an intense neutrophilic inflammatory response causing severe pathology (Meteyer et al., 2012). In this way, it is believed that the sudden return to euthermia leads to immune reconstitution inflammatory syndrome (IRIS) (Meteyer et al., 2012). Meteyer et al. (2012) draw a comparison between WNS-IRIS and HIV-IRIS in humans. They suggest that the cause of mortality mat actually be the bat attempting to rapidly fight the infection through an overactive immune response, causing severe and irreparable tissue damage (Meteyer et al., 2012). This would then lead to severe wing damage and ultimately electrolyte depletion, as discussed previously (Cryan et al., 2013). 

Conclusion

WNS is a devastating and rapidly spreading disease among North American bat populations. Underlying mechanisms of the disease have yet to be fully explained. However, it is unlikely that any one cause of mortality is acting independently. It seems more likely that the synergistic effects of elongated torpor, lower metabolic rate, decreased immunological response, and the subsequent effects of G. destructans contribute to mortality. Because of innate behaviors of certain species, such as clustered hibernation and grooming, G. destructans can spread rapidly through a hibernacula. Turner et al. (2011) created a diagram of how pathogens are able to thrive when there is both a susceptible host and a favorable environment, shown in Figure 6 (Appendix A). This allows us to better understand the multiple interactions at play in WNS-affected bats. The bats are a susceptible host because they have decreased temperature, metabolic rate, and a suspended immune response during torpor. The fungal pathogen G. destructans is able to colonize the bats because of its affinity for cold, damp environments, such as the hibernacula of bats, which provide a favorable environment (Turner et al., 2011). It is therefore not surprising that the Myotis family, especially Myotis lucifugus, is in danger of going regionally extinct within the next decade (Moore et al., 2013). Frick et al. (2010) constructed a model that first shows the probability of extinction if populations continue declining with 45% mortality. Secondly it shows the sharp decrease in overall regional population size using the same percent mortality rates, as seen in Figure 7 (Appendix A). These graphs also reveal that by decreasing the mortality from WNS alone would result in a better-sustained population. 

Future Studies

This is a relatively new topic of interest and research; therefore there are a lot of gaps in knowledge and unknowns. This is especially true when looking at the immune system of a hibernating animal attempting to fight a disease. Studying the possible effects and prevalence of IRIS in WNS-affected bats could not only yield fruitful results for decreased mortality, but also for better understanding of HIV-IRIS.

More research is needed to determine origins and European bats need to be tested with North American G. destructans in order to insure the novel pathogen hypothesis is correct. This may also help determine how far WNS could potentially spread. It is unlikely that it would spread to more tropical or warmer climates, where many megabat species live, because of G. destructan’s affinity for cold temperatures. G. destructans would not be able to reproduce and spread very quickly within that habitat range.

Most importantly it is difficult to determine the best method of conservation due to the widespread range of bats and the transferable nature of WNS. The reasons behind the mechanisms of WNS are mostly behavioral and biological, two things not easily changed by humans. Scientists are looking into the possibility of drying out affected caves in order to make G. destructans less potent. However, data collection and analysis are slow moving and by the time a solution arises bat populations in North America will have suffered extreme losses, which could create other problems when trying to re-establish the populations. 

Figure 1. Development of Geomyces destructans colonization over a 3-week period. (A + B) Shows two Myotis with white powdery, filamentous growth on the muzzle; (C) reveals the same two bats 3-weeks after (A + B) were taken and show the colonization and spread of G. destructans to the wings and torsos of the bats. (Wibblet et al., 2013)Figure 1. Development of Geomyces destructans colonization over a 3-week period. (A + B) Shows two Myotis with white powdery, filamentous growth on the muzzle; (C) reveals the same two bats 3-weeks after (A + B) were taken and show the colonization and spread of G. destructans to the wings and torsos of the bats. (Wibblet et al., 2013)

Figure 2. Wing damage caused by WNS over 3-week period. (A) No lesions can be seen, however white growth is present; (B) Bat (A) after 16 days minimal wing damage can be seen; (C) Bat (A) after 29 days shows severe wing tissue damage and lesions; (D) Bat (A) 38 days after rehabilitation by researchers presents evidence of wing healing (Meteyer et al., 2012).Figure 2. Wing damage caused by WNS over 3-week period. (A) No lesions can be seen, however white growth is present; (B) Bat (A) after 16 days minimal wing damage can be seen; (C) Bat (A) after 29 days shows severe wing tissue damage and lesions; (D) Bat (A) 38 days after rehabilitation by researchers presents evidence of wing healing (Meteyer et al., 2012).Figure 3. Metabolic rates of torpid thermoconforming small hibernators. Shows a relationship between lower body temperature and decreased metabolic rate during torpor of the bat N. geoffroyi (Geiser, 2004).Figure 3. Metabolic rates of torpid thermoconforming small hibernators. Shows a relationship between lower body temperature and decreased metabolic rate during torpor of the bat N. geoffroyi (Geiser, 2004).Figure 4. Body temperature and arousal frequency of Myotis lucifugus in the NAGd, EUGd and control conditions. Shows skin temperature for six M. lucifugus two from each group: (A) individuals inoculated with NAGd; (B) individuals inoculated with EUGd; (C) sham-inoculated controls (Warnecke et al., 2012).Figure 4. Body temperature and arousal frequency of Myotis lucifugus in the NAGd, EUGd and control conditions. Shows skin temperature for six M. lucifugus two from each group: (A) individuals inoculated with NAGd; (B) individuals inoculated with EUGd; (C) sham-inoculated controls (Warnecke et al., 2012).

Figure 5. Total circulating leukocytes in both WNS-affected and unaffected M. lucifugus. WBC was found to be significantly related to temperature in the affected bats (Kruskal Wallis χ2 = 7.61, p = 0.022) (Moore et al., 2013).Figure 5. Total circulating leukocytes in both WNS-affected and unaffected M. lucifugus. WBC was found to be significantly related to temperature in the affected bats (Kruskal Wallis χ2 = 7.61, p = 0.022) (Moore et al., 2013).Figure 6. The disease triangle, showing the interrelationships between hosts, pathogens, and environment. A disease (WNS) occurs when a specific pathogen (G. destructans) infects susceptible hosts (hibernating bats) under certain environmental conditions (cold, damp hibernacula, in which bats use torpor and effectively supress their immune systems, allowing relatively unchecked fungal growth) (Turner et al., 2011).Figure 6. The disease triangle, showing the interrelationships between hosts, pathogens, and environment. A disease (WNS) occurs when a specific pathogen (G. destructans) infects susceptible hosts (hibernating bats) under certain environmental conditions (cold, damp hibernacula, in which bats use torpor and effectively supress their immune systems, allowing relatively unchecked fungal growth) (Turner et al., 2011).Figure 7. Projected model of Myotis lucifugus regional extinction and population decline. (A) shows the probability of extinction if populations continue declining with 45% mortality; (B) shows the sharp decrease in overall regional population size using the same percent mortality rates (Frick et al., 2010).Figure 7. Projected model of Myotis lucifugus regional extinction and population decline. (A) shows the probability of extinction if populations continue declining with 45% mortality; (B) shows the sharp decrease in overall regional population size using the same percent mortality rates (Frick et al., 2010).Table 1. Average concentrations of sodium and chloride ions in the whole blood of hibernating Myotis. As severity of skin damage increases the levels of Na+ and Cl- decrease, possibly due to hypotonic dehydration (Cryan et al., 2013).Table 1. Average concentrations of sodium and chloride ions in the whole blood of hibernating Myotis. As severity of skin damage increases the levels of Na+ and Cl- decrease, possibly due to hypotonic dehydration (Cryan et al., 2013).

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