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White-nose Syndrome (WNS) is an emergent mycosis in North America that is caused by a severe cutaneous infection with the fungus Pseudogymnoascus destructans (Pd) during hibernation. Pseudogymnoascus destructans (Pd) was first observed in North America at a single site during the winter of 2006–2007 and has since spread to 39 U.S. States and 7 Canadian provinces. This fungus was introduced to North America from Europe, where it is endemic. WNS has thus far been observed to occur only in hibernating bats and has caused the populations of 4 North American bat species to decline by more than 84% within 7 years. Field studies have revealed that 4 other North American bat species are not afflicted with WNS when hibernating in areas where Pd occurs. The physiological and biochemical adaptations that permit some bat species to resist Pd infections are starting to be elucidated but are still poorly understood. A total of 47 different bat species are found in North America, about half of which hibernate during the winter. The potential future effects of WNS on 13 of these hibernating bat species remains to be determined.

Background White-nose Syndrome (WNS) is a mycosis caused by a cutaneous infection with the fungus Pseudogymnoascus destructans (Pd). It produces hibernation mortality rates of 75–98% in 4 bats: Myotis lucifugus, M. septentrionalis, M. sodalis, and Perimyotis subflavus. These high mortality rates were observed during the first several years after the arrival of P. destructans at a hibernation site. Mortality is caused by a 60% decrease in torpor bout duration, which results in a premature depletion of depot fat prior to spring. Results Little is known about the long-term effects of Pd on torpor and mortality, thus we conducted a 9-year study on M. lucifugus at 5 of the hibernation sites where Pd first appeared in North America during the winter of 2007–08. The M. lucifugus hibernating at one of these sites one year after the arrival of Pd (2008–09) had: a) a mean torpor bout duration of 7.6 d, b) no depot fat reserves by March, and c) an apparent over-winter mortality rate of 88%. The M. lucifugus hibernating at this same site 6–9 years after the arrival of Pd, in contrast, had: a) a mean torpor bout duration of 14.7 d, b) depot fat remaining in March, and c) an apparent mortality rate of 50%. The number of M. lucifugus hibernating at 2 of these sites has consistently increased since 2010 and is now more than 3.0-fold higher than the number remaining after the winter of 2008–09. Conclusions These findings indicate that this population of M. lucifugus has evolved mechanisms to hibernate well in the presence of Pd, thus reducing over-winter mortality.

White nose syndrome (WNS) is caused by the psychrophilic fungus Pseudogymnoascus destructans that can grow in the environment saprotrophically or parasitically by infecting hibernating bats. Infections are pathological in many species of North American bats, disrupting hibernation and causing mortality. To determine what fungal pathways are involved in infection of living tissue, we examined fungal gene expression using RNA-Seq. We compared P. destructans gene expression when grown in culture to that during infection of a North American bat species, Myotis lucifugus, that shows high WNS mortality. Cultured P. destructans was grown at 10 to 14 C and P. destructans growing in vivo was presumably exposed to temperatures ranging from 4 to 8 C during torpor and up to 37 C during periodic arousals. We found that when P. destructans is causing WNS, the most significant differentially expressed genes were involved in heat shock responses, cell wall remodeling, and micronutrient acquisition. These results indicate that this fungal pathogen responds to host-pathogen interactions by regulating gene expression in ways that may contribute to evasion of host responses. Alterations in fungal cell wall structures could allow P. destructans to avoid detection by host pattern recognition receptors and antibody responses. This study has also identified several fungal pathways upregulated during WNS infection that may be candidates for mitigating infection pathology. By identifying host-specific pathogen responses, these observations have important implications for host-pathogen evolutionary relationships in WNS and other fungal diseases.

Numerous free fatty acids (FFAs) are known to have potent antifungal effects. The mammalian epidermis contains both FFAs and multiple classes of fatty acid esters, including 1-monoacylglycerols and wax esters. We thus hypothesized that wax esters and 1-monoacylglycerols composed of antifungal fatty acids would also have antifungal properties. We tested this hypothesis by examining the effects of 1-monoacylglycerols, 1,3-diacylglycerols, and wax esters on the growth of Pseudogymnoascus destructans (Pd), the fungus that causes White-nose Syndrome (WNS) in North American bats by invading their epidermis. Laboratory experiments with Pd cultures demonstrated that: (a) three 1-monoacylglycerols (1-monopalmitolein, 1-monoolein, and 1-monolinolein), as well as, (b) two wax esters, behenyl oleate and behenyl palmitoleate, profoundly inhibit Pd growth. The normal growth cycle of Pd was interrupted by addition of two cholesterol esters to the media as well. A bat species resistant to cutaneous Pd infections has these 1-monoacylglycerols in the epidermis, and another Pd resistant bat species has these wax esters in the sebum, thus cutaneous lipid composition is one factor which enables some bats to avoid WNS. Our experiments also revealed that the fatty acid esters which inhibit Pd growth are not hydrolyzed by the lipases secreted by this fungus, whereas the esters that do not inhibit Pd growth are hydrolyzed.

White Nose Syndrome (WNS) greatly increases the over-winter mortality of little brown (Myotis lucifugus), Indiana (Myotis sodalis), northern (Myotis septentrionalis), and tricolored (Perimyotis subflavus) bats. It is caused by a cutaneous infection with the fungus Pseudogymnoascus destructans (Pd). Big brown bats (Eptesicus fuscus) are much more resistant to cutaneous infection with Pd, however. We thus conducted analyses of wing epidermis from hibernating E. fuscus and M. lucifugus to determine their fatty acid compositions, and laboratory Pd culture experiments at 4.0–13.4°C to determine the effects of these fatty acids on Pd growth. Our analyses revealed that the epidermis of both bat species contain the same 7 fatty acid types (14:0, 15:0, 16:0. 16:1, 18:0, 18:1, & 18:2), but the epidermis of M. lucifugus contains: a) more stearic (18:0) acid, b) less palmitoleic (16:1) acid, c) less myristic (14:0) acid, and, d) less oleic (18:1) acid than that of E. fuscus. The growth of Pd was inhibited by: a) myristic and stearic acids at 10.5–13.4°C, but not at 4.0–5.0°C, b) oleic acid at 5.0–10.6°C, c) palmitoleic acid, and, d) linoleic (18:2) acid at 5.0–10.6°C. One set of factors that enables E. fuscus to better resist cutaneous P. destructans infections (and thus WNS) therefore appears to be the relatively higher myristic, palmitoleic, and oleic acid contents of the epidermis.

Golden-mantled ground squirrels (Spermophilus lateralis) in the laboratory are most likely to enter torpor when the linoleic acid content of their diet is 33–62 mg/g diet. Diets of free-ranging ground squirrels vary in linoleic acid content throughout summer; however, they often do not have constant linoleic acid content for more than a month. The influence of natural short-term ( 62 mg/g) levels during early-summer feeding. But increasing diet l...

SYNOPSIS. A diet that has high levels of polyunsaturated fatty acids enhances mammalian torpor. Polyunsaturated fatty acids are not synthesized by mammals, but are incorporated into both membrane and storage lipids when they occur in the diet. Polyunsaturated fatty acids also undergo autoxidation more readily than other fatty acids, thereby producing highly toxic lipid peroxides. Lipid peroxidation increases during torpor. Natural selection in mammalian hibernators should thus have favored the evolution of dietary preferences that maximize hibernation ability while simultaneously minimizing the degree of lipid peroxidation during torpor. This hypothesis was tested in laboratory experiments and field studies involving golden-mantled ground squirrels ( Spermophilus lateralis ). We found that the intake of polyunsaturated fatty acids isrestricted during the fall and autoxidation in tissues occurs mostly during the later phases of hibernation.

Six bat species in northeastern North America spend winter hibernating in caves/mines. Hibernation is characterized by multi-day periods of torpor, when metabolic rates are reduced. Body fat levels increase from 7 to 27% in the fall, just prior to the onset of hibernation by bats. Some groups of hibernating bats in northeastern North America display abnormal behaviors and increased overwinter mortality called white nose syndrome (WNS). Large numbers of dead bats are found in hibernation sites affected by WNS. Previous research has demonstrated that the increased over-mortality is due to the depletion of depot fat reserves by February, which is in turn caused by greatly shortened torpor bouts. Published studies on hibernating mammals have revealed that moderately high levels of polyunsaturated fatty acids (PUFAs) are required in the diet during the fall for torpor. We thus predicted that bats suffering from WNS have lower levels of PUFAs in their fall diets than those that do not suffer from WNS. We tested this hypothesis by analyzing white adipose tissue (WAT) samples from: (a) little brown bats (Myotis lucifugus) collected from different hibernation sites and (b) big brown bats (Eptesicus fuscus), a species not greatly affected by WNS. Our analyses reveal that M. lucifugus populations prone to WNS have significantly less of the PUFA α-linolenic acid in their fall diets than those where WNS does not occur. The fall diets of E. fuscus contain significantly more of the PUFA linoleic acid than those of M. lucifugus from the same site. Our findings thus support the hypothesis that bats suffering from WNS have lower levels of PUFAs in their fall diets.

Golden-mantled ground squirrels (Spermophilus lateralis) are small herbi- vores that hibernate during winter. Previous experiments have demonstrated that a high polyunsaturate diet enhances hibernation by ground squirrels. Polyunsaturated fatty acids cannot be synthesized by mammals, but they are commonly produced by plants. It was therefore predicted that ground squirrels, during the 2 mo prior to hibernation, select diet items of a relatively high polyunsaturate content. This hypothesis was tested in a series of laboratory diet selection experiments involving S. lateralis and semisynthetic diets of different polyunsaturate contents. The stomach contents of free-ranging S. lateralis were also analyzed for polyunsaturate content. As predicted, the squirrels preferred semisynthetic diets of relatively higher polyunsaturate contents. Stomach content analyses suggest that the dietary lipids of free-ranging ground squirrels have high polyunsaturate contents. These results indicate that polyunsaturate content may influence the foraging behavior of ground squirrels.

Golden-mantled ground squirrels (Spermophilus lateralis) are herbivores that hibernate during winter. A physiological limitation on hibernation may be the melting point of stored fat, since lipids must be fluid to be metabolizable and the body temperatures maintained during torpor are often 25° C below the melting point of mammalian fats. Previous analyses of depot fats from hibernating squirrel species revealed that they contained large amounts of polyunsaturated fatty acids, which greatly reduced the melting points of these lipids. Because polyunsaturated fatty acids cannot be synthesized by mammals but are commonly produced by plants, it was predicted that high dietary levels of polyunsaturates were required for proper hibernation. This hypothesis was tested in laboratory feeding and hibernation experiments involving captive S. lateralis maintained on diets of different polyunsaturate contents. Squirrels fed a high polyunsaturate diet (1) were more likely to hibernate, (2) had lower mortality during hi...

Ground squirrels are small herbivores that hibernate during winter. The ecological–nutritional limitations on hibernation are virtually unknown, but one constraint may be the melting point of stored fat. Lipids must be fluid to be metabolizable, and body temperatures maintained during hibernation are usually 30 °C below the melting point of typical mammalian fats. Fats containing greater amounts of unsaturated fatty acids, however, have correspondingly lower melting points. White adipose tissue was sampled from free-ranging Belding's ground squirrels, Spermophilus beldingi, during both the summer and fall. The lipids were twice as unsaturated as those of other rodent species, most of the increased unsaturation being due to the accumulation of plant-produced polyunsaturated fatty acids derived from the animals' diet. The melting points of S. beldingi fats were consequently 25 °C lower than those of other mammals. These results suggest that ground squirrels may depend upon their plant diet for the polyunsaturates necessary to produce the lipids with low melting points that are needed for hibernation.

Laboratory experiments have demonstrated that the amount of polyunsaturated fatty acids (PUFAs) in the diet before hibernation influences patterns of mammalian torpor. The hibernation ability of ground squirrels is greatest (longest torpor bouts, greatest number of animals entering torpor) when the PUFA content of their fall diets is 33-74 mg/g, under laboratory conditions. The extent to which natural fall diets both (a) vary in PUFA content and (b) influence the torpor patterns of free-ranging populations of hibernating mammals is unknown, however. We conducted a 3-yr study on the diet PUFA contents and subsequent hibernation patterns of free-ranging arctic ground squirrels (Spermophilus parryii) in the Brooks Range of Alaska. We found that the PUFA contents of fall diets varied more than threefold among individuals. Our study also revealed that arctic ground squirrels that consumed a moderate-PUFA (33-74 mg/g) diet had (a) longer torpor bouts, (b) fewer arousals from torpor, (c) shorter arousal periods, (d) more days in torpor, and (e) greater probability of persisting in the population than those that consumed a high-PUFA (74 mg/g) diet during the fall. No animals were demonstrated to have consumed a diet representing low-PUFA (33 mg/g) values. Our study is therefore the first to demonstrate that estimated dietary PUFA levels of a free-ranging hibernator influence subsequent torpor patterns.

Tocopherols (Vitamin E) are antioxidants that intercept the reactive oxygen species that initiate lipid peroxidation. Plants produce four tocopherol isomers, with α-tocopherol being the most biologically active. All mammals require tocopherols in their diets. Field and laboratory studies were conducted to determine the influence of diet α-tocopherol content on torpor by ground squirrels (Spermophilus lateralis). The diets of free-ranging ground squirrels contained both α and γ-tocopherols, with α-tocopherol being the most abundant. Natural diet atocopherol levels ranged from 1.5 to 34.1 µg/g. Laboratory experiments, however, revealed that squirrels fed diets with α-tocopherol contents above or below 12 µg/g were: 1) were less likely to enter torpor, and, 2) were less likely to survive torpor than those fed a diet containing 12 µg atocopherol/g. A low α-tocopherol diet is thus actually required for proper torpor.

Changes in blood flow during torpor-arousal cycles in mammalian hibernators could increase the risk of oxidative stress to sensitive tissues. We used thirteen-lined ground squirrels (Spermophilus tridecemlineatus) to determine the effect of hibernation on glutathione (GSH) redox balance and immune cell populations in the intestine, a tissue highly susceptible to oxidative stress secondary to ischemia-reperfusion injury. Ratios of reduced:oxidized GSH were lower in intestine of hibernators compared with summer-active squirrels, an effect most pronounced for torpid animals. Numbers of intraepithelial lymphocytes were increased in torpid hibernators compared with summer squirrels or hibernators undergoing arousal or interbout euthermia. These results suggest that intestinal GSH redox balance is altered during the hibernation season to a more oxidized state, and this is accompanied by changes in intestinal mucosal immune cells that may play a protective role for stressed enterocytes.

White-tailed prairie dogs (Cynomys leucurus) are spontaneous hibernators that enter torpor each fall, whereas black-tailed prairie dogs (C. ludovicianus) hibernate facultatively only when food- or water-stressed during the winter. The body masses of both species greatly increase during the fall feeding period, with most of this gain in the form of depot fat. Body fat is utilized during winter fasting and/or hibernation. We measured the activities of fatty acid synthase (FAS), ATP-citrate lyase (ACL), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), and hormone-sensitive lipase (HSL) in the tissues of both C.leucurus (hibernating and euthermic) and C. ludovicianus (euthermic only) under controlled conditions. The activities of FAS, ACL, and G6PDH in the liver all decreased during hibernation. The activities of ME and G6PDH in white adipose tissue (WAT) were also reduced during hibernation. Euthermic C. leucurus and euthermic C. ludovicianus differed only in brown adipose (BAT) ACL and WAT G6PDH activities. No significant differences in HSL activities were found between these two species or between euthermic and hibernating animals. These results suggest that this seasonal body fat cycle is due, at least in part, to seasonal variations in the activities of FAS, ME, ACL, and G6PDH that affect the rate of fatty acid synthesis. This study also demonstrates that spontaneous hibernators do not have a greater capacity to synthesize fatty acids during the fall than facultative hibernators, as previously suggested.

Golden-mantled ground squirrels (Spermophilus lateralis) are herbivores that hibernate during winter. Although little is known about the nutritional/physiological constraints on hibernation, numerous studies have demonstrated that increasing the amount of linoleic acid (a polyunsaturated fatty acid) in the diet enhances hibernation. This is probably because high linoleic acid diets reduce the melting points of the depot fats produced for hibernation which makes them more metabolizable at low body temperatures. This suggests that a major limitation on hibernation may be obtaining enough linoleic acid in the diet for proper hibernation. In all previous studies, however, the amount of linoleic acid in the diets of free-ranging animals was either not considered, or the range of dietary linoleic acid contents in the experiments was less than that of natural diets. It is thus not known whether the amount of linoleic acid available to hibernators under natural conditions actually limits their torpor patterns. A series of laboratory feeding and hibernation experiments were conducted with S. lateralis and artificial diets with different linoleic acid contents that were either below or above the linoleic acid content of the natural diet. The results demonstrated that when dietary linoleic acid contents are either below or above natural levels, hibernation ability is greatly reduced. Hibernation ability was reduced when the squirrels were maintained on a high linoleic acid diet probably by the production of toxic lipid peroxides in brown adipose tissues. The results indicate that there is an optimal level of dietary linoleic acid for proper hibernation, and this is equal to that of the natural diet. The amount of linoleic acid available in the diet thus does not limit hibernation under normal natural conditions.

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

Kangaroo rats (family Heteromyidae) are primarily granivores. These desert rodents do not drink free water, but depend on performed water in their diet and water produced by oxidation of food (metabolic water). Oxidation of different nutrients produces different net amounts of metabolic water. At low humidities, oxidation of carbohydrates produces a net metabolic water gain, lipid metabolism results in a net water loss, and protein metabolism produces a severe water loss, mainly through urination. Under humid conditions, carbohydrate metabolism again produces a net water gain, lipid oxidation produces a large net metabolic water gain, and although this is slightly reduced, protein metabolism still results in a large water loss. Diet composition therefore influences heteromyid water balance. Preference experiments involving semisynthetic diets that were energetically equivalent tested the influence of net metabolic water production on diet preferences of Dipodomys spectabilis. At low humidities, kangaroo rats in a positive water balance preferred: (1) intermediate—protein, (2) high—lipid, and (3) high—carbohydrate diets. When water stressed, the rodents preferred low—protein diets, and the preference for high—lipid diets decreased. At high humidities, the rodents always preferred high—lipid diets. These results indicate that when energy contents were equivalent, kangaroo rats usually preferred diets that produce the greatest net metabolic water gain.

Kangaroo rats are mainly granivores, and some species store large amounts of seeds in underground caches. These desert rodents do not drink free water under natural conditions but instead rely on preformed water in their diet and metabolic water produced by food oxidation. Moisture content may therefore influence kangaroo rat seed selection. Feeding experiments were conducted to determine both the effects of water content in seed preferences by Dipodomys spectabilis and the minimal preformed water requirements of these rodents. Results demonstrate that the minimal seed water content for a positive water balance is 10%-11% wet mass, under laboratory conditions. These rodents can sense a 6.05% difference in water content and always prefer the moistest seeds available, even when selecting among seeds containing over twice the minimal water necessary for a positive balance. These results indicate that kangaroo rats do not ingest only enough preformed water to meet their requirements but instead maximize water...

Desert rodents of the family Heteromyidae are mainly seed eaters and some species of kangaroo rats (Dipodomys) store large quantities of seeds in underground caches (Shaw, 1934; Vorhies and Taylor, 1922). Approximately 23 species of molds have been discovered growing in caches of D. spectabilis (Reichman et al., 1985), about half of which produce a variety of mycotoxins (Wyllie and Morehouse, 1978). Subterranean fungi are included in diets of a variety of mammals including mice, voles, rats, lemmings (Muridae); chipmunks and squirrels (Sciuridae); and rabbits (Leporidae; Fogel and Trappe, 1978); some aquatic insect larvae eat slightly moldy leaves (Arsuffi and Suberkropp, 1984). In all of these, however, the fungal species involved are unlike those found in the caches of D. spectabilis in that they do not produce mycotoxins and most have a symbiotic relationship with the animals that ingest them; the molds produce specific fruiting bodies that the animals ingest, and through feeding the animals disperse fungal spores. Previous studies of diet choice conducted with pocket mice (Perognathus intermedius) and kangaroo rats (D. spectabilis) suggest that the order selected with respect to moldiness is: slightly moldy > nonmoldy > heavily moldy (Rebar and Reichman, 1983; Reichman and Rebar, 1985). Slightly moldy seeds, as defined by the authors, are those at the stage of fungal colonization where only hyphae are present, whereas heavily moldy seeds are extensively covered with both conidia and hyphae (Reichman, pers. comm.). When toxic fungi reach the stage of conidial production, they also begin to produce mycotoxins (Wyllie and Morehouse, 1978), and heavily moldy seeds contain mycotoxins (Reichman and Rebar, 1985). Reichman and Rebar (1985) suggested that heteromyids might select slightly moldy seeds because these seeds may possess beneficial features associated with fungal colonization (e.g., increased nutrition or digestibility) not found in nonmoldy seeds, but they do not contain the mycotoxins produced by heavily moldy seeds. The authors indicated, however, that seed choices observed in most of their experiments may have been influenced in part by differences in moisture content of seeds. In their experiments, seeds of different moldiness levels were produced by inoculating barley seeds with fungal spores and incubating the seeds at 100% relative humidity for various lengths of time. Seeds kept in high humidities gradually imbibe water over a period of days (Christian and Lederle, 1984; Morton and MacMillen, 1982). Experiments in which husked barley seeds were incubated at 100% relative humidity indicate that the moldy seeds produced by Reichman and Rebar (1985) may have contained as much as three times as much water as nonmoldy seeds used (Frank, 1988). Seeds presented to the rodents, consequently, differed greatly in water content as well as moldiness, except in a final experiment performed by the authors in which moisture levels in seeds were controlled. Heteromyid rodents do not drink water, but rely upon preformed water in their diet and metabolic water produced by the oxidation of food (Schmidt-Nielsen, 1964, 1972). Feeding trials with D. merriami (Frank, 1988) demonstrated that kangaroo rats can detect differences in moisture content in seeds as small as 10% and that they select seeds that contain the most water. Therefore, the slightly moldy seeds may have been selected only because they were more moist than nonmoldy seeds, but not yet moldy enough to be toxic. Kangaroo-rat burrows and caches in the field usually are more humid than the external environment (Kay and Whitford, 1978; Schmidt-Nielsen and Schmidt-Nielsen, 1950). Humid conditions within rodent burrows are ideal for fungal colonization of seeds because fungi require high humidities to grow (Martin and Gilman, 1976), thus seeds in kangaroo rat caches often may be both moldy and moist. Laboratory experiments suggest that D. spectabilis caches nonmoldy seeds preferentially in areas of high relative humidity, but when they become slightly moldy, they are moved to low humidities that inhibit further mold growth (Reichman et al., 1986). The rodents may be moving their caches to different humidities to increase water content and to control moldiness of seeds. To better understand the influence of moldiness level on

Kangaroo rats (family Heteromyidae) are mainly seed eaters. Diet-choice experiments were performed to determine the effect of water content on seed selection by Dipodomys merriami . Kangaroo rats selected the most moist seeds available, and responded to exceedingly small differences in water content of seeds.