White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats.

Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding.

Eurasian Red Squirrels Show Little Seasonal Variation in Metabolism in Food-Enriched Habitat.

Energy expenditure and ambient temperature (Ta) are intrinsically linked through changes in an animal's metabolic rate. While the nature of this relationship is stable, the breadth of change in thermoregulatory cost varies with body size and physiological acclimatization to season. To explore seasonal metabolic changes of small mammals, we studied a population of Eurasian red squirrels (Sciurus vulgaris) in a seminatural environment with a year-round supply of natural and supplemented food. In each season we measured the metabolic rate of wild-caught red squirrels, using open-flow respirometry, and hypothesized that individuals would make adjustments to contend with seasonal weather conditions. In comparison to summer animals, we predicted that winter squirrels would show (1) an increase in metabolic rate within the thermoneutral zone, (2) a decrease in the lower critical temperature of the thermoneutral zone, (3) a shallower slope of resting metabolic rate with decreasing Ta, and (4) lower thermal conductance. Surprisingly, we observed only minor changes in resting metabolic rate, and energetic modeling suggested that the scope of change was unlikely to be of ecological consequence. Hair area density was higher in winter than in summer, corresponding to a slightly elevated thermal conductance in summer, while body mass was reasonably constant year-round. We conclude that the scope of physiological seasonal adaptation is minimal when food is abundant and that squirrels instead rely on adjustments in activity to reduce exposure to low Ta. We suggest that this may explain the squirrel's success in a wide range of habitats, including urban areas, which require a rapid and flexible response to environmental changes and may indicate the capacity of other small mammal species to cope with environmental disturbance.

A persistently infecting coronavirus in hibernating Myotis lucifugus, the North American little brown bat.

Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats (Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.

Primate Torpor Expression: Ghost of the Climatic Past.

Torpor, the controlled depression of virtually all bodily function during scarce periods, was verified in primates under free-ranging conditions less than two decades ago. The large variety of different torpor patterns found both within and among closely related species is particularly remarkable. To help unravel the cause of these variable patterns, our review investigates primate torpor use within an evolutionary framework. First, we provide an overview of heterothermic primate species, focusing on the Malagasy lemurs, and discuss their use of daily torpor or hibernation in relation to habitat type and climatic conditions. Second, we investigate environmental characteristics that may have been involved in shaping the high variability of torpor expression found in lemurs today. Third, we examine potential triggers for torpor use in lemurs. We propose the "torpor refugia hypothesis" to illustrate how disparate primate torpor patterns possibly evolved in response to environmental cues during glacial periods, when animals were restricted to different refuge habitats along riverine corridors. For example, individuals enduring harsher conditions at higher altitudes likely developed seasonal hibernation, whereas those inhabiting lower elevation river catchments might have coped with unfavorable conditions by employing daily torpor. The ultimate stimuli triggering torpor use today likely differ between the different habitats of Madagascar. The broad diversity of torpor patterns in lemurs among closely related species, both within the same and in distinctly different habitat types, provides an ideal base for research into the stimuli for torpor use in endotherms in general. Our hypothesis highlights the importance of considering the environmental conditions under which ecosystems and species evolved when trying to explain physiological adaptations seen today.

White-Nose Syndrome Disease Severity and a Comparison of Diagnostic Methods.

White-nose syndrome is caused by the fungus Pseudogymnoascus destructans and has killed millions of hibernating bats in North America but the pathophysiology of the disease remains poorly understood. Our objectives were to (1) assess non-destructive diagnostic methods for P. destructans infection compared to histopathology, the current gold-standard, and (2) to evaluate potential metrics of disease severity. We used data from three captive inoculation experiments involving 181 little brown bats (Myotis lucifugus) to compare histopathology, quantitative PCR (qPCR), and ultraviolet fluorescence as diagnostic methods of P. destructans infection. To assess disease severity, we considered two histology metrics (wing area with fungal hyphae, area of dermal necrosis), P. destructans fungal load (qPCR), ultraviolet fluorescence, and blood chemistry (hematocrit, sodium, glucose, pCO2, and bicarbonate). Quantitative PCR was most effective for early detection of P. destructans, while all three methods were comparable in severe infections. Correlations among hyphae and necrosis scores, qPCR, ultraviolet fluorescence, blood chemistry, and hibernation duration indicate a multi-stage pattern of disease. Disruptions of homeostasis occurred rapidly in late hibernation. Our results provide valuable information about the use of non-destructive techniques for monitoring, and provide novel insight into the pathophysiology of white-nose syndrome, with implications for developing and implementing potential mitigation strategies.

Glycerophospholipid Profiles of Bats with White-Nose Syndrome.

Pseudogymnoascus destructans is an ascomycetous fungus responsible for the disease dubbed white-nose syndrome (WNS) and massive mortalities of cave-dwelling bats. The fungus infects bat epidermal tissue, causing damage to integumentary cells and pilosebaceous units. Differences in epidermal lipid composition caused by P. destructans infection could have drastic consequences for a variety of physiological functions, including innate immune efficiency and water retention. While bat surface lipid and stratum corneum lipid composition have been described, the differences in epidermal lipid content between healthy tissue and P. destructans-infected tissue have not been documented. In this study, we analyzed the effect of wing damage from P. destructans infection on the epidermal polar lipid composition (glycerophospholipids [GPs] and sphingomyelin) of little brown bats (Myotis lucifugus). We hypothesized that infection would lead to lower levels of total lipid or higher oxidized lipid product proportions. Polar lipids from three damaged and three healthy wing samples were profiled by electrospray ionization tandem mass spectrometry. We found lower total broad lipid levels in damaged tissue, specifically ether-linked phospholipids, lysophospholipids, phosphatidylcholine, and phosphatidylethanolamine. Thirteen individual GP species from four broad GP classes were present in higher amounts in healthy tissue. Six unsaturated GP species were absent in damaged tissue. Our results confirm that P. destructans infection leads to altered lipid profiles. Clinical signs of WNS may include lower lipid levels and lower proportions of unsaturated lipids due to cellular and glandular damage.

Conspecific disturbance contributes to altered hibernation patterns in bats with white-nose syndrome.

The emerging wildlife disease white-nose syndrome (WNS) affects both physiology and behaviour of hibernating bats. Infection with the fungal pathogen Pseudogymnoascus destructans (Pd), the first pathogen known to target torpid animals, causes an increase in arousal frequency during hibernation, and therefore premature depletion of energy stores. Infected bats also show a dramatic decrease in clustering behaviour over the winter. To investigate the interaction between disease progression and torpor expression we quantified physiological (i.e., timing of arousal, rewarming rate) and behavioural (i.e., arousal synchronisation, clustering) aspects of rewarming events over four months in little brown bats (Myotis lucifugus) experimentally inoculated with Pd. We tested two competing hypotheses: 1) Bats adjust arousal physiology adaptively to help compensate for an increase in energetically expensive arousals. This hypothesis predicts that infected bats should increase synchronisation of arousals with colony mates to benefit from social thermoregulation and/or that solitary bats will exhibit faster rewarming rates than clustered individuals because rewarming costs fall as rewarming rate increases. 2) As for the increase in arousal frequency, changes in arousal physiology and clustering behaviour are maladaptive consequences of infection. This hypothesis predicts no effect of infection or clustering behaviour on rewarming rate and that disturbance by normothermic bats contributes to the overall increase in arousal frequency. We found that arousals of infected bats became more synchronised than those of controls as hibernation progressed but the pattern was not consistent with social thermoregulation. When a bat rewarmed from torpor, it was often followed in sequence by up to seven other bats in an arousal "cascade". Moreover, rewarming rate did not differ between infected and uninfected bats, was not affected by clustering and did not change over time. Our results support our second hypothesis and suggest that disturbance, not social thermoregulation, explains the increased synchronisation of arousals. Negative pathophysiological effects of WNS on energy conservation may therefore be compounded by maladaptive changes in behaviour of the bats, accelerating fat depletion and starvation.

Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans.

Recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern Americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. However, despite these catastrophic events little Information is available on bat defences or how they interact with their pathogens. Even less is known about the response of bats to infection during torpor or long-term hibernation. Using tissue samples collected at the termination of an experiment to explore the pathogenesis of White Nose Syndrome in Little Brown Bats, we determined if hibernating bats infected with the fungus Pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. Lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. However, we were unable to obtain sufficient amounts of RNA from these sites. We therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. We found that bats responded to infection with a significant increase in lungs of transcripts for Cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. In conclusion, hibernating bats can respond to experimental P. destructans infection by activating expression of innate immune response genes.

Pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality.

White-nose syndrome is devastating North American bat populations but we lack basic information on disease mechanisms. Altered blood physiology owing to epidermal invasion by the fungal pathogen Geomyces destructans (Gd) has been hypothesized as a cause of disrupted torpor patterns of affected hibernating bats, leading to mortality. Here, we present data on blood electrolyte concentration, haematology and acid-base balance of hibernating little brown bats, Myotis lucifugus, following experimental inoculation with Gd. Compared with controls, infected bats showed electrolyte depletion (i.e. lower plasma sodium), changes in haematology (i.e. increased haematocrit and decreased glucose) and disrupted acid-base balance (i.e. lower CO2 partial pressure and bicarbonate). These findings indicate hypotonic dehydration, hypovolaemia and metabolic acidosis. We propose a mechanistic model linking tissue damage to altered homeostasis and morbidity/mortality.

Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome.

White-nose syndrome (WNS) is an emerging disease of hibernating bats associated with cutaneous infection by the fungus Geomyces destructans (Gd), and responsible for devastating declines of bat populations in eastern North America. Affected bats appear emaciated and one hypothesis is that they spend too much time out of torpor during hibernation, depleting vital fat reserves required to survive the winter. The fungus has also been found at low levels on bats throughout Europe but without mass mortality. This finding suggests that Gd is either native to both continents but has been rendered more pathogenic in North America by mutation or environmental change, or that it recently arrived in North America as an invader from Europe. Thus, a causal link between Gd and mortality has not been established and the reason for its high pathogenicity in North America is unknown. Here we show that experimental inoculation with either North American or European isolates of Gd causes WNS and mortality in the North American bat, Myotis lucifugus. In contrast to control bats, individuals inoculated with either isolate of Gd developed cutaneous infections diagnostic of WNS, exhibited a progressive increase in the frequency of arousals from torpor during hibernation, and were emaciated after 3-4 mo. Our results demonstrate that altered torpor-arousal cycles underlie mortality from WNS and provide direct evidence that Gd is a novel pathogen to North America from Europe.

Summer and winter torpor use by a free-ranging marsupial.

Torpor is usually associated with low ambient temperatures (T(a)) in winter, but in some species it is also used in summer, often in response to limited food availability. Since the seasonal expression of torpor of both placental and marsupial hibernators in the wild is poorly documented by quantitative data, we investigated torpor and activity patterns of the eastern pygmy-possum Cercartetus nanus (17.4 g) over two seasons. We used radio telemetry to track animals during winter (n=4) and summer (n=5) in a warm-temperate habitat and found that torpor was used in both seasons. In winter all animals entered periods of short-term hibernation (from 5 to 20 days) containing individual torpor bouts of up to 5.9 days. In summer, torpor bouts were always <1 day in duration, only used by males and were not related to daily mean T(a). Pygmy-possums entered torpor at night as T(a) cooled, and rewarmed during the afternoon as T(a) increased. Individuals interspersed torpor bouts with nocturnal activity and the percentage of the night animals were active was the same in summer and winter. Our study provides the first information on torpor patterns in free-ranging C. nanus, and shows that the use of torpor throughout the year is important for energy management in this species.

Basking behaviour in relation to energy use and food availability in one of the smallest marsupials.

Although several mammals have been observed to bask in the sun, little is known about this behaviour or its energetic consequences. We investigated the importance of basking behaviour for one of the smallest marsupials, Planigale gilesi (9g). Metabolic rates of captive planigales (n=6) exposed to simulated natural conditions with access to a radiant heat source were measured. Basking behaviour as a function of food availability was quantified using a video camera installed within the planigales' home cages (n=7). All planigales basked during respirometry measurements, reducing resting energy expenditure by 58% at an ambient temperature of 15 degrees C, which reflects conditions in their nesting sites in the wild during winter. Basking behaviour in home cages was displayed by all but one planigale; food withdrawal either triggered basking or it caused a significant increase in basking duration. Our study demonstrates the effectiveness of basking for reducing energy expenditure in one of the smallest marsupials, supporting recent findings on the importance of behavioural thermoregulation in small mammals in general.

The energetics of basking behaviour and torpor in a small marsupial exposed to simulated natural conditions.

Limited information is available on basking behaviour in torpid mammals and its energetic consequences. We investigated the effects of physiological and behavioural strategies on the energetics of the fat-tailed dunnart (Sminthopsis crassicaudata). Metabolic rate and body temperature during torpor, basking and rest were measured over 24 h in response to simulated environmental conditions: (a) constant ambient temperature (T(a)) of 15 degrees C, (b) constant T(a) of 15 degrees C with access to a radiant heat lamp, (c) a T(a) cycle (range 15-31 degrees C), and (d) a T(a) cycle with access to a radiant heat lamp. When a radiant heat source was provided, all dunnarts (n = 16) basked during all measurements, which resulted in energy savings of up to 74% during rest. Overall, torpor was used on 59% of measurements with a maximum duration of 16.2 h and reductions in metabolic rate of 90% compared to normothermic values. Torpid dunnarts actively moved from a shaded area to position themselves under the heat lamp with body temperatures as low as 17.5 degrees C and thereby reduced rewarming costs by 66%. We demonstrated, for the first time in the laboratory, that torpid animals actively move to a heat source to bask, and that this behaviour results in considerable energy savings. Our finding supports the view that basking during normothermia and rewarming from torpor substantially reduces energetic requirements, which may be important for the survival of small dasyurids living on limited resources in the Australian arid zone.

Torpor and basking in a small arid zone marsupial.

The high energetic cost associated with endothermic rewarming from torpor is widely seen as a major disadvantage of torpor. We tested the hypothesis that small arid zone marsupials, which have limited access to energy in the form of food but ample access to solar radiation, employ basking to facilitate arousal from torpor and reduce the costs of rewarming. We investigated torpor patterns and basking behaviour in free-ranging fat-tailed dunnarts Sminthopsis crassicaudata (10 g) in autumn and winter using small, internal temperature-sensitive transmitters. Torpid animals emerged from their resting sites in cracking soil at approximately 1000 h with body temperatures as low as 14.6 degrees C and positioned themselves in the sun throughout the rewarming process. On average, torpor duration in autumn was shorter, and basking was less pronounced in autumn than in winter. These are the first observations of basking during rewarming in S. crassicaudata and only the second direct evidence of basking in a torpid mammal for the reduction of energetic costs during arousal from torpor and normothermia. Our findings suggest that although overlooked in the past, basking may be widely distributed amongst heterothermic mammals. Therefore, the energetic benefits from torpor use in wild animals may currently be underestimated.