Comparison of Brain Transcriptome of the Greater Horseshoe Bats (Rhinolophus ferrumequinum) in Active and Torpid Episodes

Hibernation is an energy-saving strategy which is widely adopted by heterothermic mammals to survive in the harsh environment. The greater horseshoe bat (Rhinolophus ferrumequinum) can hibernate for a long period in the hibernation season. However, the global gene expression changes between hibernation and non-hibernation season in the greater horseshoe bat remain largely unknown. We herein reported a comprehensive survey of differential gene expression in the brain between winter hibernating and summer active greater horseshoe bats using next-generation sequencing technology. A total of 90,314,174 reads were generated and we identified 1,573 differentially expressed genes between active and torpid states. Interestingly, we found that differentially expressed genes are over-represented in some GO categories (such as metabolic suppression, cellular stress responses and oxidative stress), which suggests neuroprotective strategies might play an important role in hibernation control mechanisms. Our results determined to what extent the brain tissue of the greater horseshoe bats differ in gene expression between summer active and winter hibernating states and provided comprehensive insights into the adaptive mechanisms of bat hibernation.     

The Relationship between Cutaneous Water Loss and Thermoregulatory State in Kuhl's Pipistrelle Pipistrellus kuhlii, a Vespertillionid Bat

Abstract Total evaporative water loss is the sum of respiratory water loss (RWL) and cutaneous water loss (CWL) and constitutes the main avenue of water loss in bats. Because bats fly and have large surface-to-volume ratios, they potentially have high rates of RWL and CWL. Most species of small insectivorous bats have the ability to reduce their body temperature (T(b)) at rest, which substantially reduces energy expenditure and water loss. We hypothesized that bats reduce evaporative water loss during bouts of deep hypothermia (torpor) by decreasing RWL and CWL. We measured T(b), RWL, CWL, and resting metabolic rate (RMR) in Kuhl's pipistrelle Pipistrellus kuhlii, a small insectivorous bat. In support of our hypothesis, we found that RWL decreased with decreasing RMR. We found that CWL was lower in torpid individuals than in normothermic bats; however, bats in deep torpor had similar or higher CWL than bats in shallow torpor, suggesting that they exert a less effective physiological control over CWL when in deep torpor. Because insectivorous bats spend most of their lives in torpor or hibernation, the regulation of CWL in different heterothermic states has relevant ecological and evolutionary consequences.     

Identification and expression of microRNA in the brain of hibernating bats, Myotis lucifugus

Recent research has highlighted roles for non-coding RNA i7n the regulation of stress tolerance in bats. In this study, we propose that microRNA could also play an important role in neuronal maintenance during hibernation. To explore this possibility, RT-PCR was employed to investigate the expression of eleven microRNAs from the brain tissue of euthermic control and torpid bats. Results show that eight microRNAs (miR-21, -29b, -103, -107, -124a, -132, -183 and -501) increased (1.2–1.9 fold) in torpid bats, while the protein expression of Dicer, a microRNA processing enzyme, did not significantly change during torpor. Bioinformatic analysis of the differentially expressed microRNA suggests that these microRNAs are mainly involved in two processes: (1) focal adhesion and (2) axon guidance. To determine the extent of microRNA sequence conservation in the bat, we successfully identified bat microRNA from sequence alignments against known mouse (Mus musculus) microRNA. We successfully identified 206 conserved pre-microRNA sequences, leading to the identification of 344 conserved mature microRNA sequences. Sequence homology of the identified sequences was found to be 94.76 ± 3.95% and 98.87 ± 2.24% for both pre- and mature microRNAs, respectively. Results suggest that brain function related to the differentiation of neurons and adaptive neuroprotection may be under microRNA control during bat hibernation.     

Adaptive evolution of Leptin in heterothermic bats

Heterothermy (hibernation and daily torpor) is a key strategy that animals use to survive in harsh conditions and is widely employed by bats, which are found in diverse habitats and climates. Bats comprise more than 20% of all mammals and although heterothermy occurs in divergent lineages of bats, suggesting it might be an ancestral condition, its evolutionary history is complicated by complex phylogeographic patterns. Here, we use Leptin, which regulates lipid metabolism and is crucial for thermogenesis of hibernators, as molecular marker and combine physiological, molecular and biochemical analyses to explore the possible evolutionary history of heterothermy in bat. The two tropical fruit bats examined here were homeothermic; in contrast, the two tropical insectivorous bats were clearly heterothermic. Molecular evolutionary analyses of the Leptin gene revealed positive selection in the ancestors of all bats, which was maintained or further enhanced the lineages comprising mostly heterothermic species. In contrast, we found evidence of relaxed selection in homeothermic species. Biochemical assays of bat Leptin on the activity on adipocyte degradation revealed that Leptin in heterothermic bats was more lipolytic than in homeothermic bats. This shows that evolutionary sequence changes in this protein are indeed functional and support the interpretation of our physiological results and the molecular evolutionary analyses. Our combined data strongly support the hypothesis that heterothermy is the ancestral state of bats and that this involved adaptive changes in Leptin. Subsequent loss of heterothermy in some tropical lineages of bats likely was associated with range and dietary shifts.     

Effects of reproductive condition,roost microclimate,and weather patterns on summer torpor use by a vespertilionid bat

A growing number of mammal species are recognized as heterothermic, capable of maintaining a high‐core body temperature or entering a state of metabolic suppression known as torpor. Small mammals can achieve large energetic savings when torpid, but they are also subject to ecological costs. Studying torpor use in an ecological and physiological context can help elucidate relative costs and benefits of torpor to different groups within a population. We measured skin temperatures of 46 adult Rafinesque's big‐eared bats (Corynorhinus rafinesquii) to evaluate thermoregulatory strategies of a heterothermic small mammal during the reproductive season. We compared daily average and minimum skin temperatures as well as the frequency, duration, and depth of torpor bouts of sex and reproductive classes of bats inhabiting day‐roosts with different thermal characteristics. We evaluated roosts with microclimates colder (caves) and warmer (buildings) than ambient air temperatures, as well as roosts with intermediate conditions (trees and rock crevices). Using Akaike's information criterion (AIC), we found that different statistical models best predicted various characteristics of torpor bouts. While the type of day‐roost best predicted the average number of torpor bouts that bats used each day, current weather variables best predicted daily average and minimum skin temperatures of bats, and reproductive condition best predicted average torpor bout depth and the average amount of time spent torpid each day by bats. Finding that different models best explain varying aspects of heterothermy illustrates the importance of torpor to both reproductive and nonreproductive small mammals and emphasizes the multifaceted nature of heterothermy and the need to collect data on numerous heterothermic response variables within an ecophysiological context.     

Modulation of Gene Expression in Key Survival Pathways During Daily Torpor in the Gray Mouse Lemur,Microcebus murinus

A variety of mammals employ torpor as an energy-saving strategy in environments of marginal or severe stress either on a daily basis during their inactive period or on a seasonal basis during prolonged...     

Environmental conditions, rather than season, determine torpor use and temperature selection in large mouse-eared bats (Myotis myotis)

We tested whether food availability, thermal environment and time of year affect torpor use and temperature selection in the large mouse-eared bat (Myotis myotis) in summer and winter. Food-deprived bats were torpid longer than bats offered food ad libitum. Bats placed in a gradient of low (0 degrees C-25 degrees C) ambient temperatures (T(a)) spent more time in torpor than bats in a gradient of high (7 degrees C-43 degrees C) T(a)'s. However, we did not observe seasonal variations in the use of torpor. Moreover, even when food deprived in winter, bats never entered prolonged torpor at T(a)'s characteristic of their natural hibernation. Instead, bats preferred shallow torpor at relatively high T(a), but they always maintained a difference between body and ambient temperatures of less than 2 degrees C. Calculations based on respirometric measurements of metabolic rate showed that food deprived bats spent less energy per unit of time in torpor than fed individuals, even when they entered torpor at higher T(a)'s. We conclude that T(a) likely serves as a signal of food availability and daily torpor is apparently an adaptation to unpredictable changes in food availability, such as its decrease in summer or its increase in winter. Thus, we interpret hibernation to be a second step in the evolution of heterothermy in bats, which allows survival in seasonal environments.     

Brazilian sleeping beauty: First evidence of hibernation by a bat in South America

Torpor and hibernation are energy-saving heterothermic strategies widespread among mammals, and bats have reports of hibernation in North America, Europe, Asia, Oceania, and Africa. However, to date, there was no evidence of hibernation by bats in South America. We tagged four individuals of Eptesicus brasiliensis and one remained torpid for 13 consecutive days in a tree cavity roost in southern Brazil, showing evidence that bats can hibernate in South America.     

Telomerase activity in the bats <Emphasis Type="Italic">Hipposideros armiger</Emphasis> and <Emphasis Type="Italic">Rousettus leschenaultia</Emphasis>

Telomerase activity was examined in two species of bat, Hipposideros armiger and Rousettus leschenaultia, which have similar body mass and lifespan but differ in use of hibernation. We found that telomerase activity was present in all tissues sampled, but it was greater in metabolically active tissues such as liver, spleen, and kidney. Of special interest is the raised activity found in the heterothermic bat H. armiger, and the hibernating bats having raised values for spleen, heart, and kidney. These findings show that maintenance of high levels of telomerase is an essential part of the regulation of cellular activities during hibernation.     

Reversible depression of oxygen consumption in isolated liver mitochondria during hibernation

The biochemical mechanisms by which hibernators cool as they enter torpor are not fully understood. In order to examine whether rates of substrate oxidation vary as a function of hibernation, liver mitochondria were isolated from telemetered ground squirrels (Spermophilus lateralis) in five phases of their annual hibernation cycle: summer active, and torpid, interbout aroused, entrance, and arousing hibernators. Rates of state 3 and state 4 respiration were measured in vitro at 25 degrees C. Relative to mitochondria from summer-active animals, rates of state 3 respiration were significantly depressed in mitochondria from torpid animals yet fully restored during interbout arousals. These findings indicate that a depression of ADP-dependent respiration in liver mitochondria occurs during torpor and is reversed during the interbout arousals to euthermia. Because this inhibition was determined to be temporally independent of entrance and arousal, it is unlikely that active suppression of state 3 respiration causes entrance into torpor by facilitating metabolic depression. In contrast to the observed depression of state 3 respiration in torpid animals, state 4 respiration did not differ significantly among any of the five groups, suggesting that alterations in proton leak are not contributing appreciably to downregulation of respiration in hibernation.     

Torpor-associated fluctuations in surfactant activity in Gould's wattled bat

The primary function of pulmonary surfactant is to reduce the surface tension (ST) created at the air-liquid interface in the lung. Surfactant is a complex mixture of lipids and proteins and its function is influenced by physiological parameters such as metabolic rate, body temperature and breathing. In the microchiropteran bat Chalinolobus gouldii these parameters fluctuate throughout a 24 h period. Here we examine the surface activity of surfactant from warm-active and torpid bats at both 24 degrees C and 37 degrees C to establish whether alterations in surfactant composition correlate with changes in surface activity. Bats were housed in a specially constructed bat room at Adelaide University, at 24 degrees C and on a 8:16 h light:dark cycle. Surfactant was collected from bats sampled during torpor (2535 degrees C). Alterations in the lipid composition of surfactant occur with changes in the activity cycle. Most notable is an increase in surfactant cholesterol (Chol) with decreases in body temperature [Codd et al., Physiol. Biochem. Zool. 73 (2000) 605-612]. Surfactant from active bats was more surface active at higher temperatures, indicated by lower ST(min) and less film area compression required to reach ST(min) at 37 degrees C than at 24 degrees C. Conversely, surfactant from torpid bats was more active at lower temperatures, indicated by lower ST(min) and less area compression required to reach ST(min) at 24 degrees C than at 37 degrees C. Alterations in the Chol content of bat surfactant appear to be crucial to allow it to achieve low STs during torpor.     

Hibernation and torpor in tropical and subtropical bats in relation to energetics, extinctions, and the evolution of endothermy

Torpor, the most effective means of energy conservation available to endotherms, is still widely viewed as a specific adaptation in a few high-latitude, cold-climate endotherms with no adaptive function in warm regions. Nevertheless, a growing number of diverse terrestrial mammals and birds from low latitudes (0-30°), including species from tropical and subtropical regions, are heterothermic and employ torpor. Use of torpor is especially important for bats because they are small, expend large amounts of energy when active, rely on a fluctuating food supply, and have only a limited capacity for storage of fat. Patterns of torpor in tropical/subtropical bats are highly variable, but short bouts of torpor with relatively high body temperatures (T(b)) are most common. Hibernation (a sequence of multiday bouts of torpor) has been reported for free-ranging subtropical tree-dwelling vespertilionids, cave-dwelling hipposiderids, and house-dwelling molossids. The observed range of minimum T(b) is ～6-30 °C, and the reduction of energy expenditure through the use of torpor, in comparison to normothermic values, ranges from 50 to 99%. Overall, torpor in the tropics/subtropics has been reported for 10 out of the currently recognized 18 bat families, which contain 1079 species, or 96.7% of all bats. Although it is unlikely that all of these are heterothermic, the large majority probably will be. Frequent use of torpor, including hibernation in diverse groups of tropical/subtropical bats, suggests that heterothermy is an ancestral chiropteran trait. Although data especially from the field are still scarce, it is likely that torpor, highly effective in reducing requirements for energy and water even under warm conditions, plays a crucial role in the long-term survival of the majority of small tropical and subtropical bats. Discovering how bats achieve this provides numerous opportunities for exiting new research.     

Winter diet analysis in Rhinolophus euryale (Chiroptera)

We investigated the winter food of Mediterranean horseshoe bats (Rhinolophus euryale) in four winter cave roosts in southern Slovakia and northern Hungary and investigated the relationship between food and ambient temperature. The bats were active during the whole winter period and they produced excrement throughout the entire hibernation period, even when outside temperatures dropped below zero. The guano was in two forms, containing (1) prey items and (2) non-prey items. The identifiable items belonged to lepidopteran species, but only one was identified, on the basis of the genital fragments, the moth Colotois pennaria, which was the main prey species in autumn and early winter. Our results shed light on the extraordinarily high level of activity in this bat species during winter hibernation, which in temperate regions is a strategy that enables bats to survive when prey is reduced or absent. In R. euryale, the torpor in the course of hibernation is not continuous and our results help to explain how energy losses caused by bat movements are covered.     

Antioxidant defenses, longevity and ecophysiology of South American bats

Microchiropteran bats sustain very high oxygen consumption rates when active, but they also exhibit drastic daily drops in oxygen consumption when torpid. In addition, bats are also characterized by an extraordinary longevity considering their body mass and high specific metabolic rate when compared to other mammals of related size. Therefore, they consist of a very interesting group regarding the free radical theory of aging. The present study was carried out to measure the antioxidant defenses of several tissues of five South American bat species, attempting to correlate the antioxidant status, ecophysiology and longevity. Superoxide dismutase (SOD) and catalase (CAT) activities in blood, liver and kidney were higher compared to other tissues. The contents of alpha-tocopherol and beta-carotene found in liver, heart, kidneys, and pectoral muscles were one to two orders of magnitude higher than those usually found in rat and mouse liver. Also, these contents in liver were generally inversely related to lipoperoxidation measured as TBARS contents. Blood GSH contents and the activities of SOD and CAT were higher in torpid Sturnira lillium compared to active ones, thus suggesting that the elevation of such antioxidants might be daily modulated to minimize the oxidative stress related to the transition from torpid to active state in bats. The lower ROS generation reported in the literature for other bat species, their high constitutive antioxidant defenses, and the daily energy sparing associated with torpor appear to be closely related to their ecophysiological adaptations and to their extended longevity.     

Periodic fluctuations in the pulmonary surfactant system in Gould's wattled bat (Chalinolobus gouldii)

Pulmonary surfactant is a mixture of phospholipids, neutral lipids, and proteins that controls the surface tension of the fluid lining the lung. Surfactant amounts and composition are influenced by such physiological parameters as metabolic rate, activity, body temperature, and ventilation. Microchiropteran bats experience fluctuations in these parameters throughout their natural daily cycle of activity and torpor. The activity cycle of the microchiropteran bat Chalinolobus gouldii was studied over a 24-h period. Bats were maintained in a room at constant ambient temperature (24 degrees C) on an 8L : 16D cycle. Diurnal changes in the amount and composition of surfactant were measured at 4-h intervals throughout a 24-h period. The C. gouldii were most active at 2 a.m. and were torpid at 2 p.m. Alveolar surfactant increased 1.5-fold immediately after arousal. The proportion of disaturated phospholipid remained constant, while surfactant cholesterol levels increased 1.5-fold during torpor. Alveolar cholesterol in C. gouldii was six times lower than in other mammals. Cholesterol appears to function in maintaining surfactant fluidity during torpor in this species of bat.