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.     

Antioxidant Defenses in the Brains of Bats during Hibernation

Hibernation is a strategy used by some mammals to survive a cold winter. Small hibernating mammals, such as squirrels and hamsters, use species- and tissue-specific antioxidant defenses to cope with oxidative insults during hibernation. Little is known about antioxidant responses and their regulatory mechanisms in hibernating bats. We found that the total level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the brain of each of the two distantly related hibernating bats M. ricketti and R. ferrumequinum at arousal was lower than that at torpid or active state. We also found that the levels of malondialdehyde (product of lipid peroxidation) of the two hibernating species of bats were significantly lower than those of non-hibernating bats R. leschenaultia and C. sphinx. This observation suggests that bats maintain a basal level of ROS/RNS that does no harm to the brain during hibernation. Results of Western blotting showed that hibernating bats expressed higher amounts of antioxidant proteins than non-hibernating bats and that M. ricketti bats upregulated the expression of some enzymes to overcome oxidative stresses, such as superoxide dismutase, glutathione reductase, and catalase. In contrast, R. ferrumequinum bats maintained a relatively high level of superoxide dismutase 2, glutathione reductase, and thioredoxin-2 throughout the three different states of hibernation cycles. The levels of glutathione (GSH) were higher in M. ricketti bats than in R. ferrumequinum bats and were significantly elevated in R. ferrumequinum bats after torpor. These data suggest that M. ricketti bats use mainly antioxidant enzymes and R. ferrumequinum bats rely on both enzymes and low molecular weight antioxidants (e.g., glutathione) to avoid oxidative stresses during arousal. Furthermore, Nrf2 and FOXOs play major roles in the regulation of antioxidant defenses in the brains of bats during hibernation. Our study revealed strategies used by bats against oxidative insults during hibernation.     

河南济源人工引水渠隧道蝙蝠冬眠生态学特征

2017-2020年期间,每年1月份对河南省济源市邵原镇布袋沟水库人工引水渠隧道内蝙蝠进行冬眠生态学特征调查,共发现2科5属7种蝙蝠在此冬眠,包括马铁菊头蝠（Rhinolophus ferrumequinum）、小菊头蝠（R.pusillus）、华南水鼠耳蝠（Myotis laniger）、白腹管鼻蝠（Murina leucogaster）、金管鼻蝠（Mu.aurata）、奥氏长耳蝠（Plecotus ognevi）和亚洲宽耳蝠（Barbastella leucomelas）。马铁菊头蝠是优势种（约52%-73%的冬眠个体）,其次是小菊头蝠（约19%-37%）、华南水鼠耳蝠（约5%-8%）,其余蝙蝠物种数量不足3%。2017-2020年冬眠蝙蝠个体总数呈增长趋势,但仍少于早期报道的数量。有42个隧道每年均有蝙蝠冬眠,而且不同年度冬眠数量也不尽相同。通过多元线性回归分析发现,隧道长度可能是影响蝙蝠冬眠栖息场所选择的主要影响因子（Adjusted R²=0.208,P=0.001）。每个隧道内,蝙蝠具有不同的冬眠栖点位置,约4/5的蝙蝠选择温暖且环境相对稳定的隧道深处（> 30 m）作为冬眠栖点,超过95%的个体选择长度> 60 m的隧道冬眠。蝙蝠具有不同的冬眠方式,绝大多数个体采用独栖方式进行冬眠（> 90%）,少数采用聚集方式。不同的冬眠栖点和冬眠方式可能有利于冬眠成本优化。此外,栖点温度与蝙蝠体温之间呈显著正相关（R²=0.98,P < 0.001）,而且蝙蝠冬眠期间的栖点温度具有种内和种间差异。研究结果为我国蝙蝠种群保护和冬眠场所管理提供科学依据。     

Impact of predation by a cave-dwelling bat, Rhinolophus ferrumequinum, on the diapausing population of a troglophilic moth, Goniocraspidum preyeri

The predation pressure of the greater horseshoe bat, Rhinolophus ferrumequinum, on the diapausing population of the noctuid moth, Goniocraspidum preyeri, was examined at an abandoned mine in central Japan. These bats did not prey on the moths in summer. The number of moths preyed on was largest in March. More than 60% of the diapausing moths were eaten by only three or four bats, which accounted for over 90% of all the moth deaths. This moth was an important source of food at the end of the hibernating period when the bat had already used most of its stored subcutaneous fat. The predation on G. preyeri may have raised the overwintering success rate of R. ferrumequinum.     

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.     

Adaptation of Phenylalanine and Tyrosine Catabolic Pathway to Hibernation in Bats

Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation.     

Hibernation in warm hibernacula by free-ranging Formosan leaf-nosed bats, <Emphasis Type="Italic">Hipposideros terasensis</Emphasis>, in subtropical Taiwan

The subtropical Formosan leaf-nosed bats, Hipposideros terasensis (Hipposideridae), show little activity during winter. It has never been determined whether in winter they exhibit hibernation and multi-day periods of low body temperature. The objectives of this study were to understand the winter activity pattern of H. terasensis and to examine whether it enters hibernation during winter. We monitored the skin temperature (T _sk) of nine free-ranging H. terasensis by attaching temperature-sensitive transmitters during the winters of 2007–2008 and 2008–2009. The results showed that H. terasensis entered hibernation from late December to early March. H. terasensis, however, differs from temperate hibernating bats in several ways: (1) it is capable of hibernation at roost temperature (T _r) and T _sk > 20°C; (2) hibernation at high T _r and T _sk does not lead to a relatively high arousal frequency; and (3) adults do not increase body mass in autumn prior to hibernation. To test the hypothesis that H. terasensis feeds frequently during the hibernation period to compensate for the high energetic demands of hibernating in warm hibernacula, we recorded the number and timing of bats that emerged from and entered into a hibernaculum, which contained more than 1,000 bats. From 30 December 2007 to 29 February 2008, an average of only 8.4 bats (<1%) per night (29 nights) emerged from the hibernaculum. Adult bats lost an average of 13–14% of body mass during an approximately 70-day hibernation period. We suggest that H. terasensis might have remarkably low torpid metabolic rates during hibernation.     

Seasonal variation in use of winter roosts by five bat species in south-east Finland

We studied seasonal variation in the use of winter roosts by five bat species (Eptesicus nilssonii, Myotis brandtii/mystacinus, Myotis daubentonii and Plecotus auritus) in south-east Finland during the winters of 2003/2004 and 2004/2005. At the beginning of the bat hibernation season all species used higher temperatures and humidity than by the season’s end. Hibernacula were at their coldest in mid-hibernation season and became warmer towards the end of the season. However, no species hibernated in warmer locations at the end of the season than in mid-season. Results suggest that bats tend to use different strategies throughout the hibernation season, minimizing the cost of hibernation early in the season by hibernating in warmer locations and minimizing energy expenditure later in the season by hibernating in colder locations. M. brandtii/mystacinus were found in locations with stable temperature and humidity, moving to increasingly stable conditions (chambers, crevices, clusters, ceiling) towards spring. All other species hibernated in more variable microclimates throughout the hibernation season.     

Normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum,with special reference to nose leaf formation

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a “nose leaf,” a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15–16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.     

Histochemical and biochemical plasticity of muscle fibers in the little brown bat (Myotis lucifugus)

Summary Fiber composition, and glycolytic and oxidative capacities of the pectoralis, gastrocnemius, and cardiac muscles from active and hibernating little brown bats (Myotis lucifugus) was studied. The data were used to test two hypotheses: First, since hibernating bats maintain the capability of flight and make use of leg muscles to maintain a roosting position all winter, the fiber composition of the pectoralis and gastrocnemius muscles should not change with season. Second, we tested the hypothesis of Ianuzzo et al. (in press), who propose that the oxidative potential of mammalian cardiac muscle should increase with increasing heart rate while glycolytic potential should not. Our results indicate that the fiber composition of the pectoralis muscle was uniformly fast-twitch oxidative (FO)_ regardless of the time of year, as predicted. However, the gastrocnemius muscle exhibited a change in FO composition from 83% in active to 61% in hibernating animals. Contrary to the variable change in histochemical properties with metabolic state, a trend of reduced maximal oxidative (CS) and glycolytic (PFK) potential during hibernation in both flight and leg muscles was apparent. The oxidative potential of flight and leg muscles decreased by 15.2% and 56.5%, respectively, while the glycolytic potential of the same muscles decreased by 23.5% and 60.5%, respectively. As predicted, the glycolytic potential of cardiac muscle remained constant between active and hibernating bats, although there was a significant decrease (22.0%) in oxidative potential during hibernation.Abbreviations FO fast-twitch oxidative - FG fast-twitch glycolytic - SO slow-twitch oxidative - Vmax maximal enzyme activity - PFK phosphofructokinase - CS citrate synthase     

Free-Ranging Little Brown Myotis (Myotis lucifugus) Heal from Wing Damage Associated with White-Nose Syndrome

White-nose syndrome (WNS) is having an unprecedented impact on hibernating bat populations in the eastern United States. While most studies have focused on widespread mortality observed at winter hibernacula, few have examined the consequences of wing damage that has been observed among those bats that survive hibernation. Given that WNS-related wing damage may lead to life-threatening changes in wing function, we tested the hypothesis that reduced abundance of free-ranging little brown myotis (Myotis lucifugus) with severe wing damage as the summer progresses is due to healing of wing tissue. Photographs of captured and recaptured adult females were examined for wing damage and healing rates were calculated for each category of wing damage index (WDI = 0–3). We found that free-ranging bats with severe wing damage were able to heal to a lower WDI score within 2 weeks. Bats with the most severe wing damage had faster healing rates than did individuals with less damage. We also found a significant relationship between body condition and WDI for adult females captured in the early weeks of the active season. Our results support the hypothesis that some bats can heal from severe wing damage during the active season, and thus may not experience increased mortality associated with reduced functions of wings. We urge researchers and wildlife managers to use caution when interpreting data on WDI to assess the impact of WNS on bat populations, especially during the later months of the active season.     

Anticipating white‐nose syndrome in the Southern Hemisphere: Widespread conditions favourable to Pseudogymnoascus destructans pose a serious risk to Australia's bat fauna

There is a serious concern that white‐nose syndrome (WNS), a fungal disease causing severe population declines in North American bats, could soon threaten bats on the Australian continent. Despite an ‘almost certain' risk of incursion within the next ten years, and high virulence in naïve bat populations, we remain uncertain about the vulnerability of Australian bats to WNS. In this study, we intersected occurrences for the 27 cave roosting bat species in Australia with interpolated data on mean annual surface temperature, which provides a proxy for thermal conditions within a cave and hence its suitability for growth by the fungal pathogen Pseudogymnoascus destructans. Our analysis identifies favourable roost thermal conditions within 30–100% of the ranges of eight bat species across south‐eastern Australia, including for seven species already listed as threatened with extinction. These results demonstrate the potential for widespread exposure to P. destructans and suggest that WNS could pose a serious risk to the conservation of Australia's bat fauna. The impacts of exposure to P. destructans will depend, however, on the sensitivity of bats to developing WNS, and a more comprehensive vulnerability assessment is currently prevented by a lack of information on the hibernation biology of Australian bats. Thus, given the clear potential for widespread exposure of Australia's bats to P. destructans demonstrated by our study, two specific policy actions seem justified: (i) urgent implementation of border controls that identify and decontaminate cave‐associated fomites and (ii) dedicated funding to enable research on key aspects of bat winter behaviour and hibernation physiology. Further, as accidental translocation of this fungus could also pose a risk to other naïve bat faunas in cooler regions of southern Africa and South America, we argue that a proactive, globally coordinated approach is required to understand and mitigate the potential impacts of WNS spreading to Southern Hemisphere bats.     

Lactate Dehydrogenase Isozymes in the Tissues of Hibernating Bats (Chiroptera)

A comparative electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes has been carried out in the homogenates of the tissues of cardiac and skeletal muscles, liver, kidneys and lungs of five species of hibernating bats of the order Chiroptera: the northern bat Eptesicus nilssonii Keyserling and Blasius, the brown long-eared bat Plecotus auritus L., Brandt’s bat Myotis brandtii Eversmann, Daubenton’s bat Myotis daubentonii Kuhl, and the whiskered bat Myotis mystacinus Kuhl, which live in Karelia near the northern border of their distribution area. High contents of aerobic lactate dehydrogenase 1 and lactate dehydrogenase 2 isozymes have been detected in the skeletal muscle of the studied bats. The lactate dehydrogenase isozyme spectra of the tissues of kidneys and skeletal muscles from the smaller representatives of bats (the whiskered and Brandt’s bats) contained the highest content of H subunits among the studied species. In contrast, the predominance of M subunits has been revealed in the lactate dehydrogenase isozyme spectra of the kidneys of the northern and the brown long-eared bats. The discovered interspecies differences are discussed in the context of the adaptation of bats to hibernation.     

Evaporative water loss is a plausible explanation for mortality of bats from white-nose syndrome

White-nose syndrome (WNS) has caused alarming declines of North American bat populations in the 5 years since its discovery. Affected bats appear to starve during hibernation, possibly because of disruption of normal cycles of torpor and arousal. The importance of hydration state and evaporative water loss (EWL) for influencing the duration of torpor bouts in hibernating mammals recently led to "the dehydration hypothesis," that cutaneous infection of the wing membranes of bats with the fungus Geomyces destructans causes dehydration which in turn, increases arousal frequency during hibernation. This hypothesis predicts that uninfected individuals of species most susceptible to WNS, like little brown bats (Myotis lucifugus), exhibit high rates of EWL compared to less susceptible species. We tested the feasibility of this prediction using data from the literature and new data quantifying EWL in Natterer's bats (Myotis nattereri), a species that is, like other European bats, sympatric with G. destructans but does not appear to suffer significant mortality from WNS. We found that little brown bats exhibited significantly higher rates of normothermic EWL than did other bat species for which comparable EWL data are available. We also found that Natterer's bats exhibited significantly lower rates of EWL, in both wet and dry air, compared with values predicted for little brown bats exposed to identical relative humidity (RH). We used a population model to show that the increase in EWL required to cause the pattern of mortality observed for WNS-affected little brown bats was small, equivalent to a solitary bat hibernating exposed to RH of ～95%, or clusters hibernating in ～87% RH, as opposed to typical near-saturation conditions. Both of these results suggest the dehydration hypothesis is plausible and worth pursuing as a possible explanation for mortality of bats from WNS.