Neurons that regulate mouse torpor.

The advent of endothermy, which is achieved through the continuous homeostatic regulation of body temperature and metabolism1,2, is a defining feature of mammalian and avian evolution. However, when challenged by food deprivation or harsh environmental conditions, many mammalian species initiate adaptive energy-conserving survival strategies-including torpor and hibernation-during which their body temperature decreases far below its homeostatic set-point3-5. How homeothermic mammals initiate and regulate these hypothermic states remains largely unknown. Here we show that entry into mouse torpor, a fasting-induced state with a greatly decreased metabolic rate and a body temperature as low as 20 °C6, is regulated by neurons in the medial and lateral preoptic area of the hypothalamus. We show that restimulation of neurons that were activated during a previous bout of torpor is sufficient to initiate the key features of torpor, even in mice that are not calorically restricted. Among these neurons we identify a population of glutamatergic Adcyap1-positive cells, the activity of which accurately determines when mice naturally initiate and exit torpor, and the inhibition of which disrupts the natural process of torpor entry, maintenance and arousal. Taken together, our results reveal a specific neuronal population in the mouse hypothalamus that serves as a core regulator of torpor. This work forms a basis for the future exploration of mechanisms and circuitry that regulate extreme hypothermic and hypometabolic states, and enables genetic access to monitor, initiate, manipulate and study these ancient adaptations of homeotherm biology.

Mitochondrial microRNA profiles are altered in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation.

Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system.NEW & NOTEWORTHY During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs.

Torpor use in the wild by one of the world's largest bats.

Torpor is widespread among bats presumably because most species are small, and torpor greatly reduces their high mass-specific resting energy expenditure, especially in the cold. Torpor has not been recorded in any bat species larger than 50 g, yet in theory could be beneficial even in the world's largest bats (flying-foxes; Pteropus spp.) that are exposed to adverse environmental conditions causing energy bottlenecks. We used temperature telemetry to measure body temperature in wild-living adult male grey-headed flying-foxes (P. poliocephalus; 799 g) during winter in southern Australia. We found that all individuals used torpor while day-roosting, with minimum body temperature reaching 27°C. Torpor was recorded following a period of cool, wet and windy weather, and on a day with the coldest maximum air temperature, suggesting it is an adaptation to reduce energy expenditure during periods of increased thermoregulatory costs and depleted body energy stores. A capacity for torpor among flying-foxes has implications for understanding their distribution, behavioural ecology and life history. Furthermore, our discovery increases the body mass of bats known to use torpor by more than tenfold and extends the documented use of this energy-saving strategy under wild conditions to all bat superfamilies, with implications for the evolutionary maintenance of torpor among bats and other mammals.

Heart rate monitoring reveals differential seasonal energetic trade-offs in male noctule bats.

Understanding how animals meet their daily energy requirements is critical in our rapidly changing world. Small organisms with high metabolic rates can conserve stored energy when food availability is low or increase energy intake when energetic requirements are high, but how they balance this in the wild remains largely unknown. Using miniaturized heart rate transmitters, we continuously quantified energy expenditure, torpor use and foraging behaviour of free-ranging male bats (Nyctalus noctula) in spring and summer. In spring, bats used torpor extensively, characterized by lowered heart rates and consequently low energy expenditures. In contrast, in summer, bats consistently avoided torpor, even though they could have used this low-energy mode. As a consequence, daytime heart rates in summer were three times as high compared with the heart rates in spring. Daily energy use increased by 42% during summer, despite lower thermogenesis costs at higher ambient temperatures. Likely, as a consequence, bats nearly doubled their foraging duration. Overall, our results indicate that summer torpor avoidance, beneficial for sperm production and self-maintenance, comes with a high energetic cost. The ability to identify and monitor such vulnerable energetic life-history stages is particularly important to predict how species will deal with increasing temperatures and changes in their resource landscapes.

c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

ROS and hypoxia signaling regulate periodic metabolic arousal during insect dormancy to coordinate glucose, amino acid, and lipid metabolism.

Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.

Nightly reductions in body temperature and effect of transmitter attachment method in free-living welcome swallows (Hirundoneoxena).

Small birds and mammals face similar energetic challenges, yet use of torpor to conserve energy while resting is considered less common among birds, especially within the most specious order Passeriformes. We conducted the first study to record the natural thermoregulatory physiology of any species from the family Hirundinidae, which we predicted would use torpor because of their specialised foraging by aerial pursuit of flying insects, that are less active during cold or windy weather. We used temperature telemetry on wild-living welcome swallows (Hirundo neoxena, 13 to 17 g) and found that skin temperature declined during nightly resting by an average by 5 °C, from daytime minima of 41.0 ± 0.8 °C to nightly minima of 36.3 ± 0.8 °C, and by a maximum of 8 °C to a minimum recorded skin temperature of 32.0 °C. The extent of reduction in skin temperature was greater on cold nights and following windy daytime (foraging) periods. Further, we found that transmitters glued directly to the skin between feather tracts (i.e., an apterium) provided a less variable and probably also more accurate reflection of body temperature than transmitters applied over closely trimmed feathers. A moderate decrease in skin temperature, equivalent to shallow torpor, would provide energy savings during rest. Yet, deeper torpor was not observed, despite a period of extreme rainfall that presumedly decreased foraging success. Further studies are needed to understand the resting thermoregulatory energetics of swallows under different environmental conditions. We advocate the importance of measuring thermal biology in wild-living birds to increase our knowledge of the physiology and ecological importance of torpor among passerine birds.

Warm spells in winter affect the equilibrium between winter phenotypes.

Each phenotype is a product of the interaction of the genes and the environment. Although winter phenotype in seasonal mammals is heritable, its development may be modified by external conditions. In today's world, global climate change and increasing frequency of unpredictable weather events may affect the dynamic equilibrium between phenotypes. We tested the effect of changes in ambient temperature during acclimation to short photoperiod on the development of winter phenotypes in three generations of Siberian hamsters (Phodopus sungorus). Based on seasonal changes in fur colour, body mass, and expression of daily torpor we distinguished three different winter phenotypes: responding, non-responding, and partially-responding to short photoperiod. We found that warm spells in winter can increase the proportion of non-responding individuals in the population, while stable winter conditions can increase photoresponsiveness among the offspring of non-responders. We conclude that the polymorphism of winter phenotype is an inherent characteristic of the Siberian hamster population but the development of winter phenotype is not fixed but rather a plastic response to the environmental conditions.

Sex-specific heterothermy patterns in wintering captive Microcebus murinus do not translate into differences in energy balance.

The physiological mechanisms of responses to stressors are at the core of ecophysiological studies that examine the limits of an organism's flexibility. Interindividual variability in these physiological responses can be particularly important and lead to differences in the stress response among population groups, which can affect population dynamics. Some observations of intersexual differences in heterothermy raise the question of whether there is a difference in energy management between the sexes. In this study, we assessed male and female differences in mouse lemurs (Microcebus murinus), a highly seasonal malagasy primate, by measuring their physiological flexibility in response to caloric restriction and examining the subsequent impact on reproductive success. Using complementary methods aiming to describe large-scale and daily variations in body temperature throughout a 6-month winter-like short-day (SD) period, we monitored 12 males and 12 females, applying chronic 40% caloric restriction (CR) to 6 individuals in each group. We found variations in Tb modulation throughout the SD period and in response to caloric treatment that depended on sex, as females, regardless of food restriction, and CR males, only, entered deep torpor. The use of deeper torpor, however, did not translate into a lower loss of body mass in females and did not affect reproductive success. Captive conditions may have buffered the depth of torpor and minimised the positive effects of torpor on energy savings. However, the significant sex differences in heterothermy we observed may point to physiological benefits other than preservation of energy reserves.

Neurons in the Dorsomedial Hypothalamus Promote, Prolong, and Deepen Torpor in the Mouse.

Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular, projections from the preoptic area of the hypothalamus to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity-dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the preoptic area of the hypothalamus and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor.SIGNIFICANCE STATEMENT Daily heterotherms, such as mice, use torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the CNS controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states.

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be 'aroused' to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.

Adrenal gene expression dynamics support hibernation in 13-lined ground squirrels.

Hibernation is a natural model of extreme physiology in a mammal. Throughout winter, small hibernators repeatedly undergo rapid, dramatic swings in body temperature, perfusion, and oxygen delivery. To gain insight into the molecular mechanisms that support homeostasis despite the numerous challenges posed by this dynamic physiology, we collected 13-lined ground squirrel adrenal glands from at least five individuals representing six key timepoints across the year using body temperature telemetry. Differentially expressed genes were identified using RNA-seq, revealing both strong seasonal and torpor-arousal cycle effects on gene expression. Two novel findings emerge from this study. First, transcripts encoding multiple genes involved in steroidogenesis decreased seasonally. Taken together with morphometric analyses, the data are consistent with preservation of mineralocorticoids but suppression of glucocorticoid and androgen output throughout winter hibernation. Second, a temporally orchestrated, serial gene expression program unfolds across the brief arousal periods. This program initiates during early rewarming with the transient activation of a set of immediate early response (IER) genes, comprised of both transcription factors and the RNA degradation proteins that assure their rapid turnover. This pulse in turn activates a cellular stress response program to restore proteostasis comprised of protein turnover, synthesis, and folding machinery. These and other data support a general model for gene expression across the torpor-arousal cycle that is facilitated in synchrony with whole body temperature shifts; induction of the immediate early response upon rewarming activates a proteostasis program followed by a restored tissue-specific gene expression profile enabling renewal, repair, and survival of the torpid state.NEW & NOTEWORTHY This pioneer study of adrenal gland gene expression dynamics in hibernating ground squirrels leverages the power of RNA-seq on multiple precisely timed samples to demonstrate: 1) steroidogenesis is seasonally reorganized to preserve aldosterone at the expense of glucocorticoids and androgens throughout winter hibernation; 2) a serial gene expression program unfolds during each short arousal whereby immediate early response genes induce the gene expression machinery that restores proteostasis and the cell-specific expression profile before torpor reentry.

Blood transcriptomics mirror regulatory mechanisms during hibernation-a comparative analysis of the Djungarian hamster with other mammalian species.

Hibernation enables many species of the mammalian kingdom to overcome periods of harsh environmental conditions. During this physically inactive state metabolic rate and body temperature are drastically downregulated, thereby reducing energy requirements (torpor) also over shorter time periods. Since blood cells reflect the organism´s current condition, it was suggested that transcriptomic alterations in blood cells mirror the torpor-associated physiological state. Transcriptomics on blood cells of torpid and non-torpid Djungarian hamsters and QIAGEN Ingenuity Pathway Analysis (IPA) revealed key target molecules (TMIPA), which were subjected to a comparative literature analysis on transcriptomic alterations during torpor/hibernation in other mammals. Gene expression similarities were identified in 148 TMIPA during torpor nadir among various organs and phylogenetically different mammalian species. Based on TMIPA, IPA network analyses corresponded with described inhibitions of basic cellular mechanisms and immune system-associated processes in torpid mammals. Moreover, protection against damage to the heart, kidney, and liver was deduced from this gene expression pattern in blood cells. This study shows that blood cell transcriptomics can reflect the general physiological state during torpor nadir. Furthermore, the understanding of molecular processes for torpor initiation and organ preservation may have beneficial implications for humans in extremely challenging environments, such as in medical intensive care units and in space.

Ecological drivers and consequences of torpor in Andean hummingbirds.

Daily torpor allows endotherms to save energy during energetically stressful (e.g. cold) conditions. Although studies on avian torpor have mostly been conducted under laboratory conditions, information on the usage of torpor in the wild is limited to few, predominantly temperate-zone species. We studied torpor under seminatural conditions from 249 individuals from 29 hummingbird species across a 1920 m elevational gradient in the western Andes of Colombia using cloacal thermistors. Small birds were more likely to use torpor than large birds, but only at low ambient temperatures, where torpor was prolonged. We also found effects of proxy variables for body condition and energy expenditure on the use of torpor, its characteristics, and impacts. Our results suggest that context-dependency and phylogenetic variation in the probability of deploying torpor can help understand clade-wide patterns of elevational distribution in Andean hummingbirds.

Role of glucose in daily torpor of Djungarian hamsters (Phodopus sungorus): challenge of continuous in vivo blood glucose measurements.

Djungarian hamsters use daily torpor to save energy during winter. This metabolic downstate is part of their acclimatization strategy in response to short photoperiod and expressed spontaneously without energy challenges. During acute energy shortage, torpor incidence, depth, and duration can be modulated. Torpor induction might rely on glucose availability as acute metabolic energy source. To investigate this, the present study provides the first continuous in vivo blood glucose measurements of spontaneous daily torpor in short photoperiod-acclimated and fasting-induced torpor in long photoperiod-acclimated Djungarian hamsters. Glucose levels were almost identical in both photoperiods and showed a decrease during resting phase. Further decreases appeared during spontaneous daily torpor entrance, parallel with metabolic rate but before body temperature, while respiratory exchange rates were rising. During arousal, blood glucose tended to increase, and pretorpor values were reached at torpor termination. Although food-restricted hamsters underwent a considerable energetic challenge, blood glucose levels remained stable during the resting phase regardless of torpor expression. The activity phase preceding a torpor bout did not reveal changes in blood glucose that might be used as torpor predictor. Djungarian hamsters show a robust, circadian rhythm in blood glucose irrespective of season and maintain appropriate levels throughout complex acclimation processes including metabolic downstates. Although these measurements could not reveal blood glucose as proximate torpor induction factor, they provide new information about glucose availability during torpor. Technical innovations like in vivo microdialysis and in vitro transcriptome or proteome analyses may help to uncover the connection between torpor expression and glucose metabolism.

High latitude northern bats (Eptesicus nilssonii) reveal adaptations to both high and low ambient temperatures.

Insectivorous bats at northern latitudes need to cope with long periods of no food for large parts of the year. Hence, bats which are resident at northern latitudes throughout the year will need to undergo a long hibernation season and a short reproductive season where foraging time is limited by extended daylight periods. Eptesicus nilssonii is the northernmost occurring bat species worldwide and hibernates locally when ambient temperatures (Ta) limit prey availability. Therefore, we investigated the energy spent maintaining normothermy at different Ta, as well as how much bats limit energy expenditure while in torpor. We found that, despite being exposed to Ta as low as 1.1°C, bats did not increase torpid metabolic rate, thus indicating that E. nilssonii can survive and hibernate at low ambient temperatures. Furthermore, we found a lower critical temperature (Tlc) of 27.8°C, which is lower than in most other vespertilionid bats, and we found no indication of any metabolic response to Ta up to 37.1°C. Interestingly, carbon dioxide production increased with increasing Ta above the Tlc, presumably caused by a release of retained CO2 in bats that remained in torpor for longer and aroused at Ta above the Tlc. Our results indicate that E. nilssonii can thermoconform at near-freezing Ta, and hence maintain longer torpor bouts with limited energy expenditure, yet also cope with high Ta when sun exposed in roosts during long summer days. These physiological traits are likely to enable the species to cope with ongoing and predicted climate change.

Saving energy via short and shallow torpor bouts.

Maintaining a high and stable body temperature as observed in most endothermic mammals and birds is energetically costly and many heterothermic species reduce their metabolic demands during energetic bottlenecks through the use of torpor. With the increasing number of heterotherms revealed in a diversity of habitats, it becomes apparent that triggers and patterns of torpor use are more variable than previously thought. Here, we report the previously overlooked use of, shallow rest-time torpor (body temperature >30 °C) in African lesser bushbabies, Galago moholi. Body core temperature of three adult male bushbabies recorded over five months showed a clear bimodal distribution with an average active modal temperature of 39.2 °C and a resting modal body temperature of 36.7 °C. Shallow torpor was observed in two out of three males (n = 29 torpor bouts) between June and August (austral winter), with body temperatures dropping to an overall minimum of 30.7 °C and calculated energy savings of up to 10%. We suggest that shallow torpor may be an ecologically important, yet mostly overlooked energy-saving strategy employed by heterothermic mammals. Our data emphasise that torpor threshold temperatures need to be used with care if we aim to fully understand the level of physiological plasticity displayed by heterothermic species.

Determining the different phases of torpor from skin- or body temperature data in heterotherms.

Technological innovations have made heat-sensitive data-loggers smaller, more efficient and less expensive, which has led to a growing body of literature that measures the skin- or body temperatures of small animals in their natural environments. Studies of this type on heterothermic endotherms have prompted much debate regarding how to best define 'torpor' expressions from skin- or body temperature data alone. We propose a new quantitative method for defining torpor 'entries', 'arousals' and 'stable torpor periods' whilst comparing the results to 'torpor bout' durations identified using only the torpor cut-off method. By decomposing a torpor bout into 'entries', 'stable torpor periods', and 'active arousals', we avoid biases introduced by using strict threshold temperature values for the onset of torpor, thereby allowing better insight into individual use of torpor. We present our method as an easy-to-use function written in R-code, offering an un-biased and consistent methodology to be applied on skin- or body temperature measurements across datasets and research groups. When testing the function on a large dataset of skin temperature data collected on three bat species in Norway (Plecotus auritus: Nind = 39; Eptesicus nilssonii: Nind = 11; Myotis brandtii: Nind = 10), we identified 461 complete torpor bouts across species. More than 40% of the torpor bouts (Nbouts = 192) did not contain stable torpor periods, because the bats aroused before they had reached a stable skin temperature level. Furthermore, only considering 'torpid' and 'euthermic' temperature values by applying strict cut-off thresholds led to potentially large underestimations of torpor bout durations compared to our quantitative determination of the onset and termination of each torpor bout. We highlight the importance of differentiating between torpor phases, especially for active arousals that can be very energetically expensive and may alter our evaluation of the actual energetic savings gained by an individual employing torpor.

Free-living Allen's hummingbirds (Selasphorus sasin) rarely use torpor while nesting.

For reproducing animals, maintaining energy balance despite thermoregulatory challenges is important for surviving and successfully raising offspring. This is especially apparent in small endotherms that exhibit high mass-specific metabolic rates and live in unpredictable environments. Many of these animals use torpor, substantially reducing their metabolic rate and often body temperature to cope with high energetic demands during non-foraging periods. In birds, when the incubating parent uses torpor, the lowered temperatures that thermally sensitive offspring experience could delay development or increase mortality risk. We used thermal imaging to noninvasively explore how nesting female hummingbirds sustain their own energy balance while effectively incubating their eggs and brooding their chicks. We located 67 active Allen's hummingbird (Selasphorus sasin) nests in Los Angeles, California and recorded nightly time-lapse thermal images at 14 of these nests for 108 nights using thermal cameras. We found that nesting females usually avoided entering torpor, with one bird entering deep torpor on two nights (2% of nights), and two other birds possibly using shallow torpor on three nights (3% of nights). We also modeled nightly energetic requirements of a bird experiencing nest temperatures vs. ambient temperature and using torpor or remaining normothermic, using data from similarly-sized broad-billed hummingbirds. Overall, we suggest that the warm environment of the nest, and possibly shallow torpor, help brooding female hummingbirds reduce their own energy requirements while prioritizing the energetic demands of their offspring.

Bat-derived cells use glucose as a cryoprotectant.

Evolution of heterothermy in environments with variable temperatures has allowed bats to survive food scarcity during seasonal climatic extremes by using torpor as a hibernation strategy. The controlled reduction of body temperature and metabolism through complex behavioural and physiological adaptations at organismal, organ, cellular and molecular levels includes the ability of tissues and cells to adapt to temperature alterations. Based on the prediction that cells of different tissues cultured in vitro would differ in their ability to withstand freezing and thawing of the medium, we determined the survival rate of bat-derived cells following exposure to -20 °C for 24 h in media with no cryoprotective agents or medium supplemented by glucose in concentration range 0-3333 mM. Cell survival rates were determined in relation to availability of glucose in the medium, organ origin, cell concentration and bat species. In general, increased glucose helped cells survive at sub-zero temperatures, though concentrations up to 80-fold higher than those found in chiropterans were needed. However, cells in glucose-free phosphate buffered saline also survived, suggesting that other mechanisms may be contributing to cell survival at low temperatures. Highest in vitro viability was observed in nervus olfactorius-derived cell cultures, with high survival rates and rapid re-growth under optimal conditions after exposure to -20 °C. Kidney cells from different bat species showed comparable overall survival rate patterns, though smaller chiropteran species appeared to utilise lower glucose levels as a cryoprotectant than larger species. Our in vitro data provide evidence that cells of heterothermic bats can survive sub-zero temperatures and that higher glucose levels in important tissues significantly improve hibernation survival at extremely low temperatures.