Oxidative and osmotolerant effects in Salvator merianae (Squamata: Teiidae) red blood cells during hibernation / Efeitos de oxidação e osmotolerância dos eritrócitos de Salvator merianae (Squamata: Teiidae) durante a hibernação

Abstract Hibernation is a natural condition of animals that lives in the temperate zone, although some tropical lizards also experience hibernation annually, such as the lizard native from South America, Salvator merianae, or "tegu" lizard. Even though physiological and metabolic characteristic associated with hibernation have been extensively studied, possible alterations in the red blood cells (RBC) integrity during this period remains unclear. Dehydration and fasting are natural consequences of hibernating for several months and it could be related to some cellular modifications. In this study, we investigated if the osmotic tolerance of RBCs of tegu lizard under hibernation is different from the cells obtained from animals while normal activity. Additionally, we indirectly investigated if the RBCs membrane of hibernating tegus could be associated with oxidation by quantifying oxidized biomolecules and the activity of antioxidant enzymes. Our findings suggest that RBCs are more fragile during the hibernation period, although we did not find evidence of an oxidative stress scenario associated with the accentuated fragility. Even though we did not exclude the possibility of oxidative damage during hibernation, we suggested that an increased RBCs volume as a consequence of hypoosmotic blood during hibernation could also affect RBCs integrity as noted.

Resumo A hibernação é uma condição natural dos animais que vivem na zona temperada, embora alguns lagartos tropicais também experenciem hibernação anualmente, como é o caso do lagarto nativo da América do Sul, Salvator merianae ou "teiú". Embora as características fisiológicas e metabólicas associadas à hibernação tenham sido amplamente estudadas, possíveis alterações na integridade das hemácias durante esse período ainda permanecem obscuras. A desidratação e o jejum são consequências naturais da hibernação por vários meses e podem estar relacionadas a algumas modificações celulares. Neste estudo, investigamos se a tolerância osmótica de hemácias do lagarto teiú sob hibernação são diferentes das células obtidas de animais em atividade normal. Além disso, investigamos indiretamente por meio da quantificação de biomoléculas oxidadas e da atividade de enzimas antioxidantes se a membrana das hemácias dos teiús em hibernação poderia estar associada à oxidação. Nossos resultados sugerem que as hemácias possuem maior fragilidade durante o período de hibernação, embora não tenhamos encontrado evidências de um cenário de estresse oxidativo associado à essa fragilidade acentuada. Embora não tenhamos excluído a possibilidade de dano oxidativo durante a hibernação, sugerimos que um aumento no volume das hemácias como consequência de sangue hipoosmótico durante a hibernação também poderia afetar a integridade de hemácias, tal como foi observado.

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.

Seasonal Variation of Immunohematological Parameters of the Peripheral Blood in the Pond Bat Myotis dasycneme (Boie, 1825) of the Urals.

Immunohematological parameters were for the first time studied in the pond bat Myotis dasycneme (Boie, 1825), the most common chiropteran species in the Urals, during seasonal periods of the annual life cycle. Multivariate nonparametric analysis of variance showed the absence of significant gender differences in red blood cell parameters (p = 0.35). Gender differences were observed in the counts of white blood cells, band neutrophils, segments neutrophils, and lymphocytes in the blood (p < 0.05). Males showed a higher development of innate immunity in summer compared with females (p < 0.05). A high lymphocyte level (50.6-53.5%) was observed in both males and females in the autumn-winter hibernation period, providing immune surveillance and specific reactive activation of the acquired adaptive immune response.

Peptidomic Analysis Reveals Seasonal Neuropeptide and Peptide Hormone Changes in the Hypothalamus and Pituitary of a Hibernating Mammal.

During the winter, hibernating mammals undergo extreme changes in physiology, which allow them to survive several months without access to food. These animals enter a state of torpor, which is characterized by decreased metabolism, near-freezing body temperatures, and a dramatically reduced heart rate. The neurochemical basis of this regulation is largely unknown. Based on prior evidence suggesting that the peptide-rich hypothalamus plays critical roles in hibernation, we hypothesized that changes in specific cell-cell signaling peptides (neuropeptides and peptide hormones) underlie physiological changes during torpor/arousal cycles. To test this hypothesis, we used a mass spectrometry-based peptidomics approach to examine seasonal changes of endogenous peptides that occur in the hypothalamus and pituitary of a model hibernating mammal, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). In the pituitary, we observed changes in several distinct peptide hormones as animals prepared for torpor in October, exited torpor in March, and progressed from spring (March) to fall (August). In the hypothalamus, we observed an overall increase in neuropeptides in October (pre-torpor), a decrease as the animal entered torpor, and an increase in a subset of neuropeptides during normothermic interbout arousals. Notable changes were observed for feeding regulatory peptides, opioid peptides, and several peptides without well-established functions. Overall, our study provides critical insight into changes in endogenous peptides in the hypothalamus and pituitary during mammalian hibernation that were not available from transcriptomic measurements. Understanding the molecular basis of the hibernation phenotype may pave the way for future efforts to employ hibernation-like strategies for organ preservation, combating obesity, and treatment of stroke.

Individual Variation in Parturition Timing within and among Years for a Bat Maternity Colony.

BACKGROUND: In monoestrous species, the timing of reproduction can have important impacts on offspring survival. For heterotherms in temperate areas, parturition timing is constrained by cold weather survival strategies, such as hibernation and torpor. Female bats that are year-round residents of temperate regions, such as little brown myotis (Myotis lucifugus), invest significantly in parental care resulting in sharp changes in behavior immediately following parturition. These behavior changes may include increases in nighttime roost revisits, which can be used to identify parturition dates for individual bats that have been passive integrated transponder (PIT) tagged and use monitored roosts. METHODS: Using a system of tagged bats and monitored roosts in Pynn's Brook and Salmonier Nature Park Newfoundland, Canada, we estimated parturition dates for 426 female M. lucifugus in at least one year, based on changes in nighttime roost revisit patterns, and quantified the variation in parturition dates within years among individuals, and within individuals among years. RESULTS: Overall, we report on a wide variation in parturition dates within years among individuals as well as year-to-year variations, both across the population and within individuals. Spring weather conditions appeared to be important influences on parturition timing. CONCLUSIONS: Changes in spring and summer temperature and extreme weather events, as expected due to ongoing climate change, may impact parturition timing, and therefore, offspring survival of temperate bats.

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.

Feeding during hibernation shifts gene expression toward active season levels in brown bears (Ursus arctos).

Hibernation in bears involves a suite of metabolical and physiological changes, including the onset of insulin resistance, that are driven in part by sweeping changes in gene expression in multiple tissues. Feeding bears glucose during hibernation partially restores active season physiological phenotypes, including partial resensitization to insulin, but the molecular mechanisms underlying this transition remain poorly understood. Here, we analyze tissue-level gene expression in adipose, liver, and muscle to identify genes that respond to midhibernation glucose feeding and thus potentially drive postfeeding metabolical and physiological shifts. We show that midhibernation feeding stimulates differential expression in all analyzed tissues of hibernating bears and that a subset of these genes responds specifically by shifting expression toward levels typical of the active season. Inferences of upstream regulatory molecules potentially driving these postfeeding responses implicate peroxisome proliferator-activated receptor gamma (PPARG) and other known regulators of insulin sensitivity, providing new insight into high-level regulatory mechanisms involved in shifting metabolic phenotypes between hibernation and active states.

Elevated plasma phospholipid n-3 docosapentaenoic acid concentrations during hibernation.

Factors for initiating hibernation are unknown, but the condition shares some metabolic similarities with consciousness/sleep, which has been associated with n-3 fatty acids in humans. We investigated plasma phospholipid fatty acid profiles during hibernation and summer in free-ranging brown bears (Ursus arctos) and in captive garden dormice (Eliomys quercinus) contrasting in their hibernation patterns. The dormice received three different dietary fatty acid concentrations of linoleic acid (LA) (19%, 36% and 53%), with correspondingly decreased alpha-linolenic acid (ALA) (32%, 17% and 1.4%). Saturated and monounsaturated fatty acids showed small differences between summer and hibernation in both species. The dormice diet influenced n-6 fatty acids and eicosapentaenoic acid (EPA) concentrations in plasma phospholipids. Consistent differences between summer and hibernation in bears and dormice were decreased ALA and EPA and marked increase of n-3 docosapentaenoic acid and a minor increase of docosahexaenoic acid in parallel with several hundred percent increase of the activity index of elongase ELOVL2 transforming C20-22 fatty acids. The highest LA supply was unexpectantly associated with the highest transformation of the n-3 fatty acids. Similar fatty acid patterns in two contrasting hibernating species indicates a link to the hibernation phenotype and requires further studies in relation to consciousness and metabolism.

Cardiac Rhythms and Variation in Hibernating Arctic Ground Squirrels.

AbstractThe dramatic decrease in heart rate (HR) during entrance into hibernation is not a mere response to the lowering of core body temperature (Tb) but a highly regulated fall, as the decrease in HR precedes the drop in Tb. This regulated fall in HR is thought to be mediated by increased cardiac parasympathetic activity. Conversely, the sympathetic nervous system is thought to drive the increase of HR during arousal. Despite this general understanding, we lack temporal information on cardiac parasympathetic regulation throughout a complete hibernation bout. The goal of this study was to fill this gap in knowledge by using Arctic ground squirrels implanted with electrocardiogram/temperature telemetry transmitters. Short-term HR variability (root mean square of successive differences [RMSSD]), an indirect measure of cardiac parasympathetic regulation, was calculated in 11 Arctic ground squirrels. RMSSD, normalized as RMSSD/RR interval (RRI), increased fourfold during early entrance (from 0.2±0.1 to 0.8±0.2, P<0.05). RMSSD/RRI peaked after HR dropped by over 90% and Tb fell by 70%. Late entrance was delineated by a decline in RMSSD/RRI while Tb continued to decrease. During arousal, HR started to increase 2 h before Tb, with a concurrent decrease in RMSSD/RRI to a new minimum. As Tb increased to a maximum during interbout arousal, HR declined, and RMSSD/RRI increased. These data suggest that activation of the parasympathetic nervous system initiates and regulates the HR decrease during entrance into hibernation and that withdrawal of parasympathetic activation initiates arousal. We conclude that cardiac parasympathetic regulation persists throughout all phases of a hibernation bout-a feature of the autonomic nervous system's regulation of hibernation that was not appreciated previously.

A multi-tissue gene expression dataset for hibernating brown bears.

OBJECTIVES: Complex physiological adaptations often involve the coordination of molecular responses across multiple tissues. Establishing transcriptomic resources for non-traditional model organisms with phenotypes of interest can provide a foundation for understanding the genomic basis of these phenotypes, and the degree to which these resemble, or contrast, those of traditional model organisms. Here, we present a one-of-a-kind gene expression dataset generated from multiple tissues of two hibernating brown bears (Ursus arctos). DATA DESCRIPTION: This dataset is comprised of 26 samples collected from 13 tissues of two hibernating brown bears. These samples were collected opportunistically and are typically not possible to attain, resulting in a highly unique and valuable gene expression dataset. In combination with previously published datasets, this new transcriptomic resource will facilitate detailed investigation of hibernation physiology in bears, and the potential to translate aspects of this biology to treat human disease.

Fatty acid metabolization and insulin regulation prevent liver injury from lipid accumulation in Himalayan marmots.

Fat storage and weight gain are dominant traits for hibernating mammals. However, excessive fat accumulation may cause liver damage. Here, we explore the lipid accumulation and metabolic processes of the Himalayan marmot (Marmota himalayana), a hibernating rodent species. We find that the unsaturated fatty acid (UFA) content in food was consistent with a large increase in the body mass of Himalayan marmots. Metagenomic analysis shows that Firmicutes Bacterium CAG:110 plays a synergistic role by synthesizing UFAs, which is demonstrated by fecal transplantation experiments, indicating that the gut microbiome promotes fat storage in Himalayan marmots for hibernation. Microscopic examination results indicate that the risk of fatty liver appears at maximum weight; however, liver function is not affected. Upregulations of UFA catabolism and insulin-like growth factor binding protein genes provide an entry point for avoiding liver injury.

Seasonal assembly of skin microbiota driven by neutral and selective processes in the greater horseshoe bat.

Skin microbiota play an important role in protecting bat hosts from the fungal pathogen Pseudogymnoascus destructans, which has caused dramatic bat population declines and extinctions. Recent studies have provided insights into the bacterial communities of bat skin, but variation in skin bacterial community structure in the context of the seasonal dynamics of fungal invasion, as well as the processes that drive such variation, remain largely unexplored. In this study, we characterized bat skin microbiota over the course of the bat hibernation and active season stages and used a neutral model of community ecology to determine the relative roles of neutral and selective processes in driving microbial community variation. Our results showed significant seasonal shifts in skin community structure, as well as less diverse microbiota in hibernation than in the active season. Skin microbiota were influenced by the environmental bacterial reservoir. During both the hibernation and active season stages, more than 78% of ASVs in bat skin microbiota were consistent with neutral distribution, implying that neutral processes, that is, dispersal or ecological drift contributing the most to shifts in skin microbiota. In addition, the neutral model showed that some ASVs were actively selected by the bats from the environmental bacterial reservoir, accounting for approximately 20% and 31% of the total community during hibernation and active season stages, respectively. Overall, this research provides insights into the assemblage of bat-associated bacterial communities and will aid in the development of conservation strategies against fungal disease.

COULD WHITE-NOSE SYNDROME MANIFEST DIFFERENTLY IN MYOTIS LUCIFUGUS IN WESTERN VERSUS EASTERN REGIONS OF NORTH AMERICA? A REVIEW OF FACTORS.

White-nose syndrome (WNS) has notably affected the abundance of Myotis lucifugus (little brown myotis) in North America. Thus far, substantial mortality has been restricted to the eastern part of the continent where the cause of WNS, the invasive fungus Pseudogymnoascus destructans, has infected bats since 2006. To date, the state of Washington is the only area in the Western US or Canada (the Rocky Mountains and further west in North America) with confirmed cases of WNS in bats, and there the disease has spread more slowly than it did in Eastern North America. Here, we review differences between M. lucifugus in western and eastern parts of the continent that may affect transmission, spread, and severity of WNS in the West and highlight important gaps in knowledge. We explore the hypothesis that western M. lucifugus may respond differently to WNS on the basis of different hibernation strategies, habitat use, and greater genetic structure. To document the effect of WNS on M. lucifugus in the West most effectively, we recommend focusing on maternity roosts for strategic disease surveillance and monitoring abundance. We further recommend continuing the challenging work of identifying hibernation and swarming sites to better understand the microclimates, microbial communities, and role in disease transmission of these sites, as well as the ecology and hibernation physiology of bats in noncavernous hibernacula.

Energy and time optimization during exit from torpor in vertebrate endotherms.

Torpor is used in small sized birds and mammals as an energy conservation trait. Considerable effort has been put towards elucidating the mechanisms underlying its entry and maintenance, but little attention has been paid regarding the exit. Firstly, we demonstrate that the arousal phase has a stereotyped dynamic: there is a sharp increase in metabolic rate followed by an increase in body temperature and, then, a damped oscillation in body temperature and metabolism. Moreover, the metabolic peak is around two-fold greater than the corresponding euthermic resting metabolic rate. We then hypothesized that either time or energy could be crucial variables to this event and constructed a model from a collection of first principles of physiology, control engineering and thermodynamics. From the model, we show that the stereotyped pattern of the arousal is a solution to save both time and energy. We extended the analysis to the scaling of the use of torpor by endotherms and show that variables related to the control system of body temperature emerge as relevant to the arousal dynamics. In this sense, the stereotyped dynamics of the arousal phase necessitates a certain profile of these variables which is not maintained as body size increases.

Sodium current preserves electrical excitability in the heart of hibernating ground squirrel (Citellus undulatus).

Hibernating mammals are capable of maintaining normal cardiac function at low temperatures. Excitability of cardiac myocytes crucially depends on the fast sodium current (INa), which is decreased in hypothermia due to both depolarization of resting membrane potential and direct negative effect of low temperature. Therefore, INa in hibernating mammals should have specific features allowing to maintain excitability of myocardium at low temperatures. The current-voltage dependence of INa, its steady-state inactivation and activation and recovery from inactivation were studied in winter hibernating (WH) and summer active (SA) ground squirrels and in rats using whole-cell patch clamp at 10 °C and 20 °C. INa peak amplitude and the parameters of steady-state activation and inactivation curves did not differ between SA and WH ground squirrels at both temperatures. However, at both temperatures strong positive shift of activation and inactivation curves by 5-12 mV was observed in both WH and SA ground squirrels if compared to rats. This peculiarity of cardiac INa in ground squirrels helps to maintain excitability in conditions of depolarized resting membrane potential. The time course of INa recovery from inactivation at 10 °C was faster in WH than in SA ground squirrels, which could ensure normal activation of myocardium during hibernation.

Liver transcriptomic and methylomic analyses identify transcriptional mitogen-activated protein kinase regulation in facultative hibernation of Syrian hamster.

Hibernation consists of alternating torpor-arousal phases, during which animals cope with repetitive hypothermia and ischaemia-reperfusion. Due to limited transcriptomic and methylomic information for facultative hibernators, we here conducted RNA and whole-genome bisulfide sequencing in liver of hibernating Syrian hamster (Mesocricetus auratus). Gene ontology analysis was performed on 844 differentially expressed genes and confirmed the shift in metabolic fuel utilization, inhibition of RNA transcription and cell cycle regulation as found in seasonal hibernators. Additionally, we showed a so far unreported suppression of mitogen-activated protein kinase (MAPK) and protein phosphatase 1 pathways during torpor. Notably, hibernating hamsters showed upregulation of MAPK inhibitors (dual-specificity phosphatases and sproutys) and reduced levels of MAPK-induced transcription factors (TFs). Promoter methylation was found to modulate the expression of genes targeted by these TFs. In conclusion, we document gene regulation between hibernation phases, which may aid the identification of pathways and targets to prevent organ damage in transplantation or ischaemia-reperfusion.

Climate change is altering the physiology and phenology of an arctic hibernator.

Climate warming is rapid in the Arctic, yet impacts to biological systems are unclear because few long-term studies linking biophysiological processes with environmental conditions exist for this data-poor region. In our study spanning 25 years in the Alaskan Arctic, we demonstrate that climate change is affecting the timing of freeze-thaw cycles in the active layer of permafrost soils and altering the physiology of arctic ground squirrels (Urocitellus parryii). Soil freeze has been delayed and, in response, arctic ground squirrels have delayed when they up-regulate heat production during torpor to prevent freezing. Further, the termination of hibernation in spring has advanced 4 days per decade in females but not males. Continued warming and phenological shifts will alter hibernation energetics, change the seasonal availability of this important prey species, and potentially disrupt intraspecific interactions.

Out of thin air: surveying tropical bat roosts through air sampling of eDNA.

Understanding roosting behaviour is essential to bat conservation and biomonitoring, often providing the most accurate methods of assessing bat population size and health. However, roosts can be challenging to survey, e.g., physically impossible to access or presenting risks for researchers. Disturbance during monitoring can also disrupt natural bat behaviour and present material risks to the population such as disrupting hibernation cycles. One solution to this is the use of non-invasive monitoring approaches. Environmental (e)DNA has proven especially effective at detecting rare and elusive species particularly in hard-to-reach locations. It has recently been demonstrated that eDNA from vertebrates is carried in air. When collected in semi-confined spaces, this airborne eDNA can provide remarkably accurate profiles of biodiversity, even in complex tropical communities. In this study, we deploy novel airborne eDNA collection for the first time in a natural setting and use this approach to survey difficult to access potential roosts in the neotropics. Using airborne eDNA, we confirmed the presence of bats in nine out of 12 roosts. The identified species matched previous records of roost use obtained from photographic and live capture methods, thus demonstrating the utility of this approach. We also detected the presence of the white-winged vampire bat (Diaemus youngi) which had never been confirmed in the area but was long suspected based on range maps. In addition to the bats, we detected several non-bat vertebrates, including the big-eared climbing rat (Ototylomys phyllotis), which has previously been observed in and around bat roosts in our study area. We also detected eDNA from other local species known to be in the vicinity. Using airborne eDNA to detect new roosts and monitor known populations, particularly when species turnover is rapid, could maximize efficiency for surveyors while minimizing disturbance to the animals. This study presents the first applied use of airborne eDNA collection for ecological analysis moving beyond proof of concept to demonstrate a clear utility for this technology in the wild.

Ground squirrels - experts in thermoregulatory adaptation.

Ground squirrels exemplify one of the most extreme forms of mammalian hibernation and a convenient model for studying its mechanisms. Their thermoregulatory system demonstrates remarkable adaptive capabilities by maintaining optimal levels of body temperature both in active and hibernation states. Here, we review recent findings and unresolved issues regarding the neural mechanisms of body temperature control in ground squirrels.