Hibernation Conditions Research Articles

Репродуктивная экология бесхвостых амфибий: влияние внутренних и внешних факторов

This research paper examines the influence of internal factors (female body weight, body condition) and external ecological factors on the reproductive parameters of females, as well as on the reproductive ecology of adult individuals of anuran amphibians. Among the most significant reproductive characteristics of females are the number of eggs in a clutch, the proportion of reproductive products to female body weight (contribution to reproduction), and the average weight of eggs. The body weight of the female and the ecological (meteorological) conditions of her activity during the year preceding spawning, as well as the hibernation conditions, have the greatest impact on the quantitative expression of these parameters. The proportion of reproductive products to female body weight in anuran amphibians usually ranges from 13 to 30%. Climate warming can directly affect the reproductive parameters of females, the survival of metamorphs, and the phenology of the spawning period.

Hibernating ground squirrels keep their mitochondria supercomplex

One of my favorite things about science fiction is when the genre takes inspiration from the natural world. For example, tales involving deep-space travel often begin with the protagonist awakening from a hibernation-like state – allowing them to get around the problem of aging as they traverse the stars. Here on Earth, some mammals use hibernation to decrease their metabolism to near-undetectable levels, which helps them survive low temperatures and lack of food during winter seasons. Achieving this kind of metabolic stasis in addition to prolonging survival after trauma are just two of the many important applications that studying hibernation could provide.One key to understanding how hibernators – such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) – lower their metabolism is to study how they adjust their mitochondria, as these organelles provide most of the energy (in the form of ATP) required for life and cellular activity. Until recently, it was believed that mitochondrial enzymes produce ATP by passing electrons around as they randomly collide into each other. But Amalie Hutchinson and colleagues from the University of Western Ontario in Canada wanted to understand whether these enzymes were consistently bound together – known as supercomplexing – and whether these associations change between seasons and as these animals lower their metabolism when they hibernate.The team captured thirteen-lined ground squirrels in Carman, MB, Canada, and brought them back to their lab in London, ON, Canada, where they were housed under conditions simulating summer temperatures (∼21°C) and natural day length in Carman. As winter approached, the scientists lowered the environmental temperature to 4°C and shortened the day length to 1 h light and 23 h dark to simulate winter hibernation conditions. They also collected mitochondria from the heart, liver and brown adipose tissue – which uses mitochondria to generate warmth – at three time points: summer, when the animals were active; when the animals had briefly arisen and were active during the winter hibernation; and during winter hibernation. The researchers then measured the amounts of supercomplexes and their protein make-up using techniques that separate mitochondrial enzymes but leave supercomplexes intact.The scientists found that rather than changing the total amount of supercomplexes between seasons or winter conditions these squirrels seem to shuttle one specific mitochondrial enzyme (complex III, which is a massive protein made up of 11 subunits) between larger and smaller supercomplexes. As these squirrels transitioned from being active in the winter to resuming hibernation, the amount of complex III in larger supercomplexes decreased in brown adipose tissue – which may be important for rapidly lowering their metabolism. In contrast, the amount of complex III in larger supercomplexes was greatest in the liver in winter, while complex III was more associated with smaller supercomplexes in the heart in the summer. These changes in heart and liver mitochondria could represent an important part of the seasonal changes in the metabolism of these animals.Hutchinson and colleagues have shown that something as remarkable as completely shutting down your metabolism could involve very subtle changes in mitochondrial supercomplexes. That said, it's probably going to be a while before we can use the lessons learned from these squirrels to safely send people into deep space. In the meantime, much remains to be learned about how these hibernators signal these shifts in enzyme interactions and their ultimate effects on mitochondrial function.

The reproductive response of Cornu aspersum to different hibernation conditions

ABSTRACT This study was conducted between February and May 2019 on mature Cornu aspersum in the Black Sea region, Turkey to investigate the effects of different hibernation situations on reproductive responses. Three snail groups were studied: NH (natural hibernation), ASH (artificial short hibernation) and ALH (artificial long hibernation). NH showed inactive behaviour and had no reproductive activity while ASH and ALH performed intensive reproduction. During the experiment, the mean spawning rate (SpR), egg number (EgN), hatching rate (HR) and offspring number (OfN) were 20.50 ± 1.69%, 100.10 ± 5.22, 85.63 ± 2.36 and 80.69 ± 5.44 in the ASH group; 23.43 ± 3.36%, 122.33 ± 7.62, 61.55 ± 10.76 and 70.26 ± 11.60 in the ALH group, respectively. In conclusion, the high temperatures in the winter months of 2019 may have caused NH snails to become reproductively inactive, or it could simply be a consequence of their life-history strategy. Snails in the ASH and ALH groups represented a resource allocation trade-off between fecundity and survival to carry the genetic information to the next generation. This study leads to a better understanding of land snail reproductive physiology and behavioural responses to different hibernation conditions.

Effect of roost management on populations trends of Rhinolophus hipposideros and Rhinolophus ferrumequinum in Britain and Ireland

After a significant population decline during the 20th century, populations of both greater and lesser horseshoe bats have increased over recent decades in Britain and populations of lesser horseshoe bats have increased in Ireland. Vincent Wildlife Trust (VWT) acquired 37 bat reserves since the 1980s with the aim to safeguard the sites and enhance the roosting and hibernation conditions in buildings that were often derelict and sub-optimal for bats. These measures have resulted in a strong population size increase of all colonies. However, populations have also been increasing throughout Britain and Ireland as a consequence of legal protection and milder winters resulting in higher survival rates. Therefore, it is not clear whether the measures that have taken place in VWT reserves have led to a greater increase than roosts that have not benefited from the same type of management. We aimed to compare population trends of horseshoe bat roosts under VWT management and non-VWT management from 1999 to 2020 in order to assess its effectiveness. For this, we analysed population trends at sites under different management types (VWT and non-VWT) for lesser horseshoe bats in Britain and Ireland and greater horseshoe bats in Britain. Our results indicated that populations in Britain under VWT management have increased by 366% (CI 225% - 580%) for greater horseshoe bats and 188% (CI 125% - 283%) for lesser horseshoe bats. Roosts that did not benefit from the same levels of management increased respectively by 164% (CI 132% - 199%) and 51% (CI 40% - 60%). In Ireland, populations of lesser horseshoe bats in VWT managed roosts increased by 217% (CI 118% - 364%) while non-VWT managed roosts remained stable (-0.44%; CI -23% - 29%). We conclude that management actions carried out by VWT of greater and lesser horseshoe bat roosts have helped populations recover at a faster rate by securing the integrity of buildings, improving access points and by providing optimal microclimatic conditions within the buildings.

Hibernation Conditions Contribute to the Differential Resistance to Cadmium between Urban and Forest Ant Colonies.

Simple SummaryThe resistance of organisms to trace metals can have a genetic or a plastic origin. Indeed, differential environmental conditions experienced before the exposure to trace metals could physiologically condition organisms and plastically enhance their subsequent resistance to trace metals. In this study on the ant Temnothorax nylanderi, we investigated whether the better cadmium resistance of urban colonies relative to forest colonies could originate from the distinct hibernation conditions that they experienced prior to cadmium exposure. We compared the ability of urban and forest colonies to resist cadmium depending on whether they had hibernated in their respective urban or forest habitats or under a laboratory common garden setup. We found that urban colonies resisted cadmium better than forest colonies when they had hibernated under a common garden. Surprisingly, this difference was not observed between urban and forest colonies that had hibernated in the field, in contrast with a previous study. One reason may be that winter was particularly mild on the year of our experiment. Our results therefore support the idea that urban colonies are genetically adapted to resist trace metals, but that this adaptation is only revealed under specific environmental conditions.Trace metals such as cadmium are found in high concentrations in urban environments. Animal and plant populations living in heavily contaminated environments could adapt to trace metals exposure. A recent study shows that urban populations of the acorn ant Temnothorax nylanderi are more resistant to cadmium than their forest counterparts. However, this study was performed using field colonies that had just come out of hibernation. Because urban and forest hibernation environments differ, the differential resistance to trace metals may originate either from differential hibernation conditions or from a different resistance baseline to cadmium. In this study, we tested these two hypotheses using laboratory common garden hibernation conditions. We let urban and forest colonies of the ant T. nylanderi hibernate under the same laboratory conditions for four months. After this hibernation period, we also collected field-hibernating colonies and we compared cadmium resistance between urban and forest colonies depending on the hibernation condition. We found a differential response to cadmium under common garden, with urban colonies displaying less larval mortality and lower size reduction of the produced individuals. This suggests a different resistance baseline of urban colonies to cadmium. However, unexpectedly, we did not detect the differential response between urban and forest colonies in the field, suggesting a more complex scenario involving both genetic and environmental influences.

Hibernation Records on the Incisor Surface in the Turkish Hamster (Mesocricetus brandti) (Rodentia, Cricetidae)

The teeth of an adult female of the Turkish hamster (Mesocricetus brandti) after hibernation in semi-natural conditions were examined to reveal a relationship between the hibernation pattern on the incisor surface and the data of a temperature logger. The female spent the winter in a burrow prepared by herself inside a wintering cage from October 2018 till May 2019. A temperature logger was implanted to the animal intraperitoneally to record body temperature at 20-min intervals. Regular bouts of hypothermia started in mid-October. The longevity of each hypothermia bout increased whereas the minimum body temperature decreased and reached its lowest value in March. Later, the process proceeded backwards until the end of the heterothermia period. In the apical region of the incisor, daily increments were wide and well-distinguished which indicates that they had formed before the onset of the heterothermal period. In the basal region, closer to the growth zone, the increments were narrow and distinct, and their number corresponded in general to the number of normothermia episodes bouts. At the very base of the incisor as well as at its apical part, typical daily increments were revealed again. However, no correlation between the longevity of normothermia bouts and the width of increments was found. These data varied greatly from two other hamsters. In the Ciscaucasian hamster (Mesocricetus raddei) the number and character of increments comply with the number and duration of normothermia bouts, in the Common hamster (Cricetus cricetus), the “hibernation zone” was not recorded on the incisor surface. The Turkish hamster demonstrate an intermediate pattern, which we explain by quite different hibernation conditions of these species.

Prosurvival roles mediated by the PERK signaling pathway effectively prevent excessive endoplasmic reticulum stress-induced skeletal muscle loss during high-stress conditions of hibernation.

Stress conditions like hypoxia, ischemia, and ischemia/reperfusion can trigger excessive endoplasmic reticulum stress (ERS), which can lead to cell apoptosis-induced skeletal muscle atrophy in non-hibernators. However, although hibernators experience multiple stress conditions during hibernation, their skeletal muscles appear to be well protected. We hypothesize that hibernators effectively avoid cell apoptosis, at least partially, by controlling ERS level. Here, we focused on the potential occurrence of ERS and how hibernators cope with it during different hibernation states. Results indicated that the protein expression levels of glucose-regulated protein 78 (GRP78), phosphorylated PKR-like ER protein kinase, phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), and activating transcription factor 4 were significantly increased during hibernation, but primarily recovered in posthibernation. In the torpor-arousal cycle, the expression levels of the above indicators were lower during inter-bout arousal (IBA) than that during late torpor (LT). However, there was no change in C/EBP homologous protein expression and no apoptosis in skeletal muscles during the different hibernation states. In conclusion, the upregulation of p-eIF2α and GRP78 were identified as two crucial mechanisms mediated by the PERK signaling pathway to alleviate elevated ERS. The downregulation of ERS during IBA may be a unique countermeasure for hibernating squirrels to prevent excessive ERS. Thus, these special anti-excessive ERS abilities of ground squirrels contribute to the prevention of skeletal muscle cell apoptosis during hibernation.

Alterations in the health of hibernating bats under pathogen pressure

In underground hibernacula temperate northern hemisphere bats are exposed to Pseudogymnoascus destructans, the fungal agent of white-nose syndrome. While pathological and epidemiological data suggest that Palearctic bats tolerate this infection, we lack knowledge about bat health under pathogen pressure. Here we report blood profiles, along with body mass index (BMI), infection intensity and hibernation temperature, in greater mouse-eared bats (Myotis myotis). We sampled three European hibernacula that differ in geomorphology and microclimatic conditions. Skin lesion counts differed between contralateral wings of a bat, suggesting variable exposure to the fungus. Analysis of blood parameters suggests a threshold of ca. 300 skin lesions on both wings, combined with poor hibernation conditions, may distinguish healthy bats from those with homeostatic disruption. Physiological effects manifested as mild metabolic acidosis, decreased glucose and peripheral blood eosinophilia which were strongly locality-dependent. Hibernating bats displaying blood homeostasis disruption had 2 °C lower body surface temperatures. A shallow BMI loss slope with increasing pathogen load suggested a high degree of infection tolerance. European greater mouse-eared bats generally survive P. destructans invasion, despite some health deterioration at higher infection intensities (dependant on hibernation conditions). Conservation measures should minimise additional stressors to conserve constrained body reserves of bats during hibernation.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 2. Control of the Akt pathway and protein translation machinery.

When faced with harsh environmental conditions, the South American marsupial, monito del monte (Dromiciops gliroides), reduces its body temperature and uses either daily torpor or multiday hibernation to survive. This study used ELISA and multiplex assays to characterize the responses to hibernation by three regulatory components of protein translation machinery [p-eIF2α(S51), p-eIF4E(S209), p-4EBP(Thr37/46)] and eight targets involved in upstream signaling control of translation [p-IGF-1R(Tyr1135/1136), PTEN(S380), p-Akt(S473), p-GSK-3α(S21), p-GSK-3β(S9), p-TSC2(S939), p-mTOR(S2448), and p70S6K(T412)]. Liver, brain and kidney were analyzed comparing control and hibernation (4days continuous torpor) conditions. In the liver, increased phosphorylation of IGF-1R, Akt, GSK-3β, TSC2, mTOR, eIF2α, and 4EBP (1.60-1.98 fold compared to control) occurred during torpor suggesting that the regulatory phosphorylation cascade and protein synthesis remained active during torpor. However, responses by brain and kidney differed; torpor resulted in increased phosphorylation of GSK-3β (2.15-4.17 fold) and TSC2 (2.03-3.65 fold), but phosphorylated Akt decreased (to 34-62% of control levels). Torpor also led to an increase in phosphorylated eIF2α (1.4 fold) content in the brain. These patterns of differential protein phosphorylation in brain and kidney were indicative of suppression of protein translation but also could suggest an increase in antioxidant and anti-apoptotic signaling during torpor. Previous studies of liver metabolism in hibernating eutherian mammals have shown that Akt kinase and its downstream signaling components play roles in facilitating hypometabolism by suppressing energy expensive anabolic processes during torpor. However, the results in this study reveal differences between eutherian and marsupial hibernators, suggesting alternative actions of liver Akt during torpor.

Sperm storage influences the potential for spontaneous acrosome reaction of the sperm in the newt Cynops pyrrhogaster.

Sperm storage is supposed to influence sperm quality, although the details remain unclear. In the present study, we found that sperm stored in a sperm storage site, the vas deferens of Cynops pyrrhogaster, spontaneously undergo acrosome reaction following incubation in Steinberg's salt solution (ST). Percentages of acrosome-reacted sperm increased time-dependently to about 60% in 24 hr. The concentration of cyclic adenosine monophosphate (cAMP) was elevated after incubating sperm in ST, while dibutylyl cAMP induced an acrosome reaction. Chelating of extracellular Ca2+ suppressed the dibutylyl cAMP-induced acrosome reaction as well as spontaneous acrosome reaction in ST. These results suggest that cAMP elevation driven by Ca2+ influx can be a cue for spontaneous acrosome reaction. Relatively low Ca2+ concentration and pH in the vas deferens were sufficient to suppress spontaneous acrosome reaction within 1 hr. In addition, the cysteine rich secretory protein 2 gene was expressed in the vas deferens, indicating that it may be involved in the continuous suppression of spontaneous acrosome reaction. Sperm that underwent spontaneous acrosome reaction in ST was significantly increased when stored in the vas deferens for longer periods, or by males experiencing temperatures in excess of 12°C during hibernation conditions. Percentages of the spontaneously acrosome-reacted sperm were found to differ among males even though they were of identical genetic background. Taken together, C. pyrrhogaster sperm possess the potential for spontaneous acrosome reaction that does not become obvious in the vas deferens, unless promoted in correlation with sperm storage.

Hypothermic neuroprotections in the brain of an echolocation bat, Hipposideros terasensis.

The present study aimed to investigate how bats protect their brain in a hypothermic situation. Formosan leaf-nosed bats (Hipposideros terasensis) were used in this study and treated under three conditions: room temperature (25±1°C), low temperature (4±1°C), and hibernation. The reactive oxygen species (ROS) levels in the blood and apoptosis-related proteins in the brain tissue were assessed and then compared among those bats under three conditions. Our results showed that the blood ROS levels of bats treated under conditions of low temperature and hibernation were significantly reduced compared with bats treated under the condition of room temperature. Both immunohistochemistry and immunoblotting expressions of hypoxia, inflammation, and apoptosis-related proteins in the brain tissue of bats treated under the condition of hibernation were significantly reduced compared with those bats treated under conditions of room temperature and low temperature. Thus, we suggested that bats can protect the brain in cold environment by reducing blood ROS levels and decreasing expressions of hypoxia, inflammation, and apoptosis-related proteins in the brain. Possible protection mechanisms involved in hypothermic adaptations need to be further clarified.

Experimental evidence for beneficial effects of projected climate change on hibernating amphibians

Amphibians are the most threatened vertebrates today, experiencing worldwide declines. In recent years considerable effort was invested in exposing the causes of these declines. Climate change has been identified as such a cause; however, the expectable effects of predicted milder, shorter winters on hibernation success of temperate-zone Amphibians have remained controversial, mainly due to a lack of controlled experimental studies. Here we present a laboratory experiment, testing the effects of simulated climate change on hibernating juvenile common toads (Bufo bufo). We simulated hibernation conditions by exposing toadlets to current (1.5 °C) or elevated (4.5 °C) hibernation temperatures in combination with current (91 days) or shortened (61 days) hibernation length. We found that a shorter winter and milder hibernation temperature increased survival of toads during hibernation. Furthermore, the increase in temperature and shortening of the cold period had a synergistic positive effect on body mass change during hibernation. Consequently, while climate change may pose severe challenges for amphibians of the temperate zone during their activity period, the negative effects may be dampened by shorter and milder winters experienced during hibernation.

Hypothalamic gene expression underlying pre-hibernation satiety.

Prior to hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) enter a hypophagic period where food consumption drops by an average of 55% in 3 weeks. This occurs naturally, while the ground squirrels are in constant environmental conditions and have free access to food. Importantly, this transition occurs before exposure to hibernation conditions (5°C and constant darkness), so the ground squirrels are still maintaining a moderate level of activity. In this study, we used the Illumina HiSeq 2000 system to sequence the hypothalamic transcriptomes of ground squirrels before and after the autumn feeding transition to examine the genes underlying this extreme change in feeding behavior. The hypothalamus was chosen because it is known to play a role in the control and regulation of food intake and satiety. Overall, our analysis identified 143 genes that are significantly differentially expressed between the two groups. Specifically, we found five genes associated with feeding behavior and obesity (VGF, TRH, LEPR, ADIPOR2, IRS2) that are all upregulated during the hypophagic period, after the feeding transition has occurred. We also found that serum leptin significantly increases in the hypophagic group. Several of the genes associated with the natural autumnal feeding decline in 13-lined ground squirrels show parallels to signaling pathways known to be disrupted in human metabolic diseases, like obesity and diabetes. In addition, many other genes were identified that could be important for the control of food consumption in other animals, including humans.

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal.

Throughout the hibernation season, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experiences extreme fluctuations in heart rate, metabolism, oxygen consumption, and body temperature, along with prolonged fasting and immobility. These conditions necessitate different functional requirements for the heart, which maintains contractile function throughout hibernation, and the skeletal muscle, which remains largely inactive. The adaptations used to maintain these contractile organs under such variable conditions serves as a natural model to study a variety of medically relevant conditions including heart failure and disuse atrophy. To better understand how two different muscle tissues maintain function throughout the extreme fluctuations of hibernation we performed Illumina HiSeq 2000 sequencing of cDNAs to compare the transcriptome of heart and skeletal muscle across the circannual cycle. This analysis resulted in the identification of 1,076 and 1,466 differentially expressed genes in heart and skeletal muscle, respectively. In both heart and skeletal muscle we identified a distinct cold-tolerant mechanism utilizing peroxisomal metabolism to make use of elevated levels of unsaturated depot fats. The skeletal muscle transcriptome also shows an early increase in oxidative capacity necessary for the altered fuel utilization and increased oxygen demand of shivering. Expression of the fetal gene expression profile is used to maintain cardiac tissue, either through increasing myocyte size or proliferation of resident cardiomyocytes, while skeletal muscle function and mass are protected through transcriptional regulation of pathways involved in protein turnover. This study provides insight into how two functionally distinct muscles maintain function under the extreme conditions of mammalian hibernation.

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus.

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h=1.70 and S0.5=6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K+ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h=1.16) and GLK affinity for substrate was reduced (S0.5=23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h=1.60, S0.5=9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h=1.20, S0.5=45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.

Rapid response of a long-lived species to improved water and grazing management: The case of the European pond turtle (Emys orbicularis) in the Camargue, France

Among human activities, the effect of habitat management by grazing on population viability is ambiguous. Indeed, beneficial effects of grazing are expected by maintaining open meadows, but overgrazing is supposed to increase mortality by trampling. Grazing has been shown to negatively impact the survival of European pond turtle (Emys orbicularis) in the Camargue. Consequently, a new management plan was defined. We investigated the consequences of this management using capture–recapture methods to estimate variations of population sizes in this managed site and a control site over a 17 years period. Results show an increase of the number of adults and juveniles on the managed site after the management change. Our results suggest that improved water management with flooding in autumn provided better hibernation conditions, and that reduced grazing intensity in autumn/winter likely decreased the risk of trampling. Population size significantly increased in less than 4 years following the management change, probably by the relaxation of density-dependence. It is an original result for a long lived-species supposed to have an important time of resilience to perturbations.

Detecting spring after a long winter: coma or slow vigilance in cold, hypoxic turtles?

Many freshwater turtle species can spend the winter submerged in ice-covered lakes by lowering their metabolism, and it has been proposed that such severe metabolic depression render these turtles comatose. This raises the question of how they can detect the arrival of spring and respond in a sensible way to sensory information during hibernation. Using evoked potentials from cold or hypoxic turtles exposed to vibration and light, we show that hibernating turtles maintain neural responsiveness to light stimuli during prolonged hypoxia. Furthermore, turtles held under hibernation conditions for 14 days increase their activity when exposed to light or elevated temperatures, but not to vibration or increased oxygen. It is concluded that hibernating turtles are not comatose, but remain vigilant during overwintering in cold hypoxia, allowing them to respond to the coming of spring and to adjust their behaviour to specific sensory inputs.

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.

Effect of starvation and hibernation on the values of five biomarkers of general and specific stress using the land snail Eobania vermiculata

Values of five biomarkers related to cell stress or pollution were evaluated in tissues of the land snail Eobania vermiculata under starvation or hibernation conditions. The biomarkers applied were lysosomal membrane stability in digestive gland cells (LMS) or in haemocytes (neutral red retention assay (NRR)), acetylcholinesterase activity (AchE; EC 3.1.1.7), metallothionein content (MTs), and cyclic AMP content (cAMP). Three groups of snails were studied that were kept under starvation, hibernation and normal conditions, respectively. The results indicated statistically lower values of LMS and NRR in snails kept under starvation or hibernation compared to control ones. Higher values of AChE activity were measured in snails under hibernation compared to controls. MT contents were statistically higher in snails under starvation compared to controls. Measurement of cAMP contents showed no significant differences among the tested groups. The values of the first four biomarkers may be affected by factors other than pollution, such as starvation or hibernation. Therefore, these factors should be taken into consideration when biomonitoring studies are performed in time intervals of hibernation or starvation.

Effect of Ambient Temperature in Neonate Aspic Vipers: Growth, Locomotor Performance and Defensive Behaviors

The impact of temperature during incubation and gestation has been tested in various reptiles; the postnatal period has been rarely investigated however. Three groups of newborn aspic vipers (Vipera aspis) were placed under contrasted thermal regimes during 7 months: (1) a cool 23°C constant regime, (2) a warm 28°C constant regime, and (3) an optimal regime with free-access to a wide range of temperatures. Later, all the snakes were placed under hibernation conditions (6°C) during 3 months. Finally all the snakes were placed in the optimal thermal regime during 2 additional months. The total duration of the experiment was of 12 months. Body mass and feeding rates were recorded weekly, body size was measured monthly. We also assessed locomotor performance and recorded several behavioral traits (e.g., defensive and predatory behaviors). As expected, snakes raised under cool temperatures exhibited low feeding rate, growth rate, body condition, and they exhibited poor locomotor performance; they also displayed marked defensive behaviors (e.g., high number of defensive bites) whilst hesitating during longer periods to bite a prey. Such behavioral effects were detected at the end of the experiment (i.e., 5 months after exposure to contrasted thermal treatments [3 months of hibernation plus 2 months of optimal regime]), revealing long term effects. Surprisingly, growth rate and locomotor performance were not different between the two other groups, warm constant 28°C versus optimal regimes (albeit several behavioral traits differed), suggesting that the access to a wide range of ambient temperatures was not a crucial factor.