An assessment of seasonal bait uptake by individual grey squirrels to develop a delivery system for oral contraceptives.

Globally, human-wildlife conflicts continue to increase, owing to human population growth and expansion. Many of these conflicts concern the impacts of invasive non-native species. In the UK, the invasive, non-native grey squirrel Sciurus carolinensis negatively affects tree health and has caused the decline of the native red squirrel Sciurus vulgaris. Oral contraceptives are being developed to manage the impacts of the grey squirrel. To be effective, contraceptives will need to be deployed at a landscape scale, and will require a delivery system that is practical and economically viable. Understanding grey squirrel feeding behaviour is important so that delivery methods can be designed so that a sufficient number of target individuals receive an effective contraceptive dose at a time of year that will ensure their infertility throughout peak times of breeding. The main aims of this study were to assess how sex, season, squirrel density and bait point density influenced; (1) the probability of a squirrel visiting a feeder and (2) the amount of bait consumed from feeders. Field trials were conducted on six woodland populations of squirrels in three seasons, with four days of bait deployment via purpose-designed squirrel-specific bait hoppers with integrated PIT-tag readers. It was possible to deliver multiple doses on most days to most male and female grey squirrels, with bait deployment more likely to be effective in spring, immediately before the second annual peak in squirrel breeding, followed by winter, immediately before the first peak in breeding. The results from this study could be used to design methods for delivering oral contraceptive baits to grey squirrels in the future and the methods used could be applied to other small mammal species and other bait delivery systems. © 2024 Crown copyright and The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

Microbial urea-nitrogen recycling in arctic ground squirrels: the effect of ambient temperature of hibernation.

Energy conservation associated with hibernation is maximized at the intersection of low body temperature (Tb), long torpor bouts, and few interbout arousals. In the arctic ground squirrel (Urocitellus parryii), energy conservation during hibernation is best achieved at ambient temperatures (Ta) around 0 °C; however, they spend the majority of hibernation at considerably lower Ta. Because arctic ground squirrels switch to mixed fuel metabolism, including protein catabolism, at extreme low Ta of hibernation, we sought to investigate how microbial urea-nitrogen recycling is used under different thermal conditions. Injecting squirrels with isotopically labeled urea (13C/15N) during hibernation at Ta's of - 16 °C and 2 °C and while active and euthermic allowed us to assess the ureolytic activity of gut microbes and the amount of liberated nitrogen incorporated into tissues. We found greater incorporation of microbially-liberated nitrogen into tissues of hibernating squirrels. Although ureolytic activity appears higher in euthermic squirrels, liberated nitrogen likely makes up a smaller percentage of the available nitrogen pool in active, fed animals. Because non-lipid fuel is a limiting factor for torpor at lower Ta in this species, we hypothesized there would be greater incorporation of liberated nitrogen in animals hibernating at - 16 °C. However, we found higher microbial-ureolytic activity and incorporation of microbially-liberated nitrogen, particularly in the liver, in squirrels hibernating at 2 °C. Likely this is because squirrels hibernating at 2 °C had higher Tb and longer interbout arousals, a combination of factors creating more favorable conditions for gut microbes to thrive and maintain greater activity while giving the host more time to absorb microbial metabolites.

The effect of temperature and breathing pattern on the surface activity of ground squirrel pulmonary surfactant.

This study investigates how hibernation affects the surface activity of pulmonary surfactant with respect to temperature and breathing pattern. Surfactant was isolated from a hibernating species, the 13-lined ground squirrel, and a homeotherm, the rabbit, and analysed for biophysical properties on a constrained sessile drop surfactometer. The results showed that surfactant from ground squirrels reduced surface tension better at low temperatures, including when mimicking episodic breathing, as compared with rabbit surfactant. In addition, low temperature adaptation was also observed using only the hydrophobic components of surfactant from ground squirrels. Overall, the data support the conclusion that ground squirrel surfactant has adapted to maintain surface activity during low temperature episodic breathing patterns, and that temperature adaptation is maintained with the hydrophobic components of the surfactant.

IGF-1 and myostatin-mediated co-regulation in skeletal muscle and bone of Daurian ground squirrels (Spermophilus dauricus) during different hibernation stages.

Muscle and bone are cooperatively preserved in Daurian ground squirrels (Spermophilus dauricus) during hibernation. As such, we hypothesized that IGF-1 and myostatin may contribute to musculoskeletal maintenance during this period. Thus, we systematically assessed changes in the protein expression levels of IGF-1 and myostatin, as well as their corresponding downstream targets, in the vastus medialis (VM) muscle and femur in Daurian ground squirrels during different stages. Group differences were determined using one-way analysis of variance (ANOVA). Results indicated that the co-localization levels of IGF-1 and its receptor (IGF-1R) increased by 50% during the pre-hibernation period (PRE) and by 35% during re-entry into torpor (RET) compared to the summer active period (SA). The phosphorylation level of FOXO1 in the VM muscle increased by 50% in the torpor (TOR) group and by 82% in the inter-bout arousal (IBA) group compared to the PRE group. The phosphorylation level of SGK-1 increased by 54% in the IBA group and by 62% in the RET group compared to the SA group. In contrast, the protein expression of IGF-1 and phosphorylation levels of PI3K, Akt, mTOR, and GSK3ß in the VM muscle showed no obvious differences among the different groups. ß-catenin protein expression was up-regulated by 84% in the RET group compared to the SA group, while the content of IGF-1 protein, correlation coefficients of IGF-1 and IGF-1R, and phosphorylation levels of PI3K, Akt, and GSK3ß in the femur showed no significant differences among groups. Regarding myostatin and its downstream targets, myostatin protein expression decreased by 70% in the RET group compared to the SA group, whereas ActRIIB protein expression and Smad2/3 phosphorylation in the VM muscle showed no obvious differences among groups. Furthermore, Smad2/3 phosphorylation decreased by 58% in the TOR group and 53% in the RET group compared to the SA group, whereas ActRIIB protein expression in the femur showed no obvious differences among groups. Overall, the observed changes in IGF-1 and myostatin expression and their downstream targets may be involved in musculoskeletal preservation during hibernation in Daurian ground squirrels.

Hypothalamic hormone deficiency enables physiological anorexia in ground squirrels during hibernation.

Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) lasts for 5-7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24-48-h periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger during IBAs. These squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons exhibit reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

High levels of mitochondrial dynamics, autophagy, and apoptosis contribute to stable testicular status in hibernating Daurian ground squirrels.

Daurian ground squirrels (Spermophilus dauricus) experience various stress states during winter hibernation, but the impact on testicular function remains unclear. This study focused on the effects of changes in testicular autophagy, apoptosis, and mitochondrial homeostasis signaling pathways at various stages on the testes of Daurian ground squirrels. Results indicated that: (1) During winter hibernation, there was a significant increase in seminiferous tubule diameter and seminiferous epithelium thickness compared to summer. Spermatogonia number and testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were higher during inter-bout arousal, suggesting that the testes remained stable during hibernation. (2) An increased number of mitochondria with intact morphology were observed during hibernation, indicating that mitochondrial homeostasis may contribute to testicular stability. (3) DNA fragmentation was evident in the testes during the hibernation and inter-bout arousal stages, with the highest level of caspase3 enzyme activity detected during inter-bout arousal, together with elevated levels of Bax/Bcl-2 and Lc3 II/Lc3 I, indicating an up-regulation of apoptosis and autophagy signaling pathways during hibernation. (4) The abundance of DRP1, MFF, OPA1, and MFN2 proteins was increased, suggesting an up-regulation of mitochondrial dynamics-related pathways. Overall, testicular autophagy, apoptosis, and mitochondrial homeostasis-related signaling pathways were notably active in the extreme winter environment. The well-maintained mitochondrial morphology may favor the production of reproductive hormones and support stable testicular morphology.

Invasive shrubs differentially alter autumnal activity for three common small-mammal species.

Seasonal variation in animal activity influences fitness and the intensity of ecological interactions (e.g., competition, predation), yet aspects of global change in the Anthropocene may catalyze shifts in seasonal activity. Invasive plants are components of global change and can modify animal daily activity, but their influence on animal seasonal activity is less understood. We examined how invasive woody shrubs (Autumn olive [Elaeagnus umbellata] and Amur honeysuckle [Lonicera maackii]) affect seasonal activity of three common small-mammal species by coupling experimental shrub removal with autumnal camera trapping for two consecutive years at six paired forest sites (total 12 plots). Eastern chipmunks (Tamias striatus) foraged more, and foraging was observed at least 20 days longer, in shrub-invaded forests. White-footed mice (Peromyscus leucopus) foraged more in invaded than cleared plots in one study year, but P. leucopus autumn activity timing did not differ between shrub-removal treatments. Fox squirrel (Sciurus niger) activity displayed year-specific responses to shrub removal suggesting intraannual cues (e.g., temperature) structure S. niger autumnal activity. Our work highlights how plant invasions can have species-specific effects on seasonal animal activity, may modify the timing of physiological processes (e.g., torpor), and could generate variation in animal-mediated interactions such as seed dispersal or granivory.

Population density and vegetation resources influence demography in a hibernating herbivorous mammal.

Demography of herbivorous mammal populations may be affected by changes in predation, population density, harvesting, and climate. Whereas numerous studies have focused on the effect of single environmental variables on individual demographic processes, attempts to integrate the consequences of several environmental variables on numerous functional traits and demographic rates are rare. Over a 32-year period, we examined how forage availability (vegetation assessed through NDVI) and population density affected the functional traits and demographic rates of a population of Columbian ground squirrels (Urocitellus columbianus), a herbivorous hibernating rodent. We focused on mean population phenology, body mass, breeding success, and survival. We found a negative effect of population density on demographic rates, including on breeding success and pup and adult survival to the next year. We found diverging effects of vegetation phenology on demographic rates: positive effects of a later start of the growing season on adult and yearling female survival, and juvenile survival, but no clear effect on male survival. Interestingly, neither population density nor vegetation affected population phenology or body condition in the following year. Vegetative growth rate had a positive influence on female mass gain (somatic investment) over a season, but both vegetative growth rate and biomass, surprisingly, had negative effects on the survival of young through their first hibernation. Thus, ground squirrels appeared to benefit more from later timing of vegetation than increases in vegetative biomass per se. Our study provides evidence for complex ecological effects of vegetation and population density on functional traits and demographic rates of small mammal populations.

'Ripple effects' of urban environmental characteristics on cognitive performances in Eurasian red squirrels.

Urban areas are expanding exponentially, leading more species of wildlife living in urban environments. Urban environmental characteristics, such as human disturbance, induce stress for many wildlife and have been shown to affect some cognitive traits, such as innovative problem-solving performance. However, because different cognitive traits have common cognitive processes, it is possible that urban environmental characteristics may directly and indirectly affect related cognitive traits (the ripple effect hypothesis). We tested the ripple effect hypothesis in urban Eurasian red squirrels residing in 11 urban areas that had different urban environmental characteristics (direct human disturbance, indirect human disturbance, areas of green coverage and squirrel population size). These squirrels were innovators who had previously repeatedly solved a food extraction task (the original task). Here, we examined whether and how urban environmental characteristics would directly and indirectly influence performance in two related cognitive traits, generalisation and (long-term) memory. The generalisation task required the innovators to apply the learned successful solutions when solving a similar but novel problem. The memory task required them to recall the learned solution of the original task after an extended period of time. Some of the selected urban environmental characteristics directly influenced the task performance, both at the population level (site) and at individual levels. Urban environmental characteristics, such as increased direct and indirect human disturbance, decreased the proportion of success in solving the generalisation task or the memory task at the population (site) level. Increased direct human disturbance and less green coverage increased the solving efficiency at individual levels. We also found an indirect effect in one of the urban environmental characteristics, indirect human disturbance, in the generalisation task, but not the memory task. Such an effect was only seen at the individual level but not at the population level; indirect human disturbance decreased the first original latency, which then decreased the generalisation latency across successes. Our results partially support the ripple effect hypothesis, suggesting that urban environmental characteristics are stressors for squirrels and have a greater impact on shaping cognitive performance than previously shown. Together, these results provide a better understanding of cognitive traits that support wildlife in adapting to urban environments.

Hit Snooze: An Imperiled Hibernator Assesses Spring Snow Conditions to Decide Whether to Terminate Hibernation or Reenter Torpor.

AbstractMany animals follow annual cycles wherein physiology and behavior change seasonally. Hibernating mammals undergo one of the most drastic seasonal alterations of physiology and behavior, the timing of which can have significant fitness consequences. The environmental cues regulating these profound phenotypic changes will heavily influence whether hibernators acclimate and ultimately adapt to climate change. Hence, identifying the cues and proximate mechanisms responsible for hibernation termination timing is critical. Northern Idaho ground squirrels (Urocitellus brunneus)-a rare, endemic species threatened with extinction-exhibit substantial variation in hibernation termination phenology, but it is unclear what causes this variation. We attached geolocators to free-ranging squirrels to test the hypothesis that squirrels assess surface conditions in spring before deciding whether to terminate seasonal heterothermy or reenter torpor. Northern Idaho ground squirrels frequently reentered torpor following a brief initial emergence from hibernacula and were more likely to do so earlier in spring or when challenged by residual snowpack. Female squirrels reentered torpor when confronted with relatively shallow snowpack upon emergence, whereas male squirrels reentered torpor in response to deeper spring snowpack. This novel behavior was previously assumed to be physiologically constrained in male ground squirrels by testosterone production required for spermatogenesis and activated by the circannual clock. Assessing surface conditions to decide when to terminate hibernation may help buffer these threatened squirrels against climate change. Documenting the extent to which other hibernators can facultatively alter emergence timing by reentering torpor after emergence will help identify which species are most likely to persist under climate change.

Long-term, medium-term and acute stress response of urban populations of Eurasian red squirrels affected by different levels of human disturbance.

Animals in urban areas often encounter novel and potentially stressful conditions. It is important to understand how wildlife cope with anthropogenic disturbance. To investigate this specific adaptation we live-trapped squirrels in two study sites in Warsaw: a forest reserve and an urban park and we estimated stress responses at three levels: long-term and medium-term stress (the level of stress hormones, i.e. cortisol and cortisone concentrations, in hair and feces) and acute reaction to human-induced stress (measured during handling with the aid of the three indices: breath rate, struggle rate, and vocalization). According to GLMM models no difference in the stress hormones level was found between the two populations. The only differences in cortisol concentrations clearly depended on the season, i.e. being higher in autumn and winter comparying to other seasons. There was no influence of sex, or reproductive status on stress hormones. Forest squirrels had significantly higher breath rates, suggesting they were more stressed by handling. There was no difference in the struggle rate between study areas, this index was mostly affected by season (i.e. being highest in winter). First-trapped squirrels vocalized less than during the subsequent trappings. Assumingly, during the first, and more stressful trapping, squirrels used 'freezing' and/or little vocalization, while during next captures they used alarm calls to warn conspecifics. Overall, we showed that the two squirrel populations differed only in terms of their breath rate. This suggests that they did not differ in medium-term and long-term stress in general, but they can differ in acute response to handling. This also suggests that both populations were similarly affected by environmental factors. The lack of clear effects may also be due to population heterogeneity. Thus, in order to assess the effects of anthropogenic stressors a broader range of indicators and diverse analytical methods, including behavioral analyses, should be employed.

A future food boom rescues the negative effects of early-life adversity on adult lifespan in a small mammal.

Early-life adversity, even when transient, can have lasting effects on individual phenotypes and reduce lifespan across species. If these effects can be mitigated by a high-quality later-life environment, then differences in future resources may explain variable resilience to early-life adversity. Using data from over 1000 wild North American red squirrels, we tested the hypothesis that the costs of early-life adversity for adult lifespan could be offset by later-life food abundance. We identified six adversities that reduced juvenile survival in the first year of life, though only one-birth date-had continued independent effects on adult lifespan. We then built a weighted early-life adversity (wELA) index integrating the sum of adversities and their effect sizes. Greater weighted early-life adversity predicted shorter adult lifespans in males and females, but a naturally occurring food boom in the second year of life ameliorated this effect. Experimental food supplementation did not replicate this pattern, despite increasing lifespan, indicating that the buffering effect of a future food boom may hinge on more than an increase in available calories. Our results suggest a non-deterministic role of early-life conditions for later-life phenotype, highlighting the importance of evaluating the consequences of early-life adversity in the context of an animal's entire life course.

Down-regulating the stress axis: Living in the present while preparing for the future.

The measurement of glucocorticoid (GC) hormones provides us with a window into the stress physiology of vertebrates and the adaptative responses they use to cope with predictable and unpredictable changes in the environment. Baseline GCs inform us about the metabolic demands they are subject to at that point in their yearly life-history stage, whereas GC changes (often increases) in response to acute challenges inform us on their capacity to cope with more immediate environmental challenges. However, baseline GC levels and the kinetics of GC responses to acute stressors can vary substantially among and within species, depending on individual characteristics (age, sex, condition, life-history stage). In addition, a thorough understanding of the stress status of an animal requires moving beyond the measurement of GCs alone by focusing on downstream measures of metabolic activation, such as oxidative stress. Here, we evaluated the changes in blood cortisol and oxidative stress markers in wild adult Columbian ground squirrels (Urocitellus columbianus), following a 30-min capture-handling stress performed in mid-late June. Measurements were taken when males were post-reproductive and preparing for hibernation and adult females were weaning litters. We found three key results. First, the time-course of GC increase was markedly slower (by an order of magnitude) than what is currently reported in the literature for most species of mammals, birds and reptiles. Second, there were marked differences in the male and female response, linked to differences in life-history stage: females close to weaning had abolished GC responses, whereas post-reproductive males did not. Third, there were mild to moderate increases in oxidative damage and decreases in oxidative defenses in response to our short-term challenge, consistent with the idea that short-term acute metabolic activation may carry physiological costs. However, these changes were not correlated to the changes in GCs, a novel result suggesting a disconnect between the hormonal stress response and oxidative damage.

Paper towel shredding as a novel, affordable, noninvasive method for detecting arousals in hibernating rodents.

Many research groups explore the regulation of hibernation or compare the physiology of heterothermic mammals between the torpid and aroused, euthermic states. Current methods for monitoring torpor (for example, infrared cameras, body temperature or heart-rate telemetry, and motion sensing) are costly, require specialized techniques, and can be invasive. Here we present an alternate method for determining torpor-bout duration that is cost-effective, noninvasive and accurate: paper towel shredding. In the winter, euthermic thirteen-lined ground squirrels will shred paper towels placed in the cage, but torpid animals will not. The presence of a shredded paper towel, indicating an arousal from torpor, is easily evaluated during routine daily monitoring. In 12 animals over 52 days, this simple technique detected 59 arousals with 100% accuracy when compared with the body temperature telemetry of the same animals. Moreover, this novel method avoids some of the drawbacks of other cheap monitoring systems such as the sawdust technique.

Ammonia Accumulation as a Proxy to Determine Cage-change Frequency in Antelope Ground Squirrels (Ammospermophilus leucurus).

Due to a lack of evidence-based standards for cage-change intervals for antelope ground squirrels (AGS, Ammospermophilus leucurus), we evaluated cage ammonia accumulation in our colony of adult, wild-caught AGS and identified factors that influenced ammonia levels. Intracage ammonia was measured daily in singly housed AGS in static caging that contained a running wheel and 1/2, 3/4, 1, or 2 quart (qt) of corncob bedding. Cages were changed when ammonia levels reached greater than 50ppm, our upper acceptable limit for ammonia based on mouse studies of ammonia aversion and toxicity. We also measured average daily water consumption over 2 wk to examine any correlation between water use and ammonia accumulation. We hypothesized that the desert-dwelling AGS would not reach intracage ammonia levels of greater than 50ppm in a 2-wk interval at any bedding volume. Our data showed that intracage ammonia was highly variable among individuals and was significantly associated with water consumption and bedding volumes. Seventeen percent of AGS on 1/2qt of bedding and 18% on 3/4qt of bedding reached greater than 50ppm ammonia before 7 d. All AGS on 1 and 2qt of bedding remained below 50ppm ammonia for 1 wk. Even when maintained on 2qt of bedding, not all AGS remained below 50ppm ammonia for 2 wk. Therefore, we concluded that the most appropriate option was weekly cage change for singly housed AGS on 1qt of bedding in static caging.

Plasticity changes in iron homeostasis in hibernating Daurian ground squirrels (Spermophilus dauricus) may counteract chronically inactive skeletal muscle atrophy.

Disuse-induced muscular atrophy is frequently accompanied by iron overload. Hibernating animals are a natural animal model for resistance to disuse muscle atrophy. In this paper, we explored changes in skeletal muscle iron content of Daurian ground squirrels (Spermophilus dauricus) during different periods of hibernation as well as the regulatory mechanisms involved. The results revealed that compared with the summer active group (SA), iron content in the soleus muscle (SOL) decreased (- 65%) in the torpor group (TOR), but returned to normal levels in the inter-bout arousal (IBA); splenic iron content increased in the TOR group (vs. SA, + 67%), decreased in the IBA group (vs. TOR, - 37%). Expression of serum hepcidin decreased in the TOR group (vs. SA, - 22%) and returned to normal levels in the IBA groups; serum ferritin increased in the TOR group (vs. SA, + 31%), then recovered in the IBA groups. Soleus muscle transferrin receptor 1 (TfR1) expression increased in the TOR group (vs. SA, + 83%), decreased in the IBA group (vs. TOR, - 30%); ferroportin 1 increased in the IBA group (vs. SA, + 55%); ferritin increased in the IBA group (vs. SA, + 42%). No significant differences in extensor digitorum longus in iron content or iron metabolism-related protein expression were observed among the groups. Significantly, all increased or decreased indicators in this study returned to normal levels after the post-hibernation group, showing remarkable plasticity. In summary, avoiding iron overload may be a potential mechanism for hibernating Daurian ground squirrels to avoid disuse induced muscular atrophy. In addition, the different skeletal muscle types exhibited unique strategies for regulating iron homeostasis.

Immune interactions and heterogeneity in transmission drives the pathogen-mediated invasion of grey squirrels in the UK.

Mathematical models highlighted the importance of pathogen-mediated invasion, with the replacement of red squirrels by squirrelpox virus (SQPV) carrying grey squirrels in the UK, a well-known example. In this study, we combine new epidemiological models, with a range of infection characteristics, with recent longitudinal field and experimental studies on the SQPV dynamics in red and grey squirrel populations to better infer the mechanistic basis of the disease interaction. A key finding is that a model with either partial immunity or waning immunity and reinfection, where individuals become seropositive on the second exposure to infection, that up to now has been shown in experimental data only, can capture the key aspects of the field study observations. By fitting to SQPV epidemic observations in isolated red squirrel populations, we can infer that SQPV transmission between red squirrels is significantly (4×) higher than the transmission between grey squirrels and as a result our model shows that disease-mediated replacement of red squirrels by greys is considerably more rapid than replacement in the absence of SQPV. Our findings recover the key results of the previous model studies, which highlights the value of simple strategic models that are appropriate when there are limited data, but also emphasise the likely complexity of immune interactions in wildlife disease and how models can help infer disease processes from field data.

Neural control of fluid homeostasis is engaged below 10°C in hibernation.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate for several months each winter without access to water,1 but the mechanisms that maintain fluid homeostasis during hibernation are poorly understood. In torpor, when body temperature (TB) reaches 4°C, squirrels decrease metabolism, slow heart rate, and reduce plasma levels of the antidiuretic hormones arginine vasopressin (AVP) and oxytocin (OXT).1 Squirrels spontaneously undergo interbout arousal (IBA) every 2 weeks, temporarily recovering an active-like metabolism and a TB of 37°C for up to 48 h.1,2 Despite the low levels of AVP and OXT during torpor, profound increases in blood pressure and heart rate during the torpor-IBA transition are not associated with massive fluid loss, suggesting the existence of a mechanism that protects against diuresis at a low TB. Here, we demonstrate that the antidiuretic hormone release pathway is activated by hypothalamic supraoptic nucleus (SON) neurons early in the torpor-arousal transition. SON neuron activity, dense-core vesicle release from the posterior pituitary, and plasma hormone levels all begin to increase before TB reaches 10°C. In vivo fiber photometry of SON neurons from hibernating squirrels, together with RNA sequencing and c-FOS immunohistochemistry, confirms that SON is electrically, transcriptionally, and translationally active to monitor blood osmolality throughout the dynamic torpor-arousal transition. Our work emphasizes the importance of the antidiuretic pathway during the torpor-arousal transition and reveals that the neurophysiological mechanism that coordinates the hormonal response to retain fluid is active at an extremely low TB, which is prohibitive for these processes in non-hibernators.

Graded alarm call behavior in wild fox squirrels (Sciurus niger).

Distress or alarm calls are vocalizations made when animals are in stressful situations or faced with a predator. Squirrels (Sciuridae) are known for being very vocal; however, most studies on alarm vocalizations are limited to ground squirrels. We investigated the acoustic behavior of the arboreal fox squirrel (Sciurus niger) under different conditions. Specifically, we tested the hypothesis that fox squirrels modify acoustic alarm behavior in response to different perceived threat levels and that this response is affected by sex and individual experience. Squirrels were trapped, and acoustic data were collected during periods in which the squirrels were alone, approached by humans, manipulated in traps, and handled by humans. Calls were categorized based on acoustic features, and we quantified the call rate (calls/s) across conditions. Threat level significantly affected vocal rate, with squirrels producing more calls overall when alone but shifting the proportion of emitted call types as threat level increased. Sex, capture history, and individual had no effect on call rate. These results suggest that fox squirrels use a graded alarm call response system to respond to threatening situations.

The effects of nickel chloride on papillary muscle contractility under normothermic and hypothermic conditions: Comparison of active and hibernating ground squirrels (Urocitellus undulatus) with Wistar rats.

Extracellular Ca2+ plays a pivotal role in the regulation of cardiac contractility under normal and extreme conditions. Here, by using nickel chloride (NiCl2), a non-specific blocker of extracellular Ca2+ influx, we studied the input of extracellular Ca2+ on the regulation of papillary muscle (PM) contractility under normal and hypothermic conditions in ground squirrels (GS), and rats. By measuring isometric force of contraction, we studied how NiCl2 affects force-frequency relationship and the rest effect in PM of these species at 30 °C and 10 °C. We found that at 30 °C 1.5 mM NiCl2 significantly reduced force of contraction across entire frequency range in active GS and rats, whereas in hibernating GS force of contraction was reduced at low and high frequency range. Additionally, NiCl2 evoked spontaneous contractility in rats but not GS PM. The rest effect was significantly reduced by NiCl2 for active GS and rats but not hibernating GS. At 10 °C, NiCl2 fully reduced contractility in active GS and, to a lesser extent, in rats, whereas in hibernating GS it was significant only at 0.3 Hz. The rest effect was significantly reduced by NiCl2 in both active and hibernating GS, whereas it was unmasked in rats that had high contractility under hypothermic conditions in control. Our results show a significant contribution of extracellular Ca2+ to myocardial contractility in GS not only in active but also in hibernating states, especially under hypothermic conditions, whereas limitation of extracellular Ca2+ influx in rats under hypothermia can play protective role for myocardial contractility.