Arctic Ground Squirrels Research Articles

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P > 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pre-hibernation diet alters skeletal muscle relaxation kinetics, but not force development in torpid arctic ground squirrels.

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as - 3°C), during which they are mostly physically inactive. Once Tb reaches ~ 15°C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~ 35°C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω-6 and the ω-6:ω-3 PUFA ratio correlate with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. We hypothesized that diet (increased ω-6:ω-3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. (1) Standard rodent chow with an ω-6:ω-3 ratio of 5:1, or (2) a balanced diet with an ω-6:ω-3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4°C, 15°C, 25°C, and 37°C to determine functional resilience. Our data show that torpid animals fed standard rodent chow have faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω-6:ω-3 PUFA ratio improves skeletal muscle performance during decreased temperatures in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Microbial gene expression during hibernation in arctic ground squirrels: greater differences across gut sections than in response to pre-hibernation dietary protein content.

Obligate seasonal hibernators fast for 5-9months depending on species yet resist muscle atrophy and emerge with little lean mass loss. The role of the gut microbiome in host nitrogen metabolism during hibernation is therefore of considerable interest, and recent studies support a role for urea nitrogen salvage (UNS) in host-protein conservation. We were interested in the effect of pre-hibernation diet on UNS and the microbial provision of essential amino acids (EAAs) during hibernation; therefore, we conducted a study whereby we fed arctic ground squirrels (Urocitellus parryii) pre-hibernation diets containing 9% vs. 18% protein and compared the expression of gut bacterial urease and amino acid (AA) metabolism genes in 4 gut sections (cecum mucosa, cecum lumen, small intestine [SI] mucosa, and SI lumen) during hibernation. We found that pre-hibernation dietary protein content did not affect expression of complete bacterial AA pathway genes during hibernation; however, several individual genes within EAA pathways were differentially expressed in squirrels fed 18% pre-hibernation dietary protein. Expression of genes associated with AA pathways was highest in the SI and lowest in the cecum mucosa. Additionally, the SI was the dominant expression site of AA and urease genes and was distinct from other sections in its overall microbial functional and taxonomic composition. Urease expression in the gut microbiome of hibernating squirrels significantly differed by gut section, but not by pre-hibernation dietary protein content. We identified two individual genes that are part of the urea cycle and involved in arginine biosynthesis, which were significantly more highly expressed in the cecum lumen and SI mucosa of squirrels fed a pre-hibernation diet containing 18% protein. Six bacterial genera were responsible for 99% of urease gene expression: Cupriavidus, Burkholderia, Laribacter, Bradhyrizobium, Helicobacter, and Yersinia. Although we did not find a strong effect of pre-hibernation dietary protein content on urease or AA metabolism gene expression during hibernation, our data do suggest the potential for pre-hibernation diet to modulate gut microbiota function during hibernation, and further investigations are warranted.

Non-shivering thermogenesis is differentially regulated during the hibernation season in Arctic ground squirrels.

Arctic ground squirrels are small mammals that experience physiological extremes during the hibernation season. Body temperature rises from 1°C to 40°C during interbout arousal and requires tight thermoregulation to maintain rheostasis. Tissues from wild-caught Arctic ground squirrels were sampled over 9months to assess the expression of proteins key to thermogenic regulation. Animals were sacrificed while aroused, and the extensor digitorum longus, diaphragm, brown adipose tissue, and white adipose tissue were probed using Western blots to assess protein expression and blood was sampled for metabolite analysis. Significant seasonal expression patterns emerged showing differential regulation. Contrary to our prediction, white adipose tissue showed no expression of uncoupling protein 1, but utilization of uncoupling protein 1 peaked in brown adipose tissue during the winter months and began to taper after terminal arousal in the spring. The opposite was true for muscular non-shivering thermogenesis. Sarco/endoplasmic reticulum calcium ATPase 1a and 2a expressions were depressed during the late hibernation season and rebounded after terminal arousal in diaphragm tissues, but only SERCA2a was differentially expressed in the extensor digitorum longus. The uncoupler, sarcolipin, was only detected in diaphragm samples and had a decreased expression during hibernation. The differential timing of these non-shivering pathways indicated distinct functions in maintaining thermogenesis which may depend on burrow temperature, availability of endogenous resources, and other seasonal activity demands on these tissues. These results could be impacted by fiber type makeup of the muscles collected, the body weight of the animal, and the date of entrance or exit from hibernation.

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.

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 to , ). 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.

Characterization and Seasonal Modulation of Adenosine A1 Receptors in the Arctic Ground Squirrel Brain.

Hibernation is an adaptation that allows animals such as the Arctic ground squirrel (AGS) to survive the absence of food or water during the winter season. Understanding mechanisms of metabolic suppression during hibernation torpor promises new therapies for critical care. The activation of the Adenosine A1 receptor (A1AR) has been shown to be necessary and sufficient for entrance into hibernation with a winter season sensitization to the agonist, but the role of the A1AR in seasonal sensitization is unknown. In the current study, we characterize the A1AR in the forebrain, hippocampus and hypothalamus of summer and torpid AGS. For the first time, we define the pharmacological characteristics of the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in the AGS brain. In addition, we test the hypothesis that increased A1AR agonist efficacy is responsible for sensitization of the A1AR during the torpor season. The resulting 35S-GTPγS binding data indicate an increase in agonist potency during torpor in two out of three brain regions. In addition to 35S-GTPγS binding, [3H]DPCPX saturation and competition assays establish for the first-time pharmacological characteristics for the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in AGS brain.

A SEROLOGIC SURVEY OF FRANCISELLA TULARENSIS EXPOSURE IN WILDLIFE ON THE ARCTIC COASTAL PLAIN OF ALASKA, USA.

Tularemia is an infectious zoonotic disease caused by one of several subspecies of Francisella tularensis bacteria. Infections by F. tularensis are common throughout the northern hemisphere and have been detected in more than 250 wildlife species. In Alaska, US, where the pathogen was first identified in 1938, studies have identified F. tularensis antibodies in a diverse suite of taxa, including insects, birds, and mammals. However, few such investigations have been conducted recently and knowledge about the current distribution and disease ecology of F. tularensis is limited, particularly in Arctic Alaska, an area undergoing rapid environmental changes from climate warming. To help address these information gaps and provide insights about patterns of exposure among wildlife, we assessed the seroprevalence of F. tularensis antibodies in mammals and tundra-nesting geese from the Arctic Coastal Plain of Alaska, 2014-17. With a commercially available slide agglutination test, we detected antibodies in 14.7% of all individuals sampled (n=722), with titers ranging from 1:20 to 1:320. We detected significant differences in seroprevalence between family groups, with Canidae (foxes, Vulpes spp.) and Sciuridae (Arctic ground squirrel, Spermophilus parryii) having the highest seroprevalence at 21.5% and 33.3%, respectively. Mean seroprevalence for Ursidae (polar bears, Ursus maritimus) was 13.3%, whereas Cervidae (caribou, Rangifer tarandus) had comparatively low seroprevalence at 6.5%. Antibodies were detected in all Anatidae species sampled, with Black Brant (Branta bernicla nigricans) having the highest seroprevalence at 13.6%. The detection of F. tularensis antibodies across multiple taxa from the Arctic Coastal Plain and its nearshore marine region provides evidence of exposure to this pathogen throughout the region and highlights the need for renewed surveillance in Alaska.

Effects of Spring Warming on Seasonal Neuroendocrinology and Activation of the Reproductive Axis in Hibernating Arctic Ground Squirrels.

Many animals adjust the timing of seasonal events, such as reproduction, molt, migration, and hibernation, in response to interannual variation and directional climate-driven changes in temperature. However, the mechanisms by which temperature influences seasonal timing are relatively under-explored. Seasonal timing involves retrograde signaling in which thyrotropin (TSH) in the pars tuberalis (PT) alters expression of thyroid hormone (TH) deiodinases (Dio2/Dio3) in tanycyte cells lining the third ventricle of the hypothalamus. This, in turn, affects the availability of triiodothyronine (T3) within the mediobasal hypothalamus-increased hypothalamic T3 restores a summer phenotype and activates the reproductive axis in long-day breeders. Recently, we showed that retrograde TH signaling is activated during late hibernation in arctic ground squirrels (Urocitellus parryii) held in constant darkness and constant ambient temperature. Sensitivity of seasonal pathways to nonphotic cues, such as temperature, is likely particularly important to hibernating species that are sequestered in hibernacula during spring. To address this issue, we exposed captive arctic ground squirrels of both sexes to an ecologically relevant increase in ambient temperature (from -6 to -1°C) late in hibernation and examined the effects of warming on the seasonal retrograde TSH/Dio/T3 signaling pathway, as well as downstream elements of the reproductive axis. We found that warmed males tended to have higher PT TSHβ expression and significantly heavier testis mass whereas the TSH/Dio/T3 signaling pathway was unaffected by warming in females, although warmed females exhibited a slight decrease in ovarian mass. Our findings suggest that temperature could have different effects on gonadal growth in male and female arctic ground squirrels, which could lead to mismatched timing in response to rapid climate change.

Hypothalamic remodeling of thyroid hormone signaling during hibernation in the arctic ground squirrel

Hibernation involves prolonged intervals of profound metabolic suppression periodically interrupted by brief arousals to euthermy, the function of which is unknown. Annual cycles in mammals are timed by a photoperiodically-regulated thyroid-hormone-dependent mechanism in hypothalamic tanycytes, driven by thyrotropin (TSH) in the pars tuberalis (PT), which regulates local TH-converting deiodinases and triggers remodeling of neuroendocrine pathways. We demonstrate that over the course of hibernation in continuous darkness, arctic ground squirrels (Urocitellus parryii) up-regulate the retrograde TSH/Deiodinase/TH pathway, remodel hypothalamic tanycytes, and activate the reproductive axis. Forcing the premature termination of hibernation by warming animals induced hypothalamic deiodinase expression and the accumulation of secretory granules in PT thyrotrophs and pituitary gonadotrophs, but did not further activate the reproductive axis. We suggest that periodic arousals may allow for the transient activation of hypothalamic thyroid hormone signaling, cellular remodeling, and re-programming of brain circuits in preparation for the short Arctic summer.

Translating PUFA omega 6:3 ratios from wild to captive hibernators (Urocitellus parryii) enhances sex-dependent mass-gain without increasing physiological stress indicators

Omega 3 polyunsaturated fatty acids (PUFAs) are well-documented for their influence on health and weight loss. Recent studies indicate omega 3 PUFAs may exert a negative impact on cellular stress and physiology in some hibernators. We asked if physiological stress indicators, lipid peroxidation and mass gain in Arctic Ground Squirrels (AGS) were negatively influenced by naturally occurring dietary omega 3 PUFA levels compared to omega 3 PUFA levels found in common laboratory diets. We found plasma fatty acid profiles of free-ranging AGS to be high in omega 3 PUFAs with balanced omega 6:3 ratios, while standard laboratory diets and plasma of captive AGS are high in omega 6 and low in omega 3 PUFAs with higher omega 6:3 ratios. Subsequently, we designed a diet to mimick free-range AGS omega 6:3 ratios in captive AGS. Groups of wild-caught juvenile AGS were either fed: (1) Mazuri Rodent Chow (Standard Rodent chow, 4.95 omega 6:3 ratio), or (2) balanced omega 6:3 chow (Balanced Diet, 1.38 omega 6:3). AGS fed the Balanced Diet had plasma omega 6:3 ratios that mimicked plasma profiles of wild AGS. Balanced Diet increased female body mass before hibernation, but did not influence levels of cortisol in plasma or levels of the lipid peroxidation product 4-HNE in brown adipose tissue. Overall, as the mass gain is critical during pre-hibernation for obligate hibernators, the results show that mimicking a fatty acid profile of wild AGS facilitates sex-dependent mass accumulation without increasing stress indicators.

In Arctic ground squirrels, diet and torpor alter skeletal muscle relaxation kinetics but not force development

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as ‐3°C), during which they are mostly physically inactive. Once Tb reaches ~15°C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~35°C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω‐6 and the ω‐6:ω‐3 PUFA ratio correlates with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. Muscular activity is characterized by contraction and relaxation mediated by calcium ions cycling released from SR stores. Ryanodine receptors (RyR1) on SR membranes release calcium ions into the cytosol, facilitating muscle contraction via actin‐myosin cross‐bridging. Whereas SERCA catalyzes Ca2+ reuptake back into the SR, releasing Ca2+ from actin/myosin filaments, completing the calcium cycle, and causing muscle relaxation. We hypothesized that diet (increased ω‐6:ω‐3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex‐vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. 1) Standard rodent chow with an ω‐6:ω‐3 ratio of 5:1, and 2) a balanced diet with an ω‐6:ω‐3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4°C, 15°C, 25°C, and 37°C to determine functional resilience. Our data show that torpid animals fed standard rodent chow appear to have an increase in SERCA activity, resulting in a significant increase in calcium mobilization from the sarcoplasm to the sarcoplasmic reticulum by the SERCA pump, allowing faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω‐6:ω‐3 PUFA ratio improves skeletal muscle performance during hypothermic stress in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Abstract TP251: CRISPR-engineered Mouse Neural Cells Expressing An Optimized Manf Variant Reveals Mechanisms Of MANF Neuroprotection

Introduction: Hibernating mammals have evolved physiological strategies to cope with harsh winters by markedly reducing their metabolic rate and core temperature. Interestingly, neural stem cells derived from Arctic ground squirrels (AGS) also possess resilient cell biological processes that preserve cell integrity during hypoxia and exposure mitochondrial toxins. We identified AGS mesencephalic astrocyte-derived neurotrophic factor (MANF) as a key cytoprotective gene in diverse survival screens of mouse neural cells expressing an AGS cDNA library. Hypothesis: We hypothesize that adaptively evolved amino acid substitution in AGS MANF confers metabolic stress resilience by modulating actions of MANF including sulfatide-binding, reducing endoplasmic reticulum (ER) stress and altering mitochondrial metabolism. Methodology: CRISPR-Cas9 knock-in (KI) gene editing was utilized to generate mouse neural cells harboring the AGS Manf allele to allow for analysis of the beneficial effects of the AGS amino acid substitutions in cell survival to metabolic stress, mitochondrial metabolism, MANF protein stability, and sulfatide binding.. We also tested AGS compared to human MANF with regard to reducing ER stress under hypoxia, hypothermia, and rotenone. Results: AGS MANF expression reduced ER stress markers compared to human MANF, and demonstrated increased stability following rotenone treatment further pointing to its important role regulating cellular adaptations to metabolic stress and imparting neuroprotection in our in vitro model. We gained key functional insights into how specific amino acid substitutions improve MANF stability to impart cytoprotection to metabolic insults. This detailed dissection of the AGS optimized adaptive stress response pathway can serve as a template for the development of new neuroprotective treatments.

Survival estimates of free-living arctic ground squirrels (Urocitellus parryii): effects of sex and biologging

Hibernation is associated with long lifespan: on average, hibernating mammals live 15% longer than nonhibernators of equivalent mass. We investigated how survival varies with sex, season, and the deployment of biologgers in arctic ground squirrels (Urocitellus parryii (Richardson, 1825)), a widely distributed northern hibernator. The duration of hibernation in arctic ground squirrels differs markedly by sex: females hibernate 30% longer each year than males, a behavioural trait that could positively affect female survival. Additionally, males engage in aggressive territorial and food cache defense in spring and fall, which may decrease survival in this sex. From 13 years of mark–recapture data, we estimated apparent survival of arctic ground squirrels in Arctic Alaska (USA) using Cormack–Jolly–Seber models in program MARK. We found that females had higher annual survival ([Formula: see text] (95% confidence interval (CI): 0.469, 0.913)) than males ([Formula: see text] (95% CI: 0.416, 0.670)), with a maximum observed lifespan (10 years) that exceeded that of males (6 years). We also show that biologger use and implantation did not significantly impact survival. Quantifying basic arctic ground squirrel demographics from this well-studied population illustrates how sex-specific hibernation parameters may influence lifespan differences in male and female arctic ground squirrels and provides support for the safety of biologging devices.

A heterothermic spectrum in hummingbirds.

Many endotherms use torpor, saving energy by a controlled reduction of their body temperature and metabolic rate. Some species (e.g. arctic ground squirrels, hummingbirds) enter deep torpor, dropping their body temperature by 23-37°C, while others can only enter shallow torpor (e.g. pigeons, 3-10°C reduction). However, deep torpor in mammals can increase predation risk (unless animals are in burrows or caves), inhibit immune function and result in sleep deprivation, so even for species that can enter deep torpor, facultative shallow torpor might help balance energy savings with these potential costs. Deep torpor occurs in three avian orders, but the trade-offs of deep torpor in birds are unknown. Although the literature hints that some bird species (mousebirds and perhaps hummingbirds) can use both shallow and deep torpor, little empirical evidence of such an avian heterothermy spectrum within species exists. We infrared imaged three hummingbird species that are known to use deep torpor, under natural temperature and light cycles, to test whether they were also capable of shallow torpor. All three species used both deep and shallow torpor, often on the same night. Depending on the species, they used shallow torpor for 5-35% of the night. The presence of a heterothermic spectrum in these bird species indicates a capacity for fine-scale physiological and genetic regulation of avian torpid metabolism.

ПИТАНИЕ КPЕЧЕТА (FALCO RUSTICOLUS, FALCONIFORMES, FALCONIDAE) НА КАМЧАТКЕ

In Kamchatka, an investment project is being implemented for the artificial reproduction of rare species of falcons, primarily gyrfalcon. For successful breeding of birds, it is necessary to know their trophic preferences in nature. The article analyzes information on the nutrition of the gyrfalcon in Kamchatka, collected over the entire history of ornithological research in the region. In the diet of the gyrfalcon, minimum 41 species of birds and 11 species of mammals are identified. The composition of the food of specific individuals is determined by their habitat in a given season of the year. During the breeding season, ptarmigans (Lagopus lagopus, L. mutus) are crucial for most gyrfalcons, and small mammals (specially American (arctic) ground squirrel Spermophilus parryi) are also important in the mountains. On migrations and in winter (especially on Kamchatka Peninsula), an important role is played by water, near-water and synanthropic species of birds. In Koryak Highland in winter an important role is played ptarmigans as usual. Occasionally they feed on fish processing waste and bait in hunters' traps.

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators.

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner.

Landscape drivers of site occupancy by remnant populations of arctic ground squirrels (Urocitellus parryii)

Landscape drivers of site occupancy by remnant populations of arctic ground squirrels (Urocitellus parryii)

Identification and molecular analysis of ixodid ticks (Acari: Ixodidae) and their pathogens in Yakutia

The purpose of the research is to identify ixodid ticks and their pathogens in the left bank area of Central Yakutia. Materials and methods. The work was carried out in 2019–2020 in the left bank area of Central Yakutia. Nine ticks were collected in 2019, and 27 ticks in 2020. We studied forest shrub stations, steppe stations, meadow field stations, near-water stations and stations of settlements. To determine faunal and ecological characteristics of ectoparasites in the territory, we used standard collection methods. The tick species was determined using morphological keys by N. A. Filippova; the determination correctness was confirmed by the PCR method. The collected ticks were studied for causative agents of babesiosis and tick-borne viral encephalitis using PCR analysis. Results and discussion. One species of ixodid ticks, Ixodes persulcatus, inhabits the left bank area of Central Yakutia. Haemaphysalis concinna was not found in Yakutia. In 2008, a natural focus of blood protozoan disease of domestic reindeer appeared for the first time in Yakutia in its central zone. Recently, an increase in the number of I. persulcatus has been observed. Tick activity is recorded from the second decade of May to the second decade of August. The ground-squirrel Spermophilus parryii is the main host for the preimaginal stages. Pathogens were not detected when ticks were examined for causative agents of babesiosis and tick-borne viral encephalitis using PCR analysisd.

Investigation of keratinase digestion to improve steroid hormone extraction from diverse keratinous tissues

Monitoring the physiology of wild populations presents many technical challenges. Blood samples, long the gold standard of wildlife endocrinology studies, cannot always be obtained. The validation and use of non-plasma samples to obtain hormone data have greatly improved access to more integrated information about an organism’s physiological state. Keratinous tissues like skin, hair, nails, feathers, or baleen store steroid hormones in physiologically relevant concentrations, are stable across decades, and can be used to retrospectively infer physiological state at prior points in time. Most protocols for steroid extraction employ physical pulverization or cutting of the sample, followed by mixing with a solvent. Such methods do produce repeatable and useful data, but low hormone yield and detectability issues can complicate research on small or rare samples. We investigated the use of keratinase, an enzyme that breaks down keratin, to improve the extraction and yield of corticosterone from vertebrate keratin tissues. Corticosterone content of keratinase-digested extracts were compared to non-keratinase extracts for baleen from three species of whale (blue, Balaenoptera musculus; bowhead, Balaena mysticetus; southern right, SRW; Eubalaena australis), shed skin from two reptiles (tegu lizard, Salvator merianae; narrow-headed garter snake, Thamnophis rufipunctatus), hair from arctic ground squirrel (AGS; Urocitellus parryii), feathers from Purple Martins (PUMA; Progne subis), and spines from the short-beaked echidna (Tachyglossus aculeatus). We tested four starting masses (10, 25, 50, 100 mg) for each sample; digestion was most complete in the 10 and 25 mg samples. A corticosterone enzyme immunoassay (EIA) was validated for all keratinase-digested extracts. In all sample types except shed skin from reptiles, keratinase digestion improved hormone yield, with PUMA feathers and blue whale baleen having the greatest increase in apparent corticosterone content (100% and 66% more hormone, respectively). The reptilian shed skin samples did not benefit from keratinase digestion, actually yielding less hormone than controls. With further optimization and refinement, keratinase digestion could greatly improve yield of steroid hormones from various wildlife epidermal tissue types, allowing more efficient use of samples and ultimately improving understanding of the endocrine physiology of wild populations.