Physiological responses to hypoxia are constrained by environmental temperature in heterothermic tenrecs.

Malagasy tenrecs are placental hibernating mammals that seal the entrances to their burrows and hibernate either singly or in groups for 8-9 months, which is likely to create a hypoxic and hypercapnic burrow environment. Therefore, we hypothesized that tenrecs are tolerant to environmental hypoxia and hypercapnia. Many hypoxia- and hypercapnia-tolerant fossorial mammals respond to hypoxia by decreasing metabolic rate and thermogenesis, and have blunted ventilatory responses to both environmental hypoxia and hypercapnia. However, tenrecs exhibit extreme metabolic and thermoregulatory plasticity, which exceeds that of most heterothermic mammals and approaches that of ectothermic reptiles. Thus, we predicted that tenrecs would have abnormal physiological responses to hypoxia and hypercapnia relative to other fossorial mammals. To test this, we exposed common tenrecs (Tenrec ecaudatus) to moderate and severe hypoxia (9 and 4% O2) or hypercapnia (5 and 10% CO2) in either 28 or 16°C while non-invasively measuring metabolic rate, thermogenesis and ventilation. We found that tenrecs exhibit robust metabolic decreases in both hypoxia and hypercapnia. Furthermore, tenrecs have blunted ventilatory responses to both hypoxia and hypercapnia, and these responses are highly temperature sensitive such that they are reduced or absent in 16°C. Thermoregulation was highly variable in 16°C but constrained in 28°C across all treatment conditions and was not impacted by hypoxia or hypercapnia, unlike in other heterothermic mammals. Taken together, our results indicate that physiological responses to hypoxia and hypercapnia in tenrecs are highly dependent on environmental temperature and differ from those of other mammalian heterotherms.

Epigenomics as a paradigm to understand the nuances of phenotypes.

Quantifying the relative importance of genomic and epigenomic modulators of phenotype is a focal challenge in comparative physiology, but progress is constrained by availability of data and analytic methods. Previous studies have linked physiological features to coding DNA sequence, regulatory DNA sequence, and epigenetic state, but few have disentangled their relative contributions or unambiguously distinguished causative effects ('drivers') from correlations. Progress has been limited by several factors, including the classical approach of treating continuous and fluid phenotypes as discrete and static across time and environment, and difficulty in considering the full diversity of mechanisms that can modulate phenotype, such as gene accessibility, transcription, mRNA processing and translation. We argue that attention to phenotype nuance, progressing to association with epigenetic marks and then causal analyses of the epigenetic mechanism, will enable clearer evaluation of the evolutionary path. This would underlie an essential paradigm shift, and power the search for links between genomic and epigenomic features and physiology. Here, we review the growing knowledge base of gene-regulatory mechanisms and describe their links to phenotype, proposing strategies to address widely recognized challenges.

Liver proteome response to torpor in a basoendothermic mammal, Tenrec ecaudatus, provides insights into the evolution of homeothermy.

Many mammals use adaptive heterothermy (e.g., torpor, hibernation) to reduce metabolic demands of maintaining high body temperature (Tb). Torpor is typically characterized by coordinated declines in Tb and metabolic rate (MR) followed by active rewarming. Most hibernators experience periods of euthermy between bouts of torpor during which homeostatic processes are restored. In contrast, the common tenrec, a basoendothermic Afrotherian mammal, hibernates without interbout arousals and displays extreme flexibility in Tb and MR. We investigated the molecular basis of this plasticity in tenrecs by profiling the liver proteome of animals that were active or torpid with high and more stable Tb (∼32°C) or lower Tb (∼14°C). We identified 768 tenrec liver proteins, of which 50.9% were differentially abundant between torpid and active animals. Protein abundance was significantly more variable in active cold and torpid compared with active warm animals, suggesting poor control of proteostasis. Our data suggest that torpor in tenrecs may lead to mismatches in protein pools due to poor coordination of anabolic and catabolic processes. We propose that the evolution of endothermy leading to a more realized homeothermy of boreoeutherians likely led to greater coordination of homeostatic processes and reduced mismatches in thermal sensitivities of metabolic pathways.

Extreme physiological plasticity in a hibernating basoendothermic mammal, Tenrec ecaudatus.

Physiological plasticity allows organisms to respond to diverse conditions. However, can being too plastic actually be detrimental? Malagasy common tenrecs, Tenrec ecaudatus, have many plesiomorphic traits and may represent a basal placental mammal. We established a laboratory population of T. ecaudatus and found extreme plasticity in thermoregulation and metabolism, a novel hibernation form, variable annual timing, and remarkable growth and reproductive biology. For instance, tenrec body temperature (Tb) may approximate ambient temperature to as low as 12°C even when tenrecs are fully active. Conversely, tenrecs can hibernate with Tb of 28°C. During the active season, oxygen consumption may vary 25-fold with little or no change in Tb During the austral winter, tenrecs are consistently torpid but the depth of torpor may vary. A righting assay revealed that Tb contributes to but does not dictate activity status. Homeostatic processes are not always linked, e.g. a hibernating tenrec experienced a ∼34% decrease in heart rate while maintaining constant body temperature and oxygen consumption rates. Tenrec growth rates vary but young may grow ∼40-fold in the 5 weeks until weaning and may possess indeterminate growth as adults. Despite all of this profound plasticity, tenrecs are surprisingly intolerant of extremes in ambient temperature (<8 or >34°C). We contend that while plasticity may confer numerous energetic advantages in consistently moderate environments, environmental extremes may have limited the success and distribution of plastic basal mammals.

Systematic approach to isolating Batrachochytrium dendrobatidis.

We developed a protocol for isolating the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) from anurans. We sampled skin tissues from 2 common treefrogs, Pseudacris regilla and P. triseriata, collected from populations with high infection prevalence. We sampled tissues from 3 anatomical ventral regions (thigh, abdomen, and foot) where the pathogen is thought to concentrate. To mitigate potential bacterial contamination, we used a unique combination of 4 antibiotics. We quantified infections on frogs as zoospore equivalents (ZE) using a swabbing approach combined with quantitative real-time polymerase chain reaction. We isolated Bd from 68.9% of frogs sampled from both species. Contamination was low (9.7% of all plates), with most contamination presumed to be fungal. We found positive correlations between successful isolation attempts and infection intensity. Our levels of isolation success were 74% for P. triseriata and 100% for P. regilla once Bd detection intensities reached ≥40 ZE. Of the 3 anatomical regions sampled in both species, we had significantly more success isolating Bd from foot tissue. Our results support published recommendations to focus sampling for Bd infection on feet, particularly webbing.

The Hibernation Continuum: Physiological and Molecular Aspects of Metabolic Plasticity in Mammals.

Mammals are often considered to be masters of homeostasis, with the ability to maintain a constant internal milieu, despite marked changes in the environment; however, many species exhibit striking physiological and biochemical plasticity in the face of environmental fluctuations. Here, we review metabolic depression and body temperature fluctuation in mammals, with a focus on the extreme example of hibernation in small-bodied eutherian species. Careful exploitation of the phenotypic plasticity of mammals with metabolic flexibility may provide the key to unlocking the molecular secrets of orchestrating and surviving reversible metabolic depression in less plastic species, including humans.

Paradoxical anaerobism in desert pupfish.

In order to estimate metabolic demands of desert pupfish for conservation purposes, we measured oxygen consumption in fish acclimated to the ecologically relevant temperatures of 28 or 33°C. For these experiments, we used fish derived from a refuge population of Devils Hole pupfish (Cyprinodon diabolis). Measurement of routine oxygen consumption (V̇O2,routine) revealed some 33°C-acclimated fish (10% of 295 assayed fish) periodically exhibited periods of no measurable oxygen consumption despite available ambient oxygen tensions that were above the critical PO2. We call this phenomenon paradoxical anaerobism. The longest observed continuous bout with no oxygen consumption was 149 min, although typical bouts were much shorter. Fish maintained normal posture and ventilation rate (>230 ventilations per minute) during paradoxical anaerobism. Fish rarely demonstrated a compensatory increase in oxygen use following a period of paradoxical anaerobism. In contrast, only one out of 262 sampled fish acclimated at 28°C spontaneously demonstrated paradoxical anaerobism. Muscle lactate concentration was not elevated during periods of paradoxical anaerobism. However, the amount of ethanol released by the 33°C-acclimated fish was 7.3 times greater than that released by the 28°C acclimation group, suggesting ethanol may be used as an alternative end product of anaerobic metabolism. Exposure to exogenous ethanol, in concentrations as low as 0.1%, produced periods of paradoxical anaerobism even in 28°C-acclimated fish.

A systems-level approach to understanding transcriptional regulation by p53 during mammalian hibernation.

Presumably to conserve energy, many mammals enter into hibernation during the winter. Homeostatic processes such as transcription and translation are virtually arrested. To further elucidate transcriptional regulation during hibernation, we studied the transcription factor p53. Here, we demonstrate that changes in liver mRNA and protein concentrations of known regulators of p53 are consistent with activation. p53 mRNA and protein concentrations are unrelated. Importantly, p53 protein concentration is increased ~2-fold during the interbout arousal that punctuates bouts of torpor. As a result, both the interbout arousal and the torpid state are characterized by high levels of nuclear-localized p53. Chromatin immunoprecipitation assays indicate that p53 binds DNA during the winter. Furthermore, p53 recruits RNA polymerase II, as indicated by nuclear run-on data. However, and consistent with previous data indicating an arrest of transcriptional elongation during torpor, p53 'activity' does not result in expected changes in target gene transcripts. These data demonstrate the importance of using a systems level-approach in understanding a complex phenotype such as mammalian hibernation. Relying on interpretations of data that are based on steady-state regulation in other systems may be misleading in the context of non-steady-state conditions such as torpor.

Cell type and cell signaling innovations underlying mammalian pregnancy.

How fetal and maternal cell types have co-evolved to enable mammalian placentation poses a unique evolutionary puzzle. Here, we present a multi-species atlas integrating single-cell transcriptomes from six species bracketing therian mammal diversity. We find that invasive trophoblasts share a gene-expression signature across eutherians, and evidence that endocrine decidual cells evolved stepwise from an immunomodulatory cell type retained in Tenrec with affinity to human decidua of menstruation. We recover evolutionary patterns in ligand-receptor signaling: fetal and maternal cells show a pronounced tendency towards disambiguation, but a predicted arms race dynamic between them is limited. We reconstruct cell communication networks of extinct mammalian ancestors, finding strong integration of fetal trophoblast into maternal networks. Together, our results reveal a dynamic history of cell type and signaling evolution. Synopsis: The fetal-maternal interface is one of the most intense loci of cell-cell signaling in the human body. Invasion of cells from the fetal placenta into the uterus, and the corresponding transformation of maternal tissues called decidualization, first evolved in the stem lineage of eutherian mammals( 1 , 2 ). Single-cell studies of the human fetal-maternal interface have provided new insight into the cell type diversity and cell-cell interactions governing this chimeric organ( 3-5 ). However, the fetal-maternal interface is also one of the most rapidly evolving, and hence most diverse, characters among mammals( 6 ), and an evolutionary analysis is missing. Here, we present and compare single-cell data from the fetal-maternal interface of species bracketing key events in mammal phylogeny: a marsupial (opossum, Monodelphis domestica ), the afrotherian Tenrec ecaudatus, and four Euarchontoglires - guinea pig and mouse (Rodentia) together with recent macaque and human data (primates) ( 4 , 5 , 7 ). We infer cell type homologies, identify a gene-expression signature of eutherian invasive trophoblast conserved over 99 million years, and discover a predecidual cell in the tenrec which suggests stepwise evolution of the decidual stromal cell. We reconstruct ancestral cell signaling networks, revealing the integration of fetal cell types into the interface. Finally, we test two long-standing theoretical predictions, the disambiguation hypothesis( 8 ) and escalation hypothesis( 9 ), at transcriptome-wide scale, finding divergence between fetal and maternal signaling repertoires but arms race dynamics restricted to a small subset of ligand-receptor pairs. In so doing, we trace the co-evolutionary history of cell types and their signaling across mammalian viviparity.

Common tenrecs (Tenrec ecaudatus) reduce oxygen consumption in hypoxia and in hypercapnia without concordant changes to body temperature or heart rate.

Common tenrecs (Tenrec ecaudatus) are fossorial mammals that use burrows during both active and hibernating seasons in Madagascar and its neighboring islands. Prevailing thought was that tenrecs hibernate for 8-9 months individually, but 13 tenrecs were removed from the same sealed burrow 1 m deep from the surface. Such group hibernation in sealed burrows presumably creates a hypoxic and/or hypercapnic environment and suggests that this placental mammal may have an increased tolerance to hypoxia and hypercapnia. Higher tolerances to hypoxia and hypercapnia have been documented for other mammals capable of hibernation and to determine if this is the case for tenrecs, we exposed them to acute hypoxia (4 h of 16 or 7% O2), progressive hypoxia (2 h of 16, 10 and 4% O2), or progressive hypercapnia (2 h of 2, 5 and 10% CO2) at cold (16 °C) or warm (28 °C) ambient temperatures (Ta). Oxygen equilibrium curves were also constructed on the whole blood of tenrecs at 10, 25, and 37 °C to determine if hemoglobin (Hb)-O2 affinity contributes to hypoxia tolerance. In animals held at 16 °C, normoxic and normocapnic levels of oxygen consumption rate ( V ˙ O 2 ), body temperature (Tb), and heart rate (HR) were highly variable between individuals. This inter-individual variation was greatly reduced in animals held at 28 °C for oxygen consumption rate and body temperature. Both hypoxia (acute and progressive) and progressive hypercapnia led to decreases in V ˙ O 2 as well as the variation in V ˙ O 2 between animals held at 16 °C. The fall in oxygen consumption rate in 7% O2 independent of changes in body temperature in tenrecs held at 16 °C is unique and not consistent with the typical hypoxic metabolic response seen in other hibernating species that depends on concomitant falls in Tb. In animals held at 28 °C, exposure to O2 levels as low as 4% and CO2 levels as high as 10% had no significant effect on V ˙ O 2 , HR, or Tb, indicative of high tolerance to both hypoxia and hypercapnia. High variation in heart rate remained between individuals in all gas compositions and at all temperatures. Tenrec Hb-O2 affinity was similar to other homeothermic placental mammals and likely does not contribute to the increased hypoxia tolerance. Ultimately, our results suggest changes in Ta dictate physiological responses to hypoxia or hypercapnia in tenrecs, responses more characteristic of reptiles than of most placental mammals. Given that numerous anatomical and physiological characteristics of tenrecs suggest that they may be representative of an ancestral placental mammal, our findings suggest the typical hypoxic metabolic response evolved later in mammalian evolution.

Extracellular digestion during hyposaline exposure in the Dungeness crab, Cancer magister, and the blue crab, Callinectes sapidus.

Extracellular digestive processes were examined in the Dungeness crab, Cancer magister and the blue crab, Callinectes sapidus, during hyposaline exposure. Both species are found in estuaries as adults, but vary in their ability to balance the cardiovascular and respiratory demands of concurrent osmoregulation and digestion. The weak osmoregulator, C. magister, is unable to balance the demands of osmoregulation and digestion. Concordant with observed decreases in oxygen consumption and mechanical digestion, proteolytic digestion within the foregut and hepatopancreas was delayed, resulting in a relative reduction of circulating amino acids post-feeding in low salinity. In contrast, the efficient osmoregulator, C. sapidus, balances the demands of osmoregulation and digestion, and mechanical digestion continues unabated in low salinity. Protease activity in the gut fluid and hepatopancreas showed either no change or a reduction over time. The transport of amino acids into the cells post-feeding is opposed by an efflux of amino acids at the cellular level, and resulted in a build up of amino acids in the hemolymph. Despite differences in the extracellular responses to low salinity exposure following feeding, both species were able to maintain high digestive efficiencies.

Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

Mitigating Apoptotic and Inflammatory Signaling via Global Caspase Inhibition in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractIn most systems, the caspase cascade is activated during cellular stress and results in inflammation and apoptosis. Hibernators experience stressors such as extremely low body temperatures, bradycardia, possible ischemia and reperfusion, and acidosis. However, widespread inflammation and apoptosis would represent an energetic expense that is incompatible with hibernation. To better understand global caspase regulation during hibernation, we employed a systems-level approach and analyzed 11 caspases in ground squirrel liver that are involved in inflammatory (caspases 1, 4, 5, 11, and 12) and apoptotic (caspases 2, 6, 7, 8, 9, and 10) pathways. Western blots revealed liberation of active forms for two inflammatory (caspases 11 and 12) and two apoptotic (caspases 6 and 9) caspases during hibernation (e.g., p15, the most active fragment of caspase 6, increased 8.26±0.70-fold in interbout-aroused animals). We used specific peptide substrates to interrogate the four seemingly activated caspases and demonstrated no expected increases in proteolytic activity. Specific targets of these four caspases were similarly not cleaved, demonstrating that initiation of caspase activation may occur without concomitant downstream effects. Similarly, we found no evidence for upstream activation for caspase 9 signaling based on permeabilization of the outer mitochondrial membrane. We contend that these caspases are suppressed after seeming activation during hibernation. Incomplete caspase signaling is effectively mitigating the induction of widespread inflammation and apoptosis during hibernation.

Torpor Does Not Influence Spatial Memory in Hibernating Golden-Mantled Ground Squirrels (Spermophilus [Callospermophilus] lateralis).

AbstractMammalian hibernation in ground squirrels is characterized by periods of torpor wherein body temperature approaches ambient temperature and metabolism is reduced to as low as 1/100th of active rates. It is unclear how hibernation affects long-term spatial memory, as tremendous remodeling of neurons is associated with torpor use. Given the suspected links between remodeling and memory formation and retention, we examined long-term spatial memory retention throughout a hibernation season. Animals were trained on a Barnes maze before entering torpor. Animals were tested for memory retention once a month throughout a hibernation season. Results indicate marked variation between individuals. Some squirrels retained memory across multiple torpor bouts, while other squirrels did not. No relationship was found between the number of torpor bouts, duration of bouts, or time spent torpid on long-term memory retention. However, that some squirrels successfully retain memory suggests that the profound remodeling of dendritic spines during torpor does not always lead to memory loss.

Preference of IRES-mediated initiation of translation during hibernation in golden-mantled ground squirrels, Spermophilus lateralis.

Mammalian hibernation involves virtual cessation of energetically consumptive processes normally vital to homeostasis, including gene transcription and protein synthesis. As animals enter torpor, the bulk of initiation of translation is blocked at a body temperature of 18°C in golden-mantled ground squirrels [Spermophilus (Callospermophilus) lateralis]. Previous data demonstrated regulation of cap-dependent initiation of translation during torpor. We asked what happens to cap-independent, specifically, internal ribosome entry site (IRES)-mediated initiation of translation during hibernation. We analyzed polysome fractions for mRNAs that are known to contain or not to contain IRES elements. Here, we show that mRNAs harboring IRES elements preferentially associate with ribosomes as a torpor bout progresses. Squirrels allowed to naturally complete a torpor cycle have a higher IRES preference index than those animals that are prematurely aroused from torpor. Data indicate that this change in preference is not associated with gene expression, i.e., change is due to change in mRNA association with ribosomes as opposed to mRNA abundance. Thus, although processes like transcription and translation are virtually arrested during torpor, ribosomes are preferentially loaded with IRES-containing transcripts when squirrels arouse from torpor and translation resumes. Differential translation of preexisting mRNAs may allow for the preferential production of key stress proteins critical for survival of physiological insults that are lethal to other mammals.

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates.

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy. This review will discuss processes to mitigate apoptosis and how these processes might be regulated in stress-tolerant vertebrates such as mammalian hibernators. As data directly addressing such issues are scarce and often conflicting, an apparently complex regulation of apoptosis seems to be at work. For example, apoptosis is mitigated during dormancy, key signaling events including the activation of caspase-3 may still occur. However, both passive, temperature-induced depression of apoptotic signaling as well as active suppression of apoptosis appear to work in synergy in these systems. In many instances cell death is prevented by simply avoiding the cellular triggers (e.g. leakage of proteins from the mitochondria or increases in intracellular calcium) that initiate apoptotic signaling. In this review we discuss what is known about programmed cell death in these under-studied models and highlight features of their physiology that likely support survival in the face of conditions that would induce cell death in typical vertebrate cells.

The gut microbiome and its potential role in paradoxical anaerobism in pupfishes of the Mojave Desert.

BACKGROUND: Pupfishes frequently enter paradoxical anaerobism in response to endogenously produced or exogenously supplied ethanol in a dose-dependent manner. To decipher the role of the gut microbiota in ethanol-associated paradoxical anaerobism, gut microbial communities were depleted using a cocktail of antibiotics and profiled using 16S rRNA gene sequencing. RESULTS: Compared to the control group (n = 12), microbiota-depleted fish (n = 12) spent more time in paradoxical anaerobism. Our analysis indicated that the bacterial phyla Proteobacteria, Fusobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Patescibacteria, and Dependentiae dominated the pupfish gut, which is consistent with other fish gut microbiota. Although the gut microbial communities with and without antibiotic treatment were similarly diverse, they were distinct and the greatest contribution to the dissimilarity (27.38%) was the common fish commensal Cetobacterium. CONCLUSIONS: This study reports the first characterization of gut microbial communities of pupfish and suggests the microbiome may play a critical role in regulating metabolic strategies that are critical for survival in extremes of temperature and oxygen concentration. We speculate that Cetobacterium, a primary fermenter, also consumes ethanol through secondary fermentation via an alcohol dehydrogenase and therefore regulates the transition from paradoxical anaerobism to aerobic respiration in fish. Given the wide distribution and abundance of Cetobacterium in warm-water fishes, this process may be of broad importance, and suggests that the microbiome be carefully considered for both conservation and aquaculture.

Bile constituents in hibernating golden-mantled ground squirrels (Spermophilus lateralis).

BACKGROUND: Golden-mantled ground squirrels (S. lateralis) are anorexic during the winter and survive by exploiting hibernation to reduce energetic demands. The liver normally plays a critical role in fueling and regulating metabolism and one might expect significant changes in hepatobiliary function with hibernation. We analyzed bile collected from animals in summer, animals in winter that were either torpid, active between bouts of torpor, or which failed to enter hibernation in order to characterize the effects of hibernation on hepatobiliary function per se. RESULTS: Surprisingly, hibernator bile did not differ from summer squirrel bile in key characteristics including [bile acids], [cholesterol], [free fatty acids], [lecithin], and osmolality. One major distinction between summer and winter squirrels was that winter squirrels experience >5 fold increases in [bilirubin]. Such an increase may have significant physiological consequences that could aid in survivorship of torpor. Animals that failed to hibernate, despite being anorexic, were very similar to summer squirrels in all measured parameters except they had lower bile acid and lecithin concentrations. CONCLUSION: The data indicate that despite extended anorexia, differences in metabolic fuel privation, and bouts of reduced body temperatures, hibernators normally do not experience broad changes in hepatobiliary function.

Bone strength is maintained after 8 months of inactivity in hibernating golden-mantled ground squirrels, Spermophilus lateralis.

Prolonged inactivity leads to disuse atrophy, a loss of muscle and bone mass. Hibernating mammals are inactive for 6-9 months per year but must return to full activity immediately after completing hibernation. This necessity for immediate recovery presents an intriguing conundrum, as many mammals require two to three times the period of inactivity to recover full bone strength. Therefore, if hibernators experience typical levels of bone disuse atrophy during hibernation, there would be inadequate time available to recover during the summer active season. We examined whether there were mechanical consequences as a result of the extended inactivity of hibernation. We dissected femur and tibia bones from squirrels in various stages of the annual hibernation cycle and measured the amount of force required to fracture these bones. Three groups were investigated; summer active animals were captured during the summer and immediately killed, animals in the 1 month detraining group were captured in the summer and killed following a 1-month period of restricted mobility, hibernating animals were killed after 8 months of inactivity. A three-point bend test was employed to measure the force required to break the bones. Apparent flexural strength and apparent flexural modulus (material stiffness) were calculated for femurs. There were no differences between groups for femur fracture force, tibia fracture force, or femur flexural strength. Femur flexural modulus was significantly less for the 1 month detraining group than for the hibernation and summer active groups. Thus, hibernators seem resistant to the deleterious effects of prolonged inactivity during the winter. However, they may be susceptible to immobilization-induced bone loss during the summer.

Population-Level Resistance to Chytridiomycosis is Life-Stage Dependent in an Imperiled Anuran.

Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.