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

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

Multi-Institutional Study of Pathologist Reading of the Programmed Cell Death Ligand-1 Combined Positive Score Immunohistochemistry Assay for Gastric or Gastroesophageal Junction Cancer.

The assessment of the expression of programmed cell death ligand-1 (PD-L1) using immunohistochemistry (IHC) has been controversial since its introduction. The methods of assessment and the range of assays and platforms contribute to confusion. Perhaps the most challenging aspect of PD-L1 IHC is the combined positive score (CPS) method of interpretation of IHC results. Although the CPS method is prescribed for more indications than any other PD-L1 scoring system, its reproducibility has never been rigorously assessed. In this study, we collected a series of 108 gastric or gastroesophageal junction cancer cases, stained them using the Food and Drug Administration-approved 22C3 assay, scanned them, and then circulated them to 14 pathologists at 13 institutions for the assessment of interpretative concordance for the CPS system. We found that higher cut points (10 or 20) performed better than a CPS of <1 or >1. We used the Observers Needed to Evaluate Subjective Tests algorithm to assess how the CPS system might perform in the real-world setting and found that the cut points of <1 or >1 showed an overall percent agreement of only 30% among the pathologist raters, with a plateau occurring at 8 raters. The raters performed better at higher cut points. However, the best cut point of <20 versus that of >20 was still disappointing, with a plateau at an overall percent agreement of 70% (at 7 raters). Although there is no ground truth for CPS, we compared the score with quantitative messenger RNA measurement and showed no relationship between the score (at any cut point) and messenger RNA amount. In summary, we showed that CPS shows high subjective variability among pathologist readers and is likely to perform poorly in the real-world setting. This system may be the root cause of the poor specificity and relatively low predictive value of IHC companion diagnostic tests for PD-1 axis therapies that use the CPS system.

Multi-institutional Assessment of Pathologist Scoring HER2 Immunohistochemistry.

The HercepTest was approved 20+ years ago as the companion diagnostic test for trastuzumab in human epidermal growth factor 2 (HER2) or ERBB2 gene-amplified/overexpressing breast cancers. Subsequent HER2 immunohistochemistry (IHC) assays followed, including the now most common Ventana 4B5 assay. Although this IHC assay has become the clinical standard, its reliability, reproducibility, and accuracy have largely been approved and accepted on the basis of concordance among small numbers of pathologists without validation in a real-world setting. In this study, we evaluated the concordance and interrater reliability of scoring HER2 IHC in 170 breast cancer biopsies by 18 breast cancer-specialized pathologists from 15 institutions. We used the Observers Needed to Evaluate Subjective Tests method to determine the plateau of concordance and the minimum number of pathologists needed to estimate interrater agreement values for large numbers of raters, as seen in the real-world setting. We report substantial discordance within the intermediate categories (<1% agreement for 1+ and 3.6% agreement for 2+) in the 4-category HER2 IHC scoring system. The discordance within the IHC 0 cases is also substantial with an overall percent agreement (OPA) of only 25% and poor interrater reliability metrics (0.49 Fleiss' kappa, 0.55 intraclass correlation coefficient). This discordance can be partially reduced by using a 3-category system (28.8% vs 46.5% OPA for 4-category and 3-category scoring systems, respectively). Observers Needed to Evaluate Subjective Tests plots suggest that the OPA for the task of determining a HER2 IHC score 0 from not 0 plateaus statistically around 59.4% at 10 raters. Conversely, at the task of scoring HER2 IHC as 3+ or not 3+ pathologists' concordance was much higher with an OPA that plateaus at 87.1% with 6 raters. This suggests that legacy HER2 IHC remains valuable for finding the patients in whom the ERBB2 gene is amplified but unacceptably discordant in assigning HER2-low or HER2-negative status for the emerging HER2-low therapies.

Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer.

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Development of an immunohistochemical assay for Siglec-15.

Siglec-15, a member of sialic-acid binding immunoglobulin type lectins, is normally expressed by myeloid cells and upregulated in some human cancers and represents a promising new target for immunotherapy. While PD-L1 blockade is an important strategy for immunotherapy, its effectiveness is limited. The expression of Siglec-15 has been demonstrated to be predominantly mutually exclusive to PD-L1 in certain cancer histologies. Thus, there is significant opportunity for Siglec-15 as an immunotherapeutic target for patients that do not respond to PD-1/PD-L1 inhibition. The aim of this study was to prospectively develop an immunohistochemical (IHC) assay for Siglec-15 to be used as a companion diagnostic for future clinical trials. Here, we create and validate an IHC assay with a novel recombinant antibody to the cytoplasmic domain of Siglec-15. To find an enriched target, this antibody was first used in a quantitative fluorescence (QIF) assay to screen a broad range of tumor histologies to determine tumor types where Siglec-15 demonstrated high expression. Based on this and previous data, we focused on development of a chromogenic IHC assay for lung cancer. Then we developed a scoring system for this assay that has high concordance amongst pathologist readers. We then use this chromogenic IHC assay to test the expression of Siglec-15 in two cohorts of NSCLC. We found that this assay shows a higher level of staining in both tumor and immune cells compared to previous QIF assays utilizing a polyclonal antibody. However, similar to that study, only a small percentage of positive Siglec-15 cases showed high expression for PD-L1. This validated assay for Siglec-15 expression may support development of a companion diagnostic assay to enrich for patients expressing the Siglec-15 target for therapy.

New insights into dietary management of polar bears (Ursus maritimus) and brown bears (U. arctos).

Although polar bears (Ursus maritimus) and brown bears (U. arctos) have been exhibited in zoological gardens for centuries, little is known about their nutritional needs. Multiple recent studies on both wild and captive polar bears and brown bears have found that they voluntarily select dietary macronutrient proportions resulting in much lower dietary protein and higher fat or digestible carbohydrate concentrations than are currently fed in most zoos. These lower protein concentrations selected by both species maximized growth rates and efficiencies of energy utilization in brown bears and may play a role in reducing kidney, liver, and cardiovascular diseases in both species. Therefore, we propose the need for the development of new dietary regimens for both species in managed care that better reflect their macronutrient needs. We developed a new kibble that is higher in fat and lower in protein than typical diets that have been fed in managed care, has a fatty acid profile more consistent with wild bear diets, and has been readily consumed by both brown bears and polar bears. The kibble can be fed as the sole diet or as part of more complex diets with additional fruits, meats, or vegetables. Because many nutritional deficiencies and related diseases can take months or years to appear, we urge caution and continued long-term monitoring of bears and their diets to ensure their optimal health.

Quantifying energetic costs and defining energy landscapes experienced by grizzly bears.

Animal movements are major determinants of energy expenditure and ultimately the cost-benefit of landscape use. Thus, we sought to understand those costs and how grizzly bears (Ursus arctos) move in mountainous landscapes. We trained captive grizzly bears to walk on a horizontal treadmill and up and down 10% and 20% slopes. The cost of moving upslope increased linearly with speed and slope angle, and this was more costly than moving horizontally. The cost of downslope travel at slower speeds was greater than the cost of traveling horizontally but appeared to decrease at higher speeds. The most efficient walking speed that minimized cost per unit distance was 1.19±0.11 m s-1 However, grizzly bears fitted with GPS collars in the Greater Yellowstone Ecosystem moved at an average velocity of 0.61±0.28 m s-1 and preferred to travel on near-horizontal slopes at twice their occurrence. When traveling uphill or downhill, grizzly bears chose paths across all slopes that were ∼54% less steep and costly than the maximum available slope. The net costs (J kg-1 m-1) of moving horizontally and uphill were the same for grizzly bears, humans and digitigrade carnivores, but those costs were 46% higher than movement costs for ungulates. These movement costs and characteristics of landscape use determined using captive and wild grizzly bears were used to understand the strategies that grizzly bears use for preying on large ungulates and the similarities in travel between people and grizzly bears that might affect the risk of encountering each other on shared landscapes.

Can offsetting the energetic cost of hibernation restore an active season phenotype in grizzly bears (Ursus arctos horribilis)?

Hibernation is characterized by depression of many physiological processes. To determine if this state is reversible in a non-food caching species, we fed hibernating grizzly bears (Ursus arctos horribilis) dextrose for 10 days to replace 53% or 100% of the estimated minimum daily energetic cost of hibernation. Feeding caused serum concentrations of glycerol and ketones (ß-hydroxybutyrate) to return to active season levels irrespective of the amount of glucose fed. By contrast, free fatty acids (FFAs) and indices of metabolic rate, such as general activity, heart rate, strength of heart rate circadian rhythm, and insulin sensitivity were restored to approximately 50% of active season levels. Body temperature was unaffected by feeding. To determine the contribution of adipose to the metabolic effects observed after glucose feeding, we cultured bear adipocytes collected at the beginning and end of the feeding and performed metabolic flux analysis. We found a ∼33% increase in energy metabolism after feeding. Moreover, basal metabolism before feeding was 40% lower in hibernation cells compared with fed cells or active cells cultured at 37°C, thereby confirming the temperature independence of metabolic rate. The partial depression of circulating FFAs with feeding likely explains the incomplete restoration of insulin sensitivity and other metabolic parameters in hibernating bears. Further depression of metabolic function is likely to be an active process. Together, the results provide a highly controlled model to examine the relationship between nutrient availability and metabolism on the hibernation phenotype in bears.

Phenological synchronization disrupts trophic interactions between Kodiak brown bears and salmon.

Climate change is altering the seasonal timing of life cycle events in organisms across the planet, but the magnitude of change often varies among taxa [Thackeray SJ, et al. (2016) Nature 535:241-245]. This can cause the temporal relationships among species to change, altering the strength of interaction. A large body of work has explored what happens when coevolved species shift out of sync, but virtually no studies have documented the effects of climate-induced synchronization, which could remove temporal barriers between species and create novel interactions. We explored how a predator, the Kodiak brown bear (Ursus arctos middendorffi), responded to asymmetric phenological shifts between its primary trophic resources, sockeye salmon (Oncorhynchus nerka) and red elderberry (Sambucus racemosa). In years with anomalously high spring air temperatures, elderberry fruited several weeks earlier and became available during the period when salmon spawned in tributary streams. Bears departed salmon spawning streams, where they typically kill 25-75% of the salmon [Quinn TP, Cunningham CJ, Wirsing AJ (2016) Oecologia 183:415-429], to forage on berries on adjacent hillsides. This prey switching behavior attenuated an iconic predator-prey interaction and likely altered the many ecological functions that result from bears foraging on salmon [Helfield JM, Naiman RJ (2006) Ecosystems 9:167-180]. We document how climate-induced shifts in resource phenology can alter food webs through a mechanism other than trophic mismatch. The current emphasis on singular consumer-resource interactions fails to capture how climate-altered phenologies reschedule resource availability and alter how energy flows through ecosystems.

Regulation of metabolism during hibernation in brown bears (Ursus arctos): Involvement of cortisol, PGC-1α and AMPK in adipose tissue and skeletal muscle.

The purpose of this study was to investigate changes in expression of known cellular regulators of metabolism during hyperphagia (Sept) and hibernation (Jan) in skeletal muscle and adipose tissue of brown bears and determine whether signaling molecules and transcription factors known to respond to changes in cellular energy state are involved in the regulation of these metabolic adaptations. During hibernation, serum levels of cortisol, glycerol, and triglycerides were elevated, and protein expression and activation of AMPK in skeletal muscle and adipose tissue were reduced. mRNA expression of the co-activator PGC-1α was reduced in all tissues in hibernation whereas mRNA expression of the transcription factor PPAR-α was reduced in the vastus lateralis muscle and adipose tissue only. During hibernation, gene expression of ATGL and CD36 was not altered; however, HSL gene expression was reduced in adipose tissue. During hibernation gene expression of the lipogenic enzyme DGAT in all tissues and the expression of the FA oxidative enzyme LCAD in the vastus lateralis muscle were reduced. Gene and protein expression of the glucose transporter GLUT4 was decreased in adipose tissue in hibernation. Our data suggest that high cortisol levels are a key adaptation during hibernation and link cortisol to a reduced activation of the AMPK/PGC-1α/PPAR-α axis in the regulation of metabolism in skeletal muscle and adipose tissue. Moreover, our results indicate that during this phase of hibernation at a time when metabolic rate is significantly reduced metabolic adaptations in peripheral tissues seek to limit the detrimental effects of unduly large energy dissipation.

Severe Epididymo-Orchitis and Encephalitis Complicating Anti-PD-1 Therapy.

BACKGROUND: Immune checkpoint inhibitors such as pembrolizumab and nivolumab have emerged as active treatment options for patients with many cancers, including metastatic melanoma, but can also cause symptomatic or life-threatening immune-related adverse events, including encephalitis. Epididymitis and orchitis are rare complications of these therapies. CASE PRESENTATION: We describe herein a patient with metastatic melanoma who developed epididymo-orchitis followed by encephalitis while receiving pembrolizumab. The patient developed testicular pain and fever after his third dose of pembrolizumab; ultrasound evaluation demonstrated bilateral epididymo-orchitis. He then developed headaches, fever, and altered mental status over the next week and was admitted to the hospital. Lumbar puncture revealed inflammatory changes consistent with meningoencephalitis; he did not improve with broad-spectrum antibiotics, and an extensive workup for infectious etiologies, including cerebrospinal fluid testing using a clinical metagenomic next-generation sequencing assay, was negative. He received high-dose steroids for suspected autoimmune encephalitis, and both his orchitis and meningoencephalitis improved rapidly after one dose. He fully recovered after a 5-week taper of oral steroids. DISCUSSION: Here, we report a case of epididymo-orchitis complicating immune checkpoint inhibitor therapy. This patient subsequently developed severe encephalitis but rapidly improved with steroids. Clinicians should be aware of rare complications of these agents. KEY POINTS: Epididymo-orchitis is a rare and potentially life-threatening complication of anti-programmed death protein 1 (anti-PD-1) therapy.For patients on anti-PD-1 therapy who develop either epididymo-orchitis or epididymitis without clear infectious cause, immune-related adverse events should be considered in the differential diagnosis.If severe, epididymo-orchitis related to anti-PD-1 therapy may be treated with high-dose corticosteroids.

Energetic costs of locomotion in bears: is plantigrade locomotion energetically economical?

Ursids are the largest mammals to retain a plantigrade posture. This primitive posture has been proposed to result in reduced locomotor speed and economy relative to digitigrade and unguligrade species, particularly at high speeds. Previous energetics research on polar bears (Ursus maritimus) found locomotor costs were more than double predictions for similarly sized quadrupedal mammals, which could be a result of their plantigrade posture or due to adaptations to their Arctic marine existence. To evaluate whether polar bears are representative of terrestrial ursids or distinctly uneconomical walkers, this study measured the mass-specific metabolism, overall dynamic body acceleration, and gait kinematics of polar bears and grizzly bears (Ursus arctos) trained to rest and walk on a treadmill. At routine walking speeds, we found polar bears and grizzly bears exhibited similar costs of locomotion and gait kinematics, but differing measures of overall dynamic body acceleration. Minimum cost of transport while walking in the two species (2.21 J kg-1 m-1) was comparable to predictions for similarly sized quadrupedal mammals, but these costs doubled (4.42 J kg-1 m-1) at speeds ≥5.4 km h-1 Similar to humans, another large plantigrade mammal, bears appear to exhibit a greater economy while moving at slow speeds.

Grizzly bear (Ursus arctos horribilis) locomotion: forelimb joint mechanics across speed in the sagittal and frontal planes.

The majority of terrestrial locomotion studies have focused on parasagittal motion and paid less attention to forces or movement in the frontal plane. Our previous research has shown that grizzly bears produce higher medial ground reaction forces (lateral pushing from the animal) than would be expected for an upright mammal, suggesting frontal plane movement may be an important aspect of their locomotion. To examine this, we conducted an inverse dynamics analysis in the sagittal and frontal planes, using ground reaction forces and position data from three high-speed cameras of four adult female grizzly bears. Over the speed range collected, the bears used walks, running walks and canters. The scapulohumeral joint, wrist and the limb overall absorb energy (average total net work of the forelimb joints, -0.97 W kg-1). The scapulohumeral joint, elbow and total net work of the forelimb joints have negative relationships with speed, resulting in more energy absorbed by the forelimb at higher speeds (running walks and canters). The net joint moment and power curves maintain similar patterns across speed as in previously studied species, suggesting grizzly bears maintain similar joint dynamics to other mammalian quadrupeds. There is no significant relationship with net work and speed at any joint in the frontal plane. The total net work of the forelimb joints in the frontal plane was not significantly different from zero, suggesting that, despite the high medial ground reaction forces, the forelimb acts as a strut in that plane.

Habitat degradation affects the summer activity of polar bears.

Understanding behavioral responses of species to environmental change is critical to forecasting population-level effects. Although climate change is significantly impacting species' distributions, few studies have examined associated changes in behavior. Polar bear (Ursus maritimus) subpopulations have varied in their near-term responses to sea ice decline. We examined behavioral responses of two adjacent subpopulations to changes in habitat availability during the annual sea ice minimum using activity data. Location and activity sensor data collected from 1989 to 2014 for 202 adult female polar bears in the Southern Beaufort Sea (SB) and Chukchi Sea (CS) subpopulations were used to compare activity in three habitat types varying in prey availability: (1) land; (2) ice over shallow, biologically productive waters; and (3) ice over deeper, less productive waters. Bears varied activity across and within habitats with the highest activity at 50-75% sea ice concentration over shallow waters. On land, SB bears exhibited variable but relatively high activity associated with the use of subsistence-harvested bowhead whale carcasses, whereas CS bears exhibited low activity consistent with minimal feeding. Both subpopulations had fewer observations in their preferred shallow-water sea ice habitats in recent years, corresponding with declines in availability of this substrate. The substantially higher use of marginal habitats by SB bears is an additional mechanism potentially explaining why this subpopulation has experienced negative effects of sea ice loss compared to the still-productive CS subpopulation. Variability in activity among, and within, habitats suggests that bears alter their behavior in response to habitat conditions, presumably in an attempt to balance prey availability with energy costs.

The bear circadian clock doesn't 'sleep' during winter dormancy.

BACKGROUND: Most biological functions are synchronized to the environmental light:dark cycle via a circadian timekeeping system. Bears exhibit shallow torpor combined with metabolic suppression during winter dormancy. We sought to confirm that free-running circadian rhythms of body temperature (Tb) and activity were expressed in torpid grizzly (brown) bears and that they were functionally responsive to environmental light. We also measured activity and ambient light exposures in denning wild bears to determine if rhythms were evident and what the photic conditions of their natural dens were. Lastly, we used cultured skin fibroblasts obtained from captive torpid bears to assess molecular clock operation in peripheral tissues. Circadian parameters were estimated using robust wavelet transforms and maximum entropy spectral analyses. RESULTS: Captive grizzly bears housed in constant darkness during winter dormancy expressed circadian rhythms of activity and Tb. The rhythm period of juvenile bears was significantly shorter than that of adult bears. However, the period of activity rhythms in adult captive bears was virtually identical to that of adult wild denning bears as was the strength of the activity rhythms. Similar to what has been found in other mammals, a single light exposure during the bear's active period delayed subsequent activity onsets whereas these were advanced when light was applied during the bear's inactive period. Lastly, in vitro studies confirmed the expression of molecular circadian rhythms with a period comparable to the bear's own behavioral rhythms. CONCLUSIONS: Based on these findings we conclude that the circadian system is functional in torpid bears and their peripheral tissues even when housed in constant darkness, is responsive to phase-shifting effects of light, and therefore, is a normal facet of torpid bear physiology.

Sub-chronic administration of LY294002 sensitizes cervical cancer cells to chemotherapy by enhancing mitochondrial JNK signaling.

Chemo-sensitization is used to improve the efficacy of chemotherapeutic agents against cancers, and understanding the precise molecular mechanisms of chemo-sensitization could lead to safer and more effective approaches to treat cancer. We have previously demonstrated that mitochondrial c-Jun N-terminal Kinase (JNK) signaling is a critical component of cell death. Mitochondrial JNK signaling is coordinated on the scaffold protein Sab. In this work, we developed a sub-chronic chemo-sensitization model by exposing HeLa cells to low-dose (2 µM) LY294002. We found that this treatment increased Sab expression on mitochondria, an effect not observed in acute exposures. To examine the role of Sab in chemo-sensitization, we ectopically expressed and silenced Sab in HeLa cells. We found that elevating Sab levels in HeLa cells increased the efficacy of chemotherapeutic agents, paclitaxel and cisplatin, while silencing Sab decreased the sensitivity of cells towards these agents. The effect of Sab-mediated signaling appeared to be dependent upon mitogen dependent protein kinases (MAPKs) as ablation of Sab's MAPK-binding motifs prevented chemo-sensitization. These results suggest that mitochondrial JNK signaling is an adaptable signaling pathway that can be enhanced or restored in cancer cells to improve therapeutic efficacy.

Grizzly bear (Ursus arctos horribilis) locomotion: gaits and ground reaction forces.

Locomotion of plantigrade generalists has been relatively little studied compared with more specialised postures even though plantigrady is ancestral among quadrupeds. Bears (Ursidae) are a representative family for plantigrade carnivorans, they have the majority of the morphological characteristics identified for plantigrade species, and they have the full range of generalist behaviours. This study compared the locomotion of adult grizzly bears (Ursus arctos horribilis Linnaeus 1758), including stride parameters, gaits and analysis of three-dimensional ground reaction forces, with that of previously studied quadrupeds. At slow to moderate speeds, grizzly bears use walks, running walks and canters. Vertical ground reaction forces demonstrated the typical M-shaped curve for walks; however, this was significantly more pronounced in the hindlimb. The rate of force development was also significantly higher for the hindlimbs than for the forelimbs at all speeds. Mediolateral forces were significantly higher than would be expected for a large erect mammal, almost to the extent of a sprawling crocodilian. There may be morphological or energetic explanations for the use of the running walk rather than the trot. The high medial forces (produced from a lateral push by the animal) could be caused by frontal plane movement of the carpus and elbow by bears. Overall, while grizzly bears share some similarities with large cursorial species, their locomotor kinetics have unique characteristics. Additional studies are needed to determine whether these characters are a feature of all bears or plantigrade species.

Wolves trigger a trophic cascade to berries as alternative food for grizzly bears.

This is a Forum article in response to: Barber-Meyer, S. (2015) Trophic cascades from wolves to grizzly bears or changing abundance of bears and alternate foods? Journal of Animal Ecology, 83, doi: 10.1111/1365-2656.12338. We used multiple data sets and study areas as well as several lines of evidence to investigate potential trophic linkages in Yellowstone National Park. Our results suggest that a trophic cascade from wolves to elk to berry production to berry consumption by grizzly bears may now be underway in the Park.

Validation of a novel cognitive bias task based on difference in quantity of reinforcement for assessing environmental enrichment.

Cognitive bias tasks purport to assess affective states via responses to ambiguous stimuli. We hypothesized that a novel cognitive bias task based on positive reinforcement using quantity differences would detect changes in affect in captive grizzly bears (Ursus arctos horribilis). We trained bears (n = 8) to respond differently (nose or paw touch) to two stimuli (light or dark gray cue cards), with responses counterbalanced across bears. The two cues signaled a small or large food reward, respectively. Responses to ambiguous probe stimuli (i.e., shades of gray) intermediate to the trained stimuli were classified as either 'optimistic,' appropriate for the larger reward, or 'pessimistic,' appropriate for the smaller reward. In Experiment 1, we explored the contrast in reward size necessary to detect a change in response across probe stimuli (large reward, 3 or 6 apple slices: small reward, 1 slice). We observed a change in response across probe stimuli, with no difference in response between reward-value groups, indicating that a contrast of 3:1 apple slices was sufficient to affect responses. In Experiment 2, we investigated cognitive bias after 2.1 h of exposure to enrichment items varying in attractiveness. Results were unaffected by enrichment type or time spent interacting with enrichments, indicating that the task failed to demonstrate criterion validity for comparing mood following exposure to different enrichment items. However, greater time spent pacing prior to testing was associated with 'optimistic' judgments. The data provide some support for use of cognitive bias tasks based on quantity differences in animal welfare assessments involving captive wildlife.

Trophic cascades from wolves to grizzly bears in Yellowstone.

We explored multiple linkages among grey wolves (Canis lupus), elk (Cervus elaphus), berry-producing shrubs and grizzly bears (Ursus arctos) in Yellowstone National Park. We hypothesized competition between elk and grizzly bears whereby, in the absence of wolves, increases in elk numbers would increase browsing on berry-producing shrubs and decrease fruit availability to grizzly bears. After wolves were reintroduced and with a reduced elk population, we hypothesized there would be an increase in the establishment of berry-producing shrubs, such as serviceberry (Amelanchier alnifolia), which is a major berry-producing plant. We also hypothesized that the percentage fruit in the grizzly bear diet would be greater after than before wolf reintroduction. We compared the frequency of fruit in grizzly bear scats to elk densities prior to wolf reintroduction during a time of increasing elk densities (1968-1987). For a period after wolf reintroduction, we calculated the percentage fruit in grizzly bear scat by month based on scats collected in 2007-2009 (n = 778 scats) and compared these results to scat data collected before wolf reintroduction. Additionally, we developed an age structure for serviceberry showing the origination year of stems in a northern range study area. We found that over a 19-year period, the percentage frequency of fruit in the grizzly diet (6231 scats) was inversely correlated (P < 0·001) with elk population size. The average percentage fruit in grizzly bear scats was higher after wolf reintroduction in July (0·3% vs. 5·9%) and August (7·8% vs. 14·6%) than before. All measured serviceberry stems accessible to ungulates originated since wolf reintroduction, while protected serviceberry growing in a nearby ungulate exclosure originated both before and after wolf reintroduction. Moreover, in recent years, browsing of serviceberry outside of the exclosure decreased while their heights increased. Overall, these results are consistent with a trophic cascade involving increased predation by wolves and other large carnivores on elk, a reduced and redistributed elk population, decreased herbivory and increased production of plant-based foods that may aid threatened grizzly bears.