Gut microbial communities of American pikas (Ochotona princeps): Evidence for phylosymbiosis and adaptations to novel diets.

Gut microbial communities provide many physiological functions to their hosts, especially in herbivorous animals. We still lack an understanding of how these microbial communities are structured across hosts in nature, especially within a given host species. Studies on laboratory mice have demonstrated that host genetics can influence microbial community structure, but that diet can overwhelm these genetic effects. We aimed to test these ideas in a natural system, the American pika (Ochotona princeps). First, pikas are high-elevation specialists with significant population structure across various mountain ranges in the USA, allowing us to investigate whether similarities in microbial communities match host genetic differences. Additionally, pikas are herbivorous, with some populations exhibiting remarkable dietary plasticity and consuming high levels of moss, which is exceptionally high in fibre and low in protein. This allows us to investigate adaptations to an herbivorous diet, as well as to the especially challenging diet of moss. Here, we inventoried the microbial communities of pika caecal pellets from various populations using 16S rRNA sequencing to investigate structuring of microbial communities across various populations with different natural diets. Microbial communities varied significantly across populations, and differences in microbial community structure were congruent with genetic differences in host population structure, a pattern known as "phylosymbiosis." Several microbial members (Ruminococcus, Prevotella, Oxalobacter and Coprococcus) were detected across all samples, and thus likely represent a "core microbiome." These genera are known to perform a number of services for herbivorous hosts such as fibre fermentation and the degradation of plant defensive compounds, and thus are likely important for herbivory in pikas. Moreover, pikas that feed on moss harboured microbial communities highly enriched in Melainabacteria. This uncultivable candidate phylum has been proposed to ferment fibre for herbivores, and thus may contribute to the ability of some pika populations to consume high amounts of moss. These findings demonstrate that both host genetics and diet can influence the microbial communities of the American pika. These animals may be novel sources of fibre-degrading microbes. Last, we discuss the implications of population-specific microbial communities for conservation efforts in this species.

Temporal vs. spatial variation in stress-associated metabolites within a population of climate-sensitive small mammals.

Temporal variation in stress might signify changes in an animal's internal or external environment, while spatial variation in stress might signify variation in the quality of the habitats that individual animals experience. Habitat-induced variations in stress might be easiest to detect in highly territorial animals, and especially in species that do not take advantage of common strategies for modulating habitat-induced stress, such as migration (escape in space) or hibernation (escape in time). Spatial and temporal variation in response to potential stressors has received little study in wild animals, especially at scales appropriate for relating stress to specific habitat characteristics. Here, we use the American pika (Ochotona princeps), a territorial small mammal, to investigate stress response within and among territories. For individually territorial animals such as pikas, differences in habitat quality should lead to differences in stress exhibited by territory owners. We indexed stress using stress-associated hormone metabolites in feces collected non-invasively from pika territories every 2 weeks from June to September 2018. We hypothesized that differences in territory quality would lead to spatial differences in mean stress and that seasonal variation in physiology or the physical environment would lead to synchronous variation across territories through time. We used linear mixed-effects models to explore spatiotemporal variation in stress using fixed effects of day-of-year and broad habitat characteristics (elevation, aspect, site), as well as local variation in habitat characteristics hypothesized to affect territory quality for this saxicolous species (talus depth, clast size, available forage types). We found that temporal variation within territories was greater than spatial variation among territories, suggesting that shared seasonal stressors are more influential than differences in individual habitat quality. This approach could be used in other wildlife studies to refine our understanding of habitat quality and its effect on individual stress levels as a driver of population decline.

Connected While Distant: Networking CUREs Across Classrooms to Create Community and Empower Students.

Connections, collaborations, and community are key to the success of individual scientists as well as transformative scientific advances. Intentionally building these components into science, technology, engineering and mathematics (STEM) education can better prepare future generations of researchers. Course-based undergraduate research experiences (CUREs) are a new, fast-growing teaching practice in STEM that expand opportunities for undergraduate students to gain research skills. Because they engage all students in a course in an authentic research experience focused on a relevant scientific problem, CUREs provide an opportunity to foster community among students while promoting critical thinking skills and positively influencing their identities as scientists. Here, we review CUREs in the biological sciences that were developed as multi-institutional networks, and highlight the benefits gained by students and instructors through participation in a CURE network. Throughout, we introduce Squirrel-Net, a network of ecology-focused and field-based CUREs that intentionally create connections among students and instructors. Squirrel-Net CUREs can also be scaffolded into the curriculum to form connections between courses, and are easily transitioned to distance-based delivery. Future assessments of networked CUREs like Squirrel-Net will help elucidate how CURE networks create community and how a cultivated research community impacts students' performance, perceptions of science, and sense of belonging. We hypothesize networked CUREs have the potential to create a broader sense of belonging among students and instructors alike, which could result in better science and more confident scientists.

When can we measure stress noninvasively? Postdeposition effects on a fecal stress metric confound a multiregional assessment.

Measurement of stress hormone metabolites in fecal samples has become a common method to assess physiological stress in wildlife populations. Glucocorticoid metabolite (GCM) measurements can be collected noninvasively, and studies relating this stress metric to anthropogenic disturbance are increasing. However, environmental characteristics (e.g., temperature) can alter measured GCM concentration when fecal samples cannot be collected immediately after defecation. This effect can confound efforts to separate environmental factors causing predeposition physiological stress in an individual from those acting on a fecal sample postdeposition. We used fecal samples from American pikas (Ochotona princeps) to examine the influence of environmental conditions on GCM concentration by (1) comparing GCM concentration measured in freshly collected control samples to those placed in natural habitats for timed exposure, and (2) relating GCM concentration in samples collected noninvasively throughout the western United States to local environmental characteristics measured before and after deposition. Our timed-exposure trials clarified the spatial scale at which exposure to environmental factors postdeposition influences GCM concentration in pika feces. Also, fecal samples collected from occupied pika habitats throughout the species' range revealed significant relationships between GCM and metrics of climate during the postdeposition period (maximum temperature, minimum temperature, and precipitation during the month of sample collection). Conversely, we found no such relationships between GCM and metrics of climate during the predeposition period (prior to the month of sample collection). Together, these results indicate that noninvasive measurement of physiological stress in pikas across the western US may be confounded by climatic conditions in the postdeposition environment when samples cannot be collected immediately after defecation. Our results reiterate the importance of considering postdeposition environmental influences on this stress metric, especially in multiregional comparisons. However, measurements of fecal GCM concentration should prove useful for population monitoring within an eco-region or when postdeposition exposure can be minimized.

Plastic pikas: Behavioural flexibility in low-elevation pikas (Ochotona princeps).

Behaviour is an important mechanism for accommodating rapid environmental changes. Understanding a species' capacity for behavioural plasticity is therefore a key, but understudied, aspect of developing tractable conservation and management plans under climate-change scenarios. Here, we quantified behavioural differences between American pikas (Ochotona princeps) living in an atypical, low-elevation habitat versus those living in a more-typical, alpine habitat. With respect to foraging strategy, low-elevation pikas spent more time consuming vegetation and less time caching food for winter, compared to high-elevation pikas. Low-elevation pikas were also far more likely to be detected in forested microhabitats off the talus than their high-elevation counterparts at midday. Finally, pikas living in the atypical habitat had smaller home range sizes compared to those in typical habitat or any previously published home ranges for this species. Our findings indicate that behavioural plasticity likely allows pikas to accommodate atypical conditions in this low-elevation habitat, and that they may rely on critical habitat factors such as suitable microclimate refugia to behaviourally thermoregulate. Together, these results suggest that behavioural adjustments are one important mechanism by which pikas can persist outside of their previously appreciated dietary and thermal niches.

Relating sub-surface ice features to physiological stress in a climate sensitive mammal, the American pika (Ochotona princeps).

The American pika (Ochotona princeps) is considered a sentinel species for detecting ecological effects of climate change. Pikas are declining within a large portion of their range, and ongoing research suggests loss of sub-surface ice as a mechanism. However, no studies have demonstrated physiological responses of pikas to sub-surface ice features. Here we present the first analysis of physiological stress in pikas living in and adjacent to habitats underlain by ice. Fresh fecal samples were collected non-invasively from two adjacent sites in the Rocky Mountains (one with sub-surface ice and one without) and analyzed for glucocorticoid metabolites (GCM). We also measured sub-surface microclimates in each habitat. Results indicate lower GCM concentration in sites with sub-surface ice, suggesting that pikas are less stressed in favorable microclimates resulting from sub-surface ice features. GCM response was well predicted by habitat characteristics associated with sub-surface ice features, such as lower mean summer temperatures. These results suggest that pikas inhabiting areas without sub-surface ice features are experiencing higher levels of physiological stress and may be more susceptible to changing climates. Although post-deposition environmental effects can confound analyses based on fecal GCM, we found no evidence for such effects in this study. Sub-surface ice features are key to water cycling and storage and will likely represent an increasingly important component of water resources in a warming climate. Fecal samples collected from additional watersheds as part of current pika monitoring programs could be used to further characterize relationships between pika stress and sub-surface ice features.

The importance of biologically relevant microclimates in habitat suitability assessments.

Predicting habitat suitability under climate change is vital to conserving biodiversity. However, current species distribution models rely on coarse scale climate data, whereas fine scale microclimate data may be necessary to assess habitat suitability and generate predictive models. Here, we evaluate disparities between temperature data at the coarse scale from weather stations versus fine-scale data measured in microhabitats required for a climate-sensitive mammal, the American pika (Ochotona princeps). We collected two years of temperature data in occupied talus habitats predicted to be suitable (high elevation) and unsuitable (low elevation) by the bioclimatic envelope approach. At low elevations, talus surface and interstitial microclimates drastically differed from ambient temperatures measured on-site and at a nearby weather station. Interstitial talus temperatures were frequently decoupled from high ambient temperatures, resulting in instantaneous disparities of over 30 °C between these two measurements. Microhabitat temperatures were also highly heterogeneous, such that temperature measurements within the same patch of talus were not more correlated than measurements at distant patches. An experimental manipulation revealed that vegetation cover may cool the talus surface by up to 10 °C during the summer, which may contribute to this spatial heterogeneity. Finally, low elevation microclimates were milder and less variable than typical alpine habitat, suggesting that, counter to species distribution model predictions, these seemingly unsuitable habitats may actually be better refugia for this species under climate change. These results highlight the importance of fine-scale microhabitat data in habitat assessments and underscore the notion that some critical refugia may be counterintuitive.

A microfluidic device to investigate axon targeting by limited numbers of purified cortical projection neuron subtypes.

While much is known about general controls over axon guidance of broad classes of projection neurons (those with long-distance axonal connections), molecular controls over specific axon targeting by distinct neuron subtypes are poorly understood. Corticospinal motor neurons (CSMN) are prototypical and clinically important cerebral cortex projection neurons; they are the brain neurons that degenerate in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases, and their injury is central to the loss of motor function in spinal cord injury. Primary culture of purified immature murine CSMN has been recently established, using either fluorescence-activated cell sorting (FACS) or immunopanning, enabling a previously unattainable level of subtype-specific investigation, but the resulting number of CSMN is quite limiting for standard approaches to study axon guidance. We developed a microfluidic system specifically designed to investigate axon targeting of limited numbers of purified CSMN and other projection neurons in culture. The system contains two chambers for culturing target tissue explants, allowing for biologically revealing axonal growth "choice" experiments. This device will be uniquely enabling for investigation of controls over axon growth and neuronal survival of many types of neurons, particularly those available only in limited numbers.

Estimating duration of infection with avidity assays: potential limitations and recommendations for improvement.

Recent infections often have higher pathogen loads. The number of recent infections can therefore be used to estimate transmission rates in a host population. Antibody avidity assays are an emerging technique to infer infection age in both domestic and wild animals. These assays have the potential to supplant intensive mark-recapture efforts for identification of recent infections, but their results may be confounded by antibody titer. We examined the effectiveness of an avidity assay for identifying recent infections of Sin Nombre virus, a hantavirus in North America that establishes a chronic infection in deer mice (Peromyscus maniculatus). We found that assay performance statistics such as sensitivity, specificity, and positive predictive value for low avidity scores were significantly improved when we accounted for antibody titer in the analyses. Without accounting for titer, avidity assays may classify samples with low titers as recent infections regardless of actual infection history, thereby overestimating the number of recent infections in a population and inflating estimates of transmission rates and/or human exposure risk. We recommend that antibody titers meet a minimum threshold for use in avidity assays, and we emphasize the importance of considering titer and dilution in the validation of newly developed avidity assays.

Long-term patterns of immune investment by wild deer mice infected with Sin Nombre virus.

Immunocompetence of animals fluctuates seasonally, However, there is little consensus on the cause of these fluctuations. Some studies have suggested that these patterns are influenced by changes in reproductive condition, whereas others have suggested that differences result from seasonal variations in energy expenditures. The objective of our study was to examine these contrasting views of immunity by evaluating seasonal patterns of immune response and reproduction in wild populations of deer mice Peromyscus maniculatus exposed to Sin Nombre virus (SNV). Over three consecutive fall (September, October, November) and three consecutive spring (March, April, May) sampling periods, we used titration enzyme-linked immunosorbent assay (ELISA) to quantify virus-specific antibody production in 48 deer mice infected with SNV. Levels of reproductive hormones were quantified using ELISA. SNV antibody titers reached their lowest level during November (geometric mean titer [GMT] = 420) and their highest levels during September (GMT = 5,545) and May (GMT = 3,582), suggesting that the immune response of deer mice to SNV has seasonal patterns. The seeming decrease in antibody titer over winter coupled with the consistency in body masses suggests that during winter, immunocompetence may be compromised to offset the energetic costs of maintenance functions, including those associated with maintaining body mass. Deer mice showed distinct sex-based differences in SNV antibody production, with males producing higher antibody titers (GMT = 3,333) than females (GMT = 1,477). Levels of reproductive hormones do not appear to influence antibody production in either males or females, as there was no correlation between estradiol concentrations and SNV antibody titer in female deer mice (r² = 0.26), nor was there a significant relationship between levels of testosterone and SNV antibody titers in males (r² = 0.28). Collectively, this study demonstrates that immunocompetence of wild deer mice is seasonally variable; however, reproduction is not the primary stressor responsible for this variation. Rather, the data suggest that deer mice may compromise immunocompetence during winter to offset other maintenance costs during this period.