Investigating the effects of acute and chronic stress on DNA damage.

A hallmark of the vertebrate stress response is a rapid increase in glucocorticoids and catecholamines; however, this does not mean that these mediators are the best, or should be the only, metric measured when studying stress. Instead, it is becoming increasingly clear that assaying a suite of downstream metrics is necessary in stress physiology. One component of this suite could be assessing double-stranded DNA damage (dsDNA damage), which has recently been shown to increase in blood with both acute and chronic stress in house sparrows (Passer domesticus). To further understand the relationship between stress and dsDNA damage, we designed two experiments to address the following questions: (1) how does dsDNA damage with chronic stress vary across tissues? (2) does the increase in dsDNA damage during acute stress come from one arm of the stress response or both? We found that (1) dsDNA damage affects tissues differently during chronic stress and (2) the hypothalamic-pituitary-adrenal axis influences dsDNA damage with acute stress, but the sympathetic-adreno-medullary system does not. Surprisingly, our data are not explained by studies on changes in hormone receptor levels with chronic stress, so the underlying mechanism remains unclear.

A mathematical representation of the reactive scope model.

Researchers have long sought to understand and predict an animal's response to stressful stimuli. Since the introduction of the concept of homeostasis, a variety of model frameworks have been proposed to describe what is necessary for an animal to remain within this stable physiological state and the ramifications of leaving it. Romero et al. (Horm Behav 55(3):375-389, 2009) introduced the reactive scope model to provide a novel conceptual framework for the stress response that assumes an animal's ability to tolerate a stressful stimulus may degrade over time in response to the stimulus. We provide a mathematical formulation for the reactive scope model using a system of ordinary differential equations and show that this model is capable of recreating existing experimental data. We also provide an experimental method that may be used to verify the model as well as several potential additions to the model. If future experimentation provides the necessary data to estimate the model's parameters, the model presented here may be used to make quantitative predictions about physiological mediator levels during a stress response and predict the onset of homeostatic overload.

The effect of infection risk on female blood transcriptomics.

Defenses against pathogens can take on many forms. For instance, behavioral avoidance of diseased conspecifics is widely documented. Interactions with these infectious conspecifics can also, however, lead to physiological changes in uninfected animals, an effect that is much less well understood. These changes in behavior and physiology are particularly important to study in a reproductive context, where they can impact reproductive decisions and offspring quality. Here, we studied how an acute (3 h) exposure to an immune-challenged male affected female blood transcriptomics and behavior. We predicted that females paired with immune-challenged males would reduce eating and drinking behaviors (as avoidance behaviors) and that their blood would show activation of immune and stress responses. We used female Japanese quail as a study system because they have been shown to respond to male traits, in terms of their own physiology and egg investment. Only two genes showed significant differential expression due to treatment, including an increase in the threonine dehydrogenase (TDH) transcript, an enzyme important for threonine breakdown. However, hundreds of genes in pathways related to activation of immune responses showed coordinated up-regulation in females exposed to immune-challenged males. Suppressed pathways revealed potential changes to metabolism and reduced responsiveness to glucocorticoids. Contrary to our prediction, we found that females paired with immune-challenged males increased food consumption. Water consumption was not changed by treatment. These findings suggest that even short exposure to diseased conspecifics can trigger both behavioral and physiological responses in healthy animals.

Background DNA damage is higher in summer than winter in both free-living and captive birds.

Although stress can cause overall damage to the genome, it is currently unknown whether normal background damage to DNA varies throughout the annual cycle. If DNA damage did vary seasonally, it would have major implications on environmental-genomic interactions. We measured background DNA double-stranded breaks using the neutral comet assay in five tissues (nucleated red blood cells, abdominal fat, hippocampus, hypothalamus, and liver) in four cohorts of house sparrows (Passer domesticus): free-living summer, captives on a summer light cycle, free-living winter, and captives on a winter light cycle. The experiment was designed to answer three questions: (1) Is red blood cell DNA damage representative of other tissues? (2) Is DNA damage in captive birds representative of DNA damage in free-living birds? (3) Does DNA damage show seasonality? We found that (1) blood is a representative tissue, (2) captive animals are representative of free-living animals, and (3) DNA damage is higher in the summer than in the winter. These data indicate that red blood cells can be an index of DNA damage throughout the body and that background levels of DNA damage show substantial seasonal variation. The latter result suggests the possibility that underlying molecular mechanisms of DNA damage and/or repair also change seasonally.

Maternal Responses in the Face of Infection Risk.

When animals are sick, their physiology and behavior change in ways that can impact their offspring. Research is emerging showing that infection risk alone can also modify the physiology and behavior of healthy animals. If physiological responses to environments with high infection risk take place during reproduction, it is possible that they lead to maternal effects. Understanding whether and how high infection risk triggers maternal effects is important to elucidate how the impacts of infectious agents extend beyond infected individuals and how, in this way, they are even stronger evolutionary forces than already considered. Here, to evaluate the effects of infection risk on maternal responses, we exposed healthy female Japanese quail to either an immune-challenged (lipopolysaccharide [LPS] treated) mate or to a healthy (control) mate. We first assessed how females responded behaviorally to these treatments. Exposure to an immune-challenged or control male was immediately followed by exposure to a healthy male, to determine whether treatment affected paternity allocation. We predicted that females paired with immune-challenged males would avoid and show aggression towards those males, and that paternity would be skewed towards the healthy male. After mating, we collected eggs over a 5-day period. As an additional control, we collected eggs from immune-challenged females mated to healthy males. We tested eggs for fertilization status, embryo sex ratio, as well as albumen corticosterone, lysozyme activity, and ovotransferrin, and yolk antioxidant capacity. We predicted that immune-challenged females would show the strongest changes in the egg and embryo metrics, and that females exposed to immune-challenged males would show intermediate responses. Contrary to our predictions, we found no avoidance of immune-challenged males and no differences in terms of paternity allocation. Immune-challenged females laid fewer eggs, with an almost bimodal distribution of sex ratio for embryos. In this group, albumen ovotransferrin was the lowest, and yolk antioxidant capacity decreased over time, while it increased in the other treatments. No differences in albumen lysozyme were found. Both females that were immune-challenged and those exposed to immune-challenged males deposited progressively more corticosterone in their eggs over time, a pattern opposed to that shown by females exposed to control males. Our results suggest that egg-laying Japanese quail may be able to respond to infection risk, but that additional or prolonged sickness symptoms may be needed for more extensive maternal responses.

Female presence does not increase testosterone but still ameliorates sickness behaviours in male Japanese quail.

Infections can dramatically modify animal behaviour. The extent of these changes depends on an animal's environment. It has been proposed that testosterone modulates the suppression of behavioural symptoms of sickness under certain reproductive contexts. To further understand the role played by testosterone in modulating sickness behaviours under reproductive contexts, we studied a species, the Japanese quail, in which female exposure rapidly decreases circulating testosterone in males. Males received either an immune challenge (lipopolysaccharide - LPS) or a control injection and their behaviours, mass change and testosterone levels were quantified in the presence or absence of a female. Both the presence of a female and LPS treatment reduced testosterone levels. LPS-treated males maintained in isolation expressed expected sickness behaviours, including increased resting (quantified as crouching) and decreased food and water intake. Despite the reduction in testosterone, when paired with females LPS-treated males showed similar amounts of mating behaviours to controls and reduced crouching. In sum, even under very low levels of testosterone, male quail had reduced sickness behaviours when exposed to females, indicating that testosterone may not be key in modulating sickness behaviours, at least in this species.

Interannual measures of nutritional stress during a marine heatwave (the Blob) differ between two North Pacific seabird species.

'The Blob', a mass of anomalously warm water in the Northeast Pacific Ocean peaking from 2014 to 2016, caused a decrease in primary productivity with cascading effects on the marine ecosystem. Among the more obvious manifestations of the event were seabird breeding failures and mass mortality events. Here, we used corticosterone in breast feathers (fCort), grown in the winter period during migration, as an indicator of nutritional stress to investigate the impact of the Blob on two sentinel Pacific auk species (family Alcidae). Feathers were collected from breeding females over 8 years from 2010 to 2017, encompassing the Blob period. Since Pacific auks replace body feathers at sea during the migratory period, measures of fCort provide an accumulated measure of nutritional stress or allostatic load during this time. Changes in diet were also measured using δ15N and δ13C values from feathers. Relative to years prior to the Blob, the primarily zooplanktivorous Cassin's auklets (Ptychoramphus aleuticus) had elevated fCort in 2014-2017, which correlated with the occurrence of the Blob and a recovery period afterwards, with relatively stable feather isotope values. In contrast, generalist rhinoceros auklets (Cerorhinca monocerata) displayed stable fCort values across years and increased δ15N values during the Blob. As marine heatwaves increase in intensity and frequency due to climate change, this study provides insight into the variable response of Pacific auks to such phenomena and suggests a means for monitoring population-level responses to climatological variation.

Facilitating Constructive Discussions of Difficult Socio-Scientific Issues.

Discussion can be an important and powerful tool in efforts to build a more diverse, equitable, and inclusive future for STEM (i.e., science, technology, engineering, and mathematics). However, facilitating discussions on difficult, complex, and often uncomfortable issues, like racism and sexism, can feel daunting. We outline a series of steps that can be used by educators to facilitate productive discussions that empower everyone to listen, contribute, learn, and ultimately act to transform STEM.

Beyond corticosterone: The acute stress response increases DNA damage in house sparrows.

Although corticosterone (Cort) has been the predominant metric used to assess acute stress in birds, it does not always accurately reflect how an animal copes with a stressor. Downstream measurements may be more reliable. In the current study, we tested the hypothesis that acute increases in DNA damage could be used to assess stressor exposure. Studies have shown DNA damage increases in response to stress-related hormones in vitro; however, this has not yet been thoroughly applied in wild animals. We exposed house sparrows (Passer domesticus) to a 30- or 120-min restraint stressor and took blood samples at 0, 30, 60, and 120 min to measure Cort, DNA damage, and uric acid. Both treatments increased DNA damage and Cort, and decreased uric acid. It thus appears that DNA damage can reflect acute stressor exposure. To improve the usability of DNA damage as a metric for stress, we also tested the impacts of sample storage on DNA damage. Leaving red blood cells on ice for up to 24 hr, only slightly influenced DNA damage. Freezing blood samples for 1-4 weeks substantially increased DNA damage. These findings emphasize the importance of reducing variation between samples by assaying them together whenever possible. Overall, these results indicate that assessing DNA damage is a valid method of assessing acute stressor exposure that is suitable for both laboratory- and field-based studies; however, additional research is needed on the molecular dynamics of nucleated red blood cells, including whether and how their DNA is repaired.

Captive house sparrows (Passer domesticus) show little evidence of seasonality of neophobia responses.

Neophobia is a behavior characterized by a reluctance to approach novel objects. We measured neophobia in captive wild house sparrows (Passer domesticus) by comparing the time it took for hungry birds to approach their normal food dish compared to one that had been modified with a novel object. We tested the behavior of animals captured at different times of year to test for seasonal variation in neophobia. One group of birds tested in July approached their food dishes much more quickly than all other birds (a matter of seconds rather than minutes whether or not there was a novel object), a response that was not repeated in a subsequent July. When this possible outlier group was removed from the analysis, approach times to novel objects were not affected by season. However, animals captured and tested in October had a stronger motivation to feed (i.e., they approached unmodified food dishes faster) than birds captured at other times of the year. If we define "neophobia" as an increase in latency to approach a novel versus an unmodified food dish, then there is little evidence for underlying seasonal variation in risk assessment, although a general motivation to feed does show a seasonal pattern.

Feather corticosterone does not correlate with environmental stressors or body condition in an endangered waterbird.

Physiological metrics are becoming popular tools for assessing individual condition and population health to inform wildlife management and conservation decisions. Corticosterone assays can provide information on how animals cope with individual and habitat-level stressors, and the recent development of feather assays is an exciting innovation that could yield important insights for conservation of wild birds. Due to the widespread enthusiasm for feather corticosterone as a potential bioindicator, studies are needed to assess the ability of this technique to detect meaningful differences in physiological stress across a variety of stressor types and intensities. We examined feather corticosterone from 144 individuals among the 13 known breeding populations of Hawaiian gallinule (Gallinula galeata sandvicensis), an endangered waterbird, on the island of O'ahu. These ecologically independent subpopulations are known to have low genetic connectivity and movement rates and differ largely across a number of important conditions, including level of predator management, human disturbance, proximity to urban development and conspecific population density. This system is well suited for assessing the performance of feather corticosterone as a bioindicator of different known habitat-level threats common to this and many other conservation-reliant species. We found no statistically significant relationship between feather corticosterone and level of predator control, level of human disturbance, gallinule population density, percent urban cover or body condition across all sites despite the substantial difference in stressor magnitude in our dataset. We did find that gallinules in habitats with larger population densities were in worse body condition. These findings suggest that feather corticosterone is not a consistent indicator of anthropogenic impacts on populations. Furthermore, they suggest that feather corticosterone may be a poor bioindicator of known habitat-level threats for Hawaiian gallinules and that it should be used with caution in other avian taxa of conservation concern.

Recovery from repeated stressors: Physiology and behavior are affected on different timescales in house sparrows.

For decades, researchers across disciplines have been captivated by classifying, diagnosing, and avoiding the consequences of chronic stress. Despite the vast body of literature this has generated, we still lack the ability to predict which individuals or populations may be susceptible to stress-related pathologies. One critical unanswered question is whether the impacts of repeated stressors are reversible, or if instead they permanently alter an individual. In this study, we exposed house sparrows (Passer domesticus) to 6 days of random, repeated stressors, permitted them 0, 1, 3, or 6 days to recover, and then assessed changes in their body mass, hypothalamic-pituitaryadrenal (HPA) axis (baseline, stress-induced corticosterone, negative feedback strength), immune function, uric acid concentrations, DNA damage levels, and perch hopping activity. Body mass did not vary between groups after recovery. We found that the HPA axis and perch hopping were not significantly impacted by the 6 days of stressors, but that uric acid and DNA damage increased. Short recovery periods tended to negatively affect the HPA axis and reduced uric acid levels, but these were reversed with longer recovery periods. Following the recovery periods, the birds experienced an additional 6 days of random stressors and their responses were assessed again. All recovery times reduced perch hopping and immune function, but paradoxically, DNA damage was highest in the birds that had the longest amount of time to recover. These results show that recovery time affects responses to subsequent chronic stress in complex ways, and highlight the importance of multimodal, interdisciplinary approaches to studying stress physiology.

Recovery periods during repeated stress impact corticosterone and behavioral responses differently in house sparrows.

A number of studies have shown that chronic stress can negatively impact both physiology and behavior in a variety of organisms. What has yet to be extensively explored is whether these changes permanently alter an animal's functioning, or if they can be reversed. In this study, we used wild-caught house sparrows (Passer domesticus) to assess how recovery periods influence the physiological and behavioral impacts of an initial four days and subsequent four days of repeated stressors. Birds were randomly assigned to a recovery group and either experienced 0, 24, or 72 h of recovery between the two sets of stressors (cage rolling and cage tapping). We measured the regulation of the hypothalamic pituitary adrenal (HPA) axis by quantifying baseline and stress-induced corticosterone as well as negative feedback strength. We also assessed behavior using neophobia trials to measure how birds altered their approach towards novel objects and their overall activity. Both behavior and corticosterone responses were assessed before the experiment, after the recovery time, and following the final 4 days of stressors. We found that birds that experienced 24 h of recovery had reduced stress-induced corticosterone, but enhanced negative feedback relative to the pre-experiment sample. Additionally, 4 days of stressors was enough to significantly reduce approach latency towards novel objects; however, pre-experiment levels returned with longer periods of recovery. Finally, recovery time did not significantly influence responses to the second 4 days of stressors. Our results indicate that brief recovery periods partially ameliorate the hormonal and behavioral effects of repeated stress.

DNA damage as an indicator of chronic stress: Correlations with corticosterone and uric acid.

Corticosterone does not change in consistent ways across species and contexts, making it challenging to use as an indicator of chronic stress. We assessed DNA damage as a potential metric that could be a more integrative stress measurement with direct links to health. We captured free-living house sparrows, took an immediate blood sample, and transferred them to the laboratory, exposing them to the chronic stress of captivity. Biweekly blood and weight samples were then taken for 4 weeks. We immediately assessed DNA damage in red blood cells using the comet assay and later quantified corticosterone. Uric acid was analyzed in a separate group of birds. We found that birds initially lost, but began to regain weight over the course of captivity. DNA damage peaked within the first 10 days of captivity, and mostly remained elevated. However, the cellular distribution of damage changed considerably over time; most cells showed low levels of damage early, a bimodal distribution of high and low DNA damage during the peak of damage, and a wide unimodal distribution of damage at the end of the 4 weeks. Furthermore, corticosterone increased and remained elevated and uric acid decreased and remained depleted over the same period. Although both a molecular (DNA damage) and an endocrine (corticosterone) marker showed similar response profiles over the 4 weeks, they were not correlated, suggesting they reflect different aspects of the underlying physiology. These data provide convincing preliminary evidence that DNA damage has potential to be an additional indicator of chronic stress.

House sparrows (Passer domesticus) adjusted hypothalamic-pituitary-adrenal axis negative feedback and perch hopping activities in response to a single repeated stimulus.

Chronic stress has been extensively studied in both laboratory and field settings; however, a conclusive and consistent phenotype has not been reached. Several studies have reported attenuation of the hypothalamic-pituitary-adrenal axis during experiments intended to cause chronic stress. We sought to determine whether this attenuation could be indicative of habituation. Importantly, we were not investigating habituation to a specific stimulus-as many stress physiology studies do-but rather we assessed how the underlying physiology and behavior changed in response to repeated stressor presentation. We exposed house sparrows (Passer domesticus) to a single stimulus twice per day at random times for 8 consecutive days. We predicted that this period of time would be long enough for the birds to determine that these acute stressors were not, in fact, dangerous and they would, therefore, acclimate. A second control group remained undisturbed for the same period of time. We measured baseline, stress-induced, negative feedback strength, and maximum production of corticosterone as well as neophobic behavior before, during, and after this 8-day experiment. When birds experienced a stimulus for 4 days, their negative feedback strength was significantly diminished, but recovered after the second 4 days. Additionally, perch hopping decreased and recovered in this same time frame. These data suggest that distinct physiological and behavioral responses arise when house sparrows are exposed to the same stressor for several consecutive days as opposed to many stressors layered on top of one another. Furthermore, they indicate that habituation-as with chronic stress-can appear differently depending on the metric being examined.

Physiological and behavioral responses of house sparrows to repeated stressors.

Despite decades of research, we still lack a complete understanding of what factors influence the transition of the necessary and adaptive acute stress response to what has become known as chronic stress. This gap in knowledge has illuminated the necessity for studies that examine the thresholds between these two sides of the stress response. Here, we determine how repeated exposure to acute stressors influences physiological and behavioral responses. In this repeated measures study, house sparrows (Passer domesticus) were exposed to a chronic stress protocol. We took physiological and behavioral measurements before, during, and after the protocol. Blood samples were used to assess four aspects of hypothalamic-pituitary-adrenal (HPA) axis function: baseline corticosterone, stress-induced corticosterone, negative feedback, and the maximal capacity to secrete corticosterone. We also assessed bacterial killing capacity and changes in uric acid concentration. Neophobia trials were used to assess behavioral changes throughout the protocol. We found no significant changes in HPA axis regulation in any of the four aspects we tested. However, we found that uric acid concentrations and neophobia significantly decreased after only four days of the chronic stress protocol, while bacterial killing capacity did not decrease until after eight days of exposure. These results indicate that different components of the stress response can be impacted by chronic stress on different timescales. Our results further indicate the importance of assessing multiple aspects of both physiology and behavior in order to understand how exposure to chronic stress may influence ability to cope with future challenges.