Coping with extreme free cortisol levels: Seasonal stress axis changes in sympatric North American flying squirrels.

Most environments exhibit predictable yearly changes, permitting animals to anticipate them. The hypothalamic-pituitary-adrenal (HPA) axis is a key physiological pathway that enables animals to cope with such changes. Monitoring glucocorticoid (the end products of the HPA axis) levels in wild animals throughout the year can improve our understanding of how this pathway responds to different conditions. For this study, we collected 18 months of data on two species of North American flying squirrels (Glaucomys sabrinus and G. volans) living in a southern Ontario forest where temperature and food availability fluctuate dramatically throughout the year. These squirrels are active year-round, nest communally, and rely on scatter hoarded foods in the winter months. Flying squirrels have extremely high levels of free plasma cortisol relative to other mammals, but it is unknown how these levels are affected by environmental and reproductive factors. For both species, our goals were to (1) validate an enzyme immunoassay (EIA) to measure their fecal glucocorticoid metabolite (FGM) concentrations and (2) assess yearly differences, seasonal changes, and the influence of sex, reproduction, and ambient temperature on FGM concentrations in each species. In the lab, we successfully validated the use of antibody 5α-pregnane-3ß, 11ß, 21-triol-20-one EIA for FGM analysis in both species. In the field, neither sex nor reproductive status (breeding condition or not) were linked to FGM concentrations in either species. FGM concentrations were higher in autumn compared to the spring and summer. There were no other seasonal differences. We discuss possible explanations for the autumn peak in FGM concentrations (increased energy expenditure and social nesting changes), as well as outline possible avenues for future research. Understanding how individuals and populations respond to environmental change is a critical goal in evolutionary ecology, particularly in the context of a rapidly changing Anthropocene.

Weathering the storm: Decreased activity and glucocorticoid levels in response to inclement weather in breeding Columbian ground squirrels.

Inclement weather can rapidly modify the thermal conditions experienced by animals, inducing changes in their behavior, body condition, and stress physiology, and affecting their survival and breeding success. For animals living in variable environments, the extent to which they have adapted to cope with inclement weather is not established, especially for hibernating species with a short active season that are constrained temporally to breed and store energy for subsequent hibernation. We examined behavioral (foraging activity) and physiological (body mass and fecal cortisol metabolites) responses of Columbian ground squirrels (Urocitellus columbianus), small hibernating rodents inhabiting open meadows in Rocky Mountains, to 3 events of inclement weather (two snow storms in May 2021 and May 2022, one heavy rainfall in June 2022). We found that individuals adapted to inclement weather conditions by (1) reducing above-ground activity, including foraging, (2) decreasing the mobilization of stored resources as indicated by a decrease in the activity of the hypothalamo-pituitary-adrenal (HPA) axis and lower fecal cortisol metabolites in the hours/days following periods of inclement weather; and (3) compensating through increased foraging and more local activity when favorable conditions resumed. As a result, body mass and growth did not decrease following short periods of inclement weather. Columbian ground squirrels were well-adapted to short periods of inclement weather, coping via modifications of their behavior and the activity of the HPA axis.

Is chronic stress a causal mechanism for small mammal population cycles? Reconciling the evidence.

Chronic stress has long been hypothesized to play a role in driving population cycles. Christian (1950) hypothesized that high population density results in chronic stress and mass "die-offs" in small mammal populations. Updated variations of this hypothesis propose that chronic stress at high population density may reduce fitness, reproduction, or program aspects of phenotype, driving population declines. We tested the effect of density on the stress axis in meadow voles (Microtus pennsylvanicus) by manipulating population density in field enclosures over three years. Using fecal corticosterone metabolites as a non-invasive measure of glucocorticoid (GC) concentrations, we found that density alone was not associated with GC differences. However, we found that the seasonal relationship of GC levels differed by density treatment, with high-density populations having elevated GC levels early in the breeding season and decreasing towards late summer. We additionally tested hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression in juvenile voles born at different densities, with the hypothesis that high density may reduce receptor expression, altering negative feedback of the stress axis. We found that females had marginally higher glucocorticoid receptor expression at high density, no effect in males, and no detectable effect of density on mineralocorticoid receptor expression in either sex. Hence, we found no evidence that high density directly impairs negative feedback in the hippocampus, but rather female offspring may be better equipped for negative feedback. We compare our findings with prior studies to attempt to disentangle the complicated relationship between density, seasonality, sex, reproduction and the stress axis.

Assessing stress in wild black-and-white colobus monkeys non-invasively.

Analysis of glucocorticoid profiles serves as a valuable, multi-faceted tool for insight into the behavior and physiology of wild populations. Recently, the measurement of fecal glucocorticoid metabolites (FCMs) has exploded in popularity due to its compatibility with noninvasive techniques and remote environments A critical first step is to perform a biological validation to ensure that the assay accurately reflect changes in FCM levels. We use an enzyme immunoassay (EIA) to perform a biological validation on samples collected from two males and six females in a wild population of Colobus vellerosus in response to three naturally occurring potential stressors. We also describe the FCM response pattern in the week following parturition in three females and examine the influence of sex, reproductive state, and time of day on the concentrations of baseline samples collected daily from 13 adult individuals over a period of four months. We validated the assay: FCM levels increase in response to natural stressors with a two-day lag. In the two days surrounding parturition, FMC levels increased. Baseline concentrations were affected by collection time and female reproductive state, with lactating females having lower concentrations than pregnant or cycling females. Thus, we successfully carried out the first validation and characterization of FCMs in a wild African colobine. This will serve as an essential foundation for future studies of C. vellerosus and similar wild primates whose objective is to investigate the role glucocorticoids play in responses to social and ecological challenges.

Maternal effects in mammals: Broadening our understanding of offspring programming.

The perinatal period is a sensitive time in mammalian development that can have long-lasting consequences on offspring phenotype via maternal effects. Maternal effects have been most intensively studied with respect to two major conditions: maternal diet and maternal stress. In this review, we shift the focus by discussing five major additional maternal cues and their influence on offspring phenotype: maternal androgen levels, photoperiod (melatonin), microbiome, immune regulation, and milk composition. We present the key findings for each of these topics in mammals, their mechanisms of action, and how they interact with each other and with the maternal influences of diet and stress. We explore their impacts in the contexts of both predictive adaptive responses and the developmental origins of disease, identify knowledge gaps and research opportunities in the field, and place a particular emphasis on the application and consideration of these effects in non-model species and natural ecological systems.

Hormones do not maketh the mole-rat: No steroid hormone signatures of subordinate behavioral phenotypes.

In some cooperatively breeding groups, individuals have distinct behavioral characteristics that are often stable and predictable across time. However, in others, as in the eusocial naked mole-rat, evidence for behavioral phenotypes is ambiguous. Here, we study whether the naked mole-rat can be divided into discrete phenotypes and if circulating hormone concentrations underpin these differences. Naked mole-rat colonies consist of a single breeding female and large numbers of non-reproductive subordinates that in some cases can exceed several hundred in a colony. The subordinates can potentially be divided into soldiers, who defend the colony; workers, who maintain it; and dispersers, who want to leave it. We established six colonies de novo, tracked them over three years, and assessed the behavior and hormone concentrations of the subordinates. We found that soldiers tended to be from earlier litters and were higher ranked compared to workers, whereas dispersers were distributed throughout litters and rankings. There was no difference in estradiol, testosterone, or dehydroepiandrosterone (DHEA) concentrations among phenotypes. Progesterone concentrations were higher in soldiers, but this difference appeared to be driven by a few individuals. Principal component analysis demonstrated that soldiers separated into a discrete category relative to workers/dispersers, with the highest ranked loadings being age, body mass, and testosterone concentrations. However, the higher testosterone in soldiers was correlated with large body size instead of strictly behavioral phenotype. Workers and dispersers have more overlap with each other and no hormonal differences. Thus the behavioral variation in subordinate naked mole-rats is likely not driven by circulating steroid hormone concentrations, but rather it may stem from alternative neural and/or neuroendocrine mechanisms.

Territorial scent-marking effects on vigilance behavior, space use, and stress in female Columbian ground squirrels.

Social environments can profoundly affect the behavior and stress physiology of group-living animals. In many territorial species, territory owners advertise territorial boundaries to conspecifics by scent marking. Several studies have investigated the information that scent marks convey about donors' characteristics (e.g., dominance, age, sex, reproductive status), but less is known about whether scents affect the behavior and stress of recipients. We experimentally tested the hypothesis that scent marking may be a potent source of social stress in territorial species. We tested this hypothesis for Columbian ground squirrels (Urocitellus columbianus) during lactation, when territorial females defend individual nest-burrows against conspecifics. We exposed lactating females, on their territory, to the scent of other lactating females. Scents were either from unfamiliar females, kin relatives (a mother, daughter, or sister), or their own scent (control condition). We expected females to react strongly to novel scents from other females on their territory, displaying increased vigilance, and higher cortisol levels, indicative of behavioral and physiological stress. We further expected females to be more sensitive to unfamiliar female scents than to kin scents, given the matrilineal social structure of this species and known fitness benefits of co-breeding in female kin groups. Females were highly sensitive to intruder (both unfamiliar and kin) scents, but not to their own scent. Surprisingly, females reacted more strongly to the scent of close kin than to the scent of unfamiliar females. Vigilance behavior increased sharply in the presence of scents; this increase was more marked for kin than unfamiliar female scents, and was mirrored by a marked 131% increase in free plasma cortisol levels in the presence of kin (but not unfamiliar female) scents. Among kin scents, lactating females were more vigilant to the scent of sisters of equal age, but showed a marked 318% increase in plasma free cortisol levels in response to the scent of older and more dominant mothers. These results suggest that scent marks convey detailed information on the identity of intruders, directly affecting the stress axis of territory holders.

A mechanism for population self-regulation: Social density suppresses GnRH expression and reduces reproductivity in voles.

Nearly 100 years ago, Charles Elton described lemming and vole population cycles as ecological models for understanding population regulation in nature. Yet, the mechanisms driving these cycles are still not fully understood. These rodent populations can continue to cycle in the absence of predation and with food supplementation, and represent a major unsolved problem in population ecology. It has been hypothesized that the social environment at high population density can drive selection for a low-reproduction phenotype, resulting in population self-regulation as an intrinsic mechanism driving the cycles. However, a physiological mechanism for this self-regulation has not been demonstrated. We manipulated population density in wild meadow voles Microtus pennsylvanicus using large-scale field enclosures over 3 years and examined reproductive performance and physiology. Within the field enclosures, we assessed the proportion of breeding animals, mass at sexual maturation, and faecal androgen and oestrogen metabolites. We then collected brain tissue from juvenile voles born at high or low density, quantified mRNA expression of gonadotropin-releasing hormone (GnRH) and oestrogen receptor alpha (ERα) and measured DNA methylation at six CpG sites in a region that was highly conserved with the mouse GnRH promoter. At high density, there was a lower proportion of reproductive animals. Juvenile voles born at high densities had reduced expression of GnRH in the hypothalamus, accompanied by marginally lower faecal sex hormone metabolites. Female juvenile voles born at high density also had higher methylation levels at two CpG sites while males did not, aligning with prior observations that females (but not males) from high-density environments retain reduced reproduction long term. Our results support a physiological basis for population self-regulation in vole cycles, as altering population density alone induced reproductive downregulation at the hypothalamic level. Our results demonstrate that altering the early-life social environment can fundamentally impact reproductive function in the brain. This, in turn, can drive population demography changes in wild animals.

The role of herbivory in the macroevolution of vertebrate hormone dynamics.

Vertebrates have high species-level variation in circulating hormone concentrations, and the functional significance of this variation is largely unknown. We tested the hypothesis that interspecific differences in hormone concentrations are partially driven by plant consumption, based on the prediction that herbivores should have higher basal hormone levels to 'outcompete' plant endocrine disruptors. We compared levels of glucocorticoids (GCs), the hormones with the most available data, across 166 species. Using phylogenetically informed comparisons, we found that herbivores had higher GC levels than carnivores. Furthermore, we found that the previously described negative relationship between GC levels and body mass only held in herbivores, not carnivores, and that the effect of diet was greatest at extreme body sizes. These findings demonstrate the far-reaching effects of diet on animal physiology, and provide evidence that herbivory influences circulating hormone concentrations. We urge future direct testing of the relationship between phytochemical load and GC levels.

The stress of being alone: Removal from the colony, but not social subordination, increases fecal cortisol metabolite levels in eusocial naked mole-rats.

In many social species, hierarchical status within the group is associated with differences in basal adrenocortical activity. We examined this relationship in naked mole-rats (Heterocephalus glaber), eusocial rodents with arguably the most extreme social hierarchies of all mammals. This species lives in colonies where breeding is restricted to one socially dominant 'queen' and her male consorts, and all other individuals are reproductively suppressed 'subordinates'. The relationship between cortisol and social status in naked mole-rats has not fully been elucidated, as prior results on this topic have been contradictory. We used non-invasive feces sampling to measure baseline cortisol levels in eight laboratory colonies of naked mole-rats, to either replicate or reject rank differences. First, we successfully validated an assay to measure fecal cortisol metabolites (FCMs). Removal from the colony for the validation experiment, either alone or with an opposite sex conspecific, induced prolonged elevation of FCM levels on a scale of days to weeks. This increase in cortisol did not prevent the removed animals from sexually maturing. In colony-housed animals, we found no relationship between rank in the social hierarchy and FCM levels. Further, queens, breeding males, and reproductively suppressed subordinates all had equivalent FCM levels. We conclude that this species shows little evidence of the 'stress of dominance' or 'stress of subordination' and that reproductive suppression in naked mole-rats is not driven by elevated cortisol levels.

Anogenital distance as a measure of male competitive ability in Rwenzori Angolan colobus.

Anogenital distance (AGD) is positively correlated to fetal androgen exposure and developmental masculinization in mammals. Independent of overall body size, AGD shows a strong positive correlation with male fertility and in rodents, AGD is a good indicator of male competitive ability and is associated with female choice. We hypothesized that AGD will also predict male competitive ability in non-human primates. To test this, we measured AGD noninvasively with a parallel laser in a wild population of Angolan colobus monkeys (Colobus angolensis ruwenzorii) in Uganda and correlated to it to their social structure. C. angolensis ruwenzorii form a multilevel society with both one-male/multifemale units (OMUs) and multimale/multifemale units (MMUs). We compared AGD in males from five OMUs and six MMUs and related it to their fecal androgen metabolite concentrations, dominance rank and body size, and to the number of females in their unit. Males in OMUs had greater access to females, so were predicted to have longer AGDs, but this was not found. AGD also did not correlate overall with mean fecal androgen metabolites in MMUs. However, AGD was correlated with dominance rank in MMUs, demonstrating that higher-ranking males in these multimale units had longer AGDs. Body size did not show the same relationship with dominance rank, suggesting that male rank was not just a reflection of absolute male size. Our findings indicate that AGD predicts male competitive ability in this species and that it may be a useful correlate throughout the non-human primates. These results also support the idea that prenatal androgen exposure increases the likelihood of the expression of behaviors that maintain high dominance rank.

Glucocorticoids and CBG during pregnancy in mammals: diversity, pattern, and function.

Pregnancy is one of the defining characteristics of placental mammals. Key in the growth and development of the fetus during pregnancy are the dynamics of glucocorticoids (GCs) and their binding protein,corticosteroid-binding globulin (CBG), which determines how much of the GCs are free and biologically active. Out of more than 5000 species of placental mammals in 19 different orders, our understanding of the dynamics of maternal GCs and CBG during pregnancy is largely limited to the detailed study of 3 groups - sheep, laboratory rodents, and humans. The assumption is often made that what we see in these few species applies to the rest. To examine this generality, we compared patterns of maternal GCs over pregnancy from all placental mammals where data is available: in the blood of 13 species from 5 different orders and in metabolites in excreta in an additional 20 species from 9 orders. We found that maternal free GCs increase by late pregnancy in most taxa. This increase is achieved by either an increase in total GC secretion or a decrease in CBG. A major exception is found in the even-toed ungulates (sheep, cows, etc.) where maternal GCs and CBG remain stable, but where the fetal adrenals mature in late pregnancy and produce the majority of their own GCs. We conclude that patterns of change in maternal GCs and CBG during pregnancy are species-specific but are alternative means to the same end: increased fetal exposure to GCs in late pregnancy, which is essential for development.

Seasonal programming, not competition or testosterone, drives stress-axis changes in a partially-semelparous mammal.

Animals must make tradeoffs between reproduction and longevity. This is particularly pronounced in male arctic ground squirrels (Urocitellus parryii), that compete aggressively for territories and mates during a three-week breeding season. Breeding males have high rates of severe wounding, high mortality rates, and high free cortisol levels, along with downstream consequences of chronic stress (weight loss, reduced immune function) that appear to contribute to their early death. The elevated cortisol levels are thought to be a result of the intense intrasexual competition. An alternative hypothesis, however, is that the hormonal change is a seasonal adaptation facilitating the tradeoff of immediate competitive advantage at the expense of long-term survival. We tested a two-part hypothesis: first, that elevated free cortisol during the breeding period is a seasonal change that will still occur in the absence of actual competition, and second, that testosterone maintains this increase. We measured plasma cortisol, corticosteroid-binding globulin, and fecal glucocorticoid metabolites in three groups: wild male ground squirrels, captive males prevented from fighting, and captive castrated males. There were no differences amongst these three groups in free and total plasma cortisol, fecal glucocorticoids, or downstream measures of chronic stress. This suggests that high free cortisol and its effects on breeding males are not a consequence of contest competition during the breeding season, but rather a generalized seasonal change. We found no evidence that testosterone plays a role in maintaining elevated free cortisol in arctic ground squirrel males.

Coping with pregnancy after 9months in the dark: Post-hibernation buffering of high maternal stress in arctic ground squirrels.

Pregnancy and lactation are key times in the life of female mammals when energetic resources must be brought to bear to produce and nurture offspring. Changes in glucocorticoid (GC) levels are central to this objective, due to their roles in modulating development and physiology and in mediating energetic tradeoffs. We examined GC changes over reproduction in a species living in a harsh seasonal environment: the arctic ground squirrel (Urocitellus parryii). Females become pregnant immediately after emerging from a ∼9month hibernation, and then must begin this pregnancy during an additional month of freezing temperatures and limited food availability. We measured plasma levels of total cortisol, corticosteroid-binding globulin (CBG), and free cortisol in unstressed females at three stages: not-visibly pregnant, visibly pregnant, and lactating. Total cortisol levels were similar in all stages, but CBG levels increased 4-fold from the not-visibly pregnant stage to visibly pregnant and lactating stage. As a result, the free cortisol fraction declined from 51% of total cortisol when females were not-visibly pregnant to only 5% when they were visibly pregnant (remaining low and stable throughout pregnancy) and 10% when they were lactating. This pattern is markedly different from that seen in other mammals, where 10% or less of GCs are free and these tend to increase during gestation. We postulate that the high free cortisol just prior to visible pregnancy is a seasonal adaptation relating either to the pronounced physiological changes the female must undergo after emerging from hibernation and immediately getting pregnant, or to the mobilization of body reserves for energy to permit pregnancy, or both. Thereafter, high CBG levels may shield the developing offspring from the negative effects of cortisol overexposure.

The Curious Case of the Naked Mole-Rat: How Extreme Social and Reproductive Adaptations Might Influence Sex Differences in the Brain.

Research in the neurobiology of sex differences is inherently influenced by the study species that are used. Some traditional animal research models, such as rats and mice, show certain sex differences in the brain that have been foundational to neurobiological research. However, subsequent work has demonstrated that these differences are not always generalizable, especially to species with different social structures and sex-associated roles or behaviors. One such example is the naked mole-rat (Heterocephalus glaber), which has an unusual social structure among mammals. Naked mole-rats live in large groups where reproduction is restricted to a dominant female, called the "queen," and often only one breeding male. All other animals in the group, the "subordinates," are socially suppressed from reproduction and remain in a prepubescent state as adults, unless they are removed from the presence of the queen. These subordinates show little to no sex differences in external morphology, neural morphology, or behavior. However, there are a suite of neurobiological differences between subordinate and breeding naked mole-rats. After naked mole-rats attain breeding status, many of the classically sexually differentiated brain regions increase in volume (paraventricular nucleus, medial amygdala, bed nucleus of the stria terminalis). There are additionally social status differences in sex hormone receptor expression in the brain, as well as other changes in gene expression, some of which also show sex differences - though not always in the predicted direction based on other rodent studies. Data from naked mole-rats show that it is critical to consider the evolved social structure of a species when studying sex differences in the brain.

An experimental analysis of density dependence in meadow voles: Within-season and delayed effects.

Wild mammal populations exhibit a variety of dynamics, ranging from fairly stable with little change in population size over time to high-amplitude cyclic or erratic fluctuations. A persistent question in population ecology is why populations fluctuate as they do. Answering this seemingly simple question has proven to be challenging. Broadly, density-dependent feedback mechanisms should allow populations to grow at low density and slow or halt growth at high density. However, experimental tests of what demographic processes result in density-dependent feedback and on what timescale have proven elusive. Here, we used replicated density perturbation experiments and capture-mark-recapture analyses to test density-dependent population growth in populations of meadow voles (Microtus pennsylvanicus) during the summer breeding season by manipulating founding population density and observing the pattern of survival, reproduction, and population growth. High population density had no consistent effect on survival rates but generally negatively influenced recruitment and population growth rates. However, these density-dependent effects varied within the breeding season and across years. Our study provides evidence that density-dependent feedback mechanisms operate at finer time scales than previously believed and that process, additively with delayed year effects, is key to understanding multiyear population demography.

Queen Pregnancy Increases Group Estradiol Levels in Cooperatively Breeding Naked Mole-Rats.

For cooperative species, there can be great value in the synchronization of physiological states to coordinate group behavioral states. This is evident in naked mole-rats (Heterocephalus glaber), which have the most extreme form of cooperative breeding in mammals. Colonies have a single reproductive female, "the queen," and 1-3 breeding males. These breeders are supported by adult "subordinates," which are all socially suppressed into a pre-pubertal state. Subordinates cooperate in colony maintenance, defense, and alloparental care. Prior work has reported that there may be social sharing of hormones among individuals in the colony because when the queen is pregnant, subordinates of both sexes develop enlarged nipples and female subordinates can develop vaginal perforation. We sought to document the hormonal changes and mechanisms behind these observations. We found that subordinate estradiol levels were elevated during the queen's pregnancy and were correlated with queen levels. To determine if this occurs by direct hormone-sharing, where group members uptake the hormones of conspecifics through excreta or the skin, we then tested whether treating a single subordinate in the colony with estradiol would induce the same effect in other colony members. It did not, which indicates that the influence on group estradiol levels may be specific to cues from the queen. These queen cues may be behavioral in nature, as we found that queens were less aggressive during pregnancy, which prior work has suggested may relax reproductive suppression of subordinates. Yet, levels of queen aggression alone were not associated, or were weakly associated, with their colony's estradiol levels, though our sample size examining this particular relationship was low. This is suggestive that additional queen cues of reproductive status, beyond just aggression, may be relevant in influencing the subordinate hormonal change, or that the relationship between aggression and colony estradiol levels is more subtle and would need to be elucidated with a larger sample size. These results have implications for how cooperative breeders coordinate reproduction and alloparental care, and how social cues can influence individual and group physiology.