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• North American flying squirrels have extremely high levels of free cortisol. • Free cortisol levels can be assessed non-invasively through fecal samples. • Free cortisol levels peaked in autumn and were not impacted by sex or reproduction. 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-3β, 11β, 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. [ABSTRACT FROM AUTHOR]

Naked mole-rats are eusocial mammals that live in hierarchies consisting of one breeding female, one to three male consorts and their reproductively suppressed offspring. A 'disperser morph' subcaste has been suggested with a subset of nonbreeders exhibiting motivation to leave their natal colony and mate with unfamiliar conspecifics. To test the hypotheses that intrinsic colony variables (e.g. population density, sex ratio, queen temperament) influence the dispersal phenotype, and that males and females differ in responsiveness to these variables, we evaluated dispersal behaviour in 17 laboratory colonies. Queen aggression was associated with the number of female, but not male, dispersers, although dispersers were not themselves targets of queen aggression. Female dispersers were more aggressive than their nondispersing sisters, although still less aggressive than queens overall. Following outpairing with an unfamiliar opposite-sex animal, dispersers and nondispersers produced litters at similar rates, demonstrating that motivation to leave the colony, and not anticipatory reproductive maturation, is the key to successful dispersal. Collectively, these data suggest that aggressive naked mole-rat queens motivate dispersal in their daughters and that female dispersers show traits consistent with successful queens (e.g. aggression). • We evaluated dispersal behaviour in 17 laboratory colonies of naked mole-rats. • Queen aggression predicted presence of female, but not male, dispersers. • However, dispersers were not direct targets of queen aggression. • Dispersers were more aggressive than nondispersers but less aggressive than queens. • Litter production did not differ between dispersers and nondispersers. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Columbian ground squirrels are montane rodents that occupy habitats in rapidly changing environments. • Activity, foraging, and space utilization decreased during inclement weather during the breeding season. • Fecal cortisol metabolite levels were lower after inclement weather than during control periods. • Body mass and body mass gain were not affected by days of inclement weather. • These ground squirrels appeared well adapted to inclement weather. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• A fecal glucocorticoid metabolite assay was validated in wild colobus monkeys. • Morning samples have higher levels than afternoon samples. • Pregnant females have higher baseline levels than lactating or cycling females. • In the two days around parturition, maternal glucocorticoids temporarily increase. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ∼9 month 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. [ABSTRACT FROM AUTHOR]

• Humans, rodents, and wild mammals share biological generalities in maternal programming. • Maternal androgens, photoperiod, microbiome and immunity influence offspring phenotype. • Milk is a rich source of maternal hormones, microbiota, immune factors and exosomes. • Milk exosomes transfer microRNA into offspring cells and may induce biological changes. • The extent of maternal programming cannot be fully understood by examining a single cue. 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. [ABSTRACT FROM AUTHOR]

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. • Fecal cortisol levels do not differ in dominant and subordinate naked mole-rats. • Separation from the colony drives an increase in cortisol levels over days to weeks. • High cortisol is not associated with reproductive suppression in this species. [ABSTRACT FROM AUTHOR]

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. • Naked mole-rat subordinates are classified into workers, dispersers and soldiers. • Soldiers tend to be born in earlier litters and rank higher in dominance. • Soldiers have higher levels of circulating progesterone. • Workers and dispersers have overlapping biological correlates. • Behavioral phenotypes are not driven by circulating hormones. [ABSTRACT FROM AUTHOR]

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. • Scent-marking is an important means of social communication in territorial species. • Scents introduced on territories of female ground squirrels increase vigilance. • Females react more strongly to the scent of female kin than unfamiliar females. • Kin scents cause a marked increase in plasma cortisol levels. • Scents of mothers cause higher cortisol elevations than sister or daughter scents. [ABSTRACT FROM AUTHOR]