Parental habituation to human disturbance over time reduces fear of humans in coyote offspring.

A fundamental tenet of maternal effects assumes that maternal variance over time should have discordant consequences for offspring traits across litters. Yet, seldom are parents observed across multiple reproductive bouts, with few studies considering anthropogenic disturbances as an ecological driver of maternal effects. We observed captive coyote (Canis latrans) pairs over two successive litters to determine whether among-litter differences in behavior (i.e., risk-taking) and hormones (i.e., cortisol and testosterone) corresponded with parental plasticity in habituation. Thus, we explicitly test the hypothesis that accumulating experiences of anthropogenic disturbance reduces parental fear across reproductive bouts, which should have disparate phenotypic consequences for first- and second-litter offspring. To quantify risk-taking behavior, we used foraging assays from 5-15 weeks of age with a human observer present as a proxy for human disturbance. At 5, 10, and 15 weeks of age, we collected shaved hair to quantify pup hormone levels. We then used a quantitative genetic approach to estimate heritability, repeatability, and between-trait correlations. We found that parents were riskier (i.e., foraged more frequently) with their second versus first litters, supporting our prediction that parents become increasingly habituated over time. Second-litter pups were also less risk-averse than their first-litter siblings. Heritability for all traits did not differ from zero (0.001-0.018); however, we found moderate support for repeatability in all observed traits (r = 0.085-0.421). Lastly, we found evidence of positive phenotypic and cohort correlations among pup traits, implying that cohort identity (i.e., common environment) contributes to the development of phenotypic syndromes in coyote pups. Our results suggest that parental habituation may be an ecological cue for offspring to reduce their fear response, thus emphasizing the role of parental plasticity in shaping their pups' behavioral and hormonal responses toward humans.

Investigation of techniques to measure cortisol and testosterone concentrations in coyote hair.

Long-term noninvasive sampling for endangered or elusive species is particularly difficult due to the challenge of collecting fecal samples before hormone metabolite desiccation, as well as the difficulty in collecting a large enough sample size from all individuals. Hair samples may provide an environmentally stable alternative that provides a long-term assessment of stress and reproductive hormone profiles for captive, zoo, and wild mammals. Here, we extracted and analyzed both cortisol and testosterone in coyote (Canis latrans) hair for the first time. We collected samples from 5-week old coyote pups (six female, six male) housed at the USDA-NWRC Predator Research Facility in Millville, UT. Each individual pup was shaved in six different locations to assess variation in concentrations by body region. We found that pup hair cortisol (F5,57.1 = 0.47, p = 0.80) and testosterone concentrations (F5,60 = 1.03, p = 0.41) did not differ as a function of body region. Male pups generally had higher cortisol concentrations than females (males = 17.71 ± 0.85 ng/g, females = 15.48 ± 0.24 ng/g; F1,57.0 = 5.06, p = 0.028). Comparatively, we did not find any differences between male and female testosterone concentrations (males = 2.86 ± 0.17 ng/g, females = 3.12 ± 0.21 ng/g; F1,60 = 1.42, p = 0.24). These techniques represent an attractive method in describing long-term stress and reproductive profiles of captive, zoo-housed, and wild mammal populations.

The ontogeny of kin-recognition mechanisms in Belding's ground squirrels.

Despite extensive research on the functions and mechanisms of kin recognition, little is known about developmental changes in the abilities mediating such recognition. Belding's ground squirrels, Urocitellus beldingi, use at least two mechanisms of kin recognition in nepotistic contexts: familiarity and phenotype matching. Because recognition templates develop from early associations with familiar kin (and/or with self), familiarity-based recognition should precede phenotype-matching recognition even though one template is thought to be used for both mechanisms. I used a cross-fostering design to produce individuals that differed in relatedness and familiarity. Two pups (one female and one male) were exchanged reciprocally between two litters within 48-h of birth. Every five days, from 15 to 30-d of age, young were exposed to bedding and oral-gland odors from their familiar foster mother and an unfamiliar unrelated female (familiarity test) and from their unfamiliar genetic mother and another unfamiliar unrelated female (phenotype-matching test). As expected, discrimination of odors based on familiarity was evident at all ages tested, whereas discrimination based on relatedness was not evident until 30-d. My results provide a first estimate for when phenotype-matching mechanisms are used by young Belding's ground squirrels, and thus when they can recognize unfamiliar kin such as older sisters or grandmothers. Belding's ground squirrels are the first species for which the development of the production, perception and action components is well understood.

Olfactory attractants and parity affect prenatal androgens and territoriality of coyote breeding pairs.

Hormones are fundamental mediators of personality traits intimately linked with reproductive success. Hence, alterations to endocrine factors may dramatically affect individual behavior that has subsequent fitness consequences. Yet it is unclear how hormonal or behavioral traits change with environmental stressors or over multiple reproductive opportunities, particularly for biparental fauna. To simulate an environmental stressor, we exposed captive coyote (Canis latrans) pairs to novel coyote odor attractants (i.e. commercial scent lures) mid-gestation to influence territorial behaviors, fecal glucocorticoid (FGMs) and fecal androgen metabolites (FAMs). In addition, we observed coyote pairs as first-time and experienced breeders to assess the influence of parity on our measures. Treatment pairs received the odors four times over a 20-day period, while control pairs received water. Odor-treated pairs scent-marked (e.g. urinated, ground scratched) and investigated odors more frequently than control pairs, and had higher FAMs when odors were provided. Pairs had higher FAMs as first-time versus experienced breeders, indicating that parity also affected androgen production during gestation. Moreover, repeatability in scent-marking behaviors corresponded with FGMs and FAMs, implying that coyote territoriality during gestation is underpinned by individually-specific hormone profiles. Our results suggest coyote androgens during gestation are sensitive to conspecific olfactory stimuli and prior breeding experience. Consequently, fluctuations in social or other environmental stimuli as well as increasing parity may acutely affect coyote traits essential to reproductive success.

Within-individual correlations reveal link between a behavioral syndrome, condition and cortisol in free-ranging Belding's ground squirrels.

Animals often exhibit consistent individual differences in behavior (i.e. animal personality) and correlations between behaviors (i.e. behavioral syndromes), yet the causes of those patterns of behavioral variation remain insufficiently understood. Many authors hypothesize that state-dependent behavior produces animal personality and behavioral syndromes. However, empirical studies assessing patterns of covariation among behavioral traits and state variables have produced mixed results. New statistical methods that partition correlations into between-individual and residual within-individual correlations offer an opportunity to more sufficiently quantify relationships among behaviors and state variables to assess hypotheses of animal personality and behavioral syndromes. In a population of wild Belding's ground squirrels (Urocitellus beldingi) we repeatedly measured activity, exploration, and response to restraint behaviors alongside glucocorticoids and nutritional condition. We used multivariate mixed models to determine whether between-individual or within-individual correlations drive phenotypic relationships among traits. Squirrels had consistent individual differences for all five traits. At the between-individual level, activity and exploration were positively correlated whereas both traits negatively correlated with response to restraint, demonstrating a behavioral syndrome. At the within-individual level, condition negatively correlated with cortisol, activity and exploration. Importantly, this indicates that although behavior is state-dependent, which may play a role in animal personality and behavioral syndromes, feedback mechanisms between condition and behavior appear not to produce consistent individual differences in behavior and correlations between them.

Development, maternal effects, and behavioral plasticity.

Behavioral, hormonal, and genetic processes interact reciprocally, and differentially affect behavior depending on ecological and social contexts. When individual differences are favored either between or within environments, developmental plasticity would be expected. Parental effects provide a rich source for phenotypic plasticity, including anatomical, physiological, and behavioral traits, because parents respond to dynamic cues in their environment and can, in turn, influence offspring accordingly. Because these inter-generational changes are plastic, parents can respond rapidly to changing environments and produce offspring whose phenotypes are well suited for current conditions more quickly than occurs with changes based on evolution through natural selection. I review studies on developmental plasticity and resulting phenotypes in Belding's ground squirrels (Urocitellus beldingi), an ideal species, given the competing demands to avoid predation while gaining sufficient weight to survive an upcoming hibernation, and the need for young to learn their survival behaviors. I will show how local environments and perceived risk of predation influence not only foraging, vigilance, and anti-predator behaviors, but also adrenal functioning, which may be especially important for obligate hibernators that face competing demands on the storage and mobilization of glucose. Mammalian behavioral development is sensitive to the social and physical environments provided by mothers during gestation and lactation. Therefore, maternal effects on offspring's phenotypes, both positive and negative, can be particularly strong.

Chronically raised glucocorticoids reduce innate immune function in Belding's ground squirrels (Urocitellus beldingi) after an immune challenge.

The hypothalamic-pituitary-adrenal (HPA) axis releases glucocorticoids (GCs), or stress hormones, during the vertebrate stress response. GCs can both enhance and suppress the immune system depending on whether the experienced stressor is acute or chronic and what aspect of immune function is measured. More research is needed to fully understand how the immune system reacts to stressors. In this study, we examined the effects of chronically raised GCs on innate immune function in Belding's ground squirrels (Urocitellus beldingi). We measured immune function with a bacteria killing ability (BKA) assay, an integrative and functional assessment of an animal's ability to clear a bacterial infection. All studies to date have examined how acute stressors or repeated social stressors impact BKA. This study is the first to our knowledge to investigate how chronically raised GCs impact BKA both before and after an immune challenge. We noninvasively raised GCs in treatment squirrels for six days and then gave them, and a group of untreated (control) squirrels, an injection of lipopolysaccharide (LPS) to stimulate their innate immune system. Treatment squirrels exhibited lower BKA after, but not before, being challenged with LPS. These results suggest that experiencing chronic stress may not be detrimental to immune functioning until an individual is challenged with an infection.

Odor-based recognition of familiar and related conspecifics: a first test conducted on captive Humboldt penguins (Spheniscus humboldti).

Studies of kin recognition in birds have largely focused on parent-offspring recognition using auditory or visual discrimination. Recent studies indicate that birds use odors during social and familial interactions and possibly for mate choice, suggesting olfactory cues may mediate kin recognition as well. Here, we show that Humboldt penguins (Spheniscus humboldti), a natally philopatric species with lifetime monogamy, discriminate between familiar and unfamiliar non-kin odors (using prior association) and between unfamiliar kin and non-kin odors (using phenotype matching). Penguins preferred familiar non-kin odors, which may be associated with the recognition of nest mates and colony mates and with locating burrows at night after foraging. In tests of kin recognition, penguins preferred unfamiliar non-kin odors. Penguins may have perceived non-kin odors as novel because they did not match the birds' recognition templates. Phenotype matching is likely the primary mechanism for kin recognition within the colony to avoid inbreeding. To our knowledge this is the first study to provide evidence of odor-based kin discrimination in a bird.

Choke or thrive? The relation between salivary cortisol and math performance depends on individual differences in working memory and math-anxiety.

In the current study, we explored how a person's physiological arousal relates to their performance in a challenging math situation as a function of individual differences in working memory (WM) capacity and math-anxiety. Participants completed demanding math problems before and after which salivary cortisol, an index of arousal, was measured. The performance of lower WM individuals did not depend on cortisol concentration or math-anxiety. For higher WM individuals high in math-anxiety, the higher their concentration of salivary cortisol following the math task, the worse their performance. In contrast, for higher WM individuals lower in math-anxiety, the higher their salivary cortisol concentrations, the better their performance. For individuals who have the capacity to perform at a high-level (higher WMs), whether physiological arousal will lead an individual to choke or thrive depends on math-anxiety.

Toward an integrative understanding of social behavior: new models and new opportunities.

Social interactions among conspecifics are a fundamental and adaptively significant component of the biology of numerous species. Such interactions give rise to group living as well as many of the complex forms of cooperation and conflict that occur within animal groups. Although previous conceptual models have focused on the ecological causes and fitness consequences of variation in social interactions, recent developments in endocrinology, neuroscience, and molecular genetics offer exciting opportunities to develop more integrated research programs that will facilitate new insights into the physiological causes and consequences of social variation. Here, we propose an integrative framework of social behavior that emphasizes relationships between ultimate-level function and proximate-level mechanism, thereby providing a foundation for exploring the full diversity of factors that underlie variation in social interactions, and ultimately sociality. In addition to identifying new model systems for the study of human psychopathologies, this framework provides a mechanistic basis for predicting how social behavior will change in response to environmental variation. We argue that the study of non-model organisms is essential for implementing this integrative model of social behavior because such species can be studied simultaneously in the lab and field, thereby allowing integration of rigorously controlled experimental manipulations with detailed observations of the ecological contexts in which interactions among conspecifics occur.

How habitat features shape ground squirrel (Urocitellus beldingi) navigation.

The purpose of this investigation was to determine whether Belding's ground squirrels (Urocitellus beldingi) from areas rich in beacons perform differently in a task of spatial memory compared with squirrels from beacon-thin areas. To assess the role of environmental experience in spatial memory, wild-born squirrels with several days of experience in the field were compared with squirrels born in a lab and with no experience in their original habitat. Over two summers, squirrels captured from beacon-dense and beacon-thin areas were tested in a radial maze interspersed with beacons, using number of trials to criterion as a measure of spatial memory. To evaluate the effect of landmark navigation, in year 2 juveniles were prevented from seeing outside the maze area. In both years squirrels from beacon-dense populations reached criterion faster than squirrels from beacon-thin populations, and a weak rearing effect was present in 1 year. Despite sex differences in adult spatial skills, no differences were found between males and females in the maze. This demonstrates variation in the navigation strategies of young U. beldingi, and highlights the need to evaluate spatial preferences as a function of population or ecology in addition to species and sex.

Alarm calls elicit predator-specific physiological responses.

Glucocorticoids regulate glucose concentrations and responses to unpredictable events, while also modulating cognition. Juvenile Belding's ground squirrels (Urocitellus beldingi) learn to respond to whistle and trill alarm calls, warning of aerial and terrestrial predators, respectively, shortly after emerging from natal burrows at one month of age. Alarm calls can cause physiological reactions and arousal, and this arousal, coupled with watching adult responses, might help juveniles learn associations between calls and behavioural responses. I studied whether young show differential cortisol responses to alarm and non-alarm calls, using playbacks of U. beldingi whistles, trills, squeals (a conspecific control vocalization) and silent controls. Trills elicited very high cortisol responses, and, using an individual's response to the silent control as baseline, only their response to a trill was significantly higher than baseline. This cortisol increase would provide glucose for extended vigilance and escape efforts, which is appropriate for evading terrestrial predators which hunt for long periods. Although whistles do not elicit a cortisol response, previous research has shown that they do result in bradycardia, which enhances attention and information processing. This is a novel demonstration of two physiological responses to two alarm calls, each appropriate to the threats represented by the calls.

Inverted-U shape relationship between cortisol and learning in ground squirrels.

Adrenal hormones regulate glucose levels, responses to unpredictable stressors and modulate cognition. Glucocorticoids can have an inverted-U shape relationship with cognition, as very low or high levels impair, whereas moderate elevations facilitate, acquisition and retention of memories. To date these relationships have been tested with humans and rodents in laboratory settings rather than with wild animals in biologically relevant contexts. This study examined whether the elevated cortisol observed in juvenile Belding's ground squirrels (Spermophilus beldingi) at natal emergence might promote both acquisition of adaptive responses to this species' two alarm calls warning of predators and memory of the spatial configuration of mothers' territories. Both experimentally increased and decreased basal cortisol levels interfere with acquisition and retention of an association between a warning call and the appropriate response compared with naturally occurring moderately elevated cortisol. Further, decreased cortisol impairs learning of a novel, complex spatial maze. Thus in the field the brief elevation of cortisol at emergence might facilitate acquisition of spatial memory of a three-dimensional environment and responses to alarm calls during a sensitive period of learning. This novel demonstration of the inverted-U shape function in a wild animal suggests that natural selection has favored a hormonal profile facilitating rapid acquisition of important survival behaviors.

Rats assess degree of relatedness from human odors.

Despite widespread interest in the evolutionary implications of human olfactory communication, the mechanisms underlying human odor production are still poorly understood. Previous studies have demonstrated that human odor cues are related to variations in the major histocompatibility complex, but it is unclear whether odors are associated with overall genotypic variation. In this study, we investigated whether more closely related humans produce more similar odor cues. To assess objective odor qualities we tested odor similarity using rats in a habituation-discrimination paradigm. Rats were first habituated to a referent human odor and were then presented with two test odors obtained from individuals related in different degrees to the referent. Investigation times for each odor were compared. Because rats investigate novel odors longer than familiar odors, we were able to determine which test odor the rats perceived as more similar to the referent human odor. For six of ten odor donor families, rats investigated the odor of the less closely related individual significantly longer than that of the more closely related individual, and investigation durations were in the expected direction for all families. These results indicate that similarity of human odor cues is associated with degree of genetic relatedness, with more closely related humans producing more similar odor cues. This study supports the hypothesis that odor cues provide information regarding degree of relatedness and may thus affect a wide variety of human behaviors, including kin preferences, nepotism, and mate choice.

Ecological and hormonal correlates of antipredator behavior in adult Belding's ground squirrels (Spermophilus beldingi).

Predator-prey relationships provide an excellent opportunity to study coevolved adaptations. Decades of theoretical and empirical research have illuminated the various behavioral adaptations exhibited by prey animals to avoid detection and capture, and recent work has begun to characterize physiological adaptations, such as immune reactions, metabolic changes, and hormonal responses to predators or their cues. A 2-year study quantified the activity budgets and antipredator responses of adult Belding's ground squirrels (Spermophilus beldingi) living in three different California habitats and likely experiencing different predation pressures. At one of these sites, which is visually closed and predators and escape burrows are difficult to see, animals responding to alarm calls remain alert longer and show more exaggerated responses than adults living in two populations that likely experience less intense predation pressure. They also spend more time alert and less time foraging than adults at the other two sites. A 4-year study using noninvasive fecal sampling of cortisol metabolites revealed that S. beldingi living in the closed site also have lower corticoid levels than adults at the other two sites. The lower corticoids likely reflect that predation risk at this closed site is predictable, and might allow animals to mount large acute cortisol responses, facilitating escape from predators and enhanced vigilance while also promoting glucose storage for the approaching hibernation. Collectively, these data demonstrate that local environments and perceived predation risk influence not only foraging, vigilance, and antipredator behaviors, but adrenal functioning as well, which may be especially important for obligate hibernators that face competing demands on glucose storage and mobilization.

Development of individually distinct recognition cues.

Despite extensive research on the functions of kin recognition, little is known about ontogenetic changes in the cues mediating such recognition. In Belding's ground squirrels, Spermophilus beldingi, secretions from oral glands are both individually distinct and kin distinct, and function in social recognition across many contexts. Behavioral studies of recognition and kin preferences suggest that these cues may change across development, particularly around the time of weaning and emergence from natal burrows (around 25 days of age). I used an habituation-discrimination task with captive S. beldingi, presenting subjects with odors collected from a pair of pups at several ages across early development. I found that at 21 days of age, but not at 7 or 14, young produce detectable odors. Odors are not individually distinct, however, until 28 days of age, after young have emerged from their burrows and begun foraging. In addition, an individual's odor continues to develop after emergence: odors produced by an individual at 20 and 40 days of age are perceived as dissimilar, yet odors produced at 28 and 40 days are treated as similar. Developmental changes in odors provide a proximate explanation for why S. beldingi littermate preferences are not consolidated until after natal emergence, and demonstrate that conspecifics must update their recognition templates as young develop.

Developmental and geographic variation in stress hormones in wild Belding's ground squirrels (Spermophilus beldingi).

Extensive research has been conducted on the role of glucocorticoids in regulating growth, mobilizing energy, responding to stressors and modulating learning and memory. However, little is known about the production of corticoids during early development in free-living animals, particularly during sensitive periods of acquisition of important behaviors. In a four-year study of Belding's ground squirrels, Spermophilus beldingi, a non-invasive assay of glucocorticoids was used to quantify age and population differences among juveniles from three California locations. Fecal-cortisol metabolites are elevated during a short period when juveniles first emerge aboveground from their natal burrows at about 4 weeks of age. This period of cortisol elevation coincides with when young are learning survival behaviors such as anti-predator responses and foraging strategies. Population differences in juvenile cortisol levels, which may reflect local variation in habitat quality and predator environments, were not evident until 2 weeks after emergence. Elevated cortisol at the age of emergence was also observed in juveniles born and reared in captivity without exposure to typical stressors that occur around the age of emergence. These results indicate that corticoids are regulated during early development, and the possible functions of age-related corticoid levels are discussed, including mobilization of glucose for natal emergence and later facilitation of growth and energy storage during the short summer before hibernation. In some species, elevated corticoids can also facilitate learning and memory, and current work is exploring whether the higher cortisol observed in all three S. beldingi populations just after emergence function to promote rapid acquisition of survival behaviors.

A validation of extraction methods for noninvasive sampling of glucocorticoids in free-living ground squirrels.

Fecal hormone assays provide a powerful tool for noninvasive monitoring of endocrine status in wild animals. In this study we validated a protocol for extracting and measuring glucocorticoids in free-living and captive Belding's ground squirrels (Spermophilus beldingi). We first compared two commonly used extraction protocols to determine which performed better with commercially available antibodies. We next verified the preferred extraction method by correlating circulating and fecal glucocorticoid measures from a group of individuals over time. For this comparison, we used both a cortisol and a corticosterone antibody to determine which had greater affinity to the fecal metabolites. Cortisol was the primary circulating glucocorticoid, but both hormones were present in well above detectable concentrations in the blood, which does not occur in other sciurids. In addition, the cortisol antibody showed greater binding with the fecal extracts than did the corticosterone antibody. Finally, we used adrenocorticotropic hormone and dexamethasone challenges to demonstrate that changes in adrenal functioning are reflected in changing fecal corticoid levels. These results suggest that our extraction protocol provides a fast, reliable assay of stress hormones in free-living ground squirrels without the confounding influence of short-term rises in glucocorticoid concentrations caused by handling and restraint stress and that it can facilitate ecological and evolutionary studies of stress in wild species.

Kin-recognition abilities and nepotism as a function of sociality.

Despite widespread interest in kin selection and nepotism, relatively little is known about the perceptual abilities of animals to recognize their relatives. Here I show that a highly nepotistic species, Belding's ground squirrels (Spermophilus beldingi), produces odours from at least two sources that correlate with relatedness ('kin labels'), and that ground squirrels can use these odours to make accurate discriminations among never before encountered ('unfamiliar') kin. Recognition odours appear to vary linearly with relatedness, rather than in an all-or-none fashion, allowing precise estimates of kinship even among distant relatives. Thus S. beldingi are able to recognize their distant kin and male kin, even though they do not treat them preferentially. I also show that a closely related species (S. lateralis) similarly produces kin labels and discriminates among kin, although it shows no evidence of kin-directed behaviour. Thus, contrary to a commonly held assumption, kin favouritism and recognition abilities can evolve independently, depending on variation in the costs and benefits of nepotism for a given species.