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Seasonally breeding animals respond to environmental cues to determine optimal conditions for reproduction. Siberian hamsters (Phodopus sungorus) primarily rely on photoperiod as a predictive cue of future energy availability. When raised in long-day photoperiods (>14 h light), supplemental cues such as food availability typically do not trigger the seasonal reproductive response of gonadal regression, which curtails reproduction in unsuitable environments. We investigated whether recognition of food availability as a cue could be altered by a nutritional challenge during development. Specifically, we predicted that hamsters receiving restricted food during development would be sensitized to food restriction (FR) as adults and undergo gonadal regression in response. Male and female hamsters were given either ad libitum (AL) food or FR from weaning until d60. The FR treatment predictably limited growth and delayed puberty in both sexes. For 5 weeks after d60, all hamsters received an AL diet to allow FR hamsters to gain mass equal to AL hamsters. Then, adult hamsters of both juvenile groups received either AL or FR for 6 weeks. Juvenile FR had lasting impacts on adult male body mass and food intake. Adult FR females exhibited decreased estrous cycling and uterine horn mass indiscriminately of juvenile food treatment, but there was little effect on male reproductive measurements. Overall, we observed a delay in puberty in response to postweaning FR, but this delay appeared not to affect seasonal reproductive responses in the long term. These findings increase our understanding of seasonal reproductive responses in a relevant environmental context.

Coordinating physiological and behavioural processes across the annual cycle is essential in enabling individuals to maximize fitness. While the mechanisms underlying seasonal reproduction and its associated behaviours are well characterized, fewer studies have examined the hormonal basis of non-reproductive social behaviours (e.g. aggression) on a seasonal time scale. Our previous work suggests that the pineal hormone melatonin facilitates a ‘seasonal switch’ in neuroendocrine regulation of aggression in male and female Siberian hamsters ( Phodopus sungorus ), specifically by acting on the adrenal glands to increase the production of the androgen dehydroepiandrosterone (DHEA) during the short-day (SD) photoperiods of the non-breeding season. Here, we provide evidence that the activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), a key enzyme within the steroidogenic pathway that mediates DHEA synthesis and metabolism, varies in a sex-specific and melatonin-dependent manner. Although both male and female hamsters displayed increased aggression in response to SDs and SD-like melatonin, only males showed an increase in adrenal 3β-HSD activity. Conversely, SD and melatonin-treated females exhibited reductions in both adrenal and neural 3β-HSD activity. Collectively, these results suggest a potential role for 3β-HSD in modulating non-breeding aggression and, more broadly, demonstrate how distinct neuroendocrine mechanisms may underlie the same behavioural phenotype in males and females.

Maternal antibiotic (ABx) exposure can significantly perturb the transfer of microbiota from mother to offspring, resulting in dysbiosis of potential relevance to neurodevelopmental disorders such as autism spectrum disorder (ASD). Studies in rodent models have found long-term neurobehavioral effects in offspring of ABx-treated dams, but ASD-relevant behavior during the early preweaning period has thus far been neglected. Here, we exposed C57BL/6J mouse dams to ABx (5 mg/ml neomycin, 1.25 μg/ml pimaricin, .075% v/v acetic acid) dissolved in drinking water from gestational day 12 through offspring postnatal day 14. A number of ASD-relevant behaviors were assayed in offspring, including ultrasonic vocalization (USV) production during maternal separation, group huddling in response to cold challenge, and olfactory-guided home orientation. In addition, we obtained measures of thermoregulatory competence in pups during and following behavioral testing. We found a number of behavioral differences in offspring of ABx-treated dams (e.g., modulation of USVs by pup weight, activity while huddling) and provide evidence that some of these behavioral effects can be related to thermoregulatory deficiencies, particularly at younger ages. Our results suggest not only that ABx can disrupt microbiomes, thermoregulation, and behavior, but that metabolic effects may confound the interpretation of behavioral differences observed after early-life ABx exposure.

Individuals of virtually all vertebrate species are exposed to annual fluctuations in the deterioration and renewal of their environments. As such, organisms have evolved to restrict energetically expensive processes and activities to a specific time of the year. Thus, the precise timing of physiology and behavior is critical for individual reproductive success and subsequent fitness. Although the majority of research on seasonality has focused on seasonal reproduction, pronounced fluctuations in other non-reproductive social behaviors, including agonistic behaviors (e.g., aggression), also occur. To date, most studies that have investigated the neuroendocrine mechanisms underlying seasonal aggression have focused on the role of photoperiod (i.e., day length); prior findings have demonstrated that some seasonally breeding species housed in short "winter-like" photoperiods display increased aggression compared with those housed in long "summer-like" photoperiods, despite inhibited reproduction and low gonadal steroid levels. While fewer studies have examined how the hormonal correlates of environmental cues regulate seasonal aggression, our previous work suggests that the pineal hormone melatonin acts to increase non-breeding aggression in Siberian hamsters (Phodopus sungorus) by altering steroid hormone secretion. This review addresses the physiological and cellular mechanisms underlying seasonal plasticity in aggressive and non-aggressive social behaviors, including a key role for melatonin in facilitating a "neuroendocrine switch" to alternative physiological mechanisms of aggression across the annual cycle. Collectively, these studies highlight novel and important mechanisms by which melatonin regulates aggressive behavior in vertebrates and provide a more comprehensive understanding of the neuroendocrine bases of seasonal social behaviors broadly.

Many animals exhibit pronounced changes in physiology and behavior on a seasonal basis, and these adaptations have evolved to promote survival and reproductive success. While the neuroendocrine pathways mediating seasonal reproduction are well-studied, far less is known about the mechanisms underlying seasonal changes in social behavior, particularly outside of the context of the breeding season. Our previous work suggests that seasonal changes in melatonin secretion are important in regulating aggression in Siberian hamsters (Phodopus sungorus); it is unclear, however, how melatonin acts via its receptors to modulate seasonal variation in social behavior. In this study, we infused a MT(1) melatonin receptor-expressing (MT(1)) or control (CON) lentivirus into the adrenal glands of male Siberian hamsters. We then housed hamsters in long-day (LD) or short-day (SD) photoperiods, administered timed melatonin or control injections, and quantified aggressive and non-aggressive social behaviors (e.g., investigation, self-grooming) following 10 weeks of treatment. LD hamsters infused with the MT(1) lentivirus had significantly higher adrenal mt1 expression than LD CON hamsters, as determined via quantitative PCR. While melatonin administration was necessary to induce SD-like reductions in body and relative reproductive mass, only LD hamsters infused with the MT(1) lentivirus displayed SD-like changes in social behavior, including increased aggression and decreased investigation and grooming. In addition, SD CON and LD hamsters infused with the MT(1) lentivirus exhibited similar relationships between adrenal mt1 expression and aggressive behavior. Together, our findings suggest a role for adrenal MT(1) receptor signaling in regulating behavior, but not energetics or reproduction in seasonally breeding species.

Animal personality is defined as behavioral tendencies that are consistent across time and contexts within an individual, but differ across individuals. Studies investigating personality typically examine individuals across short time periods or within a single life stage. Growing evidence suggests that personality may be less stable across life stages, highlighting the need to consider the effects of ontogeny on the expression of consistent behavioral traits. We investigated individual consistency in social and escape behaviors across developmental stages using Siberian hamsters (Phodopus sungorus). To determine whether individuals were consistent in these behaviors as juveniles and across developmental stages, we measured male and female social and escape behaviors twice as juveniles and once as adults. Individuals' social scores were significantly repeatable within the juvenile stage, but not across developmental stages. In contrast, escape scores were highly repeatable across developmental stages, with males' scores being more repeatable than females' scores. Our results support previous findings that personality traits, especially those associated with social behavior, are less stable across development, whereas behaviors associated with stress or coping may represent a more permanent feature of an individual's phenotype. Our results also indicate potential sex differences in long-term repeatability of personality. Considering how ontogeny affects animal personality for males and females can provide insight into the evolution and mechanisms that maintain animal personality.

On and within most sites across an animal's body live complex communities of microorganisms. These microorganisms perform a variety of important functions for their hosts, including communicating with the brain, immune system and endocrine axes to mediate physiological processes and affect individual behaviour. Microbiome research has primarily focused on the functions of the microbiome within the gastrointestinal tract (gut microbiome) using biomedically relevant laboratory species (i.e. model organisms). These studies have identified important connections between the gut microbiome and host immune, neuroendocrine and nervous systems, as well as how these connections, in turn, influence host behaviour and health. Recently, the field has expanded beyond traditional model systems as it has become apparent that the microbiome can drive differences in behaviour and diet, play a fundamental role in host fitness and influence community-scale dynamics in wild populations. In this Review, we highlight the value of conducting hypothesis-driven research in non-model organisms and the benefits of a comparative approach that assesses patterns across different species or taxa. Using social behaviour as an intellectual framework, we review the bidirectional relationship between the gut microbiome and host behaviour, and identify understudied mechanisms by which these effects may be mediated.

Aggression is a complex social behaviour that allows individuals to compete for access to limited resources (eg, mates, food and territories). Excessive or inappropriate aggression, however, has become problematic in modern societies, and current treatments are largely ineffective. Although previous work in mammals suggests that aggressive behaviour varies seasonally, seasonality is largely overlooked when developing clinical treatments for inappropriate aggression. Here, we investigated how the hormone melatonin regulates seasonal changes in neurosteroid levels and aggressive behaviour in Siberian hamsters, a rodent model of seasonal aggression. Specifically, we housed males in long-day (LD) or short-day (SD) photoperiods, administered timed s.c. melatonin injections (which mimic a SD-like signal) or control injections, and measured aggression using a resident-intruder paradigm after 9 weeks of treatment. Moreover, we quantified five steroid hormones in circulation and in brain regions associated with aggressive behaviour (lateral septum, anterior hypothalamus, medial amygdala and periaqueductal gray) using liquid chromatography-tandem mass spectrometry. SD hamsters and LD hamsters administered timed melatonin injections (LD-M) displayed increased aggression and exhibited region-specific decreases in neural dehydroepiandrosterone, testosterone and oestradiol, but showed no changes in progesterone or cortisol. Male hamsters also showed distinct associations between neurosteroids and aggressive behaviour, in which neural progesterone and dehydroepiandrosterone were positively correlated with aggression in all treatment groups, whereas neural testosterone, oestradiol and cortisol were negatively correlated with aggression only in LD-M and SD hamsters. Collectively, these results provide insight into a novel neuroendocrine mechanism of mammalian aggression, in which melatonin reduces neurosteroid levels and elevates aggressive behaviour.

Many animal species exhibit year-round aggression, a behaviour that allows individuals to compete for limited resources in their environment (eg, food and mates). Interestingly, this high degree of territoriality persists during the non-breeding season, despite low levels of circulating gonadal steroids (ie, testosterone [T] and oestradiol [E2 ]). Our previous work suggests that the pineal hormone melatonin mediates a 'seasonal switch' from gonadal to adrenal regulation of aggression in Siberian hamsters (Phodopus sungorus); solitary, seasonally breeding mammals that display increased aggression during the short, 'winter-like' days (SDs) of the non-breeding season. To test the hypothesis that melatonin elevates non-breeding aggression by increasing circulating and neural steroid metabolism, we housed female hamsters in long days (LDs) or SDs, administered them timed or mis-timed melatonin injections (mimic or do not mimic a SD-like signal, respectively), and measured aggression, circulating hormone profiles and aromatase (ARO) immunoreactivity in brain regions associated with aggressive or reproductive behaviours (paraventricular hypothalamic nucleus [PVN], periaqueductal gray [PAG] and ventral tegmental area [VTA]). Females that were responsive to SD photoperiods (SD-R) and LD females given timed melatonin injections (Mel-T) exhibited gonadal regression and reduced circulating E2 , but increased aggression and circulating dehydroepiandrosterone (DHEA). Furthermore, aggressive challenges differentially altered circulating hormone profiles across seasonal phenotypes; reproductively inactive females (ie, SD-R and Mel-T females) reduced circulating DHEA and T, but increased E2 after an aggressive interaction, whereas reproductively active females (ie, LD females, SD non-responder females and LD females given mis-timed melatonin injections) solely increased circulating E2 . Although no differences in neural ARO abundance were observed, LD and SD-R females showed distinct associations between ARO cell density and aggressive behaviour in the PVN, PAG and VTA. Taken together, these results suggest that melatonin increases non-breeding aggression by elevating circulating steroid metabolism after an aggressive encounter and by regulating behaviourally relevant neural circuits in a region-specific manner.

Although research has shown interactions between the reproductive system and energy homeostasis, it is not clear how environmental or behavioral factors may factor into these associations. Here we aimed to determine how changes in reproductive state (i.e., phase of the menstrual cycle) and other behavioral and physiological factors may influence leptin levels in healthy women, as well as how sexual activity may play a role in leptin modulation. We collected serum and saliva from 32 healthy women and measured leptin, estradiol, and progesterone. Participants also completed surveys of demographics, health and sexual behaviors, and physical activity. Leptin was predicted by meals per day and missed meals at both menses and ovulation. However, estradiol and physical activity were stronger predictors of leptin at menses, while sexual activity was a stronger predictor of leptin at ovulation. These findings suggest that predictors of serum leptin, and possibly energy storage and expenditure, vary across the menstrual cycle.

The expression of a wide range of social and affective behaviors, including aggression and investigation, as well as anxiety- and depressive-like behaviors, involves interactions among many different physiological systems, including the neuroendocrine and immune systems. Recent work suggests that the gut microbiome may also play a critical role in modulating behavior and likely functions as an important integrator across physiological systems. Microbes within the gut may communicate with the brain via both neural and humoral pathways, providing numerous avenues of research in the area of the gut-brain axis. We are now just beginning to understand the intricate relationships among the brain, microbiome, and immune system and how they work in concert to influence behavior. The effects of different forms of experience (e.g., changes in diet, immune challenge, and psychological stress) on the brain, gut microbiome, and the immune system have often been studied independently. Though because these systems do not work in isolation, it is essential to shift our focus to the connections among them as we move forward in our investigations of the gut-brain axis, the shaping of behavioral phenotypes, and the possible clinical implications of these interactions. This review summarizes the recent progress the field has made in understanding the important role the gut microbiome plays in the modulation of social and affective behaviors, as well as some of the intricate mechanisms by which the microbiome may be communicating with the brain and immune system.

Sickness is typically characterized by fever, anorexia, cachexia, and reductions in social, pleasurable, and sexual behaviors. These responses can be displayed at varying intensities both within and among individuals, and the adaptive nature of sickness responses can be demonstrated by the context-dependent nature of their expression. The study of sickness has become an important area of investigation for researchers in a wide range of areas, including psychoneuroimmunology (PNI) and ecoimmunology (EI). The general goal of PNI is to identify key interactions among the nervous, endocrine and immune systems and behavior, and how disruptions in these processes might contribute to disease states. EI, in turn, has been established more recently within the perspectives of ecology and evolutionary biology, and is aimed more at understanding natural variation in immune function and sickness responses within a broadly integrative, organismal, and evolutionary context. The goal of this review is to examine the literature on sickness from both basic and biomedical perspectives within PNI and EI and to demonstrate how the integrative study of sickness behavior can serve as an integrating agent to connect ecological and translational approaches to the study of disease. By focusing on a set of specific exemplars, including the energetics of sickness, social context, and environmental influences on sickness, we hope to accomplish the larger goal of developing a common synthetic framework to understand sickness from multiple levels of analysis and varying perspectives across the fields of PNI and EI. By applying this integrative approach to sickness, we will be able to develop a more comprehensive view of sickness as a suite of adaptive responses rather than the simply deleterious consequences of illness.

Although testosterone (T) has been characterized as universally immunosuppressive across species and sexes, recent ecoimmunology research suggests that T's immunomodulatory effects (enhancing/suppressing) depend on the organism's reproductive context. Very little is known about the immune effects of T in healthy females, and even less about how reproductive effort modulates the immune effects of T in humans. We investigated how the interaction between endogenous T and sexual activity predicted menstrual cycle-related changes in several measures of immunity: inflammation (indexed by interleukin-6, IL-6), adaptive immunity (indexed by immunoglobulin A, IgA), and functional immunity (indexed by bactericidal assay). Thirty-two healthy women (sexually abstinent, N = 17; sexually active with one male partner, N = 15) provided saliva samples at four points in the menstrual cycle: menses, follicular, ovulation, and luteal phases. Among sexually abstinent women, T was positively associated with IL-6 across the cycle; for sexually active women, however, T was positively associated with IL-6 in the luteal phase only, and negatively associated with IL-6 at ovulation. High T predicted higher IgA among women who reported infrequent intercourse, but lower IgA among women who reported very frequent intercourse. Finally, across groups, T was positively associated with greater bacterial killing at menses, but negatively associated in the luteal phase. Overall, rather than being universally immunosuppressive, T appeared to signal immunomodulation relevant to reproduction (e.g., lowering inflammation at ovulation, potentially preventing immune interference with conception). Our findings support the hypothesis that the immunomodulatory effects of endogenous T in healthy females depend on sexual and reproductive context.

Mounting a sickness response is an energetically expensive task and requires precise balancing of energy allocation to ensure pathogen clearance while avoiding compromising energy reserves. Sickness intensity has previously been shown to be modulated by food restriction, body mass, and hormonal signals of energy reserves. In the current study, we tested the hypothesis that sickness intensity is modulated by glucose availability and an endocrine signal of glucose availability, insulin. We utilized male Siberian hamsters (Phodopus sungorus) and predicted that pharmacological induction of glucoprivation with 2-deoxy-D-glucose (2-DG), a non-metabolizable glucose analog that disrupts glycolysis, would attenuate energetically expensive sickness symptoms. Alternatively, we predicted that treatment of animals with insulin would enhance energetically expensive sickness symptoms, as insulin would act as a signal of increased glucose availability. Upon experimental treatment with lipopolysaccharide (LPS), we found that glucose deprivation resulted in increased sickness-induced hypothermia as compared to control- and insulin-treated animals; however, it did not have any effects on sickness-induced anorexia or body mass loss. Insulin treatment resulted in an unexpectedly exaggerated sickness response in animals of lesser body masses; however, in animals of greater body masses, insulin actually attenuated sickness-induced body mass loss and had no effects on hypothermia or anorexia. The effects of insulin on sickness severity may be modulated by sensitivity to sickness-induced hypoglycemia. Collectively, these results demonstrate that both glucose availability and signals of glucose availability can modulate the intensity of energetically expensive sickness symptoms, but their effects differ among different sickness symptoms and are sensitive to energetic context.

Seasonally breeding animals exhibit profound physiological and behavioural responses to changes in ambient day length (photoperiod), including changes in reproductive function and territorial aggression.Species where aggression persists when gonads are regressed and circulating levels of gonadal hormones are low, such as Siberian hamsters (Phodopus sungorus) and song sparrows (Melospiza melodia), challenge the well-established framework that gonadal hormones are important mediators of aggression.A solution to this apparent paradox is that a season-specific increase in sensitivity to hormones in brain areas associated with aggression offsets low levels of gonadal hormones during periods of reproductive quiescence.To test this hypothesis, we manipulated photoperiod to induce natural fluctuations in seasonal phenotype across multiple stages of the annual reproductive cycle in female Siberian hamsters that display increased aggression during short-day reproductive quiescence, suggesting that behaviour persists independent of gonadal steroids.Females were housed in long “summer” days or short “winter” days for 10, 24 or 30 weeks to capture gonadal regression, transition back to a reproductively functional state and full gonadal recrudescence, respectively.Long-day animals maintained reproductive functionality and displayed low aggression across all time points. By week 10, short-day reproductively responsive females underwent gonadal regression and displayed increased aggression; non-responsive animals showed no such changes. At week 24, animals were in a transitional period and displayed an intermediate phenotype with respect to reproduction and aggression. By week 30, short-day females were fully recrudesced and returned to long-day-like levels of aggression.Consistent with our hypothesis, gonadally regressed females displayed decreases in 17β-oestradiol (oestradiol) levels, but site-specific increases in the abundance of brain oestrogen receptor-alpha (ERα) in regions associated with aggression, but not reproduction. Increased site-specific ERα may function as a compensatory mechanism to allow increased responsiveness to oestradiol in regulating aggression in lieu of high circulating concentrations of hormones.Collectively, these results broaden our understanding of how breeding phenology maps onto social behaviour and the mechanisms that have evolved to coordinate behaviours that occur in non-breeding contexts.

Multidirectional interactions among the immune, endocrine, and nervous systems have been demonstrated in humans and non-human animal models for many decades by the biomedical community, but ecological and evolutionary perspectives are lacking. Neuroendocrine-immune interactions can be conceptualized using a series of feedback loops, which culminate into distinct neuroendocrine-immune phenotypes. Behavior can exert profound influences on these phenotypes, which can in turn reciprocally modulate behavior. For example, the behavioral aspects of reproduction, including courtship, aggression, mate selection and parental behaviors can impinge upon neuroendocrine-immune interactions. One classic example is the immunocompetence handicap hypothesis (ICHH), which proposes that steroid hormones act as mediators of traits important for female choice while suppressing the immune system. Reciprocally, neuroendocrine-immune pathways can promote the development of altered behavioral states, such as sickness behavior. Understanding the energetic signals that mediate neuroendocrine-immune crosstalk is an active area of research. Although the field of psychoneuroimmunology (PNI) has begun to explore this crosstalk from a biomedical standpoint, the neuroendocrine-immune-behavior nexus has been relatively underappreciated in comparative species. The field of ecoimmunology, while traditionally emphasizing the study of non-model systems from an ecological evolutionary perspective, often under natural conditions, has focused less on the physiological mechanisms underlying behavioral responses. This review summarizes neuroendocrine-immune interactions using a comparative framework to understand the ecological and evolutionary forces that shape these complex physiological interactions.

We identify the significance and typical requirements of developmental analyses of the microbiome-gut-brain (MGB) in parents, offspring, and parent-offspring relations, which have particular importance for neurobehavioral outcomes in mammalian species, including humans. We call for a focus on behavioral measures of social-emotional function. Methodological approaches to interpreting relations between the microbiota and behavior are discussed.

Chemical communication is a critical component of social behavior as it facilitates social encounters, allows for evaluation of the social partner, defines territories and resources, and advertises information such as sex and physiological state of an animal. Odors provide a key source of information about the social environment to rodents; however, studies identifying chemical compounds have thus far focused primarily on few species, particularly the house mouse. Moreover, considerably less attention has been focused on how environmental factors, reproductive phenotype, and behavioral context alter these compounds outside of reproduction. We examined the effects of photoperiod, sex, and social context on chemical communication in the seasonally breeding Siberian hamster. We sampled ventral gland secretions in both male and female hamsters before and after an aggressive encounter and identified changes in a range of volatile compounds. Next, we investigated how photoperiod, reproductive phenotype, and aggression altered ventral gland volatile compound composition across the sexes. Males exhibited a more diverse chemical composition, more sex-specific volatiles, and showed higher levels of excretion compared to females. Individual volatiles were also differentially excreted across photoperiod and reproductive phenotype, as well as differentially altered in response to an aggressive encounter. Female volatile compound composition, in contrast, did not differ across photoperiods or in response to aggression. Collectively, these data contribute to a greater understanding of context-dependent changes in chemical communication in a seasonally breeding rodent.

Some seasonally-breeding animals are more aggressive during the short, “winter-like” days (SD) of the non-breeding season, despite gonadal regression and reduced circulating androgen levels. While the mechanisms underlying SD increases in aggression are not well understood, previous work from our lab suggests that pineal melatonin (MEL) and the adrenal androgen dehydroepiandrosterone (DHEA) are important in facilitating non-breeding aggression in Siberian hamsters (Phodopus sungorus). To characterize the role of MEL in modulating seasonal transitions in aggressive behavior, we housed male hamsters in long days (LD) or SD, treated them with timed MEL (M) or saline injections, and measured aggression after 3, 6, and 9 weeks. Furthermore, to assess whether MEL mediates seasonal shifts in gonadal and adrenal androgen synthesis, serum testosterone (T) and DHEA concentrations were quantified 36 h before and immediately following an aggressive encounter. LD-M and SD males exhibited similar physiological and behavioral responses to treatment. Specifically, both LD-M and SD males displayed higher levels of aggression than LD males and reduced circulating DHEA and T in response to an aggressive encounter, whereas LD males elevated circulating androgens. Interestingly, LD and SD males exhibited distinct relationships between circulating androgens and aggressive behavior, in which changes in serum T following an aggressive interaction (∆T) were negatively correlated with aggression in LD males, while ∆DHEA was positively correlated with aggression in SD males. Collectively, these findings suggest that SD males transition from synthesis to metabolism of circulating androgens following an aggressive encounter, a mechanism that is modulated by MEL.

Although it is well-established that the immune system plays an important role in the development of physiology and behavior, the gut microbiome has recently become of interest in the study of developmental origins of behavior. Studies suggest that the effects of early-life immune activation may not occur until a secondary stressor is introduced, though the precise nature and timing of the stressor may be critical in the response. Further, recent work suggests that the microbiome and the immune system develop in parallel, and therefore any perturbations to one of these systems early in life will likely affect the other. Here, we sought to determine whether early-life activation of the immune system had long-term consequences on how the gut microbiome responds to antibiotic treatment in adulthood and whether those changes influence adult same-sex social behavior. In order to test the hypothesis that an early-life immune challenge makes individuals more vulnerable to the effects of antibiotics, we mimicked an early-life infection by injecting pups at postnatal day 3 and 5 with lipopolysaccharide (LPS; cell wall component of gram-negative bacteria) or saline, and subsequently exposed the same animals to antibiotic treatment (known to influence microbial community composition and behavior) or water in adulthood. We tracked physiology across development, and paired males and females with a novel individual of the same age and sex in adulthood to score same-sex behavior (e.g., aggression, investigation, grooming) before antibiotic treatment, immediately following treatment, and after recovery from antibiotics. LPS-treated females exhibited impaired reproductive physiology and function in adulthood (e.g., smaller ovaries and abnormal estrous cycles), and female and male gut microbial communities were strongly affected by antibiotic treatment in adulthood, but only slightly affected by postnatal LPS alone. Interestingly, LPS-treated males exhibited more robust changes in their behavioral response following adult antibiotic treatment, including decreased investigation and increased grooming, suggestive of changes in anxiety-like behaviors. These data suggest that males may be more vulnerable than females to behavioral abnormalities after being predisposed to an immune challenge early in life. Collectively, these results provide novel evidence that some of the sex-specific behavioral consequences of an early-life immune challenge may not transpire until an individual is faced with a secondary challenge, and the context in which an individual is exposed can greatly influence the response.

Seasonal variation in social behavior is often accompanied by seasonal variation in communication. In mammals, how seasonal environmental cues influence aggressive vocalizations remains underexplored. Photoperiod is the primary cue coordinating seasonal responses in most temperate zone animals, including Siberian hamsters ( Phodopus sungorus ), a species that undergoes reproductive inhibition and increased aggression in winter. During same-sex aggressive encounters, hamsters emit both broadband calls (BBCs) and ultrasonic vocalizations (USVs) that indicate aggression and the vocalizer's sex, respectively; however, it is not known whether these rodents adjust specific elements of their vocal repertoire to reflect their photoperiod-induced seasonal phenotypes. To address this, we recorded vocalizations emitted during dyadic interactions between male or female pairs of hamsters housed in long or short photoperiods and measured serum testosterone levels. USV emission rate remained stable across photoperiods, but proportional use of USV subtypes varied in novel ways: ‘jump’ USVs were sensitive to seasonal phenotype, but not the vocalizer's sex, whereas ‘plain’ USVs were sensitive only to the sex of the vocalizer. BBC emission rate varied with seasonal phenotype; short-day non-reproductive hamsters produced more BBCs and demonstrated increased aggression compared with reproductive hamsters. Testosterone, however, was not related to vocalization rates. Collectively, these findings demonstrate that changes in the vocal repertoire of Siberian hamsters reflect sex, aggression, and seasonal phenotype, suggesting that both BBCs and USVs are important signals used during same-sex social encounters.

Psychosocial stress, specifically social isolation, is an important risk factor for the development of a variety of psychological and physiological disorders. Changes in immune function have been hypothesized to mediate this relationship. The current study used the prairie vole (Microtus ochrogaster) model of isolation-induced depressive-like behavior to test whether social isolation led to changes in innate immune function. Specifically, we used hemolytic complement (CH50) and bacteria killing assays to assess innate immunity, in paired or singly housed male and female prairie voles. Further, in a second experiment we tested whether females exposed to an additional short-term social stressor, a resident-intruder trial, would show changes in immune function as well as enhanced hypothalamic pituitary axis (HPA) activity as indicated by elevated plasma corticosterone levels. Socially isolated animals, regardless of sex, had significantly reduced CH50s and bacteria killing ability. Socially isolated females exposed to a resident-intruder stressor also showed reduced CH50s and bacteria killing ability as well as significant increases in aggressive behavior, however, they did not show elevated circulating corticosterone levels. Collectively, these data will help inform our understanding of the relationship between social isolation and physiological and psychological health.

Vocalizations constitute an important channel of communication for many vertebrates. Classes of vocalizations may be closely associated with particular contexts or behaviours, and variation within classes may convey information on individual identity, sex or motivational state. Rodent vocal communication has largely been studied within a reproductive context, but rodents also utter vocalizations during aggressive encounters with same-sex conspecifics. In this work we investigated same-sex vocal behaviour of Siberian hamsters, a mammalian model species for studying aggression. Males and females produced two main classes of vocalizations: high-frequency (>20 kHz), narrowband vocalizations (ultrasonic vocalizations; USVs) and lower-frequency, broadband vocalizations (broadband calls; BBCs). USVs and BBCs were further classified into subtypes based on spectrotemporal characteristics. With these classifications, we made the predictions that hamsters would utter distinct subcategories of calls, that there would be sex differences in call usage, and that BBCs would be more closely associated with aggressive behaviours than USVs. While there were no sex differences in the total number of USVs or BBCs produced by a pair, the use of vocalization subcategories varied by sex, with females uttering more variable USVs and more ‘rattle’ BBCs than males. In conjunction with these differences in vocal behaviour, the sexes also differed in aggression. Across both sexes, variation in aggressive behaviour correlated with variation in the number of ‘squeak’ BBCs and ‘rattle’ BBCs, whereas USVs were not related to aggression. Thus, BBCs constitute a distinct vocalization type with an important role in aggressive communication for hamsters.

Testosterone mediates aggression in many vertebrates. In some species, aggression remains high during the non-breeding season (e.g., winter), when testosterone levels are low. In Siberian hamsters (Phodopus sungorus), we have demonstrated photoperiodic changes in aggression with hamsters housed in short, “winter-like” days displaying significantly more territorial aggression than long-day animals, despite low levels of testosterone. The mechanisms by which photoperiod regulates aggression, however, remain largely unknown. Adrenocortical hormones (e.g., glucocorticoids) have been implicated in mediating seasonal aggression; circulating concentrations of these hormones have been correlated with aggression in some species. The goal of this study was to examine the role of cortisol and glucocorticoid receptors in mediating photoperiodic changes in aggression in male Siberian hamsters. Males were housed in long or short days and treated with either exogenous cortisol or vehicle. Circulating levels of cortisol, adrenal cortisol content, and aggression were quantified. Lastly, photoperiodic effects on glucocorticoid receptor (GR) protein levels were quantified in limbic brain regions associated with aggression, including medial prefrontal cortex, amygdala, and hippocampus. Short-day hamsters were more aggressive than long-day hamsters, however cortisol treatment did not affect aggression. Photoperiod had no effect on serum or adrenal cortisol or GR levels in the brain regions examined. Taken together, these data suggest that increases in cortisol levels do not cause increases associated with short-day aggression, and further that GR protein levels are not associated with photoperiodic changes in aggression. The results of this study contribute to our understanding of the role of adrenocortical steroids in mediating seasonal aggression. J. Exp. Zool. 323A: 331–342, 2015. © 2015 Wiley Periodicals, Inc.

The study of immunity has become an important area of investigation for researchers in a wide range of areas outside the traditional discipline of immunology. For the last several decades, psychoneuroimmunology (PNI) has strived to identify key interactions among the nervous, endocrine and immune systems and behavior. More recently, the field of ecological immunology (ecoimmunology) has been established within the perspectives of ecology and evolutionary biology, sharing with PNI an appreciation of the environmental influences on immune function. The primary goal of ecoimmunology is to understand immune function within a broadly integrative, organismal context, typically from an ultimate, evolutionary perspective. To accomplish this ecoimmunology, like PNI, has become a broadly integrative field of investigation, combining diverse approaches from evolution and ecology to endocrinology and neurobiology. The disciplines of PNI and ecoimmunology, with their unique yet complementary perspectives and methodologies, have much to offer one another. Researchers in both fields, however, remain largely unaware of each other's findings despite attempts at integration. The goal of this review is to share with psychoneuroimmunologists and other mechanistically-oriented researchers some of the core concepts and principles, as well as relevant recent findings, within ecoimmunology with the hope that this information will prove relevant to their own research programs. More broadly, our goal is to attempt to integrate both the proximate and ultimate perspectives offered by PNI and ecoimmunology respectively into a common theoretical framework for understanding neuro-endocrine-immune interactions and behavior in a larger ecological, evolutionary context.

Past research has found menstrual-cycle-related changes in functional immune response; we examined if sexual activity also changed markers of immune defense. We followed 32 naturally cycling women (15 sexually active with a partner ≥ 1 time/week, 17 sexually abstinent for the last four months) over one menstrual cycle. Participants provided serum and saliva samples at menses and ovulation, and additional saliva samples at midfollicular and midluteal phases. At each phase, participants also self-reported symptoms associated with colds, flu, pain, menstrual discomfort, and premenstrual syndrome. We tested saliva and serum for ability to kill Escherichia coli or Candida albicans, and serum for complement protein activity. For serum-mediated pathogen killing, among sexually active women only, there was a significant midcycle decrease in killing of E. coli. For saliva-mediated pathogen killing, among abstinent women only, there was a significant midcycle decrease in killing of E. coli, and midcycle increase in killing of C. albicans. Sexually active women had significantly lower complement activity than abstinent women overall. Finally, both groups reported lower physical symptoms at midcycle and higher symptoms at menses. There may be important differences in immune function between healthy women who are sexually active versus abstinent. Further replication is warranted.

Animals living in temperate climates respond to environmental cues that signal current and future resource availability to ensure that energy resources are available to support reproduction. Siberian hamsters (Phodopus sungorus) undergo robust gonadal regression in short, winter-like photoperiods as well as in response to mild food restriction in intermediate photoperiods. The goal of the present study was to investigate whether leptin is a relevant metabolic signal in regulating gonadal regression in response to diminishing food availability. Adult female hamsters housed in short-day (winter-like) or intermediate (fall-like) photoperiods received either ad libitum access to food or mild food restriction (90% of ad libitum intake) and were treated with either leptin or a vehicle for five weeks in order to determine the ability of leptin to inhibit gonadal regression. At the end of five weeks, vehicle-treated hamsters showed physiological signs associated with ongoing gonadal regression, such as decreases in body mass and food intake, cessation of estrous cycling, and small decreases in reproductive tissue mass. Leptin did not modify changes in body mass, food intake, hormone concentration, or tissue mass, but showed a tendency to support estrous cycling, particularly in response to food restriction in the intermediate photoperiod treatment. Overall, leptin appears to play a minor role in coordinating reproductive responses to multiple environmental cues, at least in the early stages of gonadal regression.

Most free-living animals have finite energy stores that they must allocate to different physiological and behavioral processes. In times of energetic stress, trade-offs in energy allocation among these processes may occur. The manifestation of trade-offs may depend on the source (e.g., glucose, lipids) and severity of energy limitation. In this study, we investigated energetic trade-offs between the reproductive and immune systems by experimentally limiting energy availability to female Siberian hamsters (Phodopus sungorus) with 2-Deoxy-D-glucose, a compound that disrupts cellular utilization of glucose. We observed how glucoprivation at two levels of severity affected allocation to reproduction and immunity. Additionally, we treated a subset of these hamsters with leptin, an adipose hormone that provides a direct signal of available fat stores, in order to determine how increasing this signal of fat stores influences glucoprivation-induced trade-offs. We observed trade-offs between the reproductive and immune systems and that these trade-offs depended on the severity of energy limitation and exogenous leptin signaling. The majority of the animals experiencing mild glucoprivation entered anestrus, whereas leptin treatment restored estrous cycling in these animals. Surprisingly, virtually all animals experiencing more severe glucoprivation maintained normal estrous cycling throughout the experiment; however, exogenous leptin resulted in lower antibody production in this group. These data suggest that variation in these trade-offs may be mediated by shifts between glucose and fatty acid utilization. Collectively, the results of the present study highlight the context-dependent nature of these trade-offs, as trade-offs induced by the same metabolic stressor can manifest differently depending on its intensity.

Immune function is an important component of host fitness, and high investment in immunity should occur when the benefits outweigh the costs, such as when risk of parasitism is high. We sampled two rodent hosts, white-footed mice (Peromyscus leucopus), and prairie voles (Microtus ochrogaster), and their tick, flea, and mite ectoparasites. A bacterial killing assay was used to measure the host's innate immune function. We hypothesized that classes of hosts (species, sexes, or age classes) with overall higher tick burdens would have a higher innate immune function as an evolutionary response to historically greater exposure. We hypothesized a weaker relationship between the fleas and mites and immune function because of high host specificity in fleas and the absence of known vector function in North American mites. Ectoparasites were significantly overdispersed on hosts. In accordance with our hypothesis, Peromyscus that had higher tick burdens also exhibited significantly higher bacterial killing ability compared to Microtus. There was no significant difference in total flea burden between rodent species and no relationship with bacterial killing ability. Microtus had higher burdens of mites in each order than Peromyscus, and female rodents had higher mite burdens than males. The benefits of maintaining high levels of innate immune factors appear to be greater than the energetic costs for Peromyscus compared to Microtus.

Although it is well known that humans and great apes are capable of engaging in consolation, an affiliative behavior directed toward distressed individuals, it has largely been assumed that this form of empathy was restricted to species possessing more complex cognitive functions. Recently, however, Burkett and colleagues (Science, 351, 375-378, 2016) have provided intriguing evidence that consolation behavior may be present in a socially monogamous rodent, the prairie vole. They also provide data to implicate the neuropeptide oxytocin in the regulation of this behavior, which suggests conserved neuroendocrine mechanisms between prairie voles and humans.

The gut microbiome is a diverse, host-specific, and symbiotic bacterial environment that is critical for mammalian survival and exerts a surprising yet powerful influence on brain and behavior. Gut dysbiosis has been linked to a wide range of physical and psychological disorders, including autism spectrum disorders and anxiety, as well as autoimmune and inflammatory disorders. A wealth of information on the effects of dysbiosis on anxiety and depression has been reported in laboratory model systems (e.g., germ-free mice); however, the effects of microbiome disruption on social behaviors (e.g., aggression) of non-model species that may be particularly important in understanding many aspects of physiology and behavior have yet to be fully explored. Here we assessed the sex-specific effects of a broad-spectrum antibiotic on the gut microbiome and its effects on social behaviors in male and female Siberian hamsters (Phodopus sungorus). In Experiment 1, we administered a broad-spectrum antibiotic on a short-term basis and found that antibiotic treatment altered the microbial communities in the gut in male and female hamsters. In Experiment 2, we tested the effects of single versus repeated antibiotic treatment (including a recovery phase) on behavior, and found that two, but not one, treatments caused marked decreases in aggressive behavior, but not other social behaviors, in males; aggression returned to normal levels following recovery. Antibiotic-treated females, in contrast, showed decreased aggression after a single treatment, with all other social behaviors unaffected. Unlike males, female aggression did not return to normal during either recovery period. The present findings demonstrate that modest antibiotic treatment results in marked disruption of the gut microbiome in hamsters, akin to research done in other rodent species and humans. Further, we show that treatment with a broad-spectrum antibiotic, which has dysbiotic effects, also has robust, sex-specific effects on aggression, a critical behavior in the survival and reproductive success of many rodent species.

Objective To examine differences in inflammation markers in sexually active versus abstinent women and observe changes in inflammation markers across the menstrual cycle. Cycle-related immune fluctuations may have evolved to reduce interference with conception. If so, reproductively active (i.e., sexually active) women should show the most variability in cytokine expression. Design Participants provided serum samples at menses and ovulation (from which cytokines were assayed) and saliva samples at menses and during follicular, ovulation, and luteal phases (from which C-reactive protein [CRP] was assayed). Participants self-reported intercourse frequency during the study. Setting Academic research laboratory. Patient(s) Thirty-two healthy, naturally cycling premenopausal women (sexually active, n=15; abstinent, n=17). Intervention(s) Observational study. Main Outcome Measure(s) Levels of proinflammatory cytokines (interleukin-6 [IL-6], interferon γ [IFN-γ], tumor necrosis factor-α [TNF-α]), an anti-inflammatory cytokine (interleukin-4 [IL-4]), and a marker of total inflammation (CRP). Result(s) Sexually active women had higher levels of all of the immune markers measured, including both pro- and anti-inflammatory cytokines, than abstinent women. Relative to sexually active women, abstinent women had less change across the menstrual cycle in levels of CRP. Among sexually active women, higher intercourse frequency predicted greater midcycle decreases in CRP, IL-6, and IFN-γ and midcycle increases in IL-4. Conclusion(s) Sexual activity may stimulate a complex interaction between pro- and anti-inflammatory cytokines that subsequently drives midcycle declines in inflammation.

Chemical communication plays an integral role in social behavior by facilitating social encounters, allowing for the evaluation of social partners, defining territories and advertising information such as species and sex. Odors provide information about the social environment for rodents and other mammals; however, studies identifying chemical compounds and their functions have thus far focused primarily on a few species. In addition, considerably less attention has been focused on how environmental factors and behavioral context alter these compounds during periods of reproductive quiescence. We examined the effects of photoperiod and social context on chemical communication in the seasonally breeding Siberian hamster which displays modest territorial aggression during long "summer-like" days, but increased aggression in short "winter-like" days. We collected urine samples from long- and short-day male hamsters to investigate how photoperiod and subsequent changes in reproductive phenotype alter urinary volatile compound profiles. Next, we identified changes in urinary compounds before and after an aggressive encounter. Male hamsters exhibited a diverse urinary profile across photoperiods; however, long-day reproductive males showed higher levels of individual compounds when compared to short-day non-reproductive males. In addition, individual compounds were altered following an aggressive encounter; some changed only in long days whereas others changed regardless of photoperiod. Further, aggression and circulating levels of testosterone were positively correlated with urinary compounds in long-, but not short-day males. These findings suggest both photoperiod- and aggression-specific physiological regulation of urinary compounds in this species and contribute to a greater understanding of chemical communication more broadly.

This article is part of a Special Issue “Neuroendocrine-Immune Axis in Health and Disease.” Effective immune responses are coordinated by interactions among the nervous, endocrine, and immune systems. Mounting immune, inflammatory, and sickness responses requires substantial energetic investments, and as such, an organism may need to balance energy allocation to these processes with the energetic demands of other competing physiological systems. The metabolic hormone leptin appears to be mediating trade-offs between the immune system and other physiological systems through its actions on immune cells and the brain. Here we review the evidence in both mammalian and non-mammalian vertebrates that suggests leptin is involved in regulating immune responses, inflammation, and sickness behaviors. Leptin has also been implicated in the regulation of seasonal immune responses, including sickness; however, the precise physiological mechanisms remain unclear. Thus, we discuss recent data in support of leptin as a mediator of seasonal sickness responses and provide a theoretical model that outlines how seasonal cues, leptin, and proinflammatory cytokines may interact to coordinate seasonal immune and sickness responses.

Siberian hamsters (Phodopus sungorus) adapt to seasonal environmental conditions with marked changes in body mass, primarily in the form of adiposity. Winter-like conditions (e.g. short days) are sufficient to decrease body mass by approximately 30% in part via reductions in food intake. The neuroendocrine mechanisms responsible for these changes are not well understood, and homeostatic orexigenic/anorexigenic systems of the hypothalamus provide little explanation. We investigated the potential role of endocannabinoids, which are known modulators of appetite and metabolism, in mediating seasonal changes in energy balance. Specifically, we housed hamsters in long or short days for 0, 3, or 9 weeks and measured endocannabinoid levels in the hypothalamus, brainstem, liver and retroperitoneal white adipose tissue (RWAT). An additional group of males housed in short days for 25 weeks were also compared with long-day controls. Following 9 weeks in short days, levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) were significantly elevated in RWAT and reduced in brainstem, although they returned to long-day levels by week 25 in short-day males that had cycled back to summer-like energy balance. Endocannabinoid levels in these tissues correlated significantly with adiposity and change in body mass. No photoperiodic changes were observed in the hypothalamus or liver; however, sex differences in 2-AG levels were found in the liver (males > females). We further tested the effects of CB(1) receptor signalling on ingestive behaviour. Five daily injections of CB(1) antagonist SR141716 significantly reduced food intake and body mass but not food hoarding. Although the CB(1) agonist arachidonyl-2-chloroethylamide did not appreciably affect either ingestive behaviour, body mass was significantly elevated following 2 days of injections. Taken altogether, these findings demonstrate that endocannabinoid levels vary with sex and photoperiod in a site-specific manner, and that altered signalling at CB(1) receptors affects energy balance in Siberian hamsters.

Central oxytocin mediates the acquisition of a filial preference for maternal odour in rat pups, manifested by their huddling preferences. The present study was designed to examine whether maternal care modulates oxytocin concentrations in rat pups and, if so, how different types of maternal contact are associated with the pups' oxytocin concentrations. Pairs of 14-day-old littermates were removed from their home cage for 1 h and then placed with a lactating foster mother for 2 h, or they remained isolated at room temperature. Enzyme immunoassays revealed that maternal care and maternal separation can differentially modulate pups' oxytocin concentrations. Both hypothalamic and serum oxytocin increased during the 1-h separation. Pups placed with a foster mother after the separation maintained the same concentrations in the hypothalamus and serum through the fostering period. By contrast, pups placed with no mother showed a further increase in hypothalamic oxytocin but serum oxytocin decreased. Behavioural analyses revealed that skin-to-skin contact with the mother, but not simple physical contact or maternal licking/grooming, was positively correlated with the pups' hypothalamic oxytocin concentrations. These neuroendocrine data match previous findings showing that skin-to-skin contact with mother facilitates the acquisition of the pups' huddling preference for a maternally-associated odour. Taken together, the present study suggests that maternal skin-to-skin contact stimulates pups' central oxytocin, at the same time as creating the conditions for inducing a preference for maternal odour and establishing a social affiliation in rat pups; the natural schedule of maternal separation and reunion may modulate pups' oxytocin concentrations, providing scaffolding for the acquisition of their filial huddling preference.

Many nontropical species undergo physiological and behavioral adaptations in response to seasonal changes in photoperiod, or day length. In most rodent species, short winter photoperiods reduce testosterone concentrations, which provoke gonadal regression and reduce testosterone-dependent behaviors such as mating and aggression. Seasonally-breeding Siberian hamsters, however, are paradoxically more aggressive in short-days, despite much reduced reproductive activity and testosterone concentrations. Nitric oxide (NO) signaling has been proposed as part of an alternate mechanism underlying this phenomenon. A reduction in neuronal nitric oxide synthase (nNOS), the enzyme responsible for synthesizing NO in the brain, is associated with increased aggression in male short-day hamsters. In the present study, we hypothesized that pharmacological inhibition of nNOS would increase aggressive behavior in long days, but not in short days because nNOS is already reduced. Adult male Siberian hamsters were housed in either long (LD 16:8 h) or short (LD 8:16 h) photoperiods for 8 weeks, then treated with either the selective nNOS inhibitor, 3-bromo-7-nitroindazole (3BrN) or oil vehicle, and subsequently tested for aggression in a resident-intruder test. Treatment with 3BrN increased attack frequency and duration in long days, but had no effect in short days. Short days also reduced testosterone concentrations, without any effect of treatment. These data provide further evidence linking reduced nNOS to elevated short-day aggression and support a role for NO signaling in this phenomenon.

Organisms must distribute sufficient energy among different and often competing physiological systems. This task can become challenging, however, as resources are often limiting, resulting in energetic trade-offs. For example, energetically based trade-offs between the reproductive and immune systems are common across taxa, yet the regulatory mechanisms underlying these trade-offs remain unclear. The adipose tissue hormone leptin is an ideal candidate for the modulation of energetic trade-offs between different physiological systems as this hormone serves as a gage of fat reserves and also modulates a range of physiological activities including the reproductive and immune processes. This article presents a review of the evidence for the role of leptin as a modulator of energetic trade-offs with the immune system and suggests its importance in disease ecology. In addition, we provide a case study of the ornate tree lizard (Urosaurus ornatus), testing whether leptin is involved in mediating a well-documented influence of energy state on the trade-off between reproductive activity and immune function. Overall, the combined results suggest that leptin serves as a proximate endocrine signal of available energy to the immune system, and therefore likely to affect susceptibility to diseases.

1. Over the past decade, there has been a substantial increase in interest in the immune system and the role it plays in the regulation of disease susceptibility, giving rise to the field of eco-immunology. 2. Eco-immunology aims to understand changes in host immune responses in the broader framework of an organism's evolutionary, ecological and life-history contexts. 3. The immune system, however, is complex and multifaceted and can be intimidating for the nonimmunologist interested in incorporating immunological questions into their research. Which immune responses should one measure and what is the biological significance of these measures? 4. The focus of this review is to describe a wide range of eco-immunology techniques, from the simple to the sophisticated, with the goal of providing researchers with a range of options to consider incorporating in their own research programs. 5. These techniques were chosen because they provide relatively straightforward, biologically meaningful assessments of immune function, many of which can be performed across a range of ecological contexts (i.e. field vs. laboratory) and in a wide range of vertebrate animals without relying on species-specific reagents. 6. By incorporating assessments of immune function into their specific research questions, animal ecologists will gain a more comprehensive understanding of organism-environment interactions.

Summary 1. Communication among cells, tissues and organ systems is essential for survival. Vertebrate and invertebrate animals rely primarily on three physiological systems for intra-organismal communication: the nervous, endocrine and immune systems. Rather than acting independently of one another, these systems communicate in an integrated fashion to coordinate suites of species-appropriate physiological and behavioural responses. 2. Our understanding of how these three systems are coordinated remains incomplete, in part because the importance of the immune system as part of this regulatory network has only recently been recognized. In contrast to the well-established integrative approach to the study of the endocrine and nervous systems, the study of immunity has traditionally occurred in relative isolation from other physiological systems. Immunity was typically considered to be largely buffered from environmental perturbations. 3. In the last several decades, however, this simplistic view has changed dramatically; we now know that a wide variety of extrinsic and intrinsic factors can affect immune responses (reviewed in: Ader, Felten & Cohen 2001). This altered perspective has led to the development of new scientific disciplines including psychoneuroimmunology (Ader & Cohen 1981) and ecological immunology (Sheldon & Verhulst 1996). 4. These new research fields focus on the connections among the endocrine, nervous and immune systems. These fields also examine how environmental factors influence interactions among the three systems, and the implications of these interactions for behaviour and host defence. A comparative approach will benefit the search for the adaptive functions of these interactions.

Most animals experience marked changes in reproductive status across development that are regulated by changes in the hypothalamo-pituitary-gonadal (HPG) axis. The upstream mechanisms regulating this axis, however, remain less well understood. The neuropeptide kisspeptin serves as a positive regulator of reproduction; the precise actions of kisspeptin on the HPG axis in animals of differing developmental and seasonal reproductive states, however, remain unresolved. Further, sex differences in response to kisspeptin have not been fully explored. In Experiment 1, we investigated whether sensitivity to a broad range of kisspeptin doses differed in adult male and female Siberian hamsters held on reproductively inhibitory or stimulatory photoperiods. In Experiment 2, we asked whether the response to kisspeptin differed across different stages of reproductive development. Males and females displayed elevated luteinizing hormone LH) in response to kisspeptin; however, the sexes differed in this response, with males showing greater LH responses to kisspeptin than females. Hamsters responded to kisspeptin across all stages of reproductive development, although the magnitude of this response differed between animals of different ages and between the sexes. Males showed significant increases in LH at an earlier development age than females; females also showed blunted LH responses during early adulthood whereas males remained relatively constant in their response to kisspeptin. These findings suggest that reproductively active and inactive hamsters are responsive to kisspeptin, but that the sexes differ in their responsiveness. Collectively, these data provide further insight into the basic actions of kisspeptin in the regulation of reproduction, and provide a potential mechanism for the regulation of differential reproductive responses between the sexes.