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Ecoimmunology utilizes techniques from traditionally laboratory-based disciplines--for example, immunology, genomics, proteomics, neuroendocrinology, and cell biology--to reveal how the immune systems of wild organisms both shape and respond to ecological and evolutionary pressures. Immunological phenotypes are embedded within a mechanistic pathway leading from genotype through physiology to shape higher-order biological phenomena. As such, "mechanisms" in ecoimmunology can refer to both the within-host processes that shape immunological phenotypes, or it can refer the ways in which different immunological phenotypes alter between-organism processes at ecological and evolutionary scales. The mechanistic questions ecoimmunologists can ask, both within-organisms and between-organisms, however, often have been limited by techniques that do not easily transfer to wild, non-model systems. Thus, a major focus in ecoimmunology has been developing and refining the available toolkit. Recently, this toolkit has been expanding at an unprecedented rate, bringing new challenges to choosing techniques and standardizing protocols across studies. By confronting these challenges, we will be able to enhance ecoimmunological inquiries into the physiological basis of life-history trade-offs; the development of low-cost biomarkers for susceptibility to disease; and the investigation of the ecophysiological underpinnings of disease ecology, behavior, and the coevolution of host-parasite systems. The technical advances in, and crossover technologies from, disciplines associated with ecoimmunology and how these advances can help us understand the mechanistic basis of immunological variability in wild species were the focus of the symposium, Methods and Mechanisms in Ecoimmunology.

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 (malesfemales). 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.

Kisspeptin, a neuropeptide product of the KiSS-1 gene, has recently been implicated in the regulation of seasonal breeding in a number of species, including Siberian hamsters. In this species, kisspeptin expression is reduced in the anteroventral periventricular nucleus (AVPV) following exposure to inhibitory day lengths, and exogenous kisspeptin activates the reproductive neuroendocrine axis of reproductively quiescent animals. Because sex steroids can impact kisspeptin expression, it is unclear whether changes in kisspeptin occur in direct response to photoperiodic cues or secondarily in response to changes in sex steroid concentrations resulting from the transition to reproductive quiescence. The present study aimed to assess the relative contributions of photoperiod and testosterone in regulating kisspeptin expression in Siberian hamsters. Animals housed in long or short day lengths for 8 weeks were either castrated or received sham surgeries. Half of the hamsters in each photoperiod were given testosterone to mimic long-day sex steroid concentrations. The results obtained indicate that kisspeptin neurones in the AVPV and arcuate nuclei were influenced by both photoperiod and testosterone. In the AVPV, removal of testosterone or exposure to inhibitory day lengths led to a marked reduction in kisspeptin-immunoreactive cells, and testosterone treatment increased cell numbers across conditions. Importantly, long-day castrates exhibited significantly more kisspeptin cells than short-day castrates or intact short-day animals with empty capsules, suggesting the influences of photoperiod, independent of gonadal steroids. In general, the opposite pattern emerged for the arcuate nuclei. Collectively, these data suggest a role for both gonadal-dependent and independent (i.e. photoperiodic) mechanisms regulating seasonal changes in kisspeptin expression in Siberian hamsters.

Siberian hamsters (Phodopus sungorus) rely on photoperiod as a primary cue to coordinate seasonally appropriate changes in physiology and behaviour. Among these seasonal changes is reduced immune function in short 'winter-like' days, compared to long 'summer-like' days. Previous evidence suggests that immune function is regulated, in part, by the sympathoadrenal system. The precise role of the sympathoadrenal system in regulating photoperiodic changes in immune function, however, remains unspecified. The goal of the present study was to examine the differential contributions of direct sympathetic innervation of immune target tissue, as well as adrenal medullary catecholamines, to photoperiodic changes in immune function in male Siberian hamsters. In Experiment 1, hamsters underwent either bilateral surgical removal of the adrenal medulla (ADMEDx), or sham surgeries, and were maintained in long (LD 16 : 8) or short days (LD 8 : 16). In Experiment 2, hamsters received either surgical denervation of the spleen, or sham surgeries, and were then housed in long or short days. Serum anti-KLH IgG concentrations and splenic norepinephrine (NE) content were determined in both experiments. Short-day hamsters had reduced humoral immunity compared to long-day hamsters. ADMEDx reduced immune function, but only in long-day hamsters. In contrast, splenic denervation reduced humoral immunity, but only in short-day hamsters. Splenic NE content was increased in short days and by ADMEDx. NE content was markedly reduced in denervated hamsters compared to sham-operated hamsters. Collectively, these results suggest that the sympathoadrenal system is associated with photoperiodic changes in immune function.

Many adaptations have evolved in small mammals to maximize survival during winter. One such coping tactic in many species is an alteration of immune function in advance of the stressful conditions of winter. Leptin is a hormone produced by adipose tissue, and in addition to its central role in energy metabolism, leptin mediates the interactions among energy allocation, immune function, and reproduction. To examine this interaction further, exogenous leptin was administered for 2 weeks via osmotic minipumps to Siberian hamsters (Phodopus sungorus) housed in long or short days for a total of 12 weeks. Short-day hamsters displayed the expected reductions in humoral immune function, body mass, fat mass, and food intake. In Exp 1, exogenous leptin counteracted the reduction in food intake and the suppression of immune function in short days. In Exp 2, when the leptin-induced increase in food intake in short-day hamsters was prevented, leptin did not enhance immune function. In most of the measured fat pads and body mass, leptin had no effect in long days. In sum, leptin administered to short-day animals caused them to respond, in many cases, like long-day animals. Taken together, these data suggest that leptin acts indirectly to mediate energy allocation to humoral immune function. Additionally, leptin appears to act differentially, according to photoperiod, to regulate both immune and energetic parameters.

Recent evidence suggests that endogenous estrogens or hormone replacement therapy can ameliorate brain damage from experimental stroke. Protective mechanisms involve enhanced cerebral vasodilation during ischemic stress as well as direct preservation of neuronal viability. We hypothesized that if the intracellular estrogen receptor subtype-alpha (ERalpha) is important to estrogen's signaling in the ischemic brain, then ERalpha-deficient (knockout) (ERalphaKO) female mice would sustain exaggerated cerebral infarction damage after middle cerebral artery occlusion.The histopathology of cresyl violet-stained tissues was evaluated after reversible middle cerebral artery occlusion (2 hours, followed by 22 hours of reperfusion) in ERalphaKO transgenic and wild-type (WT) mice (C57BL/6J background strain). End-ischemic cerebral blood flow mapping was obtained from additional female murine cohorts by using [(14)C]iodoantipyrine autoradiography.Total hemispheric tissue damage was not altered by ERalpha deficiency in female mice: 51.9+/-10.6 mm(3) in ERalphaKO versus 60.5+/-5.0 mm(3) in WT. Striatal infarction was equivalent, 12.2+/-1.7 mm(3) in ERalphaKO and 13.4+/-1.0 mm(3) in WT mice, but cortical infarction was paradoxically smaller relative to that of the WT (20.7+/-4.5 mm(3) in ERalphaKO versus 30.6+/-4.1 mm(3) in WT). Intraocclusion blood flow to the parietal cortex was higher in ERalphaKO than in WT mice, likely accounting for the reduced infarction in this anatomic area. There were no differences in stroke outcomes by region or genotype in male animals.Loss of ERalpha does not enhance tissue damage in the female animal, suggesting that estrogen inhibits brain injury by mechanisms that do not depend on activation of this receptor subtype.

Nontropical rodents rely on environmental factors to restrict breeding to a specific time of the year. Among these factors, photoperiod appears to be the primary environmental cue used for predicting optimal breeding conditions. The purpose of the present study was to characterize reproductive function, as well as photoperiodic and social responsiveness in male Peromyscus aztecus, which occupy low-latitude, high-altitude habitats. In experiment 1, adult male P. aztecus were individually housed in either long (16L:8D) or short days (8L:16D) for 10 wk. Short-day mice did not differ from long-day mice on any reproductive or nonreproductive parameter. Comparisons to related Peromyscus species suggested that relative reproductive organ size and function were reduced in both long- and short-day males. Because ad libitum food and water were available, we reasoned that males in both photoperiodic conditions lacked social stimuli. To test this hypothesis, adult male P. aztecus were housed in long days either individually or with a female conspecific in experiment 2. Mice housed with females had significantly larger relative paired testes and epididymal masses, and higher testicular sperm counts and serum testosterone levels compared to those of individually housed mice. Taken together, these results suggest that social factors may play a more prominent role than photoperiod in stimulating reproductive development in laboratory-housed P. aztecus. These results are consistent with the results found for other low-latitude rodent species and suggest that P. aztecus uses a flexible rather than obligatory breeding strategy.

BACKGROUND: Mice with targeted disruption of the gene for the neuronal isoform of nitric oxide synthase (nNOS) display exaggerated aggression. Behavioral studies of mice with targeted gene deletions suffer from the criticism that the gene product is missing not only during the assessment period but also throughout development when critical processes, including activation of compensatory mechanisms, may be affected. To address this criticism, we have assessed aggressive behavior in mice treated with a specific pharmacological inhibitor of nNOS. MATERIALS AND METHODS: Aggressive behavior, as well as brain citrulline levels, were monitored in adult male mice after treatment with a specific nNOS inhibitor, 7-nitroindazole (7-NI) (50 mg/kg i.p.), which is known to reduce NOS activity in brain homogenates by > 90%. As controls, animals were treated with a related indazole, 3-indazolinone (3-I) (50 mg/kg i.p.) that does not affect nNOS or with on oil vehicle. RESULTS: Mice treated with 7-NI displayed substantially increased aggression as compared with oil- or 3-I-injected animals when tested in two different models of aggression. Drug treatment did not affect nonspecific locomotor activities or body temperature. Immunohistochemical staining for citrulline in the brain revealed a dramatic reduction in 7-NI-treated animals. CONCLUSIONS: 7-NI augmented aggression in WT mice to levels displayed by nNOS- mice, strongly implying that nNOS is a major mediator of aggression. NOS inhibitors may have therapeutic roles in inflammatory, cardiovascular, and neurologic diseases. The substantial aggressive behavior soon after administration of an nNOS inhibitor raises concerns about adverse behavioral sequelae of such pharmacological agents.