Does testosterone underly the interplay between male traits and territorial behavior in neotropical poison frogs?

The ability of individual animals to defend a territory as well as various phenotypic and behavioral traits may be targets of sexual selection used by males to evaluate their competitors or by females to choose males. A frequent question in animal behavior is whether male traits and characteristics of their territory are correlated and what are the mechanisms that may mediate such associations when they exist. Because hormones link phenotype to behavior, by studying the role of testosterone in territoriality one may come closer to understanding the mechanisms mediating correlations or lack thereof between characteristics of territories and of males. We evaluated whether variation in characteristics of territories (size and quality) are correlated with variation in morphology, coloration, testosterone, heterozygosity, and calls in two species of poison frogs. The Amazonian frog Allobates aff. trilineatus exhibits male care and defends territories only during the breeding season, while the endangered frog Oophaga lehmanni displays maternal care and defends territories throughout the year. We found that morphological traits (body length, weight, thigh size), call activity, and testosterone levels correlated with size and various indicators of quality of the territory. However, the direction of these correlations (whether positive or negative) and which specific morphological, acoustic traits or testosterone level variables covaried depended on the species. Our findings highlight an endocrine pathway as part of the physiological machinery that may underlie the interplay between male traits and territorial behavior. We were able to identify some male traits related to territory attributes, but whether females choose males based on these traits requires further research.

Cooperative breeding alters physiological and behavioral responses to habitat fragmentation.

Animals respond to habitat alteration with changes in their behavior and physiology. These changes determine individual performance and thus precede changes in population size. They are therefore hypothesized to provide important insights into how animals cope with environmental change. Here, we investigated physiological and behavioral responses of a cooperatively breeding bird, the placid greenbul (Phyllastrephus placidus), in a severely fragmented tropical biodiversity hotspot and combined these data with remotely sensed (LiDAR) environmental data. We found that individuals had increased glucocorticoid hormone levels when breeding in territories with low native canopy cover or located within small fragments. However, when breeding with the help of subordinates, breeders in low quality territories had similar glucocorticoid levels as those in higher quality territories. Our study shows that sociality may impact how well animals cope with environmental change and contributes to our understanding of the role of glucocorticoid physiology and behavior in response to anthropogenic change.

Water-borne testosterone levels predict exploratory tendency in male poison frogs.

Hormones play a fundamental role in mediating social behaviors of animals. However, it is less well understood to what extent behavioral variation between individuals can be attributed to variation in underlying hormonal profiles. The goal of the present study was to infer if individual androgen levels, and/or the modulation thereof, can explain among-individual variation in aggressiveness, boldness and exploration. We used as a model the dart-poison frog Allobates femoralis and took repeated non-invasive water-borne hormonal samples of individual males before (baseline) and after (experimental) a series of behavioral tests for assessing aggression, boldness, and exploratory tendency. Our results show that androgen levels in A. femoralis are quite stable across the reproductive season. Repeatability in wbT baseline levels was high, while time of day, age of the frog, and trial order did not show any significant impact on measured wbT levels. In general, experimental wbT levels after behavioral tests were lower compared to the respective baseline levels. However, we identified two different patterns with regard to androgen modulation in response to behavioral testing: individuals with low baseline wbT tended to have increased wbT levels after the behavioral testing, while individuals with comparatively high baseline wbT levels rather showed a decrease in hormonal levels after testing. Our results also suggest that baseline wbT levels are linked to the personality trait exploration, and that androgen modulation is linked to boldness in A. femoralis males. These results show that differences in hormonal profiles and/or hormonal modulation in response to social challenges can indeed explain among-individual differences in behavioral traits.

Alteration of the temporal association between courtship audio and visual components affects female sexual response.

Some multimodal signals-that is, occurring in more than one sensory modality-appear to carry additional information which is not present when component signals are presented separately. To understand the function of male ring dove's (Streptopelia risoria) multimodal courtship, we used audiovisual playback of male displays to investigate female response to stimuli differing in their audiovisual timing. From natural courtship recordings, we created a shifted stimulus where audio was shifted relative to video by a fixed value and a jittered stimulus, where each call was moved randomly along the visual channel. We presented 3 groups of females with the same stimulus type, that is, control, shifted, and jittered, for 7 days. We recorded their behavior and assessed pre- and post-test blood estradiol concentration. We found that playback exposure increased estradiol levels, confirming that this technique can be efficiently used to study doves' sexual communication. Additionally, chasing behavior (indicating sexual stimulation) increased over experimental days only in the control condition, suggesting a role of multimodal timing on female response. This stresses the importance of signal configuration in multimodal communication, as additional information is likely to be contained in the temporal association between modalities.

Androgen responsiveness to simulated territorial intrusions in Allobates femoralis males: Evidence supporting the challenge hypothesis in a territorial frog.

Territoriality has been widely described across many animal taxa, where the acquisition and defence of a territory are critical for the fitness of an individual. Extensive evidence suggests that androgens are involved in the modulation of territorial behaviours in male vertebrates. Short-term increase of androgen following a territorial encounter appears to favour the outcome of a challenge. The "Challenge Hypothesis" proposed by Wingfield and colleagues outlines the existence of a positive feedback relationship between androgen and social challenges (e.g., territorial intrusions) in male vertebrates. Here we tested the challenge hypothesis in the highly territorial poison frog, Allobates femoralis, in its natural habitat by exposing males to simulated territorial intrusions in the form of acoustic playbacks. We quantified repeatedly androgen concentrations of individual males via a non-invasive water-borne sampling approach. Our results show that A. femoralis males exhibited a positive behavioural and androgenic response after being confronted to simulated territorial intrusions, providing support for the Challenge Hypothesis in a territorial frog.

Telomere shortening is associated with corticosterone stress response in adult barn swallows.

When vertebrates face stressful events, the hypothalamic-pituitary-adrenal (HPA) axis is activated, generating a rapid increase in circulating glucocorticoid (GC) stress hormones followed by a return to baseline levels. However, repeated activation of HPA axis may lead to increase in oxidative stress. One target of oxidative stress is telomeres, nucleoprotein complexes at the end of chromosomes that shorten at each cell division. The susceptibility of telomeres to oxidizing molecules has led to the hypothesis that increased GC levels boost telomere shortening, but studies on this link are scanty. We studied if, in barn swallows Hirundo rustica, changes in adult erythrocyte telomere length between 2 consecutive breeding seasons are related to corticosterone (CORT) (the main avian GC) stress response induced by a standard capture-restraint protocol. Within-individual telomere length did not significantly change between consecutive breeding seasons. Second-year individuals showed the highest increase in circulating CORT concentrations following restraint. Moreover, we found a decline in female stress response along the breeding season. In addition, telomere shortening covaried with the stress response: a delayed activation of the negative feedback loop terminating the stress response was associated with greater telomere attrition. Hence, among-individual variation in stress response may affect telomere dynamics.

Measuring salivary mesotocin in birds - Seasonal differences in ravens' peripheral mesotocin levels.

Oxytocin is involved in a broad array of social behaviours. While saliva has been used regularly to investigate the role of oxytocin in social behaviour of mammal species, so far, to our knowledge, no-one has tried to measure its homolog, mesotocin, in birds' saliva. Therefore, in this study we measured salivary mesotocin in common ravens (Corvus corax), and subsequently explored its link to three aspects of raven sociality. We trained ravens (n = 13) to voluntarily provide saliva samples and analysed salivary mesotocin with a commercial oxytocin enzyme-immunoassay kit, also suitable for mesotocin. After testing parallelism and recovery, we investigated the effect of bonding status, sex and season on mesotocin levels. We found that mesotocin was significantly more likely to be detected in samples taken during the breeding season (spring) than during the mating season (winter). In those samples in which mesotocin was detected, concentrations were also significantly higher during the breeding than during the mating season. In contrast, bonding status and sex were not found to relate to mesotocin detectability and concentrations. The seasonal differences in mesotocin correspond to behavioral patterns known to be associated with mesotocin/oxytocin, with ravens showing much more aggression during the mating season while being more tolerant of conspecifics in the breeding season. We show for the first time that saliva samples can be useful for the non-invasive determination of hormone levels in birds. However, the rate of successfully analysed samples was very low, and collection and analysis methods will benefit from further improvements.

Exposure to artificial light at night alters innate immune response in wild great tit nestlings.

The large-scale impact of urbanization on wildlife is rather well documented; however, the mechanisms underlying the effects of urban environments on animal physiology and behaviour are still poorly understood. Here, we focused on one major urban pollutant - artificial light at night (ALAN) - and its effects on the capacity to mount an innate immune response in wild great tit (Parus major) nestlings. Exposure to ALAN alters circadian rhythms of physiological processes, by disrupting the nocturnal production of the hormone melatonin. Nestlings were exposed to a light source emitting 3 lx for seven consecutive nights. Subsequently, nestlings were immune challenged with a lipopolysaccharide injection, and we measured haptoglobin and nitric oxide levels pre- and post-injection. Both haptoglobin and nitric oxide are important markers for innate immune function. We found that ALAN exposure altered the innate immune response, with nestlings exposed to ALAN having lower haptoglobin and higher nitric oxide levels after the immune challenge compared with dark-night nestlings. Unexpectedly, nitric oxide levels were overall lower after the immune challenge than before. These effects were probably mediated by melatonin, as ALAN-treated birds had on average 49% lower melatonin levels than the dark-night birds. ALAN exposure did not have any clear effects on nestling growth. This study provides a potential physiological mechanism underlying the documented differences in immune function between urban and rural birds observed in other studies. Moreover, it gives evidence that ALAN exposure affects nestling physiology, potentially causing long-term effects on physiology and behaviour, which ultimately can affect their fitness.

Temporary caging results in reduced levels of circulating melatonin in migratory robins.

The hormone melatonin, a main component of the avian circadian system, plays an important role in the physiological transitions that accompany activation of the migratory phenotype in passerine birds. Most small passerines migrate at night when circulating concentrations of melatonin are elevated. Previous work measured nocturnal melatonin levels of migratory birds only in captive animals, because free-living individuals are usually caught in the daytime. In this study, we compared nocturnal melatonin levels of European robins (Erithacus rubecula) caught during the day and held in cages overnight with those of birds that were caught at night and sampled immediately. We found that circulating melatonin at night was lower in birds held in cages compared with birds that were actively migrating. This result suggests that temporary caging affects the melatonin system and that, in nature, melatonin levels could be generally higher than those previously described by studies on captive birds.

3ß-HSD expression in the CNS of a manakin and finch.

The prohormone, dehydroepiandrosterone (DHEA) circulates in vertebrate blood with the potential for actions on target tissues including the central nervous system (CNS). Many actions of DHEA require its conversion into more active products, some of which are catalyzed by the enzyme 3ß-hydroxysteroid-dehydrogenase/isomerase (3ß-HSD). Studies of birds show both expression and activity of 3ß-HSD in brain and its importance in regulating social behavior. In oscine songbirds, 3ß-HSD is expressed at reasonably high levels in brain, possibly linked to their complex neural circuitry controlling song. Studies also indicate that circulating DHEA may serve as the substrate for neural 3ß-HSD to produce active steroids that activate behavior during non-breeding seasons. In the golden-collared manakin (Manacus vitellinus), a sub-oscine bird, low levels of courtship behavior are displayed by males when circulating testosterone levels are basal. Therefore, we asked whether DHEA circulates in blood of manakins and whether the brain expresses 3ß-HSD mRNA. Given that the spinal cord is a target of androgens and likely important in regulating acrobatic movements, we also examined expression of this enzyme in the manakin spinal cord. For comparison, we examined expression levels with those of an oscine songbird, the zebra finch (Taeniopygia guttata), a species in which brain, but not spinal cord, 3ß-HSD has been well studied. DHEA was detected in manakin blood at levels similar to that seen in other species. As described previously, 3ß-HSD was expressed in all zebra finch brain regions examined. By contrast, expression of 3ß-HSD was only detected in the manakin hypothalamus where levels were greater than zebra finches. In spinal cord, 3ß-HSD was detected in some but not all regions in both species. These data point to species differences and indicate that manakins have the substrate and neural machinery to convert circulating DHEA into potentially active androgens and/or estrogens.

Measuring short-term stress in birds: Comparing different endpoints of the endocrine-immune interface.

Stress is a collective term for certain conditions and sequences of physiological events enabling living organisms to overcome unpredictable and uncontrollable situations. The context-dependent nature, multidimensional course and large individual variability make stress responses difficult to measure. In avian species, a plethora of studies on short-term stress responses have been conducted by measuring the corticosteroid response to a standardized stress protocol. Here we aimed to test the viability of the leukocyte coping capacity (LCC), measuring oxygen radical production by leukocytes, to assess short-term stress in birds. We collected blood samples from captive house sparrows (Passer domesticus) in the two seasons of winter and spring, right after capture and 30min thereafter. In order to assess the overall physiological stress response to a standardized stressor, i.e. handling and temporary constraint, we measured LCC and additionally combined it with measures of total circulating glucocorticoids (GCs) and oxidative stress. All three methodologies detected significant changes due to the stressor albeit they were not correlated with each other and revealed different information. There was no significant change in oxidative stress levels between the two time points although the amount of relative oxidative damage as well as the anti-oxidative capacity changed significantly. We observed a significant seasonal difference in GC stress response with no difference between sexes. On the contrary, LCC measures revealed with a high individual consistency, that individuals experienced a similar magnitude of stress in both seasons with a significant difference between sexes. Total GC-levels have to be interpreted with caution regarding the assessment of short-term stress reactions. We therefore suggest to supplementary combine classical approaches for measuring stress with the immunological tool of LCC. Our results reveal LCC as a strong and reliable tool to assess short-term stress in captive house sparrows and as promising for other bird species. Collectively the study highlights the necessity to incorporate a range of physiological systems and their endpoints to measure and to assess stress reactions effectively.

Effects of human disturbance on cave-nesting seabirds: the case of the storm petrel.

Human disturbance is an important stress factor with potentially strong impact on breeding activity in animals. The consequences can be extinction of the breeding population, because disturbed animals might desert their breeding area and find no suitable substitute area. In this study, we investigated the effects of anthropogenic disturbance on a breeding population of Mediterranean storm petrels. Seabirds are increasingly used as bio-indicators for sea environmental parameters, because they are very sensitive to changing conditions. Burrowing or cave-nesting species may be particularly susceptible to human disturbance because their direct contact with humans is usually minimal or absent. First, we compared two different populations (exposed or not exposed to human disturbance) for their individual stress response to a standardized stressor (handling and keeping in a cloth bag). Second, we compared the two sub-colonies for their population-level stress response. Third, we tested experimentally whether sub-colonies of storm petrels exposed to tourism have physiological adaptations to anthropogenic disturbances. Our results indicate that storm petrels may be habituated to moderate disturbance associated with boat traffic close to the colony.

Non-migratory stonechats show seasonal changes in the hormonal regulation of non-seasonal territorial aggression.

In many birds and mammals, male territorial aggression is modulated by elevated circulating concentrations of the steroid hormone testosterone (T) during the breeding season. However, many species are territorial also during the non-breeding season, when plasma T levels are basal. The endocrine control of non-breeding territorial aggression differs considerably between species, and previous studies on wintering birds suggest differences between migratory and resident species. We investigated the endocrine modulation of territorial aggression during the breeding and non-breeding season in a resident population of European stonechats (Saxicola torquata rubicola). We recorded the aggressive response to a simulated territorial intrusion in spring and winter. Then, we compared the territorial aggression between seasons and in an experiment in which we blocked the androgenic and estrogenic action of T. We found no difference in the aggressive response between the breeding and the non-breeding season. However, similarly to what is found in migratory stonechats, the hormonal treatment decreased aggressive behaviors in resident males in the breeding season, whereas no effects were recorded in the non-breeding season. When we compared the aggressive responses of untreated birds with those obtained from migratory populations in a previous study, we found that territorial aggression of resident males was lower than that of migratory males during the breeding season. Our results show that in a resident population of stonechats T and/or its metabolites control territorial aggression in the breeding but not in the non-breeding season. In addition, our study supports the hypothesis that migratory status does modulate the intensity of aggressive behavior.

Low sex steroids, high steroid receptors: Increasing the sensitivity of the nonreproductive brain.

Male aggressive behavior is generally regulated by testosterone (T). In most temperate breeding males, aggressive behavior is only expressed during the reproductive period. At this time circulating T concentrations, brain steroid receptors, and steroid metabolic enzymes are elevated in many species relative to the nonreproductive period. Many tropical birds, however, display aggressive behavior both during the breeding and the nonbreeding season, but plasma levels of T can remain low throughout the year and show little seasonal fluctuation. Studies on the year-round territorial spotted antbird (Hylophylax n. naevioides) suggest that T nevertheless regulates aggressive behavior in both the breeding and nonbreeding season. We hypothesize that to regulate aggressive behaviors during the nonbreeding season, when T is at its minimum, male spotted antbirds increase brain sensitivity to steroids. This can be achieved by locally up-regulating androgen receptors (ARs), estrogen receptors (ERs), or the enzyme aromatase (AROM) that converts T into estradiol. We therefore compared mRNA expression of AR, ERalpha, and AROM in free- living male spotted antbirds across reproductive and nonreproductive seasons in two brain regions known to regulate both reproductive and aggressive behaviors. mRNA expression of ERalpha in the preoptic area and AR in the nucleus taeniae were elevated in male spotted antbirds during the nonbreeding season when circulating T concentrations were low. This unusual seasonal receptor regulation may represent a means for the year-round regulation of vertebrate aggressive behavior via steroids by increasing the brain's sensitivity to sex steroids during the nonbreeding season.

Developmental exposure to xenoestrogen enhances spatial learning in male rats.

Steroid hormones have profound effects on the development and function of the nervous system. Environmental estrogens or xenoestrogens are manmade or are natural compounds, which mimics the action of estrogen hormones. The experimental evidence for impairment of cognitive functions in humans and mammals following exposure to xenoestrogens has been fiercely debated. The strongest arguments against such studies have been that the route, time course, and intensity of exposure did not simulate environmental exposure, and that the chemicals tested have additional, non-estrogenic toxic effects, hindering a generalization of actual "xenoestrogenic" effects. Here we show that an environmental-like exposure to the pure estrogen, 17alpha-ethynylestradiol (EE2) during development enhances spatial learning abilities in adult male Sprague-Dawley rats. To simulate an environmental exposure, we used a very low dose (4 ng/kg/day) of EE2 equivalent to concentrations measured in European and US streams which was given orally with a non-invasive method, and we extended the treatment for the entire course of development, from conception to puberty. The animals were tested in a Morris water maze protocol at 6 months of age. Male rats treated with EE2 during development showed a faster learning during the training phase, and remembered better the position of the hidden platform in the short term. Our study demonstrates that actual levels of exposure to xenoestrogens can permanently alter cognitive abilities of a mammalian species.

Androgen and the elaborate courtship behavior of a tropical lekking bird.

In most bird species, male courtship behavior is controlled by testosterone (T) and its metabolites. In species breeding in temperate and arctic regions T circulates at high levels during a relatively short courtship period because high levels of T can be costly in terms of immunocompetence and parental care. Few studies have investigated androgen modulation of courtship behavior in tropical birds. Male golden-collared manakins (Manacus vitellinus) aggregate in leks for several months and perform spectacular, acrobatic courtship displays. Here we examined whether T is elevated in golden-collared manakins during the displaying period and if courtship behavior is modulated by androgen action on androgen receptors. We measured T levels in displaying males at the beginning of the breeding season and again, one month later. In addition, both wild and captive males were treated with the anti-androgen, flutamide, and their courtship behavior was recorded for several weeks. T levels were relatively high shortly after leks were established but decreased substantially a month later, even though the amount of courtship did not change. Flutamide reduced male courtship activity for one week, but display behavior then increased after two weeks of flutamide treatment. Our studies show that androgens modulate male manakin courtship, but the amount of courtship is not directly correlated with the concentration of circulating T. These results suggest that the relationships between androgen and courtship might differ between tropical and temperate birds.

The hormonal response of female European Stonechats to a territorial intrusion: the role of the male partner.

In many bird species, the female participates in defending a pair's breeding territory, however, the endocrine control mechanism of female aggressive behavior is largely unknown. The general statement that androgens are involved in the regulation of aggressive behavior is based on studies conducted only in males. Here, we tested whether paired female stonechats show a hormonal response to a simulated male territorial intruder. Since in males of territorial bird species androgen levels usually increase following a male-male encounter, we measured androgen-levels before and after a simulated male intrusion. In addition, we measured estradiol, the main gonadal hormone in females, and corticosterone, a stress hormone. The results show that a male intruder does not affect any of the measured hormones in females. In a second experiment, we also tested whether the endocrine state of the male partner affects the hormonal response of females to a male intruder by comparing the hormonal response of females paired with pharmacologically castrated males and females paired with control males. Females paired with pharmacologically castrated males had lower corticosterone levels both before and after the intrusion than females paired with control males. Additionally, in both groups, female corticosterone levels were increased following a male intrusion. We suggest that the differences found between females paired with pharmacologically castrated males and females paired with control males are due to differences in intra-pair interactions.

Seasonal differences in the hormonal control of territorial aggression in free-living European stonechats.

In birds, territorial aggression during the breeding season is regulated by testosterone (T). However, many bird species also express aggressive behavior during the nonbreeding season, when plasma levels of T are low. It has been suggested that during this period estrogens might play a major role in regulating territorial aggression. In the present study we compared the effects of simultaneous blockage of androgenic and estrogenic actions on territorial aggression during the breeding and nonbreeding seasons in free-living male European stonechats (Saxicola torquata rubicola). European stonechats are of particular interest since they establish territories and form pairs during both the breeding and the nonbreeding seasons. Thus territorial aggression and its endocrine control can be compared between reproductive and non-reproductive contexts. Inhibition of androgenic and estrogenic actions by simultaneous application of Flutamide and ATD reduced territorial aggression during the breeding season, but not during the nonbreeding season. Our results show that androgens and/or estrogens are involved in the endocrine control of territorial aggression in stonechats only in a reproductive context, but not in a non-reproductive one.