Adult Provisioning Influences Nestling Corticosterone Levels in Florida Scrub Jays (Aphelocoma coerulescens).

We studied Florida scrub jay (Aphelocoma coerulescens) nestlings to examine the relationship between parental feeding rates and levels of corticosterone (CORT), a metabolic and stress-related steroid hormone hypothesized to play a role in mediating begging behavior. It has been documented that nutritional deficiency results in increased glucocorticoid levels in nestling birds. Further, previous studies have found that CORT levels of Florida scrub jay nestlings are negatively correlated with parental nest attendance and provisioning rates; however, the behavioral observations were made several days before the collection of samples to assess CORT levels. Few studies have investigated whether experience immediately before sampling impacts nestling glucocorticoid levels, especially in a free-living species. By monitoring parental activity at the nest before sample collection, we found that nestling CORT levels varied as a function of parental provisioning rate and the time since their last feed. However, counter to our predictions, higher provisioning rates and more recent feedings were associated with higher CORT levels in nestlings rather than lower CORT levels. These results suggest that some aspect of parental provisioning results in increased CORT levels in nestling Florida scrub jays.

Influence of corticosterone treatment on nestling begging in Florida scrub-jays (Aphelocoma coerulescens).

Altricial young are dependent on adults for protection and food, and they display nutritional need by begging to elicit feeding from parents. Begging at high levels can be energetically expensive and attract predators; thus, an individual must balance its nutritional needs with these potential costs. Further, because a parent is limited in the amount of food it can provide, begging can contribute to both parent-offspring conflict and sibling-sibling competition. Many extrinsic and intrinsic factors may contribute to begging behavior. One intrinsic factor of interest is corticosterone (CORT), a metabolic hormone hypothesized to play a role in regulating a nestling's begging behavior. We investigated the hypothesis that increased exposure to CORT influences nestling begging behavior in an altricial species, the Florida scrub-jay (Aphelocoma coerulescens). We treated one nestling per treatment nest with a twice-daily dose of exogenous hormone via a CORT-injected waxworm, whereas a second individual received a vehicle-injected waxworm. We monitored individual nestling and adult behavior at all nests with the use of high-definition video cameras on several days during treatment. We found no difference in begging rate between CORT fed and vehicle fed nestlings within a treatment nest. Further, to determine whether CORT treatment had indirect effects on the entire brood, we monitored additional nests, in which nestlings were not manipulated. When treatment and controls were compared, overall begging rates of nestlings in treatment nests were greater than those in control nests. This result suggests that CORT treatment of an individual altered its behavior, as well as that of its siblings.

Stress-responsiveness influences baseline glucocorticoid levels: Revisiting the under 3min sampling rule.

Plasma glucocorticoid (CORT) levels collected within 3min of capture are commonly believed to reflect pre-stressor, baseline CORT levels. Differences in these "baseline" values are often interpreted as reflecting differences in health, or the amount of social and environmental stress recently experienced by an individual. When interpreting "baseline" values it is generally assumed that any effect of capture-and-handling during the initial sampling period is small enough and consistent enough among individuals to not obscure pre-capture differences in CORT levels. However, plasma CORT increases in less than 3min post-capture in many free-living, endothermic species in which timing has been assessed. In addition, the rate of CORT secretion and the maximum level attained (i.e., the degree of stress-responsiveness) during a severe stressor often differs among individuals of the same species. In Florida scrub-jays (Aphelocoma coerulescens), an individual's stress-responsiveness during a 30min post-capture stressor is correlated with CORT levels in samples collected within 1.5min of capture, suggesting there is an intrinsic connection between stress-responsiveness and pre-capture CORT levels. Although differences in stress-responsiveness accounted for just 11% of the variance in these samples, on average, higher stress-responsive jays (top third of individuals) had baseline values twice that of lower stress-responsive jays (bottom third). Further, plasma CORT levels begin to increase around 2min post-capture in this species, but the rate of increase between 2 and 3min differs markedly with CORT increasing more rapidly in jays with higher stress-responsiveness. Together, these data indicate that baseline CORT values can be influenced by an individual's stress response phenotype and the differences due to stress-responsiveness can be exaggerated during sample collection. In some cases, the effects of differences in stress-responsiveness and the increase in CORT during sample collection could obscure, or supersede, differences in pre-capture plasma CORT levels that are caused by extrinsic factors.

Testosterone Mediates Seasonal Growth of the Song Control Nuclei in a Tropical Bird.

In mid- to high-latitude songbirds, seasonal reproduction is stimulated by increasing day length accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study, we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment, we treated captive male birds with T or blank implants during the nonbreeding season. In a second experiment, we treated captive male birds with either blank implants, T-filled implants, T with flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of the brain areas high vocal center (HVC), Area X, and robust nucleus of the arcopallium (RA) were measured along with singing behavior. In summary, T stimulated growth of HVC and RA, and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by T treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together, these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher- latitude bird species even if the environmental signals differ. However, unlike most higher-latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions.

Sex differences in the long-term repeatability of the acute stress response in long-lived, free-living Florida scrub-jays (Aphelocoma coerulescens).

There is increasing evidence that individual differences in the physiological stress response are persistent traits in many animals. To test the hypothesis that the stress-induced CORT (SI-CORT) response is repeatable over the adult life span of Florida scrub-jays (Aphelocoma coerulescens), we sampled 32 male and 25 female free-living scrub-jays (aged 2-13 years) during a 9-year period (2004-2012). Each individual was sampled two to five times and samples were collected one or more years apart during the pre-breeding season (Jan-March). In addition, individuals sampled over the greatest time period (6-8 years) were analyzed separately to more closely assess long-term repeatability. SI-CORT was repeatable in females, but not males, when values were not corrected for confounding variables (agreement repeatability). However, when the year and time of day of sample collection were controlled (adjusted repeatability), SI-CORT was repeatable in both sexes. SI-CORT was also repeatable in the males and females sampled 6-8 years apart. Finally, baseline CORT levels of males, but not females, exhibited low but significant repeatability when adjusted for year. The results of this study demonstrate that differences in SI-CORT levels were repeatable within adult scrub-jays sampled up to 8 years apart. Further, the female SI-CORT response was more consistent between pre-breeding seasons than males, which may have resulted from males having higher SI-CORT plasticity in response to environmental conditions. These data support the hypothesis that the SI-CORT response of Florida scrub-jays develops before adulthood and persists throughout much, if not all, of their natural adult life span.

Variation in the gonadotrophin-releasing hormone-1 and the song control system in the tropical breeding rufous-collared sparrow (Zonotrichia capensis) is dependent on sex and reproductive state.

Seasonal breeding in temperate zone vertebrates is characterised by pronounced variation in both central and peripheral reproductive physiology as well as behaviour. In contrast, many tropical species have a comparatively longer and less of a seasonal pattern of breeding than their temperate zone counterparts. These extended, more "flexible" reproductive periods may be associate with a lesser degree of annual variation in reproductive physiology. Here we investigated variation in the neuroendocrine control of reproduction in relation to the changes in the neural song control system in a tropical breeding songbird the rufous-collared sparrows (Zonotrichia capensis). Using in situ hybridization, we show that the optical density of GnRH1 mRNA expression is relatively constant across pre-breeding and breeding states. However, males were found to have significantly greater expression compared to females regardless of breeding state. Both males and females showed marked variation in measures of peripheral reproductive physiology with greater gonadal volumes and concentrations of sex steroids in the blood (i.e. testosterone in males; estrogen in females) during the breeding season as compared to the pre-breeding season. These findings suggest that the environmental cues regulating breeding in a tropical breeding bird ultimately exert their effects on physiology at the level of the median eminence and regulate the release of GnRH1. In addition, histological analysis of the song control system HVC, RA and Area X revealed that breeding males had significantly larger volumes of these brain nuclei as compared to non-breeding males, breeding females, and non-breeding females. Females did not exhibit a significant difference in the size of song control regions across breeding states. Together, these data show a marked sex difference in the extent to which there is breeding-associated variation in reproductive physiology and brain plasticity that is dependent on the reproductive state in a tropical breeding songbird.

Female, but not male, tropical sparrows respond more strongly to the local song dialect: implications for population divergence.

In addition to the observed high diversity of species in the tropics, divergence among populations of the same species exists over short geographic distances in both phenotypic traits and neutral genetic markers. Divergence among populations suggests great potential for the evolution of reproductive isolation and eventual speciation. In birds, song can evolve quickly through cultural transmission and result in regional dialects, which can be a critical component of reproductive isolation through variation in female preference. We examined female and male behavioral responses to local and nonlocal dialects in two allopatric populations of rufous-collared sparrows (Zonotrichia capensis) in the Andes Mountains of Ecuador. Here we show that female sparrows prefer their natal song dialect to the dialect of an allopatric population that is just 25 km away and separated by an unsuitable higher-elevation habitat (pass of 4,200 m), thus providing evidence of prezygotic reproductive isolation among populations. Males showed similar territorial responses to all conspecific dialects with no consistent difference with respect to distance, making male territoriality uninformative for estimating reproductive isolation. This study provides novel evidence for culturally based prezygotic isolation over very short distances in a tropical bird.

Seasonal neuroplasticity of the song control system in tropical, flexibly, and opportunistically breeding birds.

The avian song control system is one of the primary models used to study neuroplasticity and neurogenesis in the adult vertebrate brain. A great deal of progress has been made in understanding the mechanisms controlling seasonal neuroplasticity of the song control system. However, relatively little work has been done to identify how prevalent this phenomenon is and if a diversity of environmental cues can regulate it. Photoperiod is the primary environmental cue used by mid- to high-latitude seasonally breeding birds to time growth of the song control system but many birds display flexible or opportunistic breeding patterns that are less reliant on photoperiodic cues. In addition, approximately 75% of birds are tropical and in only one such species has neuroplasticity of the song control system been studied. Our goal is to outline some of what is known and expand on the ways that studying tropical, flexibly, and opportunistically breeding birds can advance our understanding of plasticity in the song bird brain.

Relative photorefractoriness, prolactin, and reproductive regression in a flexibly breeding sonoran desert passerine, the rufous-winged sparrow, Aimophila carpalis.

We tested the hypothesis that adult male rufous-winged sparrows, Aimophila carpalis, exhibit relative photorefractoriness. This condition results in partial loss of sensitivity to photoperiod as a reproductive stimulus after prolonged exposure to long photoperiods and is similar to the mammalian condition called photoperiodic memory. Captive birds were exposed either to 8 h of light/16 h of dark per day (8L) or to 16L for 11 weeks and were then exposed either to 8L, 13L, 14L, or 16L. Testicular diameter, plasma luteinizing hormone (LH), and plasma prolactin (PRL) were measured to assess reproductive system activity in response to photostimulation. In free-living birds, testicular diameter, plasma LH, and PRL were compared in birds caught in September in a year when birds were breeding and in a year when birds were not breeding to further evaluate the role of PRL in the termination of seasonal breeding. Testes completely developed after transfer from 8L to 14L or to 16L and partially developed after transfer from 8L to 13L. However, after 11 weeks of 16L exposure, transfer to 14L caused partial regression and transfer to 13L caused complete regression of the testes. Plasma LH increased in all birds that were transferred from 8L to a longer photoperiod. PRL showed a weak response to longer photoperiod treatment and was elevated in birds after chronic 16L exposure in comparison to birds exposed to chronic 8L. These data indicate that male rufous-winged sparrows lose sensitivity to photoperiod after long photoperiod exposure consistent with the relative photorefractoriness and photoperiodic memory models. Lower PRL in birds that developed testes on 13L and 14L compared to birds that regressed testes on 13L and 14L are consistent with the hypothesis that PRL regulates relative photorefractoriness. However, PRL does not appear to regulate interannual differences in the timing of testicular regression.

Auditory stimulation of reproductive function in male Rufous-winged Sparrows, Aimophila carpalis.

Prolonged exposure to conspecific song stimulates gonadal function and reproductive hormone secretion in female birds but few studies have investigated the physiological effects of conspecific song exposure on males outside of short-term, aggressive interactions. We exposed male Rufous-winged Sparrows, Aimophila carpalis, either to conspecific song (CS Song), to heterospecific song (Black-throated Sparrow, Amphispiza bilineata; HS Song), or to no recorded song (No Song) for 59 consecutive days (two h per day). Birds were exposed to short days (8L:16D) for the first 21 days of treatment and were then transferred to long days (13L:11D) for the remaining 38 days. During long day exposure, CS Song birds experienced faster growth of testes than HS Song and No Song birds. HS Song birds also grew their testes faster than No Song birds. Plasma luteinizing hormone (LH) and testosterone did not differ between CS Song and No Song birds. However, plasma LH was higher in HS Song birds compared to other groups. There were no differences in hypothalamic immunocytochemical labeling for gonadotropin-releasing hormone, its precursor proGnRH, or gonadotropin-inhibitory hormone, nor were there differences in two song control nuclei volumes (HVC and RA) between CS Song and No Song treatment groups. Furthermore, we found no effect of heterospecific song on free-living Rufous-winged Sparrow aggressive behaviors. These data indicate that long-term exposure to auditory stimuli, such as song, can influence the reproductive system of male songbirds and different types of auditory stimuli can have differential effects on reproductive function.

Photoperiod-independent hypothalamic regulation of luteinizing hormone secretion in a free-living Sonoran desert bird, the Rufous-winged Sparrow (Aimophila carpalis).

We investigated the regulation of luteinizing hormone (LH) in the male Rufous-winged Sparrow,Aimophila carpalis, a resident of the Sonoran desert that breeds after irregular summer rains. Although the testes develop in March due to increasing photoperiod and regress in September due to decreasing photoperiod, LH does not consistently increase in the spring as in other photoperiodic birds. However, throughout the year increased plasma LH is correlated with rainfall. To investigate this rainfall-associated regulation of LH secretion, we quantified immunocytochemical labeling for gonadotropin-releasing hormone I (GnRH-I), proGnRH (the GnRH precursor), and gonadotropin-inhibitory hormone (GnIH) in the hypothalamus of free-living adult males caught before (low LH), and during (high LH) the monsoon rainy season. Compared to pre-monsoon birds, birds caught during the monsoon season had larger immunoreactive GnRH-I (GnRH-I-ir) and proGnRH-ir cell bodies, as well as fewer, less densely labeled proGnRH-ir cell bodies. Birds caught during the monsoon had fewer, less densely labeled GnIH-ir cell bodies than birds caught before the monsoon. Further, there was no GnIH-ir labeling in the median eminence on either capture dates, suggesting that GnIH is not released to the pituitary gland via the portal vein at this time of year, but there were fewer GnIH-ir fibers in the preoptic area of birds caught during the monsoon season. Our data support the hypothesis that environmental factors associated with increased rainfall during the monsoon season stimulate GnRH synthesis and release to increase LH secretion. These data also suggest that GnIH could inhibit GnRH neuronal activity prior to the monsoon season.

Plasticity of the Rufous-winged Sparrow, Aimophila carpalis, song control regions during the monsoon-associated summer breeding period.

In most temperate zone songbirds, exposure to increasing photoperiod in the spring stimulates the reproductive system and induces reproductive behaviors. Additionally, the brain regions that control singing (song control regions; SCRs) are larger during the breeding season, thus paralleling changes in the activity of the reproductive system. However, in some birds, environmental factors other than photoperiod initiate breeding. For example, free-living male Rufous-winged Sparrows develop their testes in March due to increasing photoperiod, but have relatively low plasma T until after they begin to breed, usually in July, during the monsoon period when day length is declining. We tested the hypothesis that SCRs grow and singing behavior increases after the monsoon rains begin. We captured adult male Rufous-winged Sparrows in July 2002, 7 days before and 20 days after the monsoon rains began, euthanized birds in the field, collected their brains, and measured SCR volumes from sections immunostained for the neuronal marker NeuN. In June and July 2006, we measured song rates in the field before and after the monsoon rains. SCR volumes were larger and singing behavior increased after the onset of the monsoon rains, coinciding with the initiation of breeding. Unlike in other species studied so far, SCR volumes grew as day length was decreasing. Comparative studies utilizing species that do not breed when day length is increasing may provide information on the relative contributions of various environmental factors to SCR neuroplasticity.

Environmental regulation of the reproductive system in a flexibly breeding Sonoran Desert bird, the Rufous-winged Sparrow, Aimophila carpalis.

We investigated reproductive regulation in male Rufous-winged Sparrows, Aimophila carpalis, a Sonoran Desert passerine that breeds after irregular summer rains. Field and captive data demonstrate that increased photoperiod stimulates testicular development in March and maintains it until early September. Free-living birds caught in July and placed on captive long days (16L: 8D) maintained developed testes for up to 7 months, and free-living birds caught in September, during testicular regression, redeveloped testes when placed on captive long days, indicating that these birds were still photosensitive. Captive birds on long days maintained testicular development when exposed to temperatures mimicking those occurring during regression in free-living birds. In free-living birds, testicular development was observed during spring and summer, but unless this was associated with rainfall, breeding (indicated by juveniles) did not occur. Large increases in plasma luteinizing hormone (LH) in free-living males were correlated with heavy rainfall in July/August, when the birds bred, and in November, when they did not breed. In captive birds, plasma LH concentrations were unresponsive to photoperiodic changes, but may have responded to social cues. Plasma prolactin concentrations were directly correlated with photoperiod in free-living birds, but an effect of photoperiod on prolactin secretion was not seen in captive birds. It is concluded that male Rufous-winged Sparrows use long photoperiods to stimulate and maintain testicular development, but exposure to long photoperiods does not terminate breeding by inducing absolute photorefractoriness. The specific timing of reproductive behaviors is apparently determined by elevated plasma LH coinciding with long day stimulated gonad development.