A range-wide domino effect and resetting of the annual cycle in a migratory songbird.

Latitudinal differences in timing of breeding are well documented but how such differences carry over to influence timing of events in the annual cycle of migratory birds is not well understood. We examined geographical variation in timing of events throughout the year using light-level geolocator tracking data from 133 migratory tree swallows ( Tachycineta bicolor) originating from 12 North American breeding populations. A swallow's breeding latitude influenced timing of breeding, which then carried over to affect breeding ground departure. This resulted in subsequent effects on the arrival and departure schedules at autumn stopover locations and timing of arrival at non-breeding locations. This 'domino effect' between timing events was no longer apparent by the time individuals departed for spring migration. Our range-wide analysis demonstrates the lasting impact breeding latitude can have on migration schedules but also highlights how such timing relationships can reset when individuals reside at non-breeding sites for extended periods of time.

Effects of age and reproductive experience on the distribution of prolactin and growth hormone secreting cells in the anterior pituitary of a passerine.

Plasma prolactin (PRL) is released from lactotrophs in the anterior pituitary. As plasma PRL levels rise during incubation in domestic fowl, the number of lactotrophs (PRL-immunoreactive, PRL-IR cells) increases while the number of growth hormone secreting cells, somatotrophs (GH-IR cells), declines. We measured plasma PRL levels using radioimmunoassay (RIA) and examined the distribution of lactotrophs and somatotrophs in the anterior pituitary of breeding and nonbreeding zebra finches of known ages with and without prior breeding experience using fluorescent immunohistochemistry (IHC). Plasma PRL levels were higher in breeding than in nonbreeding birds, regardless of age, sex, or previous breeding history. PRL-IR cells were localized primarily, but not exclusively, to the cephalic aspect of the anterior pituitary (AP) and along the ventral margin. Birds with prior reproductive experience had more PRL-IR cells than birds with no prior reproductive experience and breeders had slightly higher PRL-IR cell counts than did nonbreeders, but there was no correlation between the number of PRL-IR cells and plasma PRL levels. GH-IR cells were concentrated in the caudal aspect of the AP with some cells in the cephalic lobe, but numbers did not differ between any of the groups studied. An increase in PRL-IR cells corresponded with an increase in GH-IR cells. An increase in lactotroph number with reproductive experience in zebra finches may facilitate future reproductive events by allowing for more robust PRL secretion and increased reproductive success.

Consequences of immune system aging in nature: a study of immunosenescence costs in free-living Tree Swallows.

Immunosenescence, the aging of the immune system, is well documented in humans and laboratory models and is known to increase infection risk, morbidity, and mortality among the old. Immunosenescence patterns have recently been unveiled in various free-living populations, but their consequences in the wild have not been explored. We investigated the consequences of immunosenescence in free-living Tree Swallows Tachycineta bicolor through a field experiment simulating a bacterial infection (challenge with lipopolysaccharide, LPS) in females of different ages during the nestling rearing period. We assessed behavioral and physiological responses of females, as well as growth and quality of their offspring, to determine the costs associated with the simulated infection. Results of the experiment differed between the two years of study. In the first year, old females challenged with LPS lost more body mass and reduced their nest visitation rates more, and their offspring tended to grow slower compared to similarly challenged younger females. In contrast, in the second year, old females did not appear to suffer larger costs than younger ones. Interestingly, immunosenescence was only detected during the first year of the study, suggesting that it is the dysregulated immune function characteristic of immunosenescent individuals rather than age per se that can lead to higher costs of immune defense in old individuals. These results provide the first evidence of costs of immunosenescence in free-living animals and support the hypothesis that old, immunosenescent individuals pay higher costs than younger ones when faced with a challenge to their immune system. Our results also suggest that these costs are mediated by an exaggerated sickness behavior, as seen in laboratory models, and can be modulated by ecological factors such as weather conditions and food availability.

Maternal effects increase within-family variation in offspring survival.

Maternal effects are environmental components of phenotypes that complicate relationships between natural selection and evolution because they often affect phenotypes and fitness simultaneously. We studied the effects of egg size variation on juvenile survival in a population of American coots (Fulica americana). We experimentally evaluated egg size variation at three levels: across the population, within natal nests, and within foster nests. Natal nests accounted for the most variation in population egg size. Within clutches, early-laid eggs were larger than later-laid eggs, with the exception of first-laid eggs, which were small. In the fostering experiment, posthatching survival was most strongly related to egg size relative to natal siblings and natal hatching order and less so to egg size within foster nests. These effects on survival were found even though young from natal nests were neither raised together nor raised by genetic parents. These results indicate that females allocate resources unequally among offspring such that offspring from larger, early-laid eggs have higher survival than offspring from smaller, later-laid eggs, regardless of their size relative to foster siblings or to mean population egg size. These results suggest that egg size variation can be maintained through selection on maternal investment strategies and not on egg size per se.

Evolutionary ecology of endocrine-mediated life-history variation in the garter snake Thamnophis elegans.

The endocrine system plays an integral role in the regulation of key life-history traits. Insulin-like growth factor-1 (IGF-1) is a hormone that promotes growth and reproduction, and it has been implicated in the reduction of lifespan. IGF-1 is also capable of responding plastically to environmental stimuli such as resource availability and temperature. Thus pleiotropic control of life-history traits by IGF-1 could provide a mechanism for the evolution of correlated life-history traits in a new or changing environment. An ideal system in which to investigate the role of IGF-1 in life-history evolution exists in two ecotypes of the garter snake Thamnophis elegans, which derive from a single recent ancestral source but have evolved genetically divergent life-history characteristics. Snakes from meadow populations near Eagle Lake, California (USA) exhibit slower growth rates, lower annual reproductive output, and longer median adult lifespans relative to populations along the lakeshore. We hypothesized that the IGF-1 system has differentiated between these ecotypes and can account for increased growth and reproduction and reduced survival in lakeshore vs. meadow snakes. We tested for a difference in plasma IGF-1 levels in free-ranging snakes from replicate populations of each ecotype over three years. IGF-1 levels were significantly associated with adult body size, reproductive output, and season in a manner that reflects established differences in prey ecology and age/size-specific reproduction between the ecotypes. These findings are discussed in the context of theoretical expectations for a tradeoff between reproduction and lifespan that is mediated by pleiotropic endocrine mechanisms.

Individual variation in baseline and stress-induced corticosterone and prolactin levels predicts parental effort by nesting mourning doves.

Endocrine systems have an important mechanistic role in structuring life-history trade-offs. During breeding, individual variation in prolactin (PRL) and corticosterone (CORT) levels affects behavioral and physiological processes that drive trade-offs between reproduction and self-maintenance. We examined patterns in baseline (BL) and stress induced (SI; level following a standard capture-restraint protocol) levels of PRL and CORT for breeding mourning doves (Zenaida macroura). We determined whether the relationship of adult condition and parental effort to hormone levels in wild birds was consistent with life-history predictions. Both BL PRL and BL CORT level in adults were positively related to nestling weight at early nestling ages, consistent with the prediction of a positive relationship of hormone levels to current parental effort of adults and associated increased energy demand. Results are consistent with the two hormones acting together at baseline levels to limit negative effects of CORT on reproduction while maintaining beneficial effects such as increased foraging for nestling feeding. Our data did not support predictions that SI responses would vary in response to nestling or adult condition. The magnitude of CORT response in the parents to our capture-restraint protocol was negatively correlated with subsequent parental effort. Average nestling weights for adults with the highest SI CORT response were on average 10-15% lighter than expected for their age in follow-up visits after the stress event. Our results demonstrated a relationship between individual hormone levels and within population variation in parental effort and suggested that hormonal control plays an important role in structuring reproductive decisions for mourning doves.

Detection of Mycoplasma gallisepticum in House Finches ( Haemorhous mexicanus) from Arizona.

In 1994, an endemic poultry pathogen, Mycoplasma gallisepticum (MG), was identified as the causative agent of a novel disease in house finches ( Haemorhous mexicanus). After an initial outbreak in Maryland, MG spread rapidly throughout eastern North American populations of house finches. Subsequently, MG spread slowly through the northern interior of North America and then into the Pacific Northwest, finally reaching California in 2006. Until 2009, there were no reports of MG in the southwestern United States east of California. In August 2011, after reports of house finches displaying conjunctivitis characteristic of MG infection in Arizona, we trapped house finches at bird feeders in central Arizona (Tempe) and southern Arizona (Tucson and Green Valley) to assay for MG infection. Upon capture, we noted whether birds exhibited conjunctivitis, and we collected choanal swabs to test for the presence of MG DNA using PCR. We detected MG in finches captured from Green Valley (in ∼12% of birds captured), but not in finches from Tucson or Tempe. Based on resampling of house finches at these sites in July 2014, we suggest that central Arizona finches likely remain unexposed to MG. We also suggest that low urban connectivity between arid habitats of southern and central Arizona or a reduction in the prevalence of MG after its initial arrival in Arizona may be limiting the spread of MG from south to north in Arizona. In addition, the observed conjunctivitis-like signs in house finches that were negative for MG by PCR may be caused primarily by avian pox virus.

The rate of telomere loss is related to maximum lifespan in birds.

Telomeres are highly conserved regions of DNA that protect the ends of linear chromosomes. The loss of telomeres can signal an irreversible change to a cell's state, including cellular senescence. Senescent cells no longer divide and can damage nearby healthy cells, thus potentially placing them at the crossroads of cancer and ageing. While the epidemiology, cellular and molecular biology of telomeres are well studied, a newer field exploring telomere biology in the context of ecology and evolution is just emerging. With work to date focusing on how telomere shortening relates to individual mortality, less is known about how telomeres relate to ageing rates across species. Here, we investigated telomere length in cross-sectional samples from 19 bird species to determine how rates of telomere loss relate to interspecific variation in maximum lifespan. We found that bird species with longer lifespans lose fewer telomeric repeats each year compared with species with shorter lifespans. In addition, phylogenetic analysis revealed that the rate of telomere loss is evolutionarily conserved within bird families. This suggests that the physiological causes of telomere shortening, or the ability to maintain telomeres, are features that may be responsible for, or co-evolved with, different lifespans observed across species.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Immunosenescence in some but not all immune components in a free-living vertebrate, the tree swallow.

A wide diversity of free-living organisms show increases in mortality rates and/or decreases in reproductive success with advancing age. However, the physiological mechanisms underlying these demographic patterns of senescence are poorly understood. Immunosenescence, the age-related deterioration of immune function, is well documented in humans and laboratory models, and often leads to increased morbidity and mortality due to disease. However, we know very little about immunosenescence in free-living organisms. Here, we studied immunosenescence in a free-living population of tree swallows, Tachycineta bicolor, assessing three components of the immune system and using both in vivo and in vitro immunological tests. Immune function in tree swallow females showed a complex pattern with age; acquired T-cell mediated immunity declined with age, but neither acquired nor innate humoral immunity did. In vitro lymphocyte proliferation stimulated by T-cell mitogens decreased with age, suggesting that reduced T-cell function might be one mechanism underlying the immunosenescence pattern of in vivo cell-mediated response recently described for this same population. Our results provide the most thorough description of immunosenescence patterns and mechanisms in a free-living vertebrate population to date. Future research should focus on the ecological implications of immunosenescence and the potential causes of variation in patterns among species.

Telomerase activity is maintained throughout the lifespan of long-lived birds.

Telomerase is an enzyme capable of elongating telomeres, the caps at the ends of chromosomes associated with aging, lifespan and survival. We investigated tissue-level variation in telomerase across different ages in four bird species that vary widely in their life history. Telomerase activity in bone marrow may be associated with the rate of erythrocyte telomere shortening; birds with lower rates of telomere shortening and longer lifespans have higher bone marrow telomerase activity throughout life. Telomerase activity in all of the species appears to be tightly correlated with the proliferative potential of specific organs, and it is also highest in the hatchling age-class, when the proliferative demands of most organs are the highest. This study offers an alternative view to the commonly held hypothesis that telomerase activity is down-regulated in all post-mitotic somatic tissues in long-lived organisms as a tumor-protective mechanism. This highlights the need for more comparative analyses of telomerase, lifespan and the incidence of tumor formation.

A laboratory exercise to illustrate increased salivary cortisol in response to three stressful conditions using competitive ELISA.

Perceived stress activates the hypothalamus-pituitary-adrenal axis, resulting in the release of glucocorticoids into the systemic circulation. Glucocorticoids cause the elevation of blood glucose, providing the necessary energy for the organism to cope with stress. Here, we outline a laboratory exercise that uses a competitive ELISA kit to illustrate the response of salivary cortisol concentrations to three stressful conditions. Twelve undergraduate students in the General and Comparative Endocrinology course at Iowa State University were subjected to presentation stress, fasting stress, and competition stress to determine their effect on salivary cortisol concentrations. Students had elevated salivary cortisol in response to each of these stresses compared with basal conditions. These results reiterate the importance of glucocorticoids as mediators of the stress response. This study also incorporates the use of the ELISA technique, a modern laboratory tool used to determine the amount of endogenous antigens in plasma or saliva. This laboratory exercise can easily be adapted to fit into already existing physiology and endocrinology curriculums.

Periodic cooling of bird eggs reduces embryonic growth efficiency.

For many bird embryos, periodic cooling occurs when the incubating adult leaves the nest to forage, but the effects of periodic cooling on embryo growth, yolk use, and metabolism are poorly known. To address this question, we conducted incubation experiments on eggs of zebra finches (Taeniopygia guttata) that were frequently cooled and then rewarmed or were allowed to develop at a constant temperature. After 12 d of incubation, embryo mass and yolk reserves were less in eggs that experienced periodic cooling than in controls incubated constantly at 37.5 degrees Celsius. Embryos that regularly cooled to 20 degrees Celsius had higher mass-specific metabolic rates than embryos incubated constantly at 37.5 degrees Celsius. Periodic cooling delayed development and increased metabolic costs, reducing the efficiency with which egg nutrients were converted into embryo tissue. Avian embryos can tolerate periodic cooling, possibly by adjusting their physiology to variable thermal conditions, but at a cost to growth efficiency as well as rate of development. This reduction in embryo growth efficiency adds a new dimension to the fitness consequences of variation in adult nest attentiveness.

Telomeres shorten more slowly in long-lived birds and mammals than in short-lived ones.

We know very little about physiological constraints on the evolution of life-history traits in general, and, in particular, about physiological and molecular adjustments that accompany the evolution of variation in lifespan. Identifying mechanisms that underlie adaptive variation in lifespan should provide insight into the evolution of trade-offs between lifespan and other life-history traits. Telomeres, the DNA caps at the ends of linear chromosomes, usually shorten as animals age, but whether telomere rate of change is associated with lifespan is unknown. We measured telomere length in erythrocytes from five bird species with markedly different lifespans. Species with shorter lifespans lost more telomeric repeats with age than species with longer lifespans. A similar correlation is seen in mammals. Furthermore, telomeres did not shorten with age in Leach's storm-petrels, an extremely long-lived bird, but actually lengthened. This novel finding suggests that regulation of telomere length is associated not only with cellular replicative lifespan, but also with organismal lifespan, and that very long-lived organisms have escaped entirely any telomeric constraint on cellular replicative lifespan.

The natural history of telomeres: tools for aging animals and exploring the aging process.

We have been exploring the use of telomere length as a technique to age animals. If telomere restriction fragments (TRFs) shorten predictably with age in a particular tissue, then measurement of TRFs will allow estimation of ages of animals when age cannot be measured directly. This would be particularly useful in population studies where tissue samples can be collected, but age of individuals or age structure of the population is otherwise unknown. We have demonstrated that rate of change in length of TRFs from blood cells can be used to estimate age in a number of avian species. Calibration of this telomere 'clock' using known-age individuals has led to new questions regarding the importance of TRF shortening in aging and its evolution in animals with differing life spans. Our current data show a tight correlation between telomere rate of change (TROC) and maximum life span in birds, with the longest living species having the slowest TROC. In contrast, absolute length of TRFs is not correlated with maximum life span. Very long-lived Leach's storm-petrels have telomeres that in fact lengthen with age! These data suggest that in the longest-lived organisms, cellular replicative life span may not be constrained by shortening telomeres. Published data show that TRFs shorten more slowly in long-lived mammals than in short-lived ones, although for birds and mammals of similar life span, telomere shortening is faster in mammals than in birds. This corresponds with the relatively greater longevity in birds than in mammals.

Calibrating the telomere clock in common terns, Sterna hirundo.

Field biologists often work with animals for which there are no prior history. A marker of an animal's age would offer insight into how age and experience affect reproductive success and other life history parameters. We previously reported that length of telomere restriction fragments shorten predictably with age in the captive zebra finch (Taeniopygia guttata). This paper reports that telomeres can also be used to gain knowledge on the age structure of wild, long-lived common terns (Sterna hirundo). Although ages cannot be determined precisely from telomere lengths alone, birds can be classified into broad age-classes. This technique can provide useful information about the age of individuals in cases where their previous histories are unknown.

Telomerase expression is differentially regulated in birds of differing life span.

Cellular senescence caused by telomere shortening has been suggested as one potential causal agent of aging. In some tissues, telomeres are maintained by telomerase; however, telomerase promotes tumor formation, suggesting a trade-off between aging and cancer. We predicted that telomerase activity should vary directly with life span. We determined telomerase activity in bone marrow in cross-sectional samples from two short-lived bird species and two long-lived bird species. The two short-lived species had high telomerase activity as hatchlings but showed a sharp downregulation in both the young and old adults, whereas the two long-lived species had relatively high telomerase activity in bone marrow that did not decrease with age. In zebra finches, the age-related change in telomerase activity varied in different tissues. Telomerase activity increased late in life in skeletal muscle, liver, and gonad, but not in blood or bone marrow.

Telomere length provides a new technique for aging animals.

Field biologists often work with animals for which there is no prior history. A marker of an animal's age would offer insight into how age and experience affect reproductive success and other life history parameters. Telomere length shortens with age in cultured cells and mouse and human tissues. We found that lengths of telomere restriction fragments cleaved from blood cell DNA shorten predictably with age in the zebra finch (Taeniopygia guttata). If this relationship holds in other species, it should be possible, once the relationship between telomere length and age has been determined for a given species, to use blood samples to estimate ages of free-living animals. This will allow the incorporation of age into estimates of factors affecting life history parameters in cases where previous histories of animals are unknown.

Functional significance of variation in egg-yolk androgens in the American coot.

Maternally derived hormones in cleidoic eggs have been implicated in mediating growth, behavior, and social interactions among offspring. Given these widespread and significant effects, hormonal investments have the potential to greatly influence fitness of offspring. Intraspecific variation can exist at three levels (within individual eggs, among eggs within clutches, and among eggs from different females), each of which has different implications for offspring. We characterized all three levels of variation in maternally derived androgens (testosterone and androstenedione) present in yolks of American coot eggs. We found no variation in testosterone levels within eggs which suggests that embryos are exposed to constant androgen levels during development, and that field-based yolk biopsies are an appropriate way to sample eggs for this species. Within clutches, early-laid eggs had higher androgen levels than late-laid eggs, and this pattern may exacerbate negative effects of hatching asynchrony on chicks from late-hatching eggs if androgens provide chicks with a behavioral or growth advantage over chicks from eggs with lower androgen levels. American coots lay large clutches, and unequal resource allocation among offspring may be the optimal strategy for females with access to limited resources. Most of the variation in androgen levels occurred among eggs from different females. Females nesting on ponds with two other pairs laid eggs with significantly higher androgen levels than females living on ponds with fewer pairs. This suggests that increased territory defense behaviors influence the levels of androgens allocated to eggs and may be one mechanism underlying density-dependent effects on reproduction.

Extent and variability of interstitial telomeric sequences and their effects on estimates of telomere length.

Telomeres often shorten with time, although this varies between tissues, individuals and species, and their length and/or rate of change may reflect fitness and rate of senescence. Measurement of telomeres is increasingly important to ecologists, yet the relative merits of different methods for estimating telomere length are not clear. In particular the extent to which interstitial telomere sequences (ITSs), telomere repeats located away from chromosomes ends, confound estimates of telomere length is unknown. Here we present a method to estimate the extent of ITS within a species and variation among individuals. We estimated the extent of ITS by comparing the amount of label hybridized to in-gel telomere restriction fragments (TRF) before and after the TRFs were denatured. This protocol produced robust and repeatable estimates of the extent of ITS in birds. In five species, the amount of ITS was substantial, ranging from 15% to 40% of total telomeric sequence DNA. In addition, the amount of ITS can vary significantly among individuals within a species. Including ITSs in telomere length calculations always underestimated telomere length because most ITSs are shorter than most telomeres. The magnitude of that error varies with telomere length and is larger for longer telomeres. Estimating telomere length using methods that incorporate ITSs, such as Southern blot TRF and quantitative PCR analyses reduces an investigator's power to detect difference in telomere dynamics between individuals or over time within an individual.

Interrelations among immune defense indexes reflect major components of the immune system in a free-living vertebrate.

Understanding the relationships among immune components in free-living animals is a challenge in ecoimmunology, and it is important not only for selecting the immune assays to be used but also for more knowledgeable interpretation of results. In this study, we investigated the relationships among six immune defense indexes commonly used by ecoimmunologists and measured simultaneously in individual free-living tree swallows. Three main axes of variation in immune function were identified using a principal components analysis, representing variation in T-cell, B-cell, and innate immunity. Measures within each axis tended to be positively correlated among individuals, while measures in different axes were uncorrelated. A trade-off between T-cell function and B-cell function became apparent only when variation among individuals in body condition, age, and general quality was taken into account. Interestingly, the level of natural antibodies, a component of innate immunity, showed the strongest association with components of acquired B-cell function, possibly reflecting a common underlying genetic mechanism, as has been documented in poultry. Our results indicate that despite the complexity of the immune system, important insights can be gained by using the currently available assays but in a more comprehensive approach than has generally been used in the field of ecoimmunology.