Unconventional life history in a migratory shorebird: desegregating reproduction and migration.

Conventional life-history theory predicts that energy-demanding events such as reproduction and migration must be temporally segregated to avoid resource limitation. Here, we provide, to our knowledge, the first direct evidence of 'itinerant breeding' in a migratory bird, an incredibly rare breeding strategy (less than 0.1% of extant bird species) that involves the temporal overlap of migratory and reproductive periods of the annual cycle. Based on GPS-tracking of over 200 female American woodcock, most female woodcock (greater than 80%) nested more than once (some up to six times) with short re-nest intervals, and females moved northwards on average 800 km between first and second nests, and then smaller distances (ca 200+ km) between subsequent nesting attempts. Reliance on ephemeral habitat for breeding, ground-nesting and key aspects of life history that reduce both the costs of reproduction and migration probably explain the prevalence of this rare phenotype in woodcock and why itinerant breeding so rarely occurs in other bird species.

Dietary fatty acids and flight-training influence the expression of the eicosanoid hormone prostacyclin in songbirds.

Diet shifts can alter tissue fatty acid composition in birds, which is subsequently related to metabolic patterns. Eicosanoids, short-lived fatty acid-derived hormones, have been proposed to mediate these relationships but neither baseline concentrations nor the responses to diet and exercise have been measured in songbirds. We quantified a stable derivative of the vasodilatory eicosanoid prostacyclin in the plasma of male European Starlings (Sturnus vulgaris, N = 25) fed semisynthetic diets with either high (PUFA) or low (MUFA) amounts of n6 fatty acid precursors to prostacyclin. Plasma samples were taken from each bird before, immediately after, and two days following a 15-day flight-training regimen that a subset of birds (N = 17) underwent. We found elevated prostacyclin levels in flight-trained birds fed the PUFA diet compared to those fed the MUFA diet and a positive relationship between prostacyclin and body condition, indexed by fat score. Prostacyclin concentrations also significantly decreased at the final time point. These results are consistent with the proposed influences of precursor availability (i.e., dietary fatty acids) and regulatory feedback associated with exercise (i.e., fuel supply and inflammation), and suggest that prostacyclin may be an important mediator of dietary influence on songbird physiology.

Concerted phenotypic flexibility of avian erythrocyte size and number in response to dietary anthocyanin supplementation.

BACKGROUND: Endurance flight impose substantial oxidative costs on the avian oxygen delivery system. In particular, the accumulation of irreversible damage in red blood cells can reduce the capacity of blood to transport oxygen and limit aerobic performance. Many songbirds consume large amounts of anthocyanin-rich fruit, which is hypothesized to reduce oxidative costs, enhance post-flight regeneration, and enable greater aerobic capacity. While their antioxidant benefits appear most straightforward, the effects of anthocyanins on blood composition remain so far unknown. We fed thirty hand-raised European starlings (Sturnus vulgaris) two semisynthetic diets (with or without anthocyanin supplement) and manipulated the extent of flight activity in a wind tunnel (daily flying or non-flying for over two weeks) to test for their interactive effects on functionally important haematological variables. RESULTS: Supplemented birds had on average 15% more and 4% smaller red blood cells compared to non-supplemented individuals and these diet effects were independent of flight manipulation. Haemoglobin content was 7% higher in non-supplemented flying birds compared to non-flying birds, while similar haemoglobin content was observed among supplemented birds that were flown or not. Neither diet nor flight activity influenced haematocrit. CONCLUSION: The concerted adjustments suggest that supplementation generally improved antioxidant protection in blood, which could prevent the excess removal of cells from the bloodstream and may have several implications on the oxygen delivery system, including improved gas exchange and blood flow. The flexible haematological response to dietary anthocyanins may also suggest that free-ranging species preferentially consume anthocyanin-rich fruits for their natural blood doping, oxygen delivery-enhancement effects.

The effects of training, acute exercise and dietary fatty acid composition on muscle lipid oxidative capacity in European starlings.

Migratory birds undergo seasonal changes to muscle biochemistry. Nonetheless, it is unclear to what extent these changes are attributable to the exercise of flight itself versus endogenous changes. Using starlings (Sturnus vulgaris) flying in a wind tunnel, we tested the effects of exercise training, a single bout of flight and dietary lipid composition on pectoralis muscle oxidative enzymes and lipid transporters. Starlings were either unexercised or trained over 2 weeks to fly in a wind tunnel and sampled either immediately following a long flight at the end of this training or after 2 days recovery from this flight. Additionally, they were divided into dietary groups that differed in dietary fatty acid composition (high polyunsaturates versus high monounsaturates) and amount of dietary antioxidant. Trained starlings had elevated (19%) carnitine palmitoyl transferase and elevated (11%) hydroxyacyl-CoA dehydrogenase in pectoralis muscle compared with unexercised controls, but training alone had little effect on lipid transporters. Immediately following a long wind-tunnel flight, starling pectoralis had upregulated lipid transporter mRNA (heart-type fatty acid binding protein, H-FABP, 4.7-fold; fatty acid translocase, 1.9-fold; plasma membrane fatty acid binding protein, 1.6-fold), and upregulated H-FABP protein (68%). Dietary fatty acid composition and the amount of dietary antioxidants had no effect on muscle catabolic enzymes or lipid transporter expression. Our results demonstrate that birds undergo rapid upregulation of catabolic capacity that largely becomes available during flight itself, with minor effects due to training. These effects likely combine with endogenous seasonal changes to create the migratory phenotype observed in the wild.

Flight and Dietary Antioxidants Influence Antioxidant Expression and Activity in a Migratory Bird.

Ecologically relevant factors such as exercise and diet quality can directly influence how physiological systems work including those involved in maintaining oxidative balance; however, to our knowledge, no studies to date have focused on how such factors directly affect expression of key components of the endogenous antioxidant system (i.e., transcription factors, select antioxidant genes, and corresponding antioxidant enzymes) in several metabolically active tissues of a migratory songbird. We conducted a three-factor experiment that tested the following hypotheses: (H1) Daily flying over several weeks increases the expression of transcription factors NRF2 and PPARs as well as endogenous antioxidant genes (i.e., CAT, SOD1, SOD2, GPX1, GPX4), and upregulates endogenous antioxidant enzyme activities (i.e., CAT, SOD, GPx). (H2) Songbirds fed diets composed of more 18:2n-6 PUFA are more susceptible to oxidative damage and thus upregulate their endogenous antioxidant system compared with when fed diets with less PUFA. (H3) Songbirds fed dietary anthocyanins gain additional antioxidant protection and thus upregulate their endogenous antioxidant system less compared with songbirds not fed anthocyanins. Flight training increased the expression of 3 of the 6 antioxidant genes and transcription factors measured in the liver, consistent with H1, but for only one gene (SOD2) in the pectoralis. Dietary fat quality had no effect on antioxidant pathways (H2), whereas dietary anthocyanins increased the expression of select antioxidant enzymes in the pectoralis, but not in the liver (H3). These tissue-specific differences in response to flying and dietary antioxidants are likely explained by functional differences between tissues as well as fundamental differences in their turnover rates. The consumption of dietary antioxidants along with regular flying enables birds during migration to stimulate the expression of genes involved in antioxidant protection likely through increasing the transcriptional activity of NRF2 and PPARs, and thereby demonstrates for the first time that these relevant ecological factors affect the regulation of key antioxidant pathways in wild birds. What remains to be demonstrated is how the extent of these ecological factors (i.e., intensity or duration of flight, amounts of dietary antioxidants) influences the regulation of these antioxidant pathways and thus oxidative balance.

Flight training and dietary antioxidants have mixed effects on the oxidative status of multiple tissues in a female migratory songbird.

Birds, like other vertebrates, rely on a robust antioxidant system to protect themselves against oxidative imbalance caused by energy-intensive activities such as flying. Such oxidative challenges may be especially acute for females during spring migration, as they must pay the oxidative costs of flight while preparing for reproduction; however, little previous work has examined how the antioxidant system of female spring migrants responds to dietary antioxidants and the oxidative challenges of regular flying. We fed two diets to female European starlings, one supplemented with a dietary antioxidant and one without, and then flew them daily in a windtunnel for 2 weeks during the autumn and spring migration periods. We measured the activity of enzymatic antioxidants (glutathione peroxidase, superoxide dismutase and catalase), non-enzymatic antioxidant capacity (ORAC) and markers of oxidative damage (protein carbonyls and lipid hydroperoxides) in four tissues: pectoralis, leg muscle, liver and heart. Dietary antioxidants affected enzymatic antioxidant activity and lipid damage in the heart, non-enzymatic antioxidant capacity in the pectoralis, and protein damage in leg muscle. In general, birds not fed the antioxidant supplement appeared to incur increased oxidative damage while upregulating non-enzymatic and enzymatic antioxidant activity, though these effects were strongly tissue specific. We also found trends for diet×training interactions for enzymatic antioxidant activity in the heart and leg muscle. Flight training may condition the antioxidant system of females to dynamically respond to oxidative challenges, and females during spring migration may shift antioxidant allocation to reduce oxidative damage.

Dietary vitamin E reaches the mitochondria in the flight muscle of zebra finches but only if they exercise.

Whether dietary antioxidants are effective for alleviating oxidative costs associated with energy-demanding life events first requires they are successfully absorbed in the digestive tract and transported to sites associated with reactive species production (e.g. the mitochondria). Flying birds are under high energy and oxidative demands, and although birds commonly ingest dietary antioxidants in the wild, the bioavailability of these consumed antioxidants is poorly understood. We show for the first time that an ingested lipophilic antioxidant, α-tocopherol, reached the mitochondria in the flight muscles of a songbird but only if they regularly exercise (60 min of perch-to-perch flights two times in a day or 8.5 km day-1). Deuterated α-tocopherol was found in the blood of exercise-trained zebra finches within 6.5 hrs and in isolated mitochondria from pectoral muscle within 22.5 hrs, but never reached the mitochondria in caged sedentary control birds. This rapid pace (within a day) and extent of metabolic routing of a dietary antioxidant to muscle mitochondria means that daily consumption of such dietary sources can help to pay the inevitable oxidative costs of flight muscle metabolism, but only when combined with regular exercise.

Dietary linoleic acid, antioxidants, and flight training influence the activity of oxidative enzymes in European Starlings (Sturnus vulgaris).

Multiple studies have demonstrated that diet (e.g., fatty acid composition, antioxidants) and exercise training affect the metabolic performance of songbirds during aerobic activity, although the physiological mechanisms that cause such an effect remain unclear. We tested the hypothesis that elevated proportions of dietary linoleic acid (18:2n6) and amounts of dietary anthocyanins (a hydrophilic antioxidant class) influence the activity and protein expression of oxidative enzymes in flight and leg muscle of European Starlings (Sturnus vulgaris N = 96), a subset of which were flown over 15 days in a wind tunnel. Carnitine palmitoyl transferase (CPT) and citrate synthase (CS) activity displayed 18:2n6-dependent relationships with soluble protein concentration. Lactate dehydrogenase (LDH) was similarly related to protein concentration although also dependent on both dietary anthocyanins and flight training. 3-Hydroxyacyl CoA Dehydrogenase (HOAD) activity increased throughout the experiment in flight muscle, whereas this relationship was dependent on dietary anthocyanins in the leg muscle. Soluble protein concentration also increased throughout the experiment in the flight muscle, but was unrelated to date in the leg muscle, instead being influenced by both dietary anthocyanins and flight training. Training also produced additive increases in CPT and leg muscle HOAD activity. FAT/CD36 expression was related to both dietary 18:2n6 and training and changed over the course of the experiment. These results demonstrate a notable influence of our diet manipulations and flight training on the activity of these key oxidative enzymes, and particularly CPT and CS. Such influence suggests a plausible mechanism linking diet quality and metabolic performance in songbirds.

Flight training in a migratory bird drives metabolic gene expression in the flight muscle but not liver, and dietary fat quality influences select genes.

Training and diet are hypothesized to directly stimulate key molecular pathways that mediate animal performance, and flight training, dietary fats, and dietary antioxidants are likely important in modulating molecular metabolism in migratory birds. This study experimentally investigated how long-distance flight training, as well as diet composition, affected the expression of key metabolic genes in the pectoralis muscle and the liver of European starlings (Sturnus vulgaris, n = 95). Starlings were fed diets composed of either a high or low polyunsaturated fatty acid (PUFA; 18:2n-6) and supplemented with or without a water-soluble antioxidant, and one-half of these birds were flight trained in a wind-tunnel while the rest were untrained. We measured the expression of 7 (liver) or 10 (pectoralis) key metabolic genes in flight-trained and untrained birds. Fifty percent of genes involved in mitochondrial metabolism and fat utilization were upregulated by flight training in the pectoralis (P < 0.05), whereas flight training increased the expression of only one gene responsible for fatty acid hydrolysis [lipoprotein lipase (LPL)] in the liver (P = 0.04). Dietary PUFA influenced the gene expression of LPL and fat transporter fatty acid translocase (CD36) in the pectoralis and one metabolic transcription factor [peroxisome proliferator-activated receptor (PPAR)-α (PPARα)] in the liver, whereas dietary antioxidants had no effect on the metabolic genes measured in this study. Flight training initiated a simpler causal network between PPARγ coactivators, PPARs, and metabolic genes involved in mitochondrial metabolism and fat storage in the pectoralis. Molecular metabolism is modulated by flight training and dietary fat quality in a migratory songbird, indicating that these environmental factors will affect the migratory performance of birds in the wild.

Dietary antioxidants attenuate the endocrine stress response during long-duration flight of a migratory bird.

Glucocorticoids (GCs) are metabolic hormones that promote catabolic processes, which release stored energy and support high metabolic demands such as during prolonged flights of migrating birds. Dietary antioxidants (e.g. anthocyanins) support metabolism by quenching excess reactive oxygen species produced during aerobic metabolism and also by activating specific metabolic pathways. For example, similar to GCs' function, anthocyanins promote the release of stored energy, although the extent of complementarity between GCs and dietary antioxidants is not well known. If anthocyanins complement GCs functions, birds consuming anthocyanin-rich food can be expected to limit the secretion of GCs when coping with a metabolically challenging activity, avoiding the exposure to potential hormonal detrimental effects. We tested this hypothesis in European starlings (Sturnus vulgaris) flying in a wind tunnel. We compared levels of corticosterone, the main avian GC, immediately after a sustained flight and at rest for birds that were fed diets with or without an anthocyanin supplement. As predicted, we found (i) higher corticosterone after flight than at rest in both diet groups and (ii) anthocyanin-supplemented birds had less elevated corticosterone after flight than unsupplemented control birds. This provides novel evidence that dietary antioxidants attenuate the activation of the HPA axis (i.e. increased secretion of corticosterone) during long-duration flight.

The effects of dietary linoleic acid and hydrophilic antioxidants on basal, peak, and sustained metabolism in flight-trained European starlings.

Dietary micronutrients have the ability to strongly influence animal physiology and ecology. For songbirds, dietary polyunsaturated fatty acids (PUFAs) and antioxidants are hypothesized to be particularly important micronutrients because of their influence on an individual's capacity for aerobic metabolism and recovery from extended bouts of exercise. However, the influence of specific fatty acids and hydrophilic antioxidants on whole-animal performance remains largely untested. We used diet manipulations to directly test the effects of dietary PUFA, specifically linoleic acid (18:2n6), and anthocyanins, a hydrophilic antioxidant, on basal metabolic rate (BMR), peak metabolic rate (PMR), and rates of fat catabolism, lean catabolism, and energy expenditure during sustained flight in a wind tunnel in European starlings (Sturnus vulgaris). BMR, PMR, energy expenditure, and fat metabolism decreased and lean catabolism increased over the course of the experiment in birds fed a high (32%) 18:2n6 diet, while birds fed a low (13%) 18:2n6 diet exhibited the reverse pattern. Additionally, energy expenditure, fat catabolism, and flight duration were all subject to diet-specific effects of whole-body fat content. Dietary antioxidants and diet-related differences in tissue fatty acid composition were not directly related to any measure of whole-animal performance. Together, these results suggest that the effect of dietary 18:2n6 on performance was most likely the result of the signaling properties of 18:2n6. This implies that dietary PUFA influence the energetic capabilities of songbirds and could strongly influence songbird ecology, given their availability in terrestrial systems.

Energy Stores, Oxidative Balance, and Sleep in Migratory Garden Warblers (Sylvia borin) and Whitethroats (Sylvia communis) at a Spring Stopover Site.

Little is known about how songbirds modulate sleep during migratory periods. Due to the alternation of nocturnal endurance flights and diurnal refueling stopovers, sleep is likely to be a major constraint for many migratory passerine species. Sleep may help to increase the endogenous antioxidant capacity that counteracts free radicals produced during endurance flight and reduces energy expenditure. Here, we investigated the relationship between sleep behavior, food intake, and two markers of physiological condition-the amount of energy reserves and oxidative status-in two migratory songbird species, the garden warbler (Sylvia borin) and the whitethroat (Sylvia communis). In garden warblers, birds with high energy stores were more prone to sleep during the day, while this condition-dependent sleep pattern was not present in whitethroats. In both species, birds with low energy stores were more likely to sleep with their head tucked in the feathers during nocturnal sleep. Moreover, we found a positive correlation between food intake and the extent of energy reserves in garden warblers, but not in whitethroats. Finally, we did not find significant correlations between oxidative status and sleep, or oxidative status and energy stores. Despite our study was not comparative, it suggests that different species might use different strategies to manage their energy during stopover and, additionally, it raises the possibility that migrants have evolved physiological adaptations to deal with oxidative damage produced during migration.

Il pattern del sonno nei passeriformi migratori è praticamente sconosciuto. Durante la migrazione, il sonno può diventare una importante limitazione fisiologica per molte specie che alternano estenuanti voli notturni con intensi foraggiamenti diurni per recuperare le energie. Oltre a ridurre il dispendio energetico, il sonno potrebbe promuovere le capacità antiossidanti endogene che contrastano i radicali liberi che si accumulano durante i voli prolungati. In questo studio, abbiamo indagato la relazione tra sonno, assunzione di cibo, e due marcatori della condizione fisiologica­la quantità di riserve di energia e lo stato ossidativo­in due specie di passeriformi migratori, il Beccafico (Sylvia borin) e la Sterpazzola (Sylvia communis). Nei Beccafichi, gli individui con abbondanti riserve energetiche erano più inclini a dormire durante il giorno che durante la notte, mentre non è stata trovata una associazione significativa tra il pattern di sonno e le riserve energetiche nelle Sterpazzole. In entrambe le specie, gli uccelli con scarse riserve energetiche erano più propensi a dormire con la testa inserita tra le penne durante la notte. Inoltre, abbiamo trovato una correlazione positiva tra la quantità di cibo ingerito e le riserve energetiche alla cattura nei Beccafichi, ma non nelle Sterpazzole. Infine, non abbiamo trovato correlazioni significative dello stato ossidativo con il pattern del sonno e le riserve energetiche. Questi risultati suggeriscono che specie diverse potrebbero utilizzare strategie differenti per gestire la loro energia durante la sosta, e indicano che i passeriformi migratori posseggono adattamenti fisiologici per limitare i danni ossidativi prodotti durante la migrazione. Translated to Italian by Leonida Fusani and Andrea Ferretti.

Acute effects of intense exercise on the antioxidant system in birds: does exercise training help?

The acute effects of an energy-intensive activity such as exercise may alter an animal's redox homeostasis, although these short-term effects may be ameliorated by chronic exposure to that activity, or training, over time. Although well documented in mammals, how energy-intensive training affects the antioxidant system and damage by reactive species has not been investigated fully in flight-trained birds. We examined changes to redox homeostasis in zebra finches exposed to energy-intensive activity (60 min of perch-to-perch flights twice a day), and how exercise training over many weeks affected this response. We measured multiple components of the antioxidant system: an enzymatic antioxidant (glutathione peroxidase, GPx) and non-enzymatic antioxidants (measured by the OXY-adsorbent test) as well as a measure of oxidative damage (d-ROMs). At no point during the experiment did oxidative damage change. We discovered that exposure to energy-intensive exercise training did not alter baseline levels of GPx, but induced exercise-trained birds to maintain a higher non-enzymatic antioxidant status as compared with untrained birds. GPx activity was elevated above baseline in trained birds immediately after completion of the second 1 h flight on each of the three sampling days, and non-enzymatic antioxidants were acutely depleted during flight after 13 and 44 days of training. The primary effect of exercise training on the acute response of the antioxidant system to 2 h flights was increased coordination between the enzymatic (GPx) and non-enzymatic components of the antioxidant system of birds that reduced oxidative damage associated with exercise.

Sleeping Unsafely Tucked in to Conserve Energy in a Nocturnal Migratory Songbird.

Each spring and fall, millions of normally diurnal birds switch to migrating at night. Most of these are small songbirds (passerine) migrating long distances that need to alternate their migratory flights with refueling stopovers [1, 2], which can account for up to 80% of the total migratory period [3]. After a long nocturnal flight, these birds face the contrasting needs to recover sleep and refill depleted energy stores, all while vulnerable to predation [4, 5]. Here, we investigated how garden warblers at a Mediterranean stopover site modulate their sleep behavior in relation to their metabolic state. At night, garden warblers in poor metabolic condition sleep more and exhibit less migratory restlessness than birds in good condition do. In addition, rather than sleeping with their head facing forward, birds in poor condition prefer to sleep with their head turned and tucked in their feathers. We further show that sleep with the head tucked is associated with lower respiratory and metabolic rates and reduced heat loss mediated by hiding the head-the body part with the highest heat dissipation-under the feathers. However, the benefit of conserving energy while sleeping with the head tucked was countered by reduced anti-predator vigilance. Birds presented with a sound simulating the approach of a predator responded more slowly when the head was tucked than when it was untucked. Consequently, our study demonstrates that through changing their sleep position and intensity, migrating songbirds can negotiate a previously unknown trade-off between sleep-mediated energy conservation and anti-predatory vigilance.

Spatially explicit network analysis reveals multi-species annual cycle movement patterns of sea ducks.

Conservation of long-distance migratory species poses unique challenges. Migratory connectivity, that is, the extent to which groupings of individuals at breeding sites are maintained in wintering areas, is frequently used to evaluate population structure and assess use of key habitat areas. However, for species with complex or variable annual cycle movements, this traditional bimodal framework of migratory connectivity may be overly simplistic. Like many other waterfowl, sea ducks often travel to specific pre- and post-breeding sites outside their nesting and wintering areas to prepare for migration by feeding extensively and, in some cases, molting their flight feathers. These additional migrations may play a key role in population structure, but are not included in traditional models of migratory connectivity. Network analysis, which applies graph theory to assess linkages between discrete locations or entities, offers a powerful tool for quantitatively assessing the contributions of different sites used throughout the annual cycle to complex spatial networks. We collected satellite telemetry data on annual cycle movements of 672 individual sea ducks of five species from throughout eastern North America and the Great Lakes. From these data, we constructed a multi-species network model of migratory patterns and site use over the course of breeding, molting, wintering, and migratory staging. Our results highlight inter- and intra-specific differences in the patterns and complexity of annual cycle movement patterns, including the central importance of staging and molting sites in James Bay, the St. Lawrence River, and southern New England to multi-species annual cycle habitat linkages, and highlight the value of Long-tailed Ducks (Calengula haemalis) as an umbrella species to represent the movement patterns of multiple sea duck species. We also discuss potential applications of network migration models to conservation prioritization, identification of population units, and integrating different data streams.

Modeling spatiotemporal abundance of mobile wildlife in highly variable environments using boosted GAMLSS hurdle models.

Modeling organism distributions from survey data involves numerous statistical challenges, including accounting for zero-inflation, overdispersion, and selection and incorporation of environmental covariates. In environments with high spatial and temporal variability, addressing these challenges often requires numerous assumptions regarding organism distributions and their relationships to biophysical features. These assumptions may limit the resolution or accuracy of predictions resulting from survey-based distribution models. We propose an iterative modeling approach that incorporates a negative binomial hurdle, followed by modeling of the relationship of organism distribution and abundance to environmental covariates using generalized additive models (GAM) and generalized additive models for location, scale, and shape (GAMLSS). Our approach accounts for key features of survey data by separating binary (presence-absence) from count (abundance) data, separately modeling the mean and dispersion of count data, and incorporating selection of appropriate covariates and response functions from a suite of potential covariates while avoiding overfitting. We apply our modeling approach to surveys of sea duck abundance and distribution in Nantucket Sound (Massachusetts, USA), which has been proposed as a location for offshore wind energy development. Our model results highlight the importance of spatiotemporal variation in this system, as well as identifying key habitat features including distance to shore, sediment grain size, and seafloor topographic variation. Our work provides a powerful, flexible, and highly repeatable modeling framework with minimal assumptions that can be broadly applied to the modeling of survey data with high spatiotemporal variability. Applying GAMLSS models to the count portion of survey data allows us to incorporate potential overdispersion, which can dramatically affect model results in highly dynamic systems. Our approach is particularly relevant to systems in which little a priori knowledge is available regarding relationships between organism distributions and biophysical features, since it incorporates simultaneous selection of covariates and their functional relationships with organism responses.

Dynamics of Individual Fatty Acids in Muscle Fat Stores and Membranes of a Songbird and Its Functional and Ecological Importance.

Although tissue fatty acid (FA) composition has been linked to whole-animal performance (e.g., aerobic endurance, metabolic rate, postexercise recovery) in a wide range of animal taxa, we do not adequately understand the pace of changes in FA composition and its implications for the ecology of animals. Therefore, we used a C4 to C3 diet shift experiment and compound-specific δ13C analysis to estimate the turnover rates of FAs in the polar and neutral fractions of flight muscle lipids (corresponding to membranes and lipid droplets) of exercised and sedentary zebra finches (Taeniopygia guttata). Turnover was fastest for linoleic acid (LA; 18:2n6) and palmitic acid (PA; 16:0), with 95% replacement times of 10.8-17.7 d in the polar fraction and 17.2-32.8 d in the neutral fraction, but was unexpectedly slow for the long-chain polyunsaturated FAs (LC-PUFAs) arachidonic acid (20:4n6) and docosahexaenoic acid (22:6n3) in the polar fraction, with 95% replacement in 64.9-136.5 d. Polar fraction LA and PA turnover was significantly faster in exercised birds (95% replacement in 8.5-13.3 d). Our results suggest that FA turnover in intramuscular lipid droplets is related to FA tissue concentrations and that turnover does not change in response to exercise. In contrast, we found that muscle membrane FA turnover is likely driven by a combination of selective LC-PUFA retention and consumption of shorter-chain FAs in energy metabolism. The unexpectedly fast turnover of membrane-associated FAs in muscle suggests that songbirds during migration could substantially remodel their membranes within a single migration stopover, and this may have substantial implications for how the FA composition of diet affects energy metabolism of birds during migration.

Environmental cues and dietary antioxidants affect breeding behavior and testosterone of male European starlings (Sturnus vulgaris).

Environmental cues, such as photoperiod, regulate the timing of major life-history events like breeding through direct neuroendocrine control. Less known is how supplementary environmental cues (e.g., nest sites, food availability) interact to influence key hormones and behaviors involved in reproduction, specifically in migratory species with gonadal recrudescence largely occurring at breeding sites. We investigated the behavioral and physiological responses of male European starlings to the sequential addition of nest boxes and nesting material, green herbs, and female conspecifics and how these responses depend on the availability of certain antioxidants (anthocyanins) in the diet. As expected, cloacal protuberance volume and plasma testosterone of males generally increased with photoperiod. More notably, testosterone levels peaked in males fed the high antioxidant diet when both nest box and herbal cues were present, while males fed the low antioxidant diet showed no or only a muted testosterone response to the sequential addition of these environmental cues; thus our results are in agreement with the oxidation handicap hypothesis. Males fed the high antioxidant diet maintained a constant frequency of breeding behaviors over time, whereas those fed the low antioxidant diet decreased breeding behaviors as environmental cues were sequentially added. Overall, sequential addition of the environmental cues modulated physiological and behavioral measures of reproductive condition, and dietary antioxidants were shown to be a key factor in affecting the degree of response to each of these cues. Our results highlight the importance of supplementary environmental cues and key resources such as dietary antioxidants in enhancing breeding condition of males, which conceivably aid in attraction of high quality females and reproductive success.

Turnover of muscle lipids and response to exercise differ between neutral and polar fractions in a model songbird, the zebra finch.

The turnover rates of tissues and their constituent molecules give us insights into animals' physiological demands and their functional flexibility over time. Thus far, most studies of this kind have focused on protein turnover, and few have considered lipid turnover despite an increasing appreciation of the functional diversity of this class of molecules. We measured the turnover rates of neutral and polar lipids from the pectoralis muscles of a model songbird, the zebra finch (Taeniopygia guttata, N=65), in a 256 day C3/C4 diet shift experiment, with tissue samples taken at 10 time points. We also manipulated the physiological state of a subset of these birds with a 10 week flight training regimen to test the effect of exercise on lipid turnover. We measured lipid δ13C values via isotope ratio mass spectrometry (IRMS) and estimated turnover in different fractions and treatment groups with non-linear mixed-effect regression. We found a significant difference between the mean retention times (τ) of neutral and polar lipids (t119=-2.22, P=0.028), with polar lipids (τ=11.80±1.28 days) having shorter retention times than neutral lipids (τ=19.47±3.22 days). When all birds were considered, we also found a significant decrease in the mean retention time of polar lipids in exercised birds relative to control birds (difference=-2.2±1.83 days, t56=-2.37, P=0.021), but not neutral lipids (difference=4.2± 7.41 days, t56=0.57, P=0.57). A larger, more variable neutral lipid pool and the exposure of polar lipids in mitochondrial membranes to oxidative damage and increased turnover provide mechanisms consistent with our results.

Modulation of digestive enzyme activities in the avian digestive tract in relation to diet composition and quality.

In nature, birds are faced with variable food sources that may differ in composition (protein vs. carbohydrates) and quality (highly digestible material vs. indigestible fiber). Studies in passerine birds and some commercial poultry demonstrate that the gastrointestinal tract can respond to varying diet composition and quality by changing morphology and/or activities of digestive enzymes. However, studies in additional avian species are warranted to understand generalities of these trends. We first fed juvenile mallards (Anas platyrhynchos), chickens (Gallus gallus), and quails (Coturnix coturnix) on either high-carbohydrate or high-protein diets. For the most part, birds fed the high-carbohydrate diet had higher small intestinal and cecal disaccharidase activities (maltase and sucrase). However, only mallards exhibited higher small intestinal aminopeptidase-N (APN) activities when fed the high-protein diet. These results differ from passerine birds, which largely modulate small intestinal proteases, but not disaccharidases. In another trial, we fed Canada geese (Branta canadensis) diets that varied in both their protein and fiber concentrations for approximately 3.5 months. Birds fed the high-fiber diets had significantly longer small intestines and caeca compared to those fed low-fiber diets. Additionally, geese fed the high-fiber diets exhibited lower mass-specific activities of small intestinal sucrase, and higher activities of APN when summed across the small intestine and ceca. Similar to the avian species above, geese fed the high-protein diets did not exhibit flexibility in their small intestinal APN activities. Overall, these experiments demonstrate that responsiveness of the avian digestive tract to diet composition may have phylogenetic or ecological constraints. Studies on other avian taxa are needed to understand these patterns.