Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Experimental evidence that EPA and DHA are dietary requirements in a migratory shorebird, but they do not affect muscle oxidative capacity.

Dietary n-3 long chain polyunsaturated fatty acids (LCPUFAs) are hypothesized to be natural doping agents in migratory shorebirds, enabling prolonged flight by increasing membrane fluidity and oxidative capacity of the flight muscles. Animals can obtain n-3 LCPUFAs from the diet or by conversion of dietary α-linolenic acid, 18:3 n-3. However, the capacity to meet n-3 LCPUFA requirements from 18:3 n-3 varies among species. Direct tests of muscle oxidative enhancement and fatty acid conversion capacity are lacking in marine shorebirds that evolved eating diets rich in n-3 LCPUFAs. We tested whether the presence and type of dietary fatty acids influence the fatty acid composition and flight muscle oxidative capacity in western sandpipers (Calidris mauri). Sandpipers were fed diets low in n-3 PUFAs, high in 18:3 n-3, or high in n-3 LCPUFAs. Dietary fatty acid composition was reflected in multiple tissues, and low intake of n-3 LCPUFAs decreased the abundance of these fatty acids in all tissues, even with a high intake of 18:3 n-3. This suggests that 18:3 n-3 cannot replace n-3 LCPUFAs, and dietary n-3 LCPUFAs are required for sandpipers. Flight muscle indicators of enzymatic oxidative capacity and regulators of lipid metabolism did not change. However, the n-3 LCPUFA diet was associated with increased FAT/CD36 mRNA expression, potentially benefitting fatty acid transport during flight. Our study suggests that flight muscle lipid oxidation is not strongly influenced by n-3 PUFA intake. The type of dietary n-3 PUFA strongly influences the abundance of n-3 LCPUFAs in the body and could still impact whole-animal performance.

Migratory songbirds exhibit seasonal modulation of the oxygen cascade.

Migratory flight requires birds to maintain intensive aerobic exercise for many hours or days. Maintaining O2 supply to flight muscles is therefore important during migration, especially since migratory songbirds have been documented flying at altitudes greater than 5000 m above sea level, where O2 is limited. Whether songbirds exhibit seasonal plasticity of the O2 cascade to maintain O2 uptake and transport during migratory flight is not well understood. We investigated changes in the hypoxic ventilatory response, haematology and pectoralis (flight) muscle phenotype of 6 songbird species from 3 families during migratory and non-migratory conditions. Songbirds were captured during southbound migration in southern Ontario, Canada. Half of the birds were assessed during migration, and the rest were transitioned onto a winter photoperiod to induce a non-migratory phenotype and measured. All species exhibited seasonal plasticity at various stages along the O2 cascade, but not all species exhibited the same responses. Songbirds tended to be more hypoxia tolerant during migration, withstanding 5 kPa O2 and breathed more effectively through slower, deeper breaths. Warblers had a stronger haemoglobin-O2 affinity during autumn migration (decrease of ∼4.7 Torr), while the opposite was observed in thrushes (increase of ∼2.6 Torr). In the flight muscle there was an ∼1.2-fold increase in the abundance of muscle fibres with smaller fibre transverse areas during autumn migration, but no changes in capillary:fibre ratio. These adjustments would enhance O2 uptake and transport to the flight muscle. Our findings demonstrate that in the O2 cascade there is no ideal migratory phenotype for all songbirds.

Experimental ghrelin administration affects migratory behaviour in a songbird.

Twice a year, billions of birds take on drastic physiological and behavioural changes to migrate between breeding and wintering areas. On migration, most passerine birds regularly stop over along the way to rest and refuel. Endogenous energy stores are not only the indispensable fuel to complete long distance flights, but are also important peripheral signals that once integrated in the brain modulate crucial behavioural decisions, such as the decision to resume migration after a stopover. A network of hormones signals metabolic fuel availability to the brain in vertebrates, including the recently discovered gut-hormone ghrelin. Here, we show that ghrelin takes part in the control of migratory behaviour during spring migration in a wild migratory passerine. We manipulated blood concentrations of ghrelin of 53 yellow-rumped warblers (Setophaga coronata coronata) caught during stopover and automatically radio-tracked their migratory behaviour following release. We found that injections of acylated and unacylated ghrelin rapidly induced movements away from the release site, indicating that the ghrelin system acts centrally to mediate stopover departure decisions. The effects of the hormone manipulation declined within 8 h following release, and did not affect the overall rate of migration. These results provide experimental evidence for a pivotal role of ghrelin in the modulation of behavioural decisions during migration. In addition, this study offers insights into the regulatory functions of metabolic hormones in the dialogue between gut and brain in birds.

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.

Species-specific metabolic responses of songbird, shorebird, and murine cultured myotubes to n-3 polyunsaturated fatty acids.

Migratory birds may benefit from diets rich in polyunsaturated fatty acids (PUFAs) that could improve exercise performance. Previous investigations suggest that different types of birds may respond differently to PUFA. We established muscle myocyte cell culture models from muscle satellite cells of a migratory passerine songbird (yellow-rumped warbler, Setophaga coronata coronata) and a nonpasserine shorebird (sanderling, Calidris alba). We differentiated and treated avian myotubes and immortalized murine C2C12 myotubes with n-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and with monounsaturated oleic acid (OA) to compare effects on aerobic performance, metabolic enzyme activities, key fatty acid (FA) transporters, and expression of peroxisome proliferator-activated receptors (PPARs). Sanderling and C2C12 myotubes increased expression of PPARs with n-3 PUFA treatments, whereas expression was unchanged in yellow-rumped warblers. Both sanderlings and yellow-rumped warblers increased expression of fatty acid transporters, whereas C2C12 cells decreased expression following n-3 PUFA treatments. Only yellow-rumped warbler myotubes increased expression of some metabolic enzymes, whereas the sanderling and C2C12 cells were unchanged. PUFA supplementation in C2C12 myotubes increased mitochondrial respiratory chain efficiency, whereas sanderlings increased proton leak-associated respiration and maximal respiration (measurements were not made in warblers). This research indicates that songbirds and shorebirds respond differently to n-3 PUFA and provides support for the hypothesis that n-3 PUFA increase the aerobic capacity of migrant shorebird muscle, which may improve overall endurance flight performance.

Extraordinarily rapid proliferation of cultured muscle satellite cells from migratory birds.

Migratory birds experience bouts of muscle growth and depletion as they prepare for, and undertake prolonged flight. Our studies of migratory bird muscle physiology in vitro led to the discovery that sanderling (Calidris alba) muscle satellite cells proliferate more rapidly than other normal cell lines. Here we determined the proliferation rate of muscle satellite cells isolated from five migratory species (sanderling; ruff, Calidris pugnax; western sandpiper, Calidris mauri; yellow-rumped warbler, Setophaga coronata; Swainson's thrush, Catharus ustulatus) from two families (shorebirds and songbirds) and with different migratory strategies. Ruff and sanderling satellite cells exhibited rapid proliferation, with population doubling times of 9.3 ± 1.3 and 11.4 ± 2 h, whereas the remaining species' cell doubling times were greater than or equal to 24 h. The results indicate that the rapid proliferation of satellite cells is not associated with total migration distance but may be related to flight bout duration and interact with lifespan.

Using breath δ13C analysis to determine the effects of dietary carbohydrate and protein on glucose and leucine oxidation at rest in the yellow-rumped warbler (Setophaga coronata).

Fat is the major fuel for migratory flight of birds, but protein is also catabolized. Flight range could be reduced if protein is used too quickly from muscles and organs, and it is important to understand factors that influence protein catabolism. Previous correlative studies suggested high protein diets may increase protein use in flight, although a wind tunnel study with yellow-rumped warblers (Setophaga coronata) did not support this relationship. We tested the hypothesis that diet composition affects nutrient oxidation in resting, fasted yellow-rumped warblers. For method development, we gavaged or subcutaneously injected warblers with 13C labelled glucose or leucine, and measured δ13C of breath CO2 in real time using infrared laser spectrometry. Regardless of route of administration, leucine had greater instantaneous and cumulative oxidation than glucose. Compared to subcutaneous injection, gavaged birds reached maximum oxidation rate faster for leucine and glucose, respectively, had a higher maximum oxidation rate, and reached final cumulative oxidation approximately faster for leucine or glucose, respectively, indicating immediate oxidation of the substrates by the digestive system. Warblers (N = 10 each) were fed isocaloric 60% carbohydrate or 60% protein diets for minimum 2 weeks, and subcutaneously injected with 13C labelled glucose or leucine. Diet composition had little effect on oxidation kinetics except that warblers fed high-carbohydrate reached final cumulative oxidation of leucine more quickly than those fed high-protein. The findings do not support the hypothesis that high protein diets increase the oxidation of protein during negative energy states in migratory birds, and provide methodology that could be applied to test it in flight.

Measuring power input, power output and energy conversion efficiency in un-instrumented flying birds.

Cost of flight at various speeds is a crucial determinant of flight behaviour in birds. Aerodynamic models, predicting that mechanical power (Pmech) varies with flight speed in a U-shaped manner, have been used together with an energy conversion factor (efficiency) to estimate metabolic power (Pmet). Despite few empirical studies, efficiency has been assumed constant across flight speeds at 23%. Ideally, efficiency should be estimated from measurements of both Pmech and Pmet in un-instrumented flight. Until recently, progress has been hampered by methodological constraints. The main aim of this study was to evaluate recently developed techniques and estimate flight efficiency across flight speeds. We used the 13C-labelled sodium bicarbonate method (NaBi) and particle image velocimetry (PIV) to measure Pmet and Pmech in blackcaps flying in a wind tunnel. We also cross-validated measurements made by NaBi with quantitative magnetic resonance (QMR) body composition analysis in yellow-rumped warblers. We found that Pmet estimated by NaBi was ∼12% lower than corresponding values estimated by QMR. Pmet varied in a U-shaped manner across flight speeds in blackcaps, but the pattern was not statistically significant. Pmech could only be reliably measured for two intermediate speeds and estimated efficiency ranged between 14% and 22% (combining the two speeds for raw and weight/lift-specific power, with and without correction for the ∼12% difference between NaBi and QMR), which were close to the currently used default value. We conclude that NaBi and PIV are viable techniques, allowing researchers to address some of the outstanding questions regarding bird flight energetics.

Stopover refuelling, movement and departure decisions in the white-throated sparrow: The influence of intrinsic and extrinsic factors during spring migration.

Differential migration timing between sex or age classes is an example of how migratory movement strategies can differ among subgroups within a population. However, in songbirds, evidence for intrinsic differences in en route migratory behaviour is often mixed, suggesting that the local environmental context may play a role in accentuating or diminishing patterns. We evaluated how multiple intrinsic and extrinsic variables influenced refuelling rates, local movement behaviour and departure decisions in the white-throated sparrow Zonotrichia albicollis during spring migration. This species exhibits a unique genetically based plumage dimorphism, providing a unique class of individual in which to evaluate patterns and processes of differential migration, in addition to sex, age and migration distance. At a migratory stopover site, plasma metabolite analysis was used to quantify individual variation in stopover refuelling rate. In after second year adults, automated and manual radio telemetry was used to quantify daily activity timing, daily movement distances, stopover duration and departure time. Arrival timing to the stopover site was determined using capture data. Non-breeding and previous breeding/natal latitude were determined using analysis of hydrogen isotopes in claws and feathers. Males arrived at the stopover site 11 days on average before females, but no difference in migration timing was observed between plumage morph or age classes. After second year, adults with more southern previous breeding latitudes arrived at stopover earlier, whereas second year birds making their first return migration arrived at stopover in an inverse relationship to non-breeding latitude. Stopover refuelling rate did not differ between ages, sexes or plumage morphs, and daily departure probability of adults was higher under warmer temperatures and favourable tailwinds. White-striped morphs moved greater distances during stopover, initiated daily activity earlier in the morning and departed for migration earlier in the evening than tan-striped morphs. Our results show that while individual phenotype can influence some aspects of local stopover-scale movement behaviour, evidence for differential stopover behaviour was weak. Differential migration timing is unlikely to result from intrinsic differences in en route refuelling rate and departure decisions, especially because the latter is strongly influenced by meteorological conditions.

Temporal patterns of foraging by silver-haired bats during migratory stopover revealed by isotopic analyses (δ13C) of breath CO2.

The extent to which migratory bats forage at stopover sites or while in migratory flight is poorly understood. Endogenous fat stores have lower δ13C values relative to the dietary substrates from which they were synthesized, and so, the fed versus fasted state of bats should be discernable by comparing their breath δ13C at capture to that after a known period of fasting. We captured silver-haired bats (Lasionycteris noctivagans) at a stopover site at Long Point, Ontario, Canada, during spring and fall migration. We collected breath samples at capture and after fasting in captivity for 12 h, providing a fasted-state δ13C value corresponding to metabolism of fat stores. We also collected and weighed fecal pellets produced while in captivity. Breath δ13C values at capture were positively correlated with mass of feces produced. During spring migration, δ13C values of breath CO2 at capture were low and similar to fasting values, but increased with date consistent with increased foraging at stopover and reliance on exogenous dietary nutrients as the season progressed. The opposite temporal pattern was found during fall migration. Our findings suggest that bats forage during migratory stopover when environmental conditions permit despite potential time trade-offs between feeding and travel, and the energy savings resulting from torpor during roosting. This study provides insight into the eco-physiology of bat migration and shows the importance of foraging habitat for migratory bats.

Dietary polyunsaturated fatty acids influence flight muscle oxidative capacity but not endurance flight performance in a migratory songbird.

The migratory flights of birds are primarily fueled by fat; however, certain fatty acids may also enhance flight performance and the capacity to oxidize fat. The natural doping hypothesis posits that n-3 long-chain polyunsaturated fatty acids (PUFA) increase membrane fluidity and aerobic and fatty acid oxidative enzymes in the flight muscles, which enables prolonged endurance flight. Support for this hypothesis is mixed, and there is no empirical evidence for increased flight performance. We fed yellow-rumped warblers ( Setophaga coronata coronata) diets enriched in either n-3 or n-6 long-chain PUFA or low in long-chain PUFA and evaluated flight muscle metabolism and endurance performance in a wind tunnel flights lasting up to 6 h. Fatty acid profiles of muscle phospholipids confirmed enrichment of the targeted dietary fatty acids, whereas less substantial differences were observed in adipose triacylglycerol. Contrary to the predictions, feeding n-3 PUFA decreased peroxisome proliferator-activated receptors-ß mRNA abundance and muscle oxidative enzyme activities. However, changes in muscle metabolism were not reflected in whole animal performance. No differences were observed in flight performance among diet treatments in terms of endurance capacity, energy costs, or fuel composition. These measures of flight performance were more strongly influenced by body mass and flight duration. Overall, we found no support for the natural doping hypothesis in a songbird. Furthermore, we caution against extending changes in flight muscle metabolic enzymes or fatty acid composition to changes to migratory performance without empirical evidence.

Energetics of migratory bats during stopover: a test of the torpor-assisted migration hypothesis.

The torpor-assisted migration hypothesis posits that migration is facilitated in bats by the use of torpor during stopover roosting periods, and predicts that at stopover, bats regulate time in torpor facultatively so that daily energy expenditure is independent of ambient roosting temperature. Energy savings can thus be directed to migratory movement. However, direct measurements of total roosting energy expenditure in relation to ambient and body temperature are lacking. We captured migratory silver-haired bats (Lasionycteris noctivagans; ∼11 g) at Long Point, ON, Canada, in spring and autumn 2016. We used quantitative magnetic resonance analysis to measure body composition change and energy expenditure over a 12 h roosting period in a ventilated incubator at 10, 17 and 25°C. We assessed the effects of season, body mass, sex and age on energy expenditure. We found that daily energy expenditure was independent of roosting temperature, and that this was achieved by flexible use of torpor. Variation in body mass at capture was driven mainly by differences in fat, and the amount of body fat was negatively related to torpor use, particularly in spring. Season, sex and age also affected torpor use and energy expenditure, notably with pregnant females being generally fatter and using less torpor than males in spring. We estimate that stopover contributes only 15-20% to the total energy costs of migration in bats compared with 70% or more in typical birds. This study provides support for the torpor-assisted migration hypothesis, and furthers our understanding of the energy budgets of migratory bats.

Flight muscle protein damage during endurance flight is related to energy expenditure but not dietary polyunsaturated fatty acids in a migratory bird.

Migration poses many physiological challenges for birds, including sustaining high intensity aerobic exercise for hours or days. A consequence of endurance flight is the production of reactive oxygen species (ROS). ROS production may be influenced by dietary polyunsaturated fatty acids (PUFA), which, although prone to oxidative damage, may limit mitochondrial ROS production and increase antioxidant capacity. We examined how flight muscles manage oxidative stress during flight, and whether dietary long-chain PUFA influence ROS management or damage. Yellow-rumped warblers were fed diets low in PUFA, or high in long-chain n-3 or n-6 PUFA. Flight muscle was sampled from birds in each diet treatment at rest or immediately after flying for up to a maximum of 360 min in a wind tunnel. Flight increased flight muscle superoxide dismutase activity but had no effect on catalase activity. The ratio of glutathione to glutathione disulphide decreased during flight. Oxidative protein damage, indicated by protein carbonyls, increased with flight duration (Pearson r=0.4). Further examination of just individuals that flew for 360 min (N=15) indicates that oxidative damage was related more to total energy expenditure (Pearson r=0.86) than to flight duration itself. This suggests that high quality individuals with higher flight efficiency have not only lower energy costs but also potentially less oxidative damage to repair after arrival at the destination. No significant effects of dietary long-chain PUFA were observed on antioxidants or damage. Overall, flight results in oxidative stress and the degree of damage is likely driven more by energy costs than fatty acid nutrition.

Fatty acid profiles of the European migratory common noctule bat (Nyctalus noctula).

In animals, fatty acids (FA) are essential as structural components in membranes and for energy storage in adipocytes. Here, we studied the relative proportions of FA in a mammal with extreme changes in metabolic rates. Common noctule bats (Nyctalus noctula) switch from energetically demanding long-distance migration at high metabolic rates to regular torpor with extremely low metabolic rates. We found that composition of FA categories differed between adipose tissue types (white adipose tissue (WAT) vs brown adipose tissue (BAT)) and muscle tissue types (skeletal vs heart), but not between sexes. We found oleic acid to be the most abundant FA in all studied tissues. Concentrations of polyunsaturated FA (PUFA) were not always higher in muscular tissue compared with adipocyte tissue, even though high concentrations of PUFA are considered beneficial for low body temperatures in torpor. In all tissues, we observed a high content in monounsaturated fatty acids (MUFA), possibly to compensate for a low PUFA content in the diet. Ratios of ω6/ω3 were lower in the heart than in skeletal muscles of common noctules. Three FA (palmitic, oleic, and linoleic acid) accounted for about 70% of the FA in adipose tissue, which is similar to proportions observed in migrating birds, yet migrating birds generally have a higher PUFA content in muscle and adipose tissues than bats. Bats seem to contrast with other mammals in having a high MUFA content in all tissues. We conclude that FA profiles of bats differ largely from those of most cursorial mammals and instead are-with the exception of MUFA-similar to those of migrating birds.

Obese super athletes: fat-fueled migration in birds and bats.

Migratory birds are physiologically specialized to accumulate massive fat stores (up to 50-60% of body mass), and to transport and oxidize fatty acids at very high rates to sustain flight for many hours or days. Target gene, protein and enzyme analyses and recent -omic studies of bird flight muscles confirm that high capacities for fatty acid uptake, cytosolic transport, and oxidation are consistent features that make fat-fueled migration possible. Augmented circulatory transport by lipoproteins is suggested by field data but has not been experimentally verified. Migratory bats have high aerobic capacity and fatty acid oxidation potential; however, endurance flight fueled by adipose-stored fat has not been demonstrated. Patterns of fattening and expression of muscle fatty acid transporters are inconsistent, and bats may partially fuel migratory flight with ingested nutrients. Changes in energy intake, digestive capacity, liver lipid metabolism and body temperature regulation may contribute to migratory fattening. Although control of appetite is similar in birds and mammals, neuroendocrine mechanisms regulating seasonal changes in fuel store set-points in migrants remain poorly understood. Triacylglycerol of birds and bats contains mostly 16 and 18 carbon fatty acids with variable amounts of 18:2n-6 and 18:3n-3 depending on diet. Unsaturation of fat converges near 70% during migration, and unsaturated fatty acids are preferentially mobilized and oxidized, making them good fuel. Twenty and 22 carbon n-3 and n-6 polyunsaturated fatty acids (PUFA) may affect membrane function and peroxisome proliferator-activated receptor signaling. However, evidence for dietary PUFA as doping agents in migratory birds is equivocal and requires further study.

Effects of experimental manipulation of hematocrit on avian flight performance in high- and low-altitude conditions.

Despite widely held assumptions that hematocrit (Hct) is a key determinant of aerobic capacity and exercise performance, this relationship has not often been tested rigorously in birds and results to date are mixed. Migration in birds involves high-intensity exercise for long durations at various altitudes. Therefore, it provides a good model system to examine the effect of Hct on flight performance and physiological responses of exercise at high altitude. We treated yellow-rumped warblers (Setophaga coronata) with avian erythropoietin (EPO) and anti-EPO to experimentally manipulate Hct and assessed flight performance at low and high altitudes using a hypobaric wind tunnel. We showed that anti-EPO-treated birds had lower Hct than vehicle- and EPO--treated birds post-treatment. Anti-EPO-treated birds also had marginally lower exercise performance at low altitude, committing a higher number of strikes (mistakes) in the first 30 min of flight. However, anti-EPO-treated birds performed significantly better at high altitude, attaining a higher altitude in a ramped altitude challenge to 3000 m equivalent altitude, and with a longer duration of flight at high altitude. Birds exercising at high altitude showed decreased Hct, increased glucose mobilization and decreased antioxidant capacity, regardless of treatment. In summary, we provide experimental evidence that the relationship between Hct and exercise performance is dependent on altitude. Future studies should investigate whether free-living birds adaptively modulate their Hct, potentially through a combination of erythropoiesis and plasma volume regulation (i.e. hemodilution), based on the altitude they fly at during migratory flight.

The performing animal: causes and consequences of body remodeling and metabolic adjustments in red knots facing contrasting thermal environments.

Using red knots (Calidris canutus) as a model, we determined how changes in mass and metabolic activity of organs relate to temperature-induced variation in metabolic performance. In cold-acclimated birds, we expected large muscles and heart as well as improved oxidative capacity and lipid transport, and we predicted that this would explain variation in maximal thermogenic capacity (Msum). We also expected larger digestive and excretory organs in these same birds and predicted that this would explain most of the variation in basal metabolic rate (BMR). Knots kept at 5°C were 20% heavier and maintained 1.5 times more body fat than individuals kept in thermoneutral conditions (25°C). The birds in the cold also had a BMR up to 32% higher and a Msum 16% higher than birds at 25°C. Organs were larger in the cold, with muscles and heart being 9-20% heavier and digestive and excretory organs being 21-36% larger than at thermoneutrality. Rather than the predicted digestive and excretory organs, the cold-induced increase in BMR correlated with changes in mass of the heart, pectoralis, and carcass. Msum varied positively with the mass of the pectoralis, supracoracoideus, and heart, highlighting the importance of muscles and cardiac function in cold endurance. Cold-acclimated knots also expressed upregulated capacity for lipid transport across mitochondrial membranes [carnitine palmitoyl transferase (CPT)] in their pectoralis and leg muscles, higher lipid catabolism capacity in their pectoralis muscles [ß-hydroxyacyl CoA-dehydrogenase (HOAD)], and elevated oxidative capacity in their liver and kidney (citrate synthase). These adjustments may have contributed to BMR through changes in metabolic intensity. Positive relationships among Msum, CPT, and HOAD in the heart also suggest indirect constraints on thermogenic capacity through limited cardiac capacity.

Seasonal and flight-related variation of galectin expression in heart, liver and flight muscles of yellow-rumped warblers (Setophaga coronata).

Galectins, a family of multifunctional glycan-binding proteins, are proposed as biomarkers of cellular stress responses. Avian migration is an energetically challenging physical stress, which represents a physiological model of muscular endurance exercises. This study assesses change in galectin gene expression profiles associated with seasonal variation in migratory state and endurance flight in yellow-rumped warblers (Setophaga coronata). Bioinformatics analysis and real-time qPCR were used to analyse the expression of galectins in flight muscle, heart and liver tissues of 15 warblers separated into three groups of winter unflown, and fall migratory flown/unflown birds. Five transcripts similar to chicken and human galectins -1, -2, -3, -4, and -8 were identified in warbler tissues. The expression of these galectins showed no seasonal changes between two experimental groups of birds maintained under unflown winter and fall conditions indicating a minor role of galectins in preparation for migration. However, endurance flight led to a significant elevation of galectin-1 and galectin-3 mRNAs in flight muscles and galectin-3 mRNA in heart tissue while no changes were observed in liver. Different changes were observed for the level of O-GlcNAcylated proteins, which were elevated in flight muscles under winter conditions. These results suggest that secreted galectin-1 and galectin-3 may be active in repair of bird muscles during and following migratory flight and serve as molecular biomarkers of recent arrival from migratory flights in field studies.

Light oiling of feathers increases flight energy expenditure in a migratory shorebird.

Flying birds depend on their feathers to undertake most activities, and maintain them in peak condition through periodic molt and frequent preening. Even small exposures to crude oil reduce the integrity of feathers, and could impair flight performance. We trained wild western sandpipers (Calidris mauri) to perform endurance flights in a wind tunnel, and used magnetic resonance body composition analysis to measure energy expenditure after birds were exposed to weathered MC252 crude oil from the Deepwater Horizon oil spill. The cost of transport was 0.26±0.04 kJ km-1 in controls, and increased by 22% when the trailing edges of the wing and tail were oiled (<20% of body surface; considered light oiling). Additional crude oil on breast and back feathers (∼30% total surface; moderate oiling) increased the cost of transport by 45% above controls. Oiling tended to decrease flight control, and only half of moderately oiled birds completed the flight test. We then flew birds at a range of speeds to estimate basic kinematic parameters. At low speeds, light and moderately oiled birds had larger wingbeat amplitudes than controls, while moderately oiled birds showed greater wingbeat frequencies across all speeds, and a shift in optimal flight speed towards higher wind speeds. We suggest these changes reflect poorer lift production and increased drag on the wings and body. Oiling will increase the difficulty and energy costs of locomotion for daily and seasonal activities such as foraging, predator evasion, territory defense, courtship, chick provisioning, commuting and long-distance migration. These sub-lethal effects must be considered in oil spill impact assessments.