Christopher G. Guglielmo

Plasma Lipid Metabolites Provide Information on Mass Change over Several Days in Captive Western Sandpipers

Individual quality is often assessed using a static measure of body condition, such as size-corrected body mass. Plasma metabolites have the potential to provide information on the dynamics of physiological state and thus may be better measures of individual performance capacity (and fitness). We studied relationships between rate of mass change and circulating levels of triglycerides, glycerol, and β-hydroxybutyrate in captive Western Sandpipers (Calidris mauri). The rate of mass change over one and two days prior to blood sampling was positively related to residual triglyceride (controlling for body mass at sampling) and negatively related to residual glycerol and residual β-hydroxybutyrate. The relationship between metabolite level and mass change was still apparent over a seven-day interval for glycerol, but not for the other two metabolites. In a stepwise multiple regression of mass change over two days (controlling for body mass), only β-hydroxybutyrate and glycerol were entered in the model at P < 0.15. Analysis of group means for seven sampling events showed that body-mass change in a group of individuals was related to mean circulating levels of each metabolite, i.e. to a characteristic metabolite profile. Thus, it may be feasible to employ these metabolites to assess habitat quality based on animal performance (e.g. at migratory stopover sites), or to understand the effects of climatic or anthropogenic factors on the health and survival of animals.

EXTRINSIC AND INTRINSIC SOURCES OF VARIATION IN PLASMA LIPID METABOLITES OF FREE-LIVING WESTERN SANDPIPERS (CALIDRIS MAURI)

Abstract Plasma lipid metabolites may be useful indicators of mass changes in migratory birds. To test utility of plasma metabolites in field studies, we examined effects of several extrinsic (bleed time, time of day, location) and intrinsic (body mass, sex, age, migratory state) factors on plasma concentrations of triglycerides (TRIG), glycerol (GLYC), and B-OH-butyrate (BUTY) in free-living Western Sandpipers (Calidris mauri). TRIG and GLYC decreased rapidly following capture (2–20 min), whereas BUTY did not change. GLYC and BUTY were negatively correlated to body mass. TRIG was positively correlated to body mass in migrant females, but not consistently in migrant males, or in females captured on the wintering grounds. Taking into account other sources of variation, the two measures of lipid utilization (GLYC and BUTY) varied little through the year. TRIG showed the greatest potential for use in field studies. TRIG was lowest during winter, when birds were leanest, and highest during spring and fall migration, when sandpipers were gaining mass rapidly at stopovers. TRIG differed between sandpipers refuelling a two stopover sites separated by 35 km, demonstrating that populations of birds can have characteristic lipid metabolite profiles that may reflect local differences in fattening rate.

The low-affinity glucocorticoid receptor regulates feeding and lipid breakdown in the migratory Gambel's white-crowned sparrow Zonotrichia leucophrys gambelii.

SUMMARY Plasma corticosterone increases during spring migration in a variety of bird species, including the Gambels white-crowned sparrow Zonotrichia leucophrys gambelii. Corticosterone is elevated specifically in association with migratory flight, suggesting that corticosterone may promote processes such as energy mobilization and/or migratory activity. General effects of glucocorticoids support such a prediction. Because glucocorticoids exert permissive effects on food intake, corticosterone may also participate in the regulation of migratory hyperphagia. To examine the role of corticosterone during migration, we induced Gambels white-crowned sparrows to enter the migratory condition and compared food intake and locomotor activity between controls and birds injected with RU486 - an antagonist to the low-affinity glucocorticoid receptor (GR). In addition, we investigated effects of RU486 in birds that were subjected to a short-term fast. Results indicate that RU486 did not affect locomotor activity. However, consistent with its effects in mammals, RU486 suppressed food intake. Thus, hyperphagia and migratory restlessness, the two behaviors that characterize migration, may be regulated by different mechanisms. Lastly, RU486 antagonized fasting-induced lipid mobilization, as evidenced by decreased plasma free fatty acids. Thus, data on spring migrants suggest that endogenous corticosterone levels act through the GR to support hyperphagia and that the GR promotes availability of lipid fuel substrates in association with periods of energetic demand, e.g. during migratory flight.

Effect of dietary fatty acid composition on depot fat and exercise performance in a migrating songbird, the red-eyed vireo

SUMMARY Most migrating birds accumulate lipid stores as their primary source of energy for fueling long distance flights. Lipid stores of birds during migration are composed of mostly unsaturated fatty acids; whether such a fatty acid composition enhances exercise performance of birds is unknown. We tested this hypothesis by measuring metabolic rate at rest and during intense exercise in two groups of red-eyed vireos, a long-distance migratory passerine, fed either a diet containing 82% unsaturated fat (82%U), or one containing 58% unsaturated fat (58%U). Vireos fed the 82%U diet had fat stores containing (77%) unsaturated fatty acids, whereas vireos fed the 58% U diet had fat stores containing less (66%) unsaturated fatty acids. Blood metabolites measured prior to and immediately following exercise confirmed that vireos were metabolizing endogenous fat during intense exercise. Mass-specific resting metabolic rate (RMR) was similar for vireos fed the 58%U diet (2.75±0.32 ml O2 g–1 h–1) and for vireos fed the 82%U diet (2.30±0.30 ml O2 g–1 h–1). However, mass-specific peak metabolic rate (MRpeak) was 25% higher in vireos fed the 58%U diet (28.55±1.47 ml O2 g–1 h–1) than in vireos fed the 82%U diet (21.50±1.76 ml O2 g–1 h–1). Such whole-animal energetic effects of fatty acid composition of birds suggest that the energetic cost of migration in birds may be affected by the fatty acid composition of the diet.

Seasonal upregulation of fatty acid transporters in flight muscles of migratory white-throated sparrows (Zonotrichia albicollis).

SUMMARY Endurance flights of birds, some known to last several days, can only be sustained by high rates of fatty acid uptake by flight muscles. Previous research in migratory shorebirds indicates that this is made possible in part by very high concentrations of cytosolic heart-type fatty acid binding protein (H-FABP), which is substantially upregulated during migratory seasons. We investigated if H-FABP and other components of muscle fatty acid transport also increase during these seasons in a passerine species, the white-throated sparrow (Zonotrichia albicollis). Fatty acid translocase (FAT/CD36) and plasma-membrane fatty acid binding protein (FABPpm) are well characterized mammalian proteins that facilitate transport of fatty acid through the muscle membrane, and in this study they were identified for the first time in birds. We used quantitative PCR to measure mRNA of FAT/CD36, FABPpm and H-FABP and immunoblotting to measure protein expression of FABPpm and H-FABP in the pectoralis muscles of sparrows captured in migratory (spring, fall) and non-migratory (winter) seasons. During migratory seasons, mRNA expression of these genes increased 70–1000% above wintering levels, while protein expression of H-FABP and FABPpm increased 43% and 110% above wintering levels. Activities of key metabolic enzymes, 3-hydroxyacyl-CoA-dehydrogenase (HOAD), carnitine palmitoyl transferase II (CPT II), and citrate synthase (CS) also increased (90–110%) in pectoralis muscles of migrant birds. These results support the hypothesis that enhanced protein-mediated transport of fatty acids from the circulation into muscle is a key component of the changes in muscle biochemistry required for migration in birds.

Fatty acid binding protein, a major protein in the flight muscle of migrating Western Sandpipers

Migratory flight in birds is fueled primarily by fatty acid oxidation imposing a requirement for high rates of fatty acid: (a) transport; (b) uptake; and (c) delivery to intracellular sites of beta-oxidation. Muscle fatty acid binding protein (M-FABP) is a cytosolic protein involved in the intracellular transport of fatty acids. Its expression appears to be correlated with muscle fatty acid oxidation capacity. The M-FABP was isolated for the first time from a long distance migrant bird using: (i) size exclusion; (ii) anion exchange; and (iii) hydroxyapatite chromatography. M-FABP has a molecular weight of approximately 14,000 Da and an isoelectric point of pH 4.8. A partial amino acid sequence of the protein demonstrated homology to M-FABPs from other species (80% identical to human heart FABP). It was estimated that M-FABP comprises approximately 14 and 21% of total cytosolic protein of the pectoralis and heart, respectively; the highest values yet reported from any vertebrate muscle. The abundance of M-FABP in these tissues suggests that the protein may play a key role in fatty acid supply during endurance flight. Thus, it is proposed that a seasonal increase in M-FABP expression could be a component of physiological preparation for migration.

Energetics of a long-distance migrant shorebird (Philomachus pugnax) during cold exposure and running.

SUMMARY The metabolic consequences of cold exposure and exercise are not well characterized in birds. Ruff sandpipers Philomachus pugnax are migrant shorebirds traveling between Africa and Siberia for up to 30 000 km annually. Our goal was to quantify the fuel selection pattern of these remarkable athletes during shivering and terrestrial locomotion. We used indirect calorimetry and nitrogen excretion analysis to measure their rates of lipid, carbohydrate and protein oxidation at different temperatures (22, 15, 10 or 5°C) and different treadmill speeds (15, 20, 25, 30, 35 or 40 m min–1). Results show that lipid oxidation supplies nearly all the energy necessary to support shivering and running, and that the pattern of oxidative fuel selection is independent of shivering or running intensity. During shivering, total ATP production is unequally shared between lipids (82%), carbohydrates (12%) and proteins (6%). During running, lipids remain the dominant substrate (66%), with carbohydrates (29%) and proteins (5%) playing more minor roles. The prevailing use of lipids during intense shivering and high-speed running is not consistent with the fuel selection pattern observed in exercising and cold-exposed mammals. The exact mechanisms allowing birds to use lipids at extremely high rates are still largely unexplored, and quantifying the relative importance of different fuels during long-distance flight remains a major challenge for future research.

Constitutive immune function in European starlings, Sturnus vulgaris, is decreased immediately after an endurance flight in a wind tunnel

SUMMARY Life-history theory predicts that animals face a trade-off in energy allocation between performing strenuous exercise, such as migratory flight, and mounting an immune response. We experimentally tested this prediction by studying immune function in European starlings, Sturnus vulgaris, flown in a wind tunnel. Specifically, we predicted that constitutive immune function decreases in response to training and, additionally, in response to immediate exercise. We compared constitutive immune function among three groups: (1) ‘untrained’ birds that were kept in cages and were not flown; (2) ‘trained’ birds that received flight training over a 15 day period and performed a 1-4 h continuous flight, after which they rested for 48 h before being sampled; and (3) ‘post-flight’ birds that differed from the ‘trained’ group only in being sampled immediately after the final flight. A bird in our trained group represents an individual during migration that has been resting between migratory flights for at least 2 days. A bird in our post-flight group represents an individual that has just completed a migratory flight and has not yet had time to recover. Three of our four indicators (haptoglobin, agglutination and lysis) showed the predicted decrease in immune function in the post-flight group, and two indicators (haptoglobin, agglutination) showed the predicted decreasing trend from the untrained to trained to post-flight group. Haptoglobin levels were negatively correlated with flight duration. No effect of training or flight was detected on leukocyte profiles. Our results suggest that in European starlings, constitutive immune function is decreased more as a result of immediate exercise than of exercise training. Because of the recent emergence of avian-borne diseases, understanding the trade-offs and challenges faced by long-distance migrants has gained a new level of relevance and urgency.

An Integrative Assessment of the Effects of Tamarisk on Stopover Ecology of a Long-Distance Migrant Along the San Pedro River, Arizona

ABSTRACT. The riparian habitats of the arid southwestern United States provide critical stopover habitat for avian migrants, but they have been severely altered by invasive tamarisk (Tamarix spp.). The responses of birds to such habitat alterations are not well understood. We combined ecological and physiological measurements to provide an integrative assessment of how tamarisk invasion affects the stopover ecology of vernally migrating Wilsons Warblers (Wilsonia pusilla) along the San Pedro River in Arizona. Despite higher arthropod biomass in native cottonwood—willow habitat, the refueling rate of Wilsons Warblers, as measured by plasma metabolite profiling, was higher in tamarisk. Density and detections of Wilsons Warblers did not differ between habitats, but both measurements were significantly higher in cottonwood—willow habitat for the other members of the Wilsons Warbler foraging guild. Our measurements of food resources and migrant densities suggest that cottonwood—willow may provide high-quality stopover habitat. But when plasma metabolites are included, our results indicate that tamarisk offers superior habitat for refueling Wilsons Warblers, possibly because of release from high interspecific competition in cottonwood—willow. Our results demonstrate the importance of measuring refueling performance in the assessment of stopover habitat quality and the value of including plasma metabolite profiling in studies of avian stopover ecology.

Coniferyl Benzoate in Quaking Aspen (Populus tremuloides): Its Effect on Energy and Nitrogen Digestion and Retention in Ruffed Grouse (Bonasa umbellus)

Many plant secondary metabolites have been identified as feeding deterrents; however, the physiological effects of these compounds remain largely unknown. Coniferyl benzoate (CB) is the principal secondary metabolite in quaking aspen (Populus tremuloides) flower buds, a primary winter food for ruffed grouse (Bonasa umbellus), and is a feeding deterrent for grouse and other birds. We conducted mass-balance trials with captive ruffed grouse to determine the efect of this compound on dietary mass and energy assimilation. A method is described for correcting assimilation coeficients for the intake of dietary plant secondary metabolites whereby changes in a birds ability to utilize dietary mass and energy can be distinguished from artifacts due to energy and mass dilution by plant secondary metabolites. Nitrogenous excretory products were measured to determine nitrogen balance and sources of nitrogen loss. In addition, energy losses resulting from the excretion of CB detoxification products were quantified. Birds that fed on a high CB diet were in negative nitrogen balance and excreted higher amounts of ornithine and ammonium than birds on control diets. Excretion rates for ornithine and ammonium may be even higher for birds feeding on aspen buds because otherphenolic compounds must be detoxified along with CB. Coniferyl benzoate did not decrease the ability of ruffed grouse to utilize dietary energy but did decrease utilization eficiencies simply by a dilution effect Overall, assimilation eficiencies increased over the 3-mo study. Acidosis, from acidic detoxication products, and nitrogen loss may be two reasons why wild ruffed grouse avoid feeding on aspen buds containing high CB levels; however, the principal mechanism by which CB acts as a feeding deterrent is still uncertain.