It pays to follow the leader: Metabolic cost of flight is lower for trailing birds in small groups.

Many bird species commonly aggregate in flocks for reasons ranging from predator defense to navigation. Available evidence suggests that certain types of flocks-the V and echelon formations of large birds-may provide a benefit that reduces the aerodynamic cost of flight, whereas cluster flocks typical of smaller birds may increase flight costs. However, metabolic flight costs have not been directly measured in any of these group flight contexts [Zhang and Lauder, J. Exp. Biol. 226, jeb245617 (2023)]. Here, we measured the energetic benefits of flight in small groups of two or three birds and the requirements for realizing those benefits, using metabolic energy expenditure and flight position measurements from European Starlings flying in a wind tunnel. The starlings continuously varied their relative position during flights but adopted a V formation motif on average, with a modal spanwise and streamwise spacing of [0.81, 0.91] wingspans. As measured via CO2 production, flight costs for follower birds were significantly reduced compared to their individual solo flight benchmarks. However, followers with more positional variability with respect to leaders did less well, even increasing their costs above solo flight. Thus, we directly demonstrate energetic costs and benefits for group flight followers in an experimental context amenable to further investigation of the underlying aerodynamics, wake interactions, and bird characteristics that produce these metabolic effects.

SERCA uncoupling may facilitate cold acclimation in dark-eyed juncos (Junco hyemalis) without regulation by sarcolipin or phospholamban.

Homeothermic endotherms defend their body temperature in cold environments using a number of behavioral and physiological mechanisms. Maintaining a stable body temperature primarily requires heat production through shivering (ST) or non-shivering thermogenesis (NST). Although the use of NST is well established in mammalian systems, the mechanisms and extent to which NST is used in birds is poorly understood. In mammals, one well-characterized mechanism of NST is through uncoupling of Ca2+ transport from ATP hydrolysis by sarco/endoplasmic reticulum ATPase (SERCA) in the skeletal muscle, which generates heat and may contribute to Ca2+ signaling for fatigue resistance and mitochondrial biogenesis. Two small proteins-sarcolipin (SLN) and phospholamban (PLN)-are known to regulate SERCA in mammals, but recent work shows inconsistent responses of SLN to cold-acclimation in birds. In this study, we measured SERCA uncoupling in the pectoralis flight muscle of control (18°C) and cold-acclimated (-8°C) dark-eyed juncos (Junco hyemalis) that exhibited suppressed SLN transcription in the cold. We measured SERCA activity and Ca2+ uptake rates for the first time in cold-acclimated birds and found greater SERCA uncoupling in the muscle of juncos in the cold. However, SERCA uncoupling was not related to SLN or PLN transcription or measures of mitochondrial biogenesis. Nonetheless, SERCA uncoupling reduced an individual's risk of hypothermia in the cold. Therefore, while SERCA uncoupling in the cold could be indicative of NST, it does not appear to be mediated by known regulatory proteins in these birds. These results prompt interesting questions about the significance of SLN and PLN in birds and the role of SERCA uncoupling in response to environmental conditions.

Sarcolipin relates to fattening, but not sarco/endoplasmic reticulum Ca2+-ATPase uncoupling, in captive migratory gray catbirds.

In order to complete their energetically demanding journeys, migratory birds undergo a suite of physiological changes to prepare for long-duration endurance flight, including hyperphagia, fat deposition, reliance on fat as a fuel source, and flight muscle hypertrophy. In mammalian muscle, SLN is a small regulatory protein which binds to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and uncouples Ca2+ transport from ATP hydrolysis, increasing energy consumption, heat production, and cytosolic Ca2+ transients that signal for mitochondrial biogenesis, fatigue resistance and a shift to fatty acid oxidation. Using a photoperiod manipulation of captive gray catbirds (Dumetella carolinensis), we investigated whether SLN may play a role in coordinating the development of the migratory phenotype. In response to long-day photostimulation, catbirds demonstrated migratory restlessness and significant body fat stores, alongside higher SLN transcription while SERCA2 remained constant. SLN transcription was strongly correlated with h-FABP and PGC1α transcription, as well as fat mass. However, SLN was not significantly correlated with HOAD or CD36 transcripts or measurements of SERCA activity, SR membrane Ca2+ leak, Ca2+ uptake rates, pumping efficiency or mitochondrial biogenesis. Therefore, SLN may be involved in the process of storing fat and shifting to fat as a fuel, but the mechanism of its involvement remains unclear.

White-throated sparrow (Zonotrichia albicollis) liver and pectoralis flight muscle transcriptomic changes in preparation for migration.

Migratory songbirds undertake challenging journeys to reach their breeding grounds each spring. They accomplish these nonstop flapping feats of endurance through a suite of physiological changes, including the development of substantial fat stores and flight muscle hypertrophy and an increased capacity for fat catabolism. In addition, migratory birds may show large reductions in organ masses during flight, including the flight muscle and liver, which they must rapidly rebuild during their migratory stopover before replenishing their fat stores. However, the molecular basis of this capacity for rapid tissue remodeling and energetic output has not been thoroughly investigated. We performed RNA-sequencing analysis of the liver and pectoralis flight muscle of captive white-throated sparrows in experimentally photostimulated migratory and nonmigratory condition to explore the mechanisms of seasonal change to metabolism and tissue mass regulation that may facilitate these migratory journeys. Based on transcriptional changes, we propose that tissue-specific adjustments in preparation for migration may alleviate the damaging effects of long-duration activity, including a potential increase to the inflammatory response in the muscle. Furthermore, we hypothesize that seasonal hypertrophy balances satellite cell recruitment and apoptosis, while little evidence appeared in the transcriptome to support myostatin-, insulin-like growth factor 1-, and mammalian target of rapamycin-mediated pathways for muscle growth. These findings can encourage more targeted molecular studies on the unique integration of pathways that we find in the development of the migratory endurance phenotype in songbirds.NEW & NOTEWORTHY Migratory songbirds undergo significant physiological changes to accomplish their impressive migratory journeys. However, we have a limited understanding of the regulatory mechanisms underlying these changes. Here, we explore the transcriptomic changes to the flight muscle and liver of white-throated sparrows as they develop the migratory condition. We use these patterns to develop hypotheses about metabolic flexibility and tissue restructuring in preparation for migration.

Long-duration wind tunnel flights reveal exponential declines in protein catabolism over time in short- and long-distance migratory warblers.

During migration, long-distance migratory songbirds may fly nonstop for days, whereas shorter-distance migrants complete flights of 6 to 10 h. Fat is the primary fuel source, but protein is also assumed to provide a low, consistent amount of energy for flight. However, little is known about how the use of these fuel sources differs among bird species and in response to flight duration. Current models predict that birds can fly until fat stores are exhausted, with little consideration of protein's limits on flight range or duration. We captured two related migratory species-ultra long-distance blackpoll warblers (Setophaga striata) and short-distance yellow-rumped warblers (Setophaga coronata)-during fall migration and flew them in a wind tunnel to examine differences in energy expenditure, overall fuel use, and fuel mixture. We measured fat and fat-free body mass before and after flight using quantitative magnetic resonance and calculated energy expenditure from body composition changes and doubly labeled water. Three blackpolls flew voluntarily for up to 28 h-the longest wind tunnel flight to date-and ended flights with substantial fat reserves but concave flight muscle, indicating that protein loss, rather than fat, may actually limit flight duration. Interestingly, while blackpolls had significantly lower mass-specific metabolic power in flight than that of yellow-rumped warblers and fuel use was remarkably similar in both species, with consistent fat use but exceptionally high rates of protein loss at the start of flight that declined exponentially over time. This suggests that protein may be a critical, dynamic, and often overlooked fuel for long-distance migratory birds.

Short-term mercury exposure disrupts muscular and hepatic lipid metabolism in a migrant songbird.

Methylmercury (MeHg) is a global pollutant that can cause metabolic disruptions in animals and thereby potentially compromise the energetic capacity of birds for long-distance migration, but its effects on avian lipid metabolism pathways that support endurance flight and stopover refueling have never been studied. We tested the effects of short-term (14-d), environmentally relevant (0.5 ppm) dietary MeHg exposure on lipid metabolism markers in the pectoralis and livers of yellow-rumped warblers (Setophaga coronata) that were found in a previous study to have poorer flight endurance in a wind tunnel than untreated conspecifics. Compared to controls, MeHg-exposed birds displayed lower muscle aerobic and fatty acid oxidation capacity, but similar muscle glycolytic capacity, fatty acid transporter expression, and PPAR expression. Livers of exposed birds indicated elevated energy costs, lower fatty acid uptake capacity, and lower PPAR-γ expression. The lower muscle oxidative enzyme capacity of exposed birds likely contributed to their weaker endurance in the prior study, while the metabolic changes observed in the liver have potential to inhibit lipogenesis and stopover refueling. Our findings provide concerning evidence that fatty acid catabolism, synthesis, and storage pathways in birds can be dysregulated by only brief exposure to MeHg, with potentially significant consequences for migratory performance.

Migratory disposition alters lean mass dynamics and protein metabolism in migratory white-throated sparrows (Zonotrichia albicollis).

Migratory birds seasonally increase fat stores and the capacity to use fat to fuel long-distance migratory flights. However, lean mass loss also occurs during migratory flights and, if adaptive, should exhibit seasonal changes in the capacity for protein metabolism. We conducted a photoperiod manipulation using captive white-throated sparrows (Zonotrichia albicollis) to investigate seasonal changes in protein metabolism between the nonmigratory "winter" condition and after exposure to a long-day "spring" photoperiod to stimulate the migratory condition. After photostimulation, birds in the migratory condition rapidly increased fat mass and activity of fat catabolism enzymes. Meanwhile, total lean mass did not change, but birds increased the activity of protein catabolism enzymes and lost more water and lean mass during water-restricted metabolic testing. These data suggest that more protein may be catabolized during migratory seasons, corresponding with more water loss. Counter to predictions, birds in the migratory condition also showed an approximately 30-fold increase in muscle expression of sarcolipin, which binds to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and uncouples Ca2+ transport from ATP hydrolysis. Our documented changes to protein catabolism enzymes and whole animal lean mass dynamics may indicate that protein breakdown or increased protein turnover is adaptive during migration in songbirds.

Developing a Stopover-CORT hypothesis: Corticosterone predicts body composition and refueling rate in Gray Catbirds during migratory stopover.

Migratory flight is energetically challenging, requiring alternating phases of fuel catabolism and fuel accumulation, accompanied by dramatic changes in body composition and behavior. Baseline corticosterone (CORT; the primary glucocorticoid in birds) is thought to underlie transitions between fuel catabolism during flight, fuel deposition during stopover, and the initiation of migratory flight. However, studies of CORT on stopover physiology and behavior remain disparate efforts, lacking the cohesion of a general hypothesis. Here we develop a Stopover-CORT hypothesis formalizing the relationships among CORT, body condition, and refueling rate in migratory birds. First we expect body mass to increase with triglycerides (TRIG) as birds refuel. Second, based on a synthesis of previous literature, we predict a U-shaped CORT curve over the course of stopover, postulating that elevated CORT at arrival is reactive, responding to poor body condition, while CORT elevation before departure is preparative, driving changes in behavior and body condition. We tested these predictions in Gray Catbirds (Dumetella carolinensis) following a trans-Gulf flight during spring migration. We found baseline CORT was negatively correlated with body condition and TRIG, corresponding with our predictions for arriving and refueling-but not departing-birds. It is possible catbirds undergo regional habitat translocations rather than complete the entire stopover phase at our study site. We propose the Stopover-CORT hypothesis as a useful predictive framework for future studies of the mechanistic basis of stopover physiology. By studying the regulation of stopover refueling and departure, we may better understand physiological limitations to overall migration rate and improve assessments of habitat quality for refueling birds.