Daily Torpor in Birds and Mammals: Past, Present, and Future of the Field.

Torpor is an incredibly efficient energy-saving strategy that many endothermic birds and mammals use to save energy, by lowering their metabolic rates, heart rates, and typically body temperatures. Over the last few decades, the study of daily torpor-in which torpor is used for less than 24 hours per bout-has advanced rapidly. The papers in this issue cover the ecological and evolutionary drivers of torpor, as well as some of the mechanisms governing torpor use. We identified broad focus areas that need special attention: clearly defining the various parameters that indicate torpor use and identifying the genetic and neurological mechanisms regulating torpor. Recent studies on daily torpor and heterothermy, including the ones in this issue, have furthered the field immensely. We look forward to a period of immense growth in this field.

Genomic insights into metabolic flux in hummingbirds.

Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome-scale genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long- and short-read sequencing, scaffolding it using existing assemblies. We then used hybrid long- and short-read RNA sequencing of liver and muscle tissue in fasted and fed metabolic states for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and deletion of critical genes (SLC2A4, GCK) involved in glucostasis in other vertebrates. We found expression of a fructose-specific version of SLC2A5 putatively in place of insulin-sensitive SLC2A5, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds, suggesting key pathways for the rapid metabolic switch hummingbirds undergo.

Leptin Resistance Does Not Facilitate Migratory Fattening in Ruby-Throated Hummingbirds (Archilochus Colubris).

In mammals, leptin is an important energy homeostasis hormone produced by adipose tissue. Circulating leptin concentrations correlate positively with fat mass and act in a negative feedback fashion to inhibit food intake and increase energy expenditure, thereby preventing fat gain. For some species, leptin resistance is advantageous during times of year where fat gain is necessary (e.g., prior to hibernation). While the function of leptin in birds remains controversial, seasonal leptin resistance may similarly benefit migratory species. Here, we used the ruby-throated hummingbird (Archilochus colubris) to test the hypothesis that leptin resistance promotes fattening prior to migration. We predicted that during the migratory fattening period, leptin levels should correlate positively with fat mass but should not inhibit food intake or increase energy expenditure, resulting in fattening. We tracked the body (fat) mass, the concentration of leptin-like protein in the urine, and the food intake of 12 captive hummingbirds from August 2021 to January 2022. In a subset of hummingbirds, we also quantified voluntary physical activity as a proxy for energy expenditure. We found remarkable age-related variation in fattening strategies, with juveniles doubling their body fat by mid-September and adults exhibiting only a 50% increase. Changes in fat mass were strongly associated with increased food intake and reduced voluntary activity. However, we found no correlation between leptin-like protein concentration and fat mass, food intake, or voluntary activity. Since increased torpor use has been shown to accelerate migratory fattening in ruby-throated hummingbirds, we also hypothesized that leptin is a mediator of torpor use. In an experimental manipulation of circulating leptin, however, we found no change in torpor use, body fat, or food intake. Overall, our findings suggest that leptin may not act as an adipostat in hummingbirds, nor does leptin resistance regulate how hummingbirds fatten prior to migration.

Reversal of the adipostat control of torpor during migration in hummingbirds.

Many small endotherms use torpor to reduce metabolic rate and manage daily energy balance. However, the physiological 'rules' that govern torpor use are unclear. We tracked torpor use and body composition in ruby-throated hummingbirds (Archilochus colubris), a long-distance migrant, throughout the summer using respirometry and quantitative magnetic resonance. During the mid-summer, birds entered torpor at consistently low fat stores (~5% of body mass), and torpor duration was negatively related to evening fat load. Remarkably, this energy emergency strategy was abandoned in the late summer when birds accumulated fat for migration. During the migration period, birds were more likely to enter torpor on nights when they had higher fat stores, and fat gain was positively correlated with the amount of torpor used. These findings demonstrate the versatility of torpor throughout the annual cycle and suggest a fundamental change in physiological feedback between adiposity and torpor during migration. Moreover, this study highlights the underappreciated importance of facultative heterothermy in migratory ecology.

Torpor is an energy-saving strategy used by warm-blooded animals, including birds and small mammals. Similar to hibernation, although shorter in duration, torpor is a state of minimal activity, low body temperatures and reduced metabolism that helps animals conserve energy in unfavorable conditions. Some animals use torpor to survive times when food is not readily available. Hummingbirds, for example, eat nectar all day long to meet their high energy needs, but must build fat reserves to see them through their overnight fast. If they go to sleep with too little fat, they can descend into torpor to stretch out that limited energy supply and survive until morning. Many hummingbirds migrate to areas with warmer weather, where food remains available, for the winter months. The ruby-throated hummingbird (Archilochus colubris), for example, travels over 5,000 kilometers in its fall migration. Like most long-distance migrants, ruby-throated hummingbirds increase their fat stores before departing, using these stores to fuel their journey. It is thought that this bird may use torpor as a way to accelerate fat build up before its annual migration. However, it remained unclear whether hummingbirds switched from using torpor strictly in energy emergencies, to using it as strategy to prepare for migration. To shed light on this question, Eberts, Guglielmo and Welch investigated when, why and how hummingbirds save energy using torpor during the summer, and whether there are seasonal shifts in their use of torpor coinciding with migration. Eberts, Guglielmo and Welch hypothesized that a bird would initiate daily torpor if its energy stores fall below a critical level during the night, but that they may abandon this threshold (triggering torpor at higher fat levels) in late summer as a way to spare energy and gain fat before their annual migration. To test their hypotheses, Eberts, Guglielmo and Welch tracked body composition, food intake, energy expenditure and torpor use throughout summer in a group of captive ruby-throated hummingbirds. In the middle of the summer, the birds entered torpor and remained torpid for longer when they went to sleep with low fat stores. In late summer, however, the same birds were more likely to enter torpor at consistent times and when they had higher fat stores. Eberts, Guglielmo and Welch also observed that the more time birds spent in torpor, the more fat they gained. This suggests that in late summer, hummingbirds switch from using torpor as a survival strategy to using it to maximize energy savings before migration. These results clearly define the physiological rules governing torpor use in hummingbirds. They also support the long-standing assumption that torpor helps migratory species save energy and accumulate fat stores before long-haul flights.

Inter- and intra-specific variation in hair cortisol concentrations of Neotropical bats.

Quantifying hair cortisol has become popular in wildlife ecology for its practical advantages for evaluating stress. Before hair cortisol levels can be reliably interpreted, however, it is key to first understand the intrinsic factors explaining intra- and inter-specific variation. Bats are an ecologically diverse group of mammals that allow studying such variation. Given that many bat species are threatened or have declining populations in parts of their range, minimally invasive tools for monitoring colony health and identifying cryptic stressors are needed to efficiently direct conservation efforts. Here we describe intra- and inter-specific sources of variation in hair cortisol levels in 18 Neotropical bat species from Belize and Mexico. We found that fecundity is an important ecological trait explaining inter-specific variation in bat hair cortisol. Other ecological variables such as colony size, roost durability and basal metabolic rate did not explain hair cortisol variation among species. At the individual level, females exhibited higher hair cortisol levels than males and the effect of body mass varied among species. Overall, our findings help validate and accurately apply hair cortisol as a monitoring tool in free-ranging bats.

Lipid oxidation during thermogenesis in high-altitude deer mice (Peromyscus maniculatus).

When at their maximum thermogenic capacity (cold-induced V̇o2max), small endotherms reach levels of aerobic metabolism as high, or even higher, than running V̇o2max. How these high rates of thermogenesis are supported by substrate oxidation is currently unclear. The appropriate utilization of metabolic fuels that could sustain thermogenesis over extended periods may be important for survival in cold environments, like high altitude. Previous studies show that high capacities for lipid use in high-altitude deer mice may have evolved in concert with greater thermogenic capacities. The purpose of this study was to determine how lipid utilization at both moderate and maximal thermogenic intensities may differ in high- and low-altitude deer mice, and strictly low-altitude white-footed mice. We also examined the phenotypic plasticity of lipid use after acclimation to cold hypoxia (CH), conditions simulating high altitude. We found that lipids were the primary fuel supporting both moderate and maximal rates of thermogenesis in both species of mice. Lipid oxidation increased threefold in mice from 30°C to 0°C, consistent with increases in oxidation of [13C]palmitic acid. CH acclimation led to an increase in [13C]palmitic acid oxidation at 30°C but did not affect total lipid oxidation. Lipid oxidation rates at cold-induced V̇o2max were two- to fourfold those at 0°C and increased further after CH acclimation, especially in high-altitude deer mice. These are the highest mass-specific lipid oxidation rates observed in any land mammal. Uncovering the mechanisms that allow for these high rates of oxidation will aid our understanding of the regulation of lipid metabolism.

Neonicotinoid pesticides exert metabolic effects on avian pollinators.

Neonicotinoids are neurotoxic systemic insecticides applied extensively worldwide. The impacts of common neonicotinoids like imidacloprid on non-target invertebrate pollinators have been widely studied, however effects on vertebrate pollinators have received little attention. Here, we describe the first study evaluating the effects of short-term (3 d) exposure to a range of environmentally relevant concentrations ([Formula: see text] to [Formula: see text]Body Weight) of imidacloprid on wild-caught ruby-throated hummingbirds. Within 2 h of exposure, hummingbirds exhibited a significant depression in energy expenditure (up to [Formula: see text]). We did not observe significant effects on foraging behaviour measured in the subsequent 2 h to 4 h, although the effect size estimate was large (0.29). We also analyzed tissues collected 24 h after the final dose and did not observe significant effects on immune response or cholinesterase activity, although this may be related to our small sample size. We determined that hummingbirds excrete imidacloprid quickly (elimination half-life of [Formula: see text]) relative to other bird species. Hummingbirds have high energetic demands and store relatively little energy, especially during migration and breeding seasons. Therefore, changes in their metabolism following exposures to imidacloprid observed herein could bear important survivorship consequences for hummingbirds.

Micronucleus Test Reveals Genotoxic Effects in Bats Associated with Agricultural Activity.

Bats play a vital role in our ecosystems and economies as natural pest-control agents, seed dispersers, and pollinators. Agricultural intensification, however, can impact bats foraging near crops, affecting the ecosystem services they provide. Exposure to pesticides, for example, may induce chromosome breakage or missegregation that can result in micronucleus formation. Detection of micronuclei is a simple, inexpensive, and relatively minimally invasive technique commonly used to evaluate chemical genotoxicity but rarely applied to assess wildlife genotoxic effects. We evaluated the suitability of the micronucleus test as a biomarker of genotoxicity for biomonitoring field studies in bats. We collected blood samples from insectivorous bats roosting in caves surrounded by different levels of disturbance (agriculture, human settlements) in Colima and Jalisco, west central Mexico. Then, we examined the frequency of micronucleus inclusions in erythrocytes using differentially stained blood smears. Bats from caves surrounded by proportionately more (53%) land used for agriculture and irrigated year-round had higher micronucleus frequency than bats from a less disturbed site (15% agriculture). We conclude that the micronucleus test is a sensitive method to evaluate genotoxic effects in free-ranging bats and could provide a useful biomarker for evaluating risk of exposure in wild populations. Environ Toxicol Chem 2021;40:202-207. © 2020 SETAC.

Glucose transporter expression and regulation following a fast in the ruby-throated hummingbird, Archilochus colubris.

Hummingbirds, subsisting almost exclusively on nectar sugar, face extreme challenges to blood sugar regulation. The capacity for transmembrane sugar transport is mediated by the activity of facilitative glucose transporters (GLUTs) and their localisation to the plasma membrane (PM). In this study, we determined the relative protein abundance of GLUT1, GLUT2, GLUT3 and GLUT5 via immunoblot using custom-designed antibodies in whole-tissue homogenates and PM fractions of flight muscle, heart and liver of ruby-throated hummingbirds (Archilochus colubris). The GLUTs examined were detected in nearly all tissues tested. Hepatic GLUT1 was minimally present in whole-tissue homogenates and absent win PM fractions. GLUT5 was expressed in flight muscles at levels comparable to those of the liver, consistent with the hypothesised uniquely high fructose uptake and oxidation capacity of hummingbird flight muscles. To assess GLUT regulation, we fed ruby-throated hummingbirds 1 mol l-1 sucrose ad libitum for 24 h followed by either 1 h of fasting or continued feeding until sampling. We measured relative GLUT abundance and concentration of circulating sugars. Blood fructose concentration in fasted hummingbirds declined (∼5 mmol l-1 to ∼0.18 mmol l-1), while fructose-transporting GLUT2 and GLUT5 abundance did not change in PM fractions. Blood glucose concentrations remained elevated in fed and fasted hummingbirds (∼30 mmol l-1), while glucose-transporting GLUT1 and GLUT3 in flight muscle and liver PM fractions, respectively, declined in fasted birds. Our results suggest that glucose uptake capacity is dynamically reduced in response to fasting, allowing for maintenance of elevated blood glucose levels, while fructose uptake capacity remains constitutively elevated promoting depletion of blood total fructose within the first hour of a fast.

Metabolic partitioning of sucrose and seasonal changes in fat turnover rate in ruby-throated hummingbirds (Archilochus colubris).

Hummingbirds fuel their high energy needs with the fructose and glucose in their nectar diets. These sugars are used both to fuel immediate energy needs and to build fat stores to fuel future fasting periods. Fasting hummingbirds can deplete energy stores in only hours and need to be continuously replacing these stores while feeding and foraging. Whether and how hummingbirds partition dietary fructose and glucose towards immediate oxidation versus fat storage is unknown. Using a chronic stable isotope tracer methodology, we examined whether glucose or fructose is preferentially used for de novo lipogenesis in ruby-throated hummingbirds (Archilochus colubris). Potential seasonal changes were correlated with variation in the overall daily energy expenditure. We fed ruby-throated hummingbirds sucrose-based diets enriched with 13C on either the glucose or the fructose portion of the disaccharide for 5 days. Isotopic incorporation into fat stores was measured via the breath 13C signature while fasting (oxidizing fat) during the winter and summer seasons. We found greater isotopic enrichment of fat stores when glucose was labelled compared with fructose, suggesting preference for glucose as a substrate for fatty acid synthesis. We also found a seasonal effect on fat turnover rate. Faster turnover rates occurred during the summer months, when birds maintained lower body mass, fat stores and exhibited higher daily nectar intake compared with winter. This demonstrates that fat turnover rate can substantially vary with changing energy expenditure and body composition; however, the partitioning of sucrose towards de novo fatty acid synthesis remains constant.

Low ambient temperature reduces the time for fuel switching in the ruby-throated hummingbird (Archilochus colubris).

Physiological adaptations that enhance flux through the sugar oxidation cascade permit hummingbirds to rapidly switch between burning lipids when fasted to burning ingested sugars when fed. Hummingbirds may be able to exert control over the timing and extent of use of ingested sugars by varying digestive rates when under pressure to accumulate energy stores or acquire energy in response to heightened energy demands. We hypothesized that hummingbirds would modulate the timing of a switch to reliance on ingested sugars differently when facing distinct energetic demands (cool versus warm ambient temperatures). The timing of the oxidation of a single nectar meal to fuel metabolism was assessed by open-flow respirometry, while the time to first excretion following the meal was used as a proxy for digestive throughput time. As predicted, birds showed a more rapid switch in respiratory exchange ratio (RER = rate of O2 consumption/CO2 production) and excreted earlier when held at cool temperatures compared to warm. In both cases, RER peaked barely above 1.0 indicating ingested sugar fueled ≈100% of resting metabolism. Our findings suggest that energetic demands modulate the rate of fuel switching through shifts of the sugar oxidation cascade. The speed of this shift may involve decreases in gut passage times which have previously been thought to be inflexible, or may be caused by changes in circulation as a result of low ambient temperature.

Maternal predator odour exposure programs metabolic responses in adult offspring.

A cardinal feature of the reaction to stress is the promotion of energy mobilization, enabling appropriate behavioural responses. Predator odours are naturalistic and ecologically relevant stressors present over evolutionary timescales. In this study, we asked whether maternal predator odour exposure could program long-term energy mobilization in C57BL/6 mice offspring. To test this hypothesis, we measured rates of oxygen consumption in prenatally predator odour exposed mice in adulthood while controlling for levels of locomotor activity at baseline and under stress. Circulating thyroid hormone levels and the transcript abundance of key regulators of the hypothalamic-pituitary-thyroid axis within the periventricular nucleus (PVN) of the hypothalamus and in the liver, including carriers and receptors and thyrotropin-releasing hormone, were measured as endocrine mediators facilitating energy availability. Prenatally predator odour exposed mice of both sexes mobilized more energy during lower energy demand periods of the day and under stressful conditions. Further, prenatally predator odour exposed mice displayed modifications of their hypothalamic-pituitary-thyroid axis through increased circulating thyroxine and thyroid hormone receptor α within the PVN and decreased transthyretin in the liver. Overall, these results suggest that maternal exposure to predator odour is sufficient to increase long-term energy mobilization in adult offspring.

Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris.

Background: Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. Findings: We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. Conclusions: We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

Integrating morphology and kinematics in the scaling of hummingbird hovering metabolic rate and efficiency.

Wing kinematics and morphology are influential upon the aerodynamics of flight. However, there is a lack of studies linking these variables to metabolic costs, particularly in the context of morphological adaptation to body size. Furthermore, the conversion efficiency from chemical energy into movement by the muscles (mechanochemical efficiency) scales with mass in terrestrial quadrupeds, but this scaling relationship has not been demonstrated within flying vertebrates. Positive scaling of efficiency with body size may reduce the metabolic costs of flight for relatively larger species. Here, we assembled a dataset of morphological, kinematic, and metabolic data on hovering hummingbirds to explore the influence of wing morphology, efficiency, and mass on hovering metabolic rate (HMR). We hypothesize that HMR would decline with increasing wing size, after accounting for mass. Furthermore, we hypothesize that efficiency will increase with mass, similarly to other forms of locomotion. We do not find a relationship between relative wing size and HMR, and instead find that the cost of each wingbeat increases hyperallometrically while wingbeat frequency declines with increasing mass. This suggests that increasing wing size is metabolically favourable over cycle frequency with increasing mass. Further benefits are offered to larger hummingbirds owing to the positive scaling of efficiency.

The Metabolic Flexibility of Hovering Vertebrate Nectarivores.

Foraging hummingbirds and nectar bats oxidize both glucose and fructose from nectar at exceptionally high rates. Rapid sugar flux is made possible by adaptations to digestive, cardiovascular, and metabolic physiology affecting shared and distinct pathways for the processing of each sugar. Still, how these animals partition and regulate the metabolism of each sugar and whether this occurs differently between hummingbirds and bats remain unclear.

Evidence of high transport and phosphorylation capacity for both glucose and fructose in the ruby-throated hummingbird (Archilochus colubris).

Hummingbirds are able to fuel hovering flight entirely with recently ingested glucose or fructose. Among vertebrates, several steps of sugar flux from circulation to skeletal muscle are potentially rate-limiting, including transport into muscle and subsequent phosphorylation. While capacities for glucose flux are substantial, capacities for fructose flux are comparatively low. The mechanisms underlying apparent high rates of glucose and fructose oxidation in hummingbird flight muscle remain unclear. We examined relative expression of facilitative sugar transporters (GLUTs) and enzymes of fructolysis in ruby-throated hummingbird (Archilochus colubris) tissues involved in energy homeostasis and flight, via qPCR and measured hexokinase activity in pectoralis in vitro. We hypothesized that expression of these genes was upregulated in hummingbird flight muscle compared to other vertebrates. We found that hummingbird pectoralis had high relative transcript abundance of GLUT1 and GLUT5 compared to expression profiles of other vertebrates. In particular, GLUT5 expression in pectoralis was similar to that of intestine. We demonstrated minimal relative densities of fructolytic enzymes in pectoralis, suggesting that the ketohexokinase pathway does not rapidly metabolize fructose in these muscles. Instead, we found that the capacity for phosphorylation of either glucose or fructose by hexokinase is very high in pectoralis in vitro. The contributions of individual hexokinase isoforms remain to be determined. Our results further characterize the strategies by which hummingbirds, and perhaps other nectarivores, accomplish rapid sugar flux. High transport and sugar phosphorylation capacities appear to exist in flight muscle, though the enzymatic pathways that catalyze the phosphorylation of sugar in muscle remain uncertain.

The biomechanical origin of extreme wing allometry in hummingbirds.

Flying animals of different masses vary widely in body proportions, but the functional implications of this variation are often unclear. We address this ambiguity by developing an integrative allometric approach, which we apply here to hummingbirds to examine how the physical environment, wing morphology and stroke kinematics have contributed to the evolution of their highly specialised flight. Surprisingly, hummingbirds maintain constant wing velocity despite an order of magnitude variation in body weight; increased weight is supported solely through disproportionate increases in wing area. Conversely, wing velocity increases with body weight within species, compensating for lower relative wing area in larger individuals. By comparing inter- and intraspecific allometries, we find that the extreme wing area allometry of hummingbirds is likely an adaptation to maintain constant burst flight capacity and induced power requirements with increasing weight. Selection for relatively large wings simultaneously maximises aerial performance and minimises flight costs, which are essential elements of humming bird life history.

Sugar Metabolism in Hummingbirds and Nectar Bats.

Hummingbirds and nectar bats coevolved with the plants they visit to feed on floral nectars rich in sugars. The extremely high metabolic costs imposed by small size and hovering flight in combination with reliance upon sugars as their main source of dietary calories resulted in convergent evolution of a suite of structural and functional traits. These allow high rates of aerobic energy metabolism in the flight muscles, fueled almost entirely by the oxidation of dietary sugars, during flight. High intestinal sucrase activities enable high rates of sucrose hydrolysis. Intestinal absorption of glucose and fructose occurs mainly through a paracellular pathway. In the fasted state, energy metabolism during flight relies on the oxidation of fat synthesized from previously-ingested sugar. During repeated bouts of hover-feeding, the enhanced digestive capacities, in combination with high capacities for sugar transport and oxidation in the flight muscles, allow the operation of the "sugar oxidation cascade", the pathway by which dietary sugars are directly oxidized by flight muscles during exercise. It is suggested that the potentially harmful effects of nectar diets are prevented by locomotory exercise, just as in human hunter-gatherers who consume large quantities of honey.

Wingbeat kinematics and energetics during weightlifting in hovering hummingbirds across an elevational gradient.

Hummingbirds differentially modify flight kinematics in response to the type of challenge imposed. Weightlifting is associated with increases in stroke amplitude (the angle swept by the wings) to increase the angular velocity of the wings and generate the requisite lift, but only up to 160°. Conversely, flight in hypodense air is accomplished by increasing the angular velocity of the wing through increases in wingbeat frequency and stroke amplitudes, with larger increases in amplitude than seen in weightlifting flight. The kinematic differences between these two challenges may be facilitated by the lower energetic costs associated with overcoming drag and inertial forces over the wing during hypodense flight. Thus, we hypothesized that energetic expenditure is what limits the kinematics of weightlifting flight, with lower air densities permitting increases in angular velocity at comparatively lower costs. To explore the kinematic and energetic effects of air density and weightlifting on hovering flight performance, video and respirometric recordings of weightlifting were performed on four species of hummingbirds across an elevational gradient. Contrary to our hypothesis, wingbeat frequency did not vary due to elevation. Instead, wingbeat frequency seems to increase depending on the power requirements for sustaining hovering flight. Furthermore, metabolic rates during hovering increased with angular velocity alone, independent of elevation. Thus, it appears that the differential responses to flight challenges are not driven by variation in the flight media.