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.

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.

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.

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.

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.

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.

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.

Always chew your food: freshwater stingrays use mastication to process tough insect prey.

Chewing, characterized by shearing jaw motions and high-crowned molar teeth, is considered an evolutionary innovation that spurred dietary diversification and evolutionary radiation of mammals. Complex prey-processing behaviours have been thought to be lacking in fishes and other vertebrates, despite the fact that many of these animals feed on tough prey, like insects or even grasses. We investigated prey capture and processing in the insect-feeding freshwater stingray Potamotrygon motoro using high-speed videography. We find that Potamotrygon motoro uses asymmetrical motion of the jaws, effectively chewing, to dismantle insect prey. However, CT scanning suggests that this species has simple teeth. These findings suggest that in contrast to mammalian chewing, asymmetrical jaw action is sufficient for mastication in other vertebrates. We also determined that prey capture in these rays occurs through rapid uplift of the pectoral fins, sucking prey beneath the ray's body, thereby dissociating the jaws from a prey capture role. We suggest that the decoupling of prey capture and processing facilitated the evolution of a highly kinetic feeding apparatus in batoid fishes, giving these animals an ability to consume a wide variety of prey, including molluscs, fishes, aquatic insect larvae and crustaceans. We propose Potamotrygon as a model system for understanding evolutionary convergence of prey processing and chewing in vertebrates.

The cost of digestion in the fish-eating myotis (Myotis vivesi).

Flying vertebrates, such as bats, face special challenges with regards to the throughput and digestion of food. On the one hand, as potentially energy-limited organisms, bats must ingest and assimilate energy efficiently in order to satisfy high resting and active metabolic demands. On the other hand, the assimilation of nutrients must be accomplished using a digestive tract that is, compared with that of similarly sized non-flying vertebrates, significantly shorter. Despite these competing demands, and the relative breadth of dietary diversity among bats, little work has been done describing the cost of digestion, termed 'specific dynamic action' (SDA). Here, we provide the first systematic assessment of the SDA response in a bat, the fish-eating myotis (Myotis vivesi). Given the shorter digestive tract and the relatively higher resting and active metabolic rates of bats in general, and based on anecdotal published evidence, we hypothesized that the SDA response in fish-eating myotis would be dependent on meal size and both significantly more brief and intense than in small, non-flying mammals. In agreement with our hypothesis, we found that the peak metabolic rate during digestion, relative to rest, was significantly higher in these bats compared with any other mammals or vertebrates, except for some infrequently eating reptiles and amphibians. Additionally, we found that the magnitude and duration of the SDA response were related to meal size. However, we found that the duration of the SDA response, while generally similar to reported gut transit times in other small bats, was not substantially shorter than in similarly sized non-flying mammals.

Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off.

Heat exposure limits pentose phosphate pathway activity in bumblebees.

Bumblebee populations across the globe are experiencing substantial declines due to climate change, with major consequences for pollination services in both natural and agricultural settings. Using an economically important species, Bombus impatiens, we explored the physiological mechanisms that may cause susceptibility to extreme heat events. We tested the hypothesis that heat exposure limits the activity of the pentose phosphate pathway (PPP)-a parallel pathway to glycolysis that can use nectar sugar to generate antioxidant potential and combat oxidative stress. Using isotopically labelled glucose, we tracked PPP activity in B. impatiens at rest, during exercise and during a post-exercise recovery period under two different temperature regimes (22°C and 32°C). We found that the PPP is routinely used by B. impatiens at moderate temperatures, but that its activity is markedly reduced when ATP demands are high, such as during periods of exercise and heat exposure. We also exposed B. impatiens to either 22°C or 32°C for 5 hours and assessed levels of oxidative damage (lipid peroxidation, protein carbonyls) and antioxidant potential [reduced (GSH) and oxidized (GSSG) glutathione concentrations]. Interestingly, bees exhibited little oxidative damage after the thermal exposure, but we found a lower GSH:GSSG ratio in 32°C-exposed bees, reflecting lower antioxidant potential. Overall, our study demonstrates that acute heat stress severely limits PPP activity and may constrain antioxidant potential in B. impatiens. The repeated attenuation of this pathway in a warming climate may have more severe physiological consequences for this species, with potential implications for pollination services across North America.

Neuromuscular control of hovering wingbeat kinematics in response to distinct flight challenges in the ruby-throated hummingbird, Archilochus colubris.

While producing one of the highest sustained mass-specific power outputs of any vertebrate, hovering hummingbirds must also precisely modulate the activity of their primary flight muscles to vary wingbeat kinematics and modulate lift production. Although recent studies have begun to explore how pectoralis (the primary downstroke muscle) neuromuscular activation and wingbeat kinematics are linked in hummingbirds, it is unclear whether different species modulate these features in similar ways, or consistently in response to distinct flight challenges. In addition, little is known about how the antagonist, the supracoracoideus, is modulated to power the symmetrical hovering upstroke. We obtained simultaneous recordings of wingbeat kinematics and electromyograms from the pectoralis and supracoracoideus in ruby-throated hummingbirds (Archilochus colubris) hovering under the following conditions: (1) ambient air, (2) air density reduction trials, (3) submaximal load-lifting trials and (4) maximal load-lifting trials. Increased power output was achieved through increased stroke amplitude during air density reduction and load-lifting trials, but wingbeat frequency only increased at low air densities. Overall, relative electromyographic (EMG) intensity was the best predictor of stroke amplitude and is correlated with angular velocity of the wingtip. The relationship between muscle activation intensity and kinematics was independent of treatment type, indicating that reduced drag on the wings in hypodense air did not lead to high wingtip angular velocities independently of increased muscle work. EMG bursts consistently began and ended before muscle shortening under all conditions. During all sustained hovering, spike number per burst consistently averaged 1.2 in the pectoralis and 2.0 in the supracoracoideus. The number of spikes increased to 2.5-3 in both muscles during maximal load-lifting trials. Despite the relative kinematic symmetry of the hovering downstroke and upstroke, the supracoracoideus was activated ~1 ms earlier, EMG bursts were longer (~0.9 ms) and they exhibited 1.6 times as many spikes per burst. We hypothesize that earlier and more sustained activation of the supracoracoideus fibres is necessary to offset the greater compliance resulting from the presence of the supracoracoid tendon.

Very low force-generating ability and unusually high temperature dependency in hummingbird flight muscle fibers.

Hummingbird flight muscle is estimated to have among the highest mass-specific power output among vertebrates, based on aerodynamic models. However, little is known about the fundamental contractile properties of their remarkable flight muscles. We hypothesized that hummingbird pectoralis fibers generate relatively low force when activated in a tradeoff for high shortening speeds associated with the characteristic high wingbeat frequencies that are required for sustained hovering. Our objective was to measure maximal force-generating ability (maximal force/cross-sectional area, Po/CSA) in single, skinned fibers from the pectoralis and supracoracoideus muscles, which power the wing downstroke and upstroke, respectively, in hummingbirds (Calypte anna) and in another similarly sized species, zebra finch (Taeniopygia guttata), which also has a very high wingbeat frequency during flight but does not perform a sustained hover. Mean Po/CSA in hummingbird pectoralis fibers was very low - 1.6, 6.1 and 12.2 kN m(-2), at 10, 15 and 20°C, respectively. Po/CSA in finch pectoralis fibers was also very low (for both species, ~5% of the reported Po/CSA of chicken pectoralis fast fibers at 15°C). Q10-force (force generated at 20°C/force generated at 10°C) was very high for hummingbird and finch pectoralis fibers (mean=15.3 and 11.5, respectively) compared with rat slow and fast fibers (1.8 and 1.9, respectively). Po/CSA in hummingbird leg fibers was much higher than in pectoralis fibers at each temperature, and the mean Q10-force was much lower. Thus, hummingbird and finch pectoralis fibers have an extremely low force-generating ability compared with other bird and mammalian limb fibers, and an extremely high temperature dependence of force generation. However, the extrapolated maximum force-generating ability of hummingbird pectoralis fibers in vivo (~48 kN m(-2)) is substantially higher than the estimated requirements for hovering flight of C. anna. The unusually low Po/CSA of hummingbird and zebra finch pectoralis fibers may reflect a constraint imposed by a need for extremely high contraction frequencies, especially during hummingbird hovering.

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.

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.

The sugar oxidation cascade: aerial refueling in hummingbirds and nectar bats.

Most hummingbirds and some species of nectar bats hover while feeding on floral nectar. While doing so, they achieve some of the highest mass-specific V(O(2)) values among vertebrates. This is made possible by enhanced functional capacities of various elements of the 'O(2) transport cascade', the pathway of O(2) from the external environment to muscle mitochondria. Fasted hummingbirds and nectar bats fly with respiratory quotients (RQs; V(CO(2))/V(O(2))) of ~0.7, indicating that fat fuels flight in the fasted state. During repeated hover-feeding on dietary sugar, RQ values progressively climb to ~1.0, indicating a shift from fat to carbohydrate oxidation. Stable carbon isotope experiments reveal that recently ingested sugar directly fuels ~80 and 95% of energy metabolism in hover-feeding nectar bats and hummingbirds, respectively. We name the pathway of carbon flux from flowers, through digestive and cardiovascular systems, muscle membranes and into mitochondria the 'sugar oxidation cascade'. O(2) and sugar oxidation cascades operate in parallel and converge in muscle mitochondria. Foraging behavior that favours the oxidation of dietary sugar avoids the inefficiency of synthesizing fat from sugar and breaking down fat to fuel foraging. Sugar oxidation yields a higher P/O ratio (ATP made per O atom consumed) than fat oxidation, thus requiring lower hovering V(O(2)) per unit mass. We propose that dietary sugar is a premium fuel for flight in nectarivorous, flying animals.

The power of feeder-mask respirometry as a method for examining hummingbird energetics.

Many birds spend important portions of their time and energy flying. For this reason, quantification of metabolic rates during flight is of crucial importance to understanding avian energy balance. Measurement of organismal gas exchange rates using a mask enclosing the whole head or respiratory orifices has served as an important tool for studying animal energetics because it can free the rest of the body, permitting movement. Application of so-called "mask respirometry" to the study of avian forward flight energetics presents unique challenges because birds must be tethered to gas analysis equipment thus typically necessitating use of a wind tunnel. Resulting potential alterations to a study organism's behaviour, physiology, and aerodynamics have made interpretation of such studies contentious. In contrast, the study of hovering flight energetics in hummingbirds using a specialized form of mask respirometry is comparatively easy and can be done without a wind tunnel. Small size, hovering flight, and a nectarivorous diet are characteristics shared by all hummingbird species that make these birds ideally suited for this approach. Specifically, nectar feeders are modified to function as respirometry masks hummingbirds voluntarily respire into when hover-feeding. Feeder-mask based respirometry has revealed some of the highest vertebrate metabolic rates in hovering hummingbirds. In this review I discuss techniques for the successful measurement of metabolic rate using feeder-mask respirometry. I also emphasize how this technique has been used to address fundamental questions regarding avian flight energetics such as capacities for fuel use and mechanisms by which ecology, behaviour and energy balance are linked.

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.

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.

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.