Papers by Kenneth C Welch
Flying animals of different masses vary widely in body proportions, but the functional implicatio... more 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.
Hummingbirds and nectar bats coevolved with the plants they visit to feed on floral nectars rich ... more 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.
Food restriction affects the activation of the immune system although the metabolic cost associat... more Food restriction affects the activation of the immune system although the metabolic cost associated with mounting such a response has rarely been examined except in model animals. Wild animals are constantly exposed to variations in the availability of food resources and they need to balance their energy budget to fight against pathogens. We examined the effect of food restriction in the fish eating Myotis (Myotis vivesi), a species of bat that experiences periods in which foraging is limited due to ambient conditions. We tested the hypothesis that acute food restriction (∼65% restriction for 1 night) would reduce the caloric response to lipopolysaccharidae (LPS) injection compared to bats fed ad libitum. We also measured a proxy for body temperature (Tskin) and expected reduced fever development when food intake was limited. Bats on the restricted diet had similar resting metabolic rate, total caloric cost and Tskin after the LPS challenge than when fed ad libitum. However, there was a delay in the metabolic and pyrogenic responses when bats were on the restricted diet. The effect of acute food restriction in delaying the hyperthermia development in fish eating Myotis might be of importance for its capacity to fight pathogens. Similar to other bats, the fish eating Myotis can fast for several consecutive days by entering torpor and future work is warranted to understand the effect of long periods of food restriction on bat immune response.
Please cite this article as: Otálora-Ardila, Aída, Herrera M., L.Gerardo, Flores-Martínez, José J... more Please cite this article as: Otálora-Ardila, Aída, Herrera M., L.Gerardo, Flores-Martínez, José Juan, Welch, Kenneth C., The effect of short-term food restriction on the metabolic cost of the acute phase response in the fish-eating Myotis (Myotis vivesi).Mammalian Biology http://dx.
Please cite this article as: Otálora-Ardila, Aída, Herrera M., L.Gerardo, Flores-Martínez, José J... more Please cite this article as: Otálora-Ardila, Aída, Herrera M., L.Gerardo, Flores-Martínez, José Juan, Welch, Kenneth C., The effect of short-term food restriction on the metabolic cost of the acute phase response in the fish-eating Myotis (Myotis vivesi).Mammalian Biology http://dx.
Inflammation and activation of the acute phase response (APR) are energetically demanding process... more Inflammation and activation of the acute phase response (APR) are energetically demanding processes that protect against pathogens. Phytohaemagglutinin (PHA) and lipopoly-saccharide (LPS) are antigens commonly used to stimulate inflammation and the APR, respectively. We tested the hypothesis that the APR after an LPS challenge was energetically more costly than the inflammatory response after a PHA challenge in the fish-eating Myotis bat (Myotis vivesi). We measured resting metabolic rate (RMR) after bats were administered PHA and LPS. We also measured skin temperature (T skin) after the LPS challenge and skin swelling after the PHA challenge. Injection of PHA elicited swelling that lasted for several days but changes in RMR and body mass were not significant. LPS injection produced a significant increase in T skin and in RMR, and significant body mass loss. RMR after LPS injection increased by 140–185% and the total cost of the response was 6.50 kJ. Inflammation was an energetically low-cost process but the APR entailed a significant energetic investment. Examination of APR in other bats suggests that the way in which bats deal with infections might not be uniform.
Keywords: energy balance mass migration passive integrated transponder (PIT) radiofrequency ident... more Keywords: energy balance mass migration passive integrated transponder (PIT) radiofrequency identification (RFID) Many avian species fatten to fuel migratory flights. However, the amount of fat deposited prior to departure is variable depending on individual migration strategies. Despite their small size and high mass-specific metabolic rates, migratory hummingbirds at isolated meadows can fatten up to 44% in just 4 days prior to resuming migration, suggesting profound changes in energy acquisition. However, it remains to be seen whether hummingbirds fatten at the breeding grounds prior to initiating migration. Using feeder stations outfitted with radiofrequency identification readers and digital scales, we identified a subset of premigratory ruby-throated hummingbirds that exhibited significant mass gain in the 4 days leading up to migration (premigratory fattening) and identified others that did not (premigratory nonfattening). We further assessed foraging behaviour, monitored individual mass throughout the day and calculated rates of overnight mass loss to understand what behavioural variation allowed some premigratory birds to rapidly fatten. Premigratory fattening hummingbirds abandoned foraging restraint during the middle of the day, a behaviour thought to enhance aerial agility, and increased foraging effort during both the middle of the day and the evenings by increasing the duration but not the frequency of feeder visits. Groups did not differ in their morning foraging strategy. Premigratory fattening hummingbirds also lost mass overnight at reduced rates, implying that birds conserved energy to minimize the depletion of existing fat stores, possibly via increased nocturnal torpor use. Fattening hummingbirds used a two-pronged approach of increasing energy intake during specific daily periods and reducing overnight energy expenditure to achieve substantial premigratory mass gain over just 4 days. However, not all hummingbirds adopted this premigratory fuelling strategy; those that did were adults (>1 year old), suggesting that the use of a premigratory fuelling strategy may be age related.
Chewing, characterized by shearing jaw motions and high-crowned molar teeth, is considered an evo... more 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.
Hummingbirds differentially modify flight kinematics in response to the type of challenge imposed... more 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.
The carbon isotope values in the exhaled breath
of an animal mirror the carbon isotope values of ... more The carbon isotope values in the exhaled breath
of an animal mirror the carbon isotope values of the metabolic
fuels being oxidized. The measurement of stable carbon
isotopes in carbon dioxide is called 13C-breath testing
and offers a minimally invasive method to study substrate
oxidation in vivo. 13C-breath testing has been broadly used
to study human exercise, nutrition, and pathologies since
the 1970s. Owing to reduced use of radioactive isotopes
and the increased convenience and affordability of 13C-analyzers,
the past decade has witnessed a sharp increase in the
use of breath testing throughout comparative physiology—
especially to answer questions about how and when animals
oxidize particular nutrients. Here, we review the practical
aspects of 13C-breath testing and identify the strengths
and weaknesses of different methodological approaches
including the use of natural abundance versus artificiallyenriched
13C tracers. We critically compare the information
that can be obtained using different experimental protocols
such as diet-switching versus fuel-switching. We also
discuss several factors that should be considered when
designing breath testing experiments including extrinsic
versus intrinsic 13C-labelling and different approaches to
model nutrient oxidation. We use case studies to highlight
the myriad applications of 13C-breath testing in basic and
clinical human studies as well as comparative studies of
fuel use, energetics, and carbon turnover in multiple vertebrate
and invertebrate groups. Lastly, we call for increased
and rigorous use of 13C-breath testing to explore a variety
of new research areas and potentially answer long standing
questions related to thermobiology, locomotion, and
nutrition.
Flying vertebrates, such as bats, face special challenges with regards
to the throughput and dige... more 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 fisheating
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, nonflying
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.
We sought to characterize the ability of hummingbirds to fuel
their energetically expensive hover... more We sought to characterize the ability of hummingbirds to fuel
their energetically expensive hovering flight using dietary sugar
by a combination of respirometry and stable carbon isotope
techniques. Broadtailed hummingbirds (Selasphorus platycercus)
were maintained on a diet containing beet sugar with an
isotopic composition characteristic of C3 plants. Hummingbirds
were fasted and then offered a solution containing cane
sugar with an isotopic composition characteristic of C4 plants.
By monitoring the rates of CO2 production and O2 consumption,
as well as the stable carbon isotope composition of expired
CO2, we were able to estimate the relative contributions of
carbohydrate and fat, as well as the absolute rate at which
dietary sucrose was oxidized during hovering. The combination
of respirometry and carbon isotope analysis revealed that hummingbirds
initially oxidized endogenous fat following a fast and
then progressively oxidized proportionately more carbohydrates.
The contribution from dietary sources increased with
each feeding bout, and by 20 min after the first meal, dietary
sugar supported ∼74% of hovering metabolism. The ability of
hummingbirds to satisfy the energetic requirements of hovering
flight mainly with recently ingested sugar is unique among
vertebrates. Our finding provides an example of evolutionary
convergence in physiological and biochemical traits among unrelated
nectar-feeding animals.
In most vertebrates, uptake and oxidation of
circulating sugars by locomotor muscles rises with i... more In most vertebrates, uptake and oxidation of
circulating sugars by locomotor muscles rises with increasing
exercise intensity. However, uptake rate by muscle plateaus
at moderate aerobic exercise intensities and intracellular
fuels dominate at oxygen consumption rates of 50 %
of maximum or more. Further, uptake and oxidation of circulating
fructose by muscle is negligible. In contrast, hummingbirds
and nectar bats are capable of fueling expensive
hovering flight exclusively, or nearly completely, with
dietary sugar. In addition, hummingbirds and nectar bats
appear capable of fueling hovering flight completely with
fructose. Three crucial steps are believed to be rate limiting
to muscle uptake of circulating glucose or fructose in
vertebrates: (1) delivery to muscle; (2) transport into muscle
through glucose transporter proteins (GLUTs); and (3)
phosphorylation of glucose by hexokinase (HK) within the
muscle. In this review, we summarize what is known about
the functional upregulation of exogenous sugar flux at each
of these steps in hummingbirds and nectar bats. High cardiac
output, capillary density, and blood sugar levels in hummingbirds and bats enhance sugar delivery to muscles
(step 1). Hummingbird and nectar bat flight muscle
fibers have relatively small cross-sectional areas and thus
relatively high surface areas across which transport can
occur (step 2). Maximum HK activities in each species are
enough for carbohydrate flux through glycolysis to satisfy
100 % of hovering oxidative demand (step 3). However,
qualitative patterns of GLUT expression in the muscle (step
2) raise more questions than they answer regarding sugar
transport in hummingbirds and suggest major differences
in the regulation of sugar flux compared to nectar bats.
Behavioral and physiological similarities among hummingbirds,
nectar bats, and other vertebrates suggest enhanced
capacities for exogenous fuel use during exercise may be
more wide spread than previously appreciated. Further,
how the capacity for uptake and phosphorylation of circulating
fructose is enhanced remains a tantalizing unknown.
Most hummingbirds and some species of nectar bats hover while feeding on floral nectar. While doi... more 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 VO2 values among vertebrates. This is made possible by enhanced functional capacities of various
elements of the ‘O2 transport cascade’, the pathway of O2 from the external environment to muscle mitochondria. Fasted
hummingbirds and nectar bats fly with respiratory quotients (RQs; VCO2/VO2) 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’.
O2 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 VO2 per unit
mass. We propose that dietary sugar is a premium fuel for flight in nectarivorous, flying animals.
Given their high metabolic rates, nectarivorous diet, and ability to directly fuel their energeti... more Given their high metabolic rates, nectarivorous diet, and ability to directly fuel their energetically-expensive
flight using recently-ingested sugar, we tested the hypothesis that Pallas long tongued nectar bats
(Glossophaga soricina) possess flight muscles similar to those of hummingbirds with respect to enzymatic
flux capacities in bioenergetic pathways. In addition, we compared these biochemical capacities with flux
rates achieved in vivo during hovering flight. Rates of oxygen consumption (V̇O2) were measured during
hover-feeding and used to estimate rates of ATP turnover, glucose and long-chain fatty acid oxidation per unit
mass of flight muscle. Enzyme Vmax values at key steps in glucose and fatty acid oxidation obtained in vitro
from pectoralis muscle samples exceed those found in the locomotory muscles of other species of small
mammals and resemble data obtained from hummingbird flight muscles. The ability of nectar bats and
hummingbirds to hover in fed and fasted states, fueled almost exclusively by carbohydrate or fat,
respectively, allowed the estimation of fractional velocities (v/Vmax) at both the hexokinase and carnitine
palmitoyltransferase-2 steps in glucose and fatty acid oxidation, respectively. The results further support the
hypothesis of convergent evolution in biochemical and physiological traits in nectar bats and hummingbirds.
Hummingbirds and nectarivorous bats in flight display some of the highest
rates of aerobic metabo... more Hummingbirds and nectarivorous bats in flight display some of the highest
rates of aerobic metabolism among vertebrates. Analysis of the pathway of
oxygen, i.e., the “oxygen transport cascade”, reveals the concerted upregulation
of capacities for O2 flux from the external environment, through the respiratory
and cardiovascular systems, into muscle mitochondria. Pathways for aerobic energy
metabolism are highly conserved, but enzymatic capacities for carbohydrate and
fatty acid oxidation, as well as for aerobic ATP synthesis, are also upregulated in
concert. Despite evidence indicating sufficient capacities for fatty acid oxidation to
support hovering, repeated bouts of hover-feeding in hummingbirds and nectar bats
involve the oxidation of carbohydrate. Recent studies reveal that recently ingested
sugar directly fuels flight, giving rise to the concept of the “sucrose oxidation cascade”.
The ecological and bioenergetic advantages conferred by sugar oxidation
during foraging are discussed.
We investigated differences between sizes of home ranges using trapping and radiotelemetry
data f... more We investigated differences between sizes of home ranges using trapping and radiotelemetry
data for syntopic Peromyscus boylii and P. truei. Sizes of home ranges were calculated
from the minimum convex polygon of trap locations and radiotelemetry locations and
compared between individuals. The 2 estimates of home-range size were significantly correlated,
although on an average trapping home ranges were significantly smaller than sizes
of radiotelemetry home ranges. Home-range sizes from radiotelemetry were inversely correlated
with conspecific density, but home-range sizes from trapping were not. Thus, at
low density, radiotelemetry home ranges were significantly larger than trapping home ranges,
but at high density there was no difference between radiotelemetry and trapping home
ranges. These results indicate that radiotelemetry results in larger estimates of home-range
size, particularly at lower densities of conspecifics. The largest size estimates of home
ranges were from a combination of radiotelemetry and trapping data.
Hummingbird flight muscle is estimated to have among the highest mass-specific power output among... more 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 12.2kNm–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 (~48kNm–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.
While producing one of the highest sustained mass-specific power outputs of any vertebrate, hover... more 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 loadlifting
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 ~1ms earlier, EMG bursts were longer (~0.9ms) 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.
Uploads
Papers by Kenneth C Welch
of an animal mirror the carbon isotope values of the metabolic
fuels being oxidized. The measurement of stable carbon
isotopes in carbon dioxide is called 13C-breath testing
and offers a minimally invasive method to study substrate
oxidation in vivo. 13C-breath testing has been broadly used
to study human exercise, nutrition, and pathologies since
the 1970s. Owing to reduced use of radioactive isotopes
and the increased convenience and affordability of 13C-analyzers,
the past decade has witnessed a sharp increase in the
use of breath testing throughout comparative physiology—
especially to answer questions about how and when animals
oxidize particular nutrients. Here, we review the practical
aspects of 13C-breath testing and identify the strengths
and weaknesses of different methodological approaches
including the use of natural abundance versus artificiallyenriched
13C tracers. We critically compare the information
that can be obtained using different experimental protocols
such as diet-switching versus fuel-switching. We also
discuss several factors that should be considered when
designing breath testing experiments including extrinsic
versus intrinsic 13C-labelling and different approaches to
model nutrient oxidation. We use case studies to highlight
the myriad applications of 13C-breath testing in basic and
clinical human studies as well as comparative studies of
fuel use, energetics, and carbon turnover in multiple vertebrate
and invertebrate groups. Lastly, we call for increased
and rigorous use of 13C-breath testing to explore a variety
of new research areas and potentially answer long standing
questions related to thermobiology, locomotion, and
nutrition.
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 fisheating
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, nonflying
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.
their energetically expensive hovering flight using dietary sugar
by a combination of respirometry and stable carbon isotope
techniques. Broadtailed hummingbirds (Selasphorus platycercus)
were maintained on a diet containing beet sugar with an
isotopic composition characteristic of C3 plants. Hummingbirds
were fasted and then offered a solution containing cane
sugar with an isotopic composition characteristic of C4 plants.
By monitoring the rates of CO2 production and O2 consumption,
as well as the stable carbon isotope composition of expired
CO2, we were able to estimate the relative contributions of
carbohydrate and fat, as well as the absolute rate at which
dietary sucrose was oxidized during hovering. The combination
of respirometry and carbon isotope analysis revealed that hummingbirds
initially oxidized endogenous fat following a fast and
then progressively oxidized proportionately more carbohydrates.
The contribution from dietary sources increased with
each feeding bout, and by 20 min after the first meal, dietary
sugar supported ∼74% of hovering metabolism. The ability of
hummingbirds to satisfy the energetic requirements of hovering
flight mainly with recently ingested sugar is unique among
vertebrates. Our finding provides an example of evolutionary
convergence in physiological and biochemical traits among unrelated
nectar-feeding animals.
circulating sugars by locomotor muscles rises with increasing
exercise intensity. However, uptake rate by muscle plateaus
at moderate aerobic exercise intensities and intracellular
fuels dominate at oxygen consumption rates of 50 %
of maximum or more. Further, uptake and oxidation of circulating
fructose by muscle is negligible. In contrast, hummingbirds
and nectar bats are capable of fueling expensive
hovering flight exclusively, or nearly completely, with
dietary sugar. In addition, hummingbirds and nectar bats
appear capable of fueling hovering flight completely with
fructose. Three crucial steps are believed to be rate limiting
to muscle uptake of circulating glucose or fructose in
vertebrates: (1) delivery to muscle; (2) transport into muscle
through glucose transporter proteins (GLUTs); and (3)
phosphorylation of glucose by hexokinase (HK) within the
muscle. In this review, we summarize what is known about
the functional upregulation of exogenous sugar flux at each
of these steps in hummingbirds and nectar bats. High cardiac
output, capillary density, and blood sugar levels in hummingbirds and bats enhance sugar delivery to muscles
(step 1). Hummingbird and nectar bat flight muscle
fibers have relatively small cross-sectional areas and thus
relatively high surface areas across which transport can
occur (step 2). Maximum HK activities in each species are
enough for carbohydrate flux through glycolysis to satisfy
100 % of hovering oxidative demand (step 3). However,
qualitative patterns of GLUT expression in the muscle (step
2) raise more questions than they answer regarding sugar
transport in hummingbirds and suggest major differences
in the regulation of sugar flux compared to nectar bats.
Behavioral and physiological similarities among hummingbirds,
nectar bats, and other vertebrates suggest enhanced
capacities for exogenous fuel use during exercise may be
more wide spread than previously appreciated. Further,
how the capacity for uptake and phosphorylation of circulating
fructose is enhanced remains a tantalizing unknown.
the highest mass-specific VO2 values among vertebrates. This is made possible by enhanced functional capacities of various
elements of the ‘O2 transport cascade’, the pathway of O2 from the external environment to muscle mitochondria. Fasted
hummingbirds and nectar bats fly with respiratory quotients (RQs; VCO2/VO2) 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’.
O2 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 VO2 per unit
mass. We propose that dietary sugar is a premium fuel for flight in nectarivorous, flying animals.
flight using recently-ingested sugar, we tested the hypothesis that Pallas long tongued nectar bats
(Glossophaga soricina) possess flight muscles similar to those of hummingbirds with respect to enzymatic
flux capacities in bioenergetic pathways. In addition, we compared these biochemical capacities with flux
rates achieved in vivo during hovering flight. Rates of oxygen consumption (V̇O2) were measured during
hover-feeding and used to estimate rates of ATP turnover, glucose and long-chain fatty acid oxidation per unit
mass of flight muscle. Enzyme Vmax values at key steps in glucose and fatty acid oxidation obtained in vitro
from pectoralis muscle samples exceed those found in the locomotory muscles of other species of small
mammals and resemble data obtained from hummingbird flight muscles. The ability of nectar bats and
hummingbirds to hover in fed and fasted states, fueled almost exclusively by carbohydrate or fat,
respectively, allowed the estimation of fractional velocities (v/Vmax) at both the hexokinase and carnitine
palmitoyltransferase-2 steps in glucose and fatty acid oxidation, respectively. The results further support the
hypothesis of convergent evolution in biochemical and physiological traits in nectar bats and hummingbirds.
rates of aerobic metabolism among vertebrates. Analysis of the pathway of
oxygen, i.e., the “oxygen transport cascade”, reveals the concerted upregulation
of capacities for O2 flux from the external environment, through the respiratory
and cardiovascular systems, into muscle mitochondria. Pathways for aerobic energy
metabolism are highly conserved, but enzymatic capacities for carbohydrate and
fatty acid oxidation, as well as for aerobic ATP synthesis, are also upregulated in
concert. Despite evidence indicating sufficient capacities for fatty acid oxidation to
support hovering, repeated bouts of hover-feeding in hummingbirds and nectar bats
involve the oxidation of carbohydrate. Recent studies reveal that recently ingested
sugar directly fuels flight, giving rise to the concept of the “sucrose oxidation cascade”.
The ecological and bioenergetic advantages conferred by sugar oxidation
during foraging are discussed.
data for syntopic Peromyscus boylii and P. truei. Sizes of home ranges were calculated
from the minimum convex polygon of trap locations and radiotelemetry locations and
compared between individuals. The 2 estimates of home-range size were significantly correlated,
although on an average trapping home ranges were significantly smaller than sizes
of radiotelemetry home ranges. Home-range sizes from radiotelemetry were inversely correlated
with conspecific density, but home-range sizes from trapping were not. Thus, at
low density, radiotelemetry home ranges were significantly larger than trapping home ranges,
but at high density there was no difference between radiotelemetry and trapping home
ranges. These results indicate that radiotelemetry results in larger estimates of home-range
size, particularly at lower densities of conspecifics. The largest size estimates of home
ranges were from a combination of radiotelemetry and trapping data.
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 12.2kNm–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 (~48kNm–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.
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 loadlifting
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 ~1ms earlier, EMG bursts were longer (~0.9ms) 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.
of an animal mirror the carbon isotope values of the metabolic
fuels being oxidized. The measurement of stable carbon
isotopes in carbon dioxide is called 13C-breath testing
and offers a minimally invasive method to study substrate
oxidation in vivo. 13C-breath testing has been broadly used
to study human exercise, nutrition, and pathologies since
the 1970s. Owing to reduced use of radioactive isotopes
and the increased convenience and affordability of 13C-analyzers,
the past decade has witnessed a sharp increase in the
use of breath testing throughout comparative physiology—
especially to answer questions about how and when animals
oxidize particular nutrients. Here, we review the practical
aspects of 13C-breath testing and identify the strengths
and weaknesses of different methodological approaches
including the use of natural abundance versus artificiallyenriched
13C tracers. We critically compare the information
that can be obtained using different experimental protocols
such as diet-switching versus fuel-switching. We also
discuss several factors that should be considered when
designing breath testing experiments including extrinsic
versus intrinsic 13C-labelling and different approaches to
model nutrient oxidation. We use case studies to highlight
the myriad applications of 13C-breath testing in basic and
clinical human studies as well as comparative studies of
fuel use, energetics, and carbon turnover in multiple vertebrate
and invertebrate groups. Lastly, we call for increased
and rigorous use of 13C-breath testing to explore a variety
of new research areas and potentially answer long standing
questions related to thermobiology, locomotion, and
nutrition.
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 fisheating
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, nonflying
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.
their energetically expensive hovering flight using dietary sugar
by a combination of respirometry and stable carbon isotope
techniques. Broadtailed hummingbirds (Selasphorus platycercus)
were maintained on a diet containing beet sugar with an
isotopic composition characteristic of C3 plants. Hummingbirds
were fasted and then offered a solution containing cane
sugar with an isotopic composition characteristic of C4 plants.
By monitoring the rates of CO2 production and O2 consumption,
as well as the stable carbon isotope composition of expired
CO2, we were able to estimate the relative contributions of
carbohydrate and fat, as well as the absolute rate at which
dietary sucrose was oxidized during hovering. The combination
of respirometry and carbon isotope analysis revealed that hummingbirds
initially oxidized endogenous fat following a fast and
then progressively oxidized proportionately more carbohydrates.
The contribution from dietary sources increased with
each feeding bout, and by 20 min after the first meal, dietary
sugar supported ∼74% of hovering metabolism. The ability of
hummingbirds to satisfy the energetic requirements of hovering
flight mainly with recently ingested sugar is unique among
vertebrates. Our finding provides an example of evolutionary
convergence in physiological and biochemical traits among unrelated
nectar-feeding animals.
circulating sugars by locomotor muscles rises with increasing
exercise intensity. However, uptake rate by muscle plateaus
at moderate aerobic exercise intensities and intracellular
fuels dominate at oxygen consumption rates of 50 %
of maximum or more. Further, uptake and oxidation of circulating
fructose by muscle is negligible. In contrast, hummingbirds
and nectar bats are capable of fueling expensive
hovering flight exclusively, or nearly completely, with
dietary sugar. In addition, hummingbirds and nectar bats
appear capable of fueling hovering flight completely with
fructose. Three crucial steps are believed to be rate limiting
to muscle uptake of circulating glucose or fructose in
vertebrates: (1) delivery to muscle; (2) transport into muscle
through glucose transporter proteins (GLUTs); and (3)
phosphorylation of glucose by hexokinase (HK) within the
muscle. In this review, we summarize what is known about
the functional upregulation of exogenous sugar flux at each
of these steps in hummingbirds and nectar bats. High cardiac
output, capillary density, and blood sugar levels in hummingbirds and bats enhance sugar delivery to muscles
(step 1). Hummingbird and nectar bat flight muscle
fibers have relatively small cross-sectional areas and thus
relatively high surface areas across which transport can
occur (step 2). Maximum HK activities in each species are
enough for carbohydrate flux through glycolysis to satisfy
100 % of hovering oxidative demand (step 3). However,
qualitative patterns of GLUT expression in the muscle (step
2) raise more questions than they answer regarding sugar
transport in hummingbirds and suggest major differences
in the regulation of sugar flux compared to nectar bats.
Behavioral and physiological similarities among hummingbirds,
nectar bats, and other vertebrates suggest enhanced
capacities for exogenous fuel use during exercise may be
more wide spread than previously appreciated. Further,
how the capacity for uptake and phosphorylation of circulating
fructose is enhanced remains a tantalizing unknown.
the highest mass-specific VO2 values among vertebrates. This is made possible by enhanced functional capacities of various
elements of the ‘O2 transport cascade’, the pathway of O2 from the external environment to muscle mitochondria. Fasted
hummingbirds and nectar bats fly with respiratory quotients (RQs; VCO2/VO2) 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’.
O2 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 VO2 per unit
mass. We propose that dietary sugar is a premium fuel for flight in nectarivorous, flying animals.
flight using recently-ingested sugar, we tested the hypothesis that Pallas long tongued nectar bats
(Glossophaga soricina) possess flight muscles similar to those of hummingbirds with respect to enzymatic
flux capacities in bioenergetic pathways. In addition, we compared these biochemical capacities with flux
rates achieved in vivo during hovering flight. Rates of oxygen consumption (V̇O2) were measured during
hover-feeding and used to estimate rates of ATP turnover, glucose and long-chain fatty acid oxidation per unit
mass of flight muscle. Enzyme Vmax values at key steps in glucose and fatty acid oxidation obtained in vitro
from pectoralis muscle samples exceed those found in the locomotory muscles of other species of small
mammals and resemble data obtained from hummingbird flight muscles. The ability of nectar bats and
hummingbirds to hover in fed and fasted states, fueled almost exclusively by carbohydrate or fat,
respectively, allowed the estimation of fractional velocities (v/Vmax) at both the hexokinase and carnitine
palmitoyltransferase-2 steps in glucose and fatty acid oxidation, respectively. The results further support the
hypothesis of convergent evolution in biochemical and physiological traits in nectar bats and hummingbirds.
rates of aerobic metabolism among vertebrates. Analysis of the pathway of
oxygen, i.e., the “oxygen transport cascade”, reveals the concerted upregulation
of capacities for O2 flux from the external environment, through the respiratory
and cardiovascular systems, into muscle mitochondria. Pathways for aerobic energy
metabolism are highly conserved, but enzymatic capacities for carbohydrate and
fatty acid oxidation, as well as for aerobic ATP synthesis, are also upregulated in
concert. Despite evidence indicating sufficient capacities for fatty acid oxidation to
support hovering, repeated bouts of hover-feeding in hummingbirds and nectar bats
involve the oxidation of carbohydrate. Recent studies reveal that recently ingested
sugar directly fuels flight, giving rise to the concept of the “sucrose oxidation cascade”.
The ecological and bioenergetic advantages conferred by sugar oxidation
during foraging are discussed.
data for syntopic Peromyscus boylii and P. truei. Sizes of home ranges were calculated
from the minimum convex polygon of trap locations and radiotelemetry locations and
compared between individuals. The 2 estimates of home-range size were significantly correlated,
although on an average trapping home ranges were significantly smaller than sizes
of radiotelemetry home ranges. Home-range sizes from radiotelemetry were inversely correlated
with conspecific density, but home-range sizes from trapping were not. Thus, at
low density, radiotelemetry home ranges were significantly larger than trapping home ranges,
but at high density there was no difference between radiotelemetry and trapping home
ranges. These results indicate that radiotelemetry results in larger estimates of home-range
size, particularly at lower densities of conspecifics. The largest size estimates of home
ranges were from a combination of radiotelemetry and trapping data.
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 12.2kNm–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 (~48kNm–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.
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 loadlifting
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 ~1ms earlier, EMG bursts were longer (~0.9ms) 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.