Bees are thought to be strict users of carbohydrates as metabolic fuel for flight. Many insects, however, have the ability to oxidize the amino acid proline at a high rate, which is a unique feature of this group of animals. The presence of proline in the haemolymph of bees and in the nectar of plants led to the hypothesis that plants may produce proline as a metabolic reward for pollinators. We investigated flight muscle metabolism of hymenopteran species using high-resolution respirometry performed on permeabilized muscle fibres. The muscle fibres of the honeybee,Apis mellifera, do not have a detectable capacity to oxidize proline, as those from the migratory locust,Locusta migratoria, used here as an outgroup representative. The closely related bumblebee,Bombus impatiens, can oxidize proline alone and more than doubles its respiratory capacity when proline is combined with carbohydrate-derived substrates. A distant wasp species,Vespula vulgaris, exhibits the same metabolic phenotype as the bumblebee, suggesting that proline oxidation is common in hymenopterans. Using a combination of mitochondrial substrates and inhibitors, we further show that inB. impatiens, proline oxidation provides reducing equivalents and electrons directly to the electron transport system. Together, these findings demonstrate that some bee and wasp species can greatly enhance the oxidation of carbohydrates using proline as fuel for flight.

Flying insects vary greatly in body size and wing proportions, significantly impacting their flight energetics. Generally, the larger the insect, the slower its flight wingbeat frequency. However, variation in frequency is also explained by differences in wing proportions, where larger-winged insects tend to have lower frequencies. These associations affect the energy required for flight. The correlated evolution of flight form and function can be further defined using a lineage of closely related bee species varying in body mass. The decline in flight wingbeat frequency with increasing size is paralleled by the flight mass-specific metabolic rate. The specific scaling exponents observed can be predicted from the wing area allometry, where a greater increase (hyperallometry) leads to a more pronounced effect on flight energetics, and hypoallometry can lead to no change in frequency and metabolic rate across species. The metabolic properties of the flight muscles also vary with body mass and wing proportions, as observed from the activity of glycolytic enzymes and the phospholipid compositions of muscle tissue, connecting morphological differences with muscle metabolic properties. The evolutionary scaling observed across species is recapitulated within species. The static allometry observed within the bumblebee Bombus impatiens, where the wing area is proportional and isometric, affects wingbeat frequency and metabolic rate, which is predicted to decrease with an increase in size. Intraspecific variation in flight muscle tissue properties is also related to flight metabolic rate. The role of developmental processes and phenotypic plasticity in explaining intraspecific differences is central to our understanding of flight energetics. These studies provide a framework where static allometry observed within species gives rise to evolutionary allometry, connecting the evolution of size, form, and function associated with insect flight.

Although intraspecific variation in metabolic rate is associated with variation in body size, similarly sized individuals nonetheless vary greatly. At similar masses, hovering bumblebee workers (Bombus impatiens) can differ in metabolic rate up to twofold. We examined how such interindividual variation arises by studying covariation of flight metabolic rate with morphological and other physiological parameters. Body size alone explained roughly half the variation in flight metabolic rate. The remaining variation could be explained as the outcome of variation in wing morphology and possibly an association with variation in flight muscle metabolic enzyme activities. As shown using statistical models, for a given mass, higher metabolic rate was correlated with both higher thoracic temperature and higher wing stroke frequency, in turn resulting from smaller wing surface area. When organismal and cellular metabolism for a given mass were linked, variation in metabolic rate was positively correlated with the activities of trehalase and hexokinase. Altogether, covariation with morphology and other physiological parameters explains up to 75% of the variation in metabolic rate. We also investigated the role of flight experience and show that neither flight restriction nor the number of lifetime flights affected flight energetics or flight muscle phenotype. Additionally, manipulating the level of wing asymmetry increased flight wing stroke frequency, metabolic rate, and thoracic temperature, but it did not alter enzyme activity. We conclude that idiosyncrasies in body morphology largely explained interindividual variation in flight metabolic rate but flight muscle metabolic phenotype shows little variation associated with differences in flight experience.

Several insect species use the amino acid proline as a major energy substrate. Although initially thought to be limited to blood-feeding dipterans, studies have revealed this capability is more widespread. Recent work with isolated flight muscle showed that the bumblebee Bombus impatiens can oxidize proline at a high rate. However, its role as a metabolic fuel to power flight is unclear. To elucidate the extent to which proline is oxidized to power flight and how its contribution changes during flight, we profiled 14 metabolites central to energy and proline metabolism at key time points in flight muscle and abdominal tissues. Ultra-high performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (UPLC-ESI-QTOF MS) analysis revealed that proline is likely used as a sparker metabolite of the tricarboxylic acid cycle at the onset of flight, whereby it supplements the intermediates of the cycle. Carbohydrates are the major energy substrates, which is evidenced by marked decreases in abdominal glycogen stores and a lack of alanine accumulation to replenish flight muscle proline. The time course of fuel stores and metabolites changes during flight highlights homeostatic regulation of energy substrates and patterns of changes in metabolic intermediates within pathways. This study clarifies the role of proline and carbohydrate metabolism during flight in hymenopterans, such as B. impatiens.

Climate changes pose risks for bumblebee populations, which have declined relative to the growing frequency and severity of warmer temperature extremes. Bumblebees might mitigate the effects of such extreme weather through colonial behaviours. In particular, fanning behaviour to dissipate heat is an important mechanism that could reduce exposure of thermally sensitive offspring to detrimental nest temperatures (T_n). The allocation of workers towards fanning over prolonged periods could impact foraging activity that is essential for colony-sustaining resource gathering. Colony maintenance and growth could suffer as a result of nutritional and high ambient temperature (T_a) thermal stress. It remains uncertain whether a compromise occurs between thermoregulation and foraging under chronic, sublethal heat events and how colony success is impacted as a result. This study held colonies of Bombus impatiens at constant high T_a (25°C, 30°C or 35°C) for 2 weeks while quantifying the percentage of foragers, fanning incidence, nest temperature (T_n) and other metrics of colony success such as the percentage of adult emergence and offspring production. We found that foraging and adult emergence were not significantly affected by T_a, but that thermoregulation was unsuccessful at maintaining T_n despite increased fanning at 35°C. Furthermore, 35°C resulted in workers abandoning the colony and fewer offspring being produced. Our findings imply that heatwave events that exceed 30°C can negatively impact colony success through failed thermoregulation and reduced workforce production.