Journal of Comparative Physiology A

Flight control requires active sensory feedback, and insects have many sensors that help them estimate their current locomotor state, including campaniform sensilla, which are mechanoreceptors that sense strain resulting from deformation of the cuticle. Campaniform sensilla on the wing detect bending and torsional forces encountered during flight, providing input to the flight feedback control system. During flight, wings experience complex spatio-temporal strain patterns. Because campaniform sensilla detect only local strain, their placement on the wing is presumably critical for determining the overall representation of wing deformation; however, how these sensilla are distributed across wings is largely unknown. Here, we test the hypothesis that campaniform sensilla are found in stereotyped locations across individuals of Manduca sexta, a hawkmoth. We found that although campaniform sensilla are consistently found on the same veins or in the same regions of the wings, their total number and distribution can vary extensively. This suggests that there is some robustness to variation in sensory feedback in the insect flight control system. The regions where campaniform sensilla are consistently found provide clues to their functional roles, although some patterns might be reflective of developmental processes. Collectively, our results on intraspecific variation in campaniform sensilla placement on insect wings will help reshape our thinking on the utility of mechanosensory feedback for insect flight control and guide further experimental and comparative studies.

Bees rely heavily on vision during most of their interaction with the environment, but so far, visual abilities have not been included into functional investigations of these crucial pollinators. This is probably due to the lack of comprehensive and phylogenetically-controlled quantification of visual traits across species. In the present study, we used high-throughput micro-CT tools to quantify, compare and understand the diversity of visual traits of compound eyes in bumblebees. Visual systems of bumblebees were far from identical, with variations across sizes, castes and species. While phylogenetic proximity poorly supported interspecific variations, these were better explained by two ecological factors: social parasitism and habitat. The eye parameter - a metric that measures the relative investment of a compound eye into resolution or sensitivity - was lower in queens of social parasitic species than of non-parasitic species. Workers of species associated with forested habitat had distinct visual traits, including a higher eye parameter, than those of species living in open landscapes. These diverse visual traits are likely to provide selective advantages to bumblebees given their specific ecological requirements. We thus propose that social parasitism and forest habitat are drivers of the diversification of compound eyes in bumblebees. Finally, we discuss how the present study can inspire trait-based approaches in ecology and conservation biology.

Many insects can use the polarization of the skylight as a navigational cue. As shown previously, freely walking Drosophila orient along the e-vector of linearly polarized UV light presented both dorsally and ventrally. We are interested in the neuronal mechanisms leading to this behavior, and specifically how the central complex and its inputs are involved. We investigated the behavior of flies exposed to linearly polarized near-UV light (400 nm) presented dorsally. Flies walked freely in a circular, flat arena surrounded by a heat barrier. Using the GAL4-UAS genetic system, we drove the expression of the potassium inward rectifier KIR2.1 to perturb each of several different neuron types of the polarization vision pathway. Perturbing EPG compass neurons in the central complex slightly weakened average alignment and increased its variability. On the other hand, when two different GAL4 lines driving expression in the ER4m ring neurons, identified by connectomics as the major polarization inputs to the fly central complex, were perturbed, the alignment strength increased. A similar effect was observed when the inputs to ER4m, the TuBua neurons, were perturbed. We did not predict EPG and ER4m perturbations to cause opposite effects. Further investigation would be required to understand the physiological mechanisms of these contradictory behavioral effects.

AimWe aimed to understand the sublethal impacts of climate change on native bee ecology. We evaluated how flight performance, a key trait for predator escape, dispersal, and pollination, is impacted by temperature. We aimed to understand how species geographic ranges shape species thermal performance curves (TPCs) and determine species vulnerability to further warming. LocationAustralia Time periodPresent Major taxa studiedStingless bees MethodsWe tested the flight speed and acceleration of two species of stingless bees, Austroplebeia australis and Tetragonula carbonaria at seven test temperatures. We examined how the climate experienced by species throughout their ranges shapes their TPCs. Inferences were made on how sublethal increases in temperature will likely impact key ecological activities associated with flight by estimating the proportion of species geographic ranges that experience temperatures that exceed their thermal optima. ResultsSpecies TPCs reflected the thermal environments they inhabit. A. australis, which experiences greater thermal variation and hotter temperatures throughout their range, had a broader TPC and higher thermal optima. However, A. australis also had faster flight performance than T. carbonaria, rejecting the jack-of-all-trades master-of-none hypothesis. Further increases in temperature will reduce flight performance of T. carbonaria at a faster rate (due to their narrower TPCs) than A. australis, however, a larger proportion of the A. australis range is currently exposed to temperatures above their thermal optima. Main conclusionsClimates will impact species ecology before they reach their lethal limits. Our study supports the idea that species fundamental niche breadths are linked to their geographic ranges, but that trade-offs are not associated with flight performance and ability to perform across a broad range of temperatures. When assessing vulnerability to climate change, it is important to consider how temperature impacts species traits, but also the climates that species experience throughout their unique geographic ranges.

O_LIThe metabolic rate of an organism is intrinsically linked to key traits such as reproductive output and lifespan. While the drivers of individual differences in metabolic rate are poorly understood, previous research in insects has shown that metabolic rate can change substantially in the initial hours and days post-eclosion as adults. C_LIO_LIHere we repeatedly measured the resting and active metabolic rate of individual adult honeybees (Apis mellifera) for up to 48 hours from the time of eclosion. We combined flow-through respirometry with automated behaviour tracking, permitting us to obtain active (AMR) and true resting metabolic rate (RMR) from freely moving animals. We compared these recordings to the more conventional approach of obtaining resting metabolic rate by restraining animals. C_LIO_LIBoth active and resting metabolic rates and mass-specific metabolic rates increased significantly in the first 48-hours post-eclosion, whereas metabolic scope did not change. Mass-specific water loss was highest in active bees and changed non-linearly with time post-eclosion, increasing in the first 24 hours before decreasing again. A similar quadratic relationship with time was also observed for bees movement speed. Speed- and mass-specific metabolic rate and scope increased with time post-emergence, whereas speed- and mass-specific water loss did not. C_LIO_LIThe metabolic rate of restrained bees was consistently significantly higher than the true RMR at all time points, likely due to the stress associated with being restrained. Therefore, we recommend future studies of insect resting metabolic rates avoid restraining organisms to restrict movement and consider employing behaviour tracking as a means to extract metabolic rate data from periods of true rest. C_LIO_LIThis study provides important insights into the previously overlooked changes in metabolism exhibited by newly emerged honeybee workers. The high mortality rate beyond 48 hours, coupled with significant changes in metabolic rates, body mass, and water loss, underscores the importance of this early post-eclosion period for survival and metabolic stabilization. C_LI

The mutualism between bees and flowers creates strong selection on both the structure of the flower and behavior of the bee to maximize pollination and foraging success, respectively. Previous research has primarily assessed the costs of foraging by quantifying the time and accuracy of search, and handling time of the flower. However, there is little attention given to the actual success of landing, and it is often not explicitly stated whether failed landing attempts are taken into consideration. We show here that landing attempts often are unsuccessful, especially in inexperienced bees. Orientation of artificial flowers in our experiment neither influenced the preference nor landing success of a naive bumble bee forager. The presence of a labellum, often considered to serve as a landing platform, also did not influence landing success, indicating that it may mostly play a role in flower recognition or act as a nectar guide. Failed landing attempts may thus play an under-recognized role in the foraging efficiency and behavior of bees, and learning may be key in individual bee landing efficiency, not just flower recognition. Further research should aim to quantify the costs of landing failures and consider the role of experience in individual bee landing success.

Photosynthetic animals produce oxygen internally, providing an ideal lens for studying how oxygen dynamics influence thermal sensitivity. The sea slug, Elysia viridis, can retain functional chloroplasts from its food alga Bryopsis plumosa for months, but retention is limited when fed Chaetomorpha sp., limiting potential oxygenic benefits. We fed slugs each alga and exposed them to 17{degrees}C (their current yearly maximum temperature) and 22{degrees}C (the increase predicted for 2100), to examine plasticity in thermal tolerance and changes in oxygen uptake when fed and starving. We also examined slugs under increased illumination to examine a potential tradeoff between increased oxygen production, and a faster rate of chloroplast degradation. Following exposure to these conditions, we performed ramping trials, subjecting them to acute thermal stress to determine their thermal tolerance. We also measured oxygen uptake before and after ramping. We observed increases in thermal tolerance for specimens exposed to 22{degrees}C, indicating they acclimated to temperatures higher than they naturally experience. Fed slugs exhibited higher rates of oxygen consumption before exposure to acute thermal stress, and suppressed their oxygen uptake more after it, than starved slugs. Under higher light, slugs exhibited improved thermal tolerance, possibly because increased oxygen production alleviated host oxygen limitation. Accordingly, this advantage disappeared later in the starvation period when photosynthesis ceased due to chloroplast digestion. In conclusion, E. viridis can suppress metabolism to cope with heat waves, however, starvation influences a slugs thermal tolerance and oxygen uptake, so continuous access to algal food for chloroplast retention is critical when facing thermal stress. Summary StatementOxygen has been implicated in determining an ectotherms thermal sensitivity. Examining photosynthetic (and therefore oxygen-producing) sea slugs under various conditions helps elucidate how oxygen and other factors impact thermal tolerance.

The light organs of the splitfin flashlight fish Anomalops katoptron are necessary for schooling behavior, to determine nearest neighbor distance, and to feed on zooplankton under dim light conditions. Each behavior is coupled to context-dependent blink frequencies and can be regulated via mechanical occlusion of light organs. During shoaling in the laboratory individuals show moderate blink frequencies around 100 blinks per minute. In this study, we correlated bioluminescent blinks with the spatio-temporal dynamics of swimming profiles in three dimensions, using a stereoscopic, infrared camera system. Groups of flashlight fish showed intermediate levels of polarization and distances to the group centroid. Individuals showed higher swimming speeds and curved swimming profiles during light organ occlusion. The largest changes in swimming direction occurred when darkening the light organs. Before A. katoptron exposed light organs again, they adapted a nearly straight movement direction. Light organs create a strong contrast against the background. Therefore, a close combination of light signals and movement is crucial to the behavior of A. katoptron.

Antennae of insects are essential mechanosensory organs that facilitate active tactile exploration and spatial navigation. Hair-plate sensilla at the base of the antenna flagellum provide proprioceptive inputs to detect their position. In hemimetabolous insects, such as crickets, the first instar immediately after hatching also possesses antennae, but the developmental dynamics and spatial organization of antennal hair plates remain poorly understood. Here, we present a comprehensive three-dimensional analysis of the antennal hair plates in crickets across developmental stages, from the first instar to adulthood. Using scanning electron microscopy and confocal laser scanning microscopy, we demonstrated that hair plate sensilla were present from the first instars and maintained a highly stereotyped spatial arrangement throughout development. Three-dimensional quantification revealed that new sensilla added during molting were formed at specific sites within the hair plate clusters that had existed at the previous stage, maintaining the spatial pattern despite the substantial growth of the antenna. Multidimensional analyses indicated that the spatial arrangement of sensilla was consistent across individuals, suggesting that organization was genetically determined. Retrograde labeling of sensory afferents showed that sensory neurons in the hair plates converged their axons, extended axon collaterals into the ipsilateral region of the antennal mechanosensory and motor center, and ultimately projected to the subesophageal ganglion. There was no apparent difference in their projection site among hair plates, suggesting no evidence of topographic organization. Our findings highlight the conserved spatial organization of hair-plate sensilla in crickets, suggesting a robust proprioceptive system that provides reliable feedback of antennal position throughout development.

Phenotypic plasticity allows many animals to quickly respond to seasonal changes in their environment. Seasonal changes to physiological systems, such as sensory systems, may explain other more obvious changes in behaviour, often working synergistically with changes in morphology. Here we investigate if there are covarying seasonal changes to morphology, behaviour, and the visual system in the seasonally plastic butterfly Junonia coenia. To describe when seasonal wing patterns occur at our field sites in the central United States and for analysis of gene expression in eye tissue, we collected animals throughout the summer and fall in 2018, 2019, 2020, and 2021. For the first three years we also visited field sites to observe behaviour during focal watches and point counts throughout the flight period. We found that more J. coenia exhibit seasonal dark wing patterns in September and October compared to butterflies collected in previous months. This change in wing pattern correlates to an increase in basking behaviour. Eye tissues of dark fall animals and lighter summer animals exhibit different patterns of gene expression, including clock genes and genes involved in eye pigment synthesis. Subsequent analysis of monthly variation in opsin gene expression in eye tissues confirmed that opsin genes are not differentially expressed throughout the year, though period gene expression is higher in the fall, and females have higher blue opsin gene expression than males. This concurrent seasonal shift in colouration, behaviour, and underlying visual physiology indicates that J. coenia undergoes a complex shift in phenotype that encompasses more than simple changes to thermoregulation.

In Drosophila, early visual processing of motion information segregates in separate ON and OFF pathways. These pathways have been studied in the context of local directional motion detection leading to the encoding of optic flow that provides visual information for flight stabilization. Less is known about their role in detecting impending collision and generating escape behaviors. Looming, the simulated approach of an object at constant speed towards an animal, provides a powerful stimulus eliciting jump escape behaviors in stationary flies. We presented looming stimuli mimicking the approach of either a dark object on a bright background or a light object on a dark background, while inactivating neurons belonging either to the ON- or the OFF-motion detection pathways by expressing the dominant Drosophila temperature-sensitive mutant shibirets in different cells of the ON/OFF pathway. Inactivation of ON, respectively OFF, neurons led to selective decreases in escape behavior to light, resp. dark, looming stimuli. Quantitative analysis showed a nearly perfect splitting of these effects according to the ON/OFF type of the targeted neural populations. Our results suggest that Drosophila ON/OFF motion detection pathways play an important role in controlling jump escape responses according to looming stimulus polarity. They further imply that the biophysical circuits triggering Drosophila jump escape behaviors likely differ substantially from those characterized in other arthropods. SummaryInactivating fly neurons of the ON or OFF directional motion detection pathways during escape behavior selectively reduced jump responses to light and dark looming stimuli, respectively.

Adaptation to new environments is key for organisms survival, but also for their invasiveness in their introduced areas. Behaviour is considered the fastest phenotype allowing adaptation, but its plasticity can involve costs as neural development. Although individuals investment in cognition was pointed out as unnecessary for colony behaviour in eusocial insects, recent studies are highlighting the behavioural dependence on neural traits in eusocial insects. The costs of producing behavioural and neural plastic offspring could exceed the investments of eusocial insects, in which one or certain reproductives must produce multiple offspring. Thus, we wanted to analyse the link between the neuroanatomy and the behavioural variability of Linepithema humile, an invasive species organised in supercolony units containing millions of individuals, to understand its adaptive mechanisms. We repeatedly tested same aged callow workers of L. humile in behavioural tests of increasing environmental complexity and analysed the volume of their brain functional areas (neuropils) and the synaptic clusters abundance in the mushroom body calices (information processing). Given the potential large cost of plasticity, we expected to find homogeneous interindividual neuronal structures and behavioural responses. Although L. humile is considered a monomorphic species, body size conditioned behavioural and neural traits and determined individuals efficiency in exploring simple environments. Contrary to our expectations, the increase in environmental complexity revealed the behavioural plasticity of Linepithema humile workers as well as its correlation with neuropil volumes and synaptic clusters. Our results highlight the relevance of the central complex and the mushroom bodies on exploration efficiency rather than optic and olfactory lobes. Behavioural plasticity under complex environments relied on the synaptic connections of the olfactory processing area (dense lip), while individuals with higher number of synaptic connections on the visual processing area (collar) explored complex environments less efficiently. Our results suggest that behavioural differences that correlate with morphological traits might promote adaptive mechanisms in simple environments, whereas neurologically based plastic behavior may be necessary to adapt in complex environments.

Nocturnal migration is a remarkable phenomenon observed in many insect species, including moths. Migratory moths are capable of maintaining precise directional orientation during migration, as demonstrated in both laboratory and field studies, suggesting that they use multiple environmental cues for orientation and navigation. Recent studies on Australian Bogong moths revealed that these animals can use stellar cues and likely the geomagnetic field (in conjunction with local visual cues) to select and maintain population-specific migratory direction. However, the underlying orientation mechanisms used by most other migratory moths are still largely unresolved. Further, it remains unclear whether migratory moths can adjust their orientation using Earths magnetic field parameters for determining their position relative to the goal (i.e. location or map information) - an ability clearly shown in some migratory birds which respond to virtual magnetic displacements by correcting their orientation (experiments when animals are exposed to magnetic cues corresponding to other geographic locations). Here, we present results from virtual magnetic displacement experiments conducted on red underwings (Catocala nupta). In addition, we tested their orientation under simulated overcast conditions and in a vertical magnetic field to get indications whether this species relies on geomagnetic or celestial cues to maintain its population-specific migratory direction. Our results show that (1) red underwings did not compensate for virtual magnetic displacement, indicating the absence of a magnetic map; (2) they remained significantly oriented in the absence of geomagnetic information, suggesting the use of a stellar compass; and (3) there was no evidence of magnetic compass orientation in absence of any visual cues.

Flying animals often encounter winds during visually guided landings. However, how winds affect their flight control strategy during landing is unknown. Here, we investigated how sidewind affects the landing strategy, sensorimotor control, and landing performance of foraging bumblebees (Bombus terrestris). For this, we trained a hive of bumblebees to forage in a wind tunnel, and used high-speed stereoscopic videography to record 19,421 landing flight maneuvers in six sidewind speeds (0 to 3.4 m s-1), which correspond to winds encountered in nature. Bumblebees landed less often in higher windspeeds, but the landing duration from free flight was not increased by wind. We then tested how bumblebees adjusted their landing control to compensate for the adverse effects of sidewind on landing. This showed that the landing strategy in sidewind was similar to that in still air, but with important adaptations. In the highest windspeeds, more hover phases occurred than during landings in still air. The rising hover frequency did not increase landing duration because bumblebees flew faster in between hover phases. Hence, they negated the adverse effects of increased hovering in high windspeeds. Using control theory, we revealed how bumblebees integrate information from the wind-mediated mechanosensory modality with their vision-based sensorimotor control loop. The proposed multi-sensory flight control system may be commonly used by insects landing in windy conditions and it may inspire the development of landing control strategies onboard man-made flying systems. Summary statementBumblebees foraging in strong sidewinds can still land precisely on artificial flowers, allowing them to be efficient and robust pollinators in these adverse environmental conditions.

Insects living in temperate regions often accumulate a large amount of glycerol during winter to avoid freezing. This seasonal accrual of glycerol is generally produced from glycogen reserves through the pentose phosphate pathway. An alternative pathway to produce glycerol is through glycolysis, normally used for pyruvate production for eventual ATP synthesis. Aside from seasonal accumulation, some insects will also rapidly increase glycerol production as a short-term response to a sudden cold event, thereby increasing cold hardiness when necessary. In the eastern spruce budworm Choristoneura fumiferana, this plasticity in cold hardiness is locally adapted, where northern populations produce more glycerol upon cold shock. Here we investigate how glycerol is produced during the rapid plastic response to fluctuating cold conditions, and whether this pathway could be a target of local adaptation. After a period of repeated cold exposure, we found evidence of increased enzyme activity and increased mRNA abundance of several proteins associated with glycolysis, and a downregulation in expression of glucose-6-phosphate dehydrogenase, associated with pentose phosphate. Pyruvate production is prevented through downregulation of glyceraldehyde-3-phosphate dehydrogenase. We found higher overall enzyme activity and glycerol accumulation in a northern population from Alberta, although there was no evidence of an interaction effect between population and cold shock treatment. This is one the first studies to show a mechanistic basis of such plasticity in cold hardiness.

The multiple swimming bells, or nectophores, of the colonial hydrozoan Nanomia septata are capable of coordinated avoidance swims in both forward and reverse directions. Individual nectophores also contribute to slower forms of swimming during foraging. Communication between a nectophore and the rest of the colony is at cone-shaped structures in the colonys stem. The stem provides an attachment point for the nectophores and houses the simple nervous system responsible for their coordination. The stem nervous system, revealed by immunocytochemistry, has three main components: two giant axons, a distributed, polygonal nerve network and a set of FMRFamide-immunoreactive nerve tracts. Whereas the nerve network is distributed throughout the stem, the nerve tracts link specific contra-lateral nectophores. Action potentials in the giant axons spread excitation rapidly along the stem, but their connection with individual nectophores is by way of the nerve network. Anatomical evidence is provided for the location of two connecting pathways between the nerve network and the nectophore; one excites an epithelial impulse and leads to reverse swimming; the other provides excitation for forward swimming by feeding into a ganglion-like cluster of nerve cells. Excitation passes to the swimming muscle epithelium by way of a single nerve axon and a nerve ring at the nectophore margin. The work presents physiological evidence for mechanisms, such as facilitation and summation, operating within a multifunctional, bidirectional nerve network, responsible for coordinating epithelial and neural signals in an early-evolved nervous system containing both condensed and distributed units. Summary statementHow a nervous system with two giant axons, a diffuse nerve network and FMRFamide-immunoreactive nerve tracts, coordinates Nanomias multiple swimming bells to provide the colony with foraging and escape behaviours.

Insects depend on a broad olfactory perception ability that involves many sensory receptors. Social insects, in particular, use olfactory cues to maintain colony cohesion (Lenoir et al., 1999; Leonhardt et al., 2016). They recognize nestmates through colony-specific olfactory labels that individuals store as neural templates in their memory (Sherman et al., 1997). Learning continuously optimises the nestmate recognition template to keep up with the constant changes in colony labels (dEttorre and Lenoir, 2010; Errard and Hefetz, 1997; Wilgenburg et al., 2012). The template is often considered to be located in higher brain centres and potentially a product of learning (Bey et al., 2025; Brandstaetter et al., 2011; Esponda and Gordon, 2015; van Zweden and dEttorre, 2010). However, some authors suggest it might be in the neural periphery, i.e. the antennae or antennal lobes, formed by habituation or receptor adaptation (Guerrieri et al., 2009; Ozaki and Hefetz, 2014; Stroeymeyt et al., 2010). Here we investigate a potential mechanism for the construction and functioning of the colony template in the peripheral nervous system: the antennas odorant receptor (OR) profile and its dynamics. We exposed Acromyrmex echinatior leaf-cutting ants to non-nestmate odours and analysed the consequences on their behaviour and antennal gene expression. Consistent with other studies, prolonged exposure to non-nestmate odours reduced worker aggression towards the non-nestmate label, indicating habituation to the non-nestmate odour (Carlin and Holldobler, 1983; Errard and Hefetz, 1997; Stroeymeyt et al., 2010). Exposure also altered the expression of the OR genes. Notably, the OR profiles were colony-specific, mirroring the colony-specific recognition labels. When we exposed two different colonies to the same non-nestmate odour, the colony-specificity of the odorant receptor gene expression vanished. This indicates that the olfactory machinery used to perceive nestmate recognition cues is flexible and adapts to the current nest-specific olfactory environment. The OR profiles could either become more sensitive to the nestmate recognition cues by increasing the number or ORs for the nest-specific substances, or less sensitive by decreasing the expression of these ORs. The latter would in turn increase the sensitivity to non-nestmate cues. This would be a mechanism explaining habituation to nestmate recognition cues that has been described across the social insect literature.

Activation and modulation of sensory-guided behaviors by biogenic amines assure appropriate adaptations to changes in an insects environment. Given its genetic tool kit Drosophila melanogaster represents an excellent model organism to study larger networks of neurons by optophysiological methods. Here, we studied stationary crawling movements of 3rd instar larvae and revealed how the octopaminergic VUM neuron system reacts during crawling behavior and tactile stimulations. We conducted calcium imaging experiments on dissections of the isolated nervous system (missing all sensory input) and found spontaneous rhythmic wave pattern of neuronal activity in VUM neuron clusters over the range of thoracic and abdominal neuromeres in the VNC. In contrast, in vivo preparations (semi-intact animals, receiving sensory input) did not reveal such spontaneous rhythmic pattern. However, tactile stimulations activated different clusters of the VUM neuron system simultaneously in these preparations. The activation intensity of VUM neurons in the VNC was correlated with the location and degree of body wall stimulation. While VUM neuron cluster near the respective location of body wall stimulation were less activated more distant cluster showed stronger activation. Repeated gentle touch stimulations led to decreased response intensities, repeated harsh stimulations resulted in increasing intensities over trials. Optophysiological signals correlated highly with crawling behavior in freely moving larvae stimulated similarly. We conclude that the octopaminergic system is strongly coupled to the neuronal pattern generator of crawling movements and that it is simultaneously activated by physical stimulation, rather intensity than sequential coded. We hope that our work raises the interest in whole biogenic network activity and shows that octopamine release does not only underlie "the more the better" principle but instead has a more complex function in control and modulation of insects locomotion.

Floral handling can be energetically costly for bees, yet these costs are rarely measured. We provide the first direct quantification of the metabolic cost of floral buzzing in bumblebees and evaluate its ecological significance. Using flow-through respirometry synchronised with laser vibrometry, we measured carbon dioxide production during buzzing by Bombus terrestris and compared it with flight take-off, which is powered by the same thoracic muscles. Buzzing required high muscular effort, with [~]0.10 J per event and mass-specific power [~]293 W kg-{superscript 1}, and overall costs comparable to take-off because buzzing bouts are longer even though the metabolic rate is lower. Absolute metabolic rate increased with body mass, whereas intertegular span did not, implying that transient load rather than structural size better explains energetic demand in short, high-intensity behaviours. Metabolic traits were repeatable within individuals, and colony identity explained additional variance, consistent with genetic or shared environmental effects. Converting costs to nectar equivalents showed that buzzing required slightly more nectar than take-off and that requirements rose as nectar sugar concentration declined. We conclude that floral buzzing is a major, previously unquantified component of bee energy budgets that is likely to shape nectar supplementation, flower sequencing, and plant-pollinator interaction strength.

Nectarless flowers are common among flowering plants, which often retain colour-changed, rewardless flowers instead of shedding them. Yet, how these flowers influence pollinators foraging choices within an inflorescence remains unclear. We hypothesised that rewardless flowers in an inflorescence may act as "decoys", causing the rewarding flowers in the inflorescence to be perceived as more valuable by contrast. Using artificial inflorescences, we presented individual bumblebees (Bombus terrestris) with a binary choice between two equally rewarding inflorescences, one of which included additional unrewarded, differently coloured flowers. We found that the presence of rewardless flowers did not increase bees preference for neighbouring flowers, nor did it affect their overall choice between inflorescences. However, bees quickly learned to avoid the unrewarded flowers, drastically reducing visits and probing within a few foraging bouts. We review research on decoy effects in bees, and find very little support for their presence. Our findings contribute to the growing body of evidence that rewardless flowers do not induce decoy effects in bees, and highlight the need for further research into the ecological role of nectarless flowers within floral patches. It may be time to abandon the search for classical decoy effects in pollinators.