Flight Muscle Research Articles

Juvenile hormone as a key regulator for asymmetric caste differentiation in ants

Caste differentiation involves many functional traits that diverge during larval growth and metamorphosis to produce adults irreversibly adapted to reproductive division of labor. Investigating developmental differentiation is important for general biological understanding and has increasingly been explored for social phenotypes that diverge in parallel from similar genotypes. Here, we use Monomorium pharaonis ants to investigate the extent to which canalized worker development can be shifted toward gyne (virgin-queen) phenotypes by juvenile hormone (JH) treatment. We show that excess JH can activate gyne-biased development in workers so that wing-buds, ocelli, antennal and genital imaginal discs, flight muscles, and gyne-like fat bodies and brains emerge after pupation. However, ovary development remained unresponsive to JH treatment, indicating that JH-sensitive germline sequestration happens well before somatic differentiation. Our findings reveal important qualitative restrictions in the extent to which JH treatment can redirect larval development and that these constraints are independent of body size. Our findings corroborate that JH is a key hormone for inducing caste differentiation but show that this process can be asymmetric for higher colony-level germline versus somatic caste differentiation in superorganisms as defined a century ago by Wheeler. We quantified gene expression changes in response to JH treatment throughout development and identified a set of JH-sensitive genes responsible for the emergence of gyne-like somatic traits. Our study suggests that the gonadotropic role of JH in ovary maturation has shifted from the individual level in solitary insects to the colony level in an evolutionary-derived and highly polygynous superorganism like the pharaoh ant.

Bioinformatic meta-analysis of transcriptomics of developing Drosophila muscles identifies temporal regulatory transcription factors including a notch effector

The intricate mechanism of gene regulation coordinates the precise control of when, where, and to what extent genes are activated or repressed, directing the complex processes that govern cellular functions and development. Dysregulation of gene expression can lead to diseases such as autoimmune disorders, cancer, and neurodegeneration. Transcriptional regulation, especially involving transcription factors (TFs), plays a major role in controlling gene expression. This study focuses on identifying gene regulatory mechanisms that generate distinct gene expression patterns during Drosophila muscle development. Utilising a bioinformatics approach, we analysed the developmental time-point-specific transcriptomics resource generated by Spletter et al., which includes mRNA sequencing data at eight stages of indirect flight muscle (IFM) development. They had identified 40 distinct genome-wide clusters representing various temporal expression dynamics using “soft” clustering. Promoter sequences of genes in these clusters were analysed to predict novel motifs that act as TF binding sites. Comparative analysis with known motifs revealed significant overlaps, indicating shared transcriptional regulation. The physiological relevance of predicted TFs was confirmed by cross-referencing with experimental ChIP-seq data. We focused on Cluster 36, characterised by a unique bimodal temporal expression profile, and identified candidate genes Rbfox1 and zfh1 for further study. Ectopic overexpression experiments revealed that the TF Enhancer of split m8 helix- loop-helix [E(spl)m8-HLH], part of the Notch signalling pathway, acts as a transcriptional repressor for Rbfox1 and zfh1. Our findings highlight the complexity of transcriptional regulation during myogenesis and identify key TFs that could be targeted for further research in muscle development and related disorders.

Dominant myosin storage myopathy mutations disrupt striated muscles in Drosophila and the myosin tail-tail interactome of human cardiac thick filaments.

Myosin storage myopathy (MSM) is a rare skeletal muscle disorder caused by mutations in the slow muscle/β-cardiac myosin heavy chain (MHC) gene. MSM missense mutations frequently disrupt the tail's stabilizing heptad repeat motif. Disease hallmarks include subsarcolemmal hyaline-like β-MHC aggregates, muscle weakness and, occasionally, cardiomyopathy. We generated transgenic, heterozygous Drosophila to examine the dominant physiological and structural effects of the L1793P, R1845W, and E1883K MHC MSM mutations on diverse muscles. The MHC variants reduced lifespan and flight and jump abilities. Moreover, confocal and electron microscopy revealed that they provoked indirect flight muscle breaks and myofibrillar disarray/degeneration with filamentous inclusions. Incorporation of GFP-myosin enabled in situ determination of thick filament lengths, which were significantly reduced in all mutants. Semi-automated heartbeat analysis uncovered aberrant cardiac function, which worsened with age. Thus, our fly models phenocopied traits observed among MSM patients. We additionally mapped the mutations onto a recently-determined, 6Å resolution, cryo-EM structure of the human cardiac thick filament. The R1845W mutation replaces a basic arginine with a polar-neutral, bulkier tryptophan, while E1883K reverses charge at critical filament loci. Both would be expected to disrupt the core and the outer shell of the backbone structure. Replacing L1793 with a proline, a potent breaker of alpha-helices, could disturb the coiled-coil of the myosin rod and alter the tail-tail interactome. Hence, all mutations likely destabilize and weaken the filament backbone. This may trigger disease in humans, while potentially analogous perturbations are likely to yield the observed thick filament and muscle disruption in our fly models.

Dragonfly: A Master of Migration

Insect migration is vital for ecosystems, with trillions of individuals redistributing biomass and nutrients annually. Dragonflies, particularly Pantala flavescens are notable migratory insects capable of traveling over 6,000 kilometres across generations. Their migrations are influenced by resource availability, breeding needs, environmental conditions, and competition avoidance. This behaviour includes both seasonal movements and sporadic flights triggered by temperature and wind cues. Despite their well-known life cycle from aquatic nymphs to aerial adults, many aspects of their migration remain under-researched. Dragonflies exhibit diverse flight mechanisms, employing four distinct modes: counter-stroking for hovering, phased-stroking for cruising synchronized stroking for acceleration, and gliding for energy conservation. Migratory species show specific wing adaptations for improved endurance, such as elongated forewings and larger hindwings, along with sensory enhancements like enlarged thoracic setae. Specialized flight muscles allow for independent wing control, essential for complex movements. Additionally, physiological adaptations like fat accumulation and rapid development support their migratory behaviour. Recent studies also highlight nocturnal migrations, revealing unique flight patterns and adaptations. Together, these traits underscore the evolutionary success of dragonflies in diverse ecosystems.

Antagonistic-contracting high-power photo-oscillators for multifunctional actuations.

High-power autonomous soft actuators are in high demand yet face challenges related to tethered power and dedicated control. Light-driven oscillation by stimuli-responsive polymers allows for remote energy input and control autonomy, but generating high output power density is a daunting challenge requiring an advanced material design principle. Here, inspired by the flight muscle structure of insects, we develop a self-oscillator based on two antagonistically contracting photo-active layers sandwiching an inactive layer. The actuator produces an output power density of 33 W kg-1, 275-fold higher than other configurations and comparable to that of insects. Such oscillators allow for broad-wavelength operation and multifunction integration, including proprioceptive actuation and energy harvesting. We demonstrate high-performance flapping motion enabling various locomotion modes, including a wing with a thrust-to-weight ratio of 0.32. This work advances autonomous, sustained and untethered actuators for powerful robotics.

Two Forms of Thick Filament in the Flight Muscle of Drosophila melanogaster.

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila, flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus, which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.

Oxidative state is associated with migration distance, but not traits linked to flight energetics

Flight can be highly energy demanding, but its efficiency depends largely on flight style, wing shape and wing loading, and a range of morphological and lifestyle adaptations that can modify the cost of sustained flight. Such behavioural and morphological adaptations can also influence the physiological costs associated with migration. For instance, during intense flight and catabolism of reserves, lipid damage induced by pro‐oxidants increases, and to keep oxidative physiological homeostasis under control, the antioxidant machinery can be upregulated. Studies on the oxidative physiology of endurance flight have produced contradictory results, making generalization difficult, especially because multispecies studies are missing. Therefore, to explore the oxidative cost of flight and migration, we used samples collected during the breeding season from 113 European bird species and explored the associations of measures of antioxidant capacity (total antioxidant status, uric acid and glutathione concentration) and oxidative damage of lipids (malondialdehyde) with variables reflecting flight energetics (year‐round or specifically during migration) using a phylogenetic framework. We found that none of the traits predicting year‐round flight energy expenditure (flight style, wing morphology and flight muscle morphology) explained any measures of oxidative state. Our results suggest that birds endure their everyday flight exercise without or with low oxidative cost. However, oxidative damage to lipids and one component of the endogenous antioxidant system (uric acid), measured after the end of spring migration on breeding adult birds, increased with migration distance. Our results suggest that migration could have oxidative consequences that might be carried over to subsequent life‐history stages (breeding).

Energy Reserve Allocation in the Trade-Off between Migration and Reproduction in Fall Armyworm.

Striking a trade-off between migration and reproduction becomes imperative during long-range migration to ensure proper energy allocation. However, the mechanisms involved in this trade-off remain poorly understood. Here, we used a takeoff assay to distinguish migratory from non-migratory individuals in the fall armyworm, which is a major migratory insect worldwide. Migratory females displayed delayed ovarian development and flew further and faster than non-migratory females during tethered flight. Transcriptome analyses demonstrated an enrichment of fatty acid genes across successive levels of ovarian development and different migratory behaviors. Additionally, genes with roles in phototransduction and carbohydrate digestion along with absorption function were enriched in migratory females. Consistent with this, we identified increased abdominal lipids in migratory females that were mobilized to supply energy to the flight muscles in the thorax. Our study reveals that the fall armyworm faces a trade-off in allocating abdominal triglycerides between migration and reproduction during flight. The findings provide valuable insights for future research on this trade-off and highlight the key energy components involved in this strategic balance.

Transcriptional reprogramming in the entomopathogenic fungus Metarhizium brunneum and its aphid host Myzus persicae during the switch between saprophytic and parasitic lifestyles

BackgroundThe fungus Metarhizium brunneum has evolved a remarkable ability to switch between different lifestyles. It develops as a saprophyte, an endophyte establishing mutualistic relationships with plants, or a parasite, enabling its use for the control of insect pests such as the aphid Myzus persicae. We tested our hypothesis that switches between lifestyles must be accompanied by fundamental transcriptional reprogramming, reflecting adaptations to different environmental settings.ResultsWe combined high throughput RNA sequencing of M. brunneum in vitro and at different stages of pathogenesis to validate the modulation of genes in the fungus and its host during the course of infection. In agreement with our hypothesis, we observed transcriptional reprogramming in M. brunneum following conidial attachment, germination on the cuticle, and early-stage growth within the host. This involved the upregulation of genes encoding degrading enzymes and gene clusters involved in synthesis of secondary metabolites that act as virulence factors. The transcriptional response of the aphid host included the upregulation of genes potentially involved in antifungal activity, but antifungal peptides were not induced. We also observed the induction of a host flightin gene, which may be involved in wing formation and flight muscle development.ConclusionsThe switch from saprophytic to parasitic development in M. brunneum is accompanied by fundamental transcriptional reprogramming during the course of the infection. The aphid host responds to fungal infection with its own transcriptional reprogramming, reflecting its inability to express antifungal peptides but featuring the induction of genes involved in winged morphs that may enable offspring to avoid the contaminated environment.

The influence of sub-lethal neonicotinoid doses and ambient temperature on individual Apis mellifera scutellata flight efficiency

Honey bee (Apis mellifera) thermoregulation plays an integral part in their behaviour and physiology and has been shown to be vulnerable to the effects of neonicotinoid insecticides. Flight muscles are a crucial source of physiological heat as well as being vital for behavioural heat regulation, and are negatively affected by neonicotinoid insecticides. In this study, we evaluated the flight efficiency and capacity of Apis mellifera scutellata under the influence of both elevated ambient temperatures and sublethal neonicotinoid exposure. The various aspects of flight; success, distance, speed, and duration, were not notably affected by these factors. However, the honey bees’ ability to initiate a successful flight was significantly affected by neonicotinoid exposure. Such a reduction in honey bee flight capacity, and flight muscle function in general, especially under the increasing frequency and intensity of hot weather events, is cause for concern when considering legislation and use of these neonicotinoids in the agricultural and suburban setting.

Functioning of unidirectional ventilation in flying hawkmoths evaluated by pressure and oxygen measurements and X-ray video and tomography.

Flying sphingids generate unidirectional ventilation with an inflow through the anterior thoracic spiracles and an outflow through the posterior thoracic spiracles. This phenomenon was documented by the CO2 emission and tracheal air pressure in split-chamber experiments in preceding studies. In the present study, we evaluated the function of the air pump mechanism by measuring the tracheal pressure and PO2in the air sacs and monitoring the wing beat using photocells. Microelectrodes recorded the abdomen flexing muscles and abdominal transverse muscle septum. The crucial structure was the vertical mesophragma, with longitudinal flight muscles attached anteriorly and large fused metathoracic air sacs posteriorly, continuous to the first abdominal segment. Longitudinal flight muscles and abdomen lifting muscles contracted synchronously, producing positive pressure pulses within the mesothoracic air sacs. In the scutellar air sacs, the PO2with starting full flight was elevated to 18-20 kPa, with a pressure increase of 35-50 Pa. In contrast, in the metathoracic air sacs, the O2 concentration during flight could rise to 10 kPa, then decline to 5±1 kPa. The metathoracic air sacs provided compliance for ventilation by the flight muscles. The initial rise and subsequent decrease of the PO2in these posterior metathoracic air sacs indicated the unidirectional flow path of the air used. Serial X-ray frames of flying Acherontia atropos visualised the cyclic phragma movement and volume changes in the metathoracic air sacs. The results showed that the contracting dorsolongitudinal flight muscles expanded the metathoracic air sacs, acting as a suction pump.

Buzz pollination: Bee bites and floral vibrations

Certain bees bite tubular anthers while contracting their flight muscles. This floral sonication ejects pollen grains that the bees collect as larval food. Bees bite and buzz simultaneously. A new study shows that where and how they bite is essential for efficient pollen collection.

EEF1α2 is required for actin cytoskeleton homeostasis in the aging muscle.

The translation elongation factor eEF1α (eukaryotic elongation factor 1α) mediates mRNA translation by delivering aminoacyl-tRNAs to ribosomes. eEF1α also has other reported roles, including the regulation of actin dynamics. However, these distinct roles of eEF1α are often challenging to uncouple and remain poorly understood in aging metazoan tissues. The genomes of mammals and Drosophila encode two eEF1α paralogs, with eEF1α1 expressed ubiquitously and eEF1α2 expression more limited to neurons and muscle cells. Here, we report that eEF1α2 plays a unique role in maintaining myofibril homeostasis during aging in Drosophila. Specifically, we generated an eEF1α2 null allele, which was viable and showed two distinct muscle phenotypes. In young flies, the mutants had thinner myofibrils in indirect flight muscles that could be rescued by expressing eEF1α1. With aging, the muscles of the mutant flies began showing abnormal distribution of actin and myosin in muscles, but without a change in actin and myosin protein levels. This age-related phenotype could not be rescued by eEF1α1 overexpression. These findings support an unconventional role of Drosophila eEF1α2 in age-related homeostasis of muscle myofibers.

Mechanoresponsive regulation of myogenesis by the force-sensing transcriptional regulator Tono

Muscle morphogenesis is a multi-step program, starting with myoblast fusion, followed by myotube-tendon attachment and sarcomere assembly, with subsequent sarcomere maturation, mitochondrial amplification, and specialization. The correct chronological order of these steps requires precise control of the transcriptional regulators and their effectors. How this regulation is achieved during muscle development is not well understood. In a genome-wide RNAi screen in Drosophila, we identified the BTB-zinc-finger protein Tono (CG32121) as a muscle-specific transcriptional regulator. tono mutant flight muscles display severe deficits in mitochondria and sarcomere maturation, resulting in uncontrolled contractile forces causing muscle rupture and degeneration during development. Tono protein is expressed during sarcomere maturation and localizes in distinct condensates in flight muscle nuclei. Interestingly, internal pressure exerted by the maturing sarcomeres deforms the muscle nuclei into elongated shapes and changes the Tono condensates, suggesting that Tono senses the mechanical status of the muscle cells. Indeed, external mechanical pressure on the muscles triggers rapid liquid-liquid phase separation of Tono utilizing its BTB domain. Thus, we propose that Tono senses high mechanical pressure to adapt muscle transcription, specifically at the sarcomere maturation stages. Consistently, tono mutant muscles display specific defects in a transcriptional switch that represses early muscle differentiation genes and boosts late ones. We hypothesize that a similar mechano-responsive regulation mechanism may control the activity of related BTB-zinc-finger proteins that, if mutated, can result in uncontrolled force production in human muscle.

Buzz-pollinating bees deliver thoracic vibrations to flowers through periodic biting

Pollinator behavior is vital to plant-pollinator interactions, affecting the acquisition of floral rewards, patterns of pollen transfer, and plant reproductive success. During buzz pollination, bees produce vibrations with their indirect flight muscles to extract pollen from tube-like flowers. Vibrations can be transmitted to the flower via the mandibles, abdomen, legs, or thorax directly. Vibration amplitude at the flower determines the rate of pollen release and should vary with the coupling of bee and flower. This coupling often occurs through anther biting, but no studies have quantified how biting affects flower vibration. Here, we used high-speed filmography to investigate how flower vibration amplitude changes during biting in Bombus terrestris visiting two species of buzz-pollinated flowering plants: Solanum dulcamara and Solanum rostratum (Solanaceae). We found that floral buzzing drives head vibrations up to 3 times greater than those of the thorax, which doubles the vibration amplitude of the anther during biting compared with indirect vibration transmission when not biting. However, the efficiency of this vibration transmission depends on the angle at which the bee bites the anther. Variation in transmission mechanisms, combined with the diversity of vibrations across bee species, yields a rich assortment of potential strategies that bees could employ to access rewards from buzz-pollinated flowers.

Molecular changes and physiological responses involved in migratory bird fuel management and stopover decisions.

Migratory birds undertake long journeys across continents to reach breeding habitats with abundant resources. These migrations are essential for their survival and are shaped by a complex interplay of physiological adaptations, behavioral cues, and gene expression patterns. Central to migration are stopovers, critical resting points where birds replenish energy stores before continuing their journey. In this study, we integrate physiological measurements, behavioral observations, and molecular data from temporarily caged migrating Garden Warblers (Sylvia borin) to gain insights into their stopover strategies and physiological adaptations after crossing the extended ecological barrier formed by the Sahara Desert and the Mediterranean Sea. Depleted individuals, marked by low body mass and flight muscle mass, showcased remarkable plasticity in recovering and rapidly rebuilding energy stores within a short 5-day stopover. Flight muscle mass increased during this period, highlighting a dynamic trade-off between muscle rebuilding and refuelling. Notably, birds prioritizing muscle rebuilding exhibited a trade-off with the downregulation of genes related to lipid transport and metabolism and at the same time showing evidence of skeletal muscle angiogenesis. Early arrivals were more motivated to depart and exhibited higher levels of physiological stress. Our study highlights the importance of understanding the adaptive responses of birds to changing environmental conditions along their migration routes.

Chronic changes in developmental oxygen have little effect on mitochondria and tracheal density in the endothermic moth Manduca sexta.

Oxygen availability during development is known to impact the development of insect respiratory and metabolic systems. Drosophila adult tracheal density exhibits developmental plasticity in response to hypoxic or hyperoxic oxygen levels during larval development. Respiratory systems of insects with higher aerobic demands, such as those that are facultative endotherms, may be even more responsive to oxygen levels above or below normoxia during development. The moth Manduca sexta is a large endothermic flying insect that serves as a good study system to start answering questions about developmental plasticity. In this study, we examined the effect of developmental oxygen levels (hypoxia: 10% oxygen, and hyperoxia: 30% oxygen) on the respiratory and metabolic phenotype of adult moths, focusing on morphological and physiological cellular and intercellular changes in phenotype. Mitochondrial respiration rate in permeabilized and isolated flight muscle was measured in adults. We found that permeabilized flight muscle fibers from the hypoxic group had increased mitochondrial oxygen consumption, but this was not replicated in isolated flight muscle mitochondria. Morphological changes in the trachea were examined using confocal imaging. We used transmission electron microscopy to quantify muscle and mitochondrial density in the flight muscle. The respiratory morphology was not significantly different between developmental oxygen groups. These results suggest that the developing M. sexta trachea and mitochondrial respiration have limited developmental plasticity when faced with rearing at 10% or 30% oxygen.

Altering developmental oxygen exposure influences thermoregulation and flight performance of Manduca sexta.

Endothermic, flying insects are capable of some of the highest recorded metabolic rates. This high aerobic demand is made possible by the insect's tracheal system, which supplies the flight muscles with oxygen. Many studies focus on metabolic responses to acute changes in oxygen to test the limits of the insect flight metabolic system, with some flying insects exhibiting oxygen limitation in flight metabolism. These acute studies do not account for possible changes induced by developmental phenotypic plasticity in response to chronic changes in oxygen levels. The endothermic moth Manduca sexta is a model organism that is easy to raise and exhibits a high thorax temperature during flight (∼40°C). In this study, we examined the effects of developmental oxygen exposure during the larval, pupal and adult stages on the adult moth's aerobic performance. We measured flight critical oxygen partial pressure (Pcrit-), thorax temperature and thermoregulating metabolic rate to understand the extent of developmental plasticity as well as effects of developmental oxygen levels on endothermic capacity. We found that developing in hypoxia (10% oxygen) decreased thermoregulating thorax temperature when compared with moths raised in normoxia or hyperoxia (30% oxygen), when moths were warming up in atmospheres with 21-30% oxygen. In addition, moths raised in hypoxia had lower critical oxygen levels when flying. These results suggest that chronic developmental exposure to hypoxia affects the adult metabolic phenotype and potentially has implications for thermoregulatory and flight behavior.

Filamin protects myofibrils from contractile damage through changes in its mechanosensory region.

Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.

Biomechanical properties of non-flight vibrations produced by bees.

Bees use thoracic vibrations produced by their indirect flight muscles for powering wingbeats in flight, but also during mating, pollination, defence and nest building. Previous work on non-flight vibrations has mostly focused on acoustic (airborne vibrations) and spectral properties (frequency domain). However, mechanical properties such as the vibration's acceleration amplitude are important in some behaviours, e.g. during buzz pollination, where higher amplitude vibrations remove more pollen from flowers. Bee vibrations have been studied in only a handful of species and we know very little about how they vary among species. In this study, we conducted the largest survey to date of the biomechanical properties of non-flight bee buzzes. We focused on defence buzzes as they can be induced experimentally and provide a common currency to compare among taxa. We analysed 15,000 buzzes produced by 306 individuals in 65 species and six families from Mexico, Scotland and Australia. We found a strong association between body size and the acceleration amplitude of bee buzzes. Comparison of genera that buzz-pollinate and those that do not suggests that buzz-pollinating bees produce vibrations with higher acceleration amplitude. We found no relationship between bee size and the fundamental frequency of defence buzzes. Although our results suggest that body size is a major determinant of the amplitude of non-flight vibrations, we also observed considerable variation in vibration properties among bees of equivalent size and even within individuals. Both morphology and behaviour thus affect the biomechanical properties of non-flight buzzes.