Muscle development : a view from adult myogenesis in Drosophila.

In Drosophila the first wave of myogenesis occurs in the embryo to produce the larval muscles. This musculature undergoes histolysis and largely disappears during metamorphosis, while a second wave of myogenesis begins to generate the muscles of the adult fly. The core myogenic program is highly conserved among both invertebrate and vertebrate species, and Drosophila embryogenic myogenesis is a well-recognized model for identifying genes and pathways governing muscle development. The more diverse and complex adult musculature is also an attractive model to study some aspects of the myogenic process. The more intense research effort focusing on adult myogenesis since early this century has added greatly to our knowledge. We review here what we know about the development of adult muscles, from the specification and diversification of the adult muscle precursors to their final differentiation. The formation of a functional contractile unit requires integrating multiple tissue interactions. We therefore also describe how muscle cells interrelate with the tendons and the nervous and tracheal systems in the course of development.

Development of the indirect flight muscles of Aedes aegypti, a main arbovirus vector.

BACKGROUND: Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. METHODS: A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. RESULTS: At L3-L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal-ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. CONCLUSIONS: A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

An insight on Drosophila myogenesis and its assessment techniques.

Movement assisted by muscles forms the basis of various behavioural traits seen in Drosophila. Myogenesis involves developmental processes like cellular specification, differentiation, migration, fusion, adherence to tendons and neuronal innervation in a series of coordinated event well defined in body space and time. Gene regulatory networks are switched on-off, fine tuning at the right developmental stage to assist each cellular event. Drosophila is a holometabolous organism that undergoes myogenesis waves at two developmental stages, and is ideal for comparative analysis of the role of genes and genetic pathways conserved across phyla. In this review we have summarized myogenic events from the embryo to adult focussing on the somatic muscle development during the early embryonic stage and then on indirect flight muscles (IFM) formation required for adult life, emphasizing on recent trends of analysing muscle mutants and advances in Drosophila muscle biology.

Metabolic 'engines' of flight drive genome size reduction in birds.

The tendency for flying organisms to possess small genomes has been interpreted as evidence of natural selection acting on the physical size of the genome. Nonetheless, the flight-genome link and its mechanistic basis have yet to be well established by comparative studies within a volant clade. Is there a particular functional aspect of flight such as brisk metabolism, lift production or maneuverability that impinges on the physical genome? We measured genome sizes, wing dimensions and heart, flight muscle and body masses from a phylogenetically diverse set of bird species. In phylogenetically controlled analyses, we found that genome size was negatively correlated with relative flight muscle size and heart index (i.e. ratio of heart to body mass), but positively correlated with body mass and wing loading. The proportional masses of the flight muscles and heart were the most important parameters explaining variation in genome size in multivariate models. Hence, the metabolic intensity of powered flight appears to have driven genome size reduction in birds.

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size.

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs.

Energy consumption during insect flight and bioinspiration for MAV design: A review.

The excellent biological characteristics of insects provide an important source of inspiration for designing micro air vehicles (MAVs). Insect flight is an incredibly complex and energy-intensive process. Unique insect flight muscles and contraction mechanisms enable flapping at high frequencies. Moreover, the metabolic rate during flight can reach hundreds of times the resting state. Understanding energy consumption during flight is crucial for designing efficient biomimetic aircraft. This paper summarizes the structures and contraction mechanisms of insect flight muscles, explores the underlying metabolic processes, and identifies methods for energy substrate identification and detection, and discusses inspiration for biomimetic MAV design. This paper reviews energy consumption during insect flight, promotes the understanding of insect bioenergetics, and applies this information to the design of MAVs.

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

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

Myoblast proliferation during flight muscle development in Manduca sexta is unaffected by reduced neural signaling.

In holometabolous insects, metamorphosis involves restructuring the musculature to accommodate adult-specific anatomy and behaviors. Evidence from experiments on remodeled muscles, as well as those that develop de novo, suggests that signals from the nervous system support adult muscle development by controlling myoblast proliferation rate. However, the dorsolongitudinal flight muscles (DLMs) of Manduca sexta undergo a mixed developmental program involving larval muscle fibers, and it is not known if neurons play the same role in the formation of these muscles. To address this question, we have blocked the most promising candidate pathways for neural input and examined the DLMs for changes in proliferation. Our results show that DLM development does not depend on neural activity, Hedgehog signaling, or EGF signaling. It remains to be determined how DLM growth is controlled and why neurally mediated proliferation differs between individual muscles.

Experimental studies suggest differences in the distribution of thorax elasticity between insects with synchronous and asynchronous musculature.

Insects have developed diverse flight actuation mechanisms, including synchronous and asynchronous musculature. Indirect actuation, used by insects with both synchronous and asynchronous musculature, transforms thorax exoskeletal deformation into wing rotation. Though thorax deformation is often attributed exclusively to muscle tension, the inertial and aerodynamic forces generated by the flapping wings may also contribute. In this study, a tethered flight experiment was used to simultaneously measure thorax deformation and the inertial/aerodynamic forces acting on the thorax generated by the flapping wing. Compared to insects with synchronous musculature, insects with asynchronous muscle deformed their thorax 60% less relative to their thorax diameter and their wings generated 2.8 times greater forces relative to their body weight. In a second experiment, dorsalventral thorax stiffness was measured across species. Accounting for weight and size, the asynchronous thorax was on average 3.8 times stiffer than the synchronous thorax in the dorsalventral direction. Differences in thorax stiffness and forces acting at the wing hinge led us to hypothesize about differing roles of series and parallel elasticity in the thoraxes of insects with synchronous and asynchronous musculature. Specifically, wing hinge elasticity may contribute more to wing motion in insects with asynchronous musculature than in those with synchronous musculature.

The larval scaffold controls fascicle number but is not required for formation of the dorsolongitudinal flight muscles in Manduca sexta.

During metamorphosis, the dorsolongitudinal flight muscles (DLMs) of both the moth Manduca sexta and the fly Drosophila melanogaster develop from the remnants of larval muscles called larval scaffolds. Although this developmental program has been conserved across highly disparate taxa, the role of the larval scaffold remains unclear. Ablation experiments have demonstrated that the Drosophila DLM does not require the scaffold, but the resulting de novo muscles vary highly in fiber number, and their functional characteristics were not examined. To address this question in Manduca, we have surgically ablated the DLM precursors in Manduca sexta larvae and assayed the resulting DLMs in pharate adults using X-ray micro-CT and phalloidin histology. Following ablation, animals were able to form de novo DLMs with normal myofibril alignment, but these muscles had an altered shape and highly variable number of fascicles. Our results suggest that the larval scaffold is not required for DLM development in Manduca sexta, but appears to define the number of fascicles in the adult muscle, as previously found in Drosophila. Additionally, our ablated animals were able to generate flight, further suggesting that the use of a larval scaffold is a modification on the more ancestral myogenesis program.

Physiological and Population Responses of Nilaparvata lugens after Feeding on Drought-Stressed Rice.

Drought stress greatly impacts insect development and population growth. Some studies have demonstrated increased reproductive capacity in drought-stressed insects; however, physiological changes in the brown planthopper (BPH), Nilaparvata lugens (Stål), during periods of drought are unclear. In this study, BPH fed on drought- stressed rice had lower population numbers than BPH feeding on non-stressed rice. Water content, osmotic pressure of hemolymph and total amino acid content of BPH were significantly lower when BPH fed on drought-stressed rice compared to the non-stressed control; however, glucose content and glutathione S-transferase (GST) activity were significantly higher in BPH fed on drought-stressed rice. The expression of Vitellogenin and Exuperantia in BPH fed on drought-stressed rice was higher than that in BPH feeding on non-stressed control plants. The size of myofibrils and the abundance of mitochondria in BPH flight muscles were significantly lower in BPH fed on drought-stressed rice compared to non-stressed plants. These results indicate that water management impacts the physiology of BPH, which may be useful in understanding the relationship between drought stress and this damaging herbivore.

Drosophila melanogaster: A Model System to Study Distinct Genetic Programs in Myoblast Fusion.

Muscle fibers are multinucleated cells that arise during embryogenesis through the fusion of mononucleated myoblasts. Myoblast fusion is a lifelong process that is crucial for the growth and regeneration of muscles. Understanding the molecular mechanism of myoblast fusion may open the way for novel therapies in muscle wasting and weakness. Recent reports in Drosophila and mammals have provided new mechanistic insights into myoblast fusion. In Drosophila, muscle formation occurs twice: during embryogenesis and metamorphosis. A fundamental feature is the formation of a cell-cell communication structure that brings the apposing membranes into close proximity and recruits possible fusogenic proteins. However, genetic studies suggest that myoblast fusion in Drosophila is not a uniform process. The complexity of the players involved in myoblast fusion can be modulated depending on the type of muscle that is formed. In this review, we introduce the different types of multinucleated muscles that form during Drosophila development and provide an overview in advances that have been made to understand the mechanism of myoblast fusion. Finally, we will discuss conceptual frameworks in cell-cell fusion in Drosophila and mammals.

Histopathological Features of Symptomatic and Asymptomatic Honeybees Naturally Infected by Deformed Wing Virus.

Deformed wing virus (DWV) is capable of infecting honeybees at every stage of development causing symptomatic and asymptomatic infections. To date, very little is known about the histopathological lesions caused by the virus. Therefore, 40 honeybee samples were randomly collected from a naturally DWV infected hive and subjected to anatomopathological examination to discriminate between symptomatic (29) and asymptomatic (11) honeybees. Subsequently, 15 honeybee samples were frozen at -80° and analyzed by PCR and RTqPCR to determinate the presence/absence of the virus and the relative viral load, while 25 honeybee samples were analyzed by histopathological techniques. Biomolecular results showed a fragment of the expected size (69bp) of DWV in all samples and the viral load was higher in symptomatic honeybees compared to the asymptomatic group. Histopathological results showed degenerative alterations of the hypopharyngeal glands (19/25) and flight muscles (6/25) in symptomatic samples while 4/25 asymptomatic samples showed an inflammatory response in the midgut and the hemocele. Results suggest a possible pathogenic action of DWV in both symptomatic and asymptomatic honeybees, and a role of the immune response in keeping under control the virus in asymptomatic individuals.

Growth rate and locomotor performance tradeoff is not universal in birds.

Though a tradeoff between growth rate and locomotor performance has been proposed, empirical data on this relationship are still limited. Here we statistically analyze the associations of growth rate and flight ability in birds by assessing how growth rate is correlated with three wing parameters of birds: flight muscle ratio, wing aspect ratio, and wing loading. We find that fast-growing birds tended to have higher flight muscle ratios and higher wing loadings than slow-growing birds, which suggests that fast-growing birds may have better takeoff performance, but lower efficiency in maneuvering flight. Accordingly, our findings suggest that the relationship between growth rate and flight ability is more complex than a simple tradeoff. Since the hindlimbs also contribute greatly to the locomotion of birds, future investigations on the relationship between growth rate and hindlimb performance will provide more insights into the evolution of birds.

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres.

Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.

High-Stakes Decision-Making by Female Crickets (Gryllus texensis): When to Trade In Wing Muscles for Eggs.

AbstractResource-intensive traits, such as dispersal and reproduction, can be difficult to express simultaneously because of resource limitations. One solution is to switch between resource-intensive behaviors. Such phenotypic plasticity is one strategy that organisms use to funnel resources from one expensive trait to another. In crickets (Gryllus texensis), the development and maintenance of flight muscles reduce resource availability for reproduction, leading to physiological trade-offs between the two traits. Long-winged female G. texensis can histolyze their wing muscles, resulting in increased egg production, but they can then no longer fly. Using a diet that mimics food availability in the field, we found that long-winged females adopted one of the three following strategies: early reproduction, intermediate reproduction, and late reproduction. Some late reproducers maintained their flight capability until the end of their natural life span and laid few eggs. If females lost the ability to fly (i.e., their hind wings are removed), they laid eggs earlier, leading to increased reproductive output. However, other environmental cues (e.g., an increased number of mates, increased oviposition substrate quality, or a bout of dispersal flight) had no effect. Late-reproducing females laid 96% fewer eggs than early reproducers, suggesting that late reproduction exacts a huge fitness cost. Nevertheless, some females maintain their flight muscles to the end of their natural life span in both the lab and the field. We suggest that the ability to fly allows for bet hedging against an environmental catastrophe (e.g., drought or flood). This benefit may help explain the persistence of late-reproducing long-winged females, despite the cost of this choice. As climate change increases drought and flood in Texas, late dispersal may be one factor that helps this species survive in the future. An increased understanding of factors that maintain seemingly low fitness strategies can help us predict the resilience of species under climate change.

Myogenesis in Drosophila melanogaster: Dissection of Distinct Muscle Types for Molecular Analysis.

Drosophila is a useful model organism for studying the molecular signatures that define specific muscle types during myogenesis. It possesses significant genetic conservation with humans for muscle disease causing genes and a lack of redundancy that simplifies functional analysis. Traditional molecular methods can be utilized to understand muscle developmental processes such as Western blots, in situ hybridizations, RT-PCR and RNAseq, to name a few. However, one challenge for these molecular methods is the ability to dissect different muscle types. In this protocol we describe some useful techniques for extracting muscles from the pupal and adult stages of development using flight and jump muscles as an example.

A Method to Injure, Dissect and Image Indirect Flight Muscle of Drosophila.

Inducing an injury specifically to Drosophila flight muscles is a difficult task, owing to the small size of the muscles and the presence of the cuticle. The protocol described below provides an easy and reproducible method to induce injury in the Drosophila flight muscles.

The multilevel antibiotic-induced perturbations to biological systems: Early-life exposure induces long-lasting damages to muscle structure and mitochondrial metabolism in flies.

Antibiotics have been increasingly used over the past decades for human medicine, food-animal agriculture, aquaculture, and plant production. A significant part of the active molecules of antibiotics can be released into the environment, in turn affecting ecosystem functioning and biogeochemical processes. At lower organizational scales, these substances affect bacterial symbionts of insects, with negative consequences on growth and development of juveniles, and population dynamics. Yet, the multiple alterations of cellular physiology and metabolic processes have remained insufficiently explored in insects. We evaluated the effects of five antibiotics with different mode of action, i.e. ampicillin, cefradine, chloramphenicol, cycloheximide, and tetracycline, on the survival and ultrastructural organization of the flight muscles of newly emerged blow flies Chrysomya albiceps. Then, we examined the effects of different concentrations of antibiotics on mitochondrial protein content, efficiency of oxidative phosphorylation, and activity of transaminases (Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase) and described the cellular metabolic perturbations of flies treated with antibiotics. All antibiotics affected the survival of the insects and decreased the total mitochondrial protein content in a dose-dependent manner. Ultrastructural organization of flight muscles in treated flies differs dramatically compared to the control groups and severe pathological damages/structures disorganization of mitochondria appeared. The activities of mitochondrial transaminases significantly increased with increased antibiotic concentrations. The oxidation rate of pyruvate + proline from isolated mitochondria of the flight muscles of 1-day-old flies was significantly reduced at high doses of antibiotics. In parallel, the level of several metabolites, including TCA cycle intermediates, was reduced in antibiotics-treated flies. Overall, antibiotics provoked a system-wide alteration of the structure and physiology of flight muscles of the blow fly Ch. albiceps, and may have fitness consequences at the organism level. Environmental antibiotic pollution is likely to have unwanted cascading ecological effects of insect population dynamics and community structure.

Adult Muscle Formation Requires Drosophila Moleskin for Proliferation of Wing Disc-Associated Muscle Precursors.

Adult muscle precursor (AMP) cells located in the notum of the larval wing disc undergo rapid amplification and eventual fusion to generate the Drosophila melanogaster indirect flight muscles (IFMs). Here we find that loss of Moleskin (Msk) function in these wing disc-associated myoblasts reduces the overall AMP pool size, resulting in the absence of IFM formation. This myoblast loss is due to a decrease in the AMP proliferative capacity and is independent of cell death. In contrast, disruption of Msk during pupal myoblast proliferation does not alter the AMP number, suggesting that Msk is specifically required for larval AMP proliferation. It has been previously shown that Wingless (Wg) signaling maintains expression of the Vestigial (Vg) transcription factor in proliferating myoblasts. However, other factors that influence Wg-mediated myoblast proliferation are largely unknown. Here we examine the interactions between Msk and the Wg pathway in regulation of the AMP pool size. We find that a myoblast-specific reduction of Msk results in the absence of Vg expression and a complete loss of the Wg pathway readout ß-catenin/Armadillo (Arm). Moreover, msk RNA interference knockdown abolishes expression of the Wg target Ladybird (Lbe) in leg disc myoblasts. Collectively, our results provide strong evidence that Msk acts through the Wg signaling pathway to control myoblast pool size and muscle formation by regulating Arm stability or nuclear transport.