Insect Muscle Research Articles

PatternJ: an ImageJ toolset for the automated and quantitative analysis of regular spatial patterns found in sarcomeres, axons, somites, and more.

Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from early stages of development to adulthood. To understand the formation of these patterns, how they assemble and mature, and how they are affected by perturbations, a precise quantitative description of the patterns is essential. However, accessible tools that offer in-depth analysis without the need for computational skills are lacking for biologists. Here, we present PatternJ, a novel toolset to analyze regular one-dimensional patterns precisely and automatically. This toolset, to be used with the popular imaging processing program ImageJ/Fiji, facilitates the extraction of key geometric features within and between pattern repeats in static images and time-lapse series. We validate PatternJ with simulated data and test it on images of sarcomeres from insect muscles and contracting cardiomyocytes, actin rings in neurons, and somites from zebrafish embryos obtained using confocal fluorescence microscopy, STORM, electron microscopy, and brightfield imaging. We show that the toolset delivers subpixel feature extraction reliably even with images of low signal-to-noise ratio. PatternJ's straightforward use and functionalities make it valuable for various scientific fields requiring quantitative one-dimensional pattern analysis, including the sarcomere biology of muscles or the patterning of mammalian axons, speeding up discoveries with the bonus of high reproducibility.

Twist is required for muscle development of the adult legs in Henosepilachna vigintioctopunctata.

Although muscle development has been widely studied in Drosophila melanogaster, it was a great challenge to apply to developmental processes of other insect muscles. This study was focused on the functional characterization of a basic helix-loop-helix transcription factor gene twist in an herbivorous ladybird Henosepilachna vigintioctopunctata. Its transcript (Hvtwist) levels were detected in all developmental stages. RNA interference (RNAi)-aidedknockdown of Hvtwist at the penultimate larval instar stage impaired pupation, and caused a deformed adult in the legs. The tarsi were malformed and did not support the bodies in an upright position. The climbing ability was impaired. Moreover, around 50% of the impaired adults had a malformed elytrum. In addition, they consumed less foliage and did not lay eggs. A hematoxylin-eosin staining of the leg demonstrated that the tibial extensor (TE)and the tibial flexor (TF)muscles were originated from the femurs while levator and depressor muscles of the tarsus (TLand TD) were located in the tibia in the control adults, in which tarsal segments were devoid of muscles. RNAi treatment specific to Hvtwist expression markedly impaired TE and TF muscles in the femurs, and prevented the development of TL and TD muscles in the tibia. Therefore, our findings demonstrate Twist plays a vital role in the myogenesis in H. vigintioctopunctata adult legs.

Flubendiamide induced genetic and cellular damages directly influence the life cycle of the oriental leaf worm, Spodoptera litura

Indiscriminate uses of insecticide greatly damage the environment as well as non-target organisms. Thus, multiple levels of bioassays can help better management of our environment. Flubendiamide is a phthalic acid diamide insecticide that ceases the function of insect muscle leading to paralysis and death. Here, we aimed to explore the effects of Flubendiamide on the life cycle of Spodoptera litura vis-a-vis the mode of action. Fourth instar larvae of the same age (120 ± 2 h) and size were fed with different concentrations (20–80 μg/mL) of Flubendiamide for 12–72 h. We performed a pharmacokinetics study, different biochemical assays, p450, Ecdysone receptor (EcR) and other genes expression analyses by Real-Time PCR and gross damages by Dye exclusion assay and histopathology. Our results demonstrate that the mean concentration of Flubendiamide after 48 h is 9.907 μg/mL and (i) altered the molting, metamorphosis, and reproduction at 80 μg/mL (24 h) (ii) increases all oxidative stress parameters (ROS/RNS, MDA, 8OHdG), decreases oxidative defense mechanisms (SOD, CAT, GST) at 80 μg/mL (48 h) and p450 in a time and concentration-dependent manner, (iii) activates CncC/Maf apoptotic pathways at 80 μg/mL concentration at 24 h while the expression declined from 48 h onwards, (iii) downregulates the EcR expression in a time and concentration-dependent manner, which might be responsible for disturbed molting, metamorphosis, and reproduction, and (iv) increase the expression of apoptotic genes (Caspase 1, −3, and − 5), in time and concentration-dependent manner causing gross morphological and histological damages. In conclusion, indiscriminate use of this insecticide can affect the ecosystem and have the capacity to cause multiple hazardous effects on experimental organisms. Thus, it warrants further investigations to improve and optimize the integrated pest management packages, including Flubendiamide for better management.

Biomechanical properties of honeybee abdominal muscles during stretch activation

The mechanical properties of the honeybee's abdominal muscles endow its abdomen with movement flexibility to perform various activities. However, the biomechanical properties of abdominal muscles during stretch activation remain unclear. To clarify this issue, we observed the microstructures of the abdominal muscles to obtain structural information. The similarity and symmetry of abdominal muscle distribution contribute to the ability to drive abdominal movement. Combined with the segmented structure characteristics, an experimental device to measure muscle stretch measurement of honeybees was developed to investigate the mechanical properties of the abdominal muscles. During measurement, the muscles were kept in a solution to maintain a physiological environment. The mechanical properties of abdominal muscles included phases: the ascending phase with proportional increase, stable phase with slight fluctuation, and decay phase with parabolic decline. These findings indicate that the nonlinear and rate-sensitive mechanical properties of the abdominal muscles enable them to rapidly adapt to environmental changes. The stretch force and stiffness coefficient reached 0.660 ± 0.139 mN and 14.364 ± 2.961 N/m, respectively. A simplified biomechanical model of the muscle fiber considering the hierarchical microstructure was introduced, in which the mechanical properties were consistent with the experimental data. Further analysis of the effects of the activation probability and the effective range of binding sites on the mechanical properties demonstrated the critical role in force generation, revealing the mechanism of underlying muscle stretch activation in the honeybee abdomen. The findings can provide a new reference for studying the biomechanical properties of the muscles of other arthropod insects.

Investigation of chordwise functionally graded flexural rigidity in flapping wings using a two-dimensional pitch–plunge model

Insect wings are heterogeneous structures, with flexural rigidity varying one to two orders of magnitude over the wing surface. This heterogeneity influences the deformation the flapping wing experiences during flight. However, it is not well understood how this flexural rigidity gradient affects wing performance. Here, we develop a simplified 2D model of a flapping wing as a pitching, plunging airfoil using the assumed mode method and unsteady vortex lattice method to model the structural and fluid dynamics, respectively. We conduct parameter studies to explore how variable flexural rigidity affects mean lift production, power consumption and the forces required to flap the wing. We find that there is an optimal flexural rigidity distribution that maximizes lift production; this distribution generally corresponds to a 3:1 ratio between the wing’s flapping and natural frequencies, though the ratio is sensitive to flapping kinematics. For hovering flight, the optimized flexible wing produces 20% more lift and requires 15% less power compared to a rigid wing but needs 20% higher forces to flap. Even when flapping kinematics deviate from those observed during hover, the flexible wing outperforms the rigid wing in terms of aerodynamic force generation and power across a wide range of flexural rigidity gradients. Peak force requirements and power consumption are inversely proportional with respect to flexural rigidity gradient, which may present a trade-off between insect muscle size and energy storage requirements. The model developed in this work can be used to efficiently investigate other spatially variant morphological or material wing features moving forward.

A method for isolating highly purified and active mitochondria from insects

So far, methods that yield the high purity and activity of the isolated mitochondria from insects have not been reported and determined. Here, we develop methods that combine differential centrifugation and discontinuous Nycodenz density gradient centrifugation to isolate highly purified mitochondria from the thorax muscle of insects, and the methods were widely validated across three orders (Coleoptera, Hymenoptera, and Blattaria) covering four insect species using Western blot and transmission electron microscopy (TEM) analysis. The results showed the removal of the residual contamination with nonmitochondrial components such as nucleus, sarcolemma, cytosol, and endoplasmic reticulum. Furthermore, TEM, mitochondria staining, fluorescence detection, and flow cytometry analyses were employed to assess membrane integrity and activity of the isolated mitochondria. The results showed no loss of mitochondria activity/integrity after isolation. In addition, temporal dynamics in activity of the isolated mitochondria under commonly used laboratory temperature (-20 °C, 4 °C, and 25 °C) were respectively detected using a fluorescence microplate reader. The results showed that it should be avoided to store the isolated mitochondria at room temperature, and the mitochondria can meet the requirements of the most downstream experiments when they were stored at −20 °C. Overall, the study presented a method for isolating highly purified and active mitochondria from insects. This study firstly described a high-speed discontinuous density gradient centrifugation-based method that could be widely applied for mitochondria isolation in insects. The present study also provided an example to assess purity and integrity/activity of the isolated mitochondria.

Robotics-based study on the kinematic control and evolution of the steering muscle of insects

Robotics-based study on the kinematic control and evolution of the steering muscle of insects

Structural basis for diamide modulation of ryanodine receptor

Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo-voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.

Quantitative model analysis of the resting membrane potential in insect skeletal muscle: Implications for low temperature tolerance

Abiotic stressors, such as cold exposure, can depolarize insect cells substantially causing cold coma and cell death. During cold exposure, insect skeletal muscle depolarization occurs through a 2-stage process. Firstly, short-term cold exposure reduces the activity of electrogenic ion pumps, which depolarize insect muscle markedly. Secondly, during long-term cold exposure, extracellular ion homeostasis is disrupted causing further depolarization. Consequently, many cold hardy insects improve membrane potential stability during cold exposure through adaptations that secure maintenance of ion homeostasis during cold exposure. Less is known about the adaptations permitting cold hardy insects to maintain membrane potential stability during the initial phase of cold exposure, before ion balance is disrupted. To address this problem it is critical to understand the membrane components (channels and transporters) that determine the membrane potential and to examine this question the present study constructed a mathematical “charge difference” model of the insect muscle membrane potential. This model was parameterized with known literature values for ion permeabilities, ion concentrations and membrane capacitance and the model was then further developed by comparing model predictions against empirical measurements following pharmacological inhibitors of the Na+/K+ ATPase, Cl− channels and symporters. Subsequently, we compared simulated and recorded membrane potentials at 0 and 31 °C and at 10–50 mM extracellular [K+] to examine if the model could describe membrane potentials during the perturbations occurring during cold exposure. Our results confirm the importance of both Na+/K+ ATPase activity and ion-selective Na+, K+ and Cl− channels, but the model also highlights that additional electroneutral flux of Na+ and K+ is needed to describe how membrane potentials respond to temperature and [K+] in insect muscle. While considerable further work is still needed, we argue that this “charge difference” model can be used to generate testable hypotheses of how insects can preserve membrane polarization in the face of stressful cold exposure.

Chronic electrical stimulation reduces reliance on anaerobic metabolism in locust jumping muscle

Chronic electrical stimulation (CES) is a well-documented method for changing mammalian muscle from more fast-twitch to slow-twitch metabolic and contractile profiles. Although both mammalian and insect muscles have many similar anatomical and physiological properties, it is unknown if CES produces similar muscle plasticity changes in insects. To test this idea, we separated Schistocerca americana grasshoppers into two groups (n = 37 to 47): one that was subjected to CES for 180 min each day for five consecutive days and one group that was not. Each group was then electrically stimulated for a single time period (0, 5, 30, 60, or 180 min) before measuring jumping muscle lactate, a characteristic of fast-twitch type fibers. At each time point, CES led to a significantly reduced jumping muscle lactate concentration. Based on similar short-term CES mammalian studies, the reduction in lactate production was most likely due to a reduced reliance on anaerobic metabolism. Thus, longer stimulation periods should result in greater aerobic enzymatic activities, altered myosin ATPase, and shift fiber types. This is the first study to use electrical stimulation to explore insect muscle plasticity and our results show that grasshopper jumping muscle responds similarly to mammalian muscle.

Biologically inspired electrostatic artificial muscles for insect-sized robots

Millimeter-sized electrostatic film actuators, inspired by the efficient spatial arrangement of insect muscles, achieve a muscle-like power density (61 W kg−1) and enable robotic applications in which agility is needed in confined spaces. Like biological muscles, these actuators incorporate a hierarchical structure, in this case building from electrodes to arrays to laminates, and are composed primarily of flexible materials. So comprised, these actuators can be designed for a wide range of manipulation and locomotion tasks, similar to natural muscle, while being robust and compact. A typical actuator can achieve 85 mN of force with a 15 mm stroke, with a size of mm3 and mass of 92 mg. Two millimeter-sized robots, an ultra-thin earthworm-inspired robot and an intestinal-muscle-inspired endoscopic tool for tissue resection, demonstrate the utility of these actuators. The earthworm robot undertakes inspection tasks: the navigation of a 5 mm channel and a 19 mm square tube while carrying an on-board camera. The surgical tool, which conforms to the surface of the distal end of an endoscope, similar to the thin, smooth muscle that covers the intestine, completes tissue cutting and penetrating tasks. Beyond these devices, we anticipate widespread use of these actuators in soft robots, medical robots, wearable robots, and miniature autonomous systems.

Pyrrole-2 carboxamides - A novel class of insect ryanodine receptor activators

The ryanodine receptor (RyR) is an intracellular calcium channel critical to the regulation of insect muscle contraction and the target site of diamide insecticides such as chlorantraniliprole, cyantraniliprole and flubendiamide. To-date, diamides are the only known class of synthetic molecules with high potency against insect RyRs. Target-based screening of an informer library led to discovery of a novel class of RyR activators, pyrrole-2-carboxamides. Efforts to optimize receptor activity resulted in analogs with potency comparable to that of commercial diamides when tested against RyR of the fruit fly, Drosophila melanogaster. Surprisingly, testing of pyrrole-2-carboxamides in whole-insect screens showed poor insecticidal activity, which is partially attributed to differential selectivity among insect receptors and rapid detoxification.Among various lepidopteran species field resistance to diamide insecticides has been well documented and in many cases has been attributed to a single point mutation, G4946E, of the RyR gene. As with diamide insecticides, the G4946E mutation confers greatly reduced sensitivity to pyrrole-2-carboxamides. This, coupled with findings from radioligand binding studies, indicates a shared binding domain between anthranilic diamides and pyrrole-2-carboxamides.

Mechanisms of Frank-Starling law of the heart and stretch activation in striated muscles may have a common molecular origin.

Vertebrate cardiac muscle generates progressively larger systolic force when the end diastolic chamber volume is increased, a property called the "Frank-Starling Law", or "length dependent activation (LDA)". In this mechanism a larger force develops when the sarcomere length (SL) increased, and the overlap between thick and thin filament decreases, indicating increased production of force per unit length of the overlap. To account for this phenomenon at the molecular level, we examined several hypotheses: as the muscle length is increased, (1) lattice spacing decreases, (2) Ca2+ sensitivity increases, (3) titin mediated rearrangement of myosin heads to facilitate actomyosin interaction, (4) increased SL activates cross-bridges (CBs) in the super relaxed state, (5) increased series stiffness at longer SL promotes larger elementary force/CB to account for LDA, and (6) stretch activation (SA) observed in insect muscles and LDA in vertebrate muscles may have similar mechanisms. SA is also known as delayed tension or oscillatory work, and universally observed among insect flight muscles, as well as in vertebrate skeletal and cardiacmuscles. The sarcomere stiffness observed in relaxed muscles may significantly contributes to the mechanisms of LDA. In vertebrate striated muscles, the sarcomere stiffness is mainly caused by titin, a single filamentary protein spanning from Z-line to M-line and tightly associated with the myosin thick filament. In insect flight muscles, kettin connects Z-line and the thick filament to stabilize the sarcomere structure. In vertebrate cardiac muscles, titin plays a similar role, and may account for LDA and may constitute a molecular mechanism of Frank-Starling response.

Crystal Structures of Insect Ryanodine Receptor Domains

Diamide insecticides are among the top-selling pesticides, generating worldwide sales over 2 billion U.S. dollars annually. They target insect ryanodine receptors (RyRs) and cause misregulation of calcium signaling in insect muscles and neurons. However, due to heavy usage, several resistance mutations identified in insect RyRs have been reported to reduce the efficacy of the diamides, but the exact binding site of diamides and the mechanism of resistance mutations remain elusive. Two options are available to overcome the resistance: modify the diamide insecticide to target the same binding pocket in resistant RyRs or develop new compounds to target a different binding site in insect RyRs. The recent breakthrough in the structural studies of mammalian RyRs has deepened our understanding of the channel, but the structural information about insect RyRs is still scarce. Here we report four crystal structures of insect RyR domains, including N-terminal domain (NTD), SPRY2 domain, and repeat34 domain from diamondback moth (DBM), and NTD from the honeybee. Several regions in these domains showed distinguished conformations in DBM relative to honeybee and mammalian RyRs. Many of these pest-specific structural features are located in the domain-domain interfaces that would change conformation upon channel gating, making them good target for candidate insecticides. We identified several protein kinase a (PKA) phosphorylation sites clustering in a loop and a newly identified α-helix in DBM repeat34 domain, showing a pest-specific phosphorylation pattern. Interestingly, the phosphorylation of insect RyR is temperature-dependent, facilitated by the low thermal stability and dynamic structure of insect Repeat34 domain. The accurate structural information of insect RyRs would provide valuable templates for the design of insecticidal molecules.

Metamorphosis in Insect Muscle: Insights for Engineering Muscle-Based Actuators.

One of the major limitations to advancing the development of soft robots is the absence of lightweight, effective soft actuators. While synthetic systems, such as pneumatics and shape memory alloys, have created important breakthroughs in soft actuation, they typically rely on large external power sources and some rigid components. Muscles provide an ideal actuator for soft constructs, as they are lightweight, deformable, biodegradable, silent, and powered by energy-dense hydrocarbons such as glucose. Vertebrate cell lines and embryonic cultures have allowed critical foundational work to this end, but progress there is limited by the difficulty of identifying individual pathways in embryonic development, and the divergence of immortal cell lines from these normal developmental programs. An alternative to culturing muscles from embryonic cells is to exploit the advantages of species with metamorphic stages. In these animals, muscles develop from a predefined pool of myoblasts with well-characterized contacts to other tissues. In addition, the endocrine triggers for development into adult muscles are often known and tractable for experimental manipulation. This is particularly true for metamorphic muscle development in holometabolous insects, which provide exciting new avenues for tissue engineering. Using insect tissues for actuator development confers additional benefits; insect muscles are more robust to varying pH, temperature, and oxygenation than are vertebrate cells. Given that biohybrid robots are likely to be used in ambient conditions and changing environments, this sort of hardiness is likely to be required for practical use. In this study, we summarize key processes and signals in metamorphic muscle development, drawing attention to those pathways that offer entry points for manipulation. By focusing on lessons learned from in vivo insect development, we propose that future culture designs will be able to use more systematic, hypothesis-driven approaches to optimizing engineered muscle. Impact statement This review summarizes our current understanding of metamorphic muscle development in insects. It provides a framework for engineering muscle-based actuators that can be used in robotic applications in a wide range of ambient conditions. The focus is on identifying key processes that might be manipulated to solve current challenges in controlling tissue development such as myoblast proliferation, myotube formation and fusion, cytoskeletal alignment, myotendinous attachment and full differentiation. An important goal is to gather findings that cross disciplinary boundaries and to promote the development of better bioactuators for nonclinical applications.

Evolution of Flight Muscle Contractility and Energetic Efficiency.

The powered flight of animals requires efficient and sustainable contractions of the wing muscles of various flying species. Despite their high degree of phylogenetic divergence, flight muscles in insects and vertebrates are striated muscles with similarly specialized sarcomeric structure and basic mechanisms of contraction and relaxation. Comparative studies examining flight muscles together with other striated muscles can provide valuable insights into the fundamental mechanisms of muscle contraction and energetic efficiency. Here, we conducted a literature review and data mining to investigate the independent emergence and evolution of flight muscles in insects, birds, and bats, and the likely molecular basis of their contractile features and energetic efficiency. Bird and bat flight muscles have different metabolic rates that reflect differences in energetic efficiencies while having similar contractile machinery that is under the selection of similar natural environments. The significantly lower efficiency of insect flight muscles along with minimized energy expenditure in Ca2+ handling is discussed as a potential mechanism to increase the efficiency of mammalian striated muscles. A better understanding of the molecular evolution of myofilament proteins in the context of physiological functions of invertebrate and vertebrate flight muscles can help explore novel approaches to enhance the performance and efficiency of skeletal and cardiac muscles for the improvement of human health.

CDNA Cloning and Expression Analysis of Troponin C from Gryllus bimaculatus (Orthoptera: Gryllidae)

Few reports have described the effect of starvation on insect muscle physiology and development. This study examined the role of troponin C (TnC) in the skeletal muscles and digestive tracts of the two-spotted cricket (Gryllus bimaculatus; Orthoptera: Gryllidae) during starvation and re-feeding. The TnC gene of G. bimaculatus (GrybiTnC) was cloned and found to encode a 153-amino-acid protein with a theoretical isoelectric point of 4.02 and molecular weight of 17,368.23 Da. Homology analyses revealed that GrybiTnC exhibited high amino acid similarities with TnCs from other insects, showing sequence identities ranging from ∼54 to 75%. GrybiTnC has two Ca2+-binding sites and five EF-hand motifs, similar to most TnC proteins. GrybiTnC gene expression was examined under starvation and re-feeding conditions in three different skeletal muscle and digestive tract regions. During starvation, GrybiTnC gene expression was unchanged in muscle but upregulated in the foregut and hindgut. Whereas GrybiTnC gene expression in muscle was similar under re-feeding and starvation conditions, it was upregulated in the foregut and midgut by re-feeding with carbohydrates and lipids and downregulated in the hindgut by re-feeding with carbohydrates, proteins and lipids.

Identification, Localization in the Central Nervous System and Novel Myostimulatory Effect of Allatostatins in Tenebrio molitor Beetle.

Allatostatins (ASTs) are pleiotropic insect neuropeptides that are potent myoinhibitors of muscle contractions. In this study, we identified and immunolocalized peptides from the MIP/AST and PISCF/AST families in the nervous system of a model beetle, Tenebrio molitor. Neurons containing MIPs were immunolocalized in the brains of adults and the ventral nerve cords of larvae, pupae and imagines of this species as well as in the retrocerebral complex. PISCFs were immunolocalized in the ventral nerve cord of all stages as well as the brain of the adult beetle. Faint signals were also observed in the corpus allatum but not in the corpus cardiacum. The results allowed us to deduce the sequences of three neuropeptides belonging to MIP/ASTs, Tenmo-MIP4—NWGQFGXWa, Tenmo-MIP5—SKWDNFRGSWa and Tenmo-MIP6—EPAWSNLKGIWa, and one peptide from the PISCF/AST family, QSRYXQCYFNPISCX. Furthermore, we showed for the first time myostimulatory action of endogenous MIP/ASTs. Tenmo-MIP5 caused dose-dependent stimulation of the contractile activity of the beetle oviduct muscles, showing a sigmoidal curve up to 81.20% at the 10−8 M concentration, and the EC50 value for the myostimulatory effect of this peptide was 8.50 × 10−12 M. This is the first report of myostimulatory action of an endogenous myoinhibitory peptide in insect muscles.

β-Sheet Nanocrystals Dictate Water Responsiveness of Bombyx Mori Silk.

Water-responsive (WR) materials that strongly swell and shrink in response to changes in relative humidity (RH) have shown a great potential to serve as high-energy actuators for soft robotics and new energy-harvesting systems. However, the design criteria governing the scalable and high-efficiency WR actuation remain unclear, and thus inhibit further development of WR materials for practical applications. Nature has provided excellent examples of WR materials that contain stiff nanocrystalline structures that can be crucial to understand the fundamentals of WR behavior. This work reports that regenerated Bombyx (B.) mori silk can be processed to increase β-sheet crystallinity, which dramatically increases the WR energy density to 1.6 MJ m-3 , surpassing that of all known natural muscles, including mammalian muscles and insect muscles. Interestingly, the maximum water sorption decreases from 80.4% to 19.2% as the silk's β-sheet crystallinity increases from 19.7% to 57.6%, but the silk's WR energy density shows an eightfold increase with higher fractions of β-sheets. The findings of this study suggest that high crystallinity of silk reduces energy dissipation and translates the chemical potential of water-induced pressure to external loads more efficiently during the hydration/dehydration processes. Moreover, the availability of B. mori silk opens up possibilities for simple and scalable modification and production of powerful WR actuators.

The glutamic acid-rich–long C-terminal extension of troponin T has a critical role in insect muscle functions

The troponin complex regulates the Ca2+ activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid-rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+ buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.