Functional annotation of insecta transcriptomes: A cautionary tale from Lepidoptera.

Functional annotation is a critical step in the analysis of genomic data, as it provides insight into the function of individual genes and the pathways in which they participate. Currently, there is no consensus on the best computational approach for assigning functional annotation. This study compares three functional annotation methods (BLAST, eggNOG-Mapper, and InterProScan) in their ability to assign Gene Ontology terms in two species of Insecta with differing levels of annotation, Bombyx mori and Manduca sexta. The methods were compared for their annotation coverage, number of term assignments, term agreement and non-overlapping terms. Here we show that there are large discrepancies in gene ontology term assignment among the three computational methods, which could lead to confounding interpretations of data and non-comparable results. This study provide insight into the strengths and weaknesses of each computational method and highlight the need for more standardized methods of functional annotation.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ('Myomatrix arrays') that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a 'motor unit,' during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and identifying pathologies of the motor system.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ("Myomatrix arrays") that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a "motor unit", during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and in identifying pathologies of the motor system.

A virtuous cycle between invertebrate and robotics research: perspective on a decade of Living Machines research.

Many invertebrates are ideal model systems on which to base robot design principles due to their success in solving seemingly complex tasks across domains while possessing smaller nervous systems than vertebrates. Three areas are particularly relevant for robot designers: Research on flying and crawling invertebrates has inspired new materials and geometries from which robot bodies (their morphologies) can be constructed, enabling a new generation of softer, smaller, and lighter robots. Research on walking insects has informed the design of new systems for controlling robot bodies (their motion control) and adapting their motion to their environment without costly computational methods. And research combining wet and computational neuroscience with robotic validation methods has revealed the structure and function of core circuits in the insect brain responsible for the navigation and swarming capabilities (their mental faculties) displayed by foraging insects. The last decade has seen significant progress in the application of principles extracted from invertebrates, as well as the application of biomimetic robots to model and better understand how animals function. This Perspectives paper on the past 10 years of the Living Machines conference outlines some of the most exciting recent advances in each of these fields before outlining lessons gleaned and the outlook for the next decade of invertebrate robotic research.

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.

In vitro Insect Fat Cultivation for Cellular Agriculture Applications.

Cell-cultured fat could provide important elements of flavor, nutrition, and texture to enhance the quality and therefore expand consumer adoption of alternative meat products. In contrast to cells from livestock animals, insect cells have been proposed as a relatively low-cost and scalable platform for tissue engineering and muscle cell-derived cultured meat production. Furthermore, insect fat cells have long been cultured and characterized for basic biology and recombinant protein production but not for food production. To develop a food-relevant approach to insect fat cell cultivation and tissue engineering, Manduca sexta cells were cultured and induced to accumulate lipids in 2D and 3D formats within decellularized mycelium scaffolding. The resultant in vitro fat tissues were characterized and compared to in vivo fat tissue data by imaging, lipidomics, and texture analyses. The cells exhibited robust lipid accumulation when treated with a 0.1 mM soybean oil emulsion and had "healthier" fat profiles, as measured by the ratio of unsaturated to saturated fatty acids. Mycelium scaffolding provided a simple, food-grade approach to support the 3D cell cultures and lipid accumulation. This approach provides a low-cost, scalable, and nutritious method for cultured fat production.

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.

Proleg retractor muscles in Manduca sexta larvae are segmentally different, suggesting anteroposterior specialization.

Manduca sexta larvae are an important model system for studying the neuromechanics of soft body locomotion. They climb on plants using the abdominal prolegs to grip and maneuver in any orientation and on different surfaces. The prolegs grip passively with an array of cuticular hooks, and grip release is actively controlled by retractor muscles inserted into the soft planta membrane at the proleg tip. Until now, the principal planta retractor muscles (PPRMs) in each body segment were thought to be a single fiber bundle originating on the lateral body wall. Here, using high resolution X-ray microtomography of intact animals, we show that the PPRM is a more complex muscle consisting of multiple contractile fibers originating at several distinct sites on the proleg. Furthermore, we show that there are segmental differences in the number and size of some of these fiber groups which suggests that the prolegs may operate differently along the anterior-posterior axis.

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.

Metal or muscle? The future of biologically inspired robots.

Biology has inspired the development of agile robots, and it is now teaching us how to grow machines from living cells.

The control of nocifensive movements in the caterpillar Manduca sexta.

In response to a noxious stimulus on the abdomen, caterpillars lunge their head towards the site of stimulation. This nocifensive 'strike' behavior is fast (∼0.5 s duration), targeted and usually unilateral. It is not clear how the fast strike movement is generated and controlled, because caterpillar muscle develops peak force relatively slowly (∼1 s) and the baseline hemolymph pressure is low (<2 kPa). Here, we show that strike movements are largely driven by ipsilateral muscle activation that propagates from anterior to posterior segments. There is no sustained pre-strike muscle activation that would be expected for movements powered by the rapid release of stored elastic energy. Although muscle activation on the ipsilateral side is correlated with segment shortening, activity on the contralateral side consists of two phases of muscle stimulation and a marked decline between them. This decrease in motor activity precedes rapid expansion of the segment on the contralateral side, presumably allowing the body wall to stretch more easily. The subsequent increase in contralateral motor activation may slow or stabilize movements as the head reaches its target. Strike behavior is therefore a controlled fast movement involving the coordination of muscle activity on each side and along the length of the body.

Stepping pattern changes in the caterpillar Manduca sexta: the effects of orientation and substrate.

Most animals can successfully travel across cluttered, uneven environments and cope with enormous changes in surface friction, deformability and stability. However, the mechanisms used to achieve such remarkable adaptability and robustness are not fully understood. Even more limited is the understanding of how soft, deformable animals such as tobacco hornworm Manduca sexta (caterpillars) can control their movements as they navigate surfaces that have varying stiffness and are oriented at different angles. To fill this gap, we analyzed the stepping patterns of caterpillars crawling on two different types of substrate (stiff and soft) and in three different orientations (horizontal and upward/downward vertical). Our results show that caterpillars adopt different stepping patterns (i.e. different sequences of transition between the swing and stance phases of prolegs in different body segments) based on substrate stiffness and orientation. These changes in stepping pattern occur more frequently in the upward vertical orientation. The results of this study suggest that caterpillars can detect differences in the material properties of the substrate on which they crawl and adjust their behavior to match those properties.

Nociceptive neurons respond to multimodal stimuli in Manduca sexta.

The caterpillar Manduca sexta produces a highly stereotyped strike behavior in response to noxious thermal or mechanical stimuli to the abdomen. This rapid movement is targeted to the site of the stimulus, but the identity of the nociceptive sensory neurons are currently unknown. It is also not known whether both mechanical and thermal stimuli are detected by the same neurons. Here, we show that the likelihood of a strike increases with the strength of the stimulus and that activity in nerves innervating the body wall increases rapidly in response to noxious stimuli. Mechanical and thermal stimuli to the dorsal body wall activate the same sensory unit, suggesting it represents a multimodal neuron. This is further supported by the effects of rapidly repeated thermal or mechanical stimuli, which cause a depression of neuronal responsiveness that is generalized across modalities. Mapping the receptive fields of neurons responding to strong thermal stimuli indicates that these multimodal, nociceptive units are produced by class γ multidendritic neurons in the body wall.

Local and generalized sensitization of thermally evoked defensive behavior in caterpillars.

In addition to camouflage and chemical toxicity, many caterpillars defend themselves against predators with sudden sharp movements. For smaller species, these movements propel the body away from the threat, but in larger caterpillars, such as the tobacco hornworm, Manduca sexta, the movement is a defensive strike targeted to a noxious stimulus on the abdomen. Previously, strikes have been studied using mechanical stimulation like poking or pinching the insect, but such stimuli are hard to control. They also introduce mechanical perturbations that interfere with measurements of the behavior. We have now established that strike behavior can be evoked using infra-red lasers to provide a highly localized and repeatable heat stimulus. The latency from the end of an effective stimulus to the start of head movement decreased with repeated stimuli and this effect generalized to other stimulus locations indicating a centrally mediated component of sensitization. The tendency to strike increased with two successive subthreshold stimuli. When delivered to different locations or to a single site, this split-pulse stimulation revealed an additional site-specific sensitization that has not previously been described in Manduca. Previous work shows that strong stimuli increases the effectiveness of sensory stimulation by activating a long-lasting muscarinic cation current in motoneurons. Injection of muscarinic cholinergic antagonists, scopolamine methyl bromide or quinuclidinyl benzilate, only decreased the strike probability evoked by paired stimuli at two locations and not at a single site. This strongly suggests a role of muscarinic acetylcholine receptors in the generalized sensitization of nociceptive responses in caterpillars.

Quantifying Dynamic Shapes in Soft Morphologies.

Soft materials are driving the development of a new generation of robots that are intelligent, versatile, and adept at overcoming uncertainties in their everyday operation. The resulting soft robots are compliant and deform readily to change shape. In contrast to rigid-bodied robots, the shape of soft robots cannot be described easily. A numerical description is needed to enable the understanding of key features of shape and how they change as the soft body deforms. It can also quantify similarity between shapes. In this article, we use a method based on elliptic Fourier descriptors to describe soft deformable morphologies. We perform eigenshape analysis on the descriptors to extract key features that change during the motion of soft robots, showing the first analysis of this type on dynamic systems. We apply the method to both biological and soft robotic systems, which include the movement of a passive tentacle, the crawling movement of two species of caterpillar (Manduca sexta and Sphacelodes sp.), the motion of body segments in the M. sexta, and a comparison of the motion of a soft robot with that of a microorganism (euglenoid, Eutreptiella sp.). In the case of the tentacle, we show that the method captures differences in movement in varied media. In the caterpillars, the method illuminates a prominent feature of crawling, the extension of the terminal proleg. In the comparison between the robot and euglenoids, our method quantifies the similarity in shape to ∼85%. Furthermore, we present a possible method of extending the analysis to three-dimensional shapes.

In Vitro Insect Muscle for Tissue Engineering Applications.

Tissue engineering is primarily associated with medical disciplines, and research has thus focused on mammalian cells. For applications where clinical relevance is not a constraint, it is useful to evaluate the potential of alternative cell sources to form tissues in vitro. Specifically, skeletal muscle tissue engineering for bioactuation and cultured foods could benefit from the incorporation of invertebrate cells because of their less stringent growth requirements and other versatile features. Here, we used a Drosophila muscle cell line to demonstrate the benefits of insect cells relative to those derived from vertebrates. The cells were adapted to serum-free media, transitioned between adherent and suspension cultures, and manipulated with hormones. Furthermore, we analyzed edible scaffolds to support cell adhesion and assayed cellular protein and minerals to evaluate nutrition potential. The insect muscle cells exhibited advantageous growth patterns and hold unique functionality for tissue engineering applications beyond the medical realm.

Caterpillar Climbing: Robust, Tension-Based Omni-Directional Locomotion.

Animals that must transition from horizontal to inclined or vertical surfaces typically change their locomotion strategy to compensate for the relative shift in gravitational forces. The species that have been studied have stiff articulated skeletons that allow them to redistribute ground reaction forces (GRFs) to control traction. Most also change their stepping patterns to maintain stability as they climb. In contrast, caterpillars, most of which are highly scansorial, soft-bodied, and lack rigid support or joints, can move with the same general kinematics in all orientations. In this study, we measure the GRFs exerted by the abdominal prolegs of Manduca sexta (Linnaeus) during locomotion. We show that, despite the orthogonal shift in gravitational forces, caterpillars use the same tension-based environmental skeleton strategy to crawl horizontally and to climb vertically. Furthermore, the transition from horizontal to vertical surfaces does not seem to require a change in gait; instead gravitational loading is used to help maintain a stance-phase body tension against which the muscles can pull the body upwards.

The neuromechanics of proleg grip release.

Because soft animals are deformable, their locomotion is particularly affected by external forces and they are expected to face challenges controlling movements in different environments and orientations. We have used the caterpillar Manduca sexta to study neuromechanical strategies of soft-bodied scansorial locomotion. Manduca locomotion critically depends on the timing of proleg grip release, which is mediated by the principal planta retractor muscle and its single motoneuron, PPR. During upright crawling, PPR firing frequency increases approximately 0.6 s before grip release but during upside-down crawling, this activity begins significantly earlier, possibly pre-tensioning the muscle. Under different loading conditions the timing of PPR activity changes relative to the stance/swing cycle. PPR motor activity is greater during upside-down crawling but these frequency changes are too small to produce significant differences in muscle force. Detailed observation of the proleg tip show that it swells before the retractor muscle is activated. This small movement is correlated with the activation of more posterior body segments, suggesting that it results from indirect mechanical effects. The timing and direction of this proleg displacement implies that proleg grip release is a dynamic interplay of mechanics and active neural control.

Biohybrid actuators for robotics: A review of devices actuated by living cells.

Actuation is essential for artificial machines to interact with their surrounding environment and to accomplish the functions for which they are designed. Over the past few decades, there has been considerable progress in developing new actuation technologies. However, controlled motion still represents a considerable bottleneck for many applications and hampers the development of advanced robots, especially at small length scales. Nature has solved this problem using molecular motors that, through living cells, are assembled into multiscale ensembles with integrated control systems. These systems can scale force production from piconewtons up to kilonewtons. By leveraging the performance of living cells and tissues and directly interfacing them with artificial components, it should be possible to exploit the intricacy and metabolic efficiency of biological actuation within artificial machines. We provide a survey of important advances in this biohybrid actuation paradigm.