Electromagnetic field exposure affects the calling song, phonotaxis, and level of biogenic amines in crickets.

The electromagnetic field (EMF) is ubiquitous in the environment, constituting a well-known but poorly understood stressor. Few studies have been conducted on insect responses to EMF, although they are an excellent experimental model and are of great ecological importance. In our work, we tested the effects of EMF (50 Hz, 7 mT) on the cricket Gryllus bimaculatus: the male calling song pattern, female mate choice, and levels of biogenic amines in the brain. Exposure of males to EMF increased the number and shortened the period of chips in their calling song (by 2.7% and 5% relative to the control song, respectively), but not the sound frequency. Aged (3-week-old) females were attracted to both natural and EMF-modified male signals, whereas young (1-week-old, virgin) females responded only to the modified signal, suggesting its higher attractance. Stress response of males to EMF may be responsible for the change in the calling song, as suggested by the changes in the amine levels in their brains: an increase in dopamine (by 50% relative to the control value), tyramine (65%), and serotonin (25%) concentration and a decrease in octopamine level (by 25%). These findings indicate that G. bimaculatus responds to EMF, like stressful conditions, which may change the condition and fitness of exposed individuals, disrupt mate selection, and, in consequence, affect the species' existence.

Photoreceptor genes in a trechine beetle, Trechiama kuznetsovi, living in the upper hypogean zone.

To address how organisms adapt to a new environment, subterranean organisms whose ancestors colonized subterranean habitats from surface habitats have been studied. Photoreception abilities have been shown to have degenerated in organisms living in caves and calcrete aquifers. Meanwhile, the organisms living in a shallow subterranean environment, which are inferred to reflect an intermediate stage in an evolutionary pathway to colonization of a deeper subterranean environment, have not been studied well. In the present study, we examined the photoreception ability in a trechine beetle, Trechiama kuznetsovi, which inhabits the upper hypogean zone and has a vestigial compound eye. By de novo assembly of genome and transcript sequences, we were able to identify photoreceptor genes and phototransduction genes. Specifically, we focused on opsin genes, where one long wavelength opsin gene and one ultraviolet opsin gene were identified. The encoded amino acid sequences had neither a premature stop codon nor a frameshift mutation, and appeared to be subject to purifying selection. Subsequently, we examined the internal structure of the compound eye and nerve tissue in the adult head, and found potential photoreceptor cells in the compound eye and nerve bundle connected to the brain. The present findings suggest that T. kuznetsovi has retained the ability of photoreception. This species represents a transitional stage of vision, in which the compound eye regresses, but it may retain the ability of photoreception using the vestigial eye.

Embodied latch mechanism of the mandible to power at ultra-high speed in the trap-jaw ant Odontomachus kuroiwae.

Rapid movements of limbs and appendages, faster than those produced by simple muscle contraction alone, are generated through mechanical networks consisting of springs and latches. The latch plays a central role in these spring-loaded mechanisms, but the structural details of the latch are not always known. The mandibles of the trap-jaw ant Odontomachus kuroiwae closes the mandible extremely quickly to capture prey or to perform mandible-powered defensive jumps to avoid potential threats. The jump is mediated by a mechanical spring and latch system embodied in the mandible. An ant can strike the tip of the mandible onto the surface of an obstacle (prey, predator or ground) in order to bounce its body away from potential threats. The angular velocity of the closing mandible was 2.3×104 rad s-1 (1.3×106 deg s-1). Latching of the joint is a key mechanism to aid the storage of energy required to power the ballistic movements of the mandibles. We have identified the fine structure of two latch systems on the mandible forming a 'ball joint' using an X-ray micro-computational tomography system (X-ray micro-CT) and X-ray live imaging with a synchrotron. Here, we describe the surface of the inner section of the socket and a projection on the lip of the ball. The X-ray live imaging and movements of the 3D model show that the ball with a detent ridge slipped into a socket and over the socket ridge before snapping back at the groove edge. Our results give insight into the complex spring-latch systems that underpin ultra-fast movements in biological systems.

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.

Fold analysis of crumpled sheets using microcomputed tomography.

Hand-crumpled paper balls involve intricate structure with a network of creases and vertices, yet show simple scaling properties, which suggests self-similarity of the structure. We investigate the internal structure of crumpled papers by the microcomputed tomography (micro-CT) without destroying or unfolding them. From the reconstructed three-dimensional (3D) data, we examine several power laws for the crumpled square sheets of paper of the sizes L=50-300 mm and obtain the mass fractal dimension D_{M}=2.7±0.1 by the relation between the mass and the radius of gyration of the balls and the fractal dimension 2.5≲d_{f}≲2.8 for the internal structure of each crumpled paper ball by the box counting method in the real space and the structure factors in the Fourier space. The data for the paper sheets are consistent with D_{M}=d_{f}, suggesting that the self-similarity in the structure of each crumpled ball gives rise to the similarity among the balls with different sizes. We also examine the cellophane sheets and the aluminium foils of the size L=200 mm and obtain 2.6≲d_{f}≲2.8 for both of them. The micro-CT also allows us to reconstruct 3D structure of a line drawn on the crumpled sheets of paper. The Hurst exponent for the root-mean-square displacement along the line is estimated as H≈0.9 for the length scale shorter than the scale of the radius of gyration, beyond which the line structure becomes more random with H∼0.5.

A symbiotic aphid selfishly manipulates attending ants via dopamine in honeydew.

Symbiotic relationships are widespread in nature, but the mechanisms maintaining these relationships remain to be elucidated because symbiosis incurs a maintenance cost to each participant, which lowers its reproductive rate. In host-parasite relationships, parasites are known to manipulate the host's behavior selfishly, and there is an arms race between them. Selfish manipulations also occur in symbiosis, but the effects of selfish manipulations on symbiosis are not fully understood. Here, we show that an ant-associated aphid manipulates attending ants to receive stronger protection. Aphid honeydew regurgitated by ants contains dopamine (DA). The ants showed low aggressiveness before contact with the aphids, but it rose after contact. Administration of DA to the ants increased ant aggressiveness as the concentration increased, while an antagonist of DA inhibited this effect. The other 3 amines showed no effect on aggressiveness. A previous study showed that attending ants selfishly manipulate aphids by increasing the reproductive rate of green morph to obtain high-quality honeydew. These results suggest that mutual selfish manipulation benefits both participants and is likely to strengthen symbiosis. The selfishness of each participant may contribute to sustaining this symbiosis because their selfishness increases their long-term fitness.

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.

Descending and Ascending Signals That Maintain Rhythmic Walking Pattern in Crickets.

The cricket is one of the model animals used to investigate the neuronal mechanisms underlying adaptive locomotion. An intact cricket walks mostly with a tripod gait, similar to other insects. The motor control center of the leg movements is located in the thoracic ganglia. In this study, we investigated the walking gait patterns of the crickets whose ventral nerve cords were surgically cut to gain an understanding of how the descending signals from the head ganglia and ascending signals from the abdominal nervous system into the thoracic ganglia mediate the initiation and coordination of the walking gait pattern. Crickets whose paired connectives between the brain and subesophageal ganglion (SEG) (circumesophageal connectives) were cut exhibited a tripod gait pattern. However, when one side of the circumesophageal connectives was cut, the crickets continued to turn in the opposite direction to the connective cut. Crickets whose paired connectives between the SEG and prothoracic ganglion were cut did not walk, whereas the crickets exhibited an ordinal tripod gait pattern when one side of the connectives was intact. Crickets whose paired connectives between the metathoracic ganglion and abdominal ganglia were cut initiated walking, although the gait was not a coordinated tripod pattern, whereas the crickets exhibited a tripod gait when one side of the connectives was intact. These results suggest that the brain plays an inhibitory role in initiating leg movements and that both the descending signals from the head ganglia and the ascending signals from the abdominal nervous system are important in initiating and coordinating insect walking gait patterns.

Extremely Low-Frequency Electromagnetic Fields Entrain Locust Wingbeats.

Extremely low-frequency electromagnetic fields (ELF EMFs) have been shown to impact the behavior and physiology of insects. Recent studies have highlighted the need for more research to determine more specifically how they affect flying insects. Here, we ask how locust flight is affected by acute exposure to 50 Hz EMFs. We analyzed the flights of individual locusts tethered between a pair of copper wire coils generating EMFs of various frequency using high-speed video recording. The mean wingbeat frequency of tethered locusts was 18.92 ± 0.27 Hz. We found that acute exposure to 50 Hz EMFs significantly increased absolute change in wingbeat frequency in a field strength-dependent manner, with greater field strengths causing greater changes in wingbeat frequency. The effect of EMFs on wingbeat frequency depended on the initial wingbeat frequency of a locust, with locusts flying at a frequency lower than 20 Hz increasing their wingbeat frequency, while locusts flying with a wingbeat frequency higher than 20 Hz decreasing their wingbeat frequency. During the application of 50 Hz EMF, the wingbeat frequency was entrained to a 2:5 ratio (two wingbeat cycles to five EMF cycles) of the applied EMF. We then applied a range of ELF EMFs that were close to normal wingbeat frequency and found that locusts entrained to the exact frequency of the applied EMF. These results show that exposure to ELF EMFs lead to small but significant changes in wingbeat frequency in locusts. We discuss the biological implications of the coordination of insect flight in response to electromagnetic stimuli. © 2021 Bioelectromagnetics Society.

Leg amputation modifies coordinated activation of the middle leg muscles in the cricket Gryllus bimaculatus.

Insects alter their walking pattern in order to respond to demands of an ever-changing environment, such as varying ground surface textures. They also exhibit resilient and flexible ability to retain the capacity to walk even after substantial changes in their body properties, e.g. leg amputation. While the motor control paradigm governing the inter-leg coordination in such adaptive walking has been extensively described in past studies, the mechanism remains unknown. Here, we examined this question by using the cricket (Gryllus bimaculatus), which shows a tetrapod/tripod gait on a flat surfaces, like many other insects. We performed leg amputation experiments to investigate modifications of leg movements and coordination of muscle activities. We simultaneously recorded (1) the leg movements, locomotion velocity, and body rotation and (2) the leg movements and leg muscles activities before and after leg amputation. Crickets displayed adaptive coordination of leg movement patterns in response to amputations. The activation timings of levator muscles in both middle legs tended to synchronize in phase when both legs were amputated at the coxatrochanteral joint. This supports the hypothesis that an intrinsic contralateral connection within the mesothoracic ganglion exists, and that mechanosensory feedback from the legs override this connection, resulting in the anti-phase movement of a normal gait.

Serotonergic control in initiating defensive responses to unexpected tactile stimuli in the trap-jaw ant Odontomachus kuroiwae.

The decision to express either a defensive response or an escape response to a potential threat is crucial for insects to survive. This study investigated an aminergic mechanism underlying defensive responses to unexpected touch in an ant that has powerful mandibles, the so-called trap-jaw. The mandibles close extremely quickly and are used as a weapon during hunting. Tactile stimulation to the abdomen elicited quick forward movements in a dart escape in 90% of the ants in a colony. Less than 10% of the ants responded with a quick defensive turn towards the source of stimulation. To reveal the neuronal mechanisms underlying this defensive behavior, the effect of brain biogenic amines on the responses to tactile stimuli were investigated. The levels of octopamine (OA), dopamine (DA) and serotonin (5HT) in the brain were significantly elevated in ants that responded with a defensive turn to the unexpected stimulus compared with ants that responded with a dart escape. Oral administration of DA and 5HT demonstrated that both amines contributed to the initiation of a defensive response. Oral administration of l-DOPA weakly affected the initiation of the defensive turn, while 5-hydroxy-l-tryptophan (5HTP) strongly affected the initiation of defensive behavior. Oral administration of ketanserin, a 5HT antagonist, inhibited the initiation of the defensive turn in aggressive workers, abolishing the effects of both 5HT and 5HTP on the initiation of turn responses. These results indicate that 5HTergic control in the nervous system is a key for the initiation of defensive behavior in the trap-jaw ant.

Earwig fan designing: Biomimetic and evolutionary biology applications.

Technologies to fold structures into compact shapes are required in multiple engineering applications. Earwigs (Dermaptera) fold their fanlike hind wings in a unique, highly sophisticated manner, granting them the most compact wing storage among all insects. The structural and material composition, in-flight reinforcement mechanisms, and bistable property of earwig wings have been previously studied. However, the geometrical rules required to reproduce their complex crease patterns have remained uncertain. Here we show the method to design an earwig-inspired fan by considering the flat foldability in the origami model, as informed by X-ray microcomputed tomography imaging. As our dedicated designing software shows, the earwig fan can be customized into artificial deployable structures of different sizes and configurations for use in architecture, aerospace, mechanical engineering, and daily use items. Moreover, the proposed method is able to reconstruct the wing-folding mechanism of an ancient earwig relative, the 280-million-year-old Protelytron permianum This allows us to propose evolutionary patterns that explain how extant earwigs acquired their wing-folding mechanism and to project hypothetical, extinct transitional forms. Our findings can be used as the basic design guidelines in biomimetic research for harnessing the excellent engineering properties of earwig wings, and demonstrate how a geometrical designing method can reveal morphofunctional evolutionary constraints and predict plausible biological disparity in deep time.

The Role of Serotonin in the Influence of Intense Locomotion on the Behavior Under Uncertainty in the Mollusk Lymnaea stagnalis.

The role of serotonin in the immediate and delayed influence of physical exercise on brain functions has been intensively studied in mammals. Recently, immediate effects of intense locomotion on the decision-making under uncertainty were reported in the Great Pond snail, Lymnaea stagnalis (Korshunova et al., 2016). In this animal, serotonergic neurons control locomotion, and serotonin modulates many processes underlying behavior, including cognitive ones (memory and learning). Whether serotonin mediates the behavioral effects of intense locomotion in mollusks, as it does in vertebrates, remains unknown. Here, the delayed facilitating effects of intense locomotion on the decision-making in the novel environment are described in Lymnaea. Past exercise was found to alter the metabolism of serotonin, namely the content of serotonin precursor and its catabolites in the cerebral and pedal ganglia, as measured by high-performance liquid chromatography. The immediate and delayed effects of exercise on serotonin metabolism were different. Moreover, serotonin metabolism was regulated differently in different ganglia. Pharmacological manipulations of the serotonin content and receptor availability suggests that serotonin is likely to be responsible for the locomotor acceleration in the test of decision-making under uncertainty performed after exercise. However, the exercise-induced facilitation of decision-making (manifested in a reduced number of turns during the orienting behavior) cannot be attributed to the effects of serotonin.

A general model of locomotion of brittle stars with a variable number of arms.

Typical brittle stars have five radially symmetrical arms that coordinate to move the body in a certain direction. However, some species have a variable number of arms, which is a unique trait since intact animals normally have a fixed number of limbs. How does a single species manage different numbers of appendages for adaptive locomotion? We herein describe locomotion in Ophiactis brachyaspis with four, five, six and seven arms to propose a common rule for the movement of brittle stars with different numbers of arms. For this, we mechanically stimulated one arm of individuals to analyse escape direction and arm movement. By gathering quantitative indices and employing Bayesian statistical modelling, we noted a pattern: regardless of the total number of arms, an anterior position emerges at one of the second neighbouring arms to a mechanically stimulated arm, while arms adjacent to the anterior one synchronously work as left and right rowers. We propose a model in which an afferent signal runs clockwise or anticlockwise along the nerve ring while linearly counting how many arms it passes through. With this model, the question on how 'left and right' emerges in a radially symmetrical body via a decentralized system is answered.

Microinjection support system for small biological subjects.

In biological research, various experiments such as behavioral experiments and physiological ones are often conducted with pharmacologically treated animals. In such experiments, it is necessary to inject the same volume of solution into numerous small animals, such as insects to prepare several experimental subjects. However, repeating manual injections is burdensome, and it is also difficult to maintain injection quality and consistency. We have developed a microinjection system that can support and semiautomate the injections of small animals. The system consists of two cameras, a micromanipulator, a syringe pump, and a structural framework all operated from a personal computer to quickly inject the same volume of liquid solutions at the same position and depth into small animals. The microinjection system has sufficient extensibility for it to be used in a variety of applications.

Decoding the essential interplay between central and peripheral control in adaptive locomotion of amphibious centipedes.

Amphibious animals adapt their body coordination to compensate for changing substrate properties as they transition between terrestrial and aquatic environments. Using behavioural experiments and mathematical modelling of the amphibious centipede Scolopendra subspinipes mutilans, we reveal an interplay between descending command (brain), local pattern generation, and sensory feedback that controls the leg and body motion during swimming and walking. The elongated and segmented centipede body exhibits a gradual transition in the locomotor patterns as the animal crosses between land and water. Changing environmental conditions elicit a mechano-sensory feedback mechanism, inducing a gait change at the local segment level. The body segments operating downstream of a severed nerve cord (no descending control) can generate walking with mechano-sensory inputs alone while swimming behaviour is not recovered. Integrating the descending control for swimming initiation with the sensory feedback control for walking in a mathematical model successfully generates the adaptive behaviour of centipede locomotion, capturing the possible mechanism for flexible motor control in animals.

Decentralized Control Mechanism for Determination of Moving Direction in Brittle Stars With Penta-Radially Symmetric Body.

A brittle star, an echinoderm with penta-radially symmetric body, can make decisions about its moving direction and move adapting to various circumstances despite lacking a central nervous system and instead possessing a rather simple distributed nervous system. In this study, we aimed to elucidate the essential control mechanism underlying the determination of moving direction in brittle stars. Based on behavioral findings on brittle stars whose nervous systems were lesioned in various ways, we propose a phenomenological mathematical model. We demonstrate via simulations that the proposed model can well reproduce the behavioral findings. Our findings not only provide insights into the mechanism for the determination of moving direction in brittle stars, but also help understand the essential mechanism underlying autonomous behaviors of animals. Moreover, they will pave the way for developing fully autonomous robots that can make decisions by themselves and move adaptively under various circumstances.

Different Synchrony in Rhythmic Movement Caused by Morphological Difference between Five- and Six-armed Brittle Stars.

Physiological experiments and mathematical models have supported that neuronal activity is crucial for coordinating rhythmic movements in animals. On the other hand, robotics studies have suggested the importance of physical properties made by body structure, i.e. morphology. However, it remains unclear how morphology affects movement coordination in animals, independent of neuronal activity. To begin to understand this issue, our study reports a rhythmic movement in the green brittle star Ophiarachna incrassata. We found this animal moved five radially symmetric parts in a well-ordered unsynchronized pattern. We built a phenomenological model where internal fluid flows between the five body parts to explain the coordinated pattern without considering neuronal activity. Changing the number of the body parts from five to six, we simulated a synchronized pattern, which was demonstrated also by an individual with six symmetric parts. Our model suggests a different number in morphology makes a different fluid flow, leading to a different synchronization pattern in the animal.

Monoamines, Insulin and the Roles They Play in Associative Learning in Pond Snails.

Molluscan gastropods have long been used for studying the cellular and molecular mechanisms underlying learning and memory. One such gastropod, the pond snail Lymnaea stagnalis, exhibits long-term memory (LTM) following both classical and operant conditioning. Using Lymnaea, we have successfully elucidated cellular mechanisms of learning and memory utilizing an aversive classical conditioning procedure, conditioned taste aversion (CTA). Here, we present the behavioral changes following CTA training and show that the memory score depends on the duration of food deprivation. Then, we describe the relationship between the memory scores and the monoamine contents of the central nervous system (CNS). A comparison of learning capability in two different strains of Lymnaea, as well as the filial 1 (F1) cross from the two strains, presents how the memory scores are correlated in these populations with monoamine contents. Overall, when the memory scores are better, the monoamine contents of the CNS are lower. We also found that as the insulin content of the CNS decreases so does the monoamine contents which are correlated with higher memory scores. The present review deepens the relationship between monoamine and insulin contents with the memory score.

Flexible Coordination of Flexible Limbs: Decentralized Control Scheme for Inter- and Intra-Limb Coordination in Brittle Stars' Locomotion.

Conventional mobile robots have difficulties adapting to unpredictable environments or performing adequately after undergoing physical damages in realtime operation, unlike animals. We address this issue by focusing on brittle stars, an echinoderm related to starfish. Most brittle stars have five flexible arms, and they can coordinate among the arms (i.e., inter-arm coordination) as well as the many bodily degrees of freedom within each arm (i.e., intra-arm coordination). They can move in unpredictable environments while promptly adapting to those, and to their own physical damages (e.g., arm amputation). Our previous work focused on the inter-arm coordination by studying trimmed-arm brittle stars. Herein, we extend our previous work and propose a decentralized control mechanism that enables coupling between the inter-arm and intra-arm coordination. We demonstrate via simulations and real-world experiments with a brittle star-like robot that the behavior of brittle stars when they are intact and undergoing shortening or amputation of arms can be replicated.