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.

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.

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.

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.

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.

The function of the ophiuroid nerve ring: how a decentralized nervous system controls coordinated locomotion.

Echinoderms lack a centralized nervous control system, yet each extant echinoderm class has evolved unique and effective strategies for locomotion. Brittle stars (Ophiuroidea) stride swiftly over the seafloor by coordinating motions of their five muscular arms. Their arms consist of many repeating segments, requiring them to use a complex control system to coordinate motions among segments and between arms. We conducted in vivo experiments with brittle stars to analyze the functional role of the nerve ring, which connects the nerves in each arm. These experiments were designed to determine how the ophiuroid nervous system performs complex decision making and locomotory actions under decentralized control. Our results show that brittle star arms must be connected by the nerve ring for coordinated locomotion, but information can travel bidirectionally around the nerve ring so that it circumvents the severance. Evidence presented indicates that ophiuroids rely on adjacent nerve ring connections for sustained periodic movements. The number of arms connected via the nerve ring is correlated positively with the likelihood that the animal will show coordinated locomotion, indicating that integrated nerve ring tissue is critical for control. The results of the experiments should provide a basis for the advancement of complex artificial decentralized systems.

Effects of 5-HT and insulin on learning and memory formation in food-deprived snails.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). How well they learn and form memory depends on the degree of food deprivation. Serotonin (5-HT) plays an important role in mediating feeding, and insulin enhances the memory consolidation process following CTA training. However, the relationship between these two signaling pathways has not been addressed. We measured the 5-HT content in the central nervous system (CNS) of snails subjected to different durations of food deprivation. One-day food-deprived snails, which exhibit the best learning and memory, had the lowest 5-HT content in the CNS, whereas 5-day food-deprived snails, which do not learn, had a high 5-HT content. Immersing 1-day food-deprived snails in 5-HT impaired learning and memory by causing an increase in 5-HT content, and that the injection of insulin into these snails reversed this impairment. We conclude that insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS of Lymnaea.

Tyraminergic modulation of agonistic outcomes in crayfish.

Octopamine, a biogenic amine, modulates various behaviors, ranging from locomotion and aggression to learning and memory in invertebrates. Several studies recently demonstrated that tyramine, the biological precursor of octopamine, also affects behaviors independent of octopamine. Here we investigated the involvement of tyramine in agonistic interaction of the male crayfish Procambarus clarkii. When male crayfish fight, larger animals (3-7% difference in body length) are more likely to win. By contrast, direct injection of tyramine or octopamine counteracted the physical advantage of larger animals. Tyramine or octopamine-injected naive large animals were mostly beaten by untreated smaller naive animals. This pharmacological effect was similar to the loser effect in which subordinate larger animals are frequently beaten by smaller animals. Furthermore, loser effects were partly eliminated by either injection of epinastine, an octopamine blocker, or yohimbine, a tyramine blocker, and significantly diminished by injection of a mixture of both blockers. We also observed that tyramine levels in the subesophageal ganglion were remarkably increased in subordinate crayfish after losing a fight. These results suggest that tyramine modulates aggressive levels of crayfish and contributes to the loser effect in parallel with octopamine.

Cyclic AMP-regulated opposing and parallel effects of serotonin and dopamine on phototaxis in the Marmorkrebs (marbled crayfish).

Phototactic behaviours are observed from prokaryotes to amphibians and are a basic form of orientation. We showed that the marbled crayfish displays phototaxis in which the behavioural response reversed from negative to positive depending on external light conditions. Animals reared in a 12-L/12-D light cycle showed negative phototaxis during daytime and positive phototaxis during night-time. Animals reared under constant light conditioning showed negative phototaxis during day- and night-time, while animals reared under constant dark conditioning showed positive phototaxis during day- and night-time. Injection of serotonin leads to a reversal of negative to positive phototaxis in both light/dark-reared and light/light-reared animals while injection of dopamine induced reversed negative phototaxis in dark/dark-reared animals. Four hours of dark adaptation were enough for light/dark-reared animals to reverse phototaxis from negative to positive. Injection of a serotonin 5HT1 receptor antagonist blocked the reverse phototaxis while serotonin 5HT2 receptor antagonists had no effects. Similarly, dark/dark-reared animals reversed to showing negative phototaxis after 4 h of light adaptation. Injection of a dopamine DA1 receptor antagonist blocked this reverse phototaxis, while dopamine DA2 receptor antagonists had no effects. Injection of a cAMP analogue into light/dark-reared animals blocked reverse phototaxis after dark adaptation, while adenylate cyclase inhibitor in dark/dark-reared animals blocked reverse phototaxis after light adaptation. These results strongly suggest that serotonin mediates positive phototaxis owing to decreased cAMP levels, while dopamine-mediated negative phototaxis occurs due to increased cAMP levels. Supporting this, the ratio of serotonin to dopamine in the brain was much higher in dark/dark-reared than light/dark-reared animals.

Weak involvement of octopamine in aversive taste learning in a snail.

The pond snail Lymnaea stagnalis is capable of learning taste aversion by pairing presentations of a sucrose solution and an electric shock and consolidating it into long-term memory (LTM), which is referred to as conditioned taste aversion (CTA). We asked here if the neurotransmitter octopamine is involved in CTA. We first determined the levels of octopamine and its catabolites in the central nervous system (CNS) of snails with varying degrees of food deprivation, because CTA grades are correlated with degrees of food deprivation. We next manipulated the octopamine signaling using both an agonist and an antagonist of octopamine receptors and correlated their respective effects with CTA grades. We found that snails with the least amount of food-deprivation obtained the best CTA grade and had low levels of octopamine; whereas the most severely food-deprived snails did not form CTA and had the highest CNS octopamine levels. In modestly food-deprived snails, octopamine application increased the basal level of feeding response to a sucrose solution, and it did not obstruct CTA formation. Application of phentolamine, an octopamine receptor antagonist, to the most severely food-deprived snails decreased the basal level of feeding elicited by sucrose, but it did not enhance CTA formation. We conclude that octopamine involvement in CTA formation in Lymnaea is at best weak, and that the changes in CNS octopamine content are an epiphenomenon.

Exposure to static electric fields leads to changes in biogenic amine levels in the brains of Drosophila.

Natural and anthropogenic static electric fields are commonly found in the environment and can have both beneficial and harmful effects on many animals. Here, we asked how the fruitfly responds to these fields and what the consequences of exposure are on the levels of biogenic amines in the brain. When given a choice in a Y-tube bioassay Drosophila avoided electric fields, and the greater the field strength the more likely Drosophila were to avoid it. By comparing wild-type flies, flies with wings surgically removed and vestigial winged flies we found that the presence of intact wings was necessary to produce avoidance behaviour. We also show that Coulomb forces produced by electric fields physically lift excised wings, with the smaller wings of males being raised by lower field strengths than larger female wings. An analysis of neurochemical changes in the brains showed that a suite of changes in biogenic amine levels occurs following chronic exposure. Taken together we conclude that physical movements of the wings are used by Drosophila in generating avoidance behaviour and are accompanied by changes in the levels of amines in the brain, which in turn impact on behaviour.

Aggressive behavior in the antennectomized male cricket Gryllus bimaculatus.

Male crickets (Gryllus bimaculatus) exhibit intensively defensive aggressive behavior towards attacking males most often culminating in fighting. After the fight, the loser no longer exhibits aggressiveness in a second, separate encounter with another male; rather, the defeated male exhibits avoidance behavior. Here, we investigated the role of sensory input from the antennae in male defensive aggressive behavior. When we removed antennae from males (antennectomized males), we found that they showed little aggressiveness towards each other whereas they continued to exhibit typical fighting behavior towards an intact male. In addition, in a second encounter, antennectomized losers showed significantly higher aggressiveness towards another male than did intact losers. We further found that antennectomized crickets do not utilize visual or palpal sensory input to elicit aggressive behavior. In contrast, intact males showed aspects of aggressive behavior to male cuticular substances before and after winning a fight, and if they lost a fight they showed avoidance behavior. It thus appears that antennal sensory information is crucial in the mediation of aggressive and avoidance behaviors. However, sensory inputs from the antennae are not necessary to elicit defensive aggressive behavior but are necessary to discriminate conspecific males and initiate attacks against them.

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.

Evidence for instantaneous e-vector detection in the honeybee using an associative learning paradigm.

Many insects use the polarization pattern of the sky for obtaining compass information during orientation or navigation. E-vector information is collected by a specialized area in the dorsal-most part of the compound eye, the dorsal rim area (DRA). We tested honeybees' capability of learning certain e-vector orientations by using a classical conditioning paradigm with the proboscis extension reflex. When one e-vector orientation (CS+) was associated with sugar water, while another orientation (CS-) was not rewarded, the honeybees could discriminate CS+ from CS-. Bees whose DRA was inactivated by painting did not learn CS+. When ultraviolet (UV) polarized light (350 nm) was used for CS, the bees discriminated CS+ from CS-, but no discrimination was observed in blue (442 nm) or green light (546 nm). Our data indicate that honeybees can learn and discriminate between different e-vector orientations, sensed by the UV receptors of the DRA, suggesting that bees can determine their flight direction from polarized UV skylight during foraging. Fixing the bees' heads during the experiments did not prevent learning, indicating that they use an 'instantaneous' algorithm of e-vector detection; that is, the bees do not need to actively scan the sky with their DRAs ('sequential' method) to determine e-vector orientation.

Waggle dance effect: dancing in autumn reduces the mass loss of a honeybee colony.

A honeybee informs her nestmates about the location of a profitable food source that she has visited by means of a waggle dance: a round dance and a figure-of-eight dance for a short- and long-distance food source, respectively. Consequently, the colony achieves an effective collection of food. However, it is still not fully understood how much effect the dance behavior has on the food collection, because most of the relevant experiments have been performed only in limited locations under limited experimental conditions. Here, we examined the efficacy of the waggle dances by physically preventing bees from dancing and then analyzing the changes in daily mass of the hive as an index of daily food collection. To eliminate place- and year-specific effects, the experiments were performed under fully natural conditions in three different cities in Japan from mid September to early October in three different years. Because the experiments were performed in autumn, all six of the tested colonies lost mass on most of the experimental days. When the dance was prevented, the daily reduction in mass change was greater than when the dance was allowed, i.e. the dance inhibited the reduction of the hive mass. This indicates that dance is effective for food collection. Furthermore, clear inhibition was observed on the first two days of the experiments; after that, inhibition was no longer evident. This result suggests that the bee colony adapted to the new environment.

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.

Social interactions influence dopamine and octopamine homeostasis in the brain of the ant Formica japonica.

In ants, including Formica japonica, trophallaxis and grooming are typical social behaviors shared among nestmates. After depriving ants of either food or nestmates and then providing them with either food or nestmates, a behavioral change in type and frequency of social interactions was observed. We hypothesized that starvation and isolation affected levels of brain biogenic amines including dopamine (DA) and octopamine (OA) - neuromediators modifying various insect behaviors - and tested the relationship between brain biogenic amines and social behaviors of stressed ants. Ants starved for 7 days contained lower brain DA levels and they did not perform trophallaxis toward nestmates. Feeding starved ants sucrose solution re-established trophallaxis but not brain DA levels. The performance of trophallaxis induced recovery of brain DA content to the level of untreated ants. Ants that were isolated for 2 days displayed markedly increased OA levels, which following nestmate interactions, returned to levels similar to those of control (non-isolated) ants and ants isolated for 1 h. We conclude that: (1) starvation reduced brain DA level but had no significant effect on brain OA (trophallaxis recovered the brain DA levels), and (2) isolation increased brain OA level but had no effect on brain DA (trophallaxis and grooming events recovered the brain OA levels). We suggest that social interactions with nestmates influenced brain biogenic amine homeostasis in stressed F. japonica.

A model for group-size-dependent behaviour decisions in insects using an oscillator network.

Aggressive behaviour within pairs of male crickets leads to the establishment of a dominance hierarchy. Defeated males avoid their victorious adversaries for several hours before regaining aggressiveness. However, the defeated male does not regain aggressiveness if repeated fighting occurs. Loss of individual aggressiveness is limited by group size, which constrains the number of crickets fighting at any given time. Thus, group aggressive behaviour is modulated by an environmental factor, group size, which is ultimately determined by individual actions, i.e. fighting between two individuals. We developed a robot model to elucidate the mechanism of group-size-dependent behaviour alternation in crickets. The behaviour of individual robots was evaluated experimentally with mobile robots and the group behaviour of the robots was evaluated by computer simulation. We demonstrated that the group-size-dependent strategy in crickets could be generated by local interactions between robots, where the behaviour was governed by an oscillator and memory of the outcome of previous fights.

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.