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Most insects show circadian rhythms of which the free-running period changes in a light-dependent manner and is generally longer under constant light (LL) than under constant dark conditions in nocturnal animals. However, the mechanism underlying this LL-dependent period change remains unclear. Here, using the cricket Gryllus bimaculatus, we examined the effects of long-term LL exposure on the free-running period of locomotor rhythms. Initially, the free-running period was considerably longer than 24 h but it gradually became shorter during long-term exposure to LL. The initiallengthening and ensuing gradual shortening under long-term LL exposure were observed evenafter unilateral removal of the optic lobe. Thus, these changes in the free-running period could be attributable to a single optic lobe clock. RNA interference (RNAi)-mediated silencing of the clock genes Par domain protein 1 (Pdp1) and timeless (tim) revealed that the treatments eliminated the initial period lengthening by LL without reducing circadian photoreceptor gene expression. However, they did not affect the period shortening during long-term LL exposure. The slopes of the regression line for the period change during long-term LL for Pdp1RNAi-treated and timRNAi-treated crickets were not different from that of the dsDsRed2-treated control. These results suggest that the initial period lengthening after transfer to LL requires tim and Pdp1, while the ensuing period shortening during long-term LL exposure is caused by a mechanism independent of tim and Pdp1.

Many insects are able to use skylight e-vector patterns to deduce their heading direction. Crickets have been well known to orient themselves to certain e-vector orientations to keep their walking direction. However, it is still unknown if crickets are able to utilize polarized light information for spatial recognition. Using an experimental paradigm similar to the Morris water maze for rodents, here we examine the possibility that the cricket Gryllus bimaculatus can utilize polarized light information to find the target place. Crickets were placed in a round arena with a heated floor, a portion of which was cooled, and a cross-shaped e-vector pattern was presented from the top of the arena so that the cricket could find the cool spot by walking along the e-vector direction. When the arrangement of the e-vector pattern and the cool spot were fixed throughout the experiments, the time and the walking distance to find the cool spot were significantly decreased with increasing trials, but not when the e-vector pattern was rotated between each trial. Moreover, a model selection indicated that the visual stimulus contributed to the decrease in time and distance. To investigate the cricket's exploration patterns in the arena, a test trial in which the whole floor was uniformly heated was performed before and after the training trials. In the test trial, the crickets trained with the positionally fixed e-vector pattern showed wall-following behavior for a significantly longer time than those untrained and those trained with random e-vector patterns.

Crickets were arbitrarily fed a caffeine-containing diet during their lifetime or were orally administered caffeine in solution once during the conditioning session. These chronically or acutely treated crickets were used to test whether there were positive effects of caffeine on their learning performance. The effects of chronic exposure to caffeine on crickets' growth were also examined by measuring their body weight and counting the number of adults that emerged from larvae. Chronic treatment with relatively high concentrations of caffeine (5 mg/g and 0.5 mg/g in the diet) impaired the growth of crickets. Mortality was higher with 5 mg/g caffeine, and the survivors could not attain the final molt (eclosion). Body weight gain and eclosion rate were also negatively influenced in the group fed 0.5 mg/g caffeine. The olfactory learning paradigm was used to examine the effects of chronic caffeine treatment. Of the caffeine concentrations tested (0.5 µg/g to 0.5 mg/g), significant improvement in long-term memory (LTM) formation was observed only with 50 µg/g caffeine. Acute effects on olfactory learning were examined after oral administration of 0.1 nM to 10 µM caffeine solutions during the conditioning session. Administration of caffeine ≥ 1 nM consistently resulted in a significant improvement in LTM formation. These results suggest that chronic exposure to caffeine enhances learning performance in crickets at a specific dose (50 µg/g in the diet), although it inhibits their growth at higher doses (≥ 0.5 mg/g). In contrast, acute oral administration of caffeine in naive crickets may enhance their learning performance even at a very low (nanomolar) concentration.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 μμg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms.

We examined the compensational recovery of the response rate (relative occurrence) of the wind-evoked escape behavior in unilaterally cercus-ablated crickets (Gryllus bimaculatus) and elucidated the existence of a sensitive period for such recovery by rearing the crickets under different conditions. In one experiment, each cricket was reared in an apparatus called a walking inducer (WI) to increase the sensory input to the remaining cercus, i.e., the self-generated wind caused by walking. In another experiment, each cricket was reared in a small plastic case separate from the outside atmosphere (wind-free: WF). In this rearing condition, the cricket did not experience self-generated wind as walking was prohibited. During the recovery period after the unilateral cercus ablation, the crickets were reared under either the WI or WF condition to investigate the role of the sensory inputs on the compensational recovery of the response rate. The compensational recovery of the response rate occurred only in the crickets reared under the WI condition during the early period after the ablation. In particular, WI rearing during the first three days after the ablation resulted in the largest compensational recovery in the response rate. In contrast, no compensational recovery was observed in the crickets reared under the WF condition during the first three days. These results suggest that a sensitive period exists in which sensory inputs from the remaining cercus affect the compensational recovery of the response rate more effectively than during other periods.

We investigated the effects of visual information on wind-evoked escape behavior in the cricket, Gryllus bimaculatus. Most agitated crickets were found to retreat into a shelter made of cardboard installed in the test arena within a short time. As this behavior was thought to be a type of escape, we confirmed how a visual image of a shelter affected wind-evoked escape behavior. Irrespective of the brightness of the visual background (black or white) or the absence or presence of a shelter, escape jumps were oriented almost 180° opposite to the source of the air puff stimulus. Therefore, the direction of wind-evoked escape depends solely depended on the direction of the stimulus air puff. In contrast, the turning direction of the crickets during the escape was affected by the position of the visual image of the shelter. During the wind-evoked escape jump, most crickets turned in the direction in which a shelter was presented. This behavioral nature is presumably necessary for crickets to retreat into a shelter within a short time after their escape jump.

We found that the genitalia of the male cricket Gryllus bimaculatus are equipped with an autocleaning system. The cricket keeps its genitalia clean by removing foreign matter and endogenous waste. Morphological study showed that the membrane complex consists of a median pouch and a genital chamber floor covered by small scales, each of which has a base of approximately 10 µm in width and a fringe with 5–10 spines 3–20 µm in length. The scales are arranged symmetrically about the midline, curving gradually in the lateral direction and continuing to the lateral pouch serving as a trash container. Observation of cleaning revealed that a small piece of artificial dirt placed on the membrane complex was conveyed over a distance of 1.3 mm to the lateral pouch in 12 minutes. Inspection of the dorsal pouch just after spermatophore extrusion in the mating stage revealed that there were patchy remnants of spermatophore material on the inner surface of the pouch, but that these were evacuated in a few minutes. Surgical elimination of the median pouch caused the formation of abnormal spermatophores with the sperm tube and attachment plate being deformed. These results suggest that genital autocleaning is indispensable for the production of a normal spermatophore in the male cricket.

Three types of genital movement, their neural controls, and functional roles were investigated to gain a better understanding of the mechanism underlying autocleaning in the male cricket. The membrane complex consisting of the median pouch and genital chamber floor shows peculiar undulation that is composed of two types of movements: a right-left large shift and small crease-like movements. The large shift was caused by contraction of a pair of muscles (MPA) located anterior to the median pouch, while the crease-like movements were caused by numerous muscle fibers extending over the membrane complex. The MPA and muscle fibers were each innervated by efferent neurons in the terminal abdominal ganglion. Experiments with artificial dirt mimicking a foreign object revealed that the crease-like movements were responsible for dirt transport, while the large shift participated in sweeping the dirt into the lateral pouch as a trash container. On the other hand, the dorsal pouch serving as a template for the spermatophore showed a jerky bending movement. Simultaneous monitoring of the membrane complex and dorsal pouch activities suggested that their movements cooperate to enable the efficient evacuation of waste in the dorsal pouch. Based on the results, we conclude that genital autocleaning supports the production of the spermatophore.

Most animals exhibit circadian rhythms in various physiological and behavioral functions regulated by circadian clock that resides in brain and in many peripheral tissues. Temperature cycle is an important time cue for entrainment, even in mammals, since the daily change in body temperature is thought to be used for phase regulation of clocks in peripheral tissues. However, little is known about the mechanisms by which temperature resets the clock. In the present study, we investigated the effect of temperature on circadian activity rhythm and clock gene transcription by using the cricket, Gryllus bimaculatus. We show that temperature cycle can entrain both behavioral and transcriptional rhythms of clock genes, such as period, timeless, cryptochrome2 and cycle in the circadian pacemaker tissue, optic lobe. Under temperature cycle, phase of evening peak of locomotor activity occurred 1 h before the warm-to-cold phase transition, which is associated with earlier peaks of mRNA expression rhythm of the clock genes than that under light/dark cycles. When the temperature cycle was advanced by 6 h, behavioral rhythms re-entrained to newly phased temperature cycle after ∼16 transient cycles. The mRNA oscillation of period and timeless gained stable rhythm under phase advanced temperature cycles with a lesser number of transient cycles than cryptochrome2 and cycle. These results suggest that temperature cycle can entrain behavioral and molecular rhythms in cricket and clock genes vary in sensitivity to temperature. It is thus likely that clock genes play differential roles in resetting the clock with environmental temperature changes.

The effects of the delay and duration of wind self-generated during walking on the compensational recovery of escape direction were investigated in unilaterally cercus-ablated crickets, Gryllus bimaculatus. Artificial self-generated winds (self-stimulations; hereafter, SSts) from a nozzle set in front of a cricket placed on a styrofoam ball for stationary walking were used for training after unilateral cercus ablation. The delay and duration of artificial SSts were separately controlled. When the stimulus duration was fixed to 100 msec, the crickets trained with artificial SSts of 1000 msec delay showed a compensational recovery of the escape direction. However, no such compensational recovery was observed in crickets trained with artificial SSts of 1200, 1500, and 2000 msec delays. The relationship between the delay and duration of artificial SSts for compensational recovery was investigated. An artificial SSt with a longer delay required a longer-duration air current to cause a recovery of the escape direction. In contrast, an artificial SSt with a shorter delay was effective even when the duration was short. On the basis of the results obtained in the present study, we propose a hypothesis to explain the initial step for the compensation, that is, how the delay and duration of SSts are traded in terms of the compensational recovery of the escape direction.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 μμg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms., Zoological Science, 33(5), pp.513-519; 2016

Wind-sensitive giant interneurons (GIs) in the cricket Gryllus bimaculatus show functional recovery after unilateral cercal ablation. Physiological properties such as threshold velocity and response magnitude (number of action potentials elicited) of GIs 8-1, 9-1, 9-2 and 9-3 to an air puff stimulus were investigated in crickets reared under the condition that permitted free walking for 6 days after unilateral cercal ablation ("6-day-free" crickets). The results were compared to those previously obtained from crickets 1 day after unilateral cercal ablation ("1-day-free" crickets) to clarify functional changes during an early 5-day period after the sensory deprivation. Each GI showed a large functional change, despite the short period after the ablation. However, the degree of physiological change was different from one GI to another and from one stimulus direction to another. The direction at which the GIs showed physiological change during the 5-day period coincided well with that at which the GIs received excitatory sensory inputs from filiform hairs on the remaining cercus. It seems that the synaptic connection or efficacy between sensory neurons of filiform hairs with particular directionality on the one remaining cercus and GIs increased during the 5-day recovery period.

The effects of rearing conditions on the functional recovery of wind-sensitive giant interneurons (GIs) after unilateral cercal ablation were investigated in the cricket, Gryllus bimaculatus. Crickets were reared in a glass vials to prohibit free walking for 14 days after unilateral cercal ablation ("14-day vial" crickets). Other crickets were reared in an apparatus called a "walking inducer" (WI) to increase the walking distance during the same 14-day period ("14-day WI" crickets). In these crickets, the response properties of GIs 8-1, 9-1, 9-2, and 9-3 to air currents from various directions were investigated. From the intensity-response curves obtained, directionality curves expressed in terms of threshold velocity and response magnitude were made independently. To understand changes in the functional recovery of GIs more thoroughly, the directional characteristics of GIs in crickets 1 day after unilateral cercal ablation ("1-day free" crickets) were also compared. Between the 1-day free and 14-day vial crickets, all the GIs showed differences in both threshold velocity and response magnitude for some stimulus directions. Between the 14-day vial and 14-day WI crickets, differences in the threshold velocities of GIs 9-1, 9-2, and 9-3, and in the response magnitudes of GIs 8-1, 9-1, and 9-3 were detected. Because the rearing condition after unilateral cercal ablation largely affects the compensatory recovery in some parameters of wind-evoked escape behavior, such as relative occurrence and escape direction, we discuss the functional differences in GIs revealed here in relation to the roles of GIs in the neural system that controls escape behavior.

An air puff stimulus evoked the swimming of an intact cricket, Gryllus bimaculatus, placed on a water surface. When only the forelegs were intact, swimming was initiated frequently, but flying was never initiated. On the other hand, flying was initiated when only the middle legs or hindlegs were intact. Therefore, the sensory inputs from the forelegs are important in the initiation of swimming and for the inhibition of flying when on the water surface. After bilateral ablation of the middle legs and hindlegs, the bilateral segments of the remaining forelegs were sequentially ablated from the distal area to the proximal area of the legs. After bilateral ablation of all tarsomeres, the relative occurrence of swimming decreased and that of flying increased. After the following ablation of the bilateral tibiae, most insects responded to an air puff stimulus by flying. Experiments performed after coating the leg surface with enamel resulted in almost the same behavioral change as that observed in the ablation experiments. These results suggest that the sensory receptors responsible for the initiation of swimming and the inhibition of flying are mainly located on the surface of the tibia and the tarsus of the forelegs. The behavioral change between swimming and walking was also studied using methylcellulose solutions of various viscosities. On the methylcellulose solution, the relative occurrence of walking in the crickets increased with an increase in the viscosity of the solution.

To identify the sensory organs that are sensitive to water stimuli in the cricket Gryllus bimaculatus, cuticular structures on the legs and the number of sensory neurons innervating them were studied. Some small hair sensilla on the legs were innervated by 2-5 sensory neurons. All such sensilla had a tiny pore at the tip of their hairs. The diameter of the pore was approximately 0.2 mum. These findings suggest that these are chemosensitive hairs (LCS: leg chemosensillum). Of the three pairs of legs, the anterior legs (forelegs) possessed the largest number of LCSs. Of the five leg segments (i.e., coxa, trochanter, femur, tibia and tarsus), the tarsus possessed the largest number of LCSs on each leg. Electrophysiological investigation by tip recording revealed that some of the LCSs contained water-receptor cells. Because the basitarsus possessed a larger number of LCSs than the other tarsomeres, the distribution of water-receptor-containing LCSs in the basitarsus of a foreleg was investigated morphologically and electrophysiologically. LCSs that contained water-receptor cells were mainly distributed on the ventral surface of the basitarsus. There were two types of water receptor that showed different response patterns to a stimulus, that is, phasic- and tonic-type water receptors. From the distribution of LCSs on the legs, the roles of these different types of water receptors in behavioral selection, that is, the initiation of swimming and the inhibition of flying, will be discussed.

After a loss against an opponent, the aggressiveness of a male cricket is significantly reduced for up to 30 minutes. This depression of aggressiveness is an important factor in the establishment and maintenance of dominance between individuals. In the present study, we investigated the functional roles of nitric oxide (NO) signaling in the depression of aggressiveness in subordinate male crickets. Pairs of male crickets, pre-injected with various NO-related reagents, were allowed to establish dominant/subordinate relationships in dyadic encounters. Opponents were separated for 15 minutes and then paired again. In second encounters, subordinate crickets pre-injected with PTIO (NO scavenger) showed agonistic behavior towards former dominant opponents. A similar effect was observed in crickets pre-injected with L-NAME (NO synthase inhibitor) or ODQ (soluble guanylate cyclase inhibitor). The effects of the latter two drugs were canceled by co-injection of NOR3 (NO donor) with L-NAME or by co-injection of 8-Br-cGMP (cGMP-analog) with ODQ. Injection of NOR3 alone prolonged the inhibition of agonistic behavior in subordinate crickets from 30 minutes to 3 hours. Our results suggest that the change in agonistic behavior observed in subordinate male crickets is closely linked to NO-mediated cGMP signaling.

Unilaterally cercus-ablated crickets, Gryllus bimaculatus, were reared in an apparatus that induced walking artificially. In the crickets that experienced different distances of enforced walking per day, the directionality of escape was investigated before and 1, 4, 7, 10, 13, and 16 days after the ablation of the cercus. The crickets that walked a longer distance per day showed a quicker and a higher degree of compensational recovery of the escape direction than the crickets that walked a shorter distance per day, even after walking the same distance. Thus, the time course and amount of compensational recovery from cercal ablation depend on when crickets experience walking during the recovery period. During the recovery period, unilaterally cercus-ablated crickets were subjected to walking at different times to determine the most effective period in which walking affects the compensational recovery of escape direction. The compensational recovery of the escape direction occurred only in crickets experiencing walking in early periods after the ablation. In particular, walking experienced 2-6 days after the ablation was most effective for the compensational recovery. On the other hand, no compensational recovery occurred in crickets experiencing walking in later periods after the ablation. These results suggest that there is a sensitive or critical period in which walking affects the compensational recovery of escape direction.

The wind-evoked escape behavior of the cricket Gryllodes sigillatus was investigated using an air puff stimulus. A high velocity air puff elicited the escape behavior in many crickets. The crickets tended to escape away from the stimulus source, but the direction was not accurately oriented 180 degrees from the stimulus. After bilateral cercal ablation, only a few crickets showed wind-evoked escape behavior, and their response rates did not increase even 19 days after ablation. Therefore, information on air motion detected by cercal filiform hairs is essential for triggering wind-evoked behavior. After unilateral cercal ablation, the 81.3% response rate of intact crickets decreased to 16.5%, that is, it decreased to almost 20% that of intact crickets. One week after unilateral cercal ablation, the response rate recovered to more than 60% that of intact crickets. However, the accuracy rate of the escape direction of G. sigillatus showed no change even immediately after the unilateral cercal ablation. Therefore, both cerci are not necessarily required to determine the escape direction. The behavioral characteristics of wind-evoked escape of G. sigillatus are compared with those of another species of cricket, Gryllus bimaculatus. The two species of cricket employ different strategies for wind-evoked escape.

The external morphologies of two cricket species, Gryllodes sigillatus and Gryllus bimaculatus, were investigated. Despite its small body length, G. sigillatus possessed longer cerci and longer cercal filiform hairs than G. bimaculatus. The estimated number of filiform hairs on a cercus was also larger in G. sigillatus than in G. bimaculatus. Wind-sensitive interneurons receiving sensory inputs from cercal filiform hairs and running in the ventral nerve cord (VNC) were investigated in G. sigillatus both morphologically and physiologically. By intracellular staining, these interneurons were proved to be morphologically homologous with previously identified giant interneurons (GIs 8-1, 9-1, 9-2, 9-3, 10-2, and 10-3) in G. bimaculatus and Acheta domesticus. In G. sigillatus, the intensity-response relationship (I-R curve) for each GI was investigated using a unidirectional air current stimulus. The stimulus was applied from 12 different directions, and an I-R curve was obtained for each stimulus direction. Each GI showed a characteristic I-R curve depending on stimulus direction. The directionality curve expressed in terms of threshold velocity showed that each GI had a distinctive directional characteristic. The functional properties of GIs in G. sigillatus, such as I-R curve, threshold velocity, and directional characteristics, were compared with those of homologous GIs in G. bimaculatus in Discussion.

Cricket calling song is a species-specific signal and plays an important role in species recognition. The role of cricket song as a pre-mating isolation mechanism should be examined in a comparative study of closely related species inhabit sympatrically. On the Japanese Archipelago, three Teleogryllus species have wide distribution areas. T. yezoemma ranges from Hokkaido Island to the Kanto region, T. emma from Hokkaido to the Kyushu region, and T. taiwanemma from south of the Kii Peninsula to the South Asian tropics. The distribution of T. emma overlaps that of T. yezoemma in the north and that of T. taiwanemma in the south. T. yezoemma and T. taiwanemma are allopatric. I compared calling songs of the three species using principal component analysis. Furthermore, three-choice playback experiments using females of the three Teleogryllus species were performed to test whether female crickets can discriminate accurately their conspecific mates on the basis of their calling song. The results showed that the allopatric species (T. yezoemma and T. taiwanemma) have similar songs that females cannot discriminate accurately, whereas the partially sympatric species (T. yezoemma and T. emma, T. emma and T. taiwanemma) have divergent songs that females can distinguish accurately. This empirical evidence suggests that calling song plays an important role as a pre-mating isolation mechanism in three species of Teleogryllus. Furthermore, I performed three-choice playback experiments for females of each species using a synthetic song in order to identify the key feature for mate choice. The results showed that females preferred the song with the conspecific pulse period, while did not show a clear preference for dominant frequency. For pulse number and inter-phrase duration, females showed open-ended preference. These results suggest that pulse period is the key feature for species-specific song recognition. Studies of acoustic animal communication have shown that temporal patterns are more important than frequency parameters for song recognition. Previous neurophysiological studies of other cricket species support these results. In addition, there were differences in female preference shape among the three species. This suggests that even closely related species evaluate key features differently, and consequently calling song diversity could have appeared in the evolutionary process.

Cuticular substances on the body surface of crickets serve as pheromones that elicit a variety of different behaviors in male crickets. Antennal contact between males and females resulted in courtship behavior, and that between two males resulted in aggressive displays. As a first step in elucidating how crickets recognize and discriminate individuals, behavioral responses of male individuals to cuticular substances of conspecific males or females were investigated. The behavioral responses of males to antennal or palpal stimulation with an isolated antenna from a male or a female were recorded. To both antennal and palpal stimulation with female antennae, the majority of males responded with courtship behavior; to stimulation with male antennae, males responded with aggressive displays. To gain insight into the chemical nature of the behaviorally relevant components, isolated antennae were washed in either n-hexane, acetone or ethanol before behavior assays. Washed antennae no longer elicited courtship or aggressive responses in males. Next, polypropylene fibers were smeared with substances from the body surface of females and used for antennal stimulation. This experiment showed that the quality and quantity of cuticular substances appear to be highly age-dependent. Significantly more males responded with courtship behavior to cuticular substances from younger females. Isolated males generally showed higher levels of aggression than males reared in groups. Grouped males also were more likely to display courtship behavior towards antennae from younger females, and aggressive behavior towards antennae from older females. These results suggest that male discrimination of mating partners depends on the nature of female cuticular substances.

The male and female genitalia are finely designed to match each other for copulation in the cricket Gryllus bimaculatus. Copulatory acts of the male, stereotyped and time-fixed, are elicited by stimulation of mechanoreceptors on particular regions of the abdomen, cerci and genitalia. Sequential execution of each motor act proceeds as a chain reaction in which one act stimulates some receptors which in turn elicits another act and so on, while the female remains immobile on the male's back. Each key stimulus for a motor act appears as a result of the male's own act, except for copulatory papilla protrusion by the female. The final sequence of spermatophore extrusion and transfer are irreversible fixed motor actions which are triggered when the female copulatory papilla stimulates the epiphallic hairs. They proceed without continual central drive from the brain, and apparently without sensory feedback. In addition, they are well coordinated with movement and posture in the entire body. Some neural mechanisms of controlling mating behavior and switching the reproductive cycle are discussed.

One of a pair of cerci was ablated in the first-, fourth- and last-instar nymphs of the cricket, Gryllus bimaculatus. The insects were then reared until the final molt, after which the intensity-response (I-R) relationships for four giant interneurons (GIs) 8-1, 9-1, 9-2 and 9-3 with regard to a controlled air current stimulus were measured. In order to examine the functional changes during postembryonic development and the differences in the physiological plasticity of GIs between nymphs and adults, the obtained I-R curves for each GI were compared with those measured in intact and unilaterally cercus-ablated adult crickets. Each GI showed a distinctive change in response magnitudes after the long-term unilateral cercal ablation. In most cases, the I-R curves for each GI in the crickets ablated from nymphal periods were different from those in the adult crickets mentioned above. Moreover, the pattern of change in response magnitude was different from GI to GI. In contrast to these observations, it was reported that some important characteristics of the wind-evoked escape behavior such as relative occurrence and escape direction in unilaterally cercus-ablated crickets investigated after a long-term rearing were almost identical with those in intact crickets. Therefore, the results obtained in the present study suggest that functional changes occur not only in GIs but also in many other neural elements in the escape-eliciting system in order to maintain the features of wind-evoked escape behavior.

Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH(2), homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents.

To elucidate neural mechanisms underlying walking and jumping in insects, motor neurons supplying femoral muscles have been identified mainly in locusts and katydids, but not in crickets. In this study, the motor innervation patterns of the metathoracic flexor and extensor tibiae muscles in the cricket, Gryllus bimaculatus were investigated by differential back-fills and nerve recordings. Whereas the extensor tibiae muscle has an innervation pattern similar to that of other orthopterans, the flexor has an innervation unique to this species. The main body of the flexor muscle is divided into the proximal, middle and distal regions, which receive morphologically unique terminations from almost non-overlapping sets of motor neurons. The proximal region is innervated by about 12 moderate-sized excitatory motor neurons and two inhibitory neurons while the middle and distal regions are innervated by three and four large excitatory motor neurons, respectively. The most-distally located accessory flexor muscle, inserting on a common flexor apodeme with the main muscle, is innervated by at least four small excitatory (slow-type) and two common inhibitory motor neurons. The two excitatory and two inhibitory motor neurons that innervate the accessory flexor muscle also innervate the proximal bundles of the main flexor muscle. This suggests that the most proximal and distal parts of the flexor muscle participate synergistically in fine motor control while the rest participates in powerful drive of tibial flexion movement.

In the air-puff-evoked escape behavior of the cricket Gryllus bimaculatus, the effect of a uni-lateral cercal ablation and the process of behavioral recovery were investigated during postembryonic development. The response rate (relative occurrence of the escape behavior in response to an air puff stimulus) and the escape direction (relative to the stimulus direction) in first-, third-, sixth- and last-instar nymphs were almost identical with those of adults. A unilateral cercal ablation in the nymphs caused a decrease in the response rate and an increase in the number of misoriented escapes as have been observed in adults. However, the effect of ablation on the response rate was less in younger insects, i.e., the escape-eliciting potential of one cercus decreased during postembryonic development. Instead, facilitation of sensory inputs from both cerci essentially occurs, thus explaining the constant response rate throughout the developmental period. The response rate of the ablated insects measu...

To understand the functional role of the insect brain in mating behavior, copulatory actions in response to a model stimulus were compared between intact and decerebrated male crickets. Decerebrated males were then tested to examine whether their copulatory actions were modifed by biogenic amines or electrical stimulation. The main difference in copulatory actions between intact and decerebrated males was in the body thrusts consisting of the protraction and retraction of the abdomen for hooking. These movements became slower after the removal of the brain, as measured by the average interval between responses, called the cycle length. The cycle length increased to twice the length in intact males within about 20 min. Intraperitoneal injection of octopamine in decerebrated males shortened the cycle length dose-dependently (10−6–10−2M) and restored it nearly to the level in intact males at 10−4–10−2M. Octopamine-mimicking agents forskolin, IBMX and synephrine, and cyclic AMP analogue DB-cyclic AMP...

The air-puff evoked escape behavior of the cricket, Gryllus bimaculatus, was investigated. Crickets almost always escaped away from the stimulus source. In an optimal condition, the mean escape direction was 162° opposite to the stimulus source. Stronger (higher velocity) air-puff elicited an escape in larger number of crickets. However, the escape direction became incorrect when the stimulus was too strong. Crickets with bilateral cercal ablation did not show any escape to an air-puff, while unilaterally ablated ones did respond to the same stimulus with an escape. However, the response rate of animals with unilateral cercal ablation was lower than that in intact animals. Although the mean escape direction of the crickets with unilateral cercal ablation was still opposite to the stimulus source, the direction was not so accurate as in intact animals. About 6 days after the unilateral cercal ablation, the response rate showed a statistically significant compensational recovery. On the other hand,...

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.

In order to determine the contribution of each cercus and its receptors in organizing the receptive field of four air-motion sensitive giant interneurons(Gls 8-1, 9-1, 9-2 and 9-3)of the cricket Gryllus bimaculatus, effects of removing the ipsilateral or contralateral cercus(referred to the side of the axons)on specific parameters of the wind-evoked responses of these neurons were investigated. All 4 Gls received only excitatory inputs from a group of filiform hairs on the ipsilateral cercus. In addition to the ipsilateral excitatory inputs, Gls 8-1 and 9-1 received weak excitatory and strong inhibitory inputs from a group of filiform hairs on the contralateral cercus. Gl 9-2 received only inhibitory inputs filiform hairs on the contralateral cercus. Gl 9-3 received excitatory inputs from filiform hairs on the contralateral cercus and no inhibitory input was confirmed. In addition to such simple excitatory and inhibitory connections, the rebound motion of cercal filiform hairs had some role in organizing the receptive fields of Gls 9-2 and 9-3. Furthermore, the possibility of using a rebound depolarization of the membrane potential for mediating the long latency response in Gls 8-1 and 9-2 will be discussed.

Lectin cytochemistry was carried out on thin sections of 6th-instar cricket testis using two GalNAc-specific lectins, Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA), and the binding sites in primary spermatocytes were surveyed. Gold particles showing DBA-binding are observed specifically in dense-body clusters. These bodies are about 100-300 nm in diameter and exhibit multilaminar or multivesicular structure. HPA can bind to the dense-body clusters and another kind of larger multivesicular structures. These bodies seem to contain some heterogeneous substances, and sometimes show an autophagosome-like structure. The ultrastructures of these organelles surveyed in conventional Epon sections confirmed the structures of these multilaminar/vesicular bodies in cricket spermatocytes, which may play certain roles in intracellular circulation and degradation of glycoconjugates.

In order to explore the functional recovery of the cercal sensory system of the cricket Gryllus bimaculatus, changes in response properties of four air-motion sensitive giant interneurons(Gls 8-1, 9-1, 9-2 and 9-3)were investigated after sensory deprivations. Velocity thresholds, response magnitudes and response latencies of each Gl to a directional air current stimulus were investigated 21 days after ipsilateral or contralateral cercal ablations. The results were compared to those measured 1 day after the same treatment(Matsuura and kanou, 1998)in order to specify the changes which occurred during the first 20 days. Each Gl showed a different pattern of change in responses according to the direction of stimulus air current ; i.e. the changes observed were direction dependent regardless of whether they were compensational or not/ Compensational changes in response magnitudes and/or response latencies were observed in Gls 8-1, 9-1 and 9-2 when air currents were applied from particular directions. As there was no regeneration of cercal filiform hairs, those changes must be caused by the changes in synaptic strength between Gls and particular sensory afferents associated with cercal filiform hairs. Such neural compensation must underlie the basis of behavioral compensation when the insects have damaged sensory apparatus.

To characterize the neural elements involved in the higher-order control of spontaneous walking in insects, we recorded extracellular spike activity in the protocerebrum of freely walking crickets (Gryllus bimaculatus). Locomotor behavior was simultaneously recorded using a newly developed motion tracking system. We focused on spike units that altered their firing patterns during walking. According to their activity patterns with reference to walking bouts, these locomotor-related spike units were classified into the following four types: continuously activated unit during walking (type 1); continuously inhibited unit during walking (type 2); transiently activated unit at the onset of walking (type 3); and transiently activated unit at the termination of walking (type 4). The type 1 unit was the most dominant group (25 out of 33 units), whereas only a few units each were recorded for types 2–4. Some of the locomotor-related units tended to change firing pattern before the onset or termination of walking bouts. Spike activity in some type 1 units was found to be closely correlated with walking speed. When firing timing was compared between unit pairs, their temporal relationships (synchronization/desynchronization) altered, depending on the behavioral state (standing/walking). Mechanical stimuli applied to the body surface elicited excitatory responses in the majority of the units. Histological observations revealed that the recorded sites were concentrated near or within the mushroom body and central complex in the protocerebrum.

An attempt was made to determine electrophysiologically and immunocytochemically the neurotransmitter at neuromuscular junctions (NMJs) in cricket abdominal muscles. One mM bath-applied Lglutamate reduced the amplitudes of large and small excitatory junctional potentials (I- and s-EJPs) reversibly by about 75% of the control on the average. It also produced a slow, transient depolarization of about 10 mV. Joro spider toxin, which is an antagonist of L-glutamate, depressed the amplitudes of I- and s-EJPs almost completely and irreversibly at 3.5 × 10−6 M. By using the antibody to glutamate, glutamate-immunoreactive processes whose configuration resembled that of NMJs revealed by nickel staining were obtained. The present results strongly suggest that the neurotransmitter at cricket NMJs is L-glutamate.

Response properties of six giant interneurons (GIs) 8-1, 9-1, 9-2, 9-3, 10-2 and 10-3 of the cricket, Gryllus bimaculatus, were investigated with a short duration unidirectional air-current stimulus which elicits escape behavior of the insects effectively. Air-currents were presented to the insects from 12 different directions in the horizontal plane in order to explore the directional characteristics of the GIs. The frequency dependency of velocity thresholds (threshold curves) of the GIs changed with the direction of stimulus air-current. Preferential directions of the GIs to the air-currents were ipsilateral-rear in Gl 8-1, ipsilateral-front in Gl 9-1, ipsilateral-rear in Gl 9-2, ipsilateral-front in Gl 9-3, ipsilateral-rear in Gl 10-2 and ipsilateral-front in Gl 10-3, with respect to the ventral nerve cord containing the axons. Intensity-response curves suggest that GIs 8-1 and 9-1 show the maximum response to air-current velocities lower than 300 mm/sec regardless of the stimulus direction. ...

We examined quantitative changes in biogenic amines in relation to effects of population density on growth and behavior in the cricket Gryllus bimaculatus, which were reared in two different densities, completely isolated solitary and 40 crowded insects. In the crowded animals, the rate of increase in body weight was slow, and also the day of imaginal moult was late, when compared to the isolated ones. Development and growth were thus suppressed in the crowded group. The body color of isolated ones was darker than that of crowded ones. Behavioral tests indicated that the isolated crickets were significantly more aggressive than the crowded ones. The levels of biogenic amines, their precursors and metabolites were examined in the brain, corpora allata, corpora cardiaca and frontal ganglion of isolated and crowded crickets using high-performance liquid chromatography with electrochemical detection. The brains of crowded crickets contained significantly higher amounts of octopamine, dopamine, and 5-hydroxytryptamine (5-HT) than those in the isolated ones. On the contrary, the level of N-acetyldopamine in the isolated crickets was significantly higher than that in the crowded ones. In crowded animals the corpora allata contained significantly higher levels of 5-HT, and the corpora cardiaca contained more octopamine. These results indicate that high population density affects aminergic systems which in turn modulate various biological events, such as development, growth and behavior.

We investigated the stridulatory courtship initiated by male-male contact after agonistic encounters and the effect of dominance status on subsequent reproductive behavior in the cricket Gryllus bimaculatus. When two male crickets were kept together in a small area, their dominance status was quickly established through fighting or non-fighting interactions. Approximately 10 min after pairing, most dominant males produced calling and/or courtship songs in the presence of subordinate males. This behavior appeared to be triggered by some contact chemicals on the body surface of the males. Stimulation using the forewing of a neutral male induced courtship in dominant males at a higher level compared with neutral males which were not previously paired with males. These observations suggest that the sexual motivation in dominant males increased because of previous agonistic interactions. In contrast, subordinate males remained silent. Stimulation using the male forewing induced stridulation to a lesser degree in subordinate males than in neutral males, suggesting decreased sexual motivation in subordinate males. Furthermore, only 40% of subordinate males exhibited courtship behavior under triadic conditions (dominant male, subordinate male and female) in contrast with 100% in male-female pairs. This result reveals that subordinate males, being less sexually motivated, are continuously suppressed in their courtship by intermittent attacks by the nearby dominant males. In the other triadic condition where the males were allowed to copulate, 65% of the dominant males copulated, while none of the subordinates did. These results suggest that dominant males have a greater chance to copulate and produce offspring.

The effects of self-generated wind on the compensational recovery of escape direction were investigated in unilaterally cercus-ablated crickets, Gryllus bimaculatus. To separate walking and self-generated wind during walking, unilaterally cercus-ablated crickets were placed on a styrofoam ball that was easily rotated by leg motion during walking. The stationary walking on the ball did not produce self-generated wind, because no body motion occurred. Crickets that were trained on the ball but given no artificial air puff for 14 days after cercal ablation did not show any compensational recovery of escape direction. Therefore, spontaneous walking itself was not sufficient to compensate the wind-evoked escape direction in the crickets. Artificial air puffs from the anterior direction synchronized with the stationary walking were effective for the compensational recovery of escape direction, but those from the posterior direction were not. As most of the spontaneous walking was directed to the forward direction, only an artificial air puff from the anterior direction coincided well with actual self-generated wind occurring during the onset of normal walking. Therefore, self-generated wind during walking seems essential for the compensational recovery of escape direction in unilaterally cercus-ablated crickets. When artificial air puffs were unsynchronized with walking, no compensational recovery was observed. This result suggests that artificial air puffs should be given just after the onset of spontaneous walking. Otherwise, the artificial air puffs may not be recognized as self-generated wind.

Male solitary animals frequently enter aggressive interactions with conspecific individuals to protect their territory or to gain access to females. After an agonistic encounter, the loser (subordinate individual) changes its behaviour from aggression to avoidance. We investigated agonistic interactions between pairs of male crickets to understand how dominance is established and maintained. Two naive males readily entered into agonistic interactions. Fights escalated in a stereotyped manner and were concluded with the establishment of dominance. If individuals were isolated after the first encounter and placed together 15 minutes later, subordinate crickets tended to avoid any further contact with the former dominant opponent. Moreover, subordinate males also avoided unfamiliar dominant and naive opponents. They displayed aggressive behaviour only towards unfamiliar subordinate opponents. This suggests that the subordinate male change their behaviour depending on the dominance status of the opponent. Dominant crickets, in contrast, displayed aggressive behaviour towards familiar as well as unfamiliar opponents. If the interval between the first and second encounter was longer than 30 minutes, the former subordinate male showed aggressive behaviour again. However, if the subordinate cricket was paired with the same opponent three consecutive times within 45 minutes, it avoided the former dominant opponent for up to 6 hours following the third encounter. Our results suggest that the maintenance of dominance in male crickets depends largely on the behavioural change of subordinate individuals. Possible mechanisms to maintain dominance are discussed.

A simple method for determination of the early stages of nymphs of the cricket, Gryllus bimaculatus, was presented. The width of the head-capsules was found to be a useful value to determine the nymphal stages. On this evidence, the testes from 3rd- to 6th-instar nymphs were investigated and histologically analyzed. The number of spermatogonia rapidly increased between the 3rd- to 5th-instars. Primary spermatocytes entered into meiotic prophase at the latest 2 days after ecdysis into the 5th-instar.

In response to an air puff stimulus, intact crickets, Gryllus bimaculatus, make an escape almost 180 degrees opposite to the stimulus source. In order to verify our previous hypothesis that a self-stimulation of the wind-sensory system is necessary for a compensational recovery of the escape direction (behavioral compensation) in unilaterally cercus-ablated crickets, we investigated the relationship between the conditions of rearing after a unilateral cercal ablation and the degree of behavioral compensation. A unilaterally cercus-ablated cricket reared in a large cage to permit free locomotion showed a significantly higher degree of recovery of escape direction compared with those reared under restrained conditions in a small glass vial. However, the degree of behavioral compensation in a cricket reared alone in a large cage was smaller than that of crickets reared in a cage of the same size with 5-6 other cercus-ablated crickets. Mutual stimulation possibly increased the extent of locomotion of crickets reared in a group and improved the degree of compensational recovery of the escape direction. To ascertain this, the distance a cricket moved during the recovery period was associated with the degree of compensational change of the escape direction. The result suggests that the degree of compensation of the escape direction clearly depended on the distance walked by the crickets. The compensation seemed not to be caused by other factors such as chemical ones in the case of group rearing because forced locomotion induced by touch stimulation on the body surface was solely effective in improving the escape direction.

The cricket Gryllus bimaculatus forms a stable olfactory memory that lasts for practically a lifetime. As a first step to elucidate the cellular mechanisms of olfactory learning and memory retention in crickets, we studied the dependency of memory retention on the de novo brain protein synthesis by injecting the protein synthesis inhibitor cycloheximide (CHX) into the head capsule. Injection of CHX inhibited (3)H-leucine incorporation into brain proteins by90% for 3 hr. Crickets were trained to associate peppermint odor with water (reward) and vanilla odor with saline solution (non-reward) and were injected with CHX before or at different times after training. Their odor preferences were tested at 2 hr, 1 day and 4 days after training. Memory retention at 2 hr after training was unaffected by CHX injection. However, the level of retention at 1 day and 4 days after training was lowered when CHX was injected 1 hour before training or at 1 hr or 6 hr after training. To study the time course of the development of CHX-sensitive memory phase, crickets that had been injected with CHX at 1 hr after training were tested at different times from 2 to 12 hr after training. The level of retention was unaffected up to 4 hr after training but significantly lowered at 5 hr after training, and the CHX-sensitive memory phase developed gradually during the next several hours. CHX dissociates two phases of olfactory memory in crickets: earlier protein synthesis-independent phase (4 hr) and later (5 hr) protein synthesis-dependent phase.

The rearing condition necessary for behavioral compensation after sensory deprivation was investigated in the cricket Gryllus bimaculatus. The right-cercus-ablated cricket was reared in a glass vial with a slightly larger diameter than the body length of the cricket. After two weeks of rearing in the vial, the air-puff-evoked escape behavior of the cricket was investigated. The response rate (relative occurrence of the escape behavior after a standard air puff) obtained was identical with that of crickets reared in a large cage. On the other hand, unlike crickets reared in a large cage, the distorted escape directional property of the cricket reared in the vial was not compensated at all. Control experiments proved that the restraint in the vial did not affect the motor system, and the air motion from environments was not essential for the compensational recovery of the escape direction. Therefore, the ablated crickets required spontaneous walking in order to compensate the directionality of their escape. A self-generated wind caused by spontaneous walking appears necessary for the crickets to realize the defect of their sensory system and to compensate the related escape behavior. A hypothesis for the compensation mechanism based on the efference copy signal is proposed.