Neurohormonal control of the mating interval in the male cricket,Gryllus bimaculatus DeGeer

Summary Between two mating acts of the male cricket (Gryllus bimaculatus), spermatophore protrusion (SP) and courtship stridulation (CS), there is a fixed time interval. This interval lasts about 1 h. During the period from SP to CS, the male cricket does not stridulate nor make any type of mating sound (post-spermatophore protrusion silence: PSPS) and tolerates external sensory stimuli. We examined the effects of injections of hemolymph and ganglia extracts on the interval. Extracts obtained from crickets which had just started CS (CS crickets) and those which had finished SP (SP crickets) were effective. The extracts were fractionated by ul trafiltration. Fractions with a molecular weight of less than 1 kdalton affected the length of the PSPS. The fractions from both the hemolymph and the mesothoracic ganglion of CS crickets shortened the PSPS. On the other hand, the fractions from the hemolymph and the brain of SP crickets lengthened the PSPS. We estimated, by gel filtration, the molecular weight of the effective fractions from the mesothoracic ganglion and the brain to be 100–200 daltons. We also examined the effects of biogenic amines on the PSPS. Octopamine shortened the PSPS, whereas serotonin lengthened it. The results in dicate that at least two neurohormones from the brain and the mesothoracic ganglion reciprocally control the elicitation of CS and provide an appropriate interval in the mating sequence of the male cricket. Octopamine and serotonin are possible candidates for these neurohormones.Abbreviations CP copulation - CS courtship stridulation - SP spermatophore protrusion - PSPS post-spermatophore protrusion silence     

Brain control of mating behavior in the male cricket Gryllus bimaculatus DeGeer: the center for inhibition of copulation actions

We re-examined the functional role of the brain and suboesophageal ganglion (SOG) in inhibiting mating behavior in the male cricket Gryllus bimaculatus DeGeer. Experiments were conducted by using mimetic stimulation to elicit copulation actions. To induce a change in the male internal state from a sexually responsive state to a sexually unresponsive state in the mating stage, noxious stimulation, head injury and leg pinching were used. Males that sustained a head injury became sexually unresponsive but became responsive as soon as the brain was removed, while males that underwent the same treatment remained unresponsive after the SOG was destroyed by sectioning. The inhibitory effects of the brain were also demonstrated by the fact that further removal of the SOG in the decerebrated males did not change their copulatory responsiveness, while removal of the brain in SOG-sectioned males markedly increased copulatory responsiveness. Furthermore, decerebrated males did not become sexually unresponsive by leg pinching, while SOG-sectioned males that had recovered sexual responsivenss, did. These results suggest that the brain, and not the SOG, plays a key role in the inhibition of copulation in the male cricket during the mating stage.     

Social isolation increases male aggression toward females in the field cricket Gryllus bimaculatus

Social isolation has often been reported to facilitate male aggressiveness in various animal species. If social isolation also escalates male aggressive behavior towards females, the mating success of the aggressive males will be low. This study evaluated the effect of social isolation on mating behavior in the field cricket Gryllus bimaculatus, which has traditionally been considered to be an asocial species. The results showed that social isolation from same-sex individuals enhanced male aggressiveness to females, and the mating success of aggressive males was reduced under the experimental conditions. More aggressive males exhibited a longer latency to court than less aggressive males. These results suggest that because male aggressiveness causes a delay in courtship, aggressive males may have reduced mating success. This demonstrated that social relationships are a critical factor affecting male mating success, even if the species is normally considered solitary.     

Behaviorally significant immobile state of so-called thanatosis in the cricket Gryllus bimaculatus DeGeer: its characterization, sensory mechanism and function

We have found that the cricket Gryllus bimaculatus shows an immobile posture, so-called thanatosis. Thanatosis was reflexly elicited by gently holding the forelegs and pronotum of the cricket. During thanatosis, the respiration rate decreased markedly while the heart rate doubled compared with the resting state. Animals in the state of extreme rigidity were unresponsive to the external disturbances but easily aroused by mechanical stimulation such as prodding. The immobile posture usually persisted for 2–4 min, but occasionally for more than 20 min, and then suddenly ended. Catalepsy was induced during thanatosis when a leg was passively forced to the extended position. For elicitation of the flexion reflex, either campaniform sensilla and femoral chordotonal organs (FCOs) in the forelegs, and spine-like sensilla on the pronotum were necessary. Among these receptors, however, only the FCOs were involved in inducing the immobile state. Centrally, the brain was indispensable for thanatosis to be maintained. In semi-natural conditions the thanatotic state did occur spontaneously while the cricket struggled to get into a small crevice according to the nature of this species. This sudden immobilization could help the cricket get out of danger of predators like reptiles and amphibians in the natural habitat.Abbreviations FCO femoral chordotonal organ - F-T femorotibial - N nerve     

Brain control of mating behavior in the male cricket Gryllus bimaculatus DeGeer: brain neurons responsible for inhibition of copulation actions

We localized brain neurons responsible for the inhibition of copulation actions in the male cricket, Gryllus bimaculatus. Males with one connective-cut between the brain and the terminal abdominal ganglion (TAG) were able to perform normal reproductive behaviors such as courtship, copulation and spermatophore protrusion. However, copulation response tests using a female dummy showed that they failed to exhibit a transient inhibitory state after application of noxious stimulation in the mating stage and to exhibit a tonic inhibitory state in the post-copulatory sexual refractory stage. A partial cut of a single connective (hemilateral connective-cut) combined with the copulation response test and an axonal backfilling demonstrated that approximately 40 brain neurons (per one connective), whose somata were located in the posterior region of the protocerebrum and mainly contralaterally to their descending axons running through the dorso-medial part of the connective, were candidates for the inhibition of copulation actions, at least in the mating stage. In addition, we report here for the first time, that copulation actions can be elicited shortly after copulation even in intact males. This unusual behavior was frequently observed in fledglings after the final molt. The results are discussed from the perspective of the operation of the brain inhibition system on the central pattern generator (CPG) for copulation.     

Two antagonistic functions of neural groups of the femoral chordotonal organ underlie thanatosis in the cricket Gryllus bimaculatus DeGeer

The cricket Gryllus bimaculatus displayed freezing (thanatosis) after struggling while the femoro-tibial joints of the walking legs were forcibly restrained. Myographic recording indicated that strong contraction of the flexor tibia muscle “leg flexion response” occurred under this restrained condition. During thanatosis, when the femoro-tibial joint was passively displaced and held for several seconds, it maintained its new position (catalepsy). Only discharge of the slow flexor units was mechanically indispensable for maintaining thanatosis and catalepsy. Differing roles of identified neuron subgroups of the femoral chordotonal organ were elucidated using this behavioral substrate. Ablation of the dorsal group neurons in the ventral scoloparium strengthened the leg flexion response and the normal resistance reflex, while ablation of the ventral group weakened both motor outputs. Ablation of the dorsal scoloparium neurons, or other main sensory nerves caused no detectable deficiency in femoro-tibial joint control. These results imply that both modes of flexor muscle activation promoted by the ventral group neurons are normally held under inhibitory control by the dorsal group. It is hypothesized that this antagonistic function causes immobilization of the femoro-tibial joint in a wide range of angles in thanatosis and catalepsy. Accepted: 12 November 1998     

Physics of directional hearing in the cricket Gryllus bimaculatus

In the cricket ear, sound acts on the external surface of the tympanum and also reaches the inner surface after travelling in at least three pathways in the tracheal system. We have determined the transmission gain of the three internal sound pathways; that is, the change of amplitude and phase angle from the entrances of the tracheal system to the inner surface of the tympanum. In addition, we have measured the diffraction and time of arrival of sound at the ear and at the three entrances at various directions of sound incidence. By combining these data we have calculated how the total driving force at the tympanum depends on the direction of sound. The results are in reasonable agreement with the directionality of the tympanal vibrations as determined with laser vibrometry.At the frequency of the calling song (4.7 kHz), the direction of the sound has little effect on the amplitudes of the sounds acting on the tympanum, but large effects on their phase angles, especially of the sound waves entering the tracheal system at the contralateral side of the body. The master parameter for causing the directionality of the ear in the forward direction is the sound wave entering the contralateral thoracic spiracle. The phase of this sound component may change by 130–140° with sound direction. The transmission of sound from the contralateral inputs is dominated by a very selective high-pass filter, and large changes in amplitude and phase are seen in the transmitted sounds when the sound frequency changes from 4 to 5 kHz. The directionality is therefore very dependent on sound frequency.The transmission gains vary considerably in different individuals, and much variation was also found in the directional patterns of the ears, especially in the effects of sounds from contralateral directions. However, the directional pattern in the frontal direction is quite robust (at least 5 dB difference between the 330° and 30° directions), so these variations have only little effect on how well the individual animals can approach singing conspecifics.Abbreviations CS contralateral spiracle - CT contralateral tympanum - IS ipsilateral spiracle - IT ipsilateral tympanum - P the vectorial sum of the sounds acting on the tympanum     

Photoperiodic modulation of circadian rhythms in the cricket Gryllus bimaculatus

The waveform and the free-running period of circadian rhythms in constant conditions are often modulated by preceding lighting conditions. We have examined the modulatory effect of variable length of light phase of a 24h light cycle on the ratio of activity (alpha) and rest phase (rho) as well as on the free-running period of the locomotor rhythm in the cricket Gryllus bimaculatus. When experienced the longer light phases, the alpha/rho-ratio was smaller and the free-running period was shorter. The magnitude of changes in alpha/rho-ratio was dependent on the number of cycles exposed, while the free-running period was changed by a single exposure, suggesting that there are separate regulatory mechanisms for the waveform and the free-running period. The neuronal activity of the optic lobe showed the alpha/rho-ratio changing with the preceding photoperiod. When different photoperiodic conditions were given to each of the two optic lobe pacemakers, the alpha/rho-ratio of a single pacemaker was rather intermediate between those of animals treated with either of the two conditions. These results suggest that the storage of the photoperiodic information occurs at least in part in the optic lobe pacemaker, and that the mutual interaction between the bilateral optic lobe pacemakers is involved in the photoperiodic modulation.     

RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus

In the Mediterranean field cricket, Gryllus bimaculatus, the action of sulfakinin (SK) gene expression on food intake, food transport in the gut and carbohydrate digestion (alpha-amylase activity) was investigated by using the RNA interference (RNAi) method. Injection of SK double-stranded (ds) RNA into the abdomen of female adults and last instar larvae led to a systemic silencing of the SK gene, as was shown by RT-PCR studies. In adults, suppression of SK gene expression was effective from the first day after injection up to at least the third day. Treatment of the adult crickets by injection or feeding of dsRNA led to a stimulation of the food intake. Assuming that the gene silencing is followed by a depletion of the SK in tissues and/or haemolymph implies an inhibitiory role of the native SK peptides on food intake. The alpha-amylase activity in vitro in the midgut tissue and in the secretions of adult females was not affected by silencing the SK gene.     

The role of the medial septum in the acoustic trachea of the cricket Gryllus bimaculatus

In the cricket Gryllus bimaculatus, it is demonstrated that the medial septum in the prothoracic trachea of the auditory system plays an important role in shaping the directional sensitivity of the ear.After perforation of the medial septum, the directional characteristic of intact animals, showing a mean right-to-left difference in sensitivity of 14 dB, becomes more omnidirectional with a mean right-to-left difference of only 7 dB. Correspondingly, the rate of change in auditory sensitivity for a sound source moving from frontal to contralateral is reduced to 0.78 dB/10° versus 1.5 dB/10° in intact animals (Figs. 2, 3).A computer simulation of phonotaxis based on these findings predicts a reduction in phonotactic performance in animals with a perforated septum. This prediction is in good quantitative agreement with experimental data (Fig. 4) and emphasizes the importance of an intact septum for effective phonotaxis in crickets.     

The role of the medial septum in the acoustic trachea of the cricket Gryllus bimaculatus

The auditory organs of the cricket which are situated in the front legs are joined together by a large transverse trachea which decisively influences their directional characteristics. The transverse trachea is medially divided by a septum. The importance of this septum for the localization of a sound source was tested by means of behavioural experiments in which the phonotactic movements of intact Gryllus bimaculatus females were compared quantitatively with those of the same specimen after perforation of the septum. The septal perforation does not noticeably influence locomotion in the absence of acoustic stimuli but selectively changes essential characteristics of phono taxis: 1) The animals walk in less straight lines. The oscillations around the mean course, typical of phonotaxis, are increased in amplitude, while the frequency decreases. 2) Course deviations from the direction of the sound source become more pronounced. 3) The threshold for phonotaxis is raised by about 10 dB. 4) Both the speed at which the animals walk and the proportion of time during which they are mobile are reduced. The results are discussed in relation to the role of the septum in the mechanism of sound localization, and with regard to its possible importance for the recognition of acoustic patterns.     

Mobilities of the cercal wind-receptor hairs of the cricket, Gryllus bimaculatus

The deflection sensitivities of cercal filiform hairs of the cricket, Gryllus bimaculatus, were determined by direct measurement. The tangential velocity of deflecting hair shafts in response to stimulus air motion was measured in situ by a laser-Doppler velocimeter with surface scattering of the shaft. The velocity of the stimulus air motion in a small wind tunnel was calibrated by the same velocimeter with smoke from a joss-stick. The mobility of the hair was obtained from former measurements with reference to the latter calibration of the single apparatus. A Gaussian white noise signal was employed as a stimulus waveform, and the stimulus-response transfer function was calculated through a cross-correlation method, which provides greater precision and wider frequency for a longer period of measurement. The mobility of hair was expressed in deflection amplitudes and phase shifts in reference to the velocity sinusoid of a stimulus at various frequencies. The measurements established the following conclusions. The wind receptor hairs comprise an array of mechanical band-pass filters whose best frequencies are inversely proportional to the length. The motion dynamics of the wind-receptor hairs have strong damping. Accepted: 24 February 1998     

Analysis of coupling between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between weakly coupled two optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording the locomotor activity, under light-dark cycles with various lengths, after the optic nerve was unilaterally severed. The activity rhythm split into two components under the light cycles different from 24 h: one was readily entrained to the light cycle and the other only loosely entrained or freeran. Additional removal of the optic lobe on the intact side resulted in a loss of the entrained component and that on the blinded side caused the reverse effect, indicating that the entrained component was driven by the pacemaker on the intact side and the other by the one on the blinded side. The synchronization between the two components was achieved only in light cycles with a limited length between 23 and 25 h. Without this range, the desynchronization of the components occurred. In the split rhythm, the phase-dependent modulation of the period of freerunning component and the mutual suppression of locomotor activity during the subjective day phase were clearly observed. The suppression was also evident in the lights-on peak that was the masking effect of light. The light cycle with dim light significantly reduced the ratio of animals with the pacemaker coupling as well as the magnitude of the period modulation. These results suggest (1) that the mutual coupling is achieved only when the difference in the periods between the two pacemakers is within an allowable range, (2) that the photic information is also involved in the mechanism of mutual coupling, and (3) that the suppression of activity occurs at the regulatory center for locomotion.Abbreviations CT circadian time - DD constant darkness - LL constant light - LD light to dark cycle - T length of light to dark cycle - freerunning period     

Expression patterns of hedgehog, wingless, and decapentaplegic during gut formation of Gryllus bimaculatus (cricket).

We observed expression patterns of hedgehog (hh), wingless (wg), and decapentaplegic (dpp) during gut development of Gryllus bimaculatus (the cricket), a typical hemimetabolous insect, and compared with those observed in Drosophila, a typical holometabolous insect. Gryllus hh(Gbhh) and Gbwg are expressed in both foregut and hindgut, while Gbdpp is expressed only in the hindgut: at the boundaries between the small and large intestine, and between the large intestine and rectum. Although the expression patterns of Gbhh and Gbwg are essentially comparable to those observed in Drosophila, the expression pattern of Gbdpp differs from those of the Drosophila dpp.     

Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus

The photo-responsiveness of 2 groups of interneurons responding to light in the protocerebrum was investigated at 2 developmental stages, the last instar nymphs and adults, in the cricket Gryllus bimaculatus. The cricket is diurnally active during the nymphal stage but becomes nocturnal as an adult. In both adults and nymphs, light-induced responses of optic lobe light-responding interneurons that conduct light information from the optic medulla to the lobula and the cerebral lobe showed a circadian rhythm peaking during the subjective night. Amplitudes of the rhythms were not significantly different between adults and nymphs, but adults showed more stable day and night states than did nymphs. The medulla bilateral neurons that interconnect the bilateral medulla areas of the optic lobe also showed circadian rhythms in their light-induced responses in both adults and nymphs. The rhythm had a clear peak and a trough in adults, and its amplitude was significantly greater than that of nymphs. These results suggest that the 2 classes of interneurons are differentially controlled by the circadian clock. The difference might be related to their functional roles in the animal's circadian behavioral organization.     

Mutual interactions between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between the bilaterally paired optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording locomotor activity, under constant light or constant red light, after the optic nerve was unilaterally severed.

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Characterization of PDF-immunoreactive neurons in the optic lobe and cerebral lobe of the cricket, Gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide playing important roles in insect circadian systems. In this study, we morphologically and physiologically characterized PDF-immunoreactive neurons in the optic lobe and the brain of the cricket Gryllus bimaculatus. PDF-immunoreactivity was detected in cells located in the proximal medulla (PDFMe cells) and those in the dorsal and ventral regions of the outer chiasma (PDFLa cells). The PDFMe cells had varicose processes spread over the frontal surface of the medulla and the PDFLa cells had varicose mesh-like innervations in almost whole lamina, suggesting their modulatory role in the optic lobe. Some of PDFMe cells had a hairpin-shaped axonal process running toward the lamina then turning back to project into the brain where they terminated at various protocerebral areas. The PDFMe cells had a low frequency spontaneous spike activity that was higher during the night and was often slightly increased by light pulses. Six pairs of PDF-immunoreactive neurons were also found in the frontal ganglion. Competitive ELISA with anti-PDF antibodies revealed daily cycling of PDF both in the optic lobe and cerebral lobe with an increase during the night that persisted in constant darkness. The physiological role of PDF is discussed based on these results.     

Location of the reproductive timer in the male cricket Gryllus bimaculatus DeGeer as revealed by local cooling of the central nervous system

The location of the reproductive timer for the post-copulatory, time-fixed, sexually refractory stage was investigated in the male cricket Gryllus bimaculatus. This stage was defined as the interval between spermatophore protrusion and recommencement of copulation or a calling song. To inactivate the central nervous system locally and reversibly, different body regions were cooled to 10°C for 20-30 min after spermatophore protrusion. A behavioural test then measured the duration of the refractory stage after males recovered from cooling. Males with the head, thorax and anterior abdomen cooled did not show a lengthening of that stage. In contrast, males with the entire abdomen or even the posterior abdominal segments containing only the 6th and terminal (7th-11th) abdominal ganglia showed a lengthening of the refractory stage up to, but not exceeding, the cooling duration. When 20-min cooling was interposed twice after spermatophore protrusion, the refractory stage was lengthened by about 40 min, indicating that interposed cooling did not reset the timer. These results are in agreement with our previous hypothesis that the reproductive timer for the refractory stage in the male cricket is located in the posterior abdominal ganglia, possibly within the terminal abdominal ganglion.