cDNA cloning and nuclear localization of the circadian neuropeptide designated as pigment-dispersing factor PDF in the cricket Gryllus bimaculatus

Pigment-dispersing factor (PDF) was recently reported to be a principal circadian neuromodulator involved in transmitting circadian rhythms of daily locomotion in insects. In Drosophila, PDF functions in some of the neurons expressing the clock genes period, timeless, Clock, and cycle, and those clock genes in turn regulate pdf gene expression. In the present study, we cloned a cDNA encoding PDF in the brain of a nocturnal insect, the cricket Gryllus bimaculatus, and found that an isolated clone (310 bp) codes for an extraordinarily short precursor protein with no definite signal sequence, but a nuclear localization signal (NLS)-like sequence instead. The cricket PDF exhibits high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to crustacean beta-PDH peptides. In the optic lobes of G. bimaculatus there are PDF-immunoreactive neurons in both the medulla and lamina neuropiles. Among the strongly immunoreactive lamina PDF neurons, on electron microscopy we identified cells exhibiting distinct staining that is not only cytoplasmic but also nuclear. When GFP-fused PDF precursor proteins were expressed in COS-7 cells, distinct translocation of the fusion protein into the nucleus was observed. This is the first finding of PDF peptide in the nucleus, which suggests a fundamental role of PDF peptide per se in the circadian clock system.     

Post-embryonic development of circadian rhythm in the cricket,Gryllus bimaculatus: A rhythm reversal

Summary Locomotor activity of the male cricketGryllus bimaculatus DeGeer was recorded from the 7th or last (8th) instar nymph. The nymph showed a diurnal rhythm (nymphal rhythm = NR), while the adult, on the contrary, was nocturnal (adult rhythm = AR) (Fig. 1). This rhythm reversal occurred suddenly 3 to 5 days after the imaginal molt, almost simultaneously with the first spermatophore formation and the start of stridulation (calling song) (Fig. 2). In addition to the antiphase relationship, both rhythms also differed in the freerunning period (tau) and wave form. Tau_scdd was significantly longer in NR (24.33 h) than in AR (23.91 h) (Fig. 3). AR was characterized by a sharp activity peak in each cycle, which NR, however, lacked (Fig. 1, 3, 6). On the basis of these differences, two possibilities are discussed; one is that NR and AR are separate oscillations and the other is that both are coupled to different phase points of one oscillation.Abbreviations LD light dark - DD constant darkness - LL constant light - NR nymphal rhythm - AR adult rhythm     

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.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

Distribution of circadian photoreceptors in the compound eye of the cricket Gryllus bimaculatus

Adult male crickets (Gryllus bimaculatus) show a nocturnal circadian locomotor rhythm, which is driven by the pacemaker in the optic lamina-medulla complex and synchronizes to the light-dark (LD) cycle received by the compound eye. To see whether there was any specially differentiated circadian photoreceptor area in the eye, we examined the effect of a partial reduction of various areas of the compound eye, in addition to a removal of the contralateral optic lamina-medulla-compound eye complex, on entrainability of the locomotor rhythm. All operated animals showed a response to the LD cycle in their locomotor rhythm, no matter which area of the eye was left intact: They either stably entrained to an LD cycle or showed a sign of weak entrainment. The capacity for stable entrainment was still retained when only 262 ommatidia were left. Transient cycles needed for re-entrainment, following a 6-hr phase advance of the LD cycle, were measured in 20 reduced-eye animals showing clear stable entrainment. They were in inverse proportion to the number of ommatidia in the reduced eye: The fewer ommatidia there were, the more transient cycles were observed (r = -0.76, p less than 0.001). These results suggest that almost the whole area of the compound eye may contain circadian photoreceptors, and that the photic information from each ommatidium may additively affect the circadian clock to entrain via neural integration mechanisms.     

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.

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     

New microsatellite loci isolated from the field cricket Gryllus bimaculatus characterized in two cricket species, Gryllus bimaculatus and Gryllus campestris

We have developed a new set of 27 polymorphic markers for each of two cricket species, Gryllus bimaculatus and Gryllus campestris. Initially, 14 published G. bimaculatus loci were tested in G. campestris; however, only five loci were polymorphic. Therefore, we isolated an additional 50 new microsatellite loci from G. bimaculatus and tested these in both species. In a minimum of 20 individuals, 27 of the new loci were polymorphic in G. bimaculatus and 25 in G. campestris.     

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.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

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     

Olfactory memory capacity of the cricket Gryllus bimaculatus

Olfactory learning in insects is a useful model for studying neural mechanisms underlying learning and memory, but memory storage capacity for olfactory learning in insects has not been studied. We investigate whether crickets are capable of simultaneously memorizing seven odour pairs. Fourteen odours were grouped into seven A/B pairs, and crickets in one group were trained to associate A odours with water reward and B odours with saline punishment for all the seven pairs. Crickets in another group were trained with the opposite stimulus arrangement. Crickets in all the groups exhibited significantly greater preference for the odours associated with water reward for all the seven odour pairs. We conclude that crickets are capable of memorizing seven odour pairs at the same time.     

Diurnal and circadian rhythm in compound eye of cricket (Gryllus bimaculatus): changes in structure and photon capture efficiency

Day-night changes in rhabdom size of compound eyes were investigated in three groups of crickets (Gryllus bimaculatus): nymphs and adult males and females. In both adults and nymphs, the rhabdoms were larger at night than during a day. In adults, the mean rhabdom occupation ratios (RORs) of ommatidial retinulae at midnight were about two times greater than the values at midday. This change contributes to control of the photon capture efficiency (PCE) of the eye according to photic environment. The RORs of adult males at midnight were higher than those of both adult females and nymphs. This suggests that the PCE of the compound eye of adult males is the greatest of all groups. Under constant darkness, day-night changes in ROR were detected only in adult males, but neither in adult females nor in nymphs. On the other hand, no day-night changes were detected in any experimental group under constant light. These results suggest that the change in rhabdom size between day and night is an adaptation to the photic environment that is controlled mainly by the light-dark (day-night) cycle. However, the change in male adults is induced by an endogenous circadian clock.     

Control of the circadian rhythm of locomotor activity in the house cricket

A detailed analysis was made of the locomotor activity of Acheta domesticus under conditions of 12 hr light and 12 hr darkness (LD 12 : 12) and of continuous darkness (DD). Under LD 12 : 12 it was found that there are three types of insects: (1) those beginning the period of increased locomotor activity immediately after darkness falls, (2) considerably before this time, and (3) considerably after this time. Under DD conditions the greater amount of the insects have a free-running rhythm shorter than 24 hr, while only a small percentage have a rhythm of more than 24 hr.Destruction of the neurosecretory cells of the pars intercerebralis by means of radio waves leads to the formation of hyperactivity and loss of locomotor activity rhythm when more than half of these cells are destroyed.Injection of reserpine into the insect's haemolymph with doses of 10 μg/g of body weight results in a reduction in locomotor activity and produces arrhythmicity for 2 to 3 days under LD 12 : 12 conditions. Under DD conditions, however, this same dose results in a total and irretrievable loss of free-running rhythm. Histological studies of the brain of crickets following injection of reserpine show a large degree of accumulation of neurosecretion in the cells of the pars intercerebralis as compared with control insects.A hypothesis is put forward as to the way in which the brain centres regulating locomotor rhythm act in crickets.     

Refuge size preference in the field cricket Gryllus bimaculatus

Size preference for artificial refuges was examined in the adult field cricket Gryllus bimaculatus under laboratory conditions. Blinded crickets were placed individually in a container consisting of a circular arena and six different-sized artificial refuges (triangular tent-like shelters). The crickets were allowed to walk freely inside the container for a constant period. Size preference was evaluated by determining cumulative stay period in each shelter. When the depth of the shelters varied from 60 to 160 mm at 20-mm intervals, and the width was fixed at 30 mm, both males and females tended to remain in relatively longer shelters (≥ 140 mm). Females, in particular, exhibited a distinct preference for the longest shelter (160 mm). The width of the shelters was then varied from 20 to 40 mm at 4-mm intervals, and the depth was fixed at 100 mm. Although males did not show selectivity to specific shelters, females tended to select a shelter with a particular width (32 mm). These results suggest that adults of G. bimaculatus have size preferences for refuges under blinded conditions. However, the preferences may involve sexual differences as well.     

Analysis of postembryonic development of locomotor activity rhythm by corpora allata implantation in the cricket gryllus bimaculatus

• 1.1. Male crickets Gryllus bimaculatus show a drastic change in circadian rhythm from nymphal diurnality to adult nocturnality, in association with an increase in activity level several days after the imaginai moult.
• 2.2. The corpora allata implantation into male 7th or 8th instar nymphs produced supernumerary instar nymphs in about 30% of the implanted animals, but did not affected the normal development in the remaining animals.
• 3.3. The majority of the supernumerary instar nymphs were diurnal and sexually inactive, although their internal reproductive organs appeared to be fully mature.
• 4.4. The supernumerary instar nymphs became nocturnal with an increase in activity level several days after the imaginai (9th) moult.
• 5.5. The roles of the nervous system in the regulation of the rhythm reversal are discussed.

     

Optic lobe circadian pacemaker sends its information to the contralateral optic lobe in the cricket Gryllus bimaculatus

The bilaterally paired optic lobe pacemakers of the cricket Gryllus bimaculatus are mutually coupled. In the present study we recorded the neural activity conveyed from the brain toward the optic lobe with a suction electrode to examine the coupling signals. The results demonstrated that the brain efferents to the optic lobe encode the circadian information: Both in constant light (LL) and constant darkness (DD), the neural activity of brain efferents showed a clear circadian rhythm with a nocturnal peak. Since the rhythm survived the severance of the contralateral optic nerve but disappeared when the contralateral optic lobe was removed, it is apparent that the rhythm originates from the contralateral optic lobe. The amplitude of the rhythm was greater in LL than in DD, suggesting that light affects the amplitude of the rhythm. This was confirmed by the fact that the light-induced response was under circadian control, being greater during the subjective night. These data suggest that the bilaterally paired optic lobe pacemakers exchange circadian information as well as light information. The data are also consistent with the results of previous behavioral experiment.Abbreviations DD constant darkness - LD light dark cycle - LL constant light     

Molecular basis of the dopaminergic system in the cricket Gryllus bimaculatus

In insects, dopamine modulates various aspects of behavior such as learning and memory, arousal and locomotion, and is also a precursor of melanin. To elucidate the molecular basis of the dopaminergic system in the field cricket Gryllus bimaculatus DeGeer, we identified genes involved in dopamine biosynthesis, signal transduction, and dopamine re-uptake in the cricket. Complementary DNA of two isoforms of tyrosine hydroxylase (TH), which convert tyrosine into l-3,4-dihydroxyphenylalanine, was isolated from the cricket brain cDNA library. In addition, four dopamine receptor genes (Dop1, Dop2, Dop3, and DopEcR) and a high-affinity dopamine transporter gene were identified. The two TH isoforms contained isoform-specific regions in the regulatory ACT domain and showed differential expression patterns in different tissues. In addition, the dopamine receptor genes had a receptor subtype-specific distribution: the Dop1, Dop2, and DopEcR genes were broadly expressed in various tissues at differential expression levels, and the Dop3 gene was restrictedly expressed in neuronal tissues and the testicles. Our findings provide a fundamental basis for understanding the dopaminergic regulation of diverse physiological processes in the cricket.     

Protein synthesis is a required process for the optic lobe circadian clock in the cricket Gryllus bimaculatus

The effects of a translation inhibitor, cycloheximide (CHX), on the circadian neuronal activity rhythm of the optic lamina-medulla compound eye complex cultured in vitro were investigated in the cricket Gryllus bimaculatus. When the complex was treated with 10(-5) M CHX for 6 h, the rhythm exhibited a marked phase shift. The magnitude and direction of the phase shift were dependent on the phase at which the complex was treated with CHX; phase delays occurred during the late subjective day to early subjective night, whereas phase advances occurred around the late subjective night. Continuous application of CHX abolished circadian rhythms of both the spontaneous neuronal activity and the visually evoked response. However, it abolished neither the spontaneous activity nor the visually evoked response. As washed with fresh medium after CHX treatment, the rhythm soon reappeared and the subsequent phase was clearly correlated to the termination time of the treatment. These results suggest that protein synthesis is also involved in the cricket optic lobe circadian clock, and that the clock-related protein synthesis may be active during the late subjective day to subjective night.