Plurisegmental neurones of the cricket Gryllus campestris L. that discharge in the rhythm of its own song

Male crickets (Gryllus campestris L.) mounted so that their wings and abdomen could move freely were induced to stridulate by brain lesion. During the song the activity of single neurones was recorded extracellularly in a cervical connective. Nine distinct spike patterns were observed. Patterns I and II tend to copy the chirp as a whole rather than the onset of the syllables (the recorded potentials of the wing-opener muscle M99 marked the syllable onset). The other patterns reflect the syllabic structure. Each, in its own way, marks the various syllables with different numbers of spikes. The delay of the spike response is different for each pattern. Some patterns, but not others, also reflect the beginning or end of the song, or the abdominal expiratory activity. One neurone also responds in correlation with muscle discharges typical of the courtship song. In some of the patterns it is evident that there is a stronger correlation with the closer muscle (M90) discharge than with the opener muscle discharge. Activation by auditory self-stimulation by way of the tympanal organs can be ruled out for all patterns. It is possible that patterns I–V are induced by afferent activity coupled to the wing movement. Patterns VI–VIII are probably copies of motor signals ascending from the thoracic song-pattern generators to the head ganglia. It is evident that the head ganglia have detailed information as to the motor output for stridulation and abdominal expiration.     

Activity of neck-muscle motoneurones during eye cleaning behaviour in the cricket Gryllus campestris

The activity of neck-muscle motoneurones which control head movements during eye cleaning behaviour was recorded from motor nerves with chronically implanted electrodes in unrestrained crickets. We show that motoneurones of the dorso-ventral muscles displayed strong activity differences between both sides of the neck, with higher discharge frequencies either ipsi- or contralateral to the direction of the head movement. Motoneurones innervating dorsal-longitudinal muscles were equally active on both sides. A single excitatory motoneurone of one dorso-ventral muscle showed a discharge pattern unequivocally related to eye cleaning. Lesions of connectives revealed that this motoneurone is monitored by interneuronal pathways from the suboesophageal ganglion although the primary sensory axons eliciting eye cleaning, project into the prothoracic ganglion.     

Auditory neurones in the brain of the cricket Gryllus bimaculatus (De Geer): Ascending interneurones

Two types of auditory interneurone which ascend from the prothoracic ganglion to the brain in the cricket Gryllus bimaculatus (De Geer) are described. Intracellular recordings were made from the axons of the neurones in the brain under closed-field stimulus conditions and the recorded cells then stained with either cobalt or Lucifer Yellow. Both neurone types—the Plurisegmental ascending low frequency neurone 1 (PALF1), and the Plurisegmental ascending high frequency neurone 1 (PAHF1)—show response characteristics which would make them well suited to encoding the conspecific calling and courtship songs respectively. Further, the projection areas of both neurone types in the brain overlap those of previously identified intraganglionic interneurones, particularly in the anterior-ventral protocerebrum, and it is suggested that an auditory neuropile may exist in this region.     

Condition dependence of a multicomponent sexual signal in the field cricket Gryllus campestris

In choosing a breeding partner, females in many animal species select between available males on the basis of several signalling traits. Some theoretical models of signalling evolution predict that multiple ornaments convey specific information on different aspects of male quality, such as current nutritional condition. We investigated the effect of nutrition on the calling song of male field crickets Gryllus campestris. This song is a multicomponent sexually selected signal. Adult males were kept on one of three feeding regimes, which resulted in significant differences in body condition between experimental groups. We found significant increases in calling rate and chirp rate and a significant decrease in interchirp duration with increasing food level. Other song characters, such as chirp duration, syllable number, chirp intensity and carrier frequency, were not affected by the food treatment. Furthermore, carrier frequency was correlated with harp area, which is an index of structural size in adult males. The calling song of the field cricket may thus serve as a multicomponent sexual signal, which contains discrete information on past growth and juvenile development as well as present nutritional condition. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Circadian oscillations outside the optic lobe in the cricket Gryllus bimaculatus

Although circadian rhythms are found in many peripheral tissues in insects, the control mechanism is still to be elucidated. To investigate the central and peripheral relationships in the circadian organization, circadian rhythms outside the optic lobes were examined in the cricket Gryllus bimaculatus by measuring mRNA levels of period (per) and timeless (tim) genes in the brain, terminal abdominal ganglion (TAG), anterior stomach, mid-gut, testis, and Malpighian tubules. Except for Malpighian tubules and testis, the tissues showed a daily rhythmic expression in either both per and tim or tim alone in LD. Under constant darkness, however, the tested tissues exhibited rhythmic expression of per and tim mRNAs, suggesting that they include a circadian oscillator. The amplitude and the levels of the mRNA rhythms varied among those rhythmic tissues. Removal of the optic lobe, the central clock tissue, differentially affected the rhythms: the anterior stomach lost the rhythm of both per and tim; in the mid-gut and TAG, tim expression became arrhythmic but per maintained rhythmic expression; a persistent rhythm with a shifted phase was observed for both per and tim mRNA rhythms in the brain. These data suggest that rhythms outside the optic lobe receive control from the optic lobe to different degrees, and that the oscillatory mechanism may be different from that of Drosophila.     

Residual circadian rhythmicity after bilateral lamina-medulla removal or optic stalk transection in the cricket, Gryllus bimaculatus

Bilateral optic stalk severance or lamina-medulla region removal were carried out in 47 adult male crickets Gryllus bimaculatus DeGeer. Effects of the operations on circadian locomotor activity were investigated under 12 h light: 12 h dark and at a constant temperature of 26°C. In the pre-operative days, 39 of the animals showed a typical nocturnal activity rhythm (normal rhythm), but the remaining 8 exhibited an atypical rhythm which is diurnal rather than nocturnal (abnormal rhythm). The operations eventually caused an arrhythmicity in all animals, suggesting that the crucial part of the central nervous system controlling the cricket circadian activity is located in the lamina-medulla region. However, in some of the post-operative crickets, the rhythm did not immediately disappear but persisted for a while: the diurnal increase of activity was observed up to 2 weeks in all 8 abnormal- and 4 normal-rhythm animals. In addition, 8 out of 39 normal-rhythm animals showed a single well-defined post-operative peak which occurred approximately in phase with the nocturnal peak prior to surgery. These results are discussed in relation to a possibility of involvement of the oscillatory structure outside the optic lobes.     

RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus

The circadian clock gene period (Gryllus bimaculatus period, Gb’per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb’per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb’per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb’per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb’per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb’per.     

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.     

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.     

Immune investment impairs growth, female reproduction and survival in the house cricket, Acheta domesticus

We investigated whether an immune response is associated with growth, female reproduction and survival costs in the house cricket. Using different intensities of challenge immune (implantation of one piece of nylon (1N) and two nylons (2N), with their respective sham-challenge and control groups) with body size and exoskeleton thickness as response variables, growth costs were determined for both sexes. A similar methodology was followed for reproduction costs, in which egg number and size, and female survival were measured as response variables. It was also determined whether mated and virgin females showed different immune responses. Body size decreased with immune challenge but only in the 2N treatment. Exoskeleton thickness increased in both sham-challenge groups and the 1N group but decreased in the 2N group. Egg number decreased more in the sham-challenge groups followed by the 1N and 2N groups. The 2N group showed the largest egg size at the end of the experiment. In these females, 2N group died first followed by the 1N, two nylon sham and one nylon sham groups. Finally, mated females showed a lower immune response than virgin females. These results are consistent with ecological immunity theory. The discovery of exoskeleton-related costs of immunity and injury may have important implications for experimental design in studies of the cost of immunity.     

The role of free amino acids in osmoregulation of cricket blood (Acheta domesticus)

The blood volume increased during normal feeding, and did not decrease during fasting at the end of the stadium. The unexpectedly high blood volume of starved crickets might be an adaptation to increase chances for moulting via stretch receptor stimulation.The amount of blood amino acids was not changed by feeding, but increased with fasting or starvation. Thus amino acid levels in the blood were not directly related to amino acid input from the gut.The blood protein concentration did not change during starvation, but the amino acid concentration was 33% higher in starved crickets that drank water as opposed to those given saline to drink. Thus amino acid levels in the blood were not related directly to blood protein concentration.The blood amino acid concentration was 19–22 mM/l in response to salt intake by feeding crickets or starved crickets drinking saline. The concentration was 32–38 mM/l when the crickets were fasting prior to and after ecdysis or when starved with water to drink during the time when they would normally be feeding. The increase of amino acids during fasting was due to a proportional increase in all amino acids augmented by a 3 × increase in tyrosine. The increase during salt depleting starvation was due to a doubling of the two predominant amino acids proline and glycine. Proline and glycine were not increased in starved crickets drinking saline, thus starvation was not the reason for the increase. This is the first instance where specific amino acids have been implicated in osmoregulation.