Distribution and morphology of descending brain neurons in the cricket Gryllus bimaculatus

The number and distribution of descending brain neurons have been investigated in the cricket. The results are based on retrograde labeling of these cells with either Lucifer yellow or Neurobiotin via whole or small split portions of the cervical connectives. Various groups of cells and single neurons have been identified, and the morphology of more than 40 cells is described. Nearly 200 descending brain neurons can be stained via one cervical connective. Their perikarya are concentrated in clusters that occur ipsi- and contralateral to the filled connective and that lie dorsal and ventral in the brain. Descending cells only arborize in the nonglomerular neuropils of the brain and never branch in the optic lobe. Cells descending ipsilaterally never arborize in the contralateral hemisphere, whereas contralateral descending neurons often branch in both hemispheres. Irrespective of soma position, cells can arborize in the ventral and/or dorsal neuropils of the brain. Neurons with somata in the protocerebrum often have branches in the deutocerebrum and vice versa. The main arborizations of the cells from the prominent ventral i5 group are found in the same part of the protocerebrum. In contrast, various cells arborize in the ventral posterior deutocerebrum, but their somata are not located in different clusters. Thus, neurons from the same cluster may, but need not necessarily, arborize in the same brain area.     

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.     

Sensory responses of descending brain neurons in the walking cricket, Gryllus bimaculatus

Sensory responses of various descending brain neurons, their modulation during standing or walking, and the correlation of such modulations with stimulus category were investigated. Stimuli involving (1) static or moving grating, artificial calling songs with (2) the conspecific and (3) an ultrasound frequency, or (4) air puffs to the cerci were presented to crickets walking in an open loop paradigm. The morphology of different descending interneurons in the brain and thoracic ganglia is described, together with their respective response properties. Some cells were excited, others inhibited by, and only some were directionally sensitive to the optomotor stimulus. Responses to artificial calling songs with conspecific and ultrasound frequency differed in the way the syllables of the sounds were coded and in the representation of ipsi- and contralateral stimuli. The majority of cells tested responded to air puffs. Stimulus representation differed among individuals of morphological types, but was very similar among individual interneurons of the morphologically homogenous i5 group. Stimuli approximating predators (air puffs, ultrasound) were usually represented during walking and standing; however, most neurons only responded to the other stimuli only during walking. These results indicate that the same neurons show different responses, and may have different functions, under different behavioral conditions.     

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.     

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     

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.     

Expression patterns of aristaless in developing appendages of Gryllus bimaculatus (cricket)

We report the isolation and expression patterns of aristaless (al), a paired-type homeobox gene, of Gryllus bimaculatus (Gb), a hemimetabola model insect. Gryllus al (Gbal) is expressed in the most distal region of developing labrum, antenna, mandible, maxilla, labium, leg, cercus, and hindgut. Gbal is also expressed in the proximal region, corresponding to the presumptive coxopodite, of the developing antenna, mandible, maxilla, labium, and leg, but not in the developing labrum, cercus, and hindgut. During development of the leg, expression of Gbal changes dynamically with the progress in leg segmentation: Gbal is expressed in order in the presumptive pretarsus, coxa, femur, tibia and tarsus before appearance of morphological segmentation.     

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.     

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.     

Topographic mapping of the axons of the femoral chordotonal organ neurons in the cricket Gryllus bimaculatus

Central projections of the femoral chordotonal organ (FCO) neurons in the cricket Gryllus bimaculatus were investigated by selectively staining small numbers of axons. The FCOs in all legs consist of partly fused ventral and dorsal scoloparia in the proximal femur. The ventral scoloparium neurons can be reliably divided into two groups: the ventral group neurons (VG), which are arranged in a sequentially smaller manner distally, and dorsal group neurons (DG), which simply aggregate in the proximal region near the dorsal scoloparium. All axons of the FCO projected to the ipsilateral half of the respective thoracic ganglion. The VG axons possessed dorso-lateral branches in the motor association neuropile and antero-ventral branches dorso-lateral to the anterior ventral association centre. However, the more proximally the somata were situated, the more medially the main neurites terminated. The DG axons showed some variations: some axons of the distally located neurons possessed dorso-lateral branches and terminated on the boundary region of the mVAC, while the other axons terminated exclusively in the medical ventral association centre (mVAC), including the ventral part, which receives auditory sensory neuron projections. All axons of the dorsal scoloparium neurons projected exclusively into the dorsal part of the mVAC; however, the ventrally located neurons projected more ventrally than did the dorsally located neurons. The above characteristics were nearly identical in the pro- and metathoracic FCOs. These results suggest that the cricket FCO axons are roughly organized in a somatotopic map and are broadly differentiated in their function.     

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.     

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.     

piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

Performance of blue- and green-sensitive photoreceptors of the cricket Gryllus bimaculatus

The compound eye of the cricket Gryllus bimaculatus contains a specialized dorsal rim area (DRA) populated by distinct blue-sensitive photoreceptors responsible for perception of polarized light. The rest of the eye is dominated by green-sensitive photoreceptors. Using patch clamp we studied dissociated ommatidia of nocturnal adults and diurnal eight-instar nymphs with the goals (1) of characterizing the biophysical properties of cricket photoreceptors in general and (2) describing the functionally dissimilar blue- and green-sensitive photoreceptors in terms of voltage-gated channel composition and signal coding. Despite different lifestyles, adult and nymph photoreceptors were indistinguishable. No significant circadian changes were observed in K⁺ currents. In contrast, prominent differences were seen between blue- and green-sensitive photoreceptors. The former were characterized by relatively low absolute sensitivity, high input resistance, slow quantum bumps with long latencies, small light-induced and K⁺ currents and low steady-state depolarization. Information rate, a measure of photoreceptor performance calculated from voltage responses to bandwidth-limited white noise-modulated light contrast, was 87 ± 8 bits s^?1 in green-sensitive photoreceptors vs. 59 ± 14 bits s^?1 in blue-sensitive photoreceptors, implying a limited role of DRA in the perception of visual contrasts. In addition, evidence of electrical coupling between photoreceptors is presented.     

The precision of auditory lateralization in the cricket, Gryllus bimaculatus

ABSTRACT. The precision of auditory lateralization was determined behaviourally for the cricket, Gryllus bimaculatus L. A forced-choice Y-maze test was devised in which the cricket, on entering the test arena, could not — in contrast to free phonotactic approaches — change its walking direction until after it had passed through a narrow wire-mesh tunnel. For a sound frequency of 4.7 kHz, matching the species' calling frequency, the minimum audible angle for correct side discrimination was 15°. For stimulus angles smaller than 15° from the longitudinal body axis, the crickets walked randomly to either side; stimulus angles greater than 25° resulted in all crickets turning correctly. These data reveal a sharply tuned lateral sensitivity for the auditory pathway of crickets, with an optimum at the species' calling frequency of 4.7 kHz (when compared with 3.5 and 6.0 kHz). The results for the forced-choice test are compared with the walking pattern during free phonotactic approaches, in order to determine the possible strategy underlying the acoustic orientation behaviour of the cricket.     

Mate preference for novel partners in the cricket Gryllus bimaculatus

Abstract .1. To benefit from the putative genetic advantages of multiple mating with multiple partners, female insects would be expected to select against mating with the same male twice when another potential partner is present.
2. This paper examined whether female Gryllus bimaculatus (Gryllidae) preferred to mate with a novel partner over a partner with which they had mated previously.
3. Females presented with a choice preferred significantly to mate with novel males over previously mated males, and preferred to do so even when the potentially confounding influence of male–male competition was controlled for.
4. The potential advantages of such a mate choice pattern and possible ways in which the choice is mediated are discussed.