Cricket Brain Research Articles

Sound processing in the cricket brain: evidence for a pulse duration filter.

The auditory system of female crickets allows them to specifically recognize and approach the species-specific male calling song, defined by sound pulses and silent intervals. Auditory brain neurons form a delay-line and coincidence detector network tuned to the pulse period of the male song. We analyzed the impact of changes in pulse duration on the behavior and the responses of the auditory neurons and the network. We confirm that the ascending neuron AN1 and the local neuron LN2 copy the temporal structure of the song. During ongoing long sound pulses, the delay-line neuron LN5 shows additional rebound responses and the coincidence detector neuron LN3 can generate additional bursts of activity, indicating that these may be driven by intrinsic oscillations of the network. Moreover, the response of the feature detector neuron LN4 is shaped by a combination of inhibitory and excitatory synaptic inputs, and LN4 responds even to long sound pulses with a short depolarization and burst of spikes, like to a sound pulse of natural duration. This response property of LN4 indicates a selective auditory pulse duration filter mechanism of the pattern recognition network, which is tuned to the duration of natural pulses. Comparing the tuning of the phonotactic behavior with the tuning of the local auditory brain neurons to the same test patterns, we find no evidence that a modulation of the phonotactic behavior is reflected at the level of the feature detector neurons. This rather suggests that steering to nonattractive pulse patterns is organized at the thoracic level.NEW & NOTEWORTHY Pulse period selectivity has been reported for the cricket delay-line and coincidence detector network, whereas pulse duration selectivity is evident from behavioral tests. Pulses of increasing duration elicit responses in the pattern recognition neurons, which do not parallel the behavioral responses and indicate additional processing mechanisms. Long sound pulses elicit rhythmic rebound activity and additional bursts, whereas the feature detector neuron reveals a pulse duration filter, expanding our understanding of the pattern recognition process.

An auditory-responsive interneuron descending from the cricket brain: a new element in the auditory pathway.

Crickets receive auditory information from their environment via ears located on the front legs. Ascending interneurons forward auditory activity to the brain, which houses a pattern recognition network for phonotaxis to conspecific calling songs and which controls negative phonotaxis to high-frequency sound pulses. Descending brain neurons, however, which are clearly involved in controlling these behaviors, have not yet been identified. We describe a descending auditory-responsive brain neuron with an arborization pattern that coincides with the ring-like auditory neuropil in the brain formed by the axonal arborizations of ascending and local interneurons, indicating its close link to auditory processing. Spiking activity of this interneuron occurs with a short latency to calling song patterns and the neuron copies the sound pulse pattern. The neuron preferentially responds to short sound pulses, but its activity appears to be independent of the calling song pattern recognition process. It also receives a weaker synaptic input in response to high-frequency pulses, which may contribute to its short latency spiking responses. This interneuron could be a crucial part in the auditory-to-motor transformation of the nervous system and contribute to the motor control of cricket auditory behavior.

Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis; now, it is known that EPO in mammal's brain plays key roles in the development, maintenance, protection, and repair of the nervous system. Also, EPO in mammals contributes to the efficient use of oxygen through the regulation of mitochondrial bioenergetic. Remarkably, a similar neuroprotective impact of recombinant human EPO (rhEPO) has been found in the brain of grasshoppers, raising questions about the evolutive origin of the EPO and its generic molecular function. The objective of this study is to show that the neuroprotective effect of rhEPO in insects involves the regulation of mitochondrial functions. The experiments were performed in crickets (Acheta domesticus). These insects were exposed under normoxia and hypoxia (5 days; 6% O2) conditions. Before experimentation, the animals were treated with EPO (30 IU/ml ‐ intra‐lymphatic injection) or PBS, as a control. The brains of the crickets were removed, and then we determined the mitochondrial respiration and production of mitochondrial ROS using our system oxygraphy ‐ 2K (ORORBOROS). Our results show that compares to normoxia; hypoxia significantly reduces mitochondrial respiration of complexes 1 and 1&2. On the other hand, the treatment of EPO in hypoxia, despite significantly increasing these parameters, does not recover the levels of mitochondrial respiration under normoxic conditions. In addition, the activity of complex IV (an indicator of the number of mitochondria) does not vary significantly between any of the treatments. Furthermore, we observed that while hypoxia did not significantly affect H2O2 production, the treatment with EPO increased ROS production under normoxic but not hypoxic conditions. Our data suggest that rhEPO regulates in some way the mitochondrial respiration and ROS production in the brain of crickets. Considering that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing, which could cause great oxidative stress due to the change in the metabolism of these animals, this molecule would have appeared as a regulator of mitochondrial functions.

A small, computationally flexible network produces the phenotypic diversity of song recognition in crickets.

How neural networks evolved to generate the diversity of species-specific communication signals is unknown. For receivers of the signals, one hypothesis is that novel recognition phenotypes arise from parameter variation in computationally flexible feature detection networks. We test this hypothesis in crickets, where males generate and females recognize the mating songs with a species-specific pulse pattern, by investigating whether the song recognition network in the cricket brain has the computational flexibility to recognize different temporal features. Using electrophysiological recordings from the network that recognizes crucial properties of the pulse pattern on the short timescale in the cricket Gryllus bimaculatus, we built a computational model that reproduces the neuronal and behavioral tuning of that species. An analysis of the model's parameter space reveals that the network can provide all recognition phenotypes for pulse duration and pause known in crickets and even other insects. Phenotypic diversity in the model is consistent with known preference types in crickets and other insects, and arises from computations that likely evolved to increase energy efficiency and robustness of pattern recognition. The model's parameter to phenotype mapping is degenerate - different network parameters can create similar changes in the phenotype - which likely supports evolutionary plasticity. Our study suggests that computationally flexible networks underlie the diverse pattern recognition phenotypes, and we reveal network properties that constrain and support behavioral diversity.

Evolutionary dynamics of sex-biased genes expressed in cricket brains and gonads.

Sex-biased gene expression, particularly sex-biased expression in the gonad, has been linked to rates of protein sequence evolution (nonsynonymous to synonymous substitutions, dN/dS) in animals. However, in insects, sex-biased expression studies remain centred on a few holometabolous species. Moreover, other major tissue types such as the brain remain underexplored. Here, we studied sex-biased gene expression and protein evolution in a hemimetabolous insect, the cricket Gryllus bimaculatus. We generated novel male and female RNA-seq data for two sexual tissue types, the gonad and somatic reproductive system, and for two core components of the nervous system, the brain and ventral nerve cord. From a genome-wide analysis, we report several core findings. Firstly, testis-biased genes had accelerated evolution, as compared to ovary-biased and unbiased genes, which was associated with positive selection events. Secondly, although sex-biased brain genes were much less common than for the gonad, they exhibited a striking tendency for rapid protein sequence evolution, an effect that was stronger for the female than male brain. Further, some sex-biased brain genes were linked to sexual functions and mating behaviours, which we suggest may have accelerated their evolution via sexual selection. Thirdly, a tendency for narrow cross-tissue expression breadth, suggesting low pleiotropy, was observed for sex-biased brain genes, suggesting relaxed purifying selection, which we speculate may allow enhanced freedom to evolve adaptive protein functional changes. The findings of rapid evolution of testis-biased genes and male and female-biased brain genes are discussed with respect to pleiotropy, positive selection and the mating biology of this cricket.

Evolution of the neural sex-determination system in insects: does fruitless homologue regulate neural sexual dimorphism in basal insects?

In the brain of holometabolous insects such as the fruit fly Drosophila melanogaster, the fruitless gene produces sex-specific gene products under the control of the sex-specific splicing cascade and contributes to the formation of the sexually dimorphic circuits. Similar sex-specific gene products of fruitless homologues have been identified in other holometabolous insects such as mosquitoes and a parasitic wasp, suggesting the fruitless-dependent neural sex-determination system is widely conserved amongst holometabolous insects. However, it remains obscure whether the fruitless-dependent neural sex-determination system is present in basal hemimetabolous insects. To address this issue, identification, characterization, and expression analyses of the fruitless homologue were conducted in the two-spotted cricket, Gryllus bimaculatus, as a model hemimetabolous insect. The Gryllus fruitless gene encodes multiple isoforms with a unique zinc finger domain, and does not encode a sex-specific gene product. The Gryllus Fruitless protein is broadly expressed in the neurones and glial cells in the brain, and there was no prominent sex-related difference in the expression levels of Gryllus fruitless isoforms. The results suggest that the Gryllus fruitless gene is not involved in the neural sex-determination in the cricket brain.

PL - 025 THE EFFECT OF SELECTED PARAMETERS ON HAND GRIP STRENGTH AMONG CRICKET PLAYERS IN PRISON DEPARTMENT

PL - 025 THE EFFECT OF SELECTED PARAMETERS ON HAND GRIP STRENGTH AMONG CRICKET PLAYERS IN PRISON DEPARTMENT

Immediate-Early Promoter-Driven Transgenic Reporter System for Neuroethological Research in a Hemimetabolous Insect.

Genes expressed in response to increased neuronal activity are widely used as activity markers in recent behavioral neuroscience. In the present study, we established transgenic reporter system for whole-brain activity mapping in the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect used in neuroethology and behavioral ecology. In the cricket brain, a homolog of early growth response-1 (Gryllus egr-B) was rapidly induced as an immediate-early gene (IEG) in response to neuronal hyperexcitability. The upstream genomic fragment of Gryllus egr-B contains potential binding sites for transcription factors regulated by various intracellular signaling pathways, as well as core promoter elements conserved across insect/crustacean egr-B homologs. Using the upstream genomic fragment of Gryllus egr-B, we established an IEG promoter-driven transgenic reporter system in the cricket. In the brain of transgenic crickets, the reporter gene (a nuclear-targeted destabilized EYFP) was induced in response to neuronal hyperexcitability. Inducible expression of reporter protein was detected in almost all neurons after neuronal hyperexcitability. Using our novel reporter system, we successfully detected neuronal activation evoked by feeding in the cricket brain. Our IEG promoter-driven activity reporting system allows us to visualize behaviorally relevant neural circuits at cellular resolution in the cricket brain.

Dendritic Ca2+ dynamics and multimodal processing in a cricket antennal interneuron.

The integration of stimuli of different modalities is fundamental to information processing within the nervous system. A descending interneuron in the cricket brain, with prominent dendrites in the deutocerebrum, receives input from three sensory modalities: touch of the antennal flagellum, strain of the antennal base, and visual stimulation. Using calcium imaging, we demonstrate that each modality drives a Ca2+ increase in a different dendritic region. Moreover, touch of the flagellum is represented in a topographic map along the neuron’s dendrites. Using intracellular recording, we investigated the effects of Ca2+ on spike shape through the application of the Ca2+ channel antagonist Cd2+ and identified probable Ca2+-dependent K+ currents.NEW & NOTEWORTHY Different dendritic regions of the cricket brain neuron DBNi1-2 showed localized Ca2+ increases when three modalities of stimulation (touch of the flagellum, strain at antennal base, and visual input) were given. Touch stimulation induces localized Ca2+ increases according to a topographic map of the antenna. Ca2+ appears to activate K+ currents in DBNi1-2.

A neural circuit for song pattern recognition in the cricket brain

Acoustic communication is based on amplitude and frequency modulation of sound signals. Temporal features of the signal require processing by central auditory neurons, the brain circuits, however that detect temporal features are poorly understood. We show how five neurons in the brain of female field crickets form an auditory feature-detector circuit for the pulse pattern of the male calling song, and exhibit properties of a delay-line and coincidence-detection mechanism. The network receives its direct input from a single ascending auditory interneuron. An internal delay that matches the pulse period of the calling song is established by a non-spiking brain neuron. In response to a sound pulse, it generates a transient inhibition that triggers a delayed rebound depolarization. The direct input and the delayed responses converge in a coincidence detector neuron, which responds best to the pulse pattern of the species-specific calling song as the rebound activation of the non-spiking neuron coincides with the response of the ascending interneuron to the subsequent sound pulse. The output of the coincidence detector neuron is further processed by a feature detector neuron to suppress unselective responses and background activity. The circuit reveals principal mechanisms of sensory processing underlying the perception of temporal auditory patterns.

Surface electrodes record and label brain neurons in insects.

We used suction electrodes to reliably record the activity of identified ascending auditory interneurons from the anterior surface of the brain in crickets. Electrodes were gently attached to the sheath covering the projection area of the ascending interneurons and the ringlike auditory neuropil in the protocerebrum. The specificity and selectivity of the recordings were determined by the precise electrode location, which could easily be changed without causing damage to the tissue. Different nonauditory fibers were recorded at other spots of the brain surface; stable recordings lasted for several hours. The same electrodes were used to deliver fluorescent tracers into the nervous system by means of electrophoresis. This allowed us to retrograde label the recorded auditory neurons and to reveal their cell body and dendritic structure in the first thoracic ganglion. By adjusting the amount of dye injected, we specifically stained the ringlike auditory neuropil in the brain, demonstrating the clusters of cell bodies contributing to it. Our data provide a proof that surface electrodes are a versatile tool to analyze neural processing in small brains of invertebrates.NEW & NOTEWORTHY We show that surface suction electrodes can be used to monitor the activity of auditory neurons in the cricket brain. They also allow delivering electrophoretically a fluorescent tracer to label the structure of the recorded neurons and the local neuropil to which the electrode was attached. This new extracellular recording and labeling technique is a versatile and useful method to explore neural processing in invertebrate sensory and motor systems.

Nitric oxide augments single Ca2+ channel currents via cGMP-dependent protein kinase in Kenyon cells isolated from the mushroom body of the cricket brain

Behavioral and pharmacological studies in insects have suggested that the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is involved in the formation of long-term memory (LTM) associated with olfactory learning. However, the target molecules of NO and the downstream signaling pathway are still not known. In this study, we investigated the action of NO on single voltage-dependent Ca2+ channels in the intrinsic neurons known as Kenyon cells within the mushroom body of the cricket brain, using the cell-attached configuration of the patch-clamp technique. Application of the NO donor S-nitrosoglutathione (GSNO) increased the open probability (NPO) of single Ca2+ channel currents. This GSNO-induced increase was blocked by ODQ, a soluble guanylate cyclase (sGC) inhibitor, suggesting that the NO generated by GSNO acts via sGC to raise cGMP levels. The membrane-permeable cGMP analog 8-Bro-cGMP also increased the NPO of single Ca2+ channel currents. Pretreatment of cells with KT5823, a protein kinase G blocker, abolished the excitatory effect of GSNO. These results suggest that NO augments the activity of single Ca2+ channels via the cGMP/PKG signaling pathway. To gain insight into the physiological role of NO, we examined the effect of GSNO on action potentials of Kenyon cells under current-clamp conditions. Application of GSNO increased the frequency of action potentials elicited by depolarizing current injections, indicating that NO acts as a modulator resulting in a stimulatory signal in Kenyon cells. We discuss the increased Ca2+ influx through these Ca2+ channels via the NO/cGMP signaling cascade in relation to the formation of olfactory LTM.

Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain

Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at −40mV, peaked at −26mV, and half-inactivated at −46.7mV. The persistent sodium current (INaP) activated at −51mV, peaked at −23mV, and half-inactivated at −30.7mV. Tetrodotoxin (TTX; 1μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd2+ (50μM) potently blocked INaP with little effect on INaf. Riluzole (>20μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at −30mV, peaked at −11.3mV, and half-inactivated at −34mV. The Ca2+ channel blocker verapamil (100μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca2+-activated K+ (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca2+ or adding verapamil or nifedipine, and increased by adding the Ca2+ agonist Bay K8644, indicating that Ca2+ entry via the L-type Ca2+ channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100μM riluzole in the bath solution. These membrane oscillations disappeared with 1μM TTX, 50μM Cd2+, replacement of external Na+ with choline, and blockage of Na+-activated K+ current (IKNa) with 50μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca2+-free medium or in the presence of verapamil were eliminated by blocking INaP with 50μM Cd2+. Taken together, these results indicate that INaP and IKNa participate in the generation of membrane oscillations and that INaP additionally participates in the generation of plateau potentials and initiation of spontaneous action potentials. ICa, through L-type Ca2+ channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels.

Targeted gene delivery in the cricket brain, using in vivo electroporation

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb′-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.

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.

Processing of ultrasound in a bush cricket's brain

The processing and categorization of conspecific and heterospecific acoustic signals is an important task of the central nervous system. In orthopteran species, carrier frequency (besides temporal cues) is one of the major discriminators. In the bush cricket species Ancistrura nigrovittata Brunner von Wattenwyl (Phaneropteridae, Barbitistini), ultrasound has potentially different meanings and may elicit vastly different behaviours depending on the context it is perceived in. In the present study, data are presented of the morphology and neuronal responses of three local brain neurones (LBNs) that respond best to ultrasound. All neurones show dense arborizations in the lateral protocerebrum, where ascending interneurones terminate. The LBN2 and LBN9 neurones are entirely restricted to one side of the brain, whereas LBN5 crosses the midline, thereby linking both hemispheres. The response maxima for LBN2 overlap closely with the peak carrier frequencies found in a species‐specific duet, which consists of sonic (16 kHz, male), as well as ultrasonic (24–28 kHz, female) sound. By contrast, LBN9 responds only to ultrasound in the range of the female reply, whereas the male song induces exceptionally long‐lasting inhibition. The LBN5 neurone shows strongest spike activity to a broad range of ultrasonic frequencies, as long as the pulse duration remains short. All three brain neurones respond to ultrasound in a unique way and may be involved in the shaping of different behavioural outcomes.

A novel GABAergic action mediated by functional coupling between GABAB-like receptor and two different high-conductance K+channels in cricket Kenyon cells

The γ-aminobutyric acid type B (GABA(B)) receptor has been shown to attenuate high-voltage-activated Ca(2+) currents and enhance voltage-dependent or inwardly rectifying K(+) currents in a variety of neurons. In this study, we report a novel coupling of GABA(B)-like receptor with two different high-conductance K(+) channels, Na(+)-activated K(+) (K(Na)) channel and Ca(2+)-activated K(+) (K(Ca)) channel, in Kenyon cells isolated from the mushroom body of the cricket brain. Single-channel activities of K(Na) and K(Ca) channels in response to bath applications of GABA and the GABA(B)-specific agonist SKF97541 were recorded with the cell-attached patch configuration. The open probability (P(o)) of both K(Na) and K(Ca) channels was found to be increased by bath application of GABA, and this increase in Po was antagonized by coapplication of the GABAB antagonist CGP54626, suggesting that GABA(B)-like receptors mediate these actions. Similarly, GABA(B)-specific agonist SKF97541 increased the Po of both K(Na) and K(Ca) channels. Perforated-patch recordings using β-escin further revealed that SKF97541 increased the amplitude of the outward currents elicited by step depolarizations. Under current-clamp conditions, SKF97541 decreased the firing frequency of spontaneous action potential (AP) and changed the AP waveform. The amplitude and duration of AP were decreased, whereas the afterhyperpolarization of AP was increased. Resting membrane potential, however, was not significantly altered by SKF97541. Taken together, these results suggest that GABA(B)-like receptor is functionally coupled with both K(Na) and K(Ca) channels and this coupling mechanism may serve to prevent AP formation and limit excitatory synaptic input.

Calling Song Recognition in Female Crickets: Temporal Tuning of Identified Brain Neurons Matches Behavior

Phonotactic orientation of female crickets is tuned to the temporal pattern of the male calling song. We analyzed the phonotactic selectivity of female crickets to varying temporal features of calling song patterns and compared it with the auditory response properties of the ascending interneuron AN1 (herein referred to as TH1-AC1) and four newly identified local brain neurons. The neurites of all brain neurons formed a ring-like branching pattern in the anterior protocerebrum that overlapped with the axonal arborizations of TH1-AC1. All brain neurons responded phasically to the sound pulses of a species-specific chirp. The spike activity of TH1-AC1 and the local interneuron, B-LI2, copied different auditory patterns regardless of their temporal structure. Two other neurons, B-LI3 and B-LC3, matched the temporal selectivity of the phonotactic responses but also responded to some nonattractive patterns. Neuron B-LC3 linked the bilateral auditory areas in the protocerebrum. One local brain neuron, B-LI4, received inhibitory as well as excitatory synaptic inputs. Inhibition was particularly pronounced for nonattractive pulse patterns, reducing its spike activity. When tested with different temporal patterns, B-LI4 exhibited bandpass response properties; its different auditory response functions significantly matched the tuning of phonotaxis. Temporal selectivity was established already for the second of two sound pulses separated by one species-specific pulse interval. Temporal pattern recognition in the cricket brain occurs within the anterior protocerebrum at the first stage of auditory processing. It is crucially linked to a change in auditory responsiveness during pulse intervals and based on fast interactions of inhibition and excitation.

Identification and expression analyses of a novel serotonin receptor gene,5-HT2β, in the field cricket,Gryllus bimaculatus

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2β gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2β gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.

Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus

Serotonin (5-HT) modulates various aspects of behaviours such as aggressive behaviour and circadian behaviour in the cricket. To elucidate the molecular basis of the cricket 5-HT system, we identified 5-HT-related genes in the field cricket Gryllus bimaculatus DeGeer. Complementary DNA of tryptophan hydroxylase and phenylalanine-tryptophan hydroxylase, which convert tryptophan into 5-hydroxy-L-tryptophan (5-HTP), and that of aromatic L-amino acid decarboxylase, which converts 5-HTP into 5-HT, were isolated from a cricket brain cDNA library. In addition, four 5-HT receptor genes (5-HT(1A) , 5-HT(1B) , 5-HT(2α) , and 5-HT(7) ) were identified. Expression analysis of the tryptophan hydroxylase gene TRH and phenylalanine-tryptophan hydroxylase gene TPH, which are selectively involved in neuronal and peripheral 5-HT synthesis in Drosophila, suggested that two 5-HT synthesis pathways co-exist in the cricket neuronal tissues. The four 5-HT receptor genes were expressed in various tissues at differential expression levels, suggesting that the 5-HT system is widely distributed in the cricket.