Diversity of intersegmental auditory neurons in a bush cricket

Various auditory interneurons of the duetting bush cricket Ancistrura nigrovittata with axons ascending to the brain are presented. In this species, more intersegmental sound-activated neurons have been identified than in any other bush cricket so far, among them a new type of ascending neuron with posterior soma in the prothoracic ganglion (AN4). These interneurons show not only morphological differences in the prothoracic ganglion and the brain, but also respond differently to carrier frequencies, intensity and direction. As a set of neurons, they show graded differences for all of these parameters. A response type not described among intersegmental neurons of crickets and other bush crickets so far is found in the AN3 neuron with a tonic response, broad frequency tuning and little directional dependence. All neurons, with the exception of AN3, respond in a relatively similar manner to the temporal patterns of the male song: phasically to high syllable repetitions and rhythmically to low syllable repetitions. The strongest coupling to the temporal pattern is found in TN1. In contrast to behavior the neuronal responses depend little on syllable duration. AN4, AN5 and TN1 respond well to the female song. AN4 (at higher intensities) and TN1 respond well to a complete duet.     

Ascending auditory interneurons in the cricketTeleogryllus commodus (Walker): comparative physiology and direct connections with afferents

Summary Ascending auditory interneurons of the cricket,Teleogryllus commodus (Walker), were investigated using simultaneous intracellular and extracellular recording in order to identify units which had previously been characterized only by extracellular recording. The morphology and physiology of the large adapting unit (LAU: Fig. 1) and of the small tonic unit (STU: Fig. 2) ofTeleogryllus correspond well to those of the ascending neuron 2 (AN2) and the ascending neuron 1 (AN1) ofGryllus (Figs. 1, 2), respectively.A summary of the ascending auditory interneurons described by various authors in 5 species of crickets is presented in order to establish common identities.Physiological evidence for direct connections between auditory afferents and the ascending auditory interneurons AN1 (STU) and AN2 (LAU) is presented. Simultaneous intracellular recordings from receptors and interneurons in response to sound as well as the activity of auditory interneurons upon electrical stimulation of the tympanal nerve reveal short and constant latencies of receptor-evoked synaptic activity in AN1 (STU) and AN2 (LAU).Abbreviations STU small tonic unit - LAU large adapting unit - AN ascending neuron - EPSP excitatory postsynaptic potential     

Morphological and physiological regeneration in the auditory system of adult <Emphasis Type="Italic">Mecopoda elongata</Emphasis> (Orthoptera: Tettigoniidae)

Orthopterans are suitable model organisms for investigations of regeneration mechanisms in the auditory system. Regeneration has been described in the auditory systems of locusts (Caelifera) and of crickets (Ensifera). In this study, we comparatively investigate the neural regeneration in the auditory system in the bush cricket Mecopoda elongata. A crushing of the tympanal nerve in the foreleg of M. elongata results in a loss of auditory information transfer. Physiological recordings of the tympanal nerve suggest outgrowing fibers 5 days after crushing. An anatomical regeneration of the fibers within the central nervous system starts 10 days after crushing. The neuronal projection reaches the target area at day 20. Threshold values to low frequency airborne sound remain high after crushing, indicating a lower regeneration capability of this group of fibers. However, within the central target area the low frequency areas are also innervated. Recordings of auditory interneurons show that the regenerating fibers form new functional connections starting at day 20 after crushing.     

Topographical organization of the auditory pathway within the prothoracic ganglion of the cricket Gryllus campestris L.

Summary The topographical organization of the prothoracic ganglion of the cricket, Gryllus campestris L., is described from horizontal, transverse, and sagittal sections of preparations specially treated to elucidate longitudinal tracts, commissures, and areas of neuropil. These structures were compared to those reported from other insect thoracic ganglia, resulting in still further evidence for a common basic morphological pattern among insect central nervous systems.Six types of auditory interneurons, all existing as mirrorimage pairs, were identified through intracellular application of the dye Lucifer yellow, and then related to several morphological patterns. Two intrasegmental neurons (ON1, ON2) are similar in location of cell bodies and course of neurites and axons; three intersegmental neurons (AN1, AN2, TN1) are likewise similar to one another. The axons of the two intrasegmental neurons cross the midline of the ganglion in the newly described omega commissure. Axons of the other four types all course within the median portion of the ventral intermediate tract and project intersegmentally.All six neuron types arborize within the ventral portion of the ring tract, the same neuropilar region in which auditory sensory neurons terminate. The ring tract is therefore considered the most important region for auditory information processing within the cricket prothoracic ganglion.     

Sound localisation in crickets

Intracellular recordings were made in the brain of the cricket Gryllus bimaculatus from an ascending auditory interneuron (AN1). Acoustic stimuli with calling song temporal pattern were delivered via earphones in a preparation with the acoustic trachea cut (attenuation of crossing sound > 30 dB). The input-output function of this cell was then determined by recording its responses to stimulation of the ipsilateral ear alone, of the contralateral ear alone and to stimulation of both ears simultaneously with the same or different carrier frequencies and intensities.This interneuron was excited by the ear ipsilateral to its axon and dendritic field and unresponsive to stimuli presented to the axon-contralateral ear alone. However, in binaural stimulation experiments, the response to a constant ipsilateral stimulus was progressively reduced as the intensity of a simultaneous contralateral stimulus was increased, above a threshold intensity.Tuning curves for threshold of this inhibition, determined in binaural stimulation experiments, indicated significant inhibition in the range 3–20 kHz with lowest threshold at 4–5 kHz. The inhibition was unaffected by sectioning of the contralateral circumoesophageal or neck connective, indicating that the inhibitory influence crosses the midline at the level of the prothoracic ganglion. Intracellular recordings from AN1 in the prothoracic ganglion confirmed that it was indeed neurally inhibited by inputs from the contralateral ear.Tuning curves for excitation of an omega neuron (ON1) by the ear ipsilateral to its soma and also the tuning of inhibition of ON1 by its contralateral ON1 partner, closely match the tuning of inhibition of AN1 and to a lesser extent, of AN2. This was taken as evidence that each AN1 is inhibited by the contralateral ON1. The significance of this interaction for directional hearing and phonotaxis is discussed.Abbreviations AP/CHP action potentials per chirp - AN1, AN2 ascending auditory interneurons 1, 2 - ON1 omega neuron 1 - ipsi ipsilateral contra contralateral - PTG prothoracic ganglion loc lateral ocellar nerve - On optic nerve an antennal nerve - coc circum-oesophageal connective so sound off     

Changes in the auditory system of locusts (Locusta migratoria and Schistocerca gregaria) after deafferentation

Deafferentation experiments during postembryonic development show morphological and/or physiological changes of receptor fibers and of identified auditory interneurons in the CNS of the locusts Locusta migratoria and Schistocerca gregaria after unilateral ablation of one tympanic organ either in the larva or the adult animal.

Directional sensitivity of wind-sensitive giant interneurons in the cave cricket Troglophilus neglectus.

Unlike the situation in most cockroach and cricket species studied so far, the wind-sensitive cerci of the cave cricket Troglophilus neglectus Krauss (Rhaphidophoridae, Orthoptera) are not oriented parallel to the body axis but perpendicular to it. The effects of this difference on the morphology, and directional sensitivity of cercal giant interneurons (GIs), were investigated. In order to test the hypothesis that the 90 degrees change in cercal orientation causes a corresponding shift in directional sensitivity of GIs, their responses in both the horizontal and vertical planes were tested. One ventral and four dorsal GIs (corresponding to GIs 9-1a and 9-2a, 9-3a, 10-2a, 10-3a of gryllid crickets) were identified. The ventral GI 9-1a of Troglophilus differed somewhat from its cricket homologue in its dendritic arborisation and its directional sensitivity in the horizontal plane. The morphology and horizontal directionality of the dorsal GIs closely resembled that of their counterparts in gryllids. In the vertical plane, the directionality of all GIs tested was similar. They were all excited mainly by wind puffs from the axon-ipsilateral quadrant. The results suggest that directional sensitivity to air currents in the horizontal plane is maintained despite the altered orientation of the cerci. This is presumably due to compensatory modifications in the directional pReferences of the filiform hairs.     

Neuronal basis of phonotactic behaviour in Tettigonia viridissima : processing of behaviourally relevant signals by auditory afferents and thoracic interneurons

Information transmission in the auditory pathway of Tettigonia viridissima was investigated using song models and artificial stimuli. Receptor cells respond tonically to song models and copy the syllable pattern within a wide intensity range. The omega-neuron responds tonically to soma-ipsilateral stimuli. Contralateral stimuli elicit IPSPs both within dendritic (ipsilateral) and axonal (contralateral) branches, thereby emphasizing directionality. Both AN1 and AN2 respond with tonic, non-adapting responses, precisely copying the syllable pattern of the song. While AN1 is excited by sonic frequencies and inhibited by ultrasonic frequencies, AN2 responds predominantly to ultrasound. The TN1 only responds to the ultrasonic components of the song, with phasic responses, which adapt quickly. In the adapted state, it responds selectively to the time pattern of the conspecific song, but not to the song patterns of two syntopic Tettigonia species. TN2, which has not been described up until now, is tuned to ultrasonic frequencies. Its responses to song models vanish after a few syllables, because of quick adaptation. The morphology is unusual with the axon running contralateral to the input site. The behavioural relevance of auditory interneurons is discussed and compared with the auditory system of crickets. Accepted: 3 November 1996     

Gleaning bat echolocation calls do not elicit antipredator behaviour in the Pacific field cricket, <Emphasis Type="Italic">Teleogryllus oceanicus</Emphasis> (Orthoptera: Gryllidae)

Bats that glean prey (capture them from surfaces) produce relatively inconspicuous echolocation calls compared to aerially foraging bats and could therefore be difficult predators to detect, even for insects with ultrasound sensitive ears. In the cricket Teleogryllus oceanicus, an auditory interneuron (AN2) responsive to ultrasound is known to elicit turning behaviour, but only when the cricket is in flight. Turning would not save a cricket from a gleaning bat so we tested the hypothesis that AN2 elicits more appropriate antipredator behaviours when crickets are on the ground. The echolocation calls of Nyctophilus geoffroyi, a sympatric gleaning bat, were broadcast to singing male and walking female T. oceanicus. Males did not cease singing and females did not pause walking more than usual in response to the bat calls up to intensities of 82 dB peSPL. Extracellular recordings from the cervical connective revealed that the echolocation calls elicited AN2 action potentials at high firing rates, indicating that the crickets could hear these stimuli. AN2 appears to elicit antipredator behaviour only in flight, and we discuss possible reasons for this context-dependent function.     

One hundred years of instability in ensiferan relationships

Although Ensifera is a major insect model group, its phylogenetic relationships have been understudied so far. Few phylogenetic hypotheses have been proposed, either with morphological or molecular data. The largest dataset ever used for phylogeny reconstruction on this group is molecular (16S rRNA, 18S rRNA and 28S rRNA sequences for 51 ensiferan species), which has been used twice with different resultant topologies. However, only one of these hypotheses has been adopted commonly as a reference classification. Here we re‐analyse this molecular dataset with different methods and parameters to test the robustness and the stability of the adopted phylogeny. Our study reveals the instability of phylogenetic relationships derived from this dataset, especially for the deepest nodes of the group, and suggests some guidelines for future studies. The comparison between the different classifications proposed in the past 70 years for Ensifera and our results allows the identification of potential monophyletic clades (katydids, mole crickets, scaly crickets + Malgasia, true crickets, leaf roller crickets, cave crickets) and the remaining unresolved clades (wetas, Jerusalem crickets and most of the highest rank clades) in Ensifera phylogeny.     

Sound localization in intact and one-eared crickets

Summary Intact and one-eared crickets,Gryllus bimaculatus, were tested for phonotactic behavior in a closed-loop and an open-loop situation and for related physiological characteristics of an identified auditory neuron pair, the left and the right AN2.Intact animals that performed phonotaxis in the closed-loop condition showed intended turning tendencies in the open-loop condition that correlated with the directional characteristics of their AN2s (Figs. 1–3).Animals in which one foreleg had been amputated during postembryonic development (one-eared regenerates) were classified according to their phonotactic performance as tracking or unoriented animals. The ability of one-eared regenerates to track a sound source was closely correlated with the direction of turning tendencies in the open-loop behavior and to specific features of their AN2 pair (Figs. 4–6).Some animals with one foreleg amputated as adults (one-eared amputees) perform stable phonotactic walking. Their open-loop behavior, however, is different from that of the tracking one- eared regenerates (Fig. 7).One-eared amputees showed stable phonotactic walking when calling song was presented from above and the sound intensity was varied according to the actual walking angle of the animal. The only orientational cue under this condition is the difference of sound intensity at different walking directions (Figs. 8–11).Different mechanisms are discussed for sound localization in one-eared regenerates and one- eared amputees based on turning tendencies which depend on the instantaneous stimulus intensity or on the intensity change between successive stimuli.     

Complex innervation of three neck muscles by motor and dorsal unpaired median neurons in crickets

Summary In the crickets, Gryllus campestris and Gryllus bimaculatus, the innervation of the dorso-ventral neck muscles M62, M57, and M59 was examined using cobalt staining via peripheral nerves and electrophysiological methods. M62 and M57 are each innervated by two motoneurons in the suboesophageal ganglion. The four motoneurons project into the median nerve to bifurcate into the transverse nerves of both sides. M62 and M57 are the only neck muscles innervated via this route. These bifurcating axon-projections are identical to those of the spiracular motoneurons in the prothoracic ganglion innervating the opener and closer muscle of the first thoracic spiracle in the cricket. The morphology of their branching pattern is described. The neck muscle M57 and the opener muscle of the first thoracic spiracle are additionally innervated by one mesothoracic motoneuron each, with similar morphology. These results suggest, that in crickets, the neck muscles M57 and M62 are homologous to spiracular muscles in the thoracic segments. The two neck muscles M62 and M59 (the posterior neighbour of M57) receive projections from a prothoracic dorsal unpaired median (DUM) neuron that also innervates dorsal-longitudinal neck muscles but not M57. In addition, one or two mesothoracic DUM neurons send axon collaterals intersegmentally to M59. This is the first demonstration of the innervation of neck muscles by DUM neurons.     

Neuroplasticity and phonotaxis in monaural adult female crickets (Gryllus bimaculatus de Geer)

Summary One foreleg was amputated at mid-femur in adultGryllus bimaculatus females. In phonotaxis tests these monaural crickets show course deviations and circling towards the intact side (Fig. 1). Mean course stability is best at 60 and 70 dB (Fig. 2). Here it differs significantly from a threshold value for orientated walking in females operated on the day of adult moult, but not in those operated two weeks later. The orientational performance improves with the interval between amputation and test (Fig. 3).Centripetal cobalt backfills reveal degeneration of tympanal nerve fibers on the amputated side (Fig. 4B, C). The mean number of intact afferents crossing the midline of the prothoracic ganglion is increased in monaural versus binaural crickets. Maximum transmidline extension is not correlated with the period of deafferentation (Fig. 5).Intracellular recording and staining of prothoracic auditory interneurons shows some axonal sprouts in ON1i (intact side) and ON2, but no significant physiological changes (Figs. 6A, D; 8A, C, E, G). Apart from axonal sprouts ON1a (amputated side) may show a few dendritic sprouts into the intact auditory neuropil (Figs. 6C, 7). Excitation in some ON1a-cells reveals functional contacts to intact auditory afferents (via crossing dendrites or possibly crossing afferents, Figs. 6e, 7, 8F). Morphological and associated physiological changes start early in AN2a (amputated side). The degree of crossing dendrites and contralateral excitation increases with postoperative age (Figs. 8H, 9).     

Coding of a sexually dimorphic song feature by auditory interneurons of grasshoppers: the role of leading inhibition

The shape of stimulus onset is a distinct feature of many acoustic communication signals. In some grasshopper species the steepness of amplitude rise of the pulses which comprise the song subunits is sexually dimorphic and a major criterion of sex recognition. Here, we describe potential mechanisms by which auditory interneurons could transmit the information on onset steepness from the metathoracic ganglion to the brain of the grasshopper. Since no single interneuron unequivocally encoded onset steepness, it appears that this information has to reside in the relative spike counts or the relative spike timing of a small group of ascending auditory interneurons. The decisive component of this mechanism seems to be the steepness-dependent leading inhibition displayed by two interneurons (AN3, AN4). The inhibition increased with increasing onset steepness, thus delayed the excitatory response, and in one interneuron even strongly reduced the spike count. Other ascending interneurons, whose responses were little affected by onset steepness, could serve as reference neurons (AN6, AN12). Thus, our results suggest that a comparison of both, spike count and first-spike timing within a small set of ascending interneurons could yield the information on signal onset steepness, that is on the sex of the sender.     

Mechanical response of the tympanal membranes of the tree cricket <Emphasis Type="Italic">Oecanthus henryi</Emphasis>

Crickets have two tympanal membranes on the tibiae of each foreleg. Among several field cricket species of the genus Gryllus (Gryllinae), the posterior tympanal membrane (PTM) is significantly larger than the anterior membrane (ATM). Laser Doppler vibrometric measurements have shown that the smaller ATM does not respond as much as the PTM to sound. Hence the PTM has been suggested to be the principal tympanal acoustic input to the auditory organ. In tree crickets (Oecanthinae), the ATM is slightly larger than the PTM. Both membranes are structurally complex, presenting a series of transverse folds on their surface, which are more pronounced on the ATM than on the PTM. The mechanical response of both membranes to acoustic stimulation was investigated using microscanning laser Doppler vibrometry. Only a small portion of the membrane surface deflects in response to sound. Both membranes exhibit similar frequency responses, and move out of phase with each other, producing compressions and rarefactions of the tracheal volume backing the tympanum. Therefore, unlike field crickets, tree crickets may have four instead of two functional tympanal membranes. This is interesting in the context of the outstanding question of the role of spiracular inputs in the auditory system of tree crickets.     

Ultrasound sensitive neurons in the cricket brain

1. The aim of this study was to identify neurons in the brain of the cricket, Teleogryllus oceanicus, that are tuned to high frequencies and to determine if these neurons are involved in the pathway controlling negative phonotaxis. In this paper we describe, both morphologically and physiologically, 20 neurons in the cricket brain which are preferentially tuned to high frequencies. 2. These neurons can be divided into two morphological classes: descending brain interneurons (DBINs) which have a posteriorly projecting axon in the circumesophageal connective and local brain neurons (LBNs) whose processes reside entirely within the brain. All the DBINs and LBNs have processes which project into one common area of the brain, the ventral brain region at the border of the protocerebrum and deutocerebrum. Some of the terminal arborizations of Int-1, an ascending ultrasound sensitive interneuron which initiates negative phonotaxis, also extend into this region. 3. Physiologically, ultrasonic sound pulses produce 3 types of responses in the DBINs and LBNs. (1) Seven DBINs and 6 LBNs are excited by ultrasound. (2) Ongoing activity in one DBIN and 5 LBNs is inhibited by ultrasound, and (3) one cell, (LBN-ei), is either excited or inhibited by ultrasound depending on the direction of the stimulus. 4. Many of the response properties of both the DBINs and LBNs to auditory stimuli are similar to those of Int-1. Specifically, the strength of the response, either excitation or inhibition, to 20 kHz sound pulses increases with increasing stimulus intensity, while the response latency generally decreases. Moreover, the thresholds to high frequencies are much lower than to low frequencies. These observations suggest that the DBINs and LBNs receive a majority of their auditory input from Int-1. However, the response latencies and directional sensitivity of only LBN-ei suggest that it is directly connected to Int-1. 5. The response of only one identified brain neuron, DBIN8, which is inhibited by 20 kHz sound pulses, is facilitated during flight compared to its response at rest. This suggests that suppression of activity in DBIN8 may be associated with ultrasound-induced negative phonotactic steering responses in flying crickets. The other DBINs and LBNs identified in this paper may also play a role in negative phonotaxis, and possibly in other cricket auditory behaviors influenced by ultrasonic frequencies.     

Mycoflora of cave cricket Troglophilus neglectus cadavers

By using different techniques and media, microfungi were isolated from cadavers of the cave cricket Troglophilus neglectus, collected on the walls of a Slovenian cave. The isolated mycobiota was consistently different for adult and larval stages of T. neglectus cadavers. Fungi isolated with the highest frequency from the larval stage were represented by five different species of the genus Mucor, the prevailing one being a newly described species, M. troglophilus. The dominating fungus isolated from the adult stage was the well-known fungal entomopathogen Beauveria bassiana. The other isolated fungi were mainly primary and secondary saprotrophic colonizers. This revised version was published online in June 2006 with corrections to the Cover Date.     

Behavioral detection of acoustic particle motion by a teleost fish (Astronotus ocellatus): sensitivity and directionality

The physiology and morphology of auditory interneurons of a fly, the parasitoid Therobia leonidei, are described for the first time. 1. The hearing threshold has been determined with summed recordings of the neck connective. Females are most sensitive in a frequency range from 16 to 40 kHz (thresholds: around 45 dB SPL). This broad hearing range matches with the peak frequencies of the song spectra of host bushcricket species. Male flies are 10–20 dB less sensitive than females. 2. The sensory cells of the prosternal tympanal organ of T. leonidei project into the thoracico-abdominal ganglion complex with arborizations in all three thoracic neuromeres. 3. Three types of ascending auditory interneurons were identified by their morphology and response properties. These have arborizations in all three thoracic neuromeres and terminate soma-contralaterally in the brain. At least three other neuron types were also identified according to response properties alone. The neurons show similar spectral tuning but different sensitivities.     

The function of auditory neurons in cricket phonotaxis

Summary The activity of auditory receptor cells and prothoracic auditory neurons of the cricket,Gryllus bimaculatus, was recorded intracellularly while the animal walked on a sphere or while passive movement was imposed on a foreleg.During walking the responses to simulated calling song is altered since (i) the auditory sensory cells and interneurons discharged impulses in the absence of sound stimuli (Figs. 1, 3) and (ii) the number of action potentials in response to sound is reduced in interneurons (Figs. 2, 3).These two effects occurred in different phases of the leg movement during walking and therefore masked, suppressed or did not affect the responses to auditory stimuli (Figs. 3, 4). Hence there is a time window within which the calling song can be detected during walking (Fig. 5).The extra excitation of receptors and interneurons is probably produced by vibration of the tympanum because (i) the excitation occurred at the same time as the leg placement (Fig. 4), (ii) during walking on only middle and hindlegs, no extra action potentials were observed (Fig. 6), (iii) in certain phases of passive movements receptor cells and interneurons were excited as long as the ipsilateral ear was not blocked (Figs. 8, 9).Suppression of auditory responses seems to be peripheral as well as central in origin because (i) it occurred at particular phases during active and passive leg movements in receptor cells and interneurons (Figs. 1, 4, 9), (ii) it disappeared if the ear was blocked during passive leg movements (Fig. 9) and (iii) it persisted if the animal walked only on the middle and hind legs (Fig. 6).     

The accessory organ,a scolopidial sensory organ,in the cave cricket Troglophilus neglectus (Orthoptera: Ensifera: Rhaphidophoridae)

Mechanoreceptor organs occur in great diversity in insect legs. This study investigates sensory organs in the leg of atympanate cave crickets (Troglophilus neglectus KRAUSS, 1879) by neuronal tracing. Previously, the subgenual and the intermediate organs were recognised in the subgenual organ complex, lacking the tympanal membranes present for example in the tibial hearing organs of Gryllidae and Tettigoniidae. We document the presence of the accessory organ in T. neglectus. This scolopidial organ is located in the posterior tibia close to the subgenual organ and can be identified by position, innervation and orientation of the dendrites of sensory neurons. The main motor nerve in the leg innervates a part of the subgenual organ and the accessory organ. The dendrites of sensory neurons in the accessory organ are characteristically bent in proximo‐dorsal direction, while the subgenual organ dendrites run distally along the longitudinal axis of the leg. The accessory organ contains 6–10 scolopidial sensilla, and no differences in neuroanatomy occur between the three thoracic leg pairs. Hence, the subgenual organ complex in cave crickets is more complex than previously known. The wider taxonomic distribution of the accessory scolopidial organ among orthopteroid insects is inconsistent, indicating its repeated losses or convergent evolution.