Many insects, including bushcrickets, use acoustic communication for mate finding and mate choice. These signals often produce simultaneous substrate vibrations, potentially aiding localization. However, independently produced vibratory signals are rarely documented in Orthoptera, despite their potential to enhance close-range communication. In the duetting bushcricket Ancistrura nigrovittata, acoustic communication has been extensively studied. We investigated whether any previously overlooked vibratory signals occur during close-range interactions in this species, and characterised their properties and interplay with acoustic signals. Combining laser vibrometry, audio and video recordings, we revealed soundless abdominal tremulation signals in both sexes during partner search and pair formation. Although subtle, these movements produce substrate vibration with amplitudes exceeding those induced by acoustic signalling (stridulation) by an order of magnitude. In both sexes, tremulation and acoustic signals can occur simultaneously or separately, demonstrating independent production mechanisms. Females combine song with tremulation during long-distance calling, use primarily tremulation after a male enters a plant, and typically cease signalling upon being found. Males add tremulation to the song after entering a plant and use it as the primary signal after partner contact. In both sexes, tremulation comprises mostly grouped pulses, emitted at a rate of about 2.5 Hz. Male tremulation exhibits a lower pulse number per train, higher dominant frequencies and amplitudes than female tremulation, and is typically accompanied by a short acoustic tick at the beginning of each pulse. Our findings reveal a previously unknown complexity of vibroacoustic communication in A. nigrovittata.

A new method for individually staining insect neurons with metal ions was described in the late 60s, closely followed by the introduction of the first bright fluorescent dye, Lucifer Yellow, for the same purpose. These milestones enabled an unprecedented level of detail regarding the neuronal basis of sensory processes such as hearing. Due to their conspicuous auditory behavior, orthopterans rapidly established themselves as a popular model for studies on hearing (first identified auditory neuron: 1974; first local auditory interneuron: 1977). Although crickets (Ensifera, Gryllidae) surpassed grasshoppers (Caelifera) as the main model taxon, surprisingly few neuronal elements have been described in crickets. More auditory neurons are described for bush crickets (Ensifera, Tettigoniidae), but due to their great biodiversity, the described auditory neurons in bush crickets are scattered over distantly related groups, hence being confounded by potential differences in the neuronal pathways themselves. Our review will outline all local auditory elements described in ensiferans so far. We will focus on one bush cricket species, Ancistrura nigrovittata (Phaneropterinae), which has the so-far highest diversity of identified auditory interneurons within Ensifera. We will present one novel and three previously described local prothoracic auditory neuron classes, comparing their morphology and aspects of sensory processing. Finally, we will hypothesize about their functions and evolutionary connections between ensiferan insects.

Detection of sound and substrate vibration is crucial for the survival and reproduction of many animals, particularly insects. Bushcrickets (Orthoptera, Tettigoniidae), developed a large mechanosensory organ complex in their legs to detect such stimuli. As demonstrated by various studies in the past, sensilla in distinct functional groups form specialized vibratory organs (the subgenual organ and the accessory organ), respond sensitively to both vibration and sound (in the intermediate organ [IO]), or mediate hearing (in the crista acustica [CA]; the tympanal hearing organ). In their recent publication, Zhantiev and Korsunovskaya addressed auditory and vibratory sensitivity in the IO and the CA in two species of bushcrickets, using single-cell recording and staining of sensory neurons from their soma in an isolated foreleg. Their main finding was that not only the IO but also the complete CA contains bimodal sensilla responding with high sensitivity to both sound and vibration, which would be a true change in the paradigm of how the auditory/vibratory sense in Orthoptera works. In addition, they revealed vibratory tuning of the IO sensilla, which differs largely from that in previous studies. We propose three major experimental causes of such discrepancies: calibration, experiments with isolated legs, and differences in the sites of recording. To judge the causes of these discrepancies more adequately, a detailed comparison of methods and a number of control experiments are needed. This will deepen our understanding of sensory adaptations and specialization of insect mechanosensory organs to stimuli entering the system by different input pathways.

Thoracic ganglia of many hearing insects house the first level of auditory processing. In bush-crickets, the largest population of local auditory neurons in the prothoracic processing centre are dorsal unpaired median (DUM) neurons. It has been suggested that DUM neurons are inhibitory using γ-aminobutyric acid (GABA) as transmitter. Immunohistochemistry reveals a population of about 35–50 GABA-positive somata in the posterior medial cluster of the prothoracic ganglion. Only very few small somata in this cluster remain unstained. At least 10 neurites from 10 neurons can be identified. Intracellularly stained auditory DUM neurons have their soma in the cluster of median GABA positive cells and most of them exhibit GABA-immunoreactivity. Responses of certain DUM neurons show obvious signs of inhibition. Application of picrotoxin (PTX), a chloride-channel blocker in insects, changes the responses of many DUM neurons. They become broader in frequency tuning and broader or narrower in temporal pattern tuning. Furthermore, inhibitory postsynaptic potentials (IPSPs) may be replaced by excitatory postsynaptic potentials. Loss of an IPSP in the rising graded potential after PTX-application leads to a significant reduction of first-spike latency. Therefore, auditory DUM neurons receive effective inhibition and are the best candidates for inhibition in DUM neurons and other auditory interneurons.

Insects with ears process sounds and respond to conspecific signals or predator cues. Axons of auditory sensory cells terminate in mechanosensory neuropils from which auditory interneurons project into (brain-) areas to prepare response behaviors. In the prothoracic ganglion of a bush-cricket, a cluster of local DUM (dorsal unpaired median) neurons has recently been described and constitutes a filter bank for carrier frequency. Here, we demonstrate that these neurons also constitute a filter bank for temporal patterns. The majority of DUM neurons showed pronounced phasic-tonic responses. The transitions from phasic to tonic activation had different time constants in different DUM neurons. Time constants of the membrane potential were shorter in most DUM neurons than in auditory sensory neurons. Patterned stimuli with known behavioral relevance evoked a broad range of responses in DUM neurons: low-pass, band-pass, and high-pass characteristics were encountered. Temporal and carrier frequency processing were not correlated. Those DUM neurons producing action potentials showed divergent processing of temporal patterns when the graded potential or the spiking was analyzed separately. The extent of membrane potential fluctuations mimicking the patterned stimuli was different between otherwise similarly responding neurons. Different kinds of inhibition were apparent and their relevance for temporal processing is discussed.

In bush-crickets the first stage of central auditory processing occurs in the prothoracic ganglion. About 15 to 50 different auditory dorsal unpaired median neurons (DUM neurons) exist but they have not been studied in any detail. These DUM neurons may be classified into seven different morphological types, although, there is only limited correlation between morphology and physiological responses. Ninety seven percent of the stained neurons were local, 3% were intersegmental. About 90% project nearly exclusively into the auditory neuropile, and 45% into restricted areas therein. Lateral extensions overlap with the axons of primary auditory sensory neurons close to their branching point. DUM neurons are typically tuned to frequencies covering the range between 2 and 50 kHz and thereby may establish a filter bank for carrier frequency. Less than 10% of DUM neurons have their branches in adjacent and more posterior regions of the auditory neuropile and are mostly tuned to low frequencies, less sensitive than the other types and respond to vibration. Thirty five percent of DUM show indications of inhibition, either through reduced responses at higher intensities, or by hyperpolarizing responses to sound. Most DUM neurons produce phasic spike responses preferably at higher intensities. Spikes may be elicited by intracellular current injection. Preliminary data suggest that auditory DUM neurons have GABA as transmitter and therefore may inhibit other auditory interneurons. From all known local auditory neurons, only DUM neurons have frequency specific responses which appear suited for local processing relevant for acoustic communication in bush crickets.

We investigated brain regions - mostly neuropils - that process auditory information relevant for the initiation of response songs of female grasshoppers Chorthippus biguttulus during bidirectional intraspecific acoustic communication. Male-female acoustic duets in the species Ch. biguttulus require the perception of sounds, their recognition as a species- and gender-specific signal and the initiation of commands that activate thoracic pattern generating circuits to drive the sound-producing stridulatory movements of the hind legs. To study sensory-to-motor processing during acoustic communication we used multielectrodes that allowed simultaneous recordings of acoustically stimulated electrical activity from several ascending auditory interneurons or local brain neurons and subsequent electrical stimulation of the recording site. Auditory activity was detected in the lateral protocerebrum (where most of the described ascending auditory interneurons terminate), in the superior medial protocerebrum and in the central complex, that has previously been implicated in the control of sound production. Neural responses to behaviorally attractive sound stimuli showed no or only poor correlation with behavioral responses. Current injections into the lateral protocerebrum, the central complex and the deuto-/tritocerebrum (close to the cerebro-cervical fascicles), but not into the superior medial protocerebrum, elicited species-typical stridulation with high success rate. Latencies and numbers of phrases produced by electrical stimulation were different between these brain regions. Our results indicate three brain regions (likely neuropils) where auditory activity can be detected with two of these regions being potentially involved in song initiation.

The sense of hearing contributes importantly to an animal's fitness. It allows detection of predators and prey and communication with conspecifics even in the dark and over large distances. Hearing organs evolved in about 20 groups of insects. Hearing is used by moths and other insects for avoiding predatory bats; by cicada, crickets/bushcrickets, moths, and grasshoppers for intraspecific communication; and by parasitic flies to locate singing hosts. Despite the variety of these insect groups, the neural processing of sound signals faces very similar fundamental challenges related to signal detection, directional processing, frequency discrimination, pattern recognition, and coping with self-generated noise. Solutions to these problems are implemented by specific network, cellular, and synaptic properties of neural circuits. Owing to their rather simple organization, insect auditory pathways can be explored and analyzed at the level of identified neurons to reveal fundamental mechanisms of auditory processing.

Currently, more and more laboratories are acquiring the capability of simultaneously detecting the extracellular activity of neuronal populations in anaesthetized and awake animals by multielectrode recordings. In insects, multielectrode recordings are challenging due to the small size of the nervous system. Nevertheless, multielectrode recordings have been successfully established in brains of cockroaches, honeybees, fruit flies and grasshoppers to study sensory processing related to mechanosensation, olfaction, vision and audition. The number of neurons which can be recorded using such multielectrode did not exceed 5 and likely depends on factors like recorded compartment of the neuron, impedance of the multielectrode, number of wires included in the multielectrode and threshold for spike detection. Signal-to-noise ratio (SNR) of the recordings obviously depends on the material and method used for production of multielectrodes. To mark the location of the recording, different methods like current-driven copper deposition, labelling with fluorescent dye and electrocoagulation of nervous tissue are used. As expected, multielectrode recordings are more difficult in freely moving compared to restricted insects due to movement artifacts and requirement for fixed placement of the multielectrode at a particular recording site in the CNS. Specific differences among different preparations and sensory systems like disentangling spike collisions in auditory stimulation increase in SNR after some time in olfactory systems and photoelectrical effect from compound eye in visual stimulation may require special attention and particular adaptations.

Several taxa of insects evolved a tympanate ear at different body positions, whereby the ear is composed of common parts: a scolopidial sense organ, a tracheal air space, and a tympanal membrane. Here, we analyzed the anatomy and physiology of the ear at the ventral prothorax of the sarcophagid fly, Emblemasoma auditrix (Soper). We used micro-computed tomography to analyze the ear and its tracheal air space in relation to the body morphology. Both tympana are separated by a small cuticular bridge, face in the same frontal direction, and are backed by a single tracheal enlargement. This enlargement is connected to the anterior spiracles at the dorsofrontal thorax and is continuous with the tracheal network in the thorax and in the abdomen. Analyses of responses of auditory afferents and interneurons show that the ear is broadly tuned, with a sensitivity peak at 5 kHz. Single-cell recordings of auditory interneurons indicate a frequency-and intensity-dependent tuning, whereby some neurons react best to 9 kHz, the peak frequency of the host's calling song. The results are compared to the convergently evolved ear in Tachinidae (Diptera).

Abstract Members of the family Phaneropteridae are well known for their acoustical duetting behaviour, used for locating and meeting a mate. In Poecilimon affinis, typically the male approaches a responding female phonotactically. A set of behavioural experiments, bioacoustic and neurophysiological measurements (some with a relatively low sample size, but not repeatable under the same circumstances) indicates the following system: the male song (92 dB SPLpeak at a distance of 1 m) is about 10 dB louder than the female song. The females respond to male signals only if these are ∼15–20 dB above their hearing threshold. The males start a phonotactic approach towards a stationary, responding female only if she is no more than ∼12 m away. Females, on the other hand, may respond to singing males up to a distance of 28m, and to more distant males with softer signals than to closer ones. A possible function of these weak signals, inaudible for the duetting male, may be to attract eavesdropping males. The communication system will work at densities as low as 0.003 females or 0.0005 males per m2.

Insect ears evolved many times independently. As a consequence, a striking diversity exists in the location, construction and behavioural implementation of ears. In this review, we first summarise what is known about the evolutionary origin of ears and the presumed precursor organs in the various insect groups. Thereafter, we focus on selective forces for making and keeping an ear: we discuss detecting and localising predators and conspecifics, including establishing new "private" channels for intraspecific communication. More advanced aspects involve judging the distance of conspecifics, or assessing individual quality from songs which makes auditory processing a means for exerting sexual selection on mating partners. We try to identify negative selective forces, mainly in the context of energy expenditure for developing and keeping an ear, but also in conjunction with acoustic communication, which incorporates risks like eavesdropping by predators and parasitoids. We then discuss balancing pressures, which might oppose optimising an ear for a specific task (when it serves different functions, for example). Subsequently, we describe various scenarios that might have led to a reduction or complete loss of ears in evolution. Finally, we describe cases of sex differences in ears and potential reasons for their appearance.

Members of the family Phaneropteridae are well known for their acoustical duetting behaviour, used for locating and meeting a mate. In Poecilimon affinis, typically the male approaches a responding female phonotactically. A set of behavioural experiments, bioacoustic and neurophysiological measurements (some with a relatively low sample size, but not repeatable under the same circumstances) indicates the following system: the male song (92 dB SPLpeak at a distance of 1 m) is about 10 dB louder than the female song. The females respond to male signals only if these are ∼15–20 dB above their hearing threshold. The males start a phonotactic approach towards a stationary, responding female only if she is no more than ∼12 m away. Females, on the other hand, may respond to singing males up to a distance of 28m, and to more distant males with softer signals than to closer ones. A possible function of these weak signals, inaudible for the duetting male, may be to attract eavesdropping males. The communication system will work at densities as low as 0.003 females or 0.0005 males per m2.

Background: Grasshoppers have been used as a model system to study the neuronal basis of insect acoustic behavior. Auditory neurons have been described from intracellular recordings. The growing interest to study population activity of neurons has been satisfied so far with artificially combining data from different individuals.New method: We for the first time used multielectrode recordings from a small grasshopper brain. We used three 12 pm tungsten wires (combined in a multielectrode) to record from local brain neurons and from a population of auditory neurons entering the brain from the thorax. Spikes of the recorded units were separated by sorting algorithms and spike collision analysis.Results: The tungsten wires enabled stable recordings with high signal to noise ratio. Due to the tight temporal coupling of auditory activity to the stimulus spike collisions were frequent and collision analysis retrieved 10-15% of additional spikes. Marking the electrode position was possible using a fluorescent dye or electrocoagulation with high current. Physiological identification of units described from intracellular recordings was hard to achieve.Comparison with existing methods: 12 pm tungsten wires gave a better signal to noise ratio than 15 pm copper wires previously used in recordings from bees' brains. Recording the population activity of auditory neurons in one individual prevents interindividual and trial-to-trial variability which otherwise reduce the validity of the analysis. Double intracellular recordings have quite low success rate and therefore are rarely achieved and their stability is much lower than that of multielectrode recordings which allows sampling of data for 30 min or more. (C) 2015 Elsevier B.V. All rights reserved.

Intracellular recordings may give valuable information about processing of a neuron and possibly its input from the network. Impalement with an electrode causes injury to the cell and depolarization from intrusion of extracellular fluid. Thus, penetration artefacts may contaminate recordings and conceal or even alter relevant information. These penetration artefacts may have the strongest impact close to the spike-generating zone near the dendrites. Recordings in axonal portions might therefore be less vulnerable while providing insufficient information about the synaptic input. In this study, we present data of five previously identified intersegmental auditory neurons of a bushcricket independently recorded in their dendrites (prothorax) and axon (brain). Generally, responses to acoustic pulses of the same parameter combination were similar within a neuronal class at the two recording sites. However, all neuronal classes showed significantly higher response variability and a tendency for higher spike activity when recorded in the dendrites. Unexpectedly, the combined activity of two neurons (Ascending Neurons 1 and 2) recorded in the brain provides a better fit to song recognition than when recorded in the thorax. Axonal recordings of T-shaped Neuron 1 revealed graded potentials originating in the brain and modulating its output in a potentially behaviourally relevant manner.

Sensory modalities typically are important for both sexes, although sex-specific functional adaptations may occur frequently. This is true for hearing as well. Consequently, distinct behavioural functions were identified for the different insect hearing systems. Here we describe a first case, where a trait of an evolutionary novelty and a highly specialized hearing organ is adaptive in only one sex. The main function of hearing of the parasitoid fly Emblemasoma auditrix is to locate the host, males of the cicada species Okanagana rimosa, by their calling song. This task is performed by female flies, which deposit larvae into the host. We show that male E. auditrix possess a hearing sense as well. The morphology of the tympanal organ of male E. auditrix is rather similar to the female ear, which is 8% broader than the male ear. In both sexes the physiological hearing threshold is tuned to 5 kHz. Behavioural tests show that males are able to orient towards the host calling song, although phonotaxis often is incomplete. However, despite extensive observations in the field and substantial knowledge of the biology of E. auditrix, no potentially adaptive function of the male auditory sense has been identified. This unique hearing system might represent an intralocus sexual conflict, as the complex sense organ and the behavioural relevant neuronal network is adaptive for only one sex. The correlated evolution of the sense organ in both sexes might impose substantial constraints on the sensory properties of the ear. Similar constraints, although hidden, might also apply to other sensory systems in which behavioural functions differ between sexes.

Syllable period (SP) selective calling song processing has been demonstrated for the prothoracic, AN2 auditory neurone that correlates very well with SP-selective phonotaxis by female cricket Gryllus bimaculatus De Geer. Both SP-selective processing by the AN2 and the phonotactic behaviour of the female exhibit substantial plasticity. Thus, the question remains as to whether the selective responses of the AN2 neurone and the selective behaviour of the female match in an individual female. The present study is designed to answer that question. The SP-selective phonotactic behaviour of individual females is evaluated, followed immediately by measuring the SP-selective responses of the same female's AN2 neurone. Very significant correlations are found between the selective responses of the AN2 neurone and the same female's selective behaviour. In 208 possible comparisons (26 females, eight behavioural and neuronal tests each), 186 resulted in matches between behaviour and neuronal processing. Dividing the SP-selective females into two groups (one group that responded phonotactically to the shortest SP tested and a second group that did not respond to this SP) resulted in significantly more selective responses to this shortest SP by the AN2 neurone in the females that responded phonotactically to the SP than for the females who did not respond to the shortest SP. The behavioural responses by these two groups to the other SPs tested are shown to be essentially identical.

Guadeloupe, the largest of the Leeward Islands, harbors three species of Pseudophyllinae (Orthoptera: Tettigoniidae) belonging to distinct tribes. This study examined the basic aspects of sound production and acousto-vibratory behavior of these species. As the songs of many Pseudophyllinae are complex and peak at high frequencies, they require high quality recordings. Wild specimens were therefore recorded ex situ. Collected specimens were used in structure-function experiments. Karukerana aguilari Bonfils (Pterophyllini) is a large species with a mirror in each tegmen and conspicuous folds over the mirror. It sings 4-6 syllables, each comprising 10-20 pulses, with several peaks in the frequency spectrum between 4 and 20 kHz. The song is among the loudest in Orthoptera (> 125 dB SPL in 10 cm distance). The folds are protective and have no function in song production. Both mirrors may work independently in sound radiation. Nesonotus reticulatus (Fabricius) (Cocconotini) produces verses from two syllables at irregular intervals. The song peaks around 20 kHz. While singing, the males often produce a tremulation signal with the abdomen at about 8-10 Hz. To our knowledge, it is the first record of simultaneous calling song and tremulation in Orthoptera. Other males reply to the tremulation with their own tremulation. Xerophyllopteryx fumosa (Brunner von Wattenwyl) (Pleminiini) is a large, bark-like species, producing a syllable of around 20 pulses. The syllables are produced with irregular rhythms (often two with shorter intervals). The song peaks around 2-3 kHz and 10 kHz. The hind wings are relatively thick and are held between the half opened tegmina during singing. Removal of the hind wings reduces song intensity by about 5 dB, especially of the low frequency component, suggesting that the hind wings have a role in amplifying the song.

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.