In invertebrates stereotypical behaviours may be controlled by command neurons. In the field cricket Gryllus bimaculatus a command neuron descending from the brain controls the generation of rhythmic wing movements underlying calling song. We analysed if a corresponding command neuron also exists in other cricket species. Our intracellular recording and stimulation experiments point to a putative command neuron for calling song in G. assimilis, although the structure of the neuron could not be revealed. When this neuron was depolarised to generate 50 AP/s the cricket raised its forewings into singing position, it started rhythmic wing movements and produced the typical species-specific calling song. Further enhancing the spike rate of the neuron increased the chirp repetition rate but not the pulse repetition rate. Blocking the spike activity of the neuron by hyperpolarizing current injection reduced the chirp repetition rate and could terminate singing activity. Our evidence indicates that the two species, G. assimilis and G. bimaculatus, have homologous neurons for controlling calling song, which may be a conserved phenotype across cricket species.

Two auditory neurons, TN-1 and ON-1, in the bush-cricket, Mecopoda elongata , have large dendritic arborisations which receive excitatory synaptic inputs from tonotopically organised axonal terminals of auditory afferents in the prothoracic ganglion. By combining intracellular microelectrode recording with calcium imaging we demonstrate that the dendrites of both neurons show a clear Ca 2+ signal in response to broad-frequency species-specific chirps. Due to the organisation of the afferents frequency specific auditory activation should lead to local Ca 2+ increases in their dendrites. In response to 20 ms sound pulses the dendrites of both neurons showed tonotopically organised Ca 2+ increases. In ON-1 we found no evidence for a tonotopic organisation of the Ca 2+ signal related to axonal spike activity or for a Ca 2+ response related to contralateral inhibition. The tonotopic organisation of the afferents may facilitate frequency-specific adaptation in these auditory neurons through localised Ca 2+ increases in their dendrites. By combining 10 and 40 kHz test pulses and adaptation series, we provide evidence for frequency-specific adaptation in TN-1 and ON-1. By reversible deactivating of the auditory afferents and removing contralateral inhibition, we show that in ON-1 spike activity and Ca 2+ responses increased but frequency-specific adaptation was not evident.

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.

In many cricket species, individuals fill sex-specific mating roles. For mate attraction in the field cricket Gryllus bimaculatus, males remain stationary and produce a species-specific calling song (senders) while females detect this signal and move towards it in a behaviour known as phonotaxis (receivers). Male G. bimaculatus have been observed responding to the calling song of conspecifics, and phonotaxis has anecdotally been reported for males in related species. Here, we aimed to quantify the tuning and intensity of male phonotaxis behaviour in G. bimaculatus by using a trackball system and established acoustic paradigms, and by comparing responses to typical female phonotaxis. The tuning to a syllable repetition interval (SRI) paradigm was similar between the sexes and both preferred sound patterns that correspond to the male calling song. Also, the responses to different chirp patterns were not different between the sexes; however, the male's absolute steering responses to attractive chirps were significantly weaker than in female phonotaxis. Our results highlight the presence of phonotactic behaviour in male G. bimaculatus, which may be relevant for keeping populations together or indicative of alternative mating strategies.(c) 2023 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.

In the bispotted field cricket auditory pulse pattern recognition of the species-specific calling song is based on a delay-line and coincidence detection network, established by the activity and synaptic connections of only 5 auditory neurons in the brain. To obtain a more detailed understanding of the network and the dynamic of the neural activity over time we analyzed the response properties of these neurons to test patterns, in which the pulse duration was kept constant while the duration of specific pulse intervals was systematically altered. We confirm that the ascending interneuron AN1 and the local interneuron LN2 copy the structure of the pulse pattern, however with limited resolution at short pulse intervals, further evident in downstream neural responses. In the non-spiking delay-line interneuron LN5 during long pulse intervals full-blown rebound potentials develop over a time course of 35-70 ms. LN5 also reveals an overall increase in its membrane potential tuned to chirps of the calling song pulse pattern. This may contribute to the pattern recognition process by driving the activity of the coincidence-detector LN3 and may indicate a further function of the delay-line neuron LN5. The activity of LN3 and of the feature detector LN4 match the tuning of the phonotactic behavior and demonstrate an increasingly sparse coding of the calling song pulse patterns as evident in the response of the feature detector LN4. The circuitry reveals a fundamental mechanism of auditory pattern recognition and demonstrates a principle of neuronal coding.

Behaviour is rooted in the organization and activity of an animal's nervous system. As male crickets use their front wings for sound production, the neural circuits underlying singing had been suggested to be housed in the thoracic ganglia. However, systematic lesion experiments of the CNS demonstrated that the abdominal nervous system is essential for their calling song behaviour. As male crickets also generate a courtship and rivalry song, we explored which parts of the abdominal central nervous system control the underlying motor patterns. A combination of systematic lesions to the abdominal nerve cord and video recording of courtship and rivalry behaviour revealed that most components of male courtship and rivalry behaviour were not affected by the lesions, except for the generation of courtship song, rivalry song, and the male's ability to copulate with the female. Any lesion to the abdominal nerve cord abolished copulations. Generation of courtship song initially failed when the connection to abdominal ganglion A6 was severed but in few males recovered after a week. For rivalry song production a central nerve cord extending to abdominal ganglion A4 was sufficient. These findings indicate that in the bispotted cricket the neural organization of courtship song is different from calling and rivalry song, while calling song and rivalry song might share a common network for generating the song patterns.

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.

We recorded the wing movements and sound signals during the production of calling, rivalry, and courtship song in the bispotted field cricket Gryllus bimaculatus. Recordings confirm that salient sound pulses during calling and rivalry song are generated during the closing movements of the wings. Wing movements for calling and rivalry song start from an elevated wing position and are performed with a very similar opening-closing movement, indicating that both types of songs may be generated by the same neuronal network. Wing movements for courtship song start from a low wing position; rapid closing movements generate high-frequency ticks and low-amplitude wing oscillations lead to low-amplitude pulses, generated during the opening and closing movements with a carrier frequency corresponding to the calling song. The two types of wing movements underlying courtship song indicate a different motor control as compared to calling song and may represent an early evolutionary phenotype.

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.

ABSTRACT For crickets, which approach singing males by phonotaxis, the female choosiness hypothesis postulates that young females should be more selective of male calling song patterns than older individuals. However, there is no information about the behavioural preferences of females over their complete adulthood. We analysed phonotaxis in female Gryllus bimaculatus throughout their entire adult lifetime and measured the impact of sound amplitude, carrier frequency and the temporal pattern of test songs on their auditory response. Females of all ages demonstrated their best responses to male calling songs with a pulse period of 34–42 ms, a carrier frequency of 4.5 kHz and a sound pressure level of 75 dB. The response profile to somewhat less optimal song types did vary with age, but not in a manner consistent with a simple loosening of selectiveness in older females. Age, however, had an effect on the overall strength of phonotaxis, as very old females showed an overall diminishing response to all song types. Our data suggest that although there are minor changes in the relative preferences of crickets to individual song elements as they age, the breadth of song patterns to which they will perform phonotaxis remains similar across age groups.

Although crickets move their front wings for sound production, the abdominal ganglia house the network of the singing central pattern generator. We compared the effects of specific lesions to the connectives of the abdominal ganglion chain on calling song activity in four different species of crickets, generating very different pulse patterns in their calling songs. In all species, singing activity was abolished after the connectives between the metathoracic ganglion complex and the first abdominal ganglion A3 were severed. The song structure was lost and males generated only single sound pulses when connectives between A3 and A4 were cut. Severing connectives between A4 and A5 had no effect in the trilling species, it led to an extension of chirps in a chirping species and to a loss of the phrase structure in two Teleogryllus species. Cutting the connectives between A5 and A6 caused no or minor changes in singing activity. In spite of the species-specific pulse patterns of calling songs, our data indicate a conserved organisation of the calling song motor pattern generating network. The generation of pulses is controlled by ganglia A3 and A4 while A4 and A5 provide the timing information for the chirp and/or phrase structure of the song.

When the amplitude modulation of species-specific acoustic signals is distorted in the transmission channel, signals become difficult to recognize by the receiver. Tolerant auditory pattern recognition systems, which after having perceived the correct species-specific signal transiently broaden their acceptance of signals, would be advantageous for animals as an adaptation to the constraints of the environment. Using a well-studied cricket species, Gryllus bimaculatus, we analysed tolerance in auditory steering responses to 'Odd' chirps, mimicking a signal distorted by the transmission channel, and control 'Silent' chirps by employing a fine-scale open-loop trackball system. Odd chirps on their own did not elicit a phonotactic response. However, when inserted into a calling song pattern with attractive Normal chirps, the females' phonotactic response toward these patterns was significantly larger than to patterns with Silent chirps. Moreover, females actively steered toward Odd chirps when these were presented within a sequence of attractive chirps. Our results suggest that crickets employ a tolerant pattern recognition system that, once activated, transiently allows responses to distorted sound patterns, as long as sufficient natural chirps are present. As pattern recognition modulates how crickets process non-attractive acoustic signals, the finding is also relevant for the interpretation of two-choice behavioural experiments.

Directional hearing is crucial for animals depending on acoustic signals to locate a mate. We focused on crickets to explore the reliability of directional information forwarded to the brain by the ascending auditory interneuron AN1, which is crucial for phonotactic behavior. We presented calling song from −45° to +45° in steps of 3° and compared the phonotactic steering of females walking on a trackball with the directional responses of AN1. Forty percent of females showed good steering behavior and changed their walking direction when the speaker passed the body’s longitudinal axis. The bilateral latency difference between right and left AN1 responses was small and may not be reliable for auditory steering. In respect to spike count, all AN1 recordings presented significant bilateral differences for angles larger than ±18°, yet 35% showed a mean significant difference of 1–3 action potentials per chirp when the frontal stimulus deviated by 3° from their length axis. For small angles, some females had a very similar AN1 activity forwarded to the brain, but the accuracy of their steering behavior was substantially different. Our results indicate a correlation between directional steering and the response strength of AN1, especially for large angles. The reliable steering of animals at small angles would have to be based on small bilateral differences of AN1 activity, if AN1 is the only source providing directional information. We discuss whether such bilateral response difference at small angles can provide a reliable measure to generate auditory steering commands descending from the brain, as pattern recognition is intensity independent.NEW & NOTEWORTHY The ascending auditory interneuron AN1 has been implicated in cricket auditory steering, but at small acoustic stimulation angles, it does not provide reliable directional information. We conclude that either the small bilateral auditory activity differences of the AN1 neurons are enhanced to generate reliable descending steering commands or, more likely, directional auditory steering is mediated via a thoracic pathway, as indicated by the reactive steering hypothesis.

Chirping male crickets combine a 30 Hz pulse pattern with a 3 Hz chirp pattern to drive the rhythmic opening-closing movements of the front wings for sound production. Lesion experiments suggest two coupled modular timer-networks located along the chain of abdominal ganglia, a network in A3 and A4 generating the pulse pattern, and a network organized along with ganglia A4-A6 controlling the generation of the chirp rhythm. We analyzed neurons of the timer-networks and their synaptic connections by intracellular recordings and staining. We identified neurons spiking in phase with the chirps and pulses, or that are inhibited during the chirps. Neurons share a similar "gestalt", regarding the position of the cell body, the dendritic arborizations and the contralateral ascending axon. Activating neurons of the pulse-timer network elicits ongoing motor activity driving the generation of pulses; this activity is not structured in the chirp pattern. Activating neurons of the chirp-timer network excites pulse-timer neurons; it drives the generation of chirps and during the chirps the pulse pattern is produced. Our results support the hypothesis that two modular networks along the abdominal ganglion chain control the cricket calling song, a pattern generating network in the mesothoracic ganglion may not be required.

The evolution of species-specific song patterns is a driving force in the speciation of acoustic communicating insects. It must be closely linked to adaptations of the neuronal network controlling the underlying singing motor activity. What are the cellular and network properties that allow generating different songs? In five cricket species, we analyzed the structure and activity of the identified abdominal ascending opener interneuron, a homologous key component of the singing central pattern generator. The structure of the interneuron, based on the position of the cell body, ascending axon, dendritic arborization pattern, and dye coupling, is highly similar across species. The neuron's spike activity shows a tight coupling to the singing motor activity. In all species, current injection into the interneuron drives artificial song patterns, highlighting the key functional role of this neuron. However, the pattern of the membrane depolarization during singing, the fine dendritic and axonal ramifications, and the number of dye-coupled neurons indicate species-specific adaptations of the neuronal network that might be closely linked to the evolution of species-specific singing. SIGNIFICANCE STATEMENT A fundamental question in evolutionary neuroscience is how species-specific behaviors arise in closely related species. We demonstrate behavioral, neurophysiological, and morphological evidence for homology of one key identified interneuron of the singing central pattern generator in five cricket species. Across-species differences of this interneuron are also observed, which might be important to the generation of the species-specific song patterns. This work offers a comprehensive and detailed comparative analysis addressing the neuronal basis of species-specific behavior.

We investigated the central nervous coordination between singing motor activity and abdominal ventilatory pumping in crickets. Fictive singing, with sensory feedback removed, was elicited by eserine-microinjection into the brain, and the motor activity underlying singing and abdominal ventilation was recorded with extracellular electrodes. During singing, expiratory abdominal muscle activity is tightly phase coupled to the chirping pattern. Occasional temporary desynchronization of the two motor patterns indicate discrete central pattern generator (CPG) networks that can operate independently. Intracellular recordings revealed a sub-threshold depolarization in phase with the ventilatory cycle in a singing-CPG interneuron, and in a ventilation-CPG interneuron an excitatory input in phase with each syllable of the chirps. Inhibitory synaptic inputs coupled to the syllables of the singing motor pattern were present in another ventilatory interneuron, which is not part of the ventilation-CPG. Our recordings suggest that the two centrally generated motor patterns are coordinated by reciprocal feedforward discharges from the singing-CPG to the ventilation-CPG and vice versa. Consequently, expiratory contraction of the abdomen usually occurs in phase with the chirps and ventilation accelerates during singing due to entrainment by the faster chirp cycle.

We studied bilateral processing in the auditory ON neurons of crickets using reversible cold-deactivation of the hearing organs by means of Peltier elements. Intracellular recordings of the neurons' activity in response to acoustic stimuli were obtained, while either the ipsilateral or the contralateral hearing organ was cold-deactivated. Afferent activity was abolished at a temperature of approximately 10°C. In ON1, contralateral inhibition had no effect on the latency and amplitude of the phasic onset activity, it enhanced the decline of the onset activity and it decreased the subsequent tonic spiking response to acoustic stimuli. As a consequence, the phasic onset activity became more salient and reciprocal inhibition may support the detection of sound pulses. Contralateral inhibition had a significant impact on the tonic ON1 response, in line with its presumed function to enhance the bilateral auditory contrast. In ON2, experiments confirmed a bilateral excitatory input, with the ipsilateral input dominating the response, and no inhibitory coupling between the ON2 neurons.

The evolutionary loss of sexual traits is widely predicted. Because sexual signals can arise from the coupling of specialized motor activity with morphological structures, disruption to a single component could lead to overall loss of function. Opportunities to observe this process and characterize any remaining signal components are rare, but could provide insight into the mechanisms, indirect costs and evolutionary consequences of signal loss. We investigated the recent evolutionary loss of a long-range acoustic sexual signal in the Hawaiian field cricket Teleogryllus oceanicus. Flatwing males carry mutations that remove sound-producing wing structures, eliminating all acoustic signalling and affording protection against an acoustically-orientating parasitoid fly. We show that flatwing males produce wing movement patterns indistinguishable from those that generate sonorous calling song in normal-wing males. Evolutionary song loss caused by the disappearance of structural components of the sound-producing apparatus has left behind the energetically costly motor behaviour underlying normal singing. These results provide a rare example of a vestigial behaviour and raise the possibility that such traits could be co-opted for novel functions.