Gekko gecko as a model organism for understanding aspects of laryngeal vocal evolution.

The ability to communicate through vocalization plays a key role in the survival of animals across all vertebrate groups. While avian reptiles have received much attention relating to their stunning sound repertoire, non-avian reptiles have been wrongfully assumed to have less elaborate vocalization types and little is known about the biomechanics of sound production and their underlying neural pathways. We investigated alarm calls of Gekko gecko using audio and cineradiographic recordings of their alarm calls. Acoustic analysis revealed three distinct call types: a sinusoidal call type (type 1), a train-like call type, characterized by distinct pulse trains (type 3), and an intermediary type, which showed both sinusoidal and pulse train components (type 2). Kinematic analysis of cineradiographic recordings showed that laryngeal movements differ significantly between respiratory and vocal behavior: during respiration, animals repeatedly moved their jaws to partially open their mouths, which was accompanied by small glottal movements. During vocalization, the glottis was pulled back, contrasting with what has previously been reported. In-vitro retrograde tracing of the nerve innervating the laryngeal constrictor and dilator muscles revealed round to fusiform motoneurons in the hindbrain-spinal cord transition ipsilateral to the labeled nerve. Taken together, our observations provide insight into the alarm calls generated by G. gecko, the biomechanics of this sound generation and the underlying organization of motoneurons involved in the generation of vocalizations. Our observations suggest that G. gecko may be an excellent non-avian reptile model organism for enhancing our understanding of the evolution of vertebrate vocalization.

Micro-CT and deep learning: Modern techniques and applications in insect morphology and neuroscience.

Advances in modern imaging and computer technologies have led to a steady rise in the use of micro-computed tomography (µCT) in many biological areas. In zoological research, this fast and non-destructive method for producing high-resolution, two- and three-dimensional images is increasingly being used for the functional analysis of the external and internal anatomy of animals. µCT is hereby no longer limited to the analysis of specific biological tissues in a medical or preclinical context but can be combined with a variety of contrast agents to study form and function of all kinds of tissues and species, from mammals and reptiles to fish and microscopic invertebrates. Concurrently, advances in the field of artificial intelligence, especially in deep learning, have revolutionised computer vision and facilitated the automatic, fast and ever more accurate analysis of two- and three-dimensional image datasets. Here, I want to give a brief overview of both micro-computed tomography and deep learning and present their recent applications, especially within the field of insect science. Furthermore, the combination of both approaches to investigate neural tissues and the resulting potential for the analysis of insect sensory systems, from receptor structures via neuronal pathways to the brain, are discussed.

Reviving the sound of a 150-year-old insect: The bioacoustics of Prophalangopsis obscura (Ensifera: Hagloidea).

Determining the acoustic ecology of extinct or rare species is challenging due to the inability to record their acoustic signals or hearing thresholds. Katydids and their relatives (Orthoptera: Ensifera) offer a model for inferring acoustic ecology of extinct and rare species, due to allometric parameters of their sound production organs. Here, the bioacoustics of the orthopteran Prophalangopsis obscura are investigated. This species is one of only eight remaining members of an ancient family with over 90 extinct species that dominated the acoustic landscape of the Jurassic. The species is known from only a single confirmed specimen-the 150-year-old holotype material housed at the London Natural History Museum. Using Laser-Doppler Vibrometry, 3D surface scanning microscopy, and known scaling relationships, it is shown that P. obscura produces a pure-tone song at a frequency of ~4.7 kHz. This frequency range is distinct but comparable to the calls of Jurassic relatives, suggesting a limitation of early acoustic signals in insects to sonic frequencies (<20 kHz). The acoustic ecology and importance of this species in understanding ensiferan evolution, is discussed.

A narrow ear canal reduces sound velocity to create additional acoustic inputs in a microscale insect ear.

Located in the forelegs, katydid ears are unique among arthropods in having outer, middle, and inner components, analogous to the mammalian ear. Unlike mammals, sound is received externally via two tympanic membranes in each ear and internally via a narrow ear canal (EC) derived from the respiratory tracheal system. Inside the EC, sound travels slower than in free air, causing temporal and pressure differences between external and internal inputs. The delay was suspected to arise as a consequence of the narrowing EC geometry. If true, a reduction in sound velocity should persist independently of the gas composition in the EC (e.g., air, [Formula: see text]). Integrating laser Doppler vibrometry, microcomputed tomography, and numerical analysis on precise three-dimensional geometries of each experimental animal EC, we demonstrate that the narrowing radius of the EC is the main factor reducing sound velocity. Both experimental and numerical data also show that sound velocity is reduced further when excess [Formula: see text] fills the EC. Likewise, the EC bifurcates at the tympanal level (one branch for each tympanic membrane), creating two additional narrow internal sound paths and imposing different sound velocities for each tympanic membrane. Therefore, external and internal inputs total to four sound paths for each ear (only one for the human ear). Research paths and implication of findings in avian directional hearing are discussed.

The Ander's organ: a mechanism for anti-predator ultrasound in a relict orthopteran.

The use of acoustics in predator evasion is a widely reported phenomenon amongst invertebrate taxa, but the study of ultrasonic anti-predator acoustics is often limited to the prey of bats. Here, we describe the acoustic function and morphology of a unique stridulatory structure - the Ander's organ - in the relict orthopteran Cyphoderris monstrosa (Ensifera, Hagloidea). This species is one of just eight remaining members of the family Prophalangopsidae, a group with a fossil record of over 90 extinct species widespread during the Jurassic period. We reveal that the sound produced by this organ has the characteristics of a broadband ultrasonic anti-predator defence, with a peak frequency of 58±15.5 kHz and a bandwidth of 50 kHz (at 10 dB below peak). Evidence from sexual dimorphism, knowledge on hearing capabilities and assessment of local predators, suggests that the signal likely targets ground-dwelling predators. Additionally, we reveal a previously undescribed series of cavities underneath the organ that probably function as a mechanism for ultrasound amplification. Morphological structures homologous in both appearance and anatomical location to the Ander's organ are observed to varying degrees in 4 of the 7 other extant members of this family, with the remaining 3 yet to be assessed. Therefore, we suggest that such structures may either be more widely present in this ancient family than previously assumed, or have evolved to serve a key function in the long-term survival of these few species, allowing them to outlive their extinct counterparts.

On the tympanic membrane impedance of the katydid Copiphora gorgonensis (Insecta: Orthoptera: Tettigoniidae).

Katydids (bush-crickets) are endowed with tympanal ears located in their forelegs' tibiae. The tympana are backed by an air-filled tube, the acoustic trachea, which transfers the sound stimulus from a spiracular opening on the thorax to the internal side of the tympanic membranes (TM). In katydids the sound stimulus reaches both the external and internal side of the membranes, and the tympanal vibrations are then transferred to the hearing organ crista acustica (CA) that contains the fluid-immersed mechanoreceptors. Hence the tympana are principally involved in transmitting and converting airborne sound into fluid vibrations that stimulate the auditory sensilla. Consequently, what is the transmission power to the CA? Are the TM tuned to a certain frequency? To investigate this, the surface normal acoustic impedance of the TM is calculated using finite-element analysis in the katydid Copiphora gorgonensis. From this, the reflectance and transmittance are obtained at the TM. Based on the impedance results obtained from the pressure recordings at TM and the velocity field calculations in the AT, in the frequency range 5-40 kHz, it is concluded that the tympana have considerably higher transmission around 23 kHz, corresponding to the dominant frequency of the male pure-tone calling song in this species.

The Auditory Mechanics of the Outer Ear of the Bush Cricket: A Numerical Approach.

Bush crickets have tympanal ears located in the forelegs. Their ears are elaborate, as they have outer-, middle-, and inner-ear components. The outer ear comprises an air-filled tube derived from the respiratory trachea, the acoustic trachea (AT), which transfers sound from the mesothoracic acoustic spiracle to the internal side of the ear drums in the legs. A key feature of the AT is its capacity to reduce the velocity of sound propagation and alter the acoustic driving forces of the tympanum (the ear drum), producing differences in sound pressure and time between the left and right sides, therefore aiding the directional hearing of the animal. It has been demonstrated experimentally that the tracheal sound transmission generates a gain of ∼15 dB and a propagation velocity of 255 ms-1, an approximately 25% reduction from free-field propagation. However, the mechanism responsible for this change in sound pressure level and velocity remains elusive. In this study, we investigate the mechanical processes behind the sound pressure gain in the AT by numerically modeling the tracheal acoustic behavior using the finite-element method and real three-dimensional geometries of the tracheae of the bush cricket Copiphora gorgonensis. Taking into account the thermoviscous acoustic-shell interaction on the propagation of sound, we analyze the effects of the horn-shaped domain, material properties of the tracheal wall, and the thermal processes on the change in sound pressure level in the AT. Through the numerical results obtained, it is discerned that the tracheal geometry is the main factor contributing to the observed pressure gain.

Complex wing motion during stridulation in the katydid Nastonotus foreli (Orthoptera: Tettigoniidae: Pseudophyllinae).

Male Katydids (Orthoptera: Tettigoniidae) rub together their specialised forewings to produce sound, a process known as stridulation. During wing closure, a lobe on the anal margin of the right forewing (a scraper), engages with a tooth-covered file on the left forewing. The movement of the scraper across the file produces vibrations which are amplified by a large wing cell adjacent to the scraper, the mirror. Katydids are known to stridulate with either sustained or interrupted sweeps of the file, generating resonant pure-tone (narrowband frequency) or non-resonant (broadband frequency) calls. However, some species can conserve some purity in their calls despite incorporating discrete pulses and silent intervals. This mechanism is exhibited by many Pseudophyllinae, such as Nastonotus spp., Cocconotus spp., Triencentrus spp. and Eubliastes spp. This study aims to measure and quantify the mechanics of wing stridulation in Nastonotus foreli, a Neotropical katydid that can produce, relatively narrowband calls at ≈20 kHz. It was predicted that this species will use a stridulatory mechanism involving elastic energy whereby the scraper bends and flicks along the file in periodic bursts. The calling behaviour and wing mechanics of seven males were studied using a combination of technologies (e.g. micro-scanning laser Doppler vibrometry, advanced microscopy, ultrasound-sensitive equipment and optical motion detectors) to quantify wing mechanics and structure. Analysis of recordings revealed no clear relationship between wing velocity and carrier frequency, and a pronounced distinction between wing velocity and scraper velocity during wing closure, suggesting that the scraper experiences considerable deformation. This is characteristic of the elastic scraper mechanism of stridulation. Curiously, N. foreli might have evolved to employ elastic energy to double the duration of the call, despite possessing muscles that can reach velocities high enough to produce the same frequency without the help of elastic energy.

Auditory sensitivity, spatial dynamics, and amplitude of courtship song in Drosophila melanogaster.

Acoustic communication is an important component of courtship in Drosophila melanogaster. It takes the form of courtship song produced by males through the unilateral extension and vibration of a wing. Following the paradigm of sender-receiver matching, song content is assumed to match tuning in the auditory system, however, D. melanogaster audition is nonlinear and tuning dependent upon signal amplitude. At low stimulus amplitudes or in the absence of sound the antenna is tuned into song frequency, but as amplitude increases the antenna's resonance is shifted up by hundreds of Hertz. Accurate measurements of song amplitude have been elusive because of the strong dependency of amplitude upon the spatial geometry between sender and receiver. Here, D. melanogaster auditory directional sensitivity and the geometric position between the courting flies are quantified. It is shown that singing occurs primarily from positions resulting in direct stimulation of the female antenna. Using this information, it is established that the majority of song is louder than theoretically predicted and at these sound levels the female antenna should not amplify or be tuned into song. The study implies that Drosophila hearing, and, in particular, its active mechanisms, could function in a broader context than previously surmised.

Structural biomechanics determine spectral purity of bush-cricket calls.

Bush-crickets (Orthoptera: Tettigoniidae) generate sound using tegminal stridulation. Signalling effectiveness is affected by the widely varying acoustic parameters of temporal pattern, frequency and spectral purity (tonality). During stridulation, frequency multiplication occurs as a scraper on one wing scrapes across a file of sclerotized teeth on the other. The frequency with which these tooth-scraper interactions occur, along with radiating wing cell resonant properties, dictates both frequency and tonality in the call. Bush-cricket species produce calls ranging from resonant, tonal calls through to non-resonant, broadband signals. The differences are believed to result from differences in file tooth arrangement and wing radiators, but a systematic test of the structural causes of broadband or tonal calls is lacking. Using phylogenetically controlled structural equation models, we show that parameters of file tooth density and file length are the best-fitting predictors of tonality across 40 bush-cricket species. Features of file morphology constrain the production of spectrally pure signals, but systematic distribution of teeth alone does not explain pure-tone sound production in this family.

Chamber music: an unusual Helmholtz resonator for song amplification in a Neotropical bush-cricket (Orthoptera, Tettigoniidae).

Animals use sound for communication, with high-amplitude signals being selected for attracting mates or deterring rivals. High amplitudes are attained by employing primary resonators in sound-producing structures to amplify the signal (e.g. avian syrinx). Some species actively exploit acoustic properties of natural structures to enhance signal transmission by using these as secondary resonators (e.g. tree-hole frogs). Male bush-crickets produce sound by tegminal stridulation and often use specialised wing areas as primary resonators. Interestingly, Acanthacara acuta, a Neotropical bush-cricket, exhibits an unusual pronotal inflation, forming a chamber covering the wings. It has been suggested that such pronotal chambers enhance amplitude and tuning of the signal by constituting a (secondary) Helmholtz resonator. If true, the intact system - when stimulated sympathetically with broadband sound - should show clear resonance around the song carrier frequency which should be largely independent of pronotum material, and change when the system is destroyed. Using laser Doppler vibrometry on living and preserved specimens, microcomputed tomography, 3D-printed models and finite element modelling, we show that the pronotal chamber not only functions as a Helmholtz resonator owing to its intact morphology but also resonates at frequencies of the calling song on itself, making song production a three-resonator system.

Functional morphology of tegmina-based stridulation in the relict species Cyphoderris monstrosa (Orthoptera: Ensifera: Prophalangopsidae).

Male grigs, bush crickets and crickets produce mating calls by tegminal stridulation: the scraping together of modified forewings functioning as sound generators. Bush crickets (Tettigoniidae) and crickets (Gryllinae) diverged some 240 million years ago, with each lineage developing unique characteristics in wing morphology and the associated mechanics of stridulation. The grigs (Prophalangopsidae), a relict lineage more closely related to bush crickets than to crickets, are believed to retain plesiomorphic features of wing morphology. The wing cells widely involved in sound production, such as the harp and mirror, are comparatively small, poorly delimited and/or partially filled with cross-veins. Such morphology is similarly observed in the earliest stridulating ensiferans, for which stridulatory mechanics remains poorly understood. The grigs, therefore, are of major importance to investigate the early evolutionary stages of tegminal stridulation, a critical innovation in the evolution of the Orthoptera. The aim of this study is to appreciate the degree of specialization on grig forewings, through identification of sound radiating areas and their properties. For well-grounded comparisons, homologies in wing venation (and associated areas) of grigs and bush crickets are re-evaluated. Then, using direct evidence, this study confirms the mirror cell, in association with two other areas (termed 'neck' and 'pre-mirror'), as the acoustic resonator in the grig Cyphoderris monstrosa Despite the use of largely symmetrical resonators, as found in field crickets, analogous features of stridulatory mechanics are observed between C. monstrosa and bush crickets. Both morphology and function in grigs represents transitional stages between unspecialized forewings and derived conditions observed in modern species.

Auditory mechanics in a bush-cricket: direct evidence of dual sound inputs in the pressure difference receiver.

The ear of the bush-cricket, Copiphora gorgonensis, consists of a system of paired eardrums (tympana) on each foreleg. In these insects, the ear is backed by an air-filled tube, the acoustic trachea (AT), which transfers sound from the prothoracic acoustic spiracle to the internal side of the eardrums. Both surfaces of the eardrums of this auditory system are exposed to sound, making it a directionally sensitive pressure difference receiver. A key feature of the AT is its capacity to reduce the velocity of sound propagation and alter the acoustic driving forces at the tympanum. The mechanism responsible for reduction in sound velocity in the AT remains elusive, yet it is deemed to depend on adiabatic or isothermal conditions. To investigate the biophysics of such multiple input ears, we used micro-scanning laser Doppler vibrometry and micro-computed X-ray tomography. We measured the velocity of sound propagation in the AT, the transmission gains across auditory frequencies and the time-resolved mechanical dynamics of the tympanal membranes in C. gorgonensis Tracheal sound transmission generates a gain of approximately 15 dB SPL, and a propagation velocity of ca 255 m s-1, an approximately 25% reduction from free field propagation. Modelling tracheal acoustic behaviour that accounts for thermal and viscous effects, we conclude that reduction in sound velocity within the AT can be explained, among others, by heat exchange between the sound wave and the tracheal walls.

Convergent evolution between insect and mammalian audition.

In mammals, hearing is dependent on three canonical processing stages: (i) an eardrum collecting sound, (ii) a middle ear impedance converter, and (iii) a cochlear frequency analyzer. Here, we show that some insects, such as rainforest katydids, possess equivalent biophysical mechanisms for auditory processing. Although katydid ears are among the smallest in all organisms, these ears perform the crucial stage of air-to-liquid impedance conversion and signal amplification, with the use of a distinct tympanal lever system. Further along the chain of hearing, spectral sound analysis is achieved through dispersive wave propagation across a fluid substrate, as in the mammalian cochlea. Thus, two phylogenetically remote organisms, katydids and mammals, have evolved a series of convergent solutions to common biophysical problems, despite their reliance on very different morphological substrates.

Sound radiation and wing mechanics in stridulating field crickets (Orthoptera: Gryllidae).

Male field crickets emit pure-tone mating calls by rubbing their wings together. Acoustic radiation is produced by rapid oscillations of the wings, as the right wing (RW), bearing a file, is swept across the plectrum borne on the left wing (LW). Earlier work found the natural resonant frequency (f(o)) of individual wings to be different, but there is no consensus on the origin of these differences. Previous studies suggested that the frequency along the song pulse is controlled independently by each wing. It has also been argued that the stridulatory file has a variable f(o) and that the frequency modulation observed in most species is associated with this variability. To test these two hypotheses, a method was developed for the non-contact measurement of wing vibrations during singing in actively stridulating Gryllus bimaculatus. Using focal microinjection of the neuroactivator eserine into the cricket's brain to elicit stridulation and micro-scanning laser Doppler vibrometry, we monitored wing vibration in actively singing insects. The results show significantly lower f(o) in LWs compared with RWs, with the LW f(o) being identical to the sound carrier frequency (N=44). But during stridulation, the two wings resonate at one identical frequency, the song carrier frequency, with the LW dominating in amplitude response. These measurements also demonstrate that the stridulatory file is a constant resonator, as no variation was observed in f(o) along the file during sound radiation. Our findings show that, as they engage in stridulation, cricket wings work as coupled oscillators that together control the mechanical oscillations generating the remarkably pure species-specific song.

Sound production during agonistic behavior of male Drosophila melanogaster.

Male Drosophila fruit flies acquire and defend territories in order to attract females for reproduction. Both, male-directed agonistic behavior and female-directed courtship consist of series of recurrent stereotypical components. Various studies demonstrated the importance of species-specific sound patterns generated by wing vibration as being critical for male courtship success. In this study we analyzed the patterns and importance of sound signals generated during agonistic interactions of male Drosophila melanogaster. In contrast to acoustic courtship signals that consist of sine and pulse patterns and are generated by one extended wing, agonistic signals lack sine-like components and are generally produced by simultaneous movements of both wings. Though intra-pulse oscillation frequencies (carrier frequency) are identical, inter-pulse intervals are twice as long and more variable in aggression signals than in courtship songs, where their precise temporal pattern serves species recognition. Acoustic signals accompany male agonistic interactions over their entire course but occur particularly often after tapping behavior which is a major way to identify the gender of the interaction partner. Since similar wing movements may either be silent or generate sound and wing movements with sound have a greater impact on the subsequent behavior of a receiver, sound producing wing movements seem to be generated intentionally to serve as a specific signal during fruit fly agonistic encounters.