What can insects teach us about hearing loss?

Over the last three decades, insects have been utilized to provide a deep and fundamental understanding of many human diseases and disorders. Here, we present arguments for insects as models to understand general principles underlying hearing loss. Despite ∼600 million years since the last common ancestor of vertebrates and invertebrates, we share an overwhelming degree of genetic homology particularly with respect to auditory organ development and maintenance. Despite the anatomical differences between human and insect auditory organs, both share physiological principles of operation. We explain why these observations are expected and highlight areas in hearing loss research in which insects can provide insight. We start by briefly introducing the evolutionary journey of auditory organs, the reasons for using insect auditory organs for hearing loss research, and the tools and approaches available in insects. Then, the first half of the review focuses on auditory development and auditory disorders with a genetic cause. The second half analyses the physiological and genetic consequences of ageing and short‐ and long‐term changes as a result of noise exposure. We finish with complex age and noise interactions in auditory systems. In this review, we present some of the evidence and arguments to support the use of insects to study mechanisms and potential treatments for hearing loss in humans. Obviously, insects cannot fully substitute for all aspects of human auditory function and loss of function, although there are many important questions that can be addressed in an animal model for which there are important ethical, practical and experimental advantages.

Most of us will probably experience some hearing loss during our life time.Age-related hearing loss is inevitable but other factors exacerbate this decline, including genetic susceptibilities, noise exposure, chemical and antibiotic effects, diet, and lifestyle.Although hearing is the perception of sound by the central nervous system, this review is focused at the level of auditory organs and the auditory nerve only.Here, we outline potential contributions to our understanding of hearing loss that are available using insect auditory organs.The ear works through a complex interaction of multiple specialized cell types, and so understanding what cells and cellular processes are responsible when auditory detection goes wrong is a truly mammoth endeavour for which we need diverse angles of experimental investigation.The idea of using insects to understand basic principles of progressive or noise-induced hearing loss is still new; only a handful of studies have been published, mainly over the last couple of years (Austin, Thomas et al., 2023;Blockley et al., 2022;Christie et al., 2013;Keder et al., 2020;Sharma et al., 2023;Warren et al., 2020).However, Drosophila in particular already has a storied history in auditory development and physiology studies (Göpfert & Robert 2003;Lehnert et al., 2013;Nadrowski et al., 2008;Roy et al., 2013).Reviewing this emerging approach will focus future investigation on the most pertinent problems and complement the greater hearing loss field.
Evolution of auditory organs.The evolution of specialized sensory cilia occurred very early after the first single-celled eukaryotes evolved (∼2 billion years ago) because it provided a competitive advantage for sensing and moving through the environment (Satir et al., 2008).Over the next ∼1.4 billion years, these cilia evolved further specializations and developmental programmes (Christie & Eberl, 2014;Fritzsch & Straka, 2014) such that, by the time of the last common ancestor of vertebrates and invertebrates ∼600 Mya (Fig. 1), many of the genes and gene pathways that orchestrate sensory cilia development and maintenance were well established.As the sensory cilia specialized, other cell types specialized with them, The earlier in time since the divergence, the more fundamental the processes that govern hearing organ physiology and genetics.
variants that contribute to phenotypic variation can be quickly identified.Finally, it should be noted that, with the advent of CRISPR technologies, many other insects beyond traditional genetic model systems can now be manipulated genetically.These insects have physiological advantages and large numbers for robust analysis.For the genetic tools to be scientifically useful, they need to be coupled with physiological approaches to characterize the phenotype and preferably localize gene-induced changes at the level of the component parts of auditory organs.
Convergent evolution of auditory organ components.
Although the ears of mammals and insects look very different, they achieve the same objective: to sensitively detect sound.In the Precambrian period, ocean-living vertebrates evolved head-based balance systems for swimming, whereas invertebrates relied on their hard exoskeleton for balance and pedalism.Nevertheless, auditory organs of mammals and insects share common principles of function, mainly as a result of convergent evolution (Fig. 2).For example, the detection of the pressure component of sound resulted in remarkably similar tympani: an evolutionary thinning of either chitin or ectoderm for insects and mammals.Developing primary auditory receptors still bear the anciently evolved (∼2 billion years ago) microtubule-based cilium that still exists in vertebrates today as a kinocilium.In the mature adult mammalian cochlea, however, the kinocilium degenerates, leaving only the numerous actin-based stereocilia.Both invertebrate and vertebrate auditory systems rely on metabolically demanding ion-pumping cells to maximize amplification using extreme electrochemical gradients in a specialized lymph.Both insect and vertebrate taxa use mechanosensitive ion channels that, on displacement, cause an influx of cations leading to depolarization.In insects, the resulting spikes are propagated to the central nervous system by the same sensory receptor neurons, whereas vertebrates have separate neurons for the task, but both have auditory nerves encapsulated by glial or Schwann cells (Fig. 2).
In addition to the homologous components of auditory organs across animal phyla, the physiological operation of auditory organs shares patterns of age-related and noise-induced decline in function (Blockley et al., 2022).Thus, there is a second reason to study insects for hearing loss.Reason 2: understanding the complex physiology of diverse auditory systems gives us novel insights of system robustness and vulnerabilities, which will apply to other multicellular complex auditory systems such as those in mammals.accessibility.In Drosophila, biophysical properties of mechanotransduction channels (such as number of channels, single channel gating force and open probability) can be quantified en masse through contactless lasers that detect movements of the antennal sound receiver in intact flies (Albert et al., 2007;Keder et al., 2020).In the bush cricket and migratory locust, the motion of tonotopic travelling waves can be optically measured along its length in vivo (Udayashankar et al., 2012;Windmill et al., 2005).In the desert locust, the transduction current can be measured from auditory receptors in intact auditory organs in response to airborne sound (Warren & Matheson, 2018).Such physiological approaches in insects are inherently high throughput, but, when combined with the genetic manipulations, offer rapid advancements in knowledge.

Auditory development
Despite their different appearance, insect and vertebrate auditory systems evolved from common developmental genetic origins.Superimposed on the common origin of basic mechanosensory functions is the diversity afforded by specialized ecological and environmental forces that have influenced evolution over the eons.The basic mechanosensory genetic toolkit shared through eukaryotic evolution comprises at least four gene superfamilies of mechanosensory ion channels, including the TRP, DEG/ENaC, Piezo and TMC superfamilies (Fritzsch et al., 2020).The specific gene or combination of genes deployed, the cellular positioning of expressed ion channels and any molecular linkages to cytoskeletal or extracellular structures are all substrates All auditory systems require the sound energy to be captured, either through tympani or antennae.Sound-induced displacements are transferred to the primary auditory receptors (grey).A steep electrochemical gradient is maintained by ion-pumping cells (red), which recycle K + and other ions.Transduction potentials are converted into spikes and propagated to the central nervous system (blue).Mammals and some insects (Andrés et al., 2016) harbour efferent input to modulate auditory sensitivity (green), including to mammalian inner hair cells (Elliott et al., 2021).
for evolution.Although variation and diversification in developmental genes and their regulation allowed for the appearance of mechanosensory organs in different anatomical locations (e.g.abdomen vs. leg, ear vs. lateral line), unique mechanical linkages were accompanied by specialization of the physiological functional genetic network, to optimize sensory responses in that location.In both insects and vertebrates, additional mechanosensory organs related to those in the auditory system may develop in diverse positions to provide a sensory input for different mechanical signals.Thus, insect chordotonal organs develop in numerous anatomical positions throughout the body in larvae, pupae or nymph, as well as the adult, serving touch, proprioception and other mechanosensory modalities.In insects, the diversity of anatomical locations and receiving structures (tympanum, antenna) belies the conservation of the sensory organ (Eberl, 1999;Field & Matheson, 1998;Hoy, 1998;Yack, 2004), namely the chordotonal organ.To achieve this diversity, the chordotonal organ developmental programme has been modified sufficiently to induce chordotonal organ precursors in new locations, sometimes at the same time as suppressing their development in more ancestral developmental lineages (Albert et al., 2020;Boekhoff-Falk & Eberl, 2014;Fritzsch et al., 2020;van Staaden & Römer, 1998).Similarly, besides the vestibular and auditory hair cells, fish have lateral line neuromast cells along the length of their bodies, as do many amphibians (Engelmann & Fritzsch, 2022).Furthermore, albeit more distantly related, mechanosensory Merkel cells share some developmental genetic machinery (Lumpkin & Caterina, 2007;Maricich et al., 2009;Woo et al., 2015;Wright et al., 2015).Thus, the ontogenetic theme of multiple diverse deployments of genetic programmes reflects the phylogenetic theme, both relying on extensive juggling of gene regulatory changes, along with gene duplication and divergence, as well as gene loss.
The shared eukaryotic developmental genetic programmes for auditory organs have been reviewed (Boekhoff-Falk & Eberl, 2014;Jarman, 2014;Jarman & Groves, 2013;Li et al., 2018;Todi et al., 2004).In both Drosophila and mouse ear development, the highly conserved global patterning gene pathways, Dpp (BMP), Wnt, Shh and the Hox genes play key roles in positioning the future auditory organs.Compared to Drosophila, mammalian ear development involves more inductive processes, such as those directed by fibroblast growth factors and retinoic acid.Layered on these global patterning processes, transcription factor cascades such as Pax2, Homothorax (Hmx), Distalless (Dlx), Extradenticle (Otx) and Cut (Cux) further pattern tissues to define locations for auditory organ specification in insects and vertebrates.Basic helix-loop-helix transcription factors, particularly in the Atonal (Atoh) family, play key roles in the specification of sense organ precursors or sensory hair cells in both taxa.Cell-cell interactions help to establish planar cell polarity (PCP) in both taxa, which is essential for correctly oriented asymmetric cell divisions.Signalling via the Notch Delta pathway ensures decisive and robust specification of cell fates, such as insect sense organ precursor vs. epithelial cell, sensory neuron vs. scolopale cell, or vertebrate hair cell vs. supporting cell.Once specified, each cell type must differentiate appropriately to its position, fate and function.In sensory cells (vertebrate hair cells or neuromasts, insect chordotonal sensory neurons), this requires remodelling of the cytoskeletal architecture, the extracellular matrix, interactions with neighbouring cells, dendritic or axonal projections, synaptic functions, and physiological machinery for its specialized role in mechanosensation.The primitive arrangement of sensory cells was probably a microtubule-based cilium, surrounded by actin-based villi (Manley & Ladher, 2008).As mammals evolved in the vertebrate lineage, their sensory transduction machinery transitioned into specialized actin-based villi.Thus, the transduction apparatus may be localized to the derivative of the primary cilium (insect chordotonal), to modified microvillar structures (vertebrate auditory and vestibular hair cells), or to both (fish and frog lateral line neuromasts).Finally, different sensory cells in an animal may be physiologically specialized to respond to different properties of sounds, including different frequencies or amplitudes.
Considering these dimensions of diversity between auditory systems, there remain strong threads of developmental homology anchored in a combination of common ancestry, sampling from shared development genetic toolkits, as well as convergent forces imposed by the fundamental properties of the very sounds being captured.Thus, discoveries in insect systems can provide an understanding of fundamental principles of auditory development.

Genetic disorders
The Hereditary Hearing Loss Homepage (Van Camp & Smith), at the time of writing, lists 124 unique human non-syndromic hearing loss genes, as well as many genes associated with syndromes that include hearing loss as a component.Large-scale mouse mutagenesis screens have identified similar numbers of deafness genes.These efforts include screens targeted specifically at hearing (Bowl et al., 2017;Ingham et al., 2019), as well as broader mutagenesis screens in which auditory dysfunction was one of multiple phenotypes tested (de Angelis et al., 2015;Groza et al., 2023).Syndromic hereditary hearing loss more frequently, but not exclusively, involves developmental patterning genes, whereas non-syndromic disorders are often more intimately involved with the specific functions of the J Physiol 602.2 auditory cells.Although developmental genetic studies in insect hearing are largely limited to Drosophila, great strides have recently been made in other insect systems at the physiological level using both pharmacological approaches and gene expression approaches (Austin, Thomas et al., 2023;French & Warren, 2021;Warren et al., 2020) with CRISPR gene knockout approaches beginning to provide genetic insight into non-model insect hearing mechanisms (Georgiades et al., 2022;Su et al., 2020).These various approaches will serve to provide a richer understanding of the variation in heritable mechanisms and their evolutionary relationship to the diversity of vertebrate auditory systems.
Structural disorders.Genetic disorders involving core cytoskeletal elements most often result in early developmental defects that are incompatible with survival to a stage at which hearing function can be assessed.Rather, mutations in cytoskeletal elements tend to focus on gene isoforms or transcripts related to the cellular specializations that evolved to form the mechanical linkages in the stimulus chain from movement of the sound receiving structures (antenna or tympanum) and activate the mechanosensitive ion channels in the sensory cells.
In vertebrates, including mammals, specialized isoforms of actin form and maintain the modified microvilli (stereocilia).Cytoskeletal disorders affecting hearing are attributed to actin, actin cross-linking proteins and unconventional myosins (Hasson et al., 1997).For example, human mutations associated with β-actin (Procaccio et al., 2006), γ -actin (Bryan et al., 2006), affect sensory hair cell stereocilia, whereas actin-binding protein diaphanous is associated with hearing in both Drosophila and mammals (Lynch et al., 1997;Schoen et al., 2010).Furthermore, myosin 7A mutations disrupt hearing in both taxa (Gibson et al., 1995;Todi et al., 2005Todi et al., , 2008;;Weil et al., 1995).Actin regulators such as Rho GTPases play important roles in establishing and maintaining correct actin configurations, the disruption of which results in hearing loss through planar cell polarity, kinocilium positioning and stereocilia formation defects (Dai et al., 2023).Besides Rho kinase (Todi et al., 2008), these represent an opportunity for exploration in insect hearing.
Mutations in ciliary proteins tend to have broader effects in mammals than in flies because flies have only a few ciliated cell types.These are primarily the type I sense organs that include chordotonal organs and spermatozoa.In vertebrate auditory systems, the ciliary derivative, the kinocilium, is required for the sensory hair cells to respond to PCP cues because mutations in PCP genes such as Vangl2 (homologous to the Drosophila gene Strabismus/Van Gogh) and Scrb1 (homologous to Drosophila gene scribble) result in misorientation of the hair cells (Jones et al., 2008;Montcouquiol et al., 2003).The kinocilium is also required for proper formation of the hair bundles (Flock & Wersall, 1962;Jones et al., 2008;Tarchini et al., 2016) in a PCP-dependent manner with conserved factors such as Inscuteable, characterized in Drosophila mechanosensory organ development (Bellaïche et al., 2001).Meanwhile, in flies, the transduction apparatus appears to be localized to the sensory cilium of chordotonal neurons, such that ciliary dysformation and dysfunction result in deafness because of loss of transduction.Active ciliary movements also appear to provide an amplification function, analogous to one component of the cochlear amplifier that in mammals arises from Prestin-mediated electromotility of the outer hair cells.In addition, both Drosophila and mammals have been shown to display transduction channel closing mechanisms that return energy to the antennal motion (Drosophila; Albert et al., 2007) or to the hair bundle (mammals; Kennedy et al., 2005).
The extracellular matrix is also important in auditory mechanosensation, including maintaining physical linkages and mechanical properties such as stiffness or elasticity in the mechanotransduction chain.Components associated with mammalian hearing loss include tectorins (Verhoeven et al., 1998), collagens (McGuirt et al., 1999) and other extracellular matrix components forming the tectorial membrane (Goodyear & Richardson, 2018), as well as Protocadherin 15 (Ahmed et al., 2001;Alagramam et al., 2001) and Cadherin 13 forming the tip links that pull on the transduction channels (Kazmierczak et al., 2007).Extracellular matrix proteins also play important roles in Drosophila hearing, with the best characterized one being NompA, a component of the dendritic cap that is directly involved in physically transmitting movement to the sensory cilium (Chung et al., 2001).
Ion channel disorders.Identification of the mechanotransduction machinery through mutant studies has not been as straightforward for hearing as for the transduction mechanisms of vision, smell or taste.Numerous candidates in vertebrates and in insects enjoyed the limelight for a time, as discussed in detail (Warren & Nowotny, 2021).In mammals, the TMC1/TMC2 channels (Pan et al., 2013(Pan et al., , 2018)), with associated subunits TMIE (Cunningham et al., 2020), CIB2 (Giese et al., 2017), LHFPL5 (Ge et al., 2018), TOMT (Cunningham et al., 2017;Erickson et al., 2017) and possibly ankyrin (Zheng & Holt, 2021), are now becoming generally accepted as the mechanotransduction channel complex in mammals (Jung & Müller, 2023).Channels in the epithelial sodium channel, TRP and Piezo families were considered as candidates because of their role as mechanosensitive channels in other systems (Coste et al., 2010;O'Hagen et al., 2005;Walker et al., 2000).Tip link proteins PCDH15 and CDH23 are tethers for gating the channel (Sakaguchi et al., 2009).Meanwhile, in Drosophila, NompC was considered the primary mechanotransduction channel for several years with its ankyrin repeats proposed as the tether, until the TRPV channel encoded by Iav/Nan genes gained attention (Lehnert et al., 2013;Li et al., 2021;Warren & Matheson, 2018).Indeed, there is supporting evidence for both models, although it is also possible that there is yet another channel protein to be identified (Albert et al., 2020;Eberl et al., 2016;Warren & Nowotny, 2021).Complicating matters is the fact that NompC homologs in fish and frogs also play roles in hearing, but are absent in amniotes (Schüler et al., 2015).As described by Fritzsch et al. (2020), genomes are evolutionarily nimble, and different members of at least four ancient families of eukaryotic mechanosensitive channels can be deployed in different mechanoreceptive organs to serve an auditory function.Nevertheless, the nature of sound and the challenges of sound localization, selection and often the amplification of relevant signals from background are common problems, and nature has solved many of these problems in different but parallel ways.For example, amplification mechanisms involve both shared and unique features between flies and mammals, in that the force of transduction channel closing altering gating compliance can provide an energy source for amplification in both systems (Howard & Hudspeth, 1988;Nadrowski et al., 2008), whereas motility-based sources involve Prestin electromotility in mammals (Zheng et al., 2000) but (presumably) ciliary dynein motility in insects (Karak et al., 2015;Kavlie et al., 2010;Möckel et al., 2012;Warren et al., 2010).Physiological hearing disorders are also caused by dysfunction of other ion channels, such as the voltage-gated Na + and K + channel in insects (Ravenscroft et al., 2023;Zhang, 2023) and K + channels and Ca 2+ channels in mammals (Baig et al., 2011;Kharkovets et al., 2000Kharkovets et al., , 2006;;Kubisch et al., 1999;Neyroud et al., 1997;Schulze-Bahr et al., 1997).In addition, gap junctions play a key role, with the GJB2 gene, encoding Connexin 43, representing one the most prevalent hereditary deafness genes (Kelsell et al., 1997).Gap junction genes in insects, called innexins, also play a key role in auditory function in Drosophila (Pézier et al., 2016).

Physiological and genetic consequences of ageing
Homeostasis is the ability to maintain function in the face of environmental perturbations including the effects of age.In this section, we focus on the effects of age, but, for humans, we cannot rule out the contribution of noise as living in the developed world appears to contribute to our hearing loss (Rosen et al., 1962) (we cover age and noise interactions in a later section).
Sound receiver and middle ear mechanics.If not maintained, articulating mechanical components are often the source of system failure.Both insect and mammalian sound-receivers (antennae and tympani) and middle ears (for mammals) go through many more articulations than other mechanical joints.Despite such use, mammalian tympani and middle ears (Fritzsch et al., 2013;Holte, 1996;Thompson et al., 1979;Uchida et al., 2000) and the Drosophila antennal sound receiver (Austin, Thomas et al., 2023;Keder et al., 2020;Sharma et al., 2023) function well into old age.Age-related structural changes occur in the mechanical components of middle ears (Carr, 2020;Ruah et al., 1991), but, if anything, these appear to be compensatory against any loss of function.The desert locust, however, has an age-related decrease in tympanal displacement (Austin, Woodrow et al., 2023), which appears to be associated with age-related sclerotization (hardening) of insect cuticle.The evolutionary pressure for desert locust hearing (at the frequency to which most of their auditory receptors are tuned) is low compared to humans and Drosophila; locusts presumably hear to help co-ordinate flight take off (Haskell, 1957) or avoid each other in flight (Boyan, 1985).We speculate that the higher evolutionary pressures for humans to hear, for predator avoidance and communication, and for Drosophila to mate have driven the evolution of compensatory mechanisms for age-related changes in the mechanical elements that capture and transfer sound to the auditory organs.
Ion pumping cells that maintain the electrochemical gradient.Historically, the most popular mechanism to explain age-related hearing loss was the decrease in the endocochlear or receptor lymph potential (Liu & Yan, 2007;Vaden et al., 2017), comprising an electrochemical gradient that is maintained across the viliated and ciliated sensory endings in mammals and insects.This shared feature is the result of common properties of sound; namely, per cycle, vibratory stimuli provide progressively B. Warren and D. Eberl J Physiol 602.2 briefer times of force to open the channels as the frequency increases.Thus, even a low frequency sound such as a 100 Hz sinusoid will pull on the channel 100 times for a maximum of only 10 ms per cycle.Both insects and vertebrates appear to solve this by use of an enlarged extracellular space enriched with K + ions to provide an enhanced electrochemical gradient to encourage ion flow during the brief open times.In both systems, this relies on a network of ion pumps and transporters, including Na + /K + -ATPase (Bartolami et al., 2011;Lang et al., 2007;Roy et al., 2013).Dysregulation of these ionic compartments can result in deafness, such as in endolymphatic hydrops (Meniere's disease), or Pendred syndrome, which is caused by mutations in the Slc26a4 transporter (Everett et al., 2001) or the KCNJ10 potassium channel (Kim & Wangemann, 2010;Wangemann et al., 2004), with Sox9 and Sox10 as transcriptional regulators of several ion transporters and channels (Szeto et al., 2022).
For humans, the number of surviving hair cells predicts the extent of hearing loss better than the state of the stria vascularis.Thus, the role of the stria may have been overestimated (Wu, O'Malley et al., 2020).Scolopale cells in insects serve the same purpose as the stria vascularis in mammals and they maintain an electrochemical gradient into older age, with help from auditory receptors that stem the flow of cations by decreasing the open probability of transduction channels (Austin, Thomas et al., 2023).This shows that, for the physiologically parallel insect auditory system, the (what are considered to be) most metabolically demanding cells function well into older age and that mechanisms (transduction channel open probability) may exist to compensate for any decline in the scolopale cells ion-pumping ability.For mammals, this may not happen in natural ageing mammals.Age-correlated decline in the function of the stria in mammalian models is not a consistent fining; only four out of seven accelerated hearing loss models show a decrease in the endocochlear potential (Cable et al., 1993;Ohlemiller, 2009).
Auditory receptors.Mammalian and insect auditory receptors have diverged dramatically but still transduce at their ciliated or 'viliated' endings with specialized transduction machinery.The detailed physiological function of insects' auditory receivers can be measured entirely non-invasively for antennal ears, such as the fruit fly and mosquitoes (Albert et al., 2007;Su et al., 2018), to give parameters at the molecular level for the transduction machinery.For tympanal ears, the transduction current and electrophysiology of auditory neurons of the desert locust can be measured in response to airborne sound (Warren & Matheson, 2018).This means that auditory receptor function can be quickly measured as a function of age in insects.For both insect and mammalian systems, the auditory receptors are a minority compared to other cell types, but they are also the most crucial.From a systems approach, it is insightful to understand how the key auditory receptors of insects, and their transduction machinery, cope with noise insults and ageing.
Yet still deeper analysis reveals age and noise-related metabolic changes in mouse hair cells that may not even present as structural differences (Majumder et al., 2019).Early work on flies shows delayed (7 days) changes in mitochondria of auditory neurons after noise exposure (Christie et al., 2013).Almost a decade later, age-dependent properties of auditory receptors have been measured in locusts, flies and mice.These include detailed electrophysiology of individual auditory receptors (Blockley et al., 2022;Jeng et al., 2021) and in vivo measurements of the mechanotransduction machinery (Keder et al., 2020).These recent analyses of the auditory receptors suggest that auditory receptors are remarkably resilient (Blockley et al., 2022;Christie et al., 2013;Jeng et al., 2021;Keder et al., 2020;Sharma et al., 2023).
For example, the resting potential remains unchanged in the inner hair cells of aged mice (Jeng et al., 2021) and in the auditory receptors of aged locusts (Blockley et al., 2022).The electrophysiological properties and morphology of auditory receptors of locusts are unchanged in the face of loud prolonged noise exposure despite a clear decrease of the performance of the auditory organ (Blockley et al., 2022;Warren et al., 2020).The magnitude of the transduction current shows no large age-related change in mouse hair cells (Jeng et al., 2021), nor does the metabolism of outer hair cells (Majumder et al., 2019).In flies, the single channel gating force of the auditory receptors for the sensitive channel population was well maintained (Keder et al., 2020) and, in locusts exposed to noise, the changes in the transduction current are hypothesized to be solely a result of the decreased maintenance of the receptor lymph and not the auditory receptor's transduction channels themselves (Warren et al., 2020).Thus, a common theme between insect and mammalian models is that the delicate process of transduction on ciliated endings is, through homeostatic mechanisms, surprisingly well maintained into old age, at least for the receptors that survive, and so we must look elsewhere for age-related or noise-induced changes in surviving auditory receptors.One ongoing puzzle is why hair cells in the high frequency end of the cochlea apoptose first.This may be linked to the stria vascularis, which, in the high frequency end of the cochlea, is under higher metabolic stress as it maintains a higher endocochlear potential.One hypothesis is that the high-frequency-end stria vascularis might produce more reactive oxygen species that end up damaging hair cells in that region, or that the higher amplification rates in outer hair cells somehow leads to their early demise.The frequency discriminating auditory organs of insects are well-placed to rapidly test such hypotheses.In the desert locust, the age-dependant decrease in auditory function is restricted to low frequencies (Gordon & Windmill, 2015), which suggests that high-frequency hearing loss may not be a result of the metabolic demands of the higher electrochemical gradients assumed to operate in the high-frequency responsive neurons, with high-frequency hearing loss in mammals being a consequence of mammal-specific physiology.
Auditory nerve.Once auditory information is transduced by the auditory receptors, it is propagated as spikes to the central nervous system through the auditory nerve.Individual axons of the auditory nerve, of both mammals and insects, have spike rates of 100−300 s -1 , placing a high demand and stress on the auditory nerve to reliably transmit them (Warren & Matheson, 2018;Yates et al., 1990).In addition, spiral ganglion neurons (SGNs) are able to maintain high spike rates because of weak adaptation; a maximal spike rate adaptation was 0.4 of maximum (Zhang et al., 2007).This trend of weak adaptation is mirrored in the locust's auditory nerve (Warren et al., 2020).A comparative approach using insects to understand trends in how high-firing nerves cope and deteriorate is insightful.For mammals, there is growing (albeit low-powered) evidence for age-related changes in the auditory nerve including thinning (Xing et al., 2012) and elongation of the length between nodes of Ranvier and decreased width in the nodes themselves (Panganiban et al., 2022).An age-accelerated mouse model appears to show a decrease in SGN number (Altschuler et al., 2015;Elliott et al., 2022), although the cause of the loss may not be a result of the demands of the axon.By contrast to these findings, embryonic Schwann cells are largely dispensable for SGN survival (Mao et al., 2014).In locusts, there was no age-related change in the number of nuclei in the auditory nerve or its width (Blockley et al., 2022).For noise exposure, there is a repeatable decrease in the thickness of the Schwann cell folds that insulate the vertebrate auditory nerve after noise exposure (Pilati et al., 2012;Tagoe et al., 2014;Wan & Corfas, 2017) where the RNA splicing regulator Quaking and its target genes play a role (Panganiban et al., 2018).The width of the locust's auditory nerve is decreased after noise exposure (Blockley et al., 2022), perhaps paralleling this pathology.Thus, at present, it appears that the auditory nerve is selectively damaged by noise but not age, although further work is clearly required to confirm an age-dependant deterioration for mammals.

Physiological and genetic responses to noise exposure, from short to long term
The programmes that control ear development and maintenance evolved before invertebrates and vertebrates last common ancestor >600 million years ago.Although the sensory organs themselves have diversified dramatically, the genetic hierarchy that controls them has functional homology that appears striking (Jarman & Groves, 2013;Wang et al., 2002), but only when neglecting their ancient evolutionary common origin.Thus, the conclusions derived from RNA sequencing and bioinformatics approaches have inherently broad applications across animals' auditory organs.In this section, we review what is known about the genetic and physiological changes in response to noise exposure.
Gene expression and physiological changes in auditory systems are dynamically altered through environmental stressors that include noise, exposure to ototoxic chemicals and one of the most pertinent: age.Insects provide a high-throughput and, for D. melanogaster, an especially genetically amenable system.Gene regulatory networks can be measured and genetic expression tweaked to probe and understand how they change.Likewise, physiological changes can also be quantified in Drosophila and locusts with amenable in vivo and ex vivo measurements (Blockley et al., 2022;Keder et al., 2020).As RNA sequencing technology advances, we J Physiol 602.2 will be able to understand changes in gene expression in unprecedented temporal detail.By using Gene Ontology terms (i.e.groups of differentially expressed genes that underlie specific cellular processes and pathways), we can identify the underlying cellular mechanisms that respond to auditory stressors.These technological genetic advances are now outstripping the rate at which physiology can be measured, but, with the high-throughput physiological measurements in insects, we stand the best chance of keeping pace.As such, insects are exceptionally well placed to test whether each temporally distinct deficit after an ototoxic insult could be treated likewise with separate specifically-timed therapeutic treatments (Jongkamonwiwat et al., 2020).

Short-term changes (up to 24 h) after noise exposure.
The first genes to change expression after noise exposure are dominated by those that code for structural proteins: collagen in mammals (Beaulac et al., 2021;Jongkamonwiwat et al., 2020) and structural glycoproteins in insects (French & Warren, 2021).These gene changes are some of the easiest to correlate with observable physiological deterioration: splayed stereocilia in hair cells and changes in tympanal displacement in the desert locust (Warren et al., 2020).Activation of the ubiquitin proteasome pathway for protein degradation is highly upregulated in mammals (Jongkamonwiwat et al., 2020) after noise exposure but weakly in insects with one ubiquitin-like protein (UFM1) (ranked in the top 40 Gene Ontology changes).One of the largest increases in gene expression in the locust's ear after noise is in a Lysozyme-like protein; a response to the cellular stress.Such lysozyme activity represents the insect's innate immune response to environmental stress.For mammals the first signature of an immune response develops after 24 h (Maeda et al., 2021).Another short-term change, in response to noise, is the temporary reduction of the endocochlear potential, which is not considered to add significantly to a permanent hearing threshold shift (Hirose & Liberman, 2003).There is also no noticeable anatomical change in the stria vascularis after noise exposure (Ward & Duvall, 1971).As in mice, the locust receptor lymph cavity potential is reduced (Blockley et al., 2022).In the locust model, this is complemented by upregulation of genes under the Gene Ontology term inorganic ion transmembrane transport (Austin, Thomas et al., 2023).The differential expression of genes involved with ion pumping are also found in cells of the lateral wall in mice (Milon et al., 2021).In locust, the equivalent of the endocochlear potential (in the receptor lymph cavity, Fig. 2) is well maintained into old age and only repeated noise exposure can permanently lower it (Blockley et al., 2022).
Separate from the stria vascularis, cells of the organ of Corti have a clear decrease in metabolism after noise and into old age (Majumder et al., 2019).This pioneering study strongly advocates an understanding of whole auditory organ metabolism and not just cells dedicated to maintaining the endocochlear potential.A very recent study in locusts suggests that whole organ metabolism dominates over that of the requirements of the ion-pumping cells that maintain the receptor lymph and the auditory neurons combined (Austin, Thomas et al., 2023).Because most noise-induced hearing loss in humans is a result of repeated exposure, it would be worth testing whether repeated noise exposure in mammalian models leads to a decreased endocochlear potential (similar to that found in the locust) or changes in the metabolism of other cells that form the vertebrate ear.
Noise exposure can cause delayed detrimental physiological changes months or years later.Thus, identifying the original cause of auditory deterioration is problematic for two reasons.First, it is costly to keep animals for prolonged durations and, second, with ageing, multiple components are affected and this profoundly obscures which primary component/s fail and their relative contribution to hearing loss.Insects have the potential to derive general principles of how complex auditory systems operate and functionally decline as a result of their genetic amenability and their experimental high-throughput.The best-known physiological change, in response to the best-known environmental stressor, noise, is loss of primary auditory receptors (in both mammals and insects), although, when this occurs months or years after a noise insult, the apoptotic trigger is most surely because of an additive effect of age.In general, ageing is considered to be an inability of homeostatic mechanisms to keep up with the cellular demands, such as reducing reactive oxygen/nitrogen species, translating proteins or maintaining DNA integrity (McHugh & Gil, 2018).In terms of DNA maintenance, the long-term consequences of noise exposure could be stored epigenetically.For example, blocking DNA methyltransferase 1 (DNMT1), either genetically or pharmacologically, results in promising improvements after noise exposure in terms of physiological performance (auditory brainstem responses), as well as against hair cell damage and auditory synapse loss (Zheng et al., 2021).It is probable that environment-induced changes occur outside of the genome and, once sufficient stress is accumulated (both through use and age), tipping point is reached and the genetic control of cell apoptosis begins.There are probably multiple cell death pathways responsible for age-related hearing loss (Wu, Ye et al., 2020) and this is probably the case for delayed apoptosis as a result of noise exposure, although obvious candidates for study are the c-Jun N-terminal kinase pathway for more general cellular stress (Wang et al., 2007) or p53 if caused by DNA damage.

Age and noise interactions
In any complex multicellular auditory system, it is insightful to understand how interactions between multiple factors, such as age and noise, alter hearing function in specific parts of the auditory system.General principles of auditory decline appear to hold between humans and insects.For humans, we have a fairly decent understanding of how age and cumulative noise exposure interact to produce hearing loss in humans (Elliott et al., 2022;Passchier-Vermeer, 1974).Age-related hearing loss dominates in later life such that even old noise-exposed individuals end up with the same hearing loss as old people not exposed to noise (Fig. 3).As a consequence, it is in the middle years of life that noise-exposed humans differ the most from their 'silent' counterparts.Because Top: extent of hearing loss in humans exposed, and not exposed, to noise.Bottom: auditory nerve response in Locusts exposed, and not exposed, to noise (note that the auditory nerve axis has been inverted for comparison).Adapted from Corso (1992) and Blockley et al. (2022).
of the sheer number of experimental units required, quantification of age and noise effects on hearing has only been repeated for one other model, the desert locust, with an uncanny similar finding (Blockley et al., 2022).Locusts exposed to 12 h of noise every 3 days lost some of their sound-evoked auditory nerve response.The nerve response was most different in the middle of their lifespan.By age 34 days, where at least half the locusts had died (as a result of old age), the difference in nerve response from non-exposed locusts was comparable.Assigning specific ear defects as a result of age and noise is overly simplistic.
In reality, age and noise interact and lead to compounding effects, which we address next.Kujawa andLiberman (2006, 2009) and Fernandez et al. (2015) pioneered some of the few longitudinal studies to examine the physiological compounding effects of age and noise on hearing loss.They quantified ribbon synapses in hair cells after noise exposure and found a sharp halving of their number directly after noise exposure, with the number of ribbon synapses remaining low even 10 weeks afterwards.By contrast, purely age-related loss accounts for only ∼25% and ∼10% of ribbon synapse loss at low and high frequencies, respectively (Kujawa & Liberman, 2009).This age-related ribbon synapse loss increases to ∼35% for accelerated ageing mouse models (Altschuler et al., 2015).Insects do not have ribbon synapses to convert transduction potentials into spikes, with this being accomplished in one auditory neuron.Their auditory nerve, however, has a dramatic and acute reduction in its width after noise, which is not compounded by age (Blockley et al., 2022).It appears that the conversion of transduction potentials into spikes presents a weak link in both systems that is especially bad for noise exposure but not so for ageing.
The loss of SGNs, sensitive to high frequencies, in mice exposed once to noise in adolescence is heavily compounded by age (Fernandez et al., 2015;Kujawa & Liberman, 2009;Yamoah et al., 2020).Exposure to noise leads to similar number of inferred SGNs compared to control 2 weeks after exposure.However, 100 weeks later, only around half the number of SGNs remain; whereas age alone (without noise exposure) leads to a ∼10% loss in SGNs sensitive to high frequencies.This age-dependent loss of SGN appears steady across a lifespan (Fernandez et al., 2015;Schuknecht & Gacek, 1993;White et al., 2000), but this has yet to be convincingly shown with sufficient experimental animal numbers.In the locust's Müller's organ, auditory receptor loss is strongly age-dependent, with a steady decline, and noise has a weak compounding effect so that, as the locust ages, the difference between control and noise-exposed groups widens (Blockley et al., 2022).Hair cell loss appears to accelerate into old age in mammals (Chincilla: Bohne et al., 1990;White et al., 2000) and birds (Ryals & Westbrook, 1988), especially for outer hair cells (Fernandez et al., 2015) and is strongly affected by noise (Chen & Fechter, 2003;Hamernik et al., 1989), but only when sufficiently severe; noise exposure at 100 dB sound pressure level in mouse models leads to little compounding effect of age of inner hair cell loss (Fernandez et al., 2015).The function of outer hair cells also appears to correlate with their loss, as inferred from acceleration of increased thresholds of distortion product otoacoustic emissions (Fernandez et al., 2015).The relative contribution of noise and age to hair cell loss, especially for periodic noise (most commonly experienced by humans) remains unknown because there are no longitudinal studies with repeated noise exposure, although studies in insects suggest a specific and distinct middle-aged pattern of hearing loss (Blockley et al., 2022).

Summary
Proportioning the physiological cause of hearing loss in the face of multiple variables is complicated, to put it mildly.However, this fundamental understanding is essential to focus and develop therapeutic treatments as a result of its wide-ranging and devastating impact on quality of life.To unravel a complex biological system requires a systems level approach at multiple points on the fundamental-to-specific spectrum (Fig. 1), from genes, to proteins, to cells and then how different cells interact to achieve function of the ear.There are a limited number of studies on insect hearing loss, which might naturally lead to the question: Are there sufficient fundamentally conserved processes or sufficient functional similarities between insect and mammalian auditory organs?At the genetic level, there is strong evidence because of the numerous homologous genes implicated in hearing loss (Li et al., 2018;Senthilan et al., 2012).Drosophila is particularly well-suited to understanding the complex genetic control and rapidly identifying gene candidates for hearing loss prevention (Keder et al., 2020).However, orthopteran insects offer advantages in our genetic understanding as their gene expression can be combined with deeper physiological insight.At the physiological level, the protein 'building materials' that evolution has employed has diverged between insects and mammals.Despite anatomical differences, both systems have evolutionarily converged on the same basic blueprint of auditory organ design: transduction channels housed on membrane protrusions on auditory receptors, powered by separate ion pumping cells and coupled to a sound receiver (Fig. 2).The limited physiological work suggests that many fundamental aspects of mammalian hearing loss are recapitulated in insects.These include auditory receptor loss, auditory nerve thinning, and patterns of age-related and noise-induced hearing loss (Blockley et al., 2022).Of course, specific findings in insects can not be simply leapfrogged into human trials.Rather, we hope that insects can tackle fundamental problems and elucidate basic processes and, in doing so, build strong foundations of scientific understanding, which can be built on in mammalian models before clinical trials in humans.

Figure 1 .
Figure 1.Evolutionary time line plotting the approximate time of evolutionary divergence of hearing modelsThe earlier in time since the divergence, the more fundamental the processes that govern hearing organ physiology and genetics.

Figure 2 .
Figure 2. Schematic of auditory systems in insects and vertebrates with functionally equivalent areas colour matchedAll auditory systems require the sound energy to be captured, either through tympani or antennae.Sound-induced displacements are transferred to the primary auditory receptors (grey).A steep electrochemical gradient is maintained by ion-pumping cells (red), which recycle K + and other ions.Transduction potentials are converted into spikes and propagated to the central nervous system (blue).Mammals and some insects(Andrés et al., 2016) harbour efferent input to modulate auditory sensitivity (green), including to mammalian inner hair cells(Elliott et al., 2021).
Figure 3.Top: extent of hearing loss in humans exposed, and not exposed, to noise.Bottom: auditory nerve response in Locusts exposed, and not exposed, to noise (note that the auditory nerve axis has been inverted for comparison).Adapted fromCorso (1992) andBlockley et al. (2022).