Journal of Comparative Physiology A

Biomechanics of hearing in katydids --Manuscript Draft--

Manuscript Number: JCPA-D-14-00131R2 Full Title: Biomechanics of hearing in katydids Article Type: S.I. : Hearing - from Physics to Ecology Section/Category: Review Abstract: have evolved a vast diversity of mechanisms to detect sounds. Auditory organs are used to detect intraspecific communicative signals and environmental sounds relevant to survival. To hear, terrestrial animals must convert the acoustic energy contained in the airborne sound pressure waves into neural signals. In mammals, spectral quality is assessed by the decomposition of incoming sound waves into elementary frequency components using a sophisticated cochlear system. Some neotropical like katydids (bushcrickets) have evolved biophysical mechanisms for auditory processing that are remarkably equivalent to those of mammals. Located on their front legs, katydid ears are small, yet are capable of performing several of the tasks usually associated with mammalian hearing. These tasks include air-to-liquid impedance conversion, signal amplification, and frequency analysis. Impedance conversion is achieved by a lever system, a mechanism functionally analogous to the mammalian middle ear ossicles, yet morphologically distinct. In katydids, the exact mechanisms supporting frequency analysis seem diverse, yet are seen to result in dispersive wave propagation phenomenologically similar to that of cochlear systems. Phylogenetically unrelated, katydids and tetrapods have evolved remarkably different structural solutions to common biophysical problems. Here, we discuss the biophysics of hearing in katydids and the variations observed across different species. Corresponding Author: Fernando Montealegre-Z, PhD University of Lincoln Lincoln, UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: University of Lincoln Corresponding Author's Secondary Institution: First Author: Fernando Montealegre-Z, PhD First Author Secondary Information: Order of Authors: Fernando Montealegre-Z, PhD Daniel Robert Order of Authors Secondary Information: Author Comments: Dear Prof. Römer, Here is the last revised version of our ms after last editorial comments. We accepted the changes suggested by the editor, and also found a few other typos to sentences that needed improvement. Please note that the ms parts that have been newly edited or modified are shown in red fonts.

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1 Biomechanics of hearing in katydids 1 2 3 2 4 3 Fernando Montealegre-Z.1*, Daniel Robert2* 5 6 4 7 8 5 1 School of Life Sciences, University of Lincoln, Joseph Banks Laboratories 9 10 6 Green Lane, Lincoln, LN6 7DL, UK 11 2 12 7 School of Biological Sciences, Life Science Building, 24 Tyndall Avenue, 13 14 8 University of Bristol, Bristol, BS8 1TQ, UK 15 9 *Authors for correspondence ([email protected]), 16 17 10 ([email protected]) 18 19 11 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2

12 Abstract: Animals have evolved a vast diversity of mechanisms to detect 1 2 13 sounds. Auditory organs are thus used to detect intraspecific communicative 3 4 14 signals and environmental sounds relevant to survival. To hear, terrestrial 5 15 animals must convert the acoustic energy contained in the airborne sound 6 7 16 pressure waves into neural signals. In mammals, spectral quality is assessed 8 9 17 by the decomposition of incoming sound waves into elementary frequency 10 11 18 components using a sophisticated cochlear system. Some neotropical insects 12 13 19 like katydids (or bushcrickets) have evolved biophysical mechanisms for 14 15 20 auditory processing that are remarkably equivalent to those of mammals. 16 21 Located on their front legs, katydid ears are small, yet are capable of 17 18 22 performing several of the tasks usually associated with mammalian hearing. 19 20 23 These tasks include air-to-liquid impedance conversion, signal amplification, 21 22 24 and frequency analysis. Impedance conversion is achieved by a lever system, 23 24 25 a mechanism functionally analogous to the mammalian middle ear ossicles, 25 26 yet morphologically distinct. In katydids, the exact mechanisms supporting 26 27 27 frequency analysis seem diverse, yet are seen to result in dispersive wave 28 29 28 propagation phenomenologically similar to that of cochlear systems. 30 31 29 Phylogenetically unrelated, katydids and tetrapods have evolved remarkably 32 33 30 different structural solutions to common biophysical problems. Here, we 34 35 31 discuss the biophysics of hearing in katydids and the variations observed 36 32 across different species. 37 38 39 33 Keywords: Cochlea. Insect hearing. Auditory mechanics. Impedance. Crista 40 41 34 acustica. 42 43 44 35 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3

36 In the kingdom many species must identify environmental sounds to 1 2 37 increase their chance of survival. Acoustic communication occurs in many 3 4 38 groups of animals, spanning the phylogeny from invertebrates to vertebrates, 5 39 and well-studied cases pertain to mammals, birds, amphibians and 6 7 40 (crustaceans and insects, Bradbury and Vehrencamp 1998). Yet, due to their 8 9 41 biological diversity, insect species constitute a large percentage of the 10 11 42 acoustic community that pervades many terrestrial habitats. Concrete 12 13 43 examples of bona fide communication are found in the cicadas, crickets, 14 15 44 katydids and grasshoppers. In these groups, males sing to attract conspecific 16 45 females, a process that opens up numerous possibilities for sexual selection 17 18 46 (Robinson and Hall 2002). As in field crickets, with few exceptions, katydid 19 20 47 males produce calls by tegminal stridulation: the scraping together of one 21 22 48 wing which possesses a vein modified with a series of small teeth against the 23 24 49 other wing which bears an edge in the anal margin that works as a scraper 25 50 (Morris 1999). However, unlike field crickets, which communicate at around 5 26 27 51 kHz, the sound frequencies exploited by katydids vary from 5 kHz to 150 kHz, 28 29 52 depending on the species (Montealegre-Z 2009; Sarria-S et al. 2014). 30 31 32 53 Besides sounds used for intraspecific communication, katydids are also 33 34 54 exposed to many other sounds that may require their attention. A large 35 36 55 number of tropical species are nocturnal and most species are a 37 56 good example of adaptation to nocturnal life. Ambient noise in typical katydid 38 39 57 habitats, tropical rainforests, increases considerably after sunset by about 20 40 41 58 decibels relative to daytime ambient noise (Lang et al. 2006). This 42 43 59 environmental noise consists of many sounds, contributing different 44 45 60 frequencies altogether. Some of these frequencies are permanent, occurring 46 61 day and night, such as the sounds of rivers (rapids and waterfalls), while other 47 48 62 sounds are temporary, transient and changeable, such as the rustlings and 49 50 63 songs of other nocturnal animals, rain, wind, etc. Thus, in addition to 51 52 64 conspecific sounds, many species can detect a wide range of frequencies. 53 54 65 This should encompass broadband sounds that are identified as potential 55 56 66 threats, such as ultrasound produced by insectivorous bats to hunt and 57 67 navigate at night in the clutter of forested environments, as well as the sound 58 59 68 produced by other nocturnal insectivorous mammals (Belwood and Morris 60 61 62 63 64 65 4

69 1987; Faure and Hoy 2000; Ramsier et al. 2012; Ratcliffe et al. 2011). The 1 2 70 Tettigoniidae ear has evolved in the context of intraspecific communication 3 4 71 and predator detection (e.g., Belwood and Morris 1987; Faure and Hoy 2000). 5 72 The aim of this paper is to present in detail the functional mechanics of 6 7 73 katydid hearing, drawing a parallel between the ear of the Tettigoniidae and 8 9 74 Tetrapods. 10 11 12 75 The basics of hearing 13 14 76 In order to detect sounds from the environment, an animal must be able to 15 16 77 convert the acoustical energy contained in sound pressure waves into 17 18 78 neuronal signals. Generally, this transduction process can be summarized as 19 20 79 follows: 1) Transformation of sound into mechanical vibration, using an 21 22 80 acousto-mechanical receiver structure like a tympanal membrane or an 23 24 81 appendage such an antenna or a long hair. 2) Coupling of this mechanical 25 82 energy to mechanosensory structures, and finally 3) mechanical and neural 26 27 83 analysis of the waveform in terms of signal frequency, amplitude and temporal 28 29 84 structure. There are several possibilities of signal amplification and filtering 30 31 85 along the hearing chain, for instance during sound capture the ear canal acts 32 33 86 as an exponential horn, boosting the sound pressure 30 to 100 fold for 34 35 87 frequencies around 3 kHz in humans. Amplification also occurs during 36 88 translocation of the mechanical energy via lever action of the ossicles in the 37 38 89 middle ear, or by motility of the mechanosensory cells (Purves et al. 2013; 39 40 90 Mhatre, this volume). 41 42 43 91 The auditory systems of many animals are capable of performing frequency 44 45 92 decomposition of complex waveforms. Such frequency analysis relies on 46 93 individual mechanosensory receptors to be responsive to a narrow range of 47 48 94 frequencies, or ideally, one frequency. Such tuning arises because of the 49 50 95 location of receptors on a physical substrate, the basilar membrane in 51 52 96 mammals, the crista acustica (CA) in katydids. In addition, tuning may also 53 54 97 result from intrinsic properties of the molecular machinery responsible for 55 56 98 signal transduction in the mechanosensory cell. Mechanically, place-specific 57 99 tuning has long been described and studied, a process called tonotopy (von 58 59 100 Bekesy 1960; Palgath Udajashankar 2012). The best known example is the 60 61 62 63 64 65 5

101 tonotopic organization of frequency sensitivity along the basilar membrane in 1 2 102 the mammalian cochlea (von Bekesy 1960; Ashmore 2008). Frequency tuning 3 4 103 within the inner ear results in part from the geometry of the basilar membrane, 5 104 which is wider and more flexible at the apex and narrower and stiffer at the 6 7 105 base. von Békésy (1960) showed that the basilar membrane vibrates 8 9 106 maximally at different positions as a function of the stimulus frequency. The 10 11 107 points responding to high frequencies are at the base of the basilar 12 13 108 membrane, and the points responding to low frequencies are at the apex, 14 15 109 giving rise to a topographical mapping of frequency, also known as tonotopy 16 110 (Robles and Ruggero 2001). In mammals, the sensory hair cells are 17 18 111 distributed in an orderly linear array along the length of the basilar membrane. 19 20 112 As a result, high frequency receptors are located at the base of the cochlea, 21 22 113 where basilar membrane stiffness is high, while low frequency receptors are 23 24 114 found at the apex, where basilar membrane stiffness is low. The mechanisms 25 115 giving rise to such mechanical frequency decomposition are still debated in 26 27 116 the details. However, it is believed that the ear's sensitivity arises from an 28 29 117 active biomechanical process, as well as from its passive resonant properties 30 31 118 (Purves et al. 2013; Mhatre, this volume). 32 33 34 119 Frequencies are thus represented along a stiffness gradient that is generally 35 36 120 regarded as smooth, but is not necessarily homogenous (Bruns and 37 121 Schmieszek 1980; Schnitzler and Denzinger 2011; Schuller and Pollak 1979). 38 39 122 In such non-smooth tonotopic systems, some frequency ranges can be 40 41 123 represented in more detail than others, a remarkable adaption often referred 42 43 124 to as the “acoustic fovea” by analogy to the visual system (Isobe and 44 45 125 Motokawa 1955). Such adaptation was unknown in invertebrate hearing, but 46 past and recent evidence suggest that an acoustic fovea might occur in the 47 126 48 127 katydid ear of some species (Oldfield 1982, Montealegre-Z et al. 2012). 49 50 51 128 Auditory systems performing frequency analysis using dedicated impedance 52 53 129 conversion and a fluid-filled dispersive medium were known only in higher 54 55 130 vertebrates like tetrapods. It was recently shown that katydids use a tetrapod- 56 57 131 like mechanism of hearing, involving the three canonical steps of hearing. The 58 132 presence of such mechanisms in insects constitutes a remarkable case of 59 60 133 convergent evolution between tetrapods and katydids (Montealegre-Z et al. 61 62 63 64 65 6

134 2012). Such convergence demonstrates that auditory sensitivity and 1 2 135 frequency analysis are possible for microscale auditory systems, using 3 4 136 analogous operating principles yet alternative morphological architecture. 5 6 137 In insects, the mechanisms that determine frequency selectivity of individual 7 8 138 auditory receptors are diverse. While many auditory insect species are known 9 10 139 to have some form of frequency selectivity, for most of them, the biophysical 11 12 140 mechanisms are little understood. 13 14 141 Generalized katydid ear anatomy 15 16 17 142 Katydids have their ear in the basal part of the fore tibia (Fig. 1). Each ear 18 19 143 presents paired eardrums; an anterior tympanal membrane (ATM) and a 20 21 144 posterior tympanal membrane (PTM), located on the proximal part of the tibia 22 23 145 in each foreleg (Fig. 1a). The tympana are partially backed by an air-filled 24 146 tube, the acoustic trachea (AcT), which extends forwards from the acoustic 25 26 147 spiracle in the prothorax through the femoral cavity of the foreleg, enters the 27 28 148 tibia (Fig. 1b), and divides into anterior and posterior branches (Bangert et al. 29 30 149 1998; Lewis 1974; Fig. 2c, d). In cross section, the katydid’s ear is 31 32 150 asymmetrical: the anterior branch occupies a large portion of the dorsal ear 33 34 151 surface (Fig. 2c, d; Rössler et al. 1994). The mechanoreceptors, comprised in 35 152 a long and thin structure, the CA, lie on the dorsal wall of this anterior tracheal 36 37 153 division, and are contained within the auditory vesicle (AV), a fluid-filled, 38 39 154 partially blind cavity (Stumpner and Nowotny 2014; Montealegre-Z et al. 2012; 40 41 155 Fig. 2c-e). Mechanoreceptors on the CA are tonotopically organized (Oldfield 42 43 156 1982), but the sensory cells are not directly in contact with the tympana as it is 44 45 157 often the case in other acoustic insects (e.g. locusts, flies and moths; Stephen 46 158 and Bennet-Clark 1982; Robert 2005; Yack 2004). 47 48 49 159 The mechanisms that together enable acute hearing and frequency selection 50 51 160 in katydid ears are presented here, step by step and in more detail. 52 53 54 161 Sound capture 55 56 162 In most vertebrates, the ear has one main input, whereby sound pressure acts 57 58 163 on the external surface of the tympanal membrane. In katydids, each ear has 59 60 164 three possible acoustic inputs: acoustic spiracle, and the two tympanal 61 62 63 64 65 7

165 membranes (Michelsen and Larsen 1978). In these insects sound can reach 1 2 166 the external surface of the tympanal membrane and/or reach the internal 3 4 167 surface of the tympanal membrane through specialised tracheal pipes. Such 5 168 tracheal ducts establish a sound passage to the acoustic (or auditory) spiracle 6 7 169 that is usually followed by a tracheal expansion known as the auditory bulla 8 9 170 (Hill and Oldfield 1981; Bailey 1991). The size of the auditory spiracle and 10 11 171 bullae vary across species, and influences sound capture and acoustic energy 12 13 172 gain. Different from gryllids (Michelsen et al. 1994b), the acoustic trachea 14 15 173 starting at the acoustic spiracle is not connected in the middle by a septum, 16 174 but the bullae are usually separated (Bailey 1990, Fig. 1a). Most Tettigoniidae 17 18 175 species exhibit vestiges of the ventilatory system as a form of a filament that 19 20 176 connects the two bullae. But in some species the connection between the two 21 22 177 acoustic bullae is open, and consists of a series of narrow channels from one 23 24 178 bulla to the other (Bailey 1990). In field crickets the two trachea are clearly 25 179 connected by a thin membrane, and this design is the anatomical basis for a 26 27 180 pressure difference receiver ear (Michelsen et al. 1994b; Hirtenlehner et al. 28 29 181 2014), however, in katydids the filament or narrow channel connections have 30 31 182 apparently no acoustic function. Some katydid species exhibit large bullae in 32 33 183 complete contact through a large surface area (Bailey 1990); the acoustic 34 35 184 adaptation of this morphology is unknown. 36 37 185 In species with large acoustic spiracles and large auditory bullae adjacent to 38 39 186 the spiracle (Fig. 1), the AcT represents the main input for sound capture 40 41 187 (Heinrich et al. 1993). In these species (e.g., Decticus albifrons, D. 42 43 188 verrucivorus (Decticinae); Tettigonia viridissima, T. cantans (Tettigoniinae); 44 45 189 Ephippiger ephippiger, Ephippigerida taeniata (Ephippigerinae); Mygalopsis 46 190 marki (Conocephalinae), Poecilimon thessalicus, P. laevissimus ), and in 47 48 191 some others, both internal and external inputs are functional and both 49 50 192 produce different gains and time delays that enhance directional hearing (Hill 51 52 193 and Oldfield 1981; Michelsen et al. 1994a). For example, in these species the 53 54 194 AcT acts as an exponential horn promoting a gain of ca. 10-30 dB depending 55 56 195 on the species. Nevertheless, it has also been found that sound propagates 57 196 with lower speed inside the trachea than it does in air. Hence, the signal 58 59 197 reaches the external surface of the tympanum travelling at normal sound 60 61 62 63 64 65 8

198 speed in air (ca. 340 m/s), while the same signal travels inside the AcT a 1 2 199 lower speed and arrives to the internal tympanal surface a few microseconds 3 4 200 later and with different vibrational phase (Schiolten et al 1981; Michelsen et al. 5 201 1994a, Michelsen and Larsen 2008). Each tympanic membrane will therefore 6 7 202 experience two events to capture a single signal: the first event (external 8 9 203 input) occurs when the tympanum collects the signal travelling at normal 10 11 204 sound speed and arriving at its external surface (with low amplitude as no 12 13 205 amplification occurs to the airborne signal prior to its arrival at the external 14 15 206 tympanal surface), the second event (tracheal input) is experienced when the 16 207 same signal travels inside the trachea at lower sound speed than the external 17 18 208 input. This delayed signal reaches the tympanal internal surface a few micro- 19 20 209 seconds later than the external input, but would exhibit high amplitude as the 21 22 210 signal has been amplified in the trachea. Both events can be observed 23 24 211 recording tympanal vibration using Laser Doppler Vibrometry (LDV- Schiolten 25 212 et al 1981; Michelsen et al. 1994a, Montealegre-Z and Robert unpublished 26 27 213 data). The AcT therefore has a vital function in directional hearing, involving 28 29 214 remarkable pressure difference mechanisms (Autrum 1940; Hill and Oldfield 30 31 215 1981; Michelsen and Larsen 2008). Although these mechanisms have been 32 33 216 studied in crickets (Michelsen et al. 1994b) and katydids (Schiolten et al. 34 35 217 1981), the neural processing of this pressure-gradient-receiver system is 36 218 poorly understood. 37 38 39 219 The benefits of a tracheal sound input in katydids may be multiple, first 40 41 220 producing sound amplification (Hill and Oldfield 1981; Heinrich et al. 1993; 42 43 221 Hoffmann and Jatho 1995; Michelsen et al. 1994a; Shen 1993) prior to its 44 45 222 capture by the tympanal membrane. This would be analogous to the role of 46 223 the mammalian pinna and ear canal. As explained above, tracheal input might 47 48 224 also enhance directional hearing in a pressure difference mechanism. 49 50 225 However, besides this sound transmission role, the AcT also serves to 51 52 226 equilibrate atmospheric pressure in both sides of the tympana, just as in the 53 54 227 Eustachian tubes in terrestrial tetrapods. 55 56 228 In species with small thoracic spiracles, such as most Pseupdophyllinae, the 57 58 229 situation appears to be different. In the external input to the 59 60 230 TMs seems to dominate the total driving force to the eardrum, at least at 61 62 63 64 65 9

231 some frequencies (Mason et al. 1991), raising the question of how directional 1 2 232 hearing is achieved using a pressure receiver only. Interestingly, earlier 3 4 233 studies on the ultrasonic neotropical species Myopophyllum speciosum, 5 234 Haenschiella ecuadorica and Typophyllum sp. (Mason et al. 1991; Morris et 6 7 235 al. 1994) suggest that the tympanal flaps act as resonating chambers which 8 9 236 affect the acoustic pressure reaching the external face of the tympanum. 10 11 237 Potentially, pressure could be altered through diffractive effects, with some 12 13 238 possible consequences for the timing at which acoustic pressure imparts force 14 15 239 on the eardrums. In the palaeotropical species Onomarchus uninotatus, a 16 240 katydid exhibiting a narrow bandwidth call with unusual low carrier frequency 17 18 241 of 3.2 kHz, acoustic partitioning between the two tympanal membranes has 19 20 242 been documented (Rajaraman et al. 2013). While the ATM acts as a low-pass 21 22 243 PTM acts as a 23 24 244 high-pass filter. The PTM which shows maximal sensitivity at several broad 25 245 frequenc This unusual feature of 26 27 246 peripheral auditory processing is poorly understood. 28 29 30 247 The role of the AcT in the Pseudophyllinae is not clear. The results of Mason 31 32 248 and Morris (1991) suggest that the acoustic spiracle and narrow trachea of 33 34 249 species using very high carriers is linked with predominant sound access via 35 36 250 the tympanal slits. But similar spiracle anatomy and narrow acoustic bullae 37 251 also occur in other Pseudophyllinae using low frequencies (e.g., Heller 1995; 38 39 252 Rajaraman et al. 2013). Thus it is not clear what effect the AcT may have on 40 41 253 auditory sensitivity and directionality. If not acoustically functional, the narrow 42 43 254 acoustic bullae in Pseudophyllinae might serve only to equilibrate atmospheric 44 45 255 pressure at both sides of the tympana. This could have been the initial 46 256 function of the tracheal system associated with the ear before adaptations to 47 48 257 collect, conduct and amplify sounds evolved. 49 50 51 258 The tettigoniidae also exhibit variation in the external morphology of the 52 53 259 tympanal organ. As mentioned above in some species the tympanal organ 54 55 260 exhibits cuticular folds surrounding the tympanum (Figs. 1 and 2c, d). Other 56 261 species present a cuticular fold surrounding the anterior tympanum only, while 57 58 262 the opposite tympanum is completely or partially exposed. In many other 59 60 263 species (e.g., most Phaneropterinae katydids) both tympanic membranes are 61 62 63 64 65 10

264 exposed. The presence of cuticular folds around the tympanam has received 1 2 265 attention by some researchers. Autrum (1940, 1963) implied that cuticular 3 4 266 folds and tympanal slits help the insect to detect the direction of the sound. 5 267 Subsequently Bailey and Stephen (1978), Stephen and Bailey (1982), Bailey 6 7 268 et al. (1988) supported Autrum's and demonstrated that the tympanal slits and 8 9 269 cuticular folds cold function as sound guides to enhance directional hearing in 10 11 270 some species. 12 13 14 271 Transmission of acoustic energy from air to fluid: the katydid middle ear 15 16 272 Once acoustic energy has been converted into tympanal mechanical 17 18 273 vibrations, the important process of impedance conversion is required to 19 20 274 enable the efficient transmission of mechanical energy from the air to the fluid 21 22 275 medium where the mechanosensory cells reside. The morphological solution 23 24 276 to this process is the hallmark of the evolution of hearing in mammals, as 25 277 middle ear ossicles are highly specialised, differ from species to species, and 26 27 278 are the key to efficient hearing. In insects, the importance of impedance 28 29 279 conversion has received little attention, as often the tympanal membrane is 30 31 280 directly connected to the chordotonal mechanosensory organ (e.g., moths, 32 33 281 locust; Field and Matheson 1998). For insect species for which signal 34 35 282 frequency composition is relevant, such as katydids, this process has been 36 283 recognised to be important (Bangert et al. 1998, ) as it holds the key to 37 38 284 efficient and frequency selective hearing. 39 40 41 285 For decades the role of the tympanal membranes in the hearing process has 42 43 286 been a topic of interest for some researchers (e.g. Michelsen and Larsen 44 45 287 1978; Oldfield 1985, Mhatre et al 2009; Notwotny et al 2010; Montealegre-Z et 46 288 al. 2012). While in the locust the tympanum exhibits different resonant 47 48 289 frequencies and vibrates with complex modes that code for travelling waves 49 50 290 and frequency selectivity (Michelsen 1971; Windmill et al. 2005), the tympanal 51 52 291 membranes of the katydid ear vibrate in a single mode (Michelsen et al. 53 54 292 1994a; Bangert et al. 1998; Nowotny et al. 2010). 55 56 293 Two models have been proposed to explain the transmission of mechanical 57 58 294 energy from the TMs to the CA in the katydid ear. Both models are based on 59 60 295 the law of levers. A lever is a movable bar that pivots on a fulcrum attached to 61 62 63 64 65 11

296 a fixed point. The ratio of the output force to the input force is the mechanical 1 2 297 amplification of the lever (Vogel 2013). Levers are relevant in biological 3 4 298 systems because they help to amplify an input force to provide a greater 5 299 output force. 6 7 8 300 The first model was proposed by Bangert et al. (1998), described as hinged- 9 10 301 flap system. Bangert and co-workers proposed an impedance conversion 11 12 302 mechanism of airborne acoustic energy to a dispersive medium, which 13 14 303 predicts a force conversion between tympanum and the elastic surface of the 15 304 tracheal wall bearing the CA. Such conversion changes the fluid space above 16 17 305 the CA. This model however does not use TP motion. Bangert et al. (1998) 18 19 306 hinge model is therefore based on a class 2 lever (a lever with the fulcrum in 20 21 307 one end, the applied force in the other end, and the resulting force in the 22 23 308 middle, Vogel 2013), in which a hinge is meant to move a load between the 24 25 309 fulcrum and the force. The force gain will be lower close to the hinge, but 26 310 effective at the location of the load (in the tympanal organ the load should be 27 28 311 an area of contact between the tympanum and the elastic dorsal wall of the 29 30 312 trachea; Fig. 2f). A similar model was supported by Nowotny et al. (2010) in 31 32 313 their studies of tympanal motion in M. elongata. In their study of M. elongata, 33 34 314 in which no middle ear was observed, Palghat Udayashankar et al. (2012) 35 36 315 conjectured that pressure waves travelling in the trachea activate vibrations of 37 316 the CA internally before activating the tympanic membranes, i.e., sound 38 39 317 enters the hearing organ at the proximal part of the leg, where low 40 41 318 frequencies are represented. Their findings imply that slow waves were 42 43 319 transmitted first to the proximal part of the CA, and from there, vibrations 44 45 320 travelled distally, and then proximally again as travelling waves. This contrasts 46 321 with the out-of-phase response between TMs and TPs observed in Copiphora 47 48 322 gorgonensis (Montealegre-Z et al. 2012), revealing a type 1 lever action (a 49 50 323 lever with the applied force in one end, the resulting force in the other, and the 51 52 324 fulcrum in the middle, Vogel 2013). 53 54 55 325 A second model of impedance conversion in the tympanal organ of the 56 326 katydid C. gorgonensis was proposed by Montealegre-Z et al. (2012). These 57 58 327 authors state that impedance conversion is a functional part of the katydid 59 60 328 ear, and is analogous to the mammalian middle-ear process. This part of 61 62 63 64 65 12

329 mechanical auditory processing is carried out by a lever-like structure, the TP 1 2 330 (Fig. 2a, b, e). The TP enables the coupling of sound-induced vibrations from 3 4 331 the TMs to the AV and CA. Montealegre-Z et al. (2012) showed that the TM 5 332 and TP are linked but are distinct structures in C. gorgonensis (Fig. 2a, b). 6 7 333 The TM is a thin membrane (6-16 µm), which presents both sides to air, while 8 9 334 the TP is thicker (20-30µm), and has one side facing air and the other applied 10 11 335 to the fluid of AV (Fig. 2c-e). Montealegre-Z et al. (2012) suggested that these 12 13 336 two structures operate like a type 1 lever model, a type of seesaw with an 14 15 337 eccentric fulcrum (Fig. 2e). Given the proportions of the TM and TP in C. 16 338 gorgonensis, such a lever system should produce a conversion ratio of 1:10 17 18 339 between effort (TM) and load (TP and fluid). 19 20 21 340 The contour of the TM is distinctly kidney shaped in the katydids studied by 22 23 341 Montealegre-Z et al. (2012), with the TP located near the dorsal curvature of 24 25 342 the shape (Fig. 2a, b). Without empirical evidence Montealegre-Z et al. (2012) 26 343 believe this specific and unusual tympanal shape serves to channel vibrations 27 28 344 to the distal part of the AV and CA via the TP. Such kidney shaped TMs in 29 30 345 some but not all katydid species may be the hallmark of impedance 31 32 346 conversion and frequency analysis. More research is needed in this area. 33 34 35 347 Bangert et al. (1998), Hummel et al. (2011), Palghat Udayashankar et al. 36 348 (2012), and Montealegre-Z et al. (2012) showed that part of the tympanum, 37 38 349 the TP, was in contact with the hemolymphatic fluid in the species studied by 39 40 350 them. A key difference between the P. denticauda, the Mecopoda and the C. 41 42 351 gorgonensis studies resides in the type of lever mechanism used to help 43 44 352 vibrations enter the fluid environment of the sensory organ proper. For P. 45 46 353 denticauda and M. elongata, the vibrations of both TM and TP are in phase 47 354 and the pivot point seems to lay on the dorsal edge of the TP. The dorsal 48 49 355 edge of the TP is hinged to the cuticle of the leg and its displacement 50 51 356 amplitudes are lower than those of the ventral part of the TM (Fig. 2f, Bangert 52 53 357 et al. 1998; Nowotny et al. 2010; Palghat Udayashankar et al. 2012). 54 55 56 358 A class 2 lever has been documented in more primitive forms of , like 57 359 Anoatostomatidae (e.g., weta, Lomas et al. 2011), and occur in 58 59 360 Prophalangopsidae (e.g., Cyphoderris spp. FMZ and DR unpublished data), 60 61 62 63 64 65 13

361 and perhaps in other ensifera with large and centralised TPs. Weta do not 1 2 362 have tracheal inputs, thus the tympanal organ seems to function as a normal 3 4 363 pressure receiver. The weta TMs are oval or nearly rounded, non-taut, and 5 364 bear a large sclerotized oval TP. This TP is not isolated from the membrane 6 7 365 contour like the TP in C. gorgonensis but is embedded within the membrane. 8 9 366 Both TMs deflect like a hinge, with the same phase as the TPs (Lomas et al. 10 11 367 2011). Different from the weta's ear, the TP of C. gorgonensis vibrates in 12 13 368 antiphase with the TMs. Large tympanal displacements produced with low 14 15 369 force exert a large force of the small area of the TP, which deflects with small 16 370 displacement (Montealegre-Z et al. 2012). This is known as mechanical 17 18 371 advantage. 19 20 21 372 The weta's mechanism of impedance conversion is unknown. It is also 22 23 373 unknown if these insects need to analyse frequencies, however in cross 24 25 374 section the tympanal organ is asymmetrical and shows a CA (Ball and Field 26 375 1981; Lomas et al. 2012). Wetas usually live in galleries, which suggests they 27 28 376 are not often exposed to insectivorous bats and perhaps that they do not need 29 30 377 to resolve a broad range of frequencies. In fact, the audiograms show their 31 32 378 frequency sensitivity is very limited and low (2.0-2.5 kHz; Field et al. 1980). 33 34 35 379 36 37 380 Spectral decomposition of the system and travelling waves: the katydid 38 39 381 inner ear 40 41 42 382 Oldfield (1985) established that the tympanic membranes do not contribute to 43 44 383 frequency selectivity in the katydid Mygalopsis marki. More recently Hummel 45 384 et al. (2011) demonstrated that tympanum motion and neuronal response are 46 47 385 not coupled directly. Thus frequency decomposition happens somewhere 48 49 386 else. 50 51 52 387 The katydid inner ear is composed of the CA (bearing the fluid-immersed 53 54 388 mechano-receptors), and the AV (containing the fluid bathing the 55 389 mechanoreceptor, Fig. 3). The coupling of the AV and CA was only recently 56 57 390 shown, and previous researchers thought of the AV as a simple continuation 58 59 391 of the hemolymph channel (HC - e.g., Schumacher 1973, 1975; Rossler et al 60 61 62 63 64 65 14

392 1994). In the species documented by Montealegre-Z et al. (2012), and some 1 2 393 others studied by the authors using the proposed methods, the AV connects 3 4 394 with the leg haemolymph supply through a narrow constriction (Fig. 3). 5 395 However in Metrioptera sphagnorum, such a connection was not observed, 6 7 396 and the AV seems to be isolated (Fig. 3a-e). In the conocephaloid katydids 8 9 397 Pancanthus pallicornis and C. gorgonensis a plug of unknown colloidal 10 11 398 material is observed in the proximal and distal ends of the AV (Fig. 3e). In a 12 13 399 similar way, the fluid contained in the AV appears not to be pure hemolymph. 14 15 400 Apolar extraction and comparison of this fluid with hemolymph taken from 16 401 different body regions suggests that the fluid might contains lipids 17 18 402 (Montealegre-Z et al. 2012). In wetas, a plesiomorphic group related to 19 20 403 modern katydids (Mugleston et al. 2013), the hemolymphatic fluid bathing the 21 22 404 mechanoreceptors is rich in lipids (Lomas et al. 2012). Although the chemical 23 24 405 composition of the AV fluid in the katydid species studied so far by the authors 25 406 is unknown, it is known that the AV is an important component of the katydid 26 27 407 tympanal organ as it provides a medium for wave propagation. The AV fluid 28 29 408 could also enhance frequency decomposition and produce an additional step 30 31 409 for signal amplification or energy localisation. In the mammalian cochlea for 32 33 410 example, the passive vibrations of the basilar membrane are the product of 34 35 411 different factors, which include not only the flexibility and mass of the basilar 36 412 membrane and organ of Corti, but also the physical properties of the adjacent 37 38 413 fluid (Robles and Ruggero 2001). 39 40 41 414 Travelling waves and mechanical tonotopy in the tympanal organ of the 42 43 415 katydid M. elongata were shown for the first time by Palghat Udayashankar et 44 45 416 al. (2012). They exposed the anterior tracheal branch containing the CA by 46 417 removing the dorsal cuticle and AV fluid (regarded as hemolymph), replaced 47 48 418 the AV fluid by insect ringer solution to avoid desiccation and stimulated the 49 50 419 ear by sound. They isolated the tympanal organ from the tracheal input using 51 52 420 a special platform, and monitored CA vibrations using LDV. Input isolation is 53 54 421 necessary here because if the dissected tympanal organ with exposed CA is 55 56 422 presented with sound in an open field, it is impossible to control for air-borne 57 423 sound reaching and stimulating the CA surface directly, producing unreliable 58 59 424 results. Palghat Udayashankar and co-workers clearly observed travelling 60 61 62 63 64 65 15

425 waves and frequency decomposition on a CA surface covered with a gentle 1 2 426 layer of insect ringer. Vibrations travel from the narrowest part (distal part) of 3 4 427 the CA to the broader proximal region; high frequencies are represented at 5 428 the narrow end, and low frequencies cells at the broader proximal end. 6 7 429 Intermediate frequencies were observed between both ends. Such gradient 8 9 430 and direction corresponds with the tonotopically ordered mechanoreceptors 10 11 431 (Oldfield 1982; Römer 1983; Stolting and Stumpner 1998). Remarkably, this 12 13 432 mechanical behaviour can be measured in the absence of the dorsal cuticle, 14 15 433 an indication that the decomposition of frequencies results from an intrinsic 16 434 mechanical property of the CA. 17 18 19 435 In the study of C. gorgonensis (Montealegre-Z et al. 2012), travelling waves 20 21 436 and mechanical tonotopy were recorded through the dorsal cuticle (see 22 23 437 discussion below). However this study showed that removal of the dorsal 24 25 438 cuticle, or simply the removal of the liquid in the vesicle subjacent to the 26 439 dorsal cuticle, is sufficient to obliterate the build-up of travelling waves and, 27 28 440 therefore the resulting tonotopic response (as measured through the dorsal 29 30 441 tibial cuticle). The differences between these lines of evidence call for more 31 32 442 studies of the tympanal organ across species of different subfamilies. 33 34 35 443 Montealegre-Z et al. (2012) designed an experiment to obtain vibrations of the 36 444 CA surface and of both TMs simultaneously to investigate the effect of TM 37 38 445 deflection on CA motion. They used a special isolating platform that isolated 39 40 446 the spiracular input from the tympanal input. In some of their experiments the 41 42 447 CA was exposed following the protocols used by Palghat Udayashankar et al. 43 44 448 (2012), and its vibration in response to acoustic stimulation recorded using 45 46 449 LDV. Tympanal vibrations were then stimulated by delivering sound uniquely 47 450 at the acoustic spiracle. In those experiments when the CA was exposed, the 48 49 451 isolating platform setup ensured that vibrations recorded from the CA surface 50 51 452 were the sole product of AcT input to the auditory system, and not of airborne 52 53 453 sound reaching the exposed dorsal surface of the CA or other adjacent 54 55 454 cuticular structures. Using this setup, three tests were performed; 1) leave the 56 455 AV intact and record AV and presumed CA activity through the dorsal cuticle 57 58 456 using LDV, 2) drain the AV of its hemolymphatic fluid through a small lateral 59 60 457 perforation using a sharp glass micro-pipette and record from the dorsal 61 62 63 64 65 16

458 cuticle using LDV; and 3) remove the dorsal cuticle and AV entirely and gain 1 2 459 direct optical access to the thin tracheal wall that bears the CA, following the 3 4 460 dissection procedure of Palghat Udayashankar et al. (2012). Vibrometric 5 461 measurements of the AV and CA recorded through the dorsal cuticle show 6 7 462 that dispersive wave propagation, and therefore frequency decomposition, 8 9 463 only occurs in the presence of an intact AV (test 1). Removing the AV fluid 10 11 464 through a lateral perforation (test 2) and recording activity though the dorsal 12 13 465 cuticle eliminates travelling waves. Removing the dorsal cuticle evacuates the 14 15 466 AV fluid by default (test 3). In C. gorgonensis this procedure eliminates 16 467 travelling waves as measured from the exposed CA surface; contrary to that 17 18 468 observed by Palghat Udayashankar et al. (2012, 2014) in M. elongata. With 19 20 469 this procedure the measurements of Montealegre-Z et al. (2012) on the 21 22 470 vibrational activity of the CA surface is irregular and unclear in most cases. 23 24 471 However, vibrational responses were observed in the middle region of the CA 25 472 surface (Montealegre-Z et al. 2012: fig S7) which was very sensitive to 23 kHz 26 27 473 (the frequency of the calling song, Montealegre-Z and Postles 2010), but no 28 29 474 obvious travelling waves were observed. Consequently, in C. gorgonensis the 30 31 475 integrity of the AV is necessary to enhance the appropriate propagation of 32 33 476 waves. 34 35 36 477 Palghat Udayashankar et al. (2012) suggest that travelling waves result from 37 478 the smooth gradient in the mechanical properties and the geometry of the 38 39 479 tracheal wall containing the CA. This conjecture is reasonable, in fact Fig. 2 40 41 480 (c, d) show that the tracheal branch holding the CA is thicker in its narrowest 42 43 481 end (distal end or high frequency region) than proximally (the low frequency 44 45 482 region). In the basilar membrane of the mammals similar gradient or 46 483 mechanical anisotropy causes the observed tonotopy (Vater and Kössl 2011). 47 48 49 484 The fact that in C. gorgonensis travelling waves are detected through the 50 51 485 cuticle when the AV is intact, suggests the AV fluid is important as a 52 53 486 dispersive medium, just like the cochlear fluids in tetrapods. In mammals for 54 55 487 instance, cochlear tonotopy can be demonstrated in vitro and by replacing the 56 488 cochlear fluids. The spatial frequency analysis in the cochlea arises from the 57 58 489 passive mechanical properties of cochlear fluids and tissues (Robles and 59 60 490 Ruggero 2001). 61 62 63 64 65 17

491 Recording CA and AV vibrations through the dorsal cuticle 1 2 3 492 Montealegre-Z et al. (2012) first reported that travelling waves could be 4 493 recorded through the dorsal cuticle using LDV. The recordings of cuticular 5 6 494 vibration of Montealegre-Z et al. (2012) also show an area that is highly 7 8 495 sensitive to frequencies in the range of the calling song of C. gorgonensis 9 10 496 (around 23 kHz, Montealegre-Z et al. 2012: fig. S5). These experiments have 11 12 497 been repeated in other katydid species of different subfamilies, and with 13 14 498 variable calling carrier frequencies, showing similar results (Fig. 4). One 15 499 wonders how and why the cuticle shielding the hearing organ vibrates. Using 16 17 500 the methods established by Montealegre-Z et al. (2012) and Montealegre-Z 18 19 501 (2014), the authors went further and conducted the following recordings in 18 20 21 502 females of C. gorgonensis: a specimen mounted in a free sound field was 22 23 503 presented with a 23 kHz pure-tone of variable sound pressures between 5 24 25 504 and 40 Pa. The amplitude responses of the TMs and dorsal cuticle were 26 505 recorded using LDV and compared. Then the tracheal input of the specimen 27 28 506 was occluded using the sound isolating platform proposed by Montealegre-Z 29 30 507 et al. (2012). In both sets of data we observed that the amplitude of the 31 32 508 vibrations recorded ‘on’ the dorsal cuticle exceeded those of the tympanic 33 34 509 membrane (Fig. 5a, b). These observations came as a surprise as the dorsal 35 cuticle in C. gorgonensis is 10-20 times thicker than the TMs themselves (Fig. 36 510 37 511 2c, d). The thickness of that dorsal wall would not allow for mechanical 38 39 512 deflections of such amplitudes. 40 41 42 513 After studying the tympanal organ of different katydid species from different 43 44 514 subfamilies using the same LDV protocols established by Montealegre-Z et al 45 46 515 (2012) we noticed that CA activity cannot be recorded by dorsal cuticle 47 516 measurements in a large number of them (Table 1). This brought us to 48 49 517 compare the cuticle structure across the species studied and found that those 50 51 518 species that allow the recording of travelling waves on the dorsal cuticle have 52 53 519 a very transparent cuticle; while those in which cuticle vibration cannot be 54 55 520 recorded using LDV exhibit a more sclerotized dorsal cuticle (Table 1). 56 57 521 In other words, the cuticle of the leg covering the CA and mechanoreceptors 58 59 522 in C. gorgonensis and a few other species is not completely sclerotized, and 60 61 62 63 64 65 18

523 therefore highly transparent to visible light. In many other species (Table 1) 1 2 524 the dorsal cuticle is opaque and/or pigmented, therefore impeding the laser 3 4 525 light to enter. One of the properties of insect cuticle is transparency and this is 5 526 particularly useful in light detection and vision. The compound eyes and ocelli 6 7 527 in all insects, and the infrared receptor organs of fire beetles are examples of 8 9 528 transparent cuticles, usually associated with the absence of exocuticle 10 11 529 (Vondran et al. 1995; Klowden 2008; Schmit 1995; Mark Klowden 2013). It is 12 13 530 unclear why in some katydids the ear dorsal cuticle is transparent. This 14 15 531 property should be further studied in katydids as it offers a unique opportunity 16 532 to access the CA and AV in a non-invasive manner. The techniques available 17 18 533 so far for accessing the CA require removal of the dorsal cuticle and AV. 19 20 21 534 One could also think that the dorsal cuticle is thin enough to allow the 22 23 535 experimenter to measure the vibrations of the AV surface, as originally 24 25 536 believed (Montealegre-Z et al. 2012). However, when measurements of the 26 537 thickness of both dorsal cuticle and tympanal membrane in C. gorgonensis 27 28 538 are compared, the dorsal cuticle is approximately 10-20x thicker than the TMs 29 30 539 in this species. Such a thick wall of small area would tend to resonate at high 31 32 540 frequencies and oscillate at low amplitudes. The hypothesis of thin walls 33 34 541 permeable to vibrations can be discarded because vibration recorded from the 35 36 542 dorsal cuticle could be obtained from several species with variable cuticle 37 543 width and transparent cuticle, but not in species with dull cuticles and variable 38 39 544 thickness (Tabe 1). Figure 3 (c, f, i) shows variable thicknesses of the ear’s 40 41 545 dorsal cuticle and TMs in three species of katydid belonging to three different 42 43 546 subfamilies, in which CA vibrations can be accessed through a transparent 44 45 547 cuticle using LDV as shown in Fig. 4. 46 47 548 These observations imply that the vibrations obtained via the dorsal cuticle 48 49 549 shown by Montealegre-Z et al. (2012) and those presented in Fig. 4 of this 50 51 550 document, might come from the katydid inner ear. The transparent cuticle and 52 53 551 translucent AV fluid seem to allow the laser light to cross through and reflect 54 55 552 from the actual CA surface. Evidence for this is that the TPs vibration is in 56 553 phase with vibration of the exposed CA, as recorded in the most sensitive 57 58 554 region at 23 kHz (which still responds to vibration after ablation of the dorsal 59 60 555 cuticle, see Montealegre-Z et al. 2012 supplementary information), and with 61 62 63 64 65 19

556 the vibrations obtained through the dorsal cuticle (Fig. 6a). The phase of the 1 2 557 ATM and PTM is always opposite to the TP and CA (Fig. 6b, c). These results 3 4 558 were presented by Montealege-Z et al. (2012, supplementary information). 5 6 559 The pressure release system in the ears of katydids 7 8 9 560 The katydid and tetrapod ears both have a mechanism of pressure release. In 10 11 561 the mammalian cochlea, the vibrations transmitted by the ossicles through the 12 13 562 oval window cause a change in pressure of the cochlear fluid. Since the fluid 14 563 is incompressible, pressure changes are released though a little membranous 15 16 564 opening, the round window, which vibrates with opposite phase to vibrations 17 18 565 entering the inner ear through the oval window. The AV in C. gorgonensis, 19 20 566 and in some other katydid species studied, is blind in the distal end, while the 21 22 567 proximal ends usually exhibits a narrow connection with the HC (Fig. 3). Since 23 24 568 the fluid is incompressible, changes in pressure in the AV cavity should be 25 569 released somewhere. Montealegre-Z et al. (2012) originally associated the 26 27 570 high resolution of travelling waves with the vibrations recorded through the 28 29 571 dorsal cuticle with a pressure release mechanism. They believed that the 30 31 572 dorsal cuticle served as a dispersive medium to release the acoustic energy in 32 33 573 the inner ear of the katydid, and that it played the role of the round window in 34 35 574 mammals. As mentioned in the previous section, the vibrations recorded on 36 575 the dorsal cuticle were likely obtained directly from the katydid ‘inner ear’, 37 38 576 perhaps from the CA surface. So the ear’s dorsal cuticle seems not to function 39 40 577 as a dispersive pressure release medium. 41 42 43 578 The ‘round window role’ in the katydid ear seems to be played by the 44 45 579 connection between AV and HC. The anatomy of the AV varies across 46 580 species and also its connection with the HC. In some species this constriction 47 48 581 is narrow but in others it is broader, but in both situations the connection 49 50 582 seems to be mediated by a soft plug of colloidal material. One would expect 51 52 583 excess vibration to be scattered towards the HC and less detected through 53 54 584 the cuticle. Among the 7000 species of katydids, it is very likely to find a range 55 56 585 of variability. 57 58 586 Acknowledgements 59 60 61 62 63 64 65 20

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798 Stumpner A, Nowotny M (2014) Neural processing in the bush-cricket auditory 1 799 pathway. In: Hedwig B (ed) Insect hearing and acoustic 2 800 communication, vol 1. Animal signals and communication. Springer 3 4 801 Berlin Heidelberg, pp 143-166. doi: 10.1007/978-3-642-40462-7_9 5 802 Vater M, Kössl M (2011) Comparative aspects of cochlear functional 6 7 803 organization in mammals. Hear Res 273 (1-2):89-99. 8 804 doi:10.1016/j.heares.2010.05.018 9 805 Vogel S (2013) Comparative Biomechanics: Life's Physical World. 2 edn. 10 11 806 Princeton University Press, New Jersey 12 807 Vondran T, Apel KH, Schmitz H (1995) The infrared receptor of Melanophila 13 14 808 acuminata De Geer (Coleoptera: Buprestidae): ultrastructural study of 15 809 a unique insect thermoreceptor and its possible descent from a hair 16 810 mechanoreceptor. Tissue Cell 27 (6): 645-658. 17 811 doi:http://dx.doi.org/10.1016/S0040-8166(05)80020-5 18 19 812 Windmill JFC, Göspfert MC, Robert D (2005) Tympanal travelling waves in 20 813 migratory locusts. J Exp Biol 208 (1): 157-168. doi:10.1242/jeb.01332 21 22 814 Yack JE (2004) The structure and function of auditory chordotonal organs in 23 815 insects. Microsc Res Tech 63 (6): 315-337. doi: 10.1002/jemt.20051 24 25 816 26 817 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 26

818 Table 1. List of katydid species and ear attributes that facilitate non-invasive 1 2 819 access to the inner ear using laser vibrometry. (*) indicates those species in 3 4 820 which tonotopy and/or travelling waves have been recorded. Dorsal cuticle 5 821 transparency was observed in the ear living individuals under light microscope 6 7 822 but not physically measured. 8 9 10 Dorsal CA 11 Loca wall vibratio Comments 12 Species Subfamily Fc (kHz) 13 lity thickn n via on cuticle 14 ess cuticle 15 16 Copiphora gorgonensis* Copiphorinae COL 22.9 135 Yes Very transparent 17 Panacanthus pallicornis* Copiphorinae COL 4.7 200 Yes Very transparent 18 19 Moderately 20 Panacanthus lacrimans* Copiphorinae COL 7.0 189 Yes transparent 21 22 Panacanthus varius* Copiphorinae COL/ECU 9.0 211 Yes Very transparent 23 Artiotonus artius* 24 Copiphorinae COL 41.2 74 Yes Very transparent 25 Artiotonus tinae* Copiphorinae ECU 36.0 60 Yes Very transparent 26 Moderately 27 Supersonus aequoreus* Listroscelidinae COL 148.7 32 Yes 28 transparent 29 Moderately 30 Supersonus undulus Listroscelidinae ECU 117.0 40 Yes 31 transparent 32 33 Mecopoda elongata Phaneropteriane INDIA 6-80 116 No Opaque 34 Gnathoclita sodalis Pseudophyllinae COL 15.6 177 No Very opaque 35 36 Nastonotus foreli Pseudophyllinae COL 22.8 183 No Very opaque 37 Parascopioricus 38 Pseudophyllinae COL 28.0 150 No Opaque 39 cordillericus 40 Onomarchus uninotatus Pseudophyllinae INDIA 3.2 140 No Opaque 41 42 Moderately Metrioptera sphagnorum* Decticinae CAN 18 & 33 55 Yes 43 transparent 44 45 823 46 47 824 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 27

825 Figure legends 1 2 3 826 Fig. 1. General anatomy of the katydid ear. a 3D reconstruction of the body 4 827 based on Micro-CT scanning techniques, showing internal and external 5 6 828 structures of the ears of an insect in dorsal view. b Lateral view of the same 7 8 829 reconstruction showing the acoustic spiracle. Act= acoustic trachea; ATM= 9 10 830 anterior tympanic membrane; PTM=posterior tympanic membrane. 11 12 13 831 Fig. 2. The impedance conversion mechanism. a Photograph of the external 14 832 ear showing the tympanal membrane (TM) and tympanal plate (TP) in 15 16 833 Copiphora gorgonensis. b Vibration map of the TM and TP amplitude as 17 18 834 monitored by LDV. c, d μCT cross sections taken at the two different regions 19 20 835 in the fore tibia, distal and proximal, as indicated by the dashed lines. e Model 21 22 836 of impedance converter, using schematic cross section (c) and lever type 1 23 24 837 analogy, as proposed by Montealegre-Z et al. (2012). f Hinge model for 25 838 vibration transmission, based on a 2nd class lever, as proposed by Bangert et 26 27 839 al. (1998). 28 29 30 840 Fig. 3. Internal anatomy of the ear of three unrelated katydid species, 31 32 841 reconstructed with micro-CT scanning techniques. a, b 3D reconstruction of 33 34 842 the tympanal organ of Supersonus aequoreus; a katydid species using a 35 843 calling song of ca. 150 kHz. C. μCT cross section of the ear shown in a. d, e 36 37 844 3D reconstruction of the tympanal organ of Panacanthus pallicornis, a katydid 38 39 845 communicating with a broadband spectrum between 5 and 25 kHz. f μCT 40 41 846 cross section of the ear shown in d. g, h Metrioptera sphagnorum, a katydid 42 43 847 using two modes of stridulation, with dominant frequencies of 17 and 33 kHz, 44 45 848 respectively. i μCT cross section of the ear shown in g. 46 47 849 Fig. 4. Tonotopic organization of frequency response as recorded on the 48 49 850 dorsal cuticle in three species of Tettigoniidae incorporated in three different 50 51 851 subfamiles. The top part of each panel shows the magnitude of the response 52 53 852 (recorded with Laser Doppler Vibrometry) to acoustic broadband stimuli 54 55 853 (produced as periodic chirps) of variable frequency ranges depending on the 56 854 species. Specimens were mounted in a special holder and exposed to a free 57 58 855 acoustic field. Magnitude response spectra on the right side of the picture 59 60 856 were obtained from the regions indicated by the blue points, and labelled with 61 62 63 64 65 28

857 a number corresponding to respective spectrum on the right side. The lower 1 2 858 part of each panel shows the analysis of a representative calling song for 3 4 859 each species. a Panacanthus pallicornis: ear stimulated by periodic chirps 5 860 between 1-40 kHz. Spectra of magnitude response averaged from 12 6 7 861 specimens (seven males and five females). Calling song recordings and 8 9 862 analysis obtained from Montealegre-Z and Morris (2004). b Supersonus 10 11 863 aequoreous: ear stimulated by periodic chirps between 1-200 kHz). Spectra of 12 13 864 magnitude response recorded from a single female specimen. Calling song 14 15 865 recordings and analysis obtained from Sarria-S et al. (2014). c Metrioptera 16 866 sphagnorum: ear stimulated by periodic chirps between 1-40 kHz). Spectra of 17 18 867 magnitude response averaged from five specimens 3 females and two males. 19 20 868 Calling song recordings and analysis obtained from Morris (2008). For details 21 22 869 of experimental protocols see Montealegre-Z et al. (2012). 23 24 25 870 Fig. 5. The gain of the AV measured through the dorsal cuticle vs. the 26 871 tympanal gain in Copiphora gorgonensis. a Gain of the ATM and AV 27 28 872 measured with the specimen exposed to a free acoustic field to variable 29 30 873 sound pressures of pure tones at 23 kHz (the carrier frequency of the species 31 32 874 call, Montealegre-Z and Postles 2010). In this natural condition, both sides of 33 34 875 the tympanum are exposed to sound. b Similar experiment as above but the 35 36 876 tracheal input has been occluded using the isolating platform described by 37 877 Montealegre-Z et al. (2012) and Montealegre-Z (2014). Note that in both 38 39 878 situations the AV gain is considerably larger than the ATM and PTM gains. 40 41 879 The normal ranges of sound pressures used by a singing male as recorded 42 43 880 with the microphone placed at 10 cm is nearly 94dB (1 Pa). N= 21 individuals 44 45 881 (10 males and 11 females). Error bars show standard deviation. 46 47 882 Fig. 6. Mechanical response of tympanal system and sensory organ (crista 48 49 883 acustica). a Time domain and phase transfer function of the vibrations 50 51 884 recorded from the dorsal cuticle (DC) and from an exposed CA after removal 52 53 885 of the hemolymphatic fluid and exposing the CA surface. Broadband acoustic 54 55 886 stimulus was delivered using the preparation described by Montealegre-Z et 56 887 al. (2012). DC and CA mechanical responses are similar in phase by differ in 57 58 888 amplitude, revealing the loss of amplification after the dorsal cuticle has been 59 60 889 removed and the hemolymphatic fluid vacated. Lower panel shows the phase 61 62 63 64 65 29

890 of the oscillations determined by Hilbert transform. The phase spectra indicate 1 2 891 no phase difference between recordings obtained from the DC and those 3 4 892 obtained on the CA surface. b Time-resolved responses of ATM, PTM and 5 893 exposed CA to a pure tone stimulus (a 23kHz, 4-cycle sound pulse). CA 6 7 894 exposure results from dorsal cuticle removal. Lower panel shows the phase of 8 9 895 the oscillations determined by Hilbert transform. ATM and PTM oscillate in 10 11 896 phase, while CA response is clearly 180° out of phase. c Time-resolved 12 13 897 oscillations of ATM and PTM plates and the CA to 23kHz. All elements 14 15 898 oscillate in phase, confirming the coupling between tympanal plates and CA. 16 899 Data obtained from Montealegre-Z et al. (2012). 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download Figure: Fig5.eps

100 a 80 60 40 20 Specimen mounted in free acoustic field elocity mm/s) 0

45 40 b AV Specimen mounted in platform 35 ATM spiracular input cancelled

Response (V PTM 30 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 Applied Sound Pressure (Pa) Figure 6 Click here to download Figure: Fig6.eps

a CA, Dorsal Cuticle vibrations b CA, ATM, PTM vibrationsc CA and tympanal plates vibrations

DC 10 ATM 2 ATM plate 15 CA PTM 1 PTM plate 10 5 CA CA 0 0 0 (nm) (nm) (nm) -10 -5 -1

-15 Displacement Displacement Displacement -10 -2 200 200 200 100 100 100 0 0 0 (deg) (deg) (deg) Phase -100 Phase -100 Phase -100 -200 -200 -200 040 80 120 160 040 80 120 160 040 80 120 160 Time (µs) Time (µs) Time (µs) Author's Response to Reviewers' Comments Click here to download Author's Response to Reviewers' Comments: Response.rtf

Dear Prof. Römer, Here is the last revised version of our ms after last editorial comments. We accepted the changes suggested by the editor, and also found a few other typos to sentences that needed improvement. Please note that the ms parts that have been newly edited or modified are shown in red fonts.

Kind regards

fer