Acoustic distortion products from the ear of a grasshopper

M. Kossla) and G. S. Boyan

Zoologisches Institut, Universitat Munchen, Luisenstrasse 14, 80333 Munchen, Germany

Received 5 January 1998; accepted for publication 26 March 1998 Distortion-product otoacoustic emissions were recorded from the tympanum of the grasshopper, Locusta migratoria. The hearing organ of this insect is in direct contact with the tympanum and does not contain sensory hair cells. 2f 1 f 2 distortions were measured for stimulus frequencies between 270 kHz. For frequencies between 39 kHz, the level of 2f 1 f 2 was 3050 dB below the stimulus level. 2f 1 f 2 threshold curves calculated from distortion growth functions at different f 2 frequencies are most sensitive between 39 kHz. These thresholds match the auditory sensitivity of low frequency receptor neurons in the ear Romer, J. Comp. Physiol. 109, 101122 1976. In contrast to vertebrates, the dependence of the 2f 1 f 2 level on the frequency ratio f 2/f 1 did not show distinct maxima for most f 2 frequencies. The distortion levels were largest for small ratios close to 1. The behavior of 2f 1 f 2 was signicantly different for stimulus frequencies below and above 10 kHz. Below 10 kHz, the thresholds were more sensitive, the slope of distortion growth curves was shallower by a factor of at least 2, and the distortion levels reversibly decreased during CO2-induced hypoxia. Nonlinear mechanical processing may therefore be a general feature of sensitive hearing organs, even if these involve very different morphologies. Our results suggest that the the ciliated dendrites of the receptor cells of the insect may play a role in distortion generation. 1998 Acoustical Society of America. S0001-49669801207-7 PACS numbers: 43.64.Jb, 43.80.Lb BLM

INTRODUCTION

Distortion-product otoacoustic emissions DPOAEs have become a valuable tool in assessing the functional properties of vertebrate ears. They are used to measure relative hearing sensitivity Gaskill and Brown, 1990; Kossl, 1992; Manley and Koppl, 1993; Faulstich et al., 1996 or hearing decits Whitehead et al., 1996; Janssen et al., 1996, to gain information about mechanical frequency tuning Brown and Kemp, 1984, and to describe the action of the so-called cochlear amplier in mammals Mills et al., 1993; Mills and Rubel, 1996; Frank and Kossl, 1996. In the mammalian cochlea, the acoustic distortions are known to depend on intact and functioning outer hair cells. If these are absent Horner et al., 1985, damaged Brown et al., 1989, or if their electro-motility is blocked by salicylate Kujawa et al., 1992, the distortion levels deteriorate markedly. There is ample evidence that force generation by outer hair cells is the basis for nonlinear mechanical amplication of low-level sound stimuli, and consequently of distortion-product otoacoustic emissions in mammals e.g., Dallos, 1992; Ruggero, 1993. In nonmammalian vertebrates there is no known hair cell motility in respect to movement of the cell body Koppl, 1995; Manley, 1995. Nonetheless, auditory sensitivity, tuning, and the properties of distortion-product otoacoustic emissions are in most respects similar to those in mammals. In these animals, the mechanics of the sensory hair bundle of the hair cells most likely generates the ears nonlinearity and mechanical distortions Howard and Hudspeth, 1988; Jaramillo et al., 1993. Indeed, it is worth noting that nonlina

Electronic mail: koessl@zi.biologie.uni-muenchen.de J. Acoust. Soc. Am. 104 (1), July 1998

ear mechanical properties are also found in mammalian hair bundles Russell et al., 1992. Thus far, otoacoustic emissions have not been investigated in invertebrates, a group whose hearing organs are very different to those of vertebrates. To explore if sensitive hearing may also involve mechanical nonlinearities in the ear of insects, we attempted to measure distortion-product otoacoustic emissions in the grasshopper. Grasshoppers are known to have well-developed hearing capabilities, particularly for frequencies between 38 kHz where auditory nerve thresholds are close to 20 dB SPL Romer, 1976. This range coincides with the frequencies of maximum sound energy in the animals communication signals Meyer and Elsner, 1996. In grasshoppers, a large tympanum is situated on each side of the rst abdominal body segment, and a receptor organ, the Mullers organ, contacts the inner surface of the tympanum Gray, 1960 Fig. 1a. The tympanum is divided into two regions based on membrane thickness. In the center of the tympanum, where the thin and the thick membranes merge, the receptor organ is in contact with sclerites that protrude from the tympanum Fig. 1b. Within the receptor organ, ciliated dendrites of primary receptor neurons Fig. 1c are in contact with attachment cells coupled to the tympanal hypodermis. The receptor cells are surrounded by supporting cells known as scolopale cells, which also contribute to the linkage between dendrite and attachment cell Gray, 1960. There are four groups of receptor cells Fig. 1a: a, b, c, d whose dendrites are in contact with different sclerites and hence receive mechanical drive from different regions of the tympanum. As shown for the closely related species Schistocerca gregaria Michelsen, 1971, the a, b, and c cells
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 FIG. 1. Schematic of the hearing organ of Locusta migratoria. a View of the inner surface of the tympanum. A receptor organ, the Mullers organ, is attached via sclerites elevated process, sytyliform body, pyriform vesicle, folded body to the center region of the tympanal membrane where the thick and the thin membrane regions merge. b Schematic of a crossection through the receptor organ showing receptor neurons and their supporting cells. c Ciliated dendrite of a receptor neuron, adapted from Gray 1960.

mainly receive input from the thick membrane, or the interaction between thick and thin membranes, and respond best to frequencies below about 10 kHz, with the b cells being most sensitive. The d cells receive input from the thin membrane, are less sensitive than the low-frequency cells, and are tuned to frequencies above 10 kHz Michelsen, 1971. The mechanical displacement of the tympanum is governed by different vibration modes, by a resonance with a fundamental frequency at about 3.84 kHz Michelsen, 1971, and by independent movement of the sclerites against each other Stephen and Bennet-Clark, 1982; Breckow and Sippel, 1985. In summary, while the grasshopper hearing organ contains fewer receptor cells about 80 than in vertebrates, its mechanics are highly sophisticated. Preliminary results from this study were presented in the form of short commu nications Kossl and Boyan, 1997, 1998.
I. MATERIALS AND METHODS

because their distortion thresholds in the range 38 kHz were more than 30 dB higher than in the other animals. These three animals were also not as alert as the others and also did not show signicant antennal movement. The acoustic measurements took place in a soundproof chamber that was heated to 28 C. An acoustic coupler, initially designed for otoacoustic emission measurement in small mammals Kossl, 1994, consisted of two adjacent

Twelve grasshoppers Locusta migratoria, of both sexes and raised in crowded laboratory cultures at 30 C, were used in the experiments. The animals were treated in strict accordance with the guidelines for animal experimentation laid down by the Deutsche Forschungsgemeinschaft. Each grasshopper was lightly anaesthetized by cooling before wings and legs were removed. Following recovery from anaesthesia the animals were pinned dorsal side up to a cork platform. Nine of the grasshoppers were alert as indicated by antennal movement and sensitive distortions could be measured from these animals; three animals were discarded
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FIG. 2. Measurement of sound transduction through the body of the grasshopper. A microphone connected to the right tympanum measured the response to white noise generated by a speaker connected to the left tympanum. The solid curve gives the measured response. Note the sharp cutoff for frequencies above about 15 kHz. The dotted curve gives the measured response with the microphone and speaker directly coupled. M. Kossl and G. S. Boyan: Grasshopper distortions 327

FIG. 3. Example of acoustic two-tone distortions measured at the tympanum of Locusta migratoria.

FIG. 4. Growth of the level of the 2f 1 f 2 distortion with increasing level of f 1 open circles and f 2 lled circles. The other stimulus was held constant at 50 dB SPL. Two cases are shown with the frequency of f 2 either at 10 kHz left or at 25 kHz right. Dotted lines give the noise level. For a constant level of f 1 of 50 dB SPL, the distortion is at a maximum at a level of f 2 of 45 left or 4070 dB SPL right. For a constant level of f 2 of 50 dB SPL, maximum distortion is elicited by levels of f 1 of 5565 dB SPL.

conical tubes for stimulation and recording. The coupler had an overall tip diameter equal to the size of the tympanum of the insect and was positioned within a distance of about 0.31 mm from the tympanum. Sensitive distortion measurements at frequencies below 10 kHz require a tightly closed acoustic system. The connection between the body surface of the locust and the walls of the coupler tip was therefore sealed with toothpaste. One coupler channel was connected to a Bruel & Kjaer 4133 microphone to measure responses up to 40 kHz, or to a Bruel & Kjaer 4135 microphone for frequencies above 40 kHz. Two additional 4133 Bruel & Kjaer microphone capsules served as loudspeakers and fed signals into the other coupler channel. Continuous pure tone stimuli were generated with a dual Hewlett-Packard 8904A synthetizer and fed into two GPIB general purpose interface bus controlled attenuators. The sound system was calibrated in situ using white noise, and sound pressure levels used in the experiments are expressed in dB SPL dB re: 2105 Pa. Stimulation and data acquisition were controlled by a Pentium PC using ASYST Keithley software. The microphone response was fed via a B&K 2610 measuring amplier into a Hewlett Packard 3651A spectrum analyzer for FFT fast Fourier transform analysis using Hanning windows. Four FFT recordings were averaged for each data point and a resolution between 75 and 0.94 Hz was used. In the latter case, and using the 4133 microphone, the average noise level for frequencies above 2 kHz ranged between 18 and 25 dB SPL. Within the analyzed frequency range of 270 kHz, distortions produced by the setup were detectable just above the baseline noise oor for high primary levels above 8391 dB SPL. To exclude any setup distortion in the present experiments, we therefore only applied stimuli of up to 80 dB SPL f 1 level and 70 dB SPL f 2 level. For a de tailed description of the setup, see Kossl 1994. Stimulation and recording of DPOAEs was performed unilaterally. In the grasshopper there is an acoustic connection between the two ears via tracheae inside the body. To test for crosstalk between the ears, we applied white noise at the left ear of one animal and recorded the response at the right ear. For this purpose a B&K 4133 microphone was
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connected via a conical coupler to the right tympanum. Another B&K 4133 microphone capsule serving as a speaker was connected to the left tympanum. Both tympana were left intact and stimulation and recording took place in a closed sound system. We found that the body of the insect basically acts as a low-pass lter with a cutoff frequency at about 15 kHz and additional slight transmission loss for frequencies below 4 kHz Fig. 2. Comparable results were obtained by Miller 1977 and Michelsen and Rohrseitz 1995 for Schistocerca gregaria, however, the cutoff frequency in their experiments was distinctly lower and there was nearly no loss at the lowest frequencies. These differences could depend on the body fat content of the animals used, or on a difference between closed versus open sound systems. Our data on Locusta migratoria resemble more the transmission characteristics of the small grasshopper Chorthippus biguttulus used in the study of Michelsen and Rohrseitz 1995. To test if the ipsilaterally recorded DPOAEs are inuenced by the contralateral ear, we destroyed the contralateral tympanum and either closed the ear with resin or left it open. In both cases the ipsilaterally recorded DPOAEs did not change within the accuracy of the measurements (2 dB). The grasshopper auditory pathway may therefore be viewed as being unilateral with respect to measurements of acoustic distortion products. For the experiments involving ventilation with CO2, a grasshopper was mounted on its side to the cork platform described above after rst removing the wings and legs. The assembly containing the grasshopper was placed vertically inside a plastic container into which CO2 was passed. In order to keep the air humidity constant, the plastic container was partly lled with warm water. The temperature of the CO2 delivered to the preparation was maintained at 28 C. An experiment began with control measurements performed with the animal exposed to normal air in the recording environment described above. The CO2 was then applied for 10 min or until the antennae of the animals assumed the depressed attitude typical for hypoxia. The experimental measurements were then carried out and lasted approximately 15 min. Subsequently a plug was removed from the container,
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FIG. 5. Level of the 2f 1 f 2 distortion as a function of frequency ratio f 2/f 1. Shown are measurements for different f 2 frequencies between 2.540 kHz in a single animal. The numbers above the traces give the f 2 frequency and the levels of both stimuli. The noise level is given by the dotted lines. For most cases, the distortion level grows with decreasing frequency ratio.

allowing the CO2 to escape. Recovery of the grasshopper from hypoxia was signalled by the antennae again assuming their normal elevated position and beginning to move freely. The identical series of control measurements was then repeated. This complete experimental procedure, including CO2 application, could be repeated up to three times with the same results described below.
II. RESULTS

Acoustic distortion products proved to be relatively easy to measure in the grasshoppers due to their large exposed tympanum. It was, however, critical to have a completely sealed sound system see above and important that the coupler tip was as perpendicular as possible to the surface of the tympanum. Before each measurement we routinely tested for
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the appearance of spontaneous otoacoustic emissions but were not able to detect any. Further, acoustic distortions could only be measured from the tympanum itself. When the coupler was directed towards any other cuticular body surface of the locust, distortions could not be recorded. The acoustic distortions also immediately disappeared when the tracheal space behind the tympanum was lled with ringer solution. Figure 3 shows an acoustic spectrum near 20 kHz with the level of f 2 adjusted 10 dB below that of f 1. As in vertebrates, the 2f 1 f 2 distortion is dominant. With higher primary levels, 3 f 12f 2, 4 f 13 f 2, and 2f 2 f 1 distortions also appear. Figure 4 shows the dependence of the 2f 1 f 2 level on the level difference between the two primaries. For this purpose, the level of either f 1 or f 2 was kept constant at 50 dB SPL and the level of the other stimulus, given by the x axis, was changed stepwise. Maximum
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FIG. 6. Growth of the level of the 2f 1 f 2 distortion with increasing stimulus levels for different f 2 frequencies in a single animal. The level of f 1 was always 10 dB above that of f 2. The numbers above the traces indicate the stimulus frequencies and the initial slope of the growth functions.

2f 1 f 2 distortion is elicited when the level of f 1 is 515 dB above that of f 2. In the following measurements, the f 1 level was therefore always chosen to be 10 dB above that of the f 2 stimulus, similar to measurements in mammals Kossl, 1992, 1994.
A. Dependence of distortion product amplitude on the frequency ratio f 2/f 1

In vertebrates, the frequency ratio f 2/f 1 is an important parameter in the measurement of distortion products. Depending on the species and the frequency range, the 2f 1 f 2 distortion product is maximal at a so-called optimum ratio ( f 2/f 1) whose value lies between about 1.1 and 1.4. This optimum ratio is considered to be due either to a secondary mechanical ltering process in the cochlea Brown et al., 1992 or to phase-dependent reections and cancellations on the basilar membrane itself Neely and Stover, 1997.
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The standard procedure for measuring the optimum ratio is to keep one stimulus frequency, normally f 2, constant, and to vary the other stimulus frequency ( f 1). Figure 5 shows that an optimum ratio could not easily be dened for most f 2 frequencies in the insect. The 2f 1 f 2 distortion either has nearly equal levels over a large range of ratios up to about 1.6 Fig. 5: f 2 at 6, 7, and 40 kHz, or there is a steady increase in distortion level with decreasing ratio Fig. 5: f 2 at 2.5, 3, 3.5, 10, 12.5, 15, and 17.5 kHz. In nearly all of these cases, very small ratios close to 1 are sufcient in inducing large distortions. At f 2 frequencies of 4 and 5 kHz the distortion level maxima and minima for certain ratios are better dened.
B. Distortion growth functions

In order to study the growth of the 2f 1 f 2 distortion product with increasing stimulus levels over different f 2 fre M. Kossl and G. S. Boyan: Grasshopper distortions 330

FIG. 7. Initial slope of the 2f 1 f 2 growth function in ve animals. Standard deviations are given by vertical bars. The horizontal bars give the respective standard deviations of f 2 in cases where slightly differing f 2 values from different animals were grouped together.

quencies, the frequency of f 1 was adjusted to a value that produced maximum distortion levels in the ratio curves see Fig. 5. This was performed for each measurement at a given f 2 frequency. Then the levels of both stimuli were increased in equal size steps with the level of f 1 always being 10 dB above that of f 2. There is a clear difference in distortion growth for f 2 frequencies below and above 10 kHz Figs. 6 and 7. For f 2 frequencies below 10 kHz, the distortion growth functions Fig. 6 are characterized by an initial slow growth for stimulus levels up to about 5060 dB SPL, followed by steeper growth for higher stimulus levels. The initial slope was determined by linear regression of the growth function data points over a stimulus range of 20 dB, starting with the rst appearance of the 2f 1 f 2 distortion at more than 3 dB above the average noise level. This slope lies between 0.46 and 0.78 Fig. 7: average values for f 2 frequencies below 10 kHz. Above 10 kHz, the initial distortion growth is steeper, ranging between 1.1 and 1.48 Fig. 7: average values. A minimum slope of 0.46 was obtained for f 2 close to 4 kHz, and a maximum slope of 1.48 appears close to 30 kHz. In some cases, the slope values were averaged for a range of f 2 values used in measurements from different animals see gure legend.
C. Distortion thresholds

FIG. 8. Distortion threshold curves in four grasshoppers. The individual traces give the level of the f 2 stimulus the f 1 level always was 10 dB higher than that of f 2 which was sufcient to induce a 2f 1 f 2 distortion of 20, 15, 10, 5, and 0 dB SPL numbers given on the right side of the isothreshold curves.

since in mammals there is strong evidence that the 2f 1 f 2 distortion is generated close to the f 2 frequency place in the cochlea. Of course, this same assumption cannot apply to the insect. We kept the reference to f 2 to allow for a better comparison with mammalian data. The threshold curves are most sensitive for f 2 frequencies between about 39 kHz, at least for threshold criteria of 10 dB SPL or lower. For higher threshold criteria, the minimum is smaller or absent. This difference reects the different slopes of distortion growth see above. In three of the four animals tested, and for low threshold criteria, the overall threshold minimum can be divided into two most sensitive regions, one at 35 kHz and the other at 79 kHz. In the fourth animal Fig. 8d a trend toward two minima is not seen for low threshold criteria but is present for high threshold criteria. There is a steep threshold increase for frequencies between 22.5 kHz and 812 kHz. Above 12 kHz, the thresholds remain fairly constant up to about 60 kHz see Fig. 8b, and are about 20 dB higher than those at the threshold minimum.
D. Effects of manipulations of the physiological state of the grasshoppers

Threshold curves for the nonlinear mechanics can be derived from 2f 1 f 2 growth functions obtained at the frequency ratios where maximum distortion is found. The 2f 1 f 2 threshold curves from four grasshoppers are shown in Fig. 8. Displayed is the f 2 level that for a given f 2/f 1 stimulus combination is sufcient to induce a small distortion of 20, 15, 10, 5, and 0 dB SPL. These iso-level thresholds were interpolated from the growth functions described above. Similar to measurements in mammals e.g., Faulstich et al., 1996, the data are referenced to the frequency of f 2,
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We rst tested if it is possible to inuence the distortion products in the grasshopper in any way, or if they are a completely passive and invulnerable characteristic of the hearing organ. For this purpose we decapitated one animal. This procedure of course does not allow us to determine the possible metabolic changes we thereby induced. It is known that ventral cord neurons in decapitated insects remain functional for a period of time Kutsch and Otto, 1972. Immediately after decapitation there was no change in the sensitivity of distortion thresholds, which indicates that the hearing organ together with its tracheal tubes remained basically intact. Changes in the distortions became apparent
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FIG. 9. The 2f 1 f 2 growth functions a and b and threshold curves c, measured before control, 2 h, and 15 h after decapitation of the animal. A threshold criterion of 20 dB SPL horizontal line in a and b was used to calculate the thresholds. The frequencies of the primary stimuli are given above the curves in a and b.

about 90 min after decapitation. Figure 9 shows changes in the slope of distortion growth functions and upward shifts of the distortion threshold curves. About 2 h after decapitation there was a pronounced loss of distortion level at the threshold minimum between 39 kHz. The corresponding growth functions became steeper Fig. 9a. A maximum threshold loss of 37 dB occurred at an f 2 frequency of 4.5 kHz. Fifteen hours after decapitation there was another large overall upward shift of the threshold, but distortions were still measurable at around 8 kHz and above 15 kHz. Taken together these data indicate that the mechanical processing of low frequencies is clearly more vulnerable than that of high frequencies above 10 kHz. In order to inuence the animalss metabolism in a more controlled way, we introduced hypoxia by ventilation with CO2. In insects, CO2 application is known to halt the movement of legs or antennae and it is used to introduce a state similar to anaesthesia in vertebrates. Insects are not killed by short-term CO2-induced hypoxia, they recover quickly after they are supplied with oxygen again. In our experiments we measured distortion growth functions before, during a 10 25-min period of CO2 ventilation, and after the CO2 was replaced with oxygen see methods. During CO2 ventilation the distortion growth functions became insensitive and shifted towards higher stimulus levels Fig. 10. In most cases the distortion level was affected equally at both low and at high stimulus levels examples in Fig. 10a, c, and e. In some cases not shown in Fig. 10 the decrease in distortion level was most pronounced at low stimulus levels. This decrease only occurred for f 2 frequencies below 10 kHz. At higher frequencies the distortion growth functions remained unchanged Fig. 10b and d. Figure 10f shows the corresponding shift in the distortion thresholds for three animals measured at a threshold criterion of 10 dB SPL. It is obvious that in the grasshopper the sensitive lowfrequency distortions are much more vulnerable to hypoxia than the high-frequency distortions elicited by stimuli above 10 kHz.
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III. DISCUSSION

The properties of DPOAEs in the grasshopper are in many respects similar to those from the verebrate ear. The

FIG. 10. ae The 2f 1 f 2 growth functions before open circles, during lled circles, and after ventilation with CO2 open squares. Dashed lines give the average noise level. The f 2 frequencies are indicated above the individual traces; for f 2 at 20 and 30 kHz there was no change in the growth functions. f Shift of the distortion threshold during CO2 application in three animals different symbols for a threshold criterion of 10 dB SPL. M. Kossl and G. S. Boyan: Grasshopper distortions 332

FIG. 11. Comparison between acoustic distortions and neuronal data. a Average isothreshold curve from the four animals in Fig. 8 for a threshold criterion of 15, 10, 5, and 0 dB SPL. b Neuronal threshold tuning curves from a b cell and a d cell Romer, 1976 c Neuronal intensity/response functions in a d cell stimulated with 25 kHz and a b cell stimulated with 4 kHz from Romer, 1976. The difference in the slope of intensity response function between the two cells is also evident in distortion slopes Fig. 7 and in the spacing of the threshold curves obtained for different threshold criteria in a.

2f 1 f 2 distortion is most prominent and can be elicited at maximum levels when the f 1 stimulus level is higher than that of f 2. This is comparable to the situation in the mammalian ear Probst et al., 1991. The difference between the 2f 1 f 2 distortion level and that of the f 1 stimulus with the level of f 2 being 10 dB lower was between 3050 dB in the most sensitive frequency region. This difference range is larger than values obtained with the same setup and paradigm in small laboratory mammals Faulstich et al., 1996; Frank and Kossl, 1996 where the minimum difference is between 1030 dB. This indicates that in the grasshopper the DPOAEs are less pronounced than in mammals by factor of about 20 dB. In terms of distortion level, the grasshopper DPOAEs are more comparable to those found in birds and lizards Manley and Koppl, 1993; Kettembeil et al., 1995. In humans, the 2f 1 f 2 levels are also lower than those measured in most laboratory mammals see, e.g., Whitehead et al., 1996. This, however, could be due to the fact that in humans the microphone coupler tube cannot be positioned as close to the tympanum as in small mammals.
A. Stimulus frequency ratio

in the cochlea of these animals suggest that in this case the small optimum ratios are a consequence of a complex double-resonator system that enhances frequency tuning Kossl and Vater, 1996; Vater and Kossl, 1996. As far as the best ratios are concerned, the mechanical processes that are responsible for a specic tuning of distortion frequencies in the grasshopper are not present for the f 2 frequencies tested. Since mechanical frequency tuning in this species is observed in the vibration patterns of specic tympanal areas Michelsen, 1971, and we always measured the response from the whole tympanum, we could have missed more localized tuning phenomena. Only in the range of 45 kHz were there larger irregularities in the dependence of the distortion levels on the frequency ratio f 2/f 1 Fig. 5. This frequency roughly corresponds to that of a tympanal resonance Michelsen, 1971.
B. Distortion threshold curves

In mammals, the dependence of the distortion level on the frequency ratio may reect secondary cochlear ltering processes in the tectorial membrane Brown et al., 1992; Allen and Fahey, 1993 and/or reections and phasedependent cancellations in the cochlea Neely and Stover, 1997. Contrasting with DPOAE measurements in mammals, the level of the 2f 1 f 2 DPOAE in grasshoppers is largest for the smallest ratios and an optimum ratio is hard to dene. This implies that the distortion level is at a maximum when the distortion frequency is close to the stimulus frequencies, as is typical of simple nonlinear resonators. For mammals, there is one exception known where the smallest ratios also induce maximum distortion, namely in bats which use pure tone echolocation calls. Here extremely small optimum ratios are found close to the call frequencies. Pronounced anatomical specializations of the basilar and tectorial membrane
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Iso-distortion thresholds are an efcient means with which to noninvasively determine the frequency dependence of cochlear sensitivity, at least with respect to nonlinear mechanical processing. In a number of vertebrate species, distortion thresholds were found to run parallel to neuronal or behavioral threshold measurements and there is a good correlation between the minima of distortion thresholds and the most sensitively perceived auditory frequencies e.g., Kossl, ppl, 1993; Faulstich et al., 1996. 1992; Manley and Ko In the grasshopper, the distortion threshold minimum between 39 kHz correlates with the most sensitive thresh old tuning of the b cells Romer 1976, compare Fig. 11a and b. The same applies to a and c cells which have higher thresholds but similar tuning to the b cells. The d cells are tuned to frequencies above 10 kHz and are less sensitive than the low-frequency receptors, a physiology which agrees with our distortion threshold data. The distortion threshold minimum also coincides with the absolute sensitivity minimum in gross auditory nerve recordings see Michelsen, 1971, and
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with maximum motion of the thick membrane measured at the tympanal insertion point for the a cells Meyer and Elsner, 1996, which are tuned to the same low-frequency range as the b cells. The slow growth of distortion levels within the frequency region of maximum sensitivity versus the steep growth for frequencies above 10 kHz corresponds to a comparable change in the slopes of neuronal intensity response functions of b versus d cells Romer 1976; Fig. 11c. The initial slope of the d cell intensity-response function stimulated with 25 kHz is a factor 2.4 steeper than that of the b cell responding to 4 kHz. A comparable difference factor 2.8 is seen in the distortion growth between f 2 frequencies of 4 and 24 kHz see Fig. 7. The b and d cells are associated with the thick and thin membranes of the tympanum, respectively see the Introduction. Therefore, our data indicate that the sensitive low-frequency distortions may be produced by the mechanics of the thick membrane, and/or the interaction between thick and thin membrane, whereas the high-frequency distortions are probably due to vibration of the thin membrane alone.
C. Possible nonlinear mechanisms

Nonlinear neural activity in receptor cells and higherorder neurons of the grasshopper was studied in detail by Sippel and Breckow 1983, 1984. They show that this nonlinearity is related to adaptation phenomena and suggest that it is largely due to electrochemical processes intrinsic to the nerve cells. One reason for this suggestion is that these authors assume from mechanical measurements at high sound pressure levels that the mechanics of the hearing organ are basically linear Breckow and Sippel, 1985. Even if the nonmonotonicity of neuronal intensity-response functions is related to intrinsic neuronal properties acting at higher sound pressure levels, the present study clearly shows that the mechanics of the tympanum of the grasshopper are nonlinear at low sound pressure levels close to the threshold of hearing. The vulnerability of the DPOAEs to hypoxia implies that they may involve a stage of the electromechanical transduction process which is metabolically dependent. As in the grasshopper, reversible hypoxia effects in vertebrates are induced within a few minutes and deterioration of the DPOAE levels of up to 2030 dB has been observed Rebillard and Lavigne-Rebillard, 1992; Manley and Koppl, 1993. In mammals, hypoxia leads to a decrease of the endocochlear potential EP which is the driving voltage for forward and reverse transduction. The time courses of changes in the DPOAEs and the EP, however, are signicantly different, and it is likely that the DPOAEs are affected both by the changed EP and by a direct inuence of hypoxia on the properties of the outer hair cells Rebillard and LavigneRebillard, 1992. In contrast to mammals, the hypoxia effects in the grasshopper are more pronounced at low stimulus frequencies than at high frequencies. The most sensitive frequency region between 39 kHz is clearly also the most vulnerable. We know of no other report which shows that hypoxia can inuence the functional properties of insect receptor organs. However, it is known that hypoxia affects spike generation and propagation in receptor neurons Hamon et al., 1988. In addition, there is evidence that the
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mechanoelectrical transduction process in locusts is strongly dependent on temperature Oldeld, 1988. The characteristic frequency of low-frequency neurons shifts up to 0.2 octaves depending on the ambient temperature. Temperature does not affect the properties of high-frequency receptor neurons, as is the case with the hypoxia effects on DPOAEs in the present study. The most likely candidate responsible for the metabolically dependent nonlinear transduction in the grasshopper is Mullers organ itself. The sclerites by which it is attached to the tympanum are known to exhibit frequency-dependent strain and rotation movements which seem to be crucial for the stimulation of the sensory dendrites Stephen and Bennet-Clark, 1982. Due to its mass and compliance, Mullers organ exhibits its own properties of oscillation and can act as a resistive load for the tympanum Stephen and Benett-Clark, 1982. In addition, there are in-phase and an tiphase movements within Mullers organ Breckow and Sip pel, 1985. The mechanics of Mullers organ change profoundly during development Breckow and Sippel, 1985, and its complex characteristics led several authors to suggest that it really is an active observer which inuences tympanal motion Michelsen, 1973; Stephen and Bennet-Clark, 1982; Breckow and Sippel, 1985. Within Mullers organ, metabolic vulnerability can occur at the level of the attachment cells, the scolopale cells, and the receptor neurons themselves see Fig. 1. Scolopale cells are known to contain actin and tropomyosin and it was suggested that the elasticity or stiffness of these cells depends on the extent of tropomyosin binding Wolfrum, 1991. The receptor cell dendrite contains a single large cilium with a long process that extends towards the cell body. An active role of the cilia in sensory transduction is described for the chordotonal organ of the grasshopper Melanoplus bivittatus Moran et al., 1977. Moran et al. suggest that an active sliding movement between adjacent doublets of the axoneme leads to a local bending in the cilium and to an enhancement of displacement at the base of the cilium. The cilium and transduction apparatus in the insect receptors might therefore be well involved in the creation of mechanical nonlinearity. As far as the hair bundles of vertebrate hair cells are concerned, it is known that their mechanics are nonlinear Howard and Hudspeth, 1988; Russell et al., 1992 and able to produce strong frequency distortions Jaramillo et al., 1993. If the same were true for the insect cilium, then the underlying mechanisms probably depend on different structures, since the insect has a true cilium, whereas the vertebrate hair bundle mainly consists of stereovilli with an actin core.
ACKNOWLEDGMENTS

For discussion of the presented data we are grateful to F. Huber, G. Manley, A. Michelsen, G. Neuweiler, U. Thurm, and M. Vater. This study was supported by the Deutsche Forschungsgemeinschaft.
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