Auditory chain reaction: Effects of sound pressure and particle motion on auditory structures in fishes.

Despite the diversity in fish auditory structures, it remains elusive how otolith morphology and swim bladder-inner ear (= otophysic) connections affect otolith motion and inner ear stimulation. A recent study visualized sound-induced otolith motion; but tank acoustics revealed a complex mixture of sound pressure and particle motion. To separate sound pressure and sound-induced particle motion, we constructed a transparent standing wave tube-like tank equipped with an inertial shaker at each end while using X-ray phase contrast imaging. Driving the shakers in phase resulted in maximised sound pressure at the tank centre, whereas particle motion was maximised when shakers were driven out of phase (180°). We studied the effects of two types of otophysic connections-i.e. the Weberian apparatus (Carassius auratus) and anterior swim bladder extensions contacting the inner ears (Etroplus canarensis)-on otolith motion when fish were subjected to a 200 Hz stimulus. Saccular otolith motion was more pronounced when the swim bladder walls oscillated under the maximised sound pressure condition. The otolith motion patterns mainly matched the orientation patterns of ciliary bundles on the sensory epithelia. Our setup enabled the characterization of the interplay between the auditory structures and provided first experimental evidence of how different types of otophysic connections affect otolith motion.

Introduction

Otoliths of modern bony fishes (Teleostei) are solid calcium carbonate biomineralisates in the inner ear and play an important role in hearing [1-4]. Teleost otoliths show a tremendous diversity in morphology, especially with respect to shape (e.g. [5-8]). Despite this known morphological diversity, it remains elusive how different otolith shapes affect sound-induced otolith motion and contribute to hearing in fish [4,9,10]. Identifying factors that determine otolith motion and thus inner ear stimulation is even more challenging in teleost taxa that evolved ancillary auditory structures (for an overview see [11,12]). These structures are morphological modifications such as connections between the gas-filled swim bladder and the inner ears (= otophysic connection), which are often correlated with a widened range of detectable frequencies (especially at frequencies > 700 Hz) and increased auditory sensitivity (e.g. [13-15]). If an otophysic connection is present, sound is thought to stimulate the inner ears along two pathways, a direct and an indirect one (Fig 1A; [16]). The direct stimulation pathway is independent of the presence of a swim bladder or any gas-filled bladder in the fish [16,17]. Sound-induced particle motion results in a to and fro motion of the fish, whereas all soft tissue is likely to move in phase with the surrounding water due to similarities in density (e.g. tail muscle tissue in fish: 1.05 g/cm3; [18]). As the otoliths are denser than the underlying sensory epithelium, they act as inertial masses and lag behind the motion of the soft tissue, thereby creating a relative motion [19,20]. This relative movement between otolith and sensory epithelium deflects the ciliary bundles of the sensory hair cells and maximizes stimulation if the bundle is pivoted in the direction of the kinocilium [21,22]. In the indirect stimulation pathway, sound pressure fluctuations compress and decompress the gas in the swim bladder, resulting in oscillating swim bladder walls [16]. The motion of swim bladder walls thereby creates local particle motion. This may stimulate the inner ears in a way similar to sound-induced particle motion, especially when the distance between swim bladder and inner ears is short due to the coupling of the two structures. The swim bladder can be directly coupled to the inner ears via anterior swim bladder extensions such as present in several sciaenid species (e.g. Bairdiella chrysoura [13], Micropogonias undulatus [23]), the holocentrid genus Myripristis [24], or the cichlid genus Etroplus [25,26]. In all otophysans such as carp or goldfish, the swim bladder is connected to the inner ears through the Weberian apparatus [27].

Fig 1

Overview of an otophysic connection represented as a scheme and as found in the cichlid Etroplus canarensis.

(A) Hypothesized direct (blue) and indirect (red) pathways of inner ear stimulation in teleost fishes. All fish, whether or not they possess a gas-filled bladder, can detect sound via the direct stimulation pathway. If the swim bladder comes close to the inner ears or is coupled to the inner ears (otophysic connection), oscillations of the swim bladder walls provoked by sound pressure can be reradiated as local particle motion which stimulates the inner ears. 3D reconstructions of the otoliths, part of the bones, and the anterior swim bladder region are superimposed on the 2D radiographs of the same individual of E. canarensis (based on a pixel size of 6.1 µm, ID17) seen in (B) lateral and (C) ventral view. Scheme in (A) adapted and modified from Schulz-Mirbach et al., 2019 [4].

Only few experimental and theoretical studies (based on mathematical modelling) have investigated the factors influencing otolith motion as well as the interplay between inner ear components and ancillary auditory structures such as otophysic connections [10,19,20,28-31]. A recent experimental study successfully visualized sound-induced in situ motion of otoliths in the orange chromide Etroplus maculatus (Bloch, 1795) (Cichlidae) using hard X-ray phase contrast imaging [30]. However, the sound field in the Plexiglas® tank equipped with a small underwater speaker (Daravoc) was complex and clearly deviated from values expected under free field conditions ([30], see also [32]). We therefore aimed to improve the tank acoustics and to develop a setup enabling experiments either under sound-induced particle motion or under sound pressure condition.

To test the newly developed setup, we chose two species representing two types of otophysic connections, i.e. goldfish, Carassius auratus (Linnaeus, 1758) (Otophysa, Cyprinidae) and the Canara pearlspot, Etroplus canarensis Day, 1877 (Cichlidae). Goldfish is well investigated with regard to inner ear and swim bladder morphology. For Etroplus, data on the gross morphology of the auditory structures are available [15,25,26,33,34]. The goldfish is characterized by an indirect swim bladder-inner ear connection via the Weberian apparatus. The Weberian apparatus is a chain of three ossicles (but see its variability in Siluriformes, [35,36]), namely tripus, intercalarium, and scaphium plus the claustrum as well as ligaments that interconnect the ossicles [37,38]. In E. canarensis, the swim bladder contacts the inner ears through two anterior swim bladder extensions [25]. In E. maculatus, each anterior swim bladder extension consists of 1) a gas-filled part contacting the bone surrounding the lagena and 2) a tissue pad that comes close to the posterior and horizontal semicircular canals and to a recessus posterior to the utricle [26]. Goldfish and E. maculatus have improved auditory abilities in terms of the detectable frequency range and relative auditory sensitivity [15,39] compared to species without swim bladders such as flatfish (Pleuronectes platessa [17]) or species that lack an otophysic connection (Holocentridae: Sargocentron, no otophysic connection vs. Myripristis, otophysic connection present [12,14]). Moreover, the goldfish is considered a “gold standard” in fish bioacoustics because its auditory abilities have been intensively studied using different behavioural and electrophysiological methods [40-43].

Here, we address the functional morphological question of how different types of otophysic connections affect otolith motion. Two formerly stated hypotheses were tested. 1) In otophysans such as goldfish, the saccule is assumed to play an important role in perceiving sound pressure. The local particle motion generated by the swim bladder and transduced via the Weberian apparatus, the sinus impar, the sinus endolymphaticus, and the transverse canal to the saccules is expected to result in a motion of the saccular otoliths that is in direct line with the flow of the endolymphatic fluid [33,44]. This otolith motion might be supported by the three wings arranged along the rostrocaudal axis of the saccular otolith [44,45]. 2) The bipartite structure of the anterior swim bladder extensions in the cichlid genus Etroplus [25,26] may facilitate the transduction of sound pressure to the utricle [26]. The modified utricular otolith and the macula utriculi in Etroplus maculatus have been interpreted in terms of a more important role of the utricle in audition [26,33].

Material and methods

Animals

Fishes (Carassius auratus, Etroplus canarensis) were purchased from French aquarium traders and kept at the animal care facility of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). For each of the three measurement sessions in 2018 (June, July, September), the animals were maintained in two 20L-plastic aquaria (5–7 individuals per aquarium) under a 12:12 h light-dark cycle. Each aquarium was equipped with an internal filter and a bubble stone to aerate the water.

Prior to each sound experiment, the test individual was deeply anesthetized and euthanized with a neutral-buffered solution of 0.4% ethyl 3-aminobenzoate methansulfonate (MS-222, Sigma Aldrich, France). After opercular movements had ceased (within ca. 5 minutes), we waited another 10–15 minutes before further handling of the fish. Standard length and body weight of the fish were then measured to the nearest millimetre using a scale paper and to the nearest 0.1 g using a Scaltec SBC22. All experiments at the ESRF were conducted in accordance with ethical guidelines, i.e. article 3, point 1 of the EU directive 2010/63, accepted by all EU countries. As no living animals were used during the imaging procedure, no ethical approval was required.

2D radiography

In total, eight specimens per species were studied with goldfish possessing a mean standard length of 62 ± 2.2 mm (ID17) and 54 ± 1.6 mm (ID19) and a body weight of 7.5 ± 1.7 g (ID17) whereas E. canarensis had a mean standard length of 51 ± 1.0 mm (ID17) and 51 ± 2.7 mm (ID19) and a body weight of 6.0 ± 0.3 g (ID17).

For each experiment, the freshly euthanized fish was placed in the tank with its head located in the centre. Imaging was performed with the fish seen in lateral (Fig 1B), dorsal (Fig 1C), and frontal views; the latter orientation (“frontal view”) was performed only in two specimens of E. canarensis. In dorsal and lateral views, sound impinged along the rostrocaudal axis of the fish, whereas, in frontal view, sound impinged on the body flanks. The fish rested on a piece of foam that was glued to a Plexiglas® holder. To provide as many degrees of freedom for the fish’s body to move in the sound field, it was fixed to the foam with two or three insect pins that were pierced subcutaneously through the body flanks (lateral and frontal views) or through the caudal peduncle (dorsal view) and through the anterior-most portion of the upper and lower jaws (lateral, dorsal, and frontal views). To minimize detrimental effects of X-ray-induced tissue damage, experiments performed in the same orientation (e.g. fish seen in lateral view) and using the same species, but different individuals, alternately started with the in phase (0°) and the out of phase condition (180°; for an explanation of the two conditions see “setup design”). Hence, we started using the in phase condition and the highest studied sound pressure level (SPL; step 1, Table 1) in one specimen and using the out of phase condition at a lower SPL (e.g. step 4, Table 1) in another individual of the same species.

Table 1

Overview of sound pressure levels (SPLs) measured at the centre of the tank and the difference of SPLs and particle acceleration levels (PALs) between the in phase (0°) and out of phase (180°) condition.

	hydrophone (Brüel & Kjær 8103)				p-a sensor (Lab Vienna)
	SPLs (dB re 1 µPa)				SPL (0°) -SPL (180°)	PAL (0°) -PAL (180°)
	ID17		ID19
ANL	117.5		118.1
step	0°	180°	0°	180°
1	> 177.2	165.5	> 177.2	157.3
2	> 177.2	161.6	177.2	155.4
3	169.6	154.6	170.7	151.1	+24.8	-8.7
4	162.6	148.6	164.3	146.2	+26.0	-8.2

Measurements were performed using a miniature hydrophone (Brüel & Kjær 8103) and the p-a sensor. The difference of SPLs was calculated from SPL values given in dB re 1 µPa, that of PALs was obtained from PAL values given in dB re 1 µm/s2. ANL, ambient (background) noise levels.

The motion of the structures was captured at an image acquisition rate of 98.99 frames per second (= fps, ID17) or 198.02 fps (ID19) when presenting the 200 Hz stimulus. The mismatch between stimulus frequency and image acquisition rate enabled sampling one oscillation (duration of 5 ms) of the 200 Hz pure tone at 50 (ID17) or 100 positions (ID19). In all experiments, the integration time was 1 ms; a pixel size of 6.1 µm (ID17) or 3.67 µm (ID19) was achieved. Images were recorded using a frame size of 2,560 x 1,100 pixels as 32-bit raw-files (ID17) or using 2,016 x 2,016 pixels as 16-bit tiff-files (ID19) with a photon energy of 70 keV (ID17: monochromatic beam, DeltaE/E approx. 10–4) or 59.2 keV (ID19), respectively. The detected photon flux was estimated at 3.5*107 photons/mm2*ms (ID17). At ID19, the field of view was not large enough to capture the region of interest in goldfish (anterior swim bladder portion, Weberian ossicles, otoliths) in one stack; thus, two overlapping stacks were produced for each condition (orientation & sound presentation).

At ID17, the detection system consisted of a PCO edge 5.5 coupled with 1x optics, yielding a final isotropic pixel size of 6.1 µm. At the entrance screen of the detection system, a 250 µm LuAG:Ce scintillator was used to convert the X-rays into visible light [46]. To achieve a sufficiently high photon flux density at ID19, the beamline was operated in the “pink beam” configuration, i.e. the white radiation from a wiggler insertion device was filtered only by Al, C and Cu attenuators as well as the mandatory Be window in the optical beam path. Accordingly, a narrow-bandwidth X-ray spectrum was available at the same position which was characterized by a high amount of photons and a homogeneous wave used by the propagation-based imaging (PBI) technique (see next paragraph). Another indirect system was used as a detector for fast micro-radioscopy: two photo-lenses in tandem-design (face-to-face, with an effective 3.0x magnification, Hasselblad) were used to project the luminescence image of a 200 µm LuAG:Ce single-crystal scintillator onto a (first generation) PCO DIMAX camera.

In conventional X-ray imaging, the contrast obtained is based on the absorption properties of the investigated object. In the case of soft tissue, the investigated materials are composed mainly by low Z elements; this limits the image contrast achievable using conventional absorption-based X-ray imaging. X-ray phase-contrast imaging (XPCI; [47]) helps overcome such limitation because it is based on detecting the phase shift induced by the presence of an object. Hence, high image contrast can also be achieved for soft tissue. The technique employed here is so-called propagation-based imaging (PBI; [48]), which is optimal whenever high spatial and temporal resolution is needed. PBI requires a highly spatially coherent beam and an optimized distance between the object and the detector in order to enhance the contrast between different interfaces thanks to Fresnel diffraction [48]. The characteristics of synchrotron radiation at ID17 and ID19 perfectly fulfil the requirements of this technique.

Setup design: Standing wave tube-like tank

The tank construction followed the standing wave tube design described by Hawkins & MacLennan (1976) [17]. Nonetheless, imaging at the synchrotron radiation facility required several modifications such as “X-ray-transparent” material (Plexiglas®) instead of steel, thin tank walls to minimize beam absorption, and a light tank weighing no more than 5 kg when filled with water to meet the maximum load requirements of the stage (especially at ID19). To minimize the adverse effects of air-bubble formation in terms of imaging and acoustics, the tank was filled with distilled water through a supply valve (inner diameter: 14.5 mm) using a thin rubber hose. The standing wave tube-like tank consisted of a horizontal Plexiglas® tube with an inner diameter of 11 cm, a length of 18 cm (volume: 1.71 l) from shaker-coupled membrane to membrane, and a wall thickness of 5 mm (for further dimensions see Fig 2C). The tank was equipped with a projector (inertial shaker) at each end of the tube (Fig 2A). The shaker-coupled membrane (diameter: 58 mm) at each end of the tube was made of a carbon disk (thickness: 1 mm; diameter: 47 mm) enclosed by a silicone membrane (thickness: 1 mm). The miniature inertial shaker 2002E (PCB Synotech) was coupled to the carbon disk by a steel screw (M3) with a length of 5 cm. During the experiments, the inertial shakers were driven either in phase (0°) to create a pressure maximum in the centre of the tank or out of phase (180°) to obtain maximum particle motion (Fig 2B).

Fig 2

Overview of the Plexiglas® standing wave tube-like setup.

(A) Tank with goldfish in lateral view; the fish is fixed to the foam-covered sample holder using three insect pins. (A) Detail of the miniature inertial shaker, the carbon disk, and the surrounding silicone membrane. (B-B) Schemes depicting the tank dimensions (inertial shakers not shown). Values in millimetres. B1-B2 drawn by Heinz Pfeiffer (University of Vienna).

Sound stimulus and characterization of tank acoustics

Pure tone stimulus

Fish were subjected to a 200 Hz pure tone generated by the two inertial shakers. We chose a 200 Hz stimulus because this frequency resulted in a successful observation of the in situ motion of otoliths in our last study [30] applying hard X-ray phase contrast imaging at the ESRF. Moreover, this enabled us to compare outcomes of the new setup design with the setup–an upright tank equipped with an underwater speaker–used in our previous study. The stimulus was generated by the software CoolEdit 2000 (Syntrillium Software Corp. Phoenix, AZ) and the internal sound card (Conexant 20561 SmartAudio HD) of a laptop (Lenovo ThinkPad T500) connected to the two inertial shakers via an amplifier (S.M.S.L. SA 36A Pro, Shenzhen ShuangMuSanLin Electronic Co., Ltd., Shenzhen, China). The stimulus was composed of two silent periods (3 s duration in the beginning, 1 s at the end) enclosing the actual sound stimulus (5 s). The 200 Hz stimulus was presented at up to 19 different SPLs (“steps”) ranging from 141 up to >177 dB re 1 µPa, four of which were used for the final analysis (Table 1). A certain “step”, e.g. step 2, is defined as the same setting of the amplifier and the internal loudspeaker of the laptop during stimulus presentation, regardless whether the shakers were driven in phase or out of phase. Thus, step 2 (at ID19) equals a SPL of 177.2 dB re 1 µPa when shakers were driven in phase and a SPL of 155.4 dB re 1 µPa when shakers were driven out of phase. SPLs decrease from steps 1 to 4 (see Table 1).

Sound pressure levels

All measurements to characterize tank acoustics were performed before or after the actual experiments. Simultaneous acoustic measurements were not possible because either the p-a sensor or the miniature hydrophone would have been affected by the X-ray beam, or the SPLs and particle acceleration levels (PALs) would have been evaluated in places other than the tank centre. At both beamlines, SPLs of the different “sound steps” (SPLs, LLSP, L frequency weighting, S time weighting) and of ambient noise present in the beamline hutches (LLeq, L frequency weighting, recording duration: 1 min) were measured using a miniature hydrophone (Brüel & Kjær 8103, sensitivity: -211 dB re 1 V/µPa) connected to a sound level meter (Brüel & Kjær 2238 Mediator) which was calibrated using a hydrophone calibrator (Brüel & Kjær 4229). The ambient noise was recorded with the miniature hydrophone connected to the laptop using the software CoolEdit 2000.

Particle acceleration levels

The sound field in the tank was characterized by measuring the SPL directly and calculating the PAL in a walk-in soundproof room at the Department of Behavioral and Cognitive Biology, University of Vienna. The SPL (LLSP) was measured with the miniature hydrophone connected to the sound level meter (details see above). In order to determine PAL in the horizontal direction, a calibrated underwater miniature acoustic pressure-acceleration (p-a) sensor (S/N 2007–001, Applied Physical Sciences Corp., Groton, CT) was placed in the centre of the tank (Fig 2B). The p-a sensor consists of two built-in units: a piezoelectric, omnidirectional hydrophone (sensitivity: -173.7 dB re 1 V/µPa) and a bi-directional accelerometer (sensitivity: -137.6 dB re 1 V/ µm/s2). The p-output and, subsequently, the a-output of the sensor, was connected to the sound level meter. SPLs were calculated in dB re 1 µPa and PALs in dB re 1 µm/s2. These are the international units for sound pressure and particle acceleration according to ISO standards (ISO 1683, 1983).

Spectra of the ambient noise and the stimulus

The ambient noise in the tank was recorded using the software CoolEdit 2000 with a miniature hydrophone (Brüel & Kjær 8103) connected to the internal sound card of the laptop via an amplifier (36B2, constructed at the University of Vienna; including an electrical grounding). The ambient noise spectrum was then analysed using the sound analysing software STX 3.7.8 (Acoustics Research Institute, Austrian Academy of Sciences, Vienna, Austria).

Tone stimuli were recorded at the Bioacoustics lab (University of Vienna) in a walk-in sound-proof room at three different “sound steps” (step no. 2–4; Table 1). All recordings were performed twice, one when the inertial shakers were driven in phase (0°) and a second time when the shakers were driven out of phase (180°). The stimulus was presented using the same devices, software, and settings described for the experiments performed at ID17 and ID19. To simultaneously record the stimulus, we used the software CoolEdit 2000 and the internal sound card of a second laptop connected to the 36B2-amplifier (again including an electrical grounding).

Image processing and analysis

Images were processed using ImageJ v. 1.52i (https://imagej.nih.gov/ij/). The 32-bit or 16-bit (unsigned) mode of the original images was retained and the background was removed from each image in the stack by subtraction (stacks from ID19) or division (stacks from ID17) of the averaged reference image (minimum intensity, ID19; average intensity, ID17), which was based on a series of 100–200 raw reference images. The reference images had been recorded without test subject or sound presentation using the same pixel size, frame rate, and integration time. After background correction, brightness and contrast were adjusted and, if necessary, all images of the stack were flipped vertically and/or horizontally to account for virtual flips of the test subject during imaging. To improve the result of the subsequent template matching procedure (see below), image stacks were reduced to 8-bit tif files and subjected to a 3D median filter with a radius of 2 pixels.

The ImageJ plugin “Template matching–Align slices in stack” corrects shifts between images within a stack using a selected region of interest (ROI) as template for slice alignment. The corrections performed in x- and y-directions for each image can be saved as x-y-coordinates. We ran this procedure with the normalized correlation coefficient method in which a match between the template (selected ROI) and a corresponding structure on the subsequent images depends on the relative intensity contrast of the pixels under the template. We chose a uniform size for selected ROIs (40 × 40 pixels; analysis of the motion of the claustrum and the sagitta in goldfish: 20 × 20 pixels), the subpixel registration activated, and a search area around the ROI set to 10–15 pixels. We studied up to 18 ROIs per specimen and orientation (i.e. dorsal, lateral, frontal view). This displacement does not necessarily reflect the true maximum displacement in micrometres of the moving structure within the ROI because those structures within ROIs highly contrasted against the background may show a greater displacement than those that display similar grey values compared to the background. Nonetheless, displacements in micrometres inferred from the pixel size can serve as an estimate of the true displacement. The mean maximum displacement at selected ROIs (i.e. morphological structures that displayed a good contrast against the background) was inferred from the image number versus displacement plots (see S1 File), focusing on the first five oscillations during each sound period. To convert these values into estimates of maximum displacements in micrometres, mean values from these plots were multiplied by the actual pixel size of 3.67 µm (ID19). To facilitate the comparison between different ROIs in the illustrations, linear trends were removed, if present, in the software PAST v. 3.15 using the “Transform–Remove trend” method and, subsequently, residuals were plotted.

In addition to analysing the motion of auditory structures using “Template matching”, we produced overlays of the mean minimum and maximum positions of the swim bladder or the Weberian ossicles (tripus) during the motion period using Adobe Photoshop® CS6. To create an overlay image, ten images of maximum and minimum displacement of the structure of interest were averaged. Then, the averaged minimum and maximum images were copied into the red channel and the green and blue channels of a RGB image, respectively.

Videos (S1–S6 Movies) of representative image stacks from ID17 and ID19 were generated in ImageJ v. 1.52i using a frame rate of 99 fps (ID17) or 198 fps (ID19) with jpeg as compression format. Subsequently, movies in avi format were labelled in Microsoft Powerpoint® and saved as mp4 files. The videos show only a short sequence of ca. 50–100 images for each silent period before and after sound presentation.

Tomography and 3D reconstruction

In order to characterize structures of interest (otoliths, swim bladder, Weberian ossicles) in a 3D vision, one specimen of each species analysed by 2D radiography experiments was subsequently imaged at ID17 using micro-computed tomography. For this purpose, the fish was put into a 40 ml plastic tube with its snout pointing toward the bottom of the tube. The tube was filled with tap water after the fish had been stabilized with pieces of foam. The imaging was performed at 70 keV, generating 2,600 angular projections equally spaced in a range between 0° and 180° using an integration time of 50 ms per frame and obtaining a voxel size of 6.1 µm. The goldfish (standard length: 61 mm, body weight: 8.1 g) was scanned, creating three partially overlapping scans (with a maximum height of a single image stack of 4.88 mm) to cover the full range of the otophysic connection and the inner ears. In E. canarensis (standard length: 51 mm, body weight: 5.6 g), inner ears and the swim bladder extensions could be captured in a single image stack.

To create one image stack for the goldfish, the three stacks were first merged in Amira® version 6.2 applying “Register images”, “Resample transformed images”, and “Merge”. Subsequently, the otoliths, the Weberian ossicles (goldfish), the anterior parts of the swim bladder as well as parts of the neurocranium, ribs, pharyngeal teeth, and vertebrae were three-dimensionally reconstructed. Structures in the goldfish and the individual of E. canarensis were labelled using the threshold tool; a different threshold setting was used for each morphological structure to avoid artificial labelling of adjacent regions with similar grey values that were not part of the structure. If necessary, sprawling structures were erased manually. The labelled structures were resampled. Then, surface rendering was performed by triangulation and the number of faces was reduced to values between 105 and 106 depending on the size and complexity of the structure. To estimate otolith volumes based on surf files, however, surfaces were generated without previous resampling or reducing the number of faces.

Results

Performance of the standing wave tube-like setup

In the hutches at the beamlines ID17 and ID19, the ambient noise (background) levels (ANLs) were distinctly lower than the SPLs measured during sound presentation (Table 1). At the same sound step, the in phase condition (0°) and the out of phase condition (180°) displayed a difference in SPLs of 14.0–21.8 dB (ID17, ID19) and 24.8–26.0 dB (lab Vienna). The p-a sensor measurements showed that the in phase condition resulted in higher SPLs and lower PALs compared to the values yielded by the out of phase condition (shown for steps 3 and 4). The ambient noise spectrum displayed highest relative amplitudes in the low-frequency range up to ca. 100 Hz (Fig 3A). The sound spectra recorded for both the in phase and the out of phase conditions showed a clear peak at the stimulus frequency, i.e. at 200 Hz (Fig 3B and 3C).

Fig 3

Noise and tone stimulus spectra recorded in the standing wave tube-like tank at ID19.

(A) Spectrum of background noise. (B-C) Spectra of the 200 Hz stimulus recorded in the tank when both inertial shakers were driven in phase (B: 0°, 177.2 dB re 1 µPa) or out of phase (C: 180°; 155.4 dB re 1 µPa). The SPL of the 200 Hz stimulus (164.3 dB re 1 µPa (0°), and 146.2 dB re 1 µPa (180°)) was distinctly above that measured for the background noise (118.1 dB re 1 µPa). Spectra were analysed at a sampling rate of 44.1 kHz, using a Hanning filter bandwidth of 10 Hz with 75% overlap. Blue arrow indicates stimulus frequency.

Motion of auditory structures depends on sound pressure and particle motion

The motion patterns of auditory structures in both species clearly differ when the shakers were driven in phase or out of phase. When shakers are driven in phase (pressure maximum in the centre of the tank), the swim bladder walls in Etroplus canarensis and goldfish oscillate, whereas no such motion is recorded when shakers are driven out of phase (180°; S1 and S2 Movies). A similar effect occurs for the tripus, intercalarium and scaphium and the sagitta in goldfish and for the lapillus and sagitta in E. canarensis; these structures show distinct motion when subjected to the in phase condition. Under the out of phase condition, structures (especially bones and otoliths) move mainly along the x-axis, which is the axis of the shaker-induced particle motion. Under the in phase condition, structures, especially the saccular and utricular otoliths in E. canarensis, move in x-direction but also reveal distinct motion along the y-axis, which corresponds to the dorsoventral (in lateral and frontal view) or mediolateral axis (in dorsal view) of the fish.

Motion of auditory structures depends on sound levels

A decrease in sound levels (steps 1 to 4) is related to a decrease in maximum displacements of the auditory structures (Fig 4). This decrease in displacement is more distinct when shakers are driven in phase versus out of phase (Fig 4A versus 4B). The maximum displacement of structures (e.g. lapillus in Etroplus canarensis) along a certain body axis also depends on the lateral, dorsal, or frontal views and the position of the “landmark” (region of interest during the template matching procedure; S1 File).

Fig 4

SPL decrease when shakers are driven in phase versus out of phase.

Maximum displacement along the dorsoventral axis of the utricular otolith (E. canarensis) decreases with decreasing SPLs when shakers are driven (A) in phase (0°) and (B) out of phase (180°). This decrease in maximum displacement is more distinct in (A) than in (B). The scheme above each plot indicates the experimental condition, i.e. the two shakers driven in phase (0°), with pressure maximum at the centre of the tank (A) or shakers driven out of phase (180°), with maximum particle motion at the centre of the tank (B). In (B), the curves of maximum displacement showed a linear trend which was removed in the software Past 3.15 for better illustration. Sound step 2, filled blue circles; sound step 3, orange diamonds; sound step 4, filled grey circles (for corresponding sound pressure and particle acceleration levels see Table 2). Schemes adapted and modified from Hawkins, 1993 [14].

Table 2

Otolith volume and estimated otolith mass in the two specimens subjected to tomography at ID17.

Species	C. auratus (SL = 61 mm)		E. canarensis (SL = 51 mm)
Otolith type	V [mm3]	m [mg]	V [mm3]	m [mg]
left asteriscus	1.397	3.55	0.079	0.20
right asteriscus	1.445	3.67	0.077	0.20
left sagitta	0.082	0.24	0.632	1.85
right sagitta	0.100	0.29	0.635	1.86
left lapillus	0.751	2.20	0.147	0.43
right lapillus	0.732	2.15	0.112	0.33

Volume data derived from 3D reconstructions of otoliths generated in the software Amira® 6.2. Estimation of otolith mass based on the density of biogenic aragonite (2.93 g/cm3: sagittae, lapilli; [49]) and biogenic vaterite (2.54 g/cm3: asterisci; [50]).

Motion in terms of preferred axes of ancillary auditory structures and otoliths

Here, we describe for each species the motion patterns of the ancillary auditory structures (swim bladder, Weberian ossicles) and the otoliths (especially sagitta and lapilli) under both experimental conditions, i.e. shakers driven in phase (0°) versus out of phase (180°). We then briefly characterize the interplay between the moving ancillary auditory structures and the otoliths during the condition with maximum sound pressure at the centre of the tank (in phase condition, 0°).

Etroplus canarensis

The morphology of the anterior swim bladder extensions and the otoliths in E. canarensis is very similar to that in E. maculatus [26,33]; the dorsally located gas-filled part of the swim bladder closely adjoins the posterior semicircular canal and the recessus located posterior to the utricle (Figs 5A, 5B, 6A and 6B).

Fig 5

Motion patterns of otoliths and the anterior swim bladder extensions in Etroplus canarensis in dorsal view.

The fish (SL = 59 mm) was subjected to the in phase (0°, B2-B3) and the out of phase (180°, B4-B5) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) that were used to depict the motion of the structures in x- (rostrocaudal; B2, B4) and y- (mediolateral; B3, B5) direction during sound presentation. In both directions (B-B), the lapillus and the anterior swim bladder extensions moved more than the sagitta and the asteriscus. All structures show distinctly less displacement in the out of phase (B-B) versus in phase mode (B2-B3).

Fig 6

Motion patterns of otoliths and the anterior swim bladder extensions in Etroplus canarensis in lateral view.

The fish (SL = 48 mm) was subjected to the in phase (0°, B2-B3) and the out of phase (180°, B4-B5) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) that were used to depict the motion of the structures in x- (rostrocaudal) and y- (dorsoventral) direction during sound presentation. (B-B) In both directions, the lapillus moved more than the sagitta and asteriscus or the swim bladder extensions. All structures show distinctly less displacement in the out of phase (B-B) versus in phase mode (B2-B3).

Swim bladder motion. When shakers are driven in phase, the anterior swim bladder extensions show a “complex” motion pattern. Some portions of the swim bladder walls move along the dorsoventral axis while other parts, at the same time, oscillate mainly along the rostrocaudal or mediolateral axes (S2 and S3 Movies). Different parts may also move along the same axis but in the opposite directions (Fig 5B). The swim bladder walls attached to the vertebral column and parts in the rostroventral portion show weak or no discernible movement (S2 and S3 Movies). When shakers are driven out of phase (180°), the walls show no clear oscillation but the whole swim bladder moves back and forth mainly along the direction of sound-induced particle motion (Figs 5B–6B versus 5B, 6B; S1B Fig versus S1A and S1D Fig versus S1C).

Otolith motion. When shakers are driven in phase, the lapilli move distinctly along all three axes, i.e. the rostrocaudal, the mediolateral, and the dorsoventral axes (Figs 5B–7B and 7B; S1C). The sagitta shows motion along the dorsoventral axis mainly in the rostral part of the otolith (S2C; S3 Movie) and to a lesser degree along the rostrocaudal axis (S2A and S2B). The dorsal view reveals that the sagitta tilts medially; this motion is most pronounced in the rostral otolith portion, indicated by the phase shift between the anterior tip, the ventral, dorsal, and posterior portions of the sagitta (S2A). In frontal view, the anterior tip of the sagitta moves in phase with the adjacent part of the neurocranium (S2C). No distinct motion pattern is evident for the asteriscus, which moves slightly along the rostrocaudal and mediolateral axes (Fig 5B). When shakers are driven out of phase (180°), the lapilli and sagitta move distinctly less than during the in phase condition (Figs 5, 6, 7B). Moreover, left and right sagittae, but especially both lapilli, move in the same direction along the mediolateral axis, i.e. left and right otoliths of the same type (e.g. both lapilli) move either to the left or the right side (S3 Movie).

Fig 7

Motion patterns of otoliths and the anterior swim bladder extensions in Etroplus canarensis in frontal view.

The fish (SL = 49 mm) was subjected to the in phase (0°, B2-B3) and the out of phase (180°, B4-B5) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) that were used to depict the motion of the structures in x- (mediolateral) and y- (dorsoventral) direction during sound presentation. In (B), the lapillus shows distinctly more motion than the sagitta and asteriscus or the swim bladder extensions along the mediolateral axis, whereas in (B3) the sagitta moves most along the dorsoventral axis. All structures are displaced distinctly less in the out of phase (B-B) versus in phase condition (B,B).

Interplay of swim bladder and otolith motion (0°, in phase condition). When the ventral wall parts of the swim bladder extensions move in ventral direction and the rostral-most part of the extensions move rostrally, the lapilli move at the same time (dorso)rostrally and laterally while the sagittae move ventrally and tilt medially.

Goldfish

Motion of the swim bladder and Weberian ossicles. When the shakers are driven in phase (0°), the anterior swim bladder walls of the goldfish oscillate along the rostrocaudal and dorsoventral axes (Fig 8C and 8D; S3C and S3D). The tripus and the intercalarium move mainly along the rostrocaudal and dorsoventral axes (Fig 8D; S3C and S3D Fig), whereas the scaphium also displays a distinct motion along the mediolateral axis (S3C and S3D Fig; S4 Movie), i.e. towards the sinus impar. In lateral view, the scaphium moves more pronouncedly along the dorsoventral axis than the claustrum (S4C and S4C Fig, S5B and S5C Fig; S5 Movie). In the out of phase condition (180°), the swim bladder walls show no clear oscillation but the whole swim bladder moves mainly along the axis of sound-induced particle motion (Fig 8C and 8D; S3C,S3C and S3D Fig, but see S3D Fig). The Weberian ossicles also move distinctly less than in the in phase condition (Fig 8C and 8D versus 8C and 8D; S3C,S3C, S3D and S3D versus S3C, S3C, S3D and S3D Fig, S4B Fig versus S4A Fig).

Fig 8

Motion patterns of the Weberian ossicles (scaphium, intercalarium, tripus) and the anterior-most swim bladder portion in goldfish in lateral view.

The fish (SL = 50 mm) was subjected to the in phase (0°, C1-C2) and the out of phase (180°, D1-D2) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) depict the motion of the structures in x- (rostrocaudal) and y- (dorsoventral) direction during sound presentation. The Weberian ossicles and the anterior swim bladder wall show distinctly less displacement when the inertial shakers are driven out of phase (D-D) versus in phase (C-C).

Comparing the left and right Weberian ossicles (S3 Fig) reveals that the amount of displacement, and whether or not a phase shift occurs, differs between left and right ossicles of the same type (see also S1 File for the scaphium).

Otolith motion. When shakers are driven in phase (0°), the sagitta moves along the rostrocaudal axis (S6C Fig, S7C, S7C and S7D Fig), tilts laterally (S7C, S7C and S7D Fig; S6 Movie) and shows distinct motion along the dorsoventral axis (Fig 9C versus 9C). The lateral tilt of the sagitta was also clearly visible when the fish was subjected to a 500 Hz pure tone stimulus (frame rate = 497.12 fps, SPL = 177.3 dB re 1µPa; S7D and S7D Fig). The asterisci and lapilli display (weak) motion along all three axes and the amount of motion is similar for both conditions (0° and 180°; Fig 9; S6 Fig); however, lapilli seen in dorsal view show a stronger rostrolateral movement under the out of phase versus the in phase condition. The sagitta does not display a clear lateral tilt under the out of phase condition and its displacement along the dorsoventral axis is distinctly smaller than under the in phase condition (Fig 9C versus 9D).

Fig 9

Motion patterns of the otoliths (lapillus, sagitta, asteriscus) in goldfish in lateral view.

The fish (SL = 50 mm) was subjected to the in phase (0°, C-C) and the out of phase (180°, D-D) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) depicted the motion of the structures in x- (rostrocaudal) and y- (dorsoventral) direction during sound presentation. The maximum displacement of lapilli and asterisci is similar regardless of whether the inertial shakers are driven in phase or out of phase. Along the dorsoventral axis, the sagitta shows less displacement in the out of phase condition (C2 vs. D2).

Interplay of the motion of all auditory structures (0°, in phase condition). When the rostral swim bladder walls move in (dorso-)rostral direction, the scaphium moves rostrodorsally and simultaneously in medial direction, while the sagitta shifts also rostrodorsally and at the same time tilts laterally. The movie (S5 Movie) shows that the sagitta tilts laterally when the scaphium moves medially towards the sinus impar.

Relationship between otolith size and otolith motion

Comparing otolith motion in both species reveals weak motion for large otoliths (asterisci, lapilli) in goldfish and light otoliths (asterisci) in E. canarensis (Table 2). In E. canarensis, lapilli move more (1.2–8.7 µm at SPL = 164.3 dB re 1 µPa) than sagittae (0.6–2.2 µm at SPL = 164.3 dB re 1 µPa). In goldfish, the lapilli are displaced less (1.0–1.8 µm at SPL 164.3 dB re 1 µPa) than the sagittae (0.6–3.9 µm at SPL 164.3 dB re 1µPa; S1 File).

When shakers are driven out of phase (180°), the difference in displacement between otolith types is much smaller; the amount of motion of sagittae and lapilli of E. canarensis ranges from 0.7–1.2 µm at SPL 146.2 dB re 1µPa and for that in goldfish ranges from 0.5–1.0 µm (sagittae) and 1.2–1.5 µm at SPL 146.2 dB re 1µPa (lapilli; S1 File).

Discussion

We successfully improved our initial setup presented in Schulz-Mirbach et al., 2018 [30] to separately study the motion of auditory structures based on sound pressure and sound-induced particle motion. First, we present models of how otolith motion patterns may stimulate different groups of ciliary bundles on the respective macula and compare the potential effects of two different types of otophysic connections on otolith motion. Our discussion focusses on the utricular and saccular otoliths because the lagenar otoliths in both species showed amplitudes too small to be reliably interpreted. In the second part, we discuss methodological aspects of the new setup.

Assumed interplay between the moving auditory structures and inner ear stimulation

The oscillating walls of the dorsal, gas-filled part of the swim bladder extension in E. canarensis may enhance the endolymph motion in the recessus posterior to the utricle (see Fig 9C in [26]), thereby enhancing lapillus motion. This is in line with the concerted forward movement of the lapillus and the swim bladder wall close to the recessus, and with a previously hypothesized stimulation of the utricle (see Fig 9C in [26]). The “swinging” motion of the lapillus may stimulate most of the sensory hair cells on the cotillus and the striola of the macula utriculi (Fig 10D; see Fig 4C in [26]). The stronger motion of the lapillus due to the contribution of the swim bladder (in phase condition) may also stimulate the sensory hair cells on the lacinia (= dorsolateral macula portion). Although the lapillus does not overlie the lacinia, a (strongly) back-and-forth “swinging” otolith might exert enough drag force on the otolithic membrane connecting otolith and macula to each other (including the lacinia), thereby resulting in a deflection of the ciliary bundles of the lacinia (see pers. comm. A. N. Popper in [26]).

Fig 10

Model of otolith and swim bladder motion in Etroplus canarensis.

Auditory structures are shown in (A) medial, (B) dorsal, (C) frontal, and (D) a perspectival view. In (D), structures are shown in rostral, medial, and dorsal aspects to visualize the assumed 3D motion of the lapillus, sagitta, and the anterior swim bladder extension (0° condition) also with regard to the underlying maculae. Black arrows: motion when fish was subjected to the in phase condition (0°); white arrows: out of phase condition (180°). For clarity, 3D reconstructions of only the otoliths and the swim bladder extension of the right side are shown in (A), (B), and (C). Green: anterior swim bladder extension; red: lapillus; purple: sagitta; yellow: asteriscus, blue: bone. Shape of maculae and the orientation patterns of ciliary bundles adapted and modified from Schulz-Mirbach et al., 2014 [33].

The sagitta motion pattern may be explained by the attachment of the anterior tip of the saccule to the surrounding bone [26], as the tip has distinctly fewer degrees of freedom to move along the dorsoventral axis than the “free” posterior part of the saccule. Accordingly, the sagitta shows a greater dorsoventral displacement in its posterior region (Fig 10A, 10C and 10D). As most ciliary bundles on the posterior portion of the macula sacculi are vertically oriented (Fig 10D, see also Fig 5C in [33]), the strong sagitta motion in this region should stimulate the sensory hair cells. Both the anterior and posterior-most portion of the macula sacculi are characterized by horizontally oriented ciliary bundles and the slight movement of the sagitta along the rostrocaudal axis (Fig 10C) might stimulate the sensory hair cells here. The tilting motion might be provoked by endolymph passing through the thin canal (see Fig 3C in [26]) connecting the saccule to the upper ear and thereby pushing the dorsal-most hump of the sagitta against the macula sacculi. The lateromedial tilt of the sagitta (Fig 10A–10D) may stimulate 1) the sensory hair cells in the “neck” of the macula sacculi, in which ciliary bundles are diagonally oriented with respect to the rostrocaudal axis and 2) those of the rostrodorsal portion covered by otolithic membrane only (see Fig 3C in [26]). In the latter case, the lateral tilt along with a backwards motion (along the rostrocaudal axis) may pull the otolithic membrane backwards and diagonally to the rostrocaudal axis resulting in a deflection of ciliary bundles not overlain by the otolith. This is in line with theoretical considerations on the stimulation of sensory hair cells not overlain by the otolith (for an overview see [4]). Whether the oscillation of the swim bladder walls is also transmitted to the neurocranium (and further transferred to the saccule at the contact site between saccular tip and the bone) remains elusive. Our data do not provide support for this pathway of local particle motion transduction (see [26]): the displacement of the neurocranium surrounding the saccule was similar for both conditions, i.e. regardless of whether the fish was subjected to the in phase (0°) or the out of phase (180°) condition.

When fish were studied in lateral or dorsal view, the main axis of the sound corresponded to the fish’s rostrocaudal axis and left and right sagittae and lapilli showed the same synchronous motion. Accordingly, if fish were alive, the relative motion between otolith and the underlying macula would be the same for the left and the right side regardless of the experimental condition applied (0° or 180°). The situation is more complex when sound impinges on the body flanks of the fish (“experiments in frontal view”). For the in phase condition (0°), left and right sagittae and lapilli showed the same (mirror-imaged) motion. Again, this means that sensory hair cells on the maculae are stimulated in the same way on the left and the right side. For the out of phase condition (180°), however, left and right lapilli both moved simultaneously either to the left or to the right side. In this case, the respective orientation groups of ciliary bundles on the left and right maculae utriculi do not experience the same shearing force. Hence, a combination of otolith motion provoked by sound-induced particle motion and sound pressure would result in a different stimulation of left and right otolith end organs (here: utricle and saccule). This interpretation is in accordance with models of the direct and indirect stimulation pathway in fish with an otophysic connection (see Fig 5 in [16]). The potential role of differences in motion patterns between left and right otoliths for the ability of fishes to localize a sound source is, however, beyond our data and remains to be studied.

The motion of the tripus, intercalarium, and scaphium provoked by the oscillating anterior swim bladder walls agrees with the movement of these ossicles hypothesized by previous studies [44,51-53]. We can also confirm the assumption [54] that the claustrum “does not” move and thus may act as a counter-bearing to the scaphium, which displays a distinct motion. In this way, the perilymphatic fluid in the part of the sinus impar enclosed by the scaphium and the claustrum would efficiently be pressed into the main anterior portion of the sinus impar. This then results in a corresponding fluid flow in the sinus endolymphaticus and the transverse canal (Fig 11B and 11C).

Fig 11

Model of otolith, Weberian ossicle, and swim bladder motion in goldfish.

Auditory structures are shown in (A) lateral, (B) dorsal, and (C) a perspectival view. In (A), a 3D scheme of the macula sacculi including the orientation pattern of ciliary bundles is shown in context with the overlying sagitta. In (B) and (C), black arrows with dotted lines indicate the motion of the hypothesized perilymphatic fluid in the sinus impar and the endolymphatic fluid in the transverse canal and the saccules (see [44,51–53]). In (C), structures are shown in rostral, medial, and dorsal aspects to visualize the assumed 3D motion of the sagitta, the Weberian ossicles, and the swim bladder (0° condition). Black arrows: motion under the in phase condition (0°), white arrows the out of phase condition (180°). White arrows in parentheses: no relative motion of structure with regard to the surrounding bones (neurocranium, vertebrae). For clarity, 3D reconstructions of the otoliths and the Weberian ossicles of only the right side are shown in (A) and (C). Green: swim bladder (anterior-most part); red: lapillus; purple: sagitta; yellow: asteriscus, light blue: claustrum; dark purple: scaphium; gold: intercalarium; dark blue: tripus. Shape of macula sacculi and orientation pattern of ciliary bundles in (A) adapted and modified from Platt, 1977 [55].

Earlier studies [44,52,53] predicted that fluid flow of the endolymph through the dorso(medially) located transverse canal into each of the saccules should “impinge” on the free (lateral) wing of the sagitta. If this was the case, the sagitta should display a lateral tilt, which is the motion we observe in our experiments with goldfish subjected to the in phase (0°) condition (Fig 11B). The fluid flow from the dorsal margin to the lateral wing of the sagitta may also enhance the dorsoventral motion of this otolith. The distinct dorsoventral motion of the sagitta probably stimulates the sensory hair cells, which possess mainly vertically oriented ciliary bundles (Fig 11A; [55]). The tilting motion may also lead to the mediolateral motion of the posterior needle-like part of the sagitta. In this posterior region of the sagitta, the macula sacculi is curved, bringing the initially vertically oriented ciliary bundles into a more mediolateral orientation [55]. Hence, sagitta motion in the posterior region would stimulate the mediolaterally oriented ciliary bundles.

In contrast to the sagitta, which shows a greater displacement under the in phase (0°) condition, the motion of the lapillus is more distinct in the out of phase (180°) condition. This supports the hypothesis of a more pronounced division of labour between the otolith end organs in otophysans [45]. This hypothesis states that the otophysan saccule is an end organ specialized to detect sound pressure, whereas the otoliths of the lagena and the utricle act as accelerometers detecting sound-induced particle motion. In our experiments, the lapillus motion was generally very small, making it difficult to determine whether there is a distinct difference in lapillus motion between the two conditions applied, i.e. 0° versus 180°.

Otolith mass and the type of otophysic connection apparently affect otolith motion

Comparing the estimated mass of the three otolith types in goldfish and E. canarensis (Table 2) with the observed motion patterns of these otoliths clearly reveals that the amount of otolith motion does not follow the simple correlation of the greater the mass the greater the displacement as predicted by mathematical modelling (see for a 200 Hz stimulus Fig 10 in [56]). In the two studied species that represent two different types of otophysic connections, otolith motion clearly depends on other additional factors. The otophysic connection enhances otolith motion in both species; nonetheless, different otolith types are affected to a different amount within the same species and also show species-specific differences. The motion of the lapillus and the sagitta in E. canarensis is clearly boosted when the swim bladder walls oscillate under the in phase (0°) condition, whereas in goldfish, only sagitta motion is enhanced by swim bladder wall oscillation and Weberian ossicle motion. Moreover, the light sagitta in goldfish moves distinctly, whereas the similarly light asteriscus in E. canarensis shows only faint movement. Finally, the “heavy” sagitta in E. canarensis moves pronouncedly, whereas the heavier lapillus and asteriscus in goldfish reveal no or only weak motion.

One potential reason for the species-specific differences is the amount of damping effect of the otophysic connection. The system in E. canarensis may enable more overmodulation at the high SPLs applied in our experiments because the anterior swim bladder extensions directly contact the thin bony lamella close to the lagena and near the exoccipital foramina approaching the posterior part of the upper ears [26]. For the otophysan type of otophysic connection (goldfish), the system is more damped because the saccules are indirectly connected to the swim bladder through the chain of Weberian ossicles [29]. Accordingly, in a more damped system, the local particle motion transmitted to accelerometer-like otoliths such as the lapillus and the asteriscus is less than in the Etroplus-type; the motion of lapilli and asterisci may be so small that it is at or beyond the limits of the spatial resolutions available in our experiments. Finally, our findings provide further support for the assumption that the otophysan saccule acts as a displacement sensor [29] in which sagitta motion mainly depends on the incoming fluid flow from the transverse canal. Sagitta motion seems to increase with increasing sound frequency based on mathematical modelling of the function of the Weberian apparatus in goldfish [29]. It remains to be studied whether the sagitta in E. canarensis is similarly influenced by fluid flow through the thin canal connecting the upper ear with the saccule.

In E. canarensis, the light asteriscus is likely to act as an accelerometer, and its mass may be too low to show a distinct motion due to the 200 Hz pure tone stimulus. At frequencies above 200 Hz, when the swim bladder contribution increases, greater local particle motion may be transmitted to the asteriscus, resulting in a stronger motion of this otolith. Note that a recent study on the plainfin midshipman (Porichthys notatus Girard, 1854) [57], a species that lacks an otophysic connection, indicates that auditory thresholds of the lagena (based on sound pressure and particle acceleration) were higher than that of the saccule in the frequency range from 85 to 505 Hz.

In both species, sagitta motion was enhanced through the oscillation of the swim bladder walls when fish were subjected to the in phase (0°) condition. Such an enhancement of otolith motion at low frequencies in goldfish agrees with previous experimental studies on tripus extirpation and swim bladder deflation [39,58] as well as with mathematical modelling of the motion of the auditory structures [29]. A swim bladder effect at 200 Hz was confirmed by Ladich & Wysocki, 2003 [58] for goldfish based on bilateral tripus extirpation. A mathematical model further predicts that the Weberian pathway of saccule stimulation has an enhancement effect over the entire range of detectable frequencies (i.e. above 100 Hz; [29]). However, in Etroplus maculatus, AEP thresholds did not differ significantly in the range from 100 to 300 Hz [15] indicating a minor swim bladder effect at 200 Hz, similar to that measured in the brown bullhead Ameiurus nebulosus (Lesueur, 1819) (Otophysa, Ictaluridae) during swim bladder deflation experiments [59]. Swim bladder deflation experiments in Etroplus maculatus and E. canarensis may help clarify the degree to which the swim bladder contributes to audition at 200 Hz.

Potential and limitations of the standing wave tube-like setup

Compared to the one-speaker setup developed by Schulz-Mirbach et al. (2018) [30], relative movements between different otolith types (sagitta and lapillus in E. canarensis), bones, and the swim bladder walls were clearly visible using the current standing wave tube-like setup (displacements in this study: up to 8.7 µm at 164 dB re 1 µPa; in [30]: ca. 6.5–13.0 µm at 158 dB re 1 µPa). The difference of at least 20 dB for measured SPLs between the in phase (0°) and out of phase condition (180°) points to a largely successful separation of the particle and pressure components of sound in our setup. This was also clearly reflected in the difference in motion of the auditory structures between these two conditions. In addition, we were able to characterize the motion patterns because of the higher spatial resolution of 3.67 µm available at ID19 versus 6.5 µm in the former study [30].

Nonetheless, several issues remain. The compliance of the Plexiglas® walls with a thickness of only 5 mm may be a problem regarding the in phase condition. Achieving a real pressure maximum with “no” particle motion at the centre of the tank would require using more rigid and stiff walls that do not oscillate during sound presentation. This can only be realized with a steel tank at a wall thickness of ca. 1 cm (see [17]). In our setup, wall thickness could not be increased because this would also substantially increase the attenuation of the X-ray beam, resulting in a poor signal-to-noise ratio [30]. Another problem of tank acoustics is related to the samples themselves. Hawkins & MacLennan (1976) [17] pointed out that the gas-filled swim bladder of a fish may alter the sound field, and to avoid this issue the authors studied a flatfish species lacking internal gas bladders (plaice, Pleuronectes platessa L., 1758). In our experiments this aspect might be, however, negligible because both studied species possess a large, two-chambered swim bladder [15].

From an imaging point of view, the “large” water body in the tank impairs a real-time observation of the moving auditory structures in the fish. At ID19, maximum image acquisition rates of up to 1 kHz (1,000 fps) can be achieved, which would be sufficient to record these motions in real time. However, such high frame rates are only possible for samples investigated in air [60,61]. It is thus important to note that our observations of moving otoliths and ancillary auditory structures are a proxy of the true motion.

High sound pressure levels and tissue damage by hard X-ray imaging might be another source of artefacts. The sound pressure levels used in our study are distinctly higher than those experienced by fish in natural habitats [42,62,63]. Yet, we are not able to detect otolith movement at a few nanometers (e.g. at auditory thresholds) due to the constraints implied by the imaging procedure. However, as the motion of the otoliths displayed a regular and reproducible pattern for different SPLs (see S1 File), we assume that the observed patterns may also hold true for biologically relevant SPLs.

To minimise detrimental effects of e.g. changed elasticity of the tissue, we avoided to conduct experiments in the same order. A comparison of histological sections of fish subjected to hard X-rays with those of control fish in future studies could help to evaluate the kind and degree of tissue damage of the inner ears and the swim bladder.

Conclusions

Even though further methodological improvements such as higher temporal resolutions are envisaged, the current setup is a suitable compromise between the intended separation of sound-induced particle motion and sound pressure on the one hand and the requirements of the X-ray based imaging procedure on the other. Our setup enabled the detailed characterization of the interplay between the otoliths and ancillary auditory structures such as anterior swim bladder extensions and Weberian ossicles. This yielded extensive experimental evidence for hypotheses on how different types of otophysic connections affect otolith motion. We were also able to draw first conclusions on how the motion patterns of the sagitta and the lapillus (the latter only in E. canarensis) stimulate the sensory hair cells on the respective macula.

The 200 Hz stimulus (0°) provoked a motion pattern of the sagitta in both species and the lapillus in Etroplus canarensis that closely fit the orientation patterns of ciliary bundles on the macula sacculi or the macula utriculi. In E. canarensis, we conclude that the specific otolith motion patterns stimulate most of the orientation groups on the respective macula under both experimental conditions (0° and 180°). The stimulation of sensory hair cells on both the macula sacculi and the macula utriculi is probably enhanced in E. canarensis because the swim bladder acts as pressure-to-displacement transducer due to the resulting greater displacement of the sagitta and the lapillus. In goldfish, the lapillus was displaced more when subjected to the out of phase (sound-induced particle motion) condition, whereas the sagitta moved more under the in phase (sound pressure) condition. We conclude that, in goldfish, this difference in otolith motion under the in phase and the out of phase condition is experimental support for a hypothesized division of labour of otolith end organs in otophysans, in which the saccule apparently plays a highly specialized role in sound pressure detection [45].

Amount of displacement of otoliths and ancillary auditory structures.

Representative output data from the template matching analysis performed in ImageJ v. 1.52i of both fish species are given, including displacement plots. Data depict motion patterns of selected “landmarks” (regions of interest 40 × 40 pixels) related to sound steps 2 to 4 for both conditions, i.e. 0° and 180°. Displacements in micrometres are given based on a pixel size of 3.67 µm (ID19).

(XLSX)

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Motion of the utricular and saccular otoliths and the swim bladder extensions in Etroplus canarensis in lateral view.

The fish was subjected to a 200 Hz pure tone stimulus when inertial shakers were driven in phase (0°). (frame rate: 99 fps; ID17, pixel size 6.1 µm).

(MP4)

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The fish was subjected to a 200 Hz pure tone stimulus when inertial shakers were driven in phase (0°) or out of phase (180°), respectively. (frame rate: 198 fps; ID19, pixel size 3.67 µm).

(MP4)

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Motion of the utricular and saccular otoliths and the swim bladder extensions in Etroplus canarensis shown in frontal view.

The fish was subjected to a 200 Hz pure tone stimulus when inertial shakers were driven in phase (0°) or out of phase (180°), respectively. (frame rate: 198 fps; ID19, pixel size 3.67 µm).

(MP4)

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Motion of the Weberian ossicles and the swim bladder in goldfish shown in dorsal view.

The fish was subjected to a 200 Hz pure tone stimulus when inertial shakers were driven in phase (0°). (frame rate: 198 fps; ID19, pixel size 3.67 µm).

(MP4)

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Motion of left and right scaphia and claustra in goldfish shown in lateral view.

The fish was subjected to a 200 Hz pure tone stimulus when inertial shakers were driven in phase (0°). (frame rate: 198 fps; ID19, pixel size 3.67 µm).

(MP4)

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Motion of the otoliths (mainly the sagittae) in goldfish shown in dorsal view.

The fish was subjected to a 200 Hz pure tone stimulus when inertial shakers were driven in phase (0°). (frame rate: 198 fps; ID19, pixel size 3.67 µm).

(MP4)

Click here for additional data file.

Motion patterns of auditory structures in Etroplus canarensis in lateral view.

Overlays of averaged maximum (red) and minimum (green) positions indicate the motion of otoliths (lapilli, sagittae) and the walls of the anterior swim bladder extensions due to the stimulus presentation. (A) and (C) depict the structures during the in phase (0°) condition of the sound stimulus, whereas (B) and (D) represent the out of phase (180°) condition. (C2) vs. (D2) illustrates that the swim bladder walls show distinct motion (shakers driven in phase) as opposed to no oscillation (shakers driven out of phase). Structures outlined in red and green indicate motion, whereas uniformly gray, white or black structures point to no or weak movement during sound presentation. The overlays in (A-B) represent an individual (SL = 51 mm) studied at ID17 (pixel size 6.1 µm, frame rate 98.9954 fps) and those in (C-D) illustrate a specimen (SL = 48 mm) imaged at ID19 (pixel size 3.67 µm, frame rate 198.02 fps).

(TIF)

Click here for additional data file.

Motion patterns of the rostral tip as well as the ventral, dorsal, and caudal margins of the sagitta in Etroplus canarensis.

Motion patterns are shown in (A) dorsal, (B) lateral, and (C) frontal views (pixel size 3.67 µm, ID19) when the fish was subjected to the in phase (0°) condition. The clear phase shift between different parts of the sagitta in all plots except in (B2) indicates the tilting movement of this otolith during sound presentation. In (C1), the rostral tip of the sagitta shows less displacement than the ventral and posterior margins and moves in phase with the adjacent part of the neurocranium, which is also seen in (B1). The respective landmark number is given in parenthesis (see also Figs 5B, 6B and 8B).

(TIF)

Click here for additional data file.

Motion patterns of the Weberian ossicles (scaphium, intercalarium, tripus) and the anterior-most swim bladder portion in goldfish in dorsal view.

The fish (SL = 56 mm) was subjected to the in phase (0°, C1-C2, D1-D2) and the out of phase (180°, C3-C4, D3-D4) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) depict the motion of the structures in x- (rostrocaudal) and y- (mediolateral) direction on the right (C) and left (D) body side during sound presentation. The Weberian ossicles and the anterior swim bladder wall show distinctly less displacement when the inertial shakers are driven out of phase (C3-C4, D3-D4) versus in phase (C1-C2, D1-D2).

(TIF)

Click here for additional data file.

Motion patterns of auditory structures in goldfish in (pseudo-)lateral view.

Overlays of averaged maximum (red) and minimum (green) positions indicate the motion of otoliths (sagittae), Weberian ossicles, and the walls of the anterior swim bladder portion due to stimulus presentation. (A) and (C) depict the structures during the in phase (0°) condition of the sound stimulus, (B) during the out of phase (180°) condition. (C2) illustrates that tripus, intercalarium, and scaphium move distinctly while the motion of the claustrum is weak. Structures outlined in red and green indicate motion, whereas uniformly grey, white, or black structures point to no or weak movement during sound presentation. (A-B) represent an individual (SL = 59 mm) studied at ID17 (pixel size 6.1 µm, frame rate 98.9954 fps) and C illustrates a specimen (SL = 50 mm) imaged at ID19 (pixel size 3.67 µm, frame rate 198.02 fps).

(TIF)

Click here for additional data file.

Motion pattern of scaphium and claustrum in two different individuals of goldfish in lateral view.

The fishes (B, SL = 50 mm; C, SL = 58 mm) were subjected to the in phase (0°) condition. (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1) and (C1). In (B, C), “landmarks” (squares of 20 × 20 pixels) depict the motion of the structures in x- (rostrocaudal) and y- (dorsoventral) direction during sound presentation. Along the rostrocaudal axis (B, C), both ossicles show a similar amount of displacement (except landmark 4 in C2). Along the dorsoventral axis (B3, C3), the scaphium in both individuals is displaced more than the claustrum.

(TIF)

Click here for additional data file.

Motion patterns of the otoliths (lapillus, sagitta, asteriscus) in goldfish in dorsal view.

The fish (SL = 56 mm) was subjected to the in phase (0°, C-C) and the out of phase (180°, D-D) conditions (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B1). (B) “Landmarks” (squares of 40 × 40 pixels) depicted the motion of the structures in x- (rostrocaudal) and y- (mediolateral) direction during sound presentation. Otoliths show a greater maximum displacement along the rostrocaudal than along the mediolateral axis. The maximum displacement of lapilli and asterisci is similar regardless of whether the inertial shakers are driven in or out of phase.

(TIF)

Click here for additional data file.

Motion pattern of the sagitta in goldfish in dorsal view.

The fish (SL = 56 mm) was subjected to the in phase condition (0°) using a 200 Hz (SPL = 177.2 dB re1 µPa; frame rate = 198.02 fps; C) or a 500 Hz pure tone stimulus (SPL = 177.3 dB re 1 µPa; frame rate = 497.512 fps; D) (pixel size 3.67 µm, ID19). (A) 3D reconstruction of the structures shown in the 2D radiograph in (B). (B) “Landmarks” (squares of 20 × 20 pixels) depict the motion of the structures in x- (rostrocaudal) and y- (mediolateral) direction during sound presentation. In (C-C), landmarks 1, 4, and 6 show a phase shift with regard to landmarks 2–3 and 5–7, indicating a tilting motion of the sagitta. In (C-C) and (D-D) this phase shift is shown for the landmarks 1 and 3 also with respect to the motion of the adjacent bone (landmark 8).

(TIF)

Click here for additional data file.

13 Jan 2020

PONE-D-19-30473

Auditory chain reaction: effects of sound pressure and particle motion on auditory structures in fishes

PLOS ONE

Dear Dr. Schulz-Mirbach,

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Additional Editor Comments (if provided):

As you can see, both reviewers were highly complemented of the value of this work and had relatively small corrections to be made before publication. Both reviewers commented on the rather high sound levels used in the experiments and I do agree with them that are are of little direct "ecological" relevance as such but do recognize they may have been needed to accurately measure the fine-scale movements in the current paper. A short note in the manuscript stating this and arguing as to whether or not there would be a linear damping of the movements with a decrease in sound levels would be helpful to rebut any criticisms in this regard. The reviewers also note that the discussion is overly long so effort should be made to reduce length here to increase readability. In particular I find you take too much space pointing out all the limitations to your study (MS pages 15 & 16) and this could be greatly reduced to maybe even one paragraph and perhaps moved to later in the discussion section. Other parts of the discussion could also be profitably condensed. Reviewer 2 also notes that the large number of figures further reduced readability so consider whether any of those could also be removed.

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Reviewer #1: Review of Schulz-Mirbach et al. manuscript titled “Auditory chain reaction: effects of sound pressure and particle motion on auditory structures in fishes”.

The manuscript is a very well written paper that investigates sound-induced motion of the otoliths within the fish inner ear end organs from two fish that have different otophysic connections of the swim bladder to the inner ear. This paper is a follow up study to their previous work but in the current paper the authors a standing wave tube-like tank to examine the motion of the inner ear otoliths under conditions where there is maximum particle motion vs maximum sound pressure. The authors provide the first experimental evidence of how the two different types of otophysic connections affect otolith motion in the fish inner ear. The subject of the paper should be of general interest to the readers of Plos One. I have only a few comments below.

1) The stimulus used to induce motion of the otoliths within the end organs was quite high ranging from 141 to >177 dB re 1 microPa. The authors reported (in the methods lines 206-207) that “step (at ID19) equals a SPL of 177.2 dB re 1 microPa when shakers were in phase and a SPL of 155.4 re 1 microPa when shakers were driven out of phase”. These sound levels seem to be out of the biologically relevant ranges. I realize that these levels were likely required in order to visualize the induced motion of the otoliths. My concern is that the induce motion and how it may activate motion of the inner ear may not be biologically relevant to natural auditory stimuli experienced by these fish in nature. To alleviate such concerns, the authors should at least address this issue and explain the limitations (I know they have to some degree in the discussion) of how more natural sound levels might induce similar motions at lower levels.

2) An interesting note on line 399-401 that states that the left and right Weberian ossicles revealed differences in the amount of displacement when subjected to sound induced motion. The authors also describe the differential motion of the otoliths based on experimental condition. Are the authors suggesting that the Weberian apparatus might afford directional sensitivity to the sound source? This would be extremely interesting if true. Also, their description of the sound induced movement of the otoliths in the Canara pearlspot cichlid may have implications for how the inner ear uses the particle motion and indirect sound pressure cues to determine sound source direction via the phase model hypothesis.

3) The authors also describe other interesting results of the “tilting motion” of the sagitta in E. canarensis. Was there any evidence for the so-called “rocking motion” of otoliths under their experimental conditions as described by Krysl et al (2012) in PloSOne.

4) Surprisingly the authors do not discuss the induced movement of the asteriscus in E. canarenis given the close proximity of the lagena to the swim bladder in the cichlid. Although sound induce movement was minimal at the experimental sound levels used, movement of this otolith could be very important in terms of the indirect detection of particle motion from the swim bladder. Fay reported that fish can detect particle motion displacement as low as 1 nm. Thus, the authors should not discount the importance the lagena and saccule given their close proximity to the swim bladder.

Reviewer #2: The study using a new apparatus to image the auditory portions of the inner ear during both sound pressure and particle motion. The design is clever and is novel in that it could image this structures during stimulation. The authors do a good job of indicating potential limitations with their procedures. The study makes a very nice overall contribution to the field.

A couple of items that need to be clarified or considered.

The fish are dead and tissue structure can break down very quickly, especially if high intensity lighting was needed and it generated heat. It would have been useful if there was postmortem examination to determine if there were any structure changes that could not be detected by imaging. For example, what was the effect of blood clotting?

The SPL were very high and not normally used in fish auditory studies. Was this due to the limitations in the imaging? I would expect to see large scale movements at these SPL that fish may never experience. How did this impact the studies and models?

I presume the test chamber is designed to get around the problems associated with auditory studies in small tanks. Can physiological studies eventually be done in these types of tanks?

I felt the discussion is far too speculative. The authors have a very nice data set and do not need to explain every variance that may be impacting the structure. I would suggest that the discussion focuses on what can definitely be determined and limit the number of speculative discussion.

I realize this is an on line journal and page limits are not a concern, however 18 figures seem a little excessive and it is hard for the reader to wade through and determine which ones are really need. I would suggest that several figures could be moved to supplemental files.

Minor

Abstract The use of the term maximum even in quotations is confusing.

29 “inner” ears. Many times the authors use the term ear, when inner ear would be more accurate

40 I think it is safe to say they are no longer “assumed”

45-46. Sentences end and start with same words

What is an improved hearing ability. This is usually termed increased sensitivity or greater frequency range

51 … whole body in motion: It is unclear the effect of hard skeletal elements. Also, the motion is oscillolatory with no net movement gain and therefore motion is not best word? Vibration?

78 The pearlspot is not considered as well investigated as the goldfish

95 The saccule has plays a very important vestibular function and therefore does not “mainly” function in audition

113 The cessation of opercular movement is not considered an endpoint of euthanasia. Many fish (including goldfish) can be easily revived after the cessation of opercular movement. There should have been an additional period following the cessation of movement to insure complete euthanasia

Methods

The image rate of 200 Hz for the 200 Hz seems relatively slow. Most video is run higher than stimulus. Please explain if this had an effect on resolving the movements

633 add reference

Table 1: delete, add info to text

**********

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Reviewer #2: No

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17 Feb 2020

Response to additional comments made by the editor:

High sound pressure levels: We carefully addressed the criticism regarding high SPLs. We think that linear damping of otolith movements with a decrease in SPLs cannot be concluded based on our data because the decrease in (otolith) oscillations was larger at higher SPLs (177.2 down to 170.7 dB) than at lower levels (170.2 down to 164.3 dB; see also please S1 File). Yet, we are not able to detect otolith movement at a few nanometers (e.g. at auditory thresholds) due to the constraints implied by the imaging procedure. However, as the motion of the otoliths displayed a regular and reproducible pattern for different SPLs (e.g. 177.2, 170.7, 164.3 dB re 1 µPa), we assume that the observed patterns may also hold true for biologically relevant SPLs.

Discussion: As recommended, we shortened the discussion by removing or shortening several paragraphs (word count, original ms version: 3,301; revised version: 2,636). In addition, we moved the (shortened) part discussing the methodology to the end of the Discussion chapter. However, to address the reviewers’ comments with regard to potential effects of tissue damage and high SPLs, we included two new (but short) paragraphs.

Figures: We removed 7 figures to the Supporting information resulting in 11 instead of 18 figures in the main text.

Response to reviewers:

Reviewer #1:

The manuscript is a very well written paper that investigates sound-induced motion of the otoliths within the fish inner ear end organs from two fish that have different otophysic connections of the swim bladder to the inner ear. This paper is a follow up study to their previous work but in the current paper the authors a standing wave tube-like tank to examine the motion of the inner ear otoliths under conditions where there is maximum particle motion vs maximum sound pressure. The authors provide the first experimental evidence of how the two different types of otophysic connections affect otolith motion in the fish inner ear. The subject of the paper should be of general interest to the readers of Plos One. I have only a few comments below.

Thank you very much for the positive feedback on our ms.

1) The stimulus used to induce motion of the otoliths within the end organs was quite high ranging from 141 to >177 dB re 1 microPa. The authors reported (in the methods lines 206-207) that “step (at ID19) equals a SPL of 177.2 dB re 1 microPa when shakers were in phase and a SPL of 155.4 re 1 microPa when shakers were driven out of phase”. These sound levels seem to be out of the biologically relevant ranges. I realize that these levels were likely required in order to visualize the induced motion of the otoliths. My concern is that the induce motion and how it may activate motion of the inner ear may not be biologically relevant to natural auditory stimuli experienced by these fish in nature. To alleviate such concerns, the authors should at least address this issue and explain the limitations (I know they have to some degree in the discussion) of how more natural sound levels might induce similar motions at lower levels.

We address this point in the Discussion to a larger extent than previously. We completely agree that the high SPLs used in our study are of no biological significance but they are necessary to detect otolith oscillations in situ using hard X-ray imaging. Spatial and temporal resolutions at tomography beamlines are constantly improved. Hence, it may be possible to characterize otolith motion at much lower SPLs and higher frequencies in future studies.

2) An interesting note on line 399-401 that states that the left and right Weberian ossicles revealed differences in the amount of displacement when subjected to sound induced motion. The authors also describe the differential motion of the otoliths based on experimental condition. Are the authors suggesting that the Weberian apparatus might afford directional sensitivity to the sound source? This would be extremely interesting if true. Also, their description of the sound induced movement of the otoliths in the Canara pearlspot cichlid may have implications for how the inner ear uses the particle motion and indirect sound pressure cues to determine sound source direction via the phase model hypothesis.

The different motion patterns of otoliths depending on experimental conditions might have implications for sound source localization. However, we refrained from discussing such a possibility because the topic of explaining the mechanisms of how fish may be able to unambiguously localize a sound source is still vividly debated. As we used high SPLs and as our setup cannot provide a 100% separation of sound-induced particle motion and sound pressure, we felt it being too speculative to interpret our data with regard to sound source localization.

3) The authors also describe other interesting results of the “tilting motion” of the sagitta in E. canarensis. Was there any evidence for the so-called “rocking motion” of otoliths under their experimental conditions as described by Krysl et al (2012) in PloSOne.

Based on our 2D radiographic imaging, it is difficult to decide whether or not otoliths such as the sagitta in E. canarensis may show a “rocking motion”. Under these imaging conditions, we observe a moving auditory structure only from a certain view often overlain by other skeletal elements like cranial bones. Capturing the motion of the otoliths in their full 3D aspect in future experiments using tomography is likely to allow for a more precise characterization of the motion pattern and may clarify whether the movement is a rocking motion sensu Krysl and colleagues.

4) Surprisingly the authors do not discuss the induced movement of the asteriscus in E. canarenis given the close proximity of the lagena to the swim bladder in the cichlid. Although sound induce movement was minimal at the experimental sound levels used, movement of this otolith could be very important in terms of the indirect detection of particle motion from the swim bladder. Fay reported that fish can detect particle motion displacement as low as 1 nm. Thus, the authors should not discount the importance the lagena and saccule given their close proximity to the swim bladder.

We agree that the close proximity of the lagena to the swimbladder extensions in Etroplus may indicate a more prominent role in audition of this otolith end organ. However, due to a rather weak contrast of asterisci compared to sagittae and lapilli (see please Fig. 6B1 vs. A) – we could hardly identify the asterisci in the radiographs when the fish was seen in lateral and frontal views – in combination with a weak overall motion of asterisci, it was very difficult to characterize the motion pattern of the lagenar otoliths. Hence, we think that our data do not allow for discussing or interpreting the motion of the lagenar otoliths.

Reviewer #2:

The study using a new apparatus to image the auditory portions of the inner ear during both sound pressure and particle motion. The design is clever and is novel in that it could image this structures during stimulation. The authors do a good job of indicating potential limitations with their procedures. The study makes a very nice overall contribution to the field.

Thank you very much for the positive feedback on our ms.

A couple of items that need to be clarified or considered.

The fish are dead and tissue structure can break down very quickly, especially if high intensity lighting was needed and it generated heat. It would have been useful if there was postmortem examination to determine if there were any structure changes that could not be detected by imaging. For example, what was the effect of blood clotting?

We would like to refer to our statement made in the Material and methods section:

“To minimize detrimental effects of X-ray-induced tissue damage, experiments performed in the same orientation (e.g. fish seen in lateral view) and using the same species, but different individuals, alternately started with the in phase (0°) and the out of phase condition (180°; for an explanation of the two conditions see “setup design”). Hence, we started using the in phase condition and the highest studied sound pressure level (SPL; step 1, Table 1) in one specimen and using the out of phase condition at a lower SPL (e.g. step 4, Table 1) in another individual of the same species.”

In the discussion of the revised version of the ms, we included a short statement that future studies should compare tissue damage on the histological level by comparing control fish with specimens subjected to hard X-ray imaging.

The SPL were very high and not normally used in fish auditory studies. Was this due to the limitations in the imaging? I would expect to see large scale movements at these SPL that fish may never experience. How did this impact the studies and models?

Yes, this is exactly the point that we had to find a compromise between the acoustics and the constraints implied by the imaging procedure to obtain a meaningful signal-to-noise ratio. We had to go for rather high SPLs because the currently achievable spatial and temporal resolution is not high enough to visualize otoliths moving only a few nanometers as would be expected for lower SPLs (e.g. at the auditory threshold). We agree that this is an important aspect and we thus briefly address this issue in the discussion in the revised version of the ms.

I presume the test chamber is designed to get around the problems associated with auditory studies in small tanks. Can physiological studies eventually be done in these types of tanks?

We think that this is a very interesting aspect. It may be feasible to measure e.g. AEPs during sound stimulation while imaging the moving auditory structures, if it was possible to subject live animals to an acceptable X-ray dosage while still yielding a good signal-to-noise ratio.

I felt the discussion is far too speculative. The authors have a very nice data set and do not need to explain every variance that may be impacting the structure. I would suggest that the discussion focuses on what can definitely be determined and limit the number of speculative discussion.

As recommended, we shortened the discussion, especially with regard to potential applications of the setup.

I realize this is an on line journal and page limits are not a concern, however 18 figures seem a little excessive and it is hard for the reader to wade through and determine which ones are really need. I would suggest that several figures could be moved to supplemental files.

As suggested, we reduced the number of figures in the main text to 11 instead of 18 and moved 7 figures to the Supporting information.

Minor

Abstract The use of the term maximum even in quotations is confusing.

Corrected as suggested. We rephrased these sentences using “maximised” instead of “maximum”.

29 “inner” ears. Many times the authors use the term ear, when inner ear would be more accurate

Corrected as suggested. We now use “inner ears” instead of just “ears” throughout the manuscript.

40 I think it is safe to say they are no longer “assumed”

Corrected as suggested. Now it reads “…and play an important role…”

45-46. Sentences end and start with same words

We rephrased this sentence to avoid at least part of the repetition. It reads now as follows: “These structures….”

What is an improved hearing ability. This is usually termed increased sensitivity or greater frequency range

We rephrased the half-sentence to be more precise/specific: “….which are often correlated with a widened range of detectable frequencies (especially at frequencies > 700 Hz) and increased auditory sensitivity.”

51 … whole body in motion: It is unclear the effect of hard skeletal elements. Also, the motion is oscillolatory with no net movement gain and therefore motion is not best word? Vibration?

We rephrased this accordingly: “….results in a to and fro motion of the fish..” to make clear that it is not a net movement into a certain direction.

78 The pearlspot is not considered as well investigated as the goldfish

We agree that goldfish and E. canarensis are not equally well investigated with regard to the auditory structures and auditory abilities. We therefore rephrased this part as follows:

“Goldfish is well investigated with regard to inner ear and swim bladder morphology. For Etroplus, data on the gross morphology of the auditory structures are available.”

95 The saccule has plays a very important vestibular function and therefore does not “mainly” function in audition

We corrected the sentence accordingly:

“1) In otophysans such as goldfish, the saccule is assumed to play an important role in perceiving sound pressure.”

113 The cessation of opercular movement is not considered an endpoint of euthanasia. Many fish (including goldfish) can be easily revived after the cessation of opercular movement. There should have been an additional period following the cessation of movement to insure complete euthanasia

As we used a strong solution of the anesthetic (0.4% buffered MS-222), opercular movements ceased rather quickly, i.e. within 5 minutes. After that we waited further 10-15 minutes before further handling of the fish. In the revised version of the ms, we clarified this accordingly.

Methods

The image rate of 200 Hz for the 200 Hz seems relatively slow. Most video is run higher than stimulus. Please explain if this had an effect on resolving the movements

We would like to refer to a section in the original version of the ms addressing this issue: “From an imaging point of view, the “large” water body in the tank impairs a real-time observation of the moving auditory structures in the fish. At ID19, maximum image acquisition rates of up to 1 kHz (1,000 fps) are possible, which would be sufficient to record these motions in real time. However, such high frame rates are only possible for samples investigated in air [60,61]. It is thus important to note that our observations of moving otoliths and ancillary auditory structures are a proxy of the true motion.”

633 add reference

Corrected as suggested. We included the respective reference, i.e. Vetter et al. (2019) J Comp Physiol A.

Table 1: delete, add info to text

As recommended, we deleted Table 1 and included the information in the text.

Submitted filename: Response to Reviewers 17022020.docx

Click here for additional data file.

4 Mar 2020

Auditory chain reaction: effects of sound pressure and particle motion on auditory structures in fishes

PONE-D-19-30473R1

Dear Dr. Schulz-Mirbach,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Reviewers' comments:

16 Mar 2020

PONE-D-19-30473R1

Auditory chain reaction: effects of sound pressure and particle motion on auditory structures in fishes

Dear Dr. Schulz-Mirbach:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Dennis M. Higgs

Academic Editor

PLOS ONE