The locust’s ear, Müller’s organ, and the scolodium.

(a) Locust’s ears are located on the first abdominal segment, hidden behind the wings. Each ear consists of a tympanic membrane with a hearing organ, Müller’s organ, directly attached. External view of the tympanic membrane (inset) shows a dark ridge (arrowhead) where Müller’s organ is located. The anatomical references used in this manuscript are defined based on the axes of the locust’s body unless stated otherwise. (b) The surface of the tympanic membrane aligns orthogonally to the antero-posterior axis of the locust body at a slight oblique angle (left, red dashed lines). Illustration of the right ear viewed from inside the body (along the orientation of the black arrow on the left panel) shows Müller’s organ located between the ‘thick’ (1) and ‘thin’ (2) regions of the tympanic membrane (right). The auditory nerve projects ventrally toward the ventral nerve cord. (c) Schematic of Müller’s organ superimposed on an X-ray micro-CT scan slice shows three groups of auditory neurons. These neurons (yellow) extend their dendrites to distinct regions where the distal ciliated end of the dendrites insert into the attachment cells (blue). The auditory neurons of Müller’s organ are classified based on their maximal frequency sensitivity. Group-I neurons respond to frequencies between 0.5 and 1.5 as well as 3.5 kHz (∼20 cells). Group-II neurons are sensitive to higher frequencies of 10–25 kHz (∼13 cells), while most auditory neurons, belong to Group-III and respond to mid-range frequencies of 2.5–4 kHz (∼46 cells). The yellow arrow marks a typical location of a scolopidium at the insertion of the scolopale cell into the attachment cells. (d) Schematic of a scolopidium indicating locations of the mechano-electrical transduction (MET) channel candidates, NompC (red) and Nanchung-Inactive (blue), near the proximal and distal ends of the ciliary dilation (top). The bottom panel shows fluorescent micrograph of the corresponding region of the dendrite and the scolopidium. The auditory neuron is filled with Dylight-488 conjugated streptavidin-neurobiotin (bottom).

Three-dimensional (3D) ultrastructure of a scolopidium.

(a) 3D reconstruction at the interface between a scolopale cell and an attachment cell using Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM). (b) Longitudinal FIB-SEM slice reveals the dendrite and its ciliated distal end enclosed within the scolopidium sheath; the cilium dilates near its insertion into the porous cap before it terminates in an electron-dense region at the border of the attachment cell. (c) Cross section at ciliary dilation shows fibrous filaments inside the membrane of the attachment cell (arrowhead). (d) Longitudinal section at the distal region of the scolopale sheath reveals that the fibrous filaments (arrowheads) observed in (c) form a cone-shaped lid covering the cap and the distal region of the ciliated dendrite. (e) Longitudinal section at the plane of the cilium shows the narrowing of the central passage, where the cilium enters (arrowhead). (f) 3D reconstruction of the cap material reveals a funnel-shaped central passage. (g) Termination of the cilium at the extreme end of the scolopale cartridge. (h) Longitudinal section at the scolopidium base reveals numerous oblique basal links between scolopale rods and the dendrite. (i) Cross section along the dashed line in (h) shows the basal links radiating from desmosome-like junctions. (j) Cross section at the ciliary dilation reveals four central fibrils (arrowheads) inside the 9-doublets axonemal structure. (k) Longitudinal section along the dashed line in (j) reveals two of the central fibrils along the length of the ciliary dilation. (l) 3D model of a complete scolopidium. The auditory neuron in the 3D model is omitted to reveal the oblique basal links.

Sound-evoked motion of Müller’s organ.

(a) Vibrometry measurements were performed using Optical Coherence Tomography (OCT) along the antero-posterior (top) or meso-lateral (bottom) axes of Müller’s organ upon 90 dB SPL sound stimulation, with the external side of the tympanic membrane facing the loudspeaker. Vertical arrows indicate the direction of the OCT beams, and the axes of vibration measured. The eyes represent the observation angles, where 2D OCT sections in (b) were reconstructed. (b) OCT sections along the two planes were reconstructed from OCT depth profiles. The symbols * and ● indicate the maximal widths of Müller’s organ at the ganglion and the styliform body, the two important landmarks used in our analysis. Yellow arrows indicate typical orientation of Group-III dendrites. (c) Ensemble amplitude and (d) phase responses to stimulated frequencies along the antero-posterior (black, N = 3, E = 3) and meso-lateral (red, N = 7, E = 12) axes. (e) Vector difference as a function of stimulated frequency, computed by comparing vibration at the position ● relative to *, as shown in (b), along the antero-posterior and meso-lateral axes. (f) Normalised positions of Group-III scolopidia between the ganglion and the styliform body (N = 16, E = 16, n = 235). The grey line indicates the outline of Müller’s organ. (g-h) Vibration maps as a function of stimulated frequency along (g) the antero-posterior and (h) meso-lateral axes. Arrowheads in (g-h) indicate the area of abrupt transitions in both the phase and amplitudes. (i-j) Comparison between relative amplitude (blue) and phase (red) responses from the position * to ● along (i) the antero-posterior (N = 3, E = 3) and (j) meso-lateral (N = 7, E = 12) axes. The relative amplitudes are selected from 1 kHz (minimal relative amplitude) and at the frequencies where the relative amplitude is largest in each axis, 4.25 kHz and 2.6 kHz, respectively. The relative distance is normalised with * as 0 and ● as 1, as shown in (b). The grey dashed line represents the average Group-III scolopidia position obtained from (f). All vibration amplitudes are shown in dB relative to a 1-nm reference vibration. For additional data, see Extended Data Fig. 2 and Source Data Fig. 3.

Sound-evoked motion of the scolopidium.

(a) Schematic of a scolopidium showing the scolopale base and the cap that are clearly visible in light microscopy. (top). Montage over a period of one cycle of sound-evoked motion of a Group-III scolopidium stimulated with 3 kHz pure tone at 90 dB SPL (bottom). At t = 0.24 ms, the cyan arrowhead indicates the cilium when it is brought into focus. The yellow lines indicate the shear near the cap and the scolopale base. The yellow arrowheads follow the region with clear deformation near the scolopale base. (b) Summary of the sound-evoked motion of the scolopidium. (c) Motion of the scolopale base (red) and cap (black) regions along (dx) and orthogonal (dy) to the ciliary axis. (d) Root-mean-square (RMS) amplitude of the scolopale base (red, N = 6, n = 22) and the cap (black, N = 6, n = 22) regions along the ciliary axis (dx). (e) RMS amplitude of the scolopale base (red, N = 6, n = 22) and the cap (black, N = 6, n = 22) regions along the orthogonal axis (dy). (f) Ratio of the axial-orthogonal motion (dx/dy) of the scolopale base (red, N = 6, n = 22) and the cap (black, N = 6, n = 22). (g) Example of distance (Δ𝑑) between the scolopale base and the cap region as a function of time over each stimulated frequency. (h) Relative distance between the scolopale base and the cap (N = 6, n = 24). (i) Shear motion along the ciliary axis at the cap region (N = 6, n = 22. For additional data, see Extended Data Fig. 3 and Source Data Fig. 4.

Transduction current upon step displacements of a scolopidium cap.

(a) Focal-stacked light microscopy image from ∼10 µm apart showing the auditory neuron (yellow), a patch recording pipette, and a stimulating pipette. (b-c) Schematics of step-displacement protocols for (b) pull-push displacements along the ciliary axis (N = 18, n = 18) and (c) up-down displacement orthogonal to the ciliary axis (N = 11, n = 11). (d-e) Transduction currents (top trace) in response to (d) pull-push and (e) up-down step-displacements with increasing amplitudes (bottom trace). Step-displacement traces below and over 100 nm in (d-e) are shown in different scales. Arrows indicate the orientation of stimulation. (f-g) Ensemble average transduction currents in response to (f) pull-push and (g) up-down step displacements. The stimulation traces are shown in (d-e). (h-i) Examples of the transduction currents in response to (h) pull-push and (i) up-down step displacement of magnitudes 2, 50, 500, and 2000 nm. (j-m) Ensemble average transduction current plotted as a logarithmic function of the step-displacement, I-log(X) curve, for (j) pull, (k) push, (l), and (m) downward displacements with their return to the offset counterparts. For additional data, see Source Data Fig. 3.

Possible gating mechanisms of MET channels in insect chordotonal neurons.

(a) Key anatomical features of Müller’s organ and the scolopidium. (b) Sound-evoked motion of Müller’s organ generates axial stretch at the level of the scolopidium. (c) Possible gating mechanisms of the mechano-electrical transduction channels in insect chordotonal neurons (left): (i) direct-stretch model, (ii) stretch-compression model, (iii) stretch-deformation model, and (iv) stretch-tilt model. Purple arrows indicate forces exerting on the scolopidium. The right panel shows magnified views of the entry of the cap’s central passage, showing forces (green arrows) exerting on the ciliary membrane.

Anatomy of the scolopidia.

(a-c) Locations of the three groups of scolopidia in Müller’s organ: Group-I scolopidia, located near the folded body; Group-II, located within the fusiform body, forming a ’bridge’ to the pyriform vesicle at the surface of the tympanic membrane; and Group-III, located on the styliform body. The overlay indicates examples of scolopale sheaths (opaque yellow) and the corresponding attachment cells (blue). Dashed lines outline the dendrites along their lengths, and yellow arrowheads mark the other visible scolopidia. (d) Oblique section of the distal region of a scolopidium reveals the full extent of the cilium. (e) Example of a potential sample preparation artifact showing the apparent detachment of a cilium from its terminal region. (f) Longitudinal section through the ciliary dilation, revealing three central fibrils (arrowhead). (g) Longitudinal section along the dashed line in (f), reveals two of the central fibrils (arrowhead) extending along the entire length of the dilation.

Locations of Group-III scolopidia and frequency response of Müller’s organ using OCT.

(a) Maximal intensity projection of a Müller’s organ labelled with DAPI superimposed with manual registration of dendrite dilations (red circles). Anatomical landmarks are indicated at the maximal width of the ganglion (cyan dashed line) and the styliform body (yellow dashed line). (b) Maximal intensity projection of auditory neurons labelled with DyLight™ 488-conjugated streptavidin neurobiotin superimposed with manual measurement of dendrite dilations’ positions (red circles) and schematic illustrating the computation of the relative distance of each dendrite dilation between the ganglion (cyan dashed line) and the styliform body (yellow dashed line). (c) Normalised positions of dendrite dilation from an example of one Müller’s organ. The normalised positions of the dendrite dilations are plotted as a function of distance across the ganglion’s width shown as cyan dashed line in (b). A black dashed line indicates the normalised position at 52% between the ganglion and the styliform body. (d) Distances between dendrite dilations and the ciliary rootlet of the corresponding scolopidium (N = 19, n = 46). (e, f) Amplitude (e) and phase (f) response of Müller’s organ along the antero-posterior axis (N = 4, E = 4) measured using OCT, plotted as a function of relative distance between the ganglion and styliform body. (g, h) Amplitude (g) and phase (h) response of Müller’s organ along the meso-lateral axis (N = 7, E = 12) measured using OCT, plotted as a function of relative distance between the ganglion and styliform body. The vertical dashed lines in (e-h) indicate the normalized position of the Group-III scolopidia. All vibration amplitudes are shown in dB relative to a 1-nm reference vibration. For additional data, see Source Data Extended Fig. 2.

Example recordings of sound-evoked motion of a scolopidium.

Raw time series amplitude traces (left traces) and their corresponding Fourier transforms (right traces) for the scolopale base and cap along (dx) and orthogonal (dy) to the ciliary axis at each stimulated frequency from 1 to 4 kHz with an increment of 0.5 kHz. (a, b) Motion of the scolopale base (a) along and (b) orthogonal to the ciliary axis. (c, d) Motion of the cap (c) along and (d) orthogonal to the ciliary axis. Pure-tone sound stimulation at 90 dB SPL.