Supplementary materials
Laser Vibrometry
The displacement of the membrane surface was recorded and using the PSV software an
animation of the membrane vibration could be created (Fig. S1). The point of the receptor
cells’ attachment was the largest displacement, as shown in Figure S1. As in previous studies,
laser vibrometry data were considered of sufficient quality when coherence exceeded 85%
[13].
Fig. S1. Laser vibrometry scans of a female greater wax moth. A Top: Greater wax moth
tympanal organ, scale 0.1 mm. Bottom: Highlighted features are the conjunctivum (blue),
tympanic membrane (green), auditory receptor attachment site (orange). B Vibrometry scans
of a female moth tympanal organ, displacements inward (green), outward (red). The site of
maximum displacement matches the auditory receptor site in A.
Ultrasonic Transducer Structure
The ultrasonic transducer used in the experiments was a custom made electrostatic air
coupled device [14]. The device was composed of an insulating PVC shoe which is encased
within an aluminium case. The front face of the transducer was a membrane made of polymer
film which had been metallised with evaporated aluminium. The membrane was stretched
over the backplate and held in place with an aluminium ring, which was screwed in place into
the aluminium case. This ring provided electrical connection between the case and the
metallised front face of the membrane.
Powering the Transducer
The transducer electronic system is shown in Figure S2. The transducer was powered with a
high voltage power supply (475R photomultiplier power supply, Brandenburg, Dudley, UK),
which provided a bias voltage. The sound signal was created using different function
generators; for the electrophysiology experiments where sound pulses were created (Arbitrary
Waveform generator, TGA12102, TTi), and for the laser vibrometry experiments where a
pure tone sine wave was used (Tektronix, Dual Channel-AFG 3102).
Fig. S2. The equipment used to power the transducer.
Transducer Power Output
The output of the transducer was measured with a microphone and preamplifier (Bruel and
Kjæl, Microphone: 4138, Preamplifier: Nexus 2690). The transducer was powered as
previously described, with the function generator output set at 5 Vpp and 10 Vpp (pp: peak to
peak). The sound pressure level was measured with the microphone 10 cm away from the
transducer. The output of the microphone was measured on an oscilloscope (Tektronix, DPO
2014), and this was recorded for frequencies between 50-120 kHz at 10 kHz intervals (see
Fig. S3). The transducer output is measured from 100-300 kHz [14], providing a known
sound stimulus at all frequencies tested.
110
5 Vpp
10 Vpp
Sound Level (dB)
100
90
80
70
60
50
60
70
80
90
Frequency (kHz)
100
110
120
Fig. S3. The sound level output of the transducer at two different driving voltage levels, 5
Vpp and 10 Vpp. The sound level (dB SPL) was recorded using a microphone, between 50120 kHz, in steps of 10 kHz.
The electrophysiology experiments’ sound stimuli, 20 ms sound pulses, were investigated to
rule out the possibility that there were other (lower) frequencies being created due to
artefacts. To test this two transducers were used, one to emit the sound and one to detect it, as
the frequencies tested were too high to use the microphone. The detected sound pulses were
recorded on an oscilloscope (Tektronix, DPO 2014). The recorded sound pulses clearly
demonstrate a correct signal at all frequencies investigated, indicating that no extra
frequencies were being produced (see Fig. S4). A FFT of the recorded sound pulses
confirmed that no other frequencies were present.
Fig. S4. Start of a sound pulse used in moth electrophysiology experiments, recorded using a
second transducer. The sine wave recorded was as expected, and so no extra frequencies were
being created. This was the same for the range of frequencies tested. The above example is a
200 kHz sound pulse.
Simultaneous Laser and Electrophysiology Experiments
To ensure the simultaneity of mechanical and neural sensitivity, a combined experiment
measured the mechanical motion of the tympanum and the electrophysiological response
whilst the ear was stimulated with ultrasound pulses. The moth was prepared as for
electrophysiology experiments; the laser vibrometer was then positioned to record the
membrane displacement. As the sampling rate was 1.024 MHz over a period of 512 ms, the
oscilloscope could not be used for these experiments due to lack of memory space, and so the
laser vibrometer acquisition system was used to record the data instead. The sound pulses
produced by the function generator and the electrophysiology voltage signal were combined
with a custom-built summing amplifier and recorded using the laser vibrometer reference
channel. The laser vibrometer voltage signal of the membrane displacements was also
recorded through the second channel. The recorded data set of the combined sound and
electrophysiology data was analysed with LabVIEW, as the two signals occupy different
frequency bands they were separated using bandpass filters (Bessel, 16th Order). Further
filtering was done of all the signals using the LabVIEW program to remove noise. The sound
stimulus was produced as was previously described for electrophysiology experiments, a
range of frequencies were used and a high sound level of 100 dB SPL was used so that the
response of the hearing organ would be easily detectable on both the laser vibrometry
recordings and electrophysiology. These time-domain recordings clearly show the onset of
tympanal motion followed by a volley of neural spikes (Fig. S5).
Fig. S5. Simultaneous recording of the mechanical and neural auditory response of a greater
wax moth. The top trace is the electrophysiological response with the sound pulses
highlighted in green. The lower trace is the averaged response to 5 sound pulses. Sound
stimulus, 200 kHz (green) elicits a mechanical motion (red). Neural activity (black trace)
appears after some latency.
References
13. Windmill J. F. C., Göpfert M. C. & Robert D. 2005 Tympanal travelling waves in
migratory locusts. J. Exp. Biol. 208, 157-168.
14. Whiteley S. M., Waters D. A., Hayward G., Pierce S. G. & Farr I. 2010 Wireless
recording of the calls of Rousettus aegyptiacus and their reproduction using
electrostatic transducers. Bioinspir. Biomim. 5, 026001.