Preparation used for recording wing vibrations in tethered singers (G. bimaculatus) stimulated with neuroactive substances. The specimen was mounted assuming a prognatous head orientation; this allowed full access to the brain, different degrees of rotation and alignment, and perpendicularity of the wing with respect to the laser beam. After the insect stopped singing, wings were extended and basally fixed with wax. The speaker was used to excite the wings with sound after the experiment and to obtain whole laser scans. Diagram not to scale. B&K, Brüel & Kjær; mic., microphone; for details, see Materials and methods.

Triggering system used for wing vibration recordings in actively stridulating insects. Two typical sound pulses of G. bimaculatus, and the associated wing movements (red outline), obtained with a motion detector, are shown to indicate the two trigger systems used. Microphone signals were used to trigger the recordings. In the first case (red rectangle) the maximum level of pulse amplitude (closing stroke) was used to trigger recordings of 2 ms (1 ms before and 1 ms after maximum amplitude of the pulse). In the second case (blue rectangle), the decaying amplitude of the last oscillations of a pulse were used to trigger a recording (same duration as previous) of the following silent opening stroke at maximum amplitude of the wings (usually 12 ms before the start of a pulse, at 23.5°C). This trigger was used to record free wing vibration in response to sound, while the animal had its wings in a singing position but disengaged.

Capture of the video image from the laser Doppler vibrometer illustrating the extended wings (axillary sclerites fixed with wax) in frontal view during measurements and the lattice of laser scanning points (N=260 points; mesh size, 170 μm; dot positioning accuracy, ∼1 μm). The condenser microphone (Mic.) was positioned on top in the middle of the wings. This setup was used to record the vibration of extended wings in a fixed position, while stimulated with sound.

Frequency modulation plots recorded from 12 specimens of G. bimaculatus. Each box represents a different specimen. Traces within a box are the mean instantaneous frequencies of single pulses picked randomly from recordings of the same specimen obtained on different days within a 10 day span. Error bars indicate standard deviation from the mean. Note that each specimen modulates its call in a particular way. Lateral ghost plots depict the spectrum of the call of each specimen, recorded on the last day. N=number of recordings, numbers in parentheses indicate the days on which recordings were made.

Comparison of calls before and after using pharmacological brain stimulation in one male G. bimaculatus. (A) Oscillograms of two pulses randomly selected from calls recorded before and after eserine injection into the brain. (B) Zero-crossing (ZC) analysis of these pulses. (C,D) Fast Fourier transform (FFT) analysis of natural and elicited calls (two chirps in each case) recorded from the same insect.

Mechanically coupled resonance and free resonance of the forewings of G. bimaculatus. (A) Spectrum of the natural calling song recorded during the experiment. (B) Wing vibration measured from the harps of both wings during stridulation using micro-scanning laser vibrometry. Spectra were normalized to the maximum value of the left wing (LW), for comparative purposes. (C) Free resonance of both wings in response to acoustic stimulation during the opening phase of the wing during stridulation. (D) Resonance of the wings in response to acoustic stimulation measured with both wings extended with axillary sclerites fixed with bee's wax. Note that in C and D the response is similar; the LW resonates at lower frequency than the right wing (RW); the magnitude of the response in both recordings is higher in the LW.

Resonances of free wing vibration in response to acoustic stimulation. (A) Mean resonance frequency (f_o) of the harps of 44 specimens. (B) Mean f_o of the mirrors of the same 44 animals. Blue and red lines show the RW and LW mean, respectively. Shaded areas indicate standard deviation in both cases.

Correlations of resonance in the forewings of G. bimaculatus. Carrier frequency of the call (f_c) as function of wing f_o for RW (A) and LW (B). Dotted lines represent 95% confidence intervals.

Amplitude response of the wings to acoustic stimulation. (A) Picture of the wings extended, illustrating the sections through which the deflections were built. (B,C) Envelope of mechanical deflections along the transects shown in A for a series of phases in the full oscillation cycle (for this specimen, the resonance of the LW was 5.125 kHz,). B, RW; C, LW.

Wing resonances measured from free and engaged wings. (A) Mean f_o of the harps of 13 specimens with wings extended, stimulated with sound. (B) Mean f_o measured from the harps of the same 13 stridulating specimens. Blue and red lines show the RW and LW mean, respectively. Shaded areas indicate standard deviation in both cases.

Vibration of the stridulatory file. (A) Picture of a RW segment including the stridulatory file and plectrum. Numbers 1–4 indicate the different file locations monitored with the laser vibrometer during acoustic stimulation. The RWs of 44 specimens were stimulated with sound and the vibrations at file regions 1–4 recorded (values in red are measurements from one specimen). (B) Amplitude of vibration of the file in the same specimen (shown as the transfer function between file displacement and sound pressure) at the f_o of the RW of this specimen (∼5.9 kHz). Yellow transect lines indicate the equivalent file position between picture and chart. Blue rectangles connect the file regions 1–4 in the picture with the respective envelope of deflection. (C) Envelope of a sound pulse produced by the same specimen for comparison with the file deflection shape. (D) Resonances recorded at the file regions, harp and short flexible region. (E) Mean resonances of 44 specimens of the same wing areas as depicted in D (Kendall's W for related samples: W=0.37, d.f.=5, χ²=8.054, P=0.154). A, file area.