(A) Tetherless flight control system (∼230 mg total) mounted on Cotinis texana (Green June Beetle) using beeswax next to a US$ 0. 25 coin. A microcontroller provided potential pulses to four stimulating wire electrodes (∅125 μm) implanted into the brain, left and right basalar muscles and posterior pronotum (counter electrode). (B) Radio flight control system (∼1.3 g total) mounted on Mecynorrhina torquata using beeswax next to a US$ 0.25 coin. The system consisted of a microcontroller, a custom PCB, a dipole antenna, a microbattery and stimulating wire electrodes (∅125 μm) implanted as in Cotinis. (C) Front and (D) tilted views of dissected Cotinis beetle head showing the brain stimulator at implant site 1, optic lobe stimulator at implant site 2. The brain stimulator was implanted along the rostral–caudal midline of the head, at the center between the left and right compound eyes. Implant site 2 was at the interior edge of each compound eye. (E) Sagittal section of thorax showing the counter electrode at implant site 3 and the basalar muscle stimulator at implant site 4. (F) Cross-section of mesothorax showing the basalar muscle stimulator sites (implant site 4 on left and right sides). The basalar muscle stimulator was implanted midway between sternum and notum of mesothorax to a depth of approximately 1 cm in rostral–caudal direction on either the left or right side of the insect. The blue letters X and bars indicate implant sites and approximate implant lengths, respectively. Mecynorrhina torquata has nearly identical, scaled anatomy to Cotinis texana.

Initiation and cessation control of Cotinis texana beetle during tethered flight; (top) audio recordings of tethered beetle, (bottom) applied potential to the brain (with counter electrode inserted into posterior pronotum). The applied potential waveform is identical to Figure 3A, but frequency varied. As the period between pulses decreased, the beetle was incapable of fully starting or stopping wing oscillation and audio amplitudes were modulated by the stimulus frequency. Audio amplitudes were normalized using mean absolute value during normal, sustained flight recorded at each individual trial. See Movies 1, 2 and 4 in Supplementary Material for flight initiations of fully tethered, weakly tethered and fully untethered Cotinis texana, respectively.

Three types of pulse trains (stimulus protocols) were investigated to elicit flight. (A) Neg + Pos: alternating 1 s duration positive and negative pulses, (B) Pos: 1 s duration positive pulses, (C) Neg: 1 s duration negative pulses. Pulse amplitude was swept from 0.1 to 5.0 V in 100 mV increments when testing for the amplitude threshold. Delay, τ₁ or τ₂, is response time from beginning of positive or negative potential pulse to beginning of wing oscillation, respectively. See Table 1 in Supplementary Material for data on stimulated flight bouts in all tested Cotinis texana.

Initiation and cessation control of Mecynorrhina torquata beetle tethered flight. (A) Alternating positive and negative potential pulses (100 Hz, see (B) for the details of the waveform) applied between left and right optic lobes initiated wing oscillations while a single pulse ceased wing oscillations; (top) audio recording of tethered beetle, (bottom) applied potential to the one side optic lobe regarding the other side optic lobe. Delay, τ₃, is response time from beginning of the multi pulse trains to beginning of the wing oscillation. Delay, τ₄, is response time from beginning of the single pulse to ending of wing oscillation. τ₃ and τ₄ for all the tested beetles are summarized in Table 2 in Supplementary Material. The sharp rise of audio amplitude at the beginning of oscillation was attributed to friction between elytra and wings when the wings were unfolded from the underneath of elytra. The whole audio amplitudes were normalized using mean absolute value calculated for the middle period of the flight time (2.5–3.7 s). (B) Pulse trains applied between left and right optic lobes. Number of waveforms was swept from 1 to 100 in one waveform increment when testing for the number of waveforms required to trigger flight initiation (Table 2 in Supplementary Material). See Movies 5–7 in Supplementary Material for flight initiation and cessation control of fully tethered (Movie 5) and fully untethered (wireless communication, Movies 6 and 7) Mecynorrhina torquata.

Elevation control of Cotinis texana beetle tethered on a custom pitching gimbal. Brain stimulus altered the gimbal pitch of the beetle. (A) Gimbal pitch angle with the mounted beetle during alternating periods of un-stimulated and stimulated flight. Horizontal bars indicate durations of the stimuli (3 s each); a 10-Hz, 3.0 V pulse train whose waveform is identical to that in Figure 3A was applied during the indicated periods. (B) Audio recording corresponding to (A). Red and black arrows indicate the beginnings and endings of the stimuli to the brain. The audio amplitudes were normalized using a mean absolute value during un-stimulated periods. Photographs of a gimbal-mounted beetle during (C) un-stimulated and (D) stimulated flight. A light-emitting diode (LED) mounted to the microcontroller acted as an indicator by blinking during stimulation. See Movies 8 and 9 in Supplementary Material for the corresponding normal and high speed video tracks, respectively.

Elevation control of a Mecynorrhina torquata beetle tethered on a custom pitching gimbal. Brain stimulus altered the gimbal pitch of the beetle (100 Hz, 2.0 V amplitude, see Figure 3A for waveform). (A) Gimbal pitch angle with the mounted beetle during alternating periods of un-stimulated and stimulated flight. Horizontal bars indicate durations of the stimuli. (B) Audio recording corresponding to (A). Red and black arrows indicate beginnings and endings of the stimuli to the brain. The sharp peaks at the arrows were attributed to the signal tones coming from function generator to output the stimulus signal to the beetle brain. The audio amplitudes were normalized using a mean absolute value during un-stimulated periods. Photographs of a gimbal-mounted beetle during (C) un-stimulated and (D) stimulated flight. See Movie 10 in Supplementary Material.

Elevation control of a free-flying Mecynorrhina torquata beetle: temporal height-change of a flying beetle (ten flight paths). Alternating positive and negative potential pulse trains at 100 Hz and 2.0 V amplitude to the brain caused the beetle to fly downward. The applied waveform was identical to that in Figure 3A, but the frequency was different (100 Hz). The median height change was 60 cm (the range was 33–129 cm). See Movie 11 in Supplementary Material.

Turn control of Cotinis texana flight. A 100-Hz and 2.0-V positive potential (vs. counter electrode at posterior pronotum) pulse train to the basalar muscle on one side of the beetle triggered a turn. Beetle mounted on a string (10 cm) was programmed with continuous sequences of left, pause, right, pause instructions; each instruction lasted 2 s. (A) Left basalar muscle stimulus generating a right turn, followed by (B) a pause during which the beetle zigged and zagged randomly, followed by (C) right basalar muscle stimulus generating a left turn. Each successive photograph consists of 10 frames; frames were taken every 0.2 s. Numbers in (A) and (C) signify the frame number. See Movie 12 in Supplementary Material.

Turn control of free-flying Mecynorrhina torquata beetle. Pulse trains at 100 Hz and 1.3 V positive potential to the left or right basalar muscle triggered turns. Ten flight paths elicited by a 0.5-s continuous stimulus to (A) right or (B) left basalar flight muscle. Each flight path is obtained after the three-dimensional digitized flight path is projected on the XY-plane (see text for detailed method). The first point of each flight path (beginning of the 0.5 s stimulus) is located at the origin of coordinate system while the last point indicates the ending of the stimulus. Different colored and shaped plots show different individual beetles’ flight paths. See Movie 13 in Supplementary Material for representative turn control in free flight.