The implantation process is important, as it affects the reliability of the experiment. The electrodes should be inserted into the muscle at a depth of 3 mm or less depending on the size of the beetle (avoiding contact with nearby muscles). If the electrodes touch the nearby muscles, undesirable motor actions and behaviors may occur owing to the contraction of nearby muscles. The two electrodes should be well aligned to ensure that no short circuits occur. When melting and reflowing beeswax using a soldering iron, the experimentalist has to be careful and solder as quickly as possible, since the muscle can be burnt by prolonged contact with high temperatures, leading to a malfunction of the muscle. Although removing the cuticle is required to access the 3Ax muscle, the insertion and sealing process takes less than one minute and was managed to minimize damage to the muscle. The insects were returned to the rearing room after the experiments and could survive for up to 3 more months (end of their life time). To maintain good performance of the beetle, the beetle should be fed and allowed to rest for 3 to 4 hr after every 20 consecutive trials as the insect can become fatigued after many consecutive (40 to 50) flight trials and may not be able to open its wings.

As for the free flight experiment, volume calibration for the motion capture system is necessary, as it affects the trajectory tracking accuracy. It is important to fill the cameras' view full of the waves of the calibration wand with an image error of less than 0.3 for all of the cameras to maintain the accuracy of the motion tracking system. In addition, the surface of the marker should be clean, or the 3D motion capture system may frequently miss the marker. After calibration, a dummy test should be carried out by waving the battery wrapped with retro-reflective tape in the defined volume to check the coverage of the motion capture system. For testing the motion tracking accuracy, we measured the distance of two markers moving in the flight arena. The markers were fixed on a carton board with a distance of 200 mm to each other. The board was moved in the entire flight arena to obtain various positions of the two markers. The standard deviation was then calculated to be 1.3 mm (n = 3,000).

The free flight test facility (Figures 1 and 4) allows us to track the position (X, Y, and Z) of a flying insect along with a timestamp. Since only a single marker is attached to the beetle and the 3D motion capture system only detects that marker, the beetle is treated as a particle or a mass point. As such, data from the flying beetle has positional information but lacks orientation. Therefore, kinematic analysis from the positional data of the beetle provides only the translational velocity and acceleration along the X, Y, and Z axes without angular velocity or angular acceleration in rotations about the yaw, pitch, and roll axes. Multiple markers fixed on a beetle (such as the one shown in Figure 6) must be used for the 3D motion capture system to treat the flying insect as a rigid body and record rotation and translation data. However, the experimentalist must take note of the contribution of these markers to the kinetics of a flying beetle, because the marker is not a small piece of tape but needs to be large enough to be detected by the camera system with minimum tracking loss. Such an arrangement and the attachment of multiple markers may significantly increase its mass and moment of inertia25. Besides, the size of the flight arena can be set as large as possible within the coverage range of the motion tracking system to reduce the constraints to the free flight behavior of the beetle. For this paper, the size of the flight arena is defined based on the maximum coverage of motion capture system (12.5 x 8 x 4 m3).

Various possibilities exist to modifying this technique along with increasing the number of markers to record the orientation of the insect as mentioned above. The stimulation of different muscles in free flight can produce various behaviors, e.g., the basalar muscle for a contralateral turn7 and 3Ax muscle for an ipsilateral turn13. In addition, certain parts of the nervous system of an insect can induce various reactions. Optic lobe stimulation can induce flight initiation7, whereas the stimulation of antennae can induce contralateral turning in a walking insect12. Furthermore, we can change the function of the backpack from being an electrical stimulator to an electromyography recorder to record the activities of an insect during its natural behavior3,26.

The free flight stimulation of the beetle helped to reveal and confirm the natural function of the 3Ax muscle by enabling observations of the instantaneous reaction of the insect freely moving in air. Such information is not available under tethered conditions11,13,27-30. The behavior of an insect is constrained under tethered conditions and may be different from that in free flight, possibly leading to an incorrect understanding of insect behavior. Thus, free flight stimulation using this technique is a strong tool for validating the hypotheses drawn from tethered experiments. Furthermore, an insect-machine hybrid system is superior to current artificial flapping robots in terms of locomotive capabilities and power consumption13,17,31,32.

Insect-machine hybrid systems may replace artificial robots in the future as they inherit the complex and flexible structure and locomotive capabilities of a living insect and reduce the production time of the fabrication process. Various locomotive capabilities can help an insect-machine hybrid system to operate more efficiently in constrained spaces that involve the combination of walking and flying, e.g., in rescue missions. In addition, insect-machine hybrid systems can potentially be used as a tool for insect control in agriculture as it may be able to blend into natural insect colonies and help to control their activities.