1. Experimental Animal

1. Prepare a plastic box to keep the pupae of male silkmoths (B. mori) until their eclosion. Put paper towels at the bottom and pieces of cardboard around the inner wall of the box (Figure 1A).
   Note: The pieces of cardboard are necessary for the adult moths to hold while extending their wings during eclosion (Figure 1A).
2. Put male silkmoth (Bombyx mori) pupae in the box and keep them in an incubator until eclosion under a 16-hr:8-hr light:dark cycle at 25 °C.
   NOTE: The male and female pupae can be discriminated by the sex markings on the abdomen (Figure 1B).
3. Collect adult male moths after eclosion and move them into a new box.
4. Keep the adult moths in an incubator under a 16-hr:8-hr light:dark cycle and decrease the temperature to 15 °C to reduce their activity before the experiment.

2. Tethering a Silkmoth

1. Fabrication of an attachment for tethering (Figure 2A)
   Note: The attachment consists of a copper wire with a strip of a thin plastic sheet at its tip. This ensures the dorsal-ventral movement of the thorax during walking (Figure 2B).
   1. Prepare a strip of a thin plastic sheet, 2 × 40 mm (thickness: 0.1 mm), and fold it in the middle.
   2. Attach the folded strip to the tip of a copper wire with an adhesive.
   3. Bend the tip of the folded strip where the thorax of a silkmoth is attached.
2. Use adult moths (2-8 days old) during the light period for the experiment.
   Note: The sensitivity to the pheromone strongly depends on the circadian clock¹⁸. Because B. mori is a diurnal moth, the experiment must be performed during the light period.
3. Gently remove all scales on the dorsal thorax (mesonotum) using a piece of wet tissue (or cotton swab) and expose the cuticle of the mesonotum (Figure 2C).
4. Paste an adhesive on the strip of plastic on the attachment and on the surface of the exposed mesonotum with a small flat-blade screwdriver and wait 5-10 min until the adhesive is no longer sticky.
   Note: The adhesive should not touch the wing hinge or the forewing tegulae (Figure 2C).
5. Bond the mesonotum to the attachment.
6. Keep the moth tethered before placing it inside the cockpit of the robot. Hold the attachment on a stand and put a piece of paper under the legs to rest the moth.

3. Insect-controlled Robot

1. Design the hardware of the insect-controlled robot based on previous works^16,17,19.
   Note: The insect-controlled robot consists of an air-supported treadmill with an optical mouse sensor to capture the insect locomotion, custom-built AVR-based microcontroller boards for processing and motor control, and two DC brushless motors (Figures 3 and 4). The robot can run on the basis of the ball rotation with 96% precision or higher, within a time delay of 200 msec. It also ensures the mobility of maximum forward speed (24.8 mm/sec) and angular velocity (96.3°/sec) of the silkmoth during pheromone tracking behavior¹⁶. The airflow of the treadmill (Figure 5A) and odor delivery system (Figure 5B) are designed for the onboard moth to walk smoothly on the ball and to acquire an odor by two antennae. The air intake and flow channel of the treadmill is separated from those of the odor delivery system to avoid contamination of the pheromone.
2. Design the software for the onboard microcontrollers based on previous works¹⁶.
   Note: The onboard microcontroller calculates the robot movements from the insect locomotion measured with an optical sensor (rotational, Δx; translational, Δy; Figure 6). The travel distance (ΔL) and turn angle (Δθ) per unit time of the robot are calculated on the basis of travel distance of each wheel (left, ΔL_L; right, ΔL_R) such as ΔL = (ΔL_L + ΔL_R) / 2 and Δθ = (ΔL_L – ΔL_R) / D_wheel, where D_wheel is the distance between the two wheels (120 mm). ΔL_L and ΔL_R are further described as ΔL_L = ΔL_x,L + ΔL_y,L and ΔL_R = ΔL_x,R + ΔL_y,R, where ΔL_x,L and ΔL_x,R are the travel distances of the wheels on the left and right sides controlled by Δx, and ΔL_y,L and ΔL_y,R are those controlled by Δy. Ideally, ΔL_x,L and ΔL_x,R are described as ΔL_x,L = -ΔL_x,R = G Δx (D_wheel / D_ball), and ΔL_y,L and ΔL_y,R are described as ΔL_y,L = ΔL_y,R = G Δy, where G is the motor gain and D_ball is the diameter of the ball (50 mm). In practice, the motor gain is independently set by each side (left or right wheel) and by each direction (forward or backward rotation) so as to calibrate the robot movement. The independent gains further allow for the setting of asymmetrical motor rotation to generate a turning bias of the robot (see step 6.1).
3. Wash the surface of a white expanded polystyrene ball (mass: approximately 2 g; diameter: 50 mm) with water to remove any possible olfactory or visual cues.
   Note: The surface of a new ball should be roughed with fine-grit sandpaper, such as P400, which ensures the grip of the legs on the ball.
4. Turn on the blower fan that supplies air at 9 V to the treadmill and floats the ball (Figure 5A). Observe the ball float approximately 2 mm from the bottom of the cup.
5. Using a screw, attach the copper wire of the attachment with the moth (see step 2) to a fixture in the cockpit of the robot (see Figure 3 inset). Make sure that the position of the middle legs is at the center of the ball (Figure 7A).
6. Adjust the vertical position of the attachment to enable the moth to walk normally on the ball. Keep the ball at the same height before and after attaching the moth (Figure 7B).
   Note: A too-low position of the attachment adds pressure on the moth and elicits backward walking to resist the pressure (Figure 7C), whereas a too-high position causes unstable walking and failures of the sensor due to changes in the vertical position of the ball (Figure 7D). To check normal walking behavior, a single-puffed pheromone stimulus is used to trigger walking in the moth (for the pheromone stimulus, see step 4). Note that the test stimulus must be minimal because previous exposure to bombykol habituates silkmoths and decreases their sensitivity (Matsuyama and Kanzaki, unpublished data).

4. Odor Source Preparation

Note: Male B. mori are sensitive to the major component of the conspecific female sex pheromone (bombykol: (E,Z)-10,12-hexadecadien-1-ol)²⁰. Any contamination of experimental equipment with bombykol elicits the odor-tracking behavior and affects the responsiveness of the moth.

1. Drop 10 µl of the bombykol solution dissolved in n-hexane (200 ng/µl) on a piece of filter paper (approximately 10 mm × 10 mm). The amount of bombykol per piece of filter paper is 2,000 ng.
   Note: To check the normal walking behavior of the moth, prepare a pheromone stimulus cartridge in this step. The cartridge is a glass Pasteur pipette with one piece of filter paper containing 2,000 ng of bombykol. Pushing a bulb puffs the air containing bombykol.

5. Odor Source Localization Experiment

1. Turn on the fan of a pulling-air-type wind tunnel (1,800 × 900 × 300 mm, L × W × H; Figure 8) and set the wind speed to 0.7 m/sec. Ensure that the temperature is more than 20 °C.
2. Set the odor source (the piece of filter paper containing bombykol) upstream of the wind tunnel.
   Note: The plume width should be confirmed prior to the experiment by using TiCl₄^17,19.
3. Turn on the microcontroller board of the robot and establish a serial connection to a PC via Bluetooth.
4. Launch a custom-made Java program called "BioSignal," which provides an interface between the PC and the robot.
   Note: The main window includes buttons for sending commands to the robot, text windows for displaying the input and output of the serial communication, and small boxes to configure parameters. The subsequent commands are sent by clicking corresponding buttons in this program, except for video capturing.
5. Click on the "about device" button to confirm the connection by sending a command to the robot via the specified COM port and check that a message is returned by the robot.
6. Click on the "memory erase" button to erase previous locomotion data left on the onboard flash memory.
7. Click on the "drivemode1" button to send the default motor gains to the robot.
   Note: The manipulations of the motor gains and the time delay between insect locomotion and robot movement are applied after this step (see steps 6.1 and 6.3, Figure 9).
8. Click on the "don't drive" button to send a command to immobilize the robot until the experiment starts.
9. Put the robot at a start position (600 mm downstream from the odor source) and turn on the switch of the motor driver board.
10. Push the recording button of the camcorder to start video capture.
11. Click on the "rec start" button to send a start command to initiate the robot with a simultaneous recording of the ball rotation on the onboard flash memory. Observe that the robot starts to move and tracks the odor plume.
12. Click on the "rec stop" and "don't drive" buttons to send commands to stop both the robot movement and the recording if the robot localizes the odor source.
13. Push the recording button of the camcorder to stop video capture.
14. Download recorded locomotion data from the onboard flash memory to the computer via a serial connection. Close the program.

6. Manipulation of the Insect-controlled Robot

Note: The timing of each manipulation is indicated in Figure 9.

1. Manipulation of motor gains
   Note: This manipulation alters the translational and rotational velocity of the robot. Asymmetrical motor gains generate a turning bias, which can be used to investigate how insects compensate for the bias¹⁷.
   1. Define the rotational gains for forward and backward rotation of the motor on each side¹⁷ (Figure 6B) by editing the configuration file named "param2.txt" using a text editor.
   2. Click on the "set param2" to read the edited configuration file in the software program. Then, click on the "drivemode2" to send the manipulated gains to the robot.
2. Inversion of the motor output
   Note: This manipulation provides a condition similar to the inversion of bilateral olfactory input (see step 6.4) and can be used to investigate the significance of bilateral olfaction. However, the inversion of motor output also inverts self-induced visual motion of an onboard moth. The impact of the inverted self-induced visual input can be evaluated by a comparison with the inverted olfactory input¹⁹.
   1. Invert the bilateral motor control by crossing the control cables for each motor.
3. Manipulation of the time delay between insect locomotion and robot movement.
   Note: This manipulation allows for the investigation of the acceptable period of time spent on sensory-motor processing for the robotic odor-tracking. The microcontroller stores the locomotion data on a buffer memory and then processes it after the specified time delay. Note that the robot has a maximal internal time delay of 200 msec; therefore, the actual time delay is expected to be the specified time delay plus 200 msec^16,17.
   1. Input a number (from 0-10) in a small box of the main window to specify a time delay from 0-1,000 msec at 100-msec steps.
   2. Click on the "set delay" button to apply the time delay.
4. Manipulation of the olfactory input.
   Note: This manipulation can be used to investigate the significance of bilateral olfactory input. The surge direction of silkmoths is biased on the higher-concentration side²².
   1. Change the gap between the suction tube tips or invert their positions to alter the difference in odor concentration acquired by each antenna.
5. Manipulation of visual input
   Note: This manipulation is to investigate the role of visual input for odor-tracking.
   1. Cover the canopy with a white paper that occludes 105° and 90° of the horizontal and vertical visual field of the onboard moth, respectively.