Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

Summary

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

Abstract

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Introduction

An insect-machine hybrid system, often referred to as a cyborg insect or biobot, is the fusion of a living insect platform with a miniature mounted electronic device. The electronic device, which is wirelessly commanded by a remote user, outputs an electrical signal to electrically stimulate neuromuscular sites in the insect via implanted wire electrodes to induce user desired motor actions and behaviors. In the early stages of this research field, researchers were limited to conducting wireless recording of the muscular action of an insect, using simple analog circuits comprised of surface-mounted components^1-3. The development of system-on-a-chip technology with radio frequency functionality enabled not only the wireless recording of neuromuscular signals but also the electrical stimulation of the neuromuscular sites in living insects. At present, a built-in radio microcontroller is small enough to be mounted on living insects without causing any obstructions to their locomotion^4-13.

The development of the built-in radio microcontroller allows researchers to determine electrical stimulation protocols to induce desired motor actions to control the locomotion of the insect of interest. On the ground, researchers have demonstrated walking control by stimulating the neuromuscular sites of cockroaches^4,12,14, spiders¹⁵, and beetles^16,17. In the air, the initiation and cessation of flight were achieved using different methods such as the stimulation of the optic lobes (the massive neural cluster of a compound eye) in beetles^7,9 and brain sub-regions in bees¹⁸, whereas turning control has been demonstrated by stimulating the antennae muscles and nervous system of the abdomens in moths^11,19 and the flight muscles of beetles^7,9,13. In most cases, a built-in radio microcontroller was integrated on a custom-designed printed circuit board to produce a miniature wireless stimulator (backpack), which was mounted on the insect of interest. This allows wireless electrical stimulation to be applied to a freely walking or flying insect. Such a microcontroller-mounted insect is what is referred to as an insect-machine hybrid system.

This study describes the experimental protocols for building an insect-machine hybrid system, wherein a living beetle is employed as the insect platform, and instructs on how to operate the robot and test its flight control systems. The third axillary sclerite (3Ax) muscle was chosen as the muscle of interest for electrical stimulation and demonstration of left or right turning control¹³. A pair of thin silver wire electrodes was implanted in both the left and right 3Ax muscles. Moreover, a backpack was mounted on the living beetle. The other ends of the wire electrode were connected to the output pins of the microcontroller. The backpack was small enough for the beetle to carry in flight. Thus, this allows an experimentalist to remotely stimulate the muscle of interest of an insect in free flight and investigate its reactions to the stimulations.

Protocol

1. Study Animal

1. Rear individual Mecynorrhina torquata beetles (6 cm, 8 g) in separate plastic containers with wood pellet bedding.
2. Feed each beetle a cup of sugar jelly (12 ml) every 3 days.
3. Keep the temperature and humidity of the rearing room at 25 °C and 60%, respectively.
4. Test the flight capability of each beetle before implanting thin wire electrodes.
   1. Gently throw a beetle into the air. If the beetle can fly for longer than 10 sec for 5 consecutive trials, conclude that the beetle has regular flight capabilities and employ it for subsequent flight experiments. To recapture the beetle, switch off all the lights in the room to make it dark. This causes the beetle to terminate flight.
      Note: A beetle spontaneously begins to fly away when released into the air. It is better to conduct the flight experiments in a large closed room such as the one shown in Figure 1 (16 x 8 x 4 m³ with a motion capture space of 12.5 x 8 x 4 m³), as a flying beetle moves very fast (approximately 3-5 m/sec) and draws large arcs when turning in the air.

2. Electrode Implantation

1. Anesthetize the beetle by placing it in a plastic container filled with CO₂ for 1 min^13,16,20-24.
2. Soften dental wax by dipping it in hot water for 10 sec. Place the anesthetized beetle on a wooden block and immobilize it with the softened dental wax. The dental wax naturally cools and solidifies within a few minutes.
3. Cut insulated silver wires (127 µm bare diameter, 178 µm diameter when coated with perfluoroalkoxy) into lengths of 25 mm to use as thin wire electrodes for implantation.
4. Expose 3 mm of bare silver by flaming the insulator at both ends of each wire.
5. Dissect the top surface of the beetle's cuticle using a fine-tipped scissor to create a small window of approximately 4 x 4 mm on the metepisternum (Figure 2c). Note: A soft brown-colored cuticle is then exposed, as shown in Figures 2c-e. The 3Ax muscle is located underneath the soft cuticle.
6. Pierce two holes on the exposed brown cuticle using an insect pin (size 00) with a distance of 2 mm between the two holes (Figure 2d).
7. Insert two wire electrodes (including one active and one return electrodes prepared in step 2.4) carefully through the holes and implant them into each 3Ax muscle at a depth of 3 mm.
8. Secure the implanted electrodes and hold them in place to avoid contact and short-circuits by dropping melted beeswax on the holes. If needed, reflow the beeswax over the cuticle by touching the beeswax with the tip of a hot soldering iron. The beeswax quickly solidifies and reinforces the implantation.
   Note: To check if the implantation is correct, the elytra of the beetle can be lifted to observe the movement of the 3Ax muscle during electrical stimulation.

3. Wireless Backpack Assembly

Note: The backpack consisted of a built-in radio microcontroller on a 4 layered FR-4 board (1.6 x 1.6 cm²). The backpack was driven by a lithium polymer microbattery (3.7 V, 350 mg, 10 mAh). The total mass of the backpack including the battery was 1.2 ± 0.26 g which is less than the payload capacity of the beetle (30% of 10 g body weight). The backpack was pre-programmed to receive wireless communications and had two output channels.

1. Clean the pronotum surface (remove the wax layer on the cuticle) using double-sided tape. Then, attach the backpack on the pronotum of the beetle with a piece of double-sided tape.
2. Connect the ends of the implanted electrodes to the outputs of the backpack.
3. Wrap retro-reflective tape around the microbattery to produce a marker for motion capture cameras to detect.
4. Attach the microbattery to the top of the backpack using a piece of double-sided tape so that the retro-reflective tape can be detected by motion capture cameras.

4. Wireless Control System

Note: In this case, the term wireless control system includes a receiver for the remote controller, a laptop computer to run the custom flight control software, a base station, the backpack, and the motion capture system.

1. Connect the base station and receiver of the remote controller to the laptop computer via USB ports.
2. Switch on the motion capture system and connect it to the laptop computer via an Ethernet port.
3. Perform volume calibration by waving the calibration wand (provided by the vendor company of the motion capture system) to fully cover the motion capture space.
   1. Open the motion capture software from the desktop of the laptop. Click and drag to select all the cameras on the "System" menu of the "Resources" panel.
   2. Click on the "3D Perspective" menu and select "Camera" to change to the camera view. Click on the "Camera" tab on the "Tools" panel to show the calibration setup. Click "Start" on the "Create Camera Masks" menu to eliminate noise from the cameras and then "Stop" after the noise is masked in blue.
   3. Click and select "5 Marker Wand & L-Frame" from the "Wand" menu and the "L-Frame" menu on the "Camera" tab. Set the "Wand Count" to 2,500, click "Start" on the "Calibrate Cameras" menu, and wave the calibration wand through the entire motion capture space. The calibration process stops when the wand count reaches 2,500.
   4. Repeat the calibration process if image error (at the bottom of the "Camera" tab of the "Tools" panel) is higher than 0.3 for any camera. After calibrating, put the wand on the floor in the middle of the motion capture space and click "Start" on the "Set Volume Origin" menu to set the origin of the motion capture space.
4. Check the coverage of the motion capture system using a dummy test to record the motion path of a marker waved by a user in the motion capture space and confirm whether the marker is detected and tracked. If the marker is frequently lost during detection, repeat volume calibration until the dummy test succeeds.
   1. Click on the "Capture" tab on the "Tools" panel and then "Start" on the "Capture" menu before waving the sample marker through the entire motion capture space to record its trajectory.
   2. After recording, click on "Runs the Reconstruct pipeline" to reconstruct the positions of the marker and check the quality of the recording.
5. Connect the terminals of the microbattery (attached to the backpack in step 3.4) to the power pins of the backpack.
6. Test the wireless communication between the laptop and the backpack using the custom flight control software. Click the "Start" command on the software and check the displayed connection status.

5. Free Flight Experiment

1. Carry out the free flight experiment in a flight arena measuring 16 x 8 x 4 m³.
2. Input the appropriate parameters to the flight control software (voltage, pulse width, frequency, and stimulation duration). Note: For demonstration, we fixed the voltage to 3 V, pulse width to 3 msec, and stimulation duration to 1 sec and varied the frequency from 60 to 100 Hz.
   1. On the software screen, type 3 for 3 V in the "Voltage" box, 1,000 for 1,000 msec in the "Stimulation Duration" box, 3 for 3 ms in the "Pulse Width" box, and a desired frequency in Hz in the "Frequency" box on the command window.
3. Release the backpack-mounted beetle into the air allowing it to fly freely within the flight arena. Manually trigger the stimulation when the beetle enters the motion capture space. Press the appropriate command button (Left or Right) on the remote to stimulate the target muscle on the left or right side of the beetle.
   Note: Once the button is pressed, the flight control software running on the laptop generates the command and sends it to the backpack. The backpack then outputs the electrical stimulus to the muscle of interest (on the left or right side).
4. Observe the beetle's reaction in real time during the stimulation and reconstruct the data using 3D graphing software.
   1. Select one of the trials recorded in the data list of the "Beetle Display" window and click "Export Panda" to copy the data of that trial to the analysis folder and run the 3D graphing module.
   2. Press "N" on the keyboard to combine the stimulus signal with the recorded trajectory. Press I to show the trajectory of the beetle with the highlighted stimulation periods.

Results

The electrode implantation procedure is presented in Figure 2. Thin silver wire electrodes were implanted into the 3Ax muscle of the beetle through small holes pierced on the soft cuticle on the muscle (Figures 2d-e). This soft cuticle is found just above the apodema of the basalar muscle after removing the anterior part of the metepisternum (Figures 2d-c). The electrodes were then secured using beeswax (...

Discussion

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 ex...

Materials

Name	Company	Catalog Number	Comments
Mecynorrhina torquata beetle	Kingdom of Beetle Taiwan		10 g, 8 cm, pay load capacity is 30% of the body mass Aproval of importing and using by Agri-Food and Veterinary Authority of Singapore (AVA; HS code: 01069000, product code: ALV002).
Wireless backpack stimulator	Custom		TI CC2431 micocontroler The board is custom made based on the GINA board from Prof. Kris Pister’s lab. The layout of GINA board can be found at    https://openwsn.atlassian.net/wiki/display/OW/GINA
Wii Remote control	Nintendo		Bluetooth remote control to send the command to the operator laptop
BeetleCommander v1.8	Custom. Maharbiz group at UC Berkeley and Sato group at NTU		Establish the wireless communication of the backpack and the operator laptop. Configure the stimulus parameters and log the positional data. Visualize the flight data.
GINA base station	Kris Pister group at UC Berkeley		TI MSP430F2618 and AT86RF231
Motion capture system	VICON	T160	8 cameras for a flight arena of 12.5 m x 8 m x 4 m
Motion capture system	VICON	T40s	12 cameras for a flight arena of 12.5 x 8 x 4 m
Micro battery	Fullriver	201013HS10C	3.7V, 10 mAh
Retro reflective tape	Reflexite	V92-1549-010150	V92 reflective tape, silver color
PFA-Insulated Silver Wire	A-M systems	786000	127 µm bare, 177.8 µm coated, 3 mm bare silver flame exposed at tips
SMT Micro Header	SAMTEC	FTSH-110-01-L-DV	0.3 mm x 6 mm, bend to make a 3 mm long slider to secure the electrode into the PCB header.
Beeswax			Secure the electrodes
Dental Wax	Vertex		Immobilize the beetle
Insect pin	ROBOZ	RS-6082-30	Size  00; 0.3 mm Rod diameter; 0.03 mm tip width; 38 mm Length  Make electrode guiding holes on cuticle
Tweezers	DUMONT	RS-5015	Pattern #5; .05 mm x .01 mm Tip Size; 110 mm Length Dissecting and implantation
Scissors	ROBOZ	RS-5620	Vannas Micro Dissecting Spring Scissors; Straight; 3mm Cutting Edge; 0.1 mm Tip Width; 3" Overall Length  Dissecting and implantation
Potable soldering iron	DAIYO	DS241	Reflow beeswax
Hotplate	CORNING	PC-400D	Melting beeswax and dental wax
Flourescent lamp	Philips	TL5 14W	Light the entire flight arena with 30 panels (60 x 60 cm2). Each panel has 3 lamps. 14 W, 549 mm x 17 mm