This video describes the process of constructing an insect-machine hybrid system for wireless electrical stimulation of the flight muscles in a freely flying insect. This method can help answer key question in insect research field, such as function of insect flight muscle in flight. The main advantage of this technique is that we can investigate the effect of activating special flight muscle on the flight of three different insect by using a tiny remote stimulator.
Through this method, kind of our insight into the function of the fly muscles in beetles. It can also be applied to other insects or other muscle groups, such as in the leg or antenna. To begin, test the flight capability of the beetles to identify ones suitable for experimentation.
Select a beetle and gently throw it into the air in a large enclosed room. To recapture a beetle, just make the room dark, which will cause it to stop flying. If the beetle flies for more than 10 seconds on five consecutive trials, then it may be used for the experiment.
First, anesthetize the beetle using a carbon dioxide chamber. Leave it there for a minute. Next, soften some dental wax in hot water for 10 seconds.
Then, place the anesthetized beetle on a wooden block, and immobilize it with the wax. Next, cut insulated silver wire into 25-millimeter lengths to function as electrodes. At both ends, expose three millimeters of bare silver wire using a flame.
Now, dissect the top surface of the beetle's cuticle with fine-tipped scissors to create a small four by four-millimeter window in the metepisternum, exposing the soft brown-colored cuticle beneath. Just beneath the cuticle is the 3AX muscle. Next, using a double-zero insect pin, pierce two holes in the brown cuticle two millimeters apart.
Then, insert a wire electrode through each hole and into each 3AX muscle. Penetrate each wire three millimeters into the tissue. Any mistake in this process can lead to long reaction or muscle deficit, so identify the muscle of interest carefully, and implant the electrode precisely.
Now, secure the electrodes using beeswax to avoid contacts and short circuits. If needed, reflow the beeswax over the cuticle by remelting it with a soldering iron. Now, to check if the implantation is correct, lift the electra of the beetle to observe the movement of the 3AX muscle while stimulating it with an electrode.
Details on the backpack device are provided in the text protocol. To attach it, first, clean the wax layer off of the pronotum surface using double-sided tape. Then, attach the backpack using double-sided tape.
Next, connect the ends of the implanted electrodes to the outputs of the backpack. Then, wrap retro-reflective tape around the microbattery, and attach it to the top of the backpack using more double-sided tape. Ensure that the terminals are accessible.
This tape-wrapped battery will function as a marker for the motion capture cameras. The 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. First, connect the base station and receiver of the remote controller to the laptop computer using USB connections.
Then, switch on the motion capture system and connect it to the laptop computer via the ethernet port. Now, in the software, perform a volume calibration using the manufacturer supplied calibration wand. In the software, click and drag to select all the cameras on the system menu of the resources panel.
Then, click on the 3D Perspective menu, and select Camera to change to the camera view. Next, click on the Camera tab on the Tools panel to show the calibration set-up, and select Start from the Create Camera Masks menu to eliminate noise from the cameras. Once the noise is masked, shown in blue, stop the process.
Now, from the Wand menu, select 5 Marker Wand L-Frame. Then, go to the L-Frame menu on the Camera tab and do the same. Next, set the wand count to 2500.
Then, 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 2500. After the calibration, put the wand on the floor in the middle of the motion capture space.
Then, click on Start on the Set Volume Origin menu to set the origin of the motion capture space. Then, start a test by clicking on the Capture tab in the Tools panel, and then clicking Start from the Capture menu. Now, check that the system works by recording the motion path of a marker while it is waved by a user.
To check the quality of the recording, click on Runs the Reconstruct Pipeline to reconstruct the positions of the marker and check the quality of the recording. If the marker is lost for distances exceeding 200 millimeters, recalibrate the system. Now, check that the system on the beetle works.
Connect the terminals of the microbattery to the power pins of the backpack. Then, click the Start command on the software, and check for a displayed connection status. If it is registering, a free flight experiment can now be made.
Carry out the free flight experiment in a flight arena. In this example, the arena measures 16 by eight by four cubic meters, with part of the arena outside of the motion tracking area. In the flight software, input the appropriate parameters, including the voltage, pulse width, frequency, and stimulation duration.
Now, release the backpack-mounted beetle into the flight arena, and wait until the beetle enters the motion capture space to manually trigger the stimulation. Press the appropriate command button on the remote to stimulate the target muscle on the left or right side of the beetle, and observe the beetle's reaction. Later, reconstruct the data using 3D graphing software.
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. In the graphing software, 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.
The activation of the 3AX muscle was found to cause a reduction in the wing beat amplitude of the ipsilateral side, thus resulting in the beetle performing an ipsilateral turn in free flight. The turning rate of the beetle was then graded as a function of the stimulation frequency. Notice that the frequency of stimulation of the right side muscle neatly coordinated with the degree of turning to the right side.
Once mastered, this technique can be done in 20 minutes if it's performed properly. While attempting this procedures, it's important to remember to identify the muscle correctly and position the implant correctly to avoid isolating his house. Following this procedure, the auto-fly muscle lie supular muscle or basular muscle, can be stimulated to produce different behavior of the insect.
After its development, this technique paves a way for researchers in the field of insect-related research to explore the function of their flight muscle of 3D flying insects.
