Name: insect-machine hybrid system: remote radio control of a freely flying beetle (mercynorrhina torquata)
Uploaded: 2016-09-02T13:00:00
Description: Watch this Scientific Journal Video about Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata at JoVE.com

This material is based on the works supported by Nanyang Assistant Professorship (NAP, M4080740), Agency for Science, Technology and Research (A*STAR) Public Sector Research Funding (PSF, M4070190), A*STAR-JST (The Japan Science and Technology Agency) joint grant (M4070198), and Singapore Ministry of Education (MOE2013-T2-2-049). The authors would like to thank Mr. Roger Tan Kay Chia, Prof. Low Kin Huat, Mr. Poon Kee Chun, Mr. Chew Hock See, Mr. Lam Kim Kheong and Dr. Mao Shixin at School of MAE for their support in setting up and maintaining the research facilities. The authors thank Prof. Michel Maharbiz (U.C. Berkeley) his advice and discussion, Prof. Kris Pister and his group (U.C. Berkeley) for their support in providing the GINA used in this study.

1. Study Animal
<ol>
	<li>Rear individual Mecynorrhina torquata beetles (6 cm, 8 g) in separate plastic containers with wood pellet bedding.</li>
	<li>Feed each beetle a cup of sugar jelly (12 ml) every 3 days.</li>
	<li>Keep the temperature and humidity of the rearing room at 25 &#176;C and 60%, respectively.</li>
	<li>Test the flight capability of each beetle before implanting thin wire electrodes.
	<ol>
		<li>Gently throw a beetle into the air. If the beetle can fly for longer than 10 sec for 5 consecutive trials, c...

<ol>
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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...

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 components1-3. 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 locomotion4-13.
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 cockroaches4,12,14, spiders15, and beetles16,17. 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 beetles7,9 and brain sub-regions in bees18, whereas turning control has been demonstrated by stimulating the antennae muscles and nervous system of the abdomens in moths11,19 and the flight muscles of beetles7,9,13. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mecynorrhina torquata beetle</td>
			<td>Kingdom of Beetle Taiwan</td>
			<td></td>
			<td>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).</td>
		</tr>
		<tr>
			<td>Wireless backpack stimulator</td>
			<td>Custom</td>
			<td></td>
			<td>TI CC2431 micocontroler 
			The board is custom made based on the GINA board from Prof. Kris Pister&#8217;s lab. The layout of GINA board can be found at&#160;&#160;&#160; https://openwsn.atlassian.net/wiki/display/OW/GINA</td>
		</tr>
		<tr>
			<td>Wii Remote control</td>
			<td>Nintendo</td>
			<td></td>
			<td>Bluetooth remote control to send the command to the operator laptop</td>
		</tr>
		<tr>
			<td>BeetleCommander v1.8</td>
			<td>Custom. Maharbiz group at UC Berkeley and Sato group at NTU</td>
			<td></td>
			<td>Establish the wireless communication of the backpack and the operator laptop. Configure the stimulus parameters and log the positional data. Visualize the flight data.</td>
		</tr>
		<tr>
			<td>GINA base station</td>
			<td>Kris Pister group at UC Berkeley</td>
			<td></td>
			<td>TI MSP430F2618 and AT86RF231</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T160</td>
			<td>8 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T40s</td>
			<td>12 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Micro battery</td>
			<td>Fullriver</td>
			<td>&#160;201013HS10C&#160;</td>
			<td>3.7V, 10 mAh</td>
		</tr>
		<tr>
			<td>Retro reflective tape</td>
			<td>Reflexite</td>
			<td>V92-1549-010150</td>
			<td>V92 reflective tape, silver color</td>
		</tr>
		<tr>
			<td>PFA-Insulated Silver Wire&#160;</td>
			<td>A-M systems</td>
			<td>786000</td>
			<td>127 &#181;m bare, 177.8 &#181;m coated, 3 mm bare silver flame exposed at tips</td>
		</tr>
		<tr>
			<td>SMT Micro Header&#160;</td>
			<td>SAMTEC</td>
			<td>FTSH-110-01-L-DV</td>
			<td>0.3 x 6 mm, bend to make a 3 mm long slider to secure the electrode into the PCB header.</td>
		</tr>
		<tr>
			<td>Beeswax</td>
			<td></td>
			<td></td>
			<td>Secure the electrodes</td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Vertex</td>
			<td></td>
			<td>Immobilize the beetle</td>
		</tr>
		<tr>
			<td>Insect pin</td>
			<td>ROBOZ</td>
			<td>RS-6082-30</td>
			<td>Size&#160; 00; 0.3mm Rod diameter; 0.03 mm tip width; 38 mm Length&#160; 
			Make electrode guiding holes on cuticle</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>DUMONT</td>
			<td>RS-5015</td>
			<td>Pattern #5; .05 X .01mm Tip Size; 110mm Length 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>ROBOZ</td>
			<td>RS-5620</td>
			<td>Vannas Micro Dissecting Spring Scissors; Straight; 3mm Cutting Edge; 0.1mm Tip Width; 3&#34; Overall Length&#160; 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Potable soldering iron</td>
			<td>DAIYO</td>
			<td>DS241</td>
			<td>Reflow beeswax</td>
		</tr>
		<tr>
			<td>Hotplate&#160;</td>
			<td>CORNING</td>
			<td>PC-400D</td>
			<td>Melting beeswax and dental wax</td>
		</tr>
		<tr>
			<td>Flourescent lamp</td>
			<td>Philips</td>
			<td>TL5 14W</td>
			<td>Light the entire flight arena with 30 panels (60 x 60 cm2). Each panel has 3 lamps. 
			14 W, 549 mm x 17 mm&#160;</td>
		</tr>
	</tbody>
</table>

insect-machine hybrid system: remote radio control of a freely flying beetle (mercynorrhina torquata)

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.

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 (...

Watch this Scientific Journal Video about Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata at JoVE.com

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

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.

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

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

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Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...