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>
	<li>Kutsch, W., Schwarz, G., Fischer, H., Kautz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wireless+Transmission+of+Muscle+Potentials+During+Free+Flight+of+a+Locust.">Wireless Transmission of Muscle Potentials During Free Flight of a Locust.</a> J. Exp. Biol. 185 (1), 367-373 (1993).</li><li>Fischer, H., Kautz, H., Kutsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Radiotelemetric+2-Channel+Unit+for+Transmission+of+Muscle+Potentials+During+Free+Flight+of+the+Desert+Locust,+Schistocerca+Gregaria.">A Radiotelemetric 2-Channel Unit for Transmission of Muscle Potentials During Free Flight of the Desert Locust, Schistocerca Gregaria.</a> J. Neurosci. Methods. 64 (1), 39-45 (1996).</li><li>Ando, N., Shimoyama, I., Kanzaki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Dual-Channel+FM+Transmitter+for+Acquisition+of+Flight+Muscle+Activities+from+the+Freely+Flying+Hawkmoth,+Agrius+Convolvuli.">A Dual-Channel FM Transmitter for Acquisition of Flight Muscle Activities from the Freely Flying Hawkmoth, Agrius Convolvuli.</a> J. Neurosci. Methods. 115 (2), 181-187 (2002).</li><li>Sanchez, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotion+control+of+hybrid+cockroach+robots.">Locomotion control of hybrid cockroach robots.</a> J. R. Soc. Interface. 12 (105), (2015).</li><li>Sato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyborg+beetle:+insect+flight+control+through+an+implantable,+tetherless+microsystem.">A cyborg beetle: insect flight control through an implantable, tetherless microsystem.</a> , 164-167 (2008).</li><li>Bozkurt, A., Gilmour, R. F., Lal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon-Assisted+Flight+of+Radio-Controlled+Insect+Biobots.">Balloon-Assisted Flight of Radio-Controlled Insect Biobots.</a> IEEE Trans. Biomed. Eng. 56 (9), 2304-2307 (2009).</li><li>Sato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+Radio+Control+of+Insect+Flight.">Remote Radio Control of Insect Flight.</a> Front. Neurosci. 3, (2009).</li><li>Daly, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pulsed+UWB+Receiver+SoC+for+Insect+Motion+Control.">A Pulsed UWB Receiver SoC for Insect Motion Control.</a> IEEE J. Solid-State Circuits. 45 (1), 153-166 (2010).</li><li>Maharbiz, M. M., Sato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyborg+Beetles.">Cyborg Beetles.</a> Sci. Am. 303 (6), 94-99 (2010).</li><li>Tsang, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+a+cyborg+moth+using+carbon+nanotube-enhanced+flexible+neuroprosthetic+probe.">Remote control of a cyborg moth using carbon nanotube-enhanced flexible neuroprosthetic probe.</a> , 39-42 (2010).</li><li>Hinterwirth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wireless+Stimulation+of+Antennal+Muscles+in+Freely+Flying+Hawkmoths+Leads+to+Flight+Path+Changes.">Wireless Stimulation of Antennal Muscles in Freely Flying Hawkmoths Leads to Flight Path Changes.</a> PloS ONE. 7 (12), (2012).</li><li>Whitmire, E., Latif, T., Bozkurt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinect-based+system+for+automated+control+of+terrestrial+insect+biobots.">Kinect-based system for automated control of terrestrial insect biobots.</a> , 1470-1473 (2013).</li><li>Sato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+Role+of+a+Coleopteran+Steering+Muscle+via+Free+Flight+Stimulation.">Deciphering the Role of a Coleopteran Steering Muscle via Free Flight Stimulation.</a> Curr. Biol. 25 (6), 798-803 (2015).</li><li>Erickson, J. C., Herrera, M., Bustamante, M., Shingiro, A., Bowen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+Stimulus+Parameters+for+Directed+Locomotion+in+Madagascar+Hissing+Cockroach+Biobot.">Effective Stimulus Parameters for Directed Locomotion in Madagascar Hissing Cockroach Biobot.</a> PLoS ONE. 10 (8), e0134348 (2015).</li><li>Zhaolin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+study+of+motion+control+patterns+for+biorobotic+spiders.">A preliminary study of motion control patterns for biorobotic spiders.</a> , 128-132 (2014).</li><li>Feng, C., Chao, Z., Hao Yu, C., Sato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect-machine+hybrid+robot:+Insect+walking+control+by+sequential+electrical+stimulation+of+leg+muscles.">Insect-machine hybrid robot: Insect walking control by sequential electrical stimulation of leg muscles.</a> , 4576-4582 (2015).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Biological+Micro+Actuator:+Graded+and+Closed-Loop+Control+of+Insect+Leg+Motion+by+Electrical+Stimulation+of+Muscles.">A Biological Micro Actuator: Graded and Closed-Loop Control of Insect Leg Motion by Electrical Stimulation of Muscles.</a> PLoS ONE. 9 (8), e105389 (2014).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromechanism+Study+of+Insect-Machine+Interface:+Flight+Control+by+Neural+Electrical+Stimulation.">Neuromechanism Study of Insect-Machine Interface: Flight Control by Neural Electrical Stimulation.</a> PLoS ONE. 9 (11), e113012 (2014).</li><li>Tsang, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+Split-Ring+Electrode+for+Insect+Flight+Biasing+Using+Multisite+Neural+Stimulation.">Flexible Split-Ring Electrode for Insect Flight Biasing Using Multisite Neural Stimulation.</a> IEEE Trans. Biomed. Eng. 57 (7), 1757-1764 (2010).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J. Insect Physiol. 46 (4), 439-442 (2000).</li><li>Cooper, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia,+Analgesia,+and+Euthanasia+of+Invertebrates.">Anesthesia, Analgesia, and Euthanasia of Invertebrates.</a> ILAR Journal. 52 (2), 196-204 (2011).</li><li>Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect neurophysiological techniques. , (2012).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA guidelines for the euthanasia of animals. , (2013).</li><li>Heath, B., West, G., Heard, D., Caulkett, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobile+Inhalant+Anesthesia+Techniques.">Mobile Inhalant Anesthesia Techniques.</a> in Zoo Animal and Wildlife Immobilization and Anesthesia. , 75-80 (2008).</li><li>Mischiati, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+models+direct+dragonfly+interception+steering.">Internal models direct dragonfly interception steering.</a> Nature. 517 (7534), 333-338 (2015).</li><li>Kutsch, W., Berger, S., Kautz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Manoeuvres+in+Free-Flying+Locusts:+Two-Channel+Radio-Telemetric+Transmission+of+Muscle+Activity.">Turning Manoeuvres in Free-Flying Locusts: Two-Channel Radio-Telemetric Transmission of Muscle Activity.</a> J. Exp. Zoolog. Part A Comp. Exp. Biol. 299 (2), 139-150 (2003).</li><li>Wang, H., Ando, N., Kanzaki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+Control+of+Free+Flight+Manoeuvres+in+a+Hawkmoth,+Agrius+Convolvuli.">Active Control of Free Flight Manoeuvres in a Hawkmoth, Agrius Convolvuli.</a> J. Exp. Biol. 211 (3), 423-432 (2008).</li><li>Sato, H., Maharbiz, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+the+remote+radio+control+of+insect+flight.">Recent developments in the remote radio control of insect flight.</a> Front. Neurosci. 4, (2010).</li><li>Tien Van, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flight+behavior+of+the+rhinoceros+beetle+Trypoxylus+dichotomus+during+electrical+nerve+stimulation.">Flight behavior of the rhinoceros beetle Trypoxylus dichotomus during electrical nerve stimulation.</a> Bioinsp. Biomim. 7 (3), 036021 (2012).</li><li>Sane, S. P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+flight+force+by+a+flapping+wing:+lift+and+drag+production.">The control of flight force by a flapping wing: lift and drag production.</a> J. Exp. Biol. 204 (15), 2607-2626 (2001).</li><li>de Croon, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+aerodynamics+and+autonomy+of+the+DelFly.">Design, aerodynamics and autonomy of the DelFly.</a> Bioinsp. Biomim. 7 (2), 025003 (2012).</li><li>Ma, K. Y., Chirarattananon, P., Fuller, S. B., Wood, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Flight+of+a+Biologically+Inspired,+Insect-Scale+Robot.">Controlled Flight of a Biologically Inspired, Insect-Scale Robot.</a> Science. 340 (6132), 603-607 (2013).</li></ol>

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 ...

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.

insect-machine-hybrid-system-remote-radio-control-freely-flying

Research

JoVE Journal

Neuroscience

12.1K Views.  Nanyang Technological University. 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,...