A.B. gratefully acknowledges the National Science Foundation for funding under Cyber Physical Systems program (1239243) and Division of Undergraduate Education (1245680); and the Defense Advanced Research Project Agency (DARPA) for supporting the earlier stages of this work. The earlier stages of this work was performed by A.B. in Prof. Amit Lal&#39;s laboratory at Cornell University. A.B. thanks Ayesa Sinha and Prof. Lal for experimental guidance and idea generation at that stage. Manduca sexta (Linnaeus 1763) were obtained from a colony maintained by the Department of Biology at Duke University, Durham, NC, USA. Moths were used within 5 days of eclosion. We would like to thank Triangle Biosystems International, especially David Juranas and Katy Millay for their excellent technical assistance and use of their Neuroware system. We also would like to thank Will Caffey for his help during experiments.

NOTE: The source of the materials and reagents required to follow the protocol is provided in the &#8220;Reagents&#8221; Table below.
1. Preparing Printed Circuit Boards (PCBs) for Recording Electrode Connection
NOTE: In order to provide a practical experimental procedure, wire electrodes are soldered to a PCB to insert these electrodes into an FFC (flexible flat cable) connector.
<ol>
	<li>Cut a 0.5x5 cm2 piece of copper clad laminate.</li>
	<li>Using ...

<ol>
	<li>Taubes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologists+and+engineers+create+a+new+generation+of+robotics+that+imitate+life.">Biologists and engineers create a new generation of robotics that imitate life.</a> Science. 288 (7), 80-83 (2000).</li><li>Duch, C., Bayline, R. J., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+development+of+the+dorsal+longitudinal+flight+muscle+and+its+innervation+in+Manduca+sexta.">Postembryonic development of the dorsal longitudinal flight muscle and its innervation in Manduca sexta.</a> Journal of Comparative Neurology. 422 (1), 1-17 (2000).</li><li>Levine, R. B., Morton, D. B., Restifo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+the+insect+nervous+system.">Remodeling of the insect nervous system.</a> Current opinion in neurobiology. 5 (1), 28-35 (1995).</li><li>Williams, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+insect+diapause:+the+role+of+the+brain+in+the+production+and+termination+of+pupal+dormancy+in+the+giant+silkworm+Platysamia+cecropia.">Physiology of insect diapause: the role of the brain in the production and termination of pupal dormancy in the giant silkworm Platysamia cecropia.</a> Bio. Bull. 90, 234-243 (1946).</li><li>Williams, C. 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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=In+vivo+electrochemical+characterization+of+a+tissue&#8211;electrode+interface+during+metamorphic+growth.">In vivo electrochemical characterization of a tissue&#8211;electrode interface during metamorphic growth.</a> IEEE Transactions on Biomedical Engineering. 58 (8), 2401-2406 (2011).</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=Insect&#8211;machine+interface+based+neurocybernetics.">Insect&#8211;machine interface based neurocybernetics.</a> IEEE Transactions on Biomedical Engineering. 56 (6), 1727-1733 (2009).</li><li>Chapman, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Insects: Structure and Function. , (1998).</li><li>Eaton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+the+head+and+thorax+of+the+adult+tobacco+hornworm,+Manduca+sexta+(Lepidoptera:Sphingidae).+I.+Skeleton+and+muscles.">Morphology of the head and thorax of the adult tobacco hornworm, Manduca sexta (Lepidoptera:Sphingidae). I. Skeleton and muscles.</a> Annals of the Entomological Society of America. 64, 437-445 (1971).</li><li>Resier, M. B., 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=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> Journal of Neuroscience Methods. 167 (2), 127-139 (2008).</li><li>Dombeck, D. A., Reiser, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+neuroscience+in+virtual+worlds.">Real neuroscience in virtual worlds.</a> Current opinion in neurobiology. 22 (1), 3-10 (2011).</li><li>Weir, P. T., 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=Flying+drosophila+orient+to+sky+polarization.">Flying drosophila orient to sky polarization.</a> Current Biology. 22 (1), 21-27 (2012).</li><li>Ristroph, L., Bergou, 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=Discovering+the+flight+autostabilizer+of+fruit+flies+by+inducing+aerial+stumbles.">Discovering the flight autostabilizer of fruit flies by inducing aerial stumbles.</a> Proceedings of the National Academy of Sciences. 107 (11), 4820-4824 (2010).</li><li>Strauss, R., Schuster, S., G&#246;tz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+artificial+visual+feedback+in+the+walking+fruit+fly+Drosophila+melanogaster.">Processing of artificial visual feedback in the walking fruit fly Drosophila melanogaster.</a> The Journal of experimental biology. 20 (9), 1281-1296 (1997).</li><li>Lindemann, J., Kern, R., Michaelis, C., Meyer, P., van Hateren, J., Egelhaaf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FliMax,+a+novel+stimulus+device+for+panoramic+and+highspeed+presentation+of+behaviourally+generated+optic+flow.">FliMax, a novel stimulus device for panoramic and highspeed presentation of behaviourally generated optic flow.</a> Vision Research. 43 (7), 779-791 (2003).</li><li>Reiser, M. B., 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=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> Journal of neuroscience methods. 167 (2), 127-139 (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 Transactions on Biomedical Engineering. 56 (9), 2304-2307 (2009).</li></ol>

There are several critical steps during the surgical insertion of the recording electrodes that affect the ability to record data in the later steps of the protocol. The recording electrodes should be inserted into the pupa one day after exhibiting wing spots on its dorsal side. If the insertion is performed two or more days after this time, the insect&#8217;s tissue will not have enough time to develop around and stabilize the inserted electrodes. This could lead to movement of the implanted electrodes and unreliable re...

Inserting electrodes, even with attached electronic systems to insects for telemetric recording applications, has been a major method to understand how neural systems function during natural flight 1. Attaching or implanting artificial systems in insects has posed many challenges involving the potential to disturb the natural flight of the insect. Superficial attachment or surgical insertion of artificial platforms on the adult insect is unreliable due to possible shifting of the inserted devices caused by body-induced inertial and stress forces. Superficially attached or surgically inserted electrodes are also prone to be rejected by the insects as a foreign body. Furthermore, the implantation operation requires the removal of scales and piles around the exoskeleton. The thick cuticle layer also needs to be penetrated for surgical innervations which could cause collateral tissue damage, thereby interfering with the natural flight of the insect. All these factors can make a surgical or superficial implantation operation a challenging and delicate task. In order to alleviate these concerns involved in externally attaching control and sensing systems to the insects, a novel methodology involving metamorphic growth will be described in this article.
The metamorphic development of holometabolic insects starts with the transformation of the larva (or nymph) into an adult with an intermediate pupal stage (Figure 1). The metamorphosis process involves an extensive tissue reprogramming including degeneration followed by remodeling. This transformation turns a terrestrial larva to an adult insect demonstrating several complex behaviors2,3.
The survival of insects after extreme parabiotic surgeries has been demonstrated where the surgeries were performed during the early metamorphic stages4,5. In these surgeries, the developmental histogenesis caused surgical wounds to be repaired in shorter durations. Following these observations, a new technique has been developed where the implantation of electrically conductive electrodes was performed during the earlier stages of metamorphic growth (Figure 1). This enables a biomechanically secure attachment on the insect6. A highly reliable interface is also secured with the insect&#8217;s neural and neuromuscular systems7. This technique is known as &#8220;Early Metamorphosis Insertion Technology&#8221; (EMIT) 8.
After the rebuilding of the entire tissue system, structures inserted in the pupa emerge with the adult insect. Flight muscle groups make up to 65% of the total thoracic body mass and, thus, is a relatively convenient target for the EMIT procedure9. During the basic wing beat, the changes in the morphology of the flight powering dorsolongitudinal (dl) and the dorsoventral (dv) muscles cause the wing articulation geometry to generate lift10. Therefore the functional coordination of dl and dv muscles has been an active research topic under flight neurophysiology. Tethering insects in electronically programmed visual environments has been the most common method for studying the neurophysiology of complex locomotory behaviors11,12. Cylindrical arenas composed of light emitting diode panels have been used for these virtual-reality environments, where flying insects are tethered in the middle and the motion is simulated by dynamically updating the surrounding panoramic visual display. In the case of smaller insects, such as fruit fly Drosophila, tethering is achieved by attaching a metal pin to the dorsal thorax of the insect and placing the pin under a permanent magnet13,14. This method only allows quantification of motor responses through visual observations with high speed cameras without any electrophysiological analysis. Moreover, this method has been inefficient to suspend the larger and heavier body of Manduca sexta. To solve this problem, we benefitted from magnetically levitating frames where light weight frames with magnets attached to their bottom are levitated through electromagnetic forces. When combined with commercially available neural amplifiers and LED arrays, this provides a platform to control flight-motor output and record the related electrophysiology of Manduca sexta.

<table border="0" cellpadding="0" cellspacing="0" width="579">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Coated stainless steel wire</td>
			<td style="width:113px;">A-M Systems</td>
			<td style="width:137px;">791900</td>
			<td style="width:135px;">0.008&#8217;&#8217; bare, 0.011&#8217;&#8217; coated, annealed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Flexible electrode wire</td>
			<td style="width:113px;"></td>
			<td style="width:137px;"></td>
			<td style="width:135px;">Litz or inductor wire can be used.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Surface-mount FFC connector</td>
			<td style="width:113px;">Hirose Connector</td>
			<td style="width:137px;">FH28E-20S-0.5SH(05)</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Tweezers</td>
			<td style="width:113px;">Grobet USA</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Clean with 70% alcohol before use on the insect.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Kim-Wipes</td>
			<td style="width:113px;">Kimberly-Clark Worldwide</td>
			<td style="width:137px;">34155</td>
			<td style="width:135px;">Any size delicate-wipe tissues can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Teflon tape</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">5 mm width Teflon tape.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Hypodermic Needle</td>
			<td style="width:113px;">Becton Dickinson &#38; Co.</td>
			<td style="width:137px;">30511</td>
			<td style="width:135px;">20-30 gauge hypodermic needle can be used. Video showed 30 gauge.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Rigid Fixation Stick</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Variety of materials can be used (e.g. coffee stirrers)</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:195px;">Insect Emergence Cage</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Plastic pet cage lined with packing paper or similar padding. Ventilation holes are needed.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Thermal Cauterizer</td>
			<td style="width:113px;">Advanced Meditech International</td>
			<td style="width:137px;">CH-HI CT2103 (tip)</td>
			<td style="width:135px;">Optional equipment used for application of dental wax.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Dental Wax</td>
			<td style="width:113px;">Orthomechanics LC., Broken Arrow, Oklahoma</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Optional material used for stabilizing the electrodes on the insect.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Magnetic Levitation Platform</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Custom designed frame fabricated in-house with 3D prototyping</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">CA40 Instant Adhesive</td>
			<td style="width:113px;">3M</td>
			<td style="width:137px;">62-3803-0330-5</td>
			<td style="width:135px;">Avoid skin contact. Use gloves when handling.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">70% Isopropyl alcohol</td>
			<td style="width:113px;">store brand</td>
			<td style="width:137px;"></td>
			<td style="width:135px;">Commercially available from many suppliers.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">PCB Etchant</td>
			<td style="width:113px;">RadioShack</td>
			<td style="width:137px;">276-1535</td>
			<td style="width:135px;">Toxic if swallowed or ingested, skin irritant&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;"></td>
			<td style="width:113px;"></td>
			<td style="width:137px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td style="width:113px;"></td>
			<td style="width:137px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Name of Reagent/Material</td>
			<td style="width:113px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:195px;">TBSI Neuroware Software</td>
			<td style="width:113px;">Triangle Biosystems International</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">NeuroWare v1.4. Software for importing .nex files obtained at Technologies, N. NeuroExplorer Code and Scripts. (2012).at http://www.neuroexplorer.com/code.html</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">TBSI Wireless Recording System</td>
			<td style="width:113px;">Triangle Biosystems International</td>
			<td style="width:137px;">W5 FI</td>
			<td style="width:135px;">USB Base station, headstage unit, charger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">16 Channel Amplifier A-C Amplifier</td>
			<td style="width:113px;">A-M Systems&#160;</td>
			<td style="width:137px;">950000</td>
			<td style="width:135px;">Model 3500 (110 V)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Oscilloscope&#160;</td>
			<td style="width:113px;">Agilent Technologies&#160;</td>
			<td style="width:137px;">DSO1014A</td>
			<td style="width:135px;">Oscilloscope, 100 MHz, 4 channel</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:195px;">Microscope</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">5x magnification microscope to assist visualization during electronics construction.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Ultrasonic Cleaner</td>
			<td style="width:113px;">ColeParmer</td>
			<td style="width:137px;">EW-08848-10</td>
			<td style="width:135px;">Ultrasonic Cleaner with Timer,&#160;</td>
		</tr>
	</tbody>
</table>

early metamorphic insertion technology for insect flight behavior monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

A schematic of the overall EMIT procedure is presented in Figure&#160;1, showing the major stages in the hawkmoth&#8217;s metamorphic cycle and the corresponding electrode insertion steps. The electrode insertion should be performed in the late pupal stage 4 to 7 days before eclosion. This allows the muscle fibers to develop around the electrodes and secure the implant in the insect.
The typical result of a completed late pupal stage insertion where the two active electrodes a...

Watch this Scientific Journal Video about Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring at JoVE.com

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

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14.4K Views.  North Carolina State University. We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.