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 a fine tip marker, draw three 0.1x5cm2 rectangle pads as etching mask patterns.</li>
	<li>Etch the exposed laminate using a PCB etchant inside a ventilated area or fume hood. Cover about 1 cm of the laminate cutout&#8217;s length with non-reactive tape. Fill a graduated beaker with at least 100 ml of PCB etchant and tape the copper laminate cutout to the inside of the graduated beaker with scotch tape. Half of the copper laminate cutout should be submerged in the PCB etchant.</li>
	<li>Place the beaker on a rotating platform for 20 min.</li>
	<li>Remove the cutout from the etchant and place it in a beaker filled with water for 10 min.</li>
	<li>Using a tissue paper, apply isopropyl alcohol and remove the markings to expose the non-etched copper pads.</li>
	<li>Cut the printed circuit board into smaller squares approximately 1 cm long.</li>
	<li>Cut two pieces of coated, annealed, stainless steel wire (0.11&#8221; coated, 0.008&#8221; bare) using a sharp blade to lengths of 3 cm each. These pieces of stainless steel wire are the active electrodes that will be inserted into the thorax of the insect.</li>
	<li>Using a blade, remove 4-5 mm of the plastic coating from each end of each wire. Use of a microscope is recommended.</li>
	<li>Cut one 0.7 cm piece of insulated stainless steel wire to create a tip extension for the ground electrode. Gently remove the coating with a blade or melt it with the heat of a soldering iron as performed in step 1.9.</li>
	<li>For the ground connection, cut one piece of flexible (litz or inductor) wire to a length of 4.5 cm.</li>
	<li>Solder the 0.7 cm piece of stainless steel prepared in step 1.10 to the ground connection wire prepared in step 1.11. An exposed stainless steel tip should be at the end of the ground connection.</li>
	<li>Tape the prepared electrode board firmly to the soldering workspace using a non-reactive tape. Use the tape to mask all but 1-2 mm of the pads on the board where the electrodes will be soldered. This masked, solder-free end of the pads will be inserted into the FFC connector described in step 4.1.</li>
	<li>Align the three electrode wires such that one end of each can be soldered to the corresponding pads on the electrode board. Apply stainless steel flux across the electrode pads for easier soldering.</li>
	<li>Solder each of the exposed electrodes on the pads.</li>
	<li>Immerse the electrodes in acetone and isopropyl alcohol for 10 min each to clean the solder residues. Use of an ultrasonic bath improves the cleaning performance.</li>
</ol>
2. Surgical Insertion to the Manduca Sexta Pupae
NOTE: The insects will be most active during the transitions between day and night. Therefore, an artificial day/night cycle should be established within an insect chamber using automatic outlet timers. These should be set to simulate a 7 hr dark and 17 hr light cycle.
<ol>
	<li>Examine the Manduca sexta pupae daily to determine an appropriate insertion time. The pupae are ready for insertion approximately one day after the wings exhibit dark spots.</li>
	<li>To anesthetize the pupae, place them in the refrigerator (4C) for around 6 hr.</li>
	<li>Prepare the insertion workspace. The workspace should include isopropyl alcohol, sharp tweezers, blades, and a 30 G hypodermic needle. As an option, cyanoacrylate adhesive may be used to enhance the electrode fixation.</li>
	<li>Sterilize the needle, tweezers, and the electrodes by dipping them into or wiping with isopropyl alcohol.</li>
	<li>Remove the pupa from the refrigerator and transfer it to the workspace.</li>
	<li>Determine the location on the thorax that corresponds to the muscle group of interest. The focus of the work in this example is the dorsoventral muscles responsible for wing upstroke movement.</li>
	<li>Using a sharp blade, gently scratch a 1x1 cm2 rectangle through the exocutical layer. Using the tweezers, slowly peel off these pieces.</li>
	<li>(Optional) Use a vacuum to remove wing hair from the exposed region of the thorax.</li>
	<li>Slowly insert the needle about 5mm into the mesothorax where the wings attach to the thorax to create two insertion points targeting the muscle group.</li>
	<li>Using tweezers, guide the two recording electrodes into the two insertion points.</li>
	<li>(Optional) To enhance the mechanical durability, clean the hair around the electrodes and generously apply cyanoacrylate adhesive around each insertion point on the thorax with a wire applicator.</li>
	<li>Prepare a cage for emergence with proper material (rough and textured) covering the walls and ceiling so that the insect may climb upon emergence. Perforated cardboard boxes or packing paper may be used.</li>
	<li>Prepare a rigid fixation stick with around 6 cm length and 2 mm diameter. Plastic stirrers, a cotton swab, or metal wires can be used for this step.</li>
	<li>Carefully slide this stick through the hole underneath the protruding proboscis.</li>
	<li>Fix both sides of the stick on the cage surface such that the pupa cannot roll around. Position the pupa inside of the cage such that the mesothorax is facing up. Extensive movement may cause damage to the electrode, loss of hemolymph, or render the insertion useless.</li>
</ol>
3. Inserting the Ground Electrode into Manduca Sexta
NOTE: The ground (reference) electrode should be inserted into the abdomen or distal parts of the thorax to avoid signal coupling. This insertion can be done either during the later stages of the pupal development or after the insect emerges. The window for the ground electrode has to be prepared in the pupal stage for either a pupal or adult stage ground electrode insertion.
<ol>
	<li>For pupal stage insertion: after peeling of the mesothoracic cuticle around the active electrode (see step 2.7), scratch another rectangle through the exocutical layer (around 0.5x0.5 cm2) on the dorsal abdomen close to the thorax using the 30 G hypodermic needle. Insert the ground electrode into this window using the technique described in section 2.</li>
	<li>For adult stage ground electrode insertion: once the insect has emerged, place it in the refrigerator at 4 &#176;C for 6 to 24 hr to immobilize. 
	The remaining steps are the same for both pupal and adult stage insertions.</li>
	<li>Prepare the insertion workspace including isopropyl alcohol, sharp tweezers, a 30 G hypodermic needle, cyanoacrylate adhesive, a piece of wire for application of glue, a thermal cauterizer (optional), and a dental wax stick (optional).</li>
	<li>Locate an insertion point approximately 1-2 cm away from the recording electrodes along the posterior abdomen.</li>
	<li>Slowly insert the needle to puncture the abdomen and provide an insertion site.</li>
	<li>Using tweezers carefully insert the ground electrode into the insertion site and apply pressure until it is 3-4 mm deep. Hold the electrode in place and use a wire to apply glue around the insertion site.</li>
	<li>(Optional) To enhance the mechanical strength, use the thermal cauterizer and collect a small (2-3 mm) bead of wax at the tip. Place the tip close to the insertion site and apply heat such that the wax surrounds the electrode and holds it firmly in place.</li>
</ol>
4. Preparation of the Adapter Board
NOTE: An adapter board is required to connect the electrode board to the wireless recording headstage through an FFC (Flat Flexible Cable) connector. For this, a board similar to the electrode board needs to be prepared by following steps 1.1 to 1.7.
<ol>
	<li>Solder a FFC connector to one end of the prepared board.</li>
	<li>Solder three 30 AWG (American Wire Gauge) hook up wires to three pads on the other end.</li>
	<li>Solder three mini connectors to the three pads on the adapter board for oscilloscope readings as described in the next stage.</li>
	<li>Solder the other end of these three wires to the headstage connector.</li>
	<li>Secure the headstage circuit board on top of the levitation frame.</li>
</ol>
5. Prerecording with Oscilloscope (Optional)
NOTE: In order to assess the reliability of the electrodes and observe the signal to noise ratio, tethered oscilloscope recordings can be obtained before deploying the wireless recording system. The mini wire connectors on the adapter board should be used for this.
<ol>
	<li>Connect the oscilloscope to an extracellular neural recording amplifier. Set the amplifier parameters to a high-pass cut-off frequency of 1 Hz, a low-pass cut-off frequency of 20 kHz, and a gain of 100.</li>
	<li>Connect each of the female mini wire connectors on the adapter board to the amplifier input channels.</li>
	<li>Remove the insect with the implanted electrode board from the cage when it is in an active state (during its dawn time). Place a piece of tissue paper under the insect for it to rest on before measurements are taken.</li>
	<li>Using tweezers, slide the electrode board into the FFC receptor on the adapter board. Observe a flat and low voltage baseline when the insect is resting and the generation of electromyogram (EMG) spikes as the insect flaps its wings. 
	NOTE: Refer to Section 6: Observing insect flight with the Wireless Recording System for representative oscilloscope results.</li>
	<li>Adjust the viewing parameters of the oscilloscope as needed. Capture the data on the oscilloscope and save the data.</li>
</ol>
6. Observing Insect Flight with the Wireless Recording System
NOTE: An electromagnetic levitation platform can be built for wireless recording of EMG signals during tethered Manduca sexta flight. The levitation platform consists of a frame designed to balance a tethering mechanism. The levitation allows the frame, and therefore the insect, to yaw during testing without the constraint of tethering wires. The frame can be rapid-prototyped using a fused deposition modeling (FDM) machine. A magnet needs to be attached to the bottom of this frame to be levitated by a series of magnets in the base platform. The insect is connected to the FFC connector suspended from the top of the frame. This levitating platform is located inside of the LED Arena which was constructed using 60 panels composed of an array of 5x7 individual LEDs. This system was based on established methods for developing an environment for visual stimulation of fruit flies15,16,17. The arena is controlled by a microcontroller allowing simulation of both clockwise and counterclockwise rotation as well as control of the rotational speed.
<ol>
	<li>Set up the wireless recording system by connecting the headstage to the adapter board connector on the levitation platform.</li>
	<li>Remove the insect from the cage when it is in an active state preferably during its dawn time.</li>
	<li>Using tweezers, carefully insert the electrode board into the FFC receptor on the levitating frame such that the insect is suspended firmly within the setup.</li>
	<li>Place the magnetic wand near the magnetic switch on the headstage to activate wireless data transmission. A blue light will come on indicating that the headstage is active.</li>
	<li>Turn off the lights in the room for complete darkness. A red lamp can be used to add lighting to the room. Open the telemetry data collection software on a computer and select the appropriate preloaded configuration file if provided. Start the data acquisition to begin viewing signals.</li>
	<li>Select the relevant user interface for observation of EMG signals on the wireless recording system to ensure a reliable wireless connection and electrode operation.</li>
	<li>Turn on all the LED Arena components: Regulated DC power supply and microcontroller. The microcontroller can adjust the rotations per minute of the cyclic light pattern and can also control the direction of the light rotation.</li>
	<li>Slowly balance the levitation platform within the arena. Align the frame above the center of the levitation base carefully, otherwise the frame will be pulled quickly to the ground possibly injuring the insect.</li>
	<li>Initiate the video recording system.</li>
	<li>Select the relevant recording tab of the software interface. Designate the recording time and file save destination. Choose the appropriate output settings to save the data. Click the start button to initiate a recording session within the software. This will save the data file which can be imported into numerical computing environments.</li>
	<li>Observe as the insect flies in the direction that corresponds with the movement of the LEDs. Reverse the direction of the LEDs and confirm that the insect reverses direction. Perform this as many times as desired.</li>
</ol>

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Authors have no conflict of interest in this study.

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>
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		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
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			<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

 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.

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

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.

Video: 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 ...

early-metamorphic-insertion-technology-for-insect-flight-behavior

Research

JoVE Journal

Behavior

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern United States. Some populations have developed resistance to management strategies including transgenic corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Knowledge of western corn rootworm dispersal is of critical importance for models of resistance evolution, spread, and mitigation. Flight behavior of an insect, especially over a long distance, is inherently difficult to observe and characterize. Flight mills provide a means to directly test developmental and physiological impacts and consequences of flight in the laboratory that cannot be obtained in field studies. In this study, flight mills were used to measure the timing of flight activity, total number of flights, and the distance, duration, and speed of flights taken by female rootworms during a 22-h test period. Sixteen flight mills were housed in an environmental chamber with programmable lighting, temperature, and humidity control. The flight mill described is of a typical design, where a flight arm is free to rotate about a central pivot. Rotation is caused by flight of an insect tethered to one end of the flight arm, and each rotation is recorded by a sensor with a time-stamp. Raw data are compiled by software, which are subsequently processed to provide summary statistics for flight parameters of interest. The most difficult task for any flight mill study is attachment of the tether to the insect with an adhesive, and the method used must be tailored to each species. The attachment must be strong enough to hold the insect in a rigid orientation and to prevent detachment during movement, while not interfering with natural wing motion during flight. The attachment process requires dexterity, finesse, and speed, making video footage of the process for rootworms of value.

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect&#8217;s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...

The Participant-Reported Implementation Update and Score (PRIUS): A Novel Method for Capturing Implementation-Related Data Over Time

"Implementation" of new initiatives in healthcare settings typically encompasses two distinct components: a "clinical intervention" plus accompanying "implementation strategies" that support putting the clinical intervention into day-to-day practice. A novel clinical intervention, for example, might consist of a new medication, a new protocol, a new device, or a new program. As clinical interventions are not self-implementing, however, they nearly always require effective implementation strategies in order to succeed. Implementation strategies set out to engage healthcare providers, staff and patients in ways that increase the likelihood of the new initiative being successfully adopted, a process that often involves behavior change and new ways of thinking by participants. One of the challenges in studying implementation is that it can be difficult to collect data about the status and progress of implementation, including participants' own perspectives and experiences concerning implementation to date. This protocol describes a novel method for collecting and analyzing data related to ongoing implementation called the Participant-Reported Implementation Update and Score, or PRIUS. The PRIUS method allows for the efficient and systematic capture of qualitative and quantitative data that can provide a detailed and nuanced account of implementation over time and from multiple viewpoints. This longitudinal method can enable researchers, as well as implementation leaders and organizational stakeholders, to monitor implementation progress more closely, conduct formative evaluation, identify improvement opportunities, and gauge the effect of any implementation changes on a rolling basis.

The Participant-Reported Implementation Update and Score PRIUS: A Novel Method for Capturing Implementation-Related Data Over Time

This protocol describes a novel method for collecting and analyzing data related to ongoing implementation called the Participant-Reported Implementation Update and Score (PRIUS). The PRIUS method allows for the efficient and systematic capture of data over time and from multiple viewpoints in healthcare settings.

"Implementation" of new initiatives in healthcare settings typically encompasses two distinct components: a "clinical intervention" plus accompanying ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&amp;#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...