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1Department of Electrical and Computer Engineering, North Carolina State University
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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.
Verderber, A., McKnight, M., Bozkurt, A. Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring. J. Vis. Exp. (89), e50901, doi:10.3791/50901 (2014).
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 “only” while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.
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’s neural and neuromuscular systems7. This technique is known as “Early Metamorphosis Insertion Technology” (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.
NOTE: The source of the materials and reagents required to follow the protocol is provided in the “Reagents” 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.
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.
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.
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.
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.
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.
A schematic of the overall EMIT procedure is presented in Figure 1, showing the major stages in the hawkmoth’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 and the ground electrodes have been inserted is shown in Figure 2.
The typical result of a completed adult stage insertion where the two active electrodes and the ground electrode have been inserted is shown in Figure 3.
The LED arena used to induce turning during flight for the Manduca sexta is shown in Figure 4. A microcontroller was programmed to allow control of the rotational speed of the LED array vertical pattern. The angular velocity of the LED pattern was set to 7.3 degrees per second. The magnetic levitation platform was placed in the center of the LED arena to allow the insect to freely turn in response to the LED array.
Figure 5 shows the muscle potential signal acquired from the dorsoventral muscles with the oscilloscope before and after wing flapping. The signal has been processed with 100 times amplification and a high-pass filter of 1 Hz and a low pass filter of 20 kHz. In the quiescent period, no muscle potentials are observed. The muscle potentials during wing flapping occur at approximately 15 Hz-20Hz.
Figure 6 shows the muscle potential signal acquired with the wireless instrumentation before and after wing flapping. In the quiescent period, no muscle potentials are observed. The muscle potentials during wing flapping occur at approximately 15Hz-20 Hz.
Figure 1. EMIT Procedure. A schematic diagram of the EMIT procedure performed on Manduca sexta, as described in the protocol.
Figure 2. Pupa Insertion. Photograph of a late stage pupa immediately after the recording electrodes were inserted using EMIT.
Figure 3. Moth Emergence. Photograph of an adult moth with implanted recording electrodes after eclosion.
Figure 4. Recording Setup. The magnetic levitation platform and LED arena used to record EMG signals from Manduca sexta flight muscles. Here a Manduca sexta is performing a yaw maneuver in response to the revolving LE pattern.
Figure 5. Oscilloscope EMG. A 2.5 sec EMG recording of a dorsoventral muscle using an amplifier and an oscilloscope.
Figure 6. Wireless EMG. A 1.9 sec EMG recording of the dorsoventral muscle using the wireless headstage recording unit and data acquisition software.
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’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 recordings in the adult stage.
It is important not to insert the recording electrodes into the pupal flight muscles at a depth of more than 5 mm. Otherwise, hemolymph will exit the insertion points and result in the development of weaker flight muscles. If hemolymph does emerge, stop the procedure and allow the pupa 24 hr to recover before attempting to insert the electrodes again. The insertion site should be cleaned thoroughly of all wing hair before the electrodes are inserted in the pupa. This prevents hair from entering the insertion holes and interfering with the electrode tissue interface.
To ensure optimal wing health in the adult moth, the insertion site should be re-cleaned of wing hair the day before eclosion using tweezers. In addition, it is recommended to use tweezers to loosen the edges of the cuticle window that was incised with the hypodermic needle to assist eclosion occurring on the following day. If any glue or hemolymph has dried near the edges of the cuticle window, the moth will not be able to inflate its wings after eclosion and this specimen will not be useful for experiments.
Although the insertion times are given in days, these may slightly vary as the timeline of the metamorphic development is a function of the rearing temperatures for poikilotherms. The provided days are for insects reared in RT If a standard 25°C insectary incubator is used, the development will be approximately 10-20% faster and the insertion times need to be adjusted accordingly.
A limitation of this study would be the rotational inertia introduced to the setup by the rapid prototyped ABS plastic levitation frame. The mass of the frame can be up to 200 grams while the mass of a moth is about 4 grams. The benefit of using an electromagnetically levitated frame is loss of frictional contact between the frame and a supporting structure. However, the use of a relatively heavy frame causes the insect to expend more energy to complete yaw maneuvers in response to the revolving LED pattern. A modification to the tethering frame used in this study could be the use of a less dense material and/or building a thinner frame to reduce the inertial loading.
The developmental changes during metamorphosis bring new capabilities to neural engineering methods to learn how insects fly It is a remarkable observation that the electrode insertion during the pupal stages results in alleviated tissue reactions with respect to the adult stage insertions. Therefore, EMIT based insertions ensure mechanical attachment of synthetic systems in or on an insect, while realizing a predictable neuromuscular interface with minimal short term effect on insect locomotory behavior. During the last two decades, robotists working on very small scale unmanned air vehicles have been inspired by insect flight. Beyond enabling a novel electrophysiological technique, EMIT procedure also allows for insect-machine-interfaces (IMI) that may provide access for neural engineers to the electrically excitable cells of the insect to control its sensory and behavioral physiology8. This has a potential to “biobotically” tame and control insect locomotion. Therefore, the specific methodology presented in this article is not only useful for studying the insect flight, but also for domesticating insects as hybrid centimeter-scale flying biobots18. An application of such a hybrid platform is to convert insects into mobile environmental sensing systems. These working animals can potentially assist humans in monitoring the co-shared ecosystems by gathering and storing environmental information.
Authors have no conflict of interest in this study.
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'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.
|Coated stainless steel wire||A-M Systems||791900||0.008’’ bare, 0.011’’ coated, annealed|
|Flexible electrode wire||Litz or inductor wire can be used.|
|Surface-mount FFC connector||Hirose Connector||FH28E-20S-0.5SH(05)|
|Tweezers||Grobet USA||Clean with 70% alcohol before use on the insect.|
|Kim-Wipes||Kimberly-Clark Worldwide||34155||Any size delicate-wipe tissues can be used.|
|Teflon tape||5 mm width Teflon tape.|
|Hypodermic Needle||Becton Dickinson & Co.||30511||20-30 G hypodermic needle can be used. Video showed 30 G.|
|Rigid fixation stick||Variety of materials can be used (e.g., coffee stirrers)|
|Insect emergence cage||Plastic pet cage lined with packing paper or similar padding. Ventilation holes are needed.|
|Thermal cauterizer||Advanced Meditech International||CH-HI CT2103 (tip)||Optional equipment used for application of dental wax.|
|Dental wax||Orthomechanics LC., Broken Arrow, Oklahoma||Optional material used for stabilizing the electrodes on the insect.|
|Magnetic levitation platform||Custom designed frame fabricated in-house with 3D prototyping.|
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