We thank Shigeru Matsuyama for providing purified bombykol. This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant numbers 22700197 and 24650090) and the Human Frontier Science Program (HFSP).

1. Experimental Animal
<ol>
	<li>Prepare a plastic box to keep the pupae of male silkmoths (B. mori) until their eclosion. Put paper towels at the bottom and pieces of cardboard around the inner wall of the box (Figure 1A). 
	Note: The pieces of cardboard are necessary for the adult moths to hold while extending their wings during eclosion (Figure 1A).</li>
	<li>Put male silkmoth (Bombyx mori) pupae in the box and keep them in an incubator until eclosion under a 16-hr:8-hr light:dark cycle at 25 &#176;C. 
	NOTE: The male and female pupae can be discriminated by the sex markings on the abdomen (Figure 1B).</li>
	<li>Collect adult male moths after eclosion and move them into a new box.</li>
	<li>Keep the adult moths in an incubator under a 16-hr:8-hr light:dark cycle and decrease the temperature to 15 &#176;C to reduce their activity before the experiment.</li>
</ol>
2. Tethering a Silkmoth
<ol>
	<li>Fabrication of an attachment for tethering (Figure 2A) 
	Note: The attachment consists of a copper wire with a strip of a thin plastic sheet at its tip. This ensures the dorsal-ventral movement of the thorax during walking (Figure 2B).
	<ol>
		<li>Prepare a strip of a thin plastic sheet, 2 &#215; 40 mm (thickness: 0.1 mm), and fold it in the middle.</li>
		<li>Attach the folded strip to the tip of a copper wire with an adhesive.</li>
		<li>Bend the tip of the folded strip where the thorax of a silkmoth is attached.</li>
	</ol>
	</li>
	<li>Use adult moths (2-8 days old) during the light period for the experiment. 
	Note: The sensitivity to the pheromone strongly depends on the circadian clock18. Because B. mori is a diurnal moth, the experiment must be performed during the light period.</li>
	<li>Gently remove all scales on the dorsal thorax (mesonotum) using a piece of wet tissue (or cotton swab) and expose the cuticle of the mesonotum (Figure 2C).</li>
	<li>Paste an adhesive on the strip of plastic on the attachment and on the surface of the exposed mesonotum with a small flat-blade screwdriver and wait 5-10 min until the adhesive is no longer sticky. 
	Note: The adhesive should not touch the wing hinge or the forewing tegulae (Figure 2C).</li>
	<li>Bond the mesonotum to the attachment.</li>
	<li>Keep the moth tethered before placing it inside the cockpit of the robot. Hold the attachment on a stand and put a piece of paper under the legs to rest the moth.</li>
</ol>
3. Insect-controlled Robot
<ol>
	<li>Design the hardware of the insect-controlled robot based on previous works16,17,19. 
	Note: The insect-controlled robot consists of an air-supported treadmill with an optical mouse sensor to capture the insect locomotion, custom-built AVR-based microcontroller boards for processing and motor control, and two DC brushless motors (Figures 3 and 4). The robot can run on the basis of the ball rotation with 96% precision or higher, within a time delay of 200 msec. It also ensures the mobility of maximum forward speed (24.8 mm/sec) and angular velocity (96.3&#176;/sec) of the silkmoth during pheromone tracking behavior16. The airflow of the treadmill (Figure 5A) and odor delivery system (Figure 5B) are designed for the onboard moth to walk smoothly on the ball and to acquire an odor by two antennae. The air intake and flow channel of the treadmill is separated from those of the odor delivery system to avoid contamination of the pheromone.</li>
	<li>Design the software for the onboard microcontrollers based on previous works16. 
	Note: The onboard microcontroller calculates the robot movements from the insect locomotion measured with an optical sensor (rotational, &#916;x; translational, &#916;y; Figure 6). The travel distance (&#916;L) and turn angle (&#916;&#952;) per unit time of the robot are calculated on the basis of travel distance of each wheel (left, &#916;LL; right, &#916;LR) such as &#916;L = (&#916;LL + &#916;LR) / 2 and &#916;&#952; = (&#916;LL - &#916;LR) / Dwheel, where Dwheel is the distance between the two wheels (120 mm). &#916;LL and &#916;LR are further described as &#916;LL = &#916;Lx,L + &#916;Ly,L and &#916;LR = &#916;Lx,R + &#916;Ly,R, where &#916;Lx,L and &#916;Lx,R are the travel distances of the wheels on the left and right sides controlled by &#916;x, and &#916;Ly,L and &#916;Ly,R are those controlled by &#916;y. Ideally, &#916;Lx,L and &#916;Lx,R are described as &#916;Lx,L = -&#916;Lx,R = G &#916;x (Dwheel / Dball), and &#916;Ly,L and &#916;Ly,R are described as &#916;Ly,L = &#916;Ly,R = G &#916;y, where G is the motor gain and Dball is the diameter of the ball (50 mm). In practice, the motor gain is independently set by each side (left or right wheel) and by each direction (forward or backward rotation) so as to calibrate the robot movement. The independent gains further allow for the setting of asymmetrical motor rotation to generate a turning bias of the robot (see step 6.1).</li>
	<li>Wash the surface of a white expanded polystyrene ball (mass: approximately 2 g; diameter: 50 mm) with water to remove any possible olfactory or visual cues. 
	Note: The surface of a new ball should be roughed with fine-grit sandpaper, such as P400, which ensures the grip of the legs on the ball.</li>
	<li>Turn on the blower fan that supplies air at 9 V to the treadmill and floats the ball (Figure 5A). Observe the ball float approximately 2 mm from the bottom of the cup.</li>
	<li>Using a screw, attach the copper wire of the attachment with the moth (see step 2) to a fixture in the cockpit of the robot (see Figure 3 inset). Make sure that the position of the middle legs is at the center of the ball (Figure 7A).</li>
	<li>Adjust the vertical position of the attachment to enable the moth to walk normally on the ball. Keep the ball at the same height before and after attaching the moth (Figure 7B). 
	Note: A too-low position of the attachment adds pressure on the moth and elicits backward walking to resist the pressure (Figure 7C), whereas a too-high position causes unstable walking and failures of the sensor due to changes in the vertical position of the ball (Figure 7D). To check normal walking behavior, a single-puffed pheromone stimulus is used to trigger walking in the moth (for the pheromone stimulus, see step 4). Note that the test stimulus must be minimal because previous exposure to bombykol habituates silkmoths and decreases their sensitivity (Matsuyama and Kanzaki, unpublished data).</li>
</ol>
4. Odor Source Preparation
Note: Male B. mori are sensitive to the major component of the conspecific female sex pheromone (bombykol: (E,Z)-10,12-hexadecadien-1-ol)20. Any contamination of experimental equipment with bombykol elicits the odor-tracking behavior and affects the responsiveness of the moth.
<ol>
	<li>Drop 10 &#181;l of the bombykol solution dissolved in n-hexane (200 ng/&#181;l) on a piece of filter paper (approximately 10 mm &#215; 10 mm). The amount of bombykol per piece of filter paper is 2,000 ng. 
	Note: To check the normal walking behavior of the moth, prepare a pheromone stimulus cartridge in this step. The cartridge is a glass Pasteur pipette with one piece of filter paper containing 2,000 ng of bombykol. Pushing a bulb puffs the air containing bombykol.</li>
</ol>
5. Odor Source Localization Experiment
<ol>
	<li>Turn on the fan of a pulling-air-type wind tunnel (1,800 &#215; 900 &#215; 300 mm, L &#215; W &#215; H; Figure 8) and set the wind speed to 0.7 m/sec. Ensure that the temperature is more than 20 &#176;C.</li>
	<li>Set the odor source (the piece of filter paper containing bombykol) upstream of the wind tunnel. 
	Note: The plume width should be confirmed prior to the experiment by using TiCl417,19.</li>
	<li>Turn on the microcontroller board of the robot and establish a serial connection to a PC via Bluetooth.</li>
	<li>Launch a custom-made Java program called &#34;BioSignal,&#34; which provides an interface between the PC and the robot. 
	Note: The main window includes buttons for sending commands to the robot, text windows for displaying the input and output of the serial communication, and small boxes to configure parameters. The subsequent commands are sent by clicking corresponding buttons in this program, except for video capturing.</li>
	<li>Click on the &#34;about device&#34; button to confirm the connection by sending a command to the robot via the specified COM port and check that a message is returned by the robot.</li>
	<li>Click on the &#34;memory erase&#34; button to erase previous locomotion data left on the onboard flash memory.</li>
	<li>Click on the &#34;drivemode1&#34; button to send the default motor gains to the robot. 
	Note: The manipulations of the motor gains and the time delay between insect locomotion and robot movement are applied after this step (see steps 6.1 and 6.3, Figure 9).</li>
	<li>Click on the &#34;don&#39;t drive&#34; button to send a command to immobilize the robot until the experiment starts.</li>
	<li>Put the robot at a start position (600 mm downstream from the odor source) and turn on the switch of the motor driver board.</li>
	<li>Push the recording button of the camcorder to start video capture.</li>
	<li>Click on the &#34;rec start&#34; button to send a start command to initiate the robot with a simultaneous recording of the ball rotation on the onboard flash memory. Observe that the robot starts to move and tracks the odor plume.</li>
	<li>Click on the &#34;rec stop&#34; and &#34;don&#39;t drive&#34; buttons to send commands to stop both the robot movement and the recording if the robot localizes the odor source.</li>
	<li>Push the recording button of the camcorder to stop video capture.</li>
	<li>Download recorded locomotion data from the onboard flash memory to the computer via a serial connection. Close the program.</li>
</ol>
6. Manipulation of the Insect-controlled Robot
Note: The timing of each manipulation is indicated in Figure 9.
<ol>
	<li>Manipulation of motor gains 
	Note: This manipulation alters the translational and rotational velocity of the robot. Asymmetrical motor gains generate a turning bias, which can be used to investigate how insects compensate for the bias17.
	<ol>
		<li>Define the rotational gains for forward and backward rotation of the motor on each side17 (Figure 6B) by editing the configuration file named &#34;param2.txt&#34; using a text editor.</li>
		<li>Click on the &#34;set param2&#34; to read the edited configuration file in the software program. Then, click on the &#34;drivemode2&#34; to send the manipulated gains to the robot.</li>
	</ol>
	</li>
	<li>Inversion of the motor output 
	Note: This manipulation provides a condition similar to the inversion of bilateral olfactory input (see step 6.4) and can be used to investigate the significance of bilateral olfaction. However, the inversion of motor output also inverts self-induced visual motion of an onboard moth. The impact of the inverted self-induced visual input can be evaluated by a comparison with the inverted olfactory input19.
	<ol>
		<li>Invert the bilateral motor control by crossing the control cables for each motor.</li>
	</ol>
	</li>
	<li>Manipulation of the time delay between insect locomotion and robot movement. 
	Note: This manipulation allows for the investigation of the acceptable period of time spent on sensory-motor processing for the robotic odor-tracking. The microcontroller stores the locomotion data on a buffer memory and then processes it after the specified time delay. Note that the robot has a maximal internal time delay of 200 msec; therefore, the actual time delay is expected to be the specified time delay plus 200 msec16,17.
	<ol>
		<li>Input a number (from 0-10) in a small box of the main window to specify a time delay from 0-1,000 msec at 100-msec steps.</li>
		<li>Click on the &#34;set delay&#34; button to apply the time delay.</li>
	</ol>
	</li>
	<li>Manipulation of the olfactory input. 
	Note: This manipulation can be used to investigate the significance of bilateral olfactory input. The surge direction of silkmoths is biased on the higher-concentration side22.
	<ol>
		<li>Change the gap between the suction tube tips or invert their positions to alter the difference in odor concentration acquired by each antenna.</li>
	</ol>
	</li>
	<li>Manipulation of visual input 
	Note: This manipulation is to investigate the role of visual input for odor-tracking.
	<ol>
		<li>Cover the canopy with a white paper that occludes 105&#176; and 90&#176; of the horizontal and vertical visual field of the onboard moth, respectively.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The most important points for the successful control of the robot by a silkmoth are letting the moth walk smoothly on the air-supported ball and the stably measuring the ball rotation. Therefore, tethering the silkmoth and mounting it on the ball at the appropriate position are the critical steps in this protocol. Inappropriate adhesion of the moth to the attachment or inappropriate positioning of the moth on the ball will cause unnatural pressure on it, which perturbs its normal walking behavior and/or causes a failure ...

Autonomous robots capable of finding an odor source can be important for the safety and security of society. They can be used for the detection of disaster victims, of drugs or explosive materials at an airport, and of hazardous material spills or leaks in the environment. At present, we rely entirely on well-trained animals (e.g., dogs) for these tasks, and robotic odor source localization has been strongly expected to relieve the workload of these animals. Finding an odor source is a challenging task for robots because odorants are distributed intermittently in an atmosphere1; therefore, continuous sampling of the odor concentration gradient is not always possible. Thus, a search strategy using intermittent odor cues is necessary for the achievement of robotic odor source localization2-4.
Odor source localization is essential for organism survival and includes tasks such as finding food, mating partners, and sites for oviposition. To overcome the difficulty in tracking patchy distributed odorants, organisms have evolved various behavioral strategies consisting of two fundamental behaviors: moving upstream during odor reception and cross-stream during cessation of odor reception5,6. These reactive strategies have been well-documented in insects and further combined with other modalities, such as wind direction and vision5-8. The insect behavioral models have also been useful examples for robotics3,9-11, in which behavioral algorithms or neural circuit models are implemented into mobile robots for the evaluation of odor source localization abilities10,12-15. From biomimetic perspectives, this "bottom-up" approach is certainly a fundamental way to develop biomimetic robots. However, the bottom-up approach is not a shortcut to obtaining a useful search strategy, because biological analyses are still ongoing, and the modeling of the sensory-motor systems behind insect behaviors has not been completed. Therefore, it is still unknown how such a biological system actually works as a controller of a robotic platform.
In this article, we demonstrate the protocol of a straightforward "top-down" approach to develop an odor-tracking mobile robot controlled by a biological system16,17. The robot is controlled by a real insect and can be regarded as a prototype of future insect-mimetic robots. In the robot's cockpit, a tethered adult male silkmoth (Bombyx mori) walked on an air-supported ball in response to the female sex pheromone, which was delivered to each antenna through air suction tubes. The ball rotations caused by the walking of the onboard moth were measured by an optical sensor and were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can investigate how the insect sensory-motor system works on the robotic platform where a pilot insect is in a closed loop between the robot and a real odor circumstance. The manipulation of the robotic hardware alters the closed loop; therefore, the insect-controlled robot is a useful platform for both engineers and biologists. For engineering, the robot represents the first steps of applying a biological model to meet the requirements for robotic tasks. For biology, the robot is an experimental platform for studying sensory-motor control under a closed loop.

<table><tbody><tr><td>Male adult silkmoth (&lt;em&gt;Bombyx mori&lt;/em&gt;)</td><td></td><td></td><td>Rear from eggs, or purchase as pupae.</td></tr><tr><td>Incubator</td><td>Panasonic</td><td>MIR-254</td><td>Store pupae or adult silkmoths at a constant temperature, 238 L.</td></tr><tr><td>Plastic box</td><td>Sunplatec</td><td>O-3</td><td>Store pupae or adult silkmoths, 299 &amp;times; 224 &amp;times; 62 mm L &amp;times; W &amp;times; H.</td></tr><tr><td>Copper wire</td><td></td><td></td><td>2-mm diameter for the attachment. Any rigid bar can be used as an alternative for making the attachment to tether a silkmoth.&amp;nbsp;</td></tr><tr><td>Plastic sheet</td><td>Kokuyo</td><td>VF-1420N</td><td>Sold as overhead projector film with thickness of 0.1 mm. Use at the tip of the attachment.</td></tr><tr><td>Forceps</td><td>As one</td><td>5SA</td><td>Remove scales on the thorax.</td></tr><tr><td>Adhesive</td><td>Konishi</td><td>G17</td><td>Bond a silkmoth to the attachment.</td></tr><tr><td>Insect-controlled robot</td><td>Custom</td><td></td><td>Bearing an air-supported treadmill, an optical sensor, custom-built AVR-based microcontroller boards, and two DC brushless motors. It is powered by 8 &amp;times; AA and 3 &amp;times; 006P batteries.</td></tr><tr><td>Microcontroller</td><td>Atmel</td><td>ATMEGA8</td><td>A component of the insect-controlled robot.</td></tr><tr><td>DC blower</td><td>Nidec</td><td>A34342-55</td><td>A component of the insect-controlled robot for floating a ball in an air-supported treadmill.&amp;nbsp;</td></tr><tr><td>DC fan</td><td>Minebea</td><td>1606KL-04W-B50</td><td>A component of the insect-controlled robot for suctioning air containing an odor.</td></tr><tr><td>Optical mouse sensor</td><td>Agilent technologies</td><td>HDNS-2000</td><td>A component of the insect-controlled robot, obtained from an optical mouse (M-GUWSRSV, Elecom, Japan).</td></tr><tr><td>Brushless motor</td><td>Maxon</td><td>EC-45</td><td>A component of the insect-controlled robot for driving a wheel.</td></tr><tr><td>White polystyrene ball</td><td></td><td></td><td>A component of the insect-controlled robot. Diameter 50 mm, mass approximately 2 g.</td></tr><tr><td>Bombykol:&lt;br/&gt;(E,Z)-10,12-hexadecadien-1-ol</td><td>Shin-Etsu chemical</td><td></td><td>Custom synthesis.</td></tr><tr><td>n-hexane</td><td>Wako</td><td>085-00416</td><td>Solvent for bombykol.</td></tr><tr><td>Wind tunnel</td><td>Custom</td><td></td><td>Pulling-air type, sized 1,800 &amp;times; 900 &amp;times; 300 mm L &amp;times; W &amp;times; H.</td></tr><tr><td>BioSignal program</td><td>Custom</td><td></td><td>A program to establish serial communication between the insect-controlled robot and a PC via Bluetooth. Used for sending commands to start/stop the robot or configuring its motor properties.&amp;nbsp;</td></tr><tr><td>Camcorder</td><td>Sony</td><td>HDR-XR520V</td><td>Capture robot movements.</td></tr></tbody></table>

insect-controlled robot: a mobile robot platform to evaluate the odor-tracking capability of an insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

We present here the basic characteristics of the insect-controlled robot required for the successful localization of an odor source. The comparison between the robot and silkmoths, the effectiveness of the odor delivery system, and the significance of accurate bilateral olfactory and visual inputs are examined.
The comparison of odor-tracking behaviors between freely-walking moths and the insect-controlled robot is shown in <str...

Watch this Scientific Journal Video about Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect at JoVE.com

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

insect-controlled-robot-mobile-robot-platform-to-evaluate-odor

Research

JoVE Journal

Neuroscience

14.6K Views.  The University of Tokyo.  De mogelijkheid een geurbron lokaliseren is noodzakelijk voor insecten overleven en zal gelden voor geurstof-volgen is. Het insect bestuurbare robot wordt aangedreven door een feitelijke silkmoth en stelt ons in staat om de geur volgen vermogen van insecten te evalueren door middel van een robot platform. 

Video: Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

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 ...

Behavior

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their applicability in environmental and security monitoring and search-and-rescue operations. Small drones with commercial metal-oxide-semiconductor (MOX) gas sensors have been developed for odor source localization; however, their real-time-odor-detection performance has proven inadequate. However, biosensing technologies based on insect olfactory systems exhibit relatively high sensitivity, selectivity, and real-time response with respect to odorant molecules compared to commercial MOX gas sensors. In such devices, excised insect antennae function as portable odorant biosensor elements and have been found to deliver excellent sensing performance. This study presents experimental protocols for odorant-molecule detection in the air using a small autonomous bio-hybrid drone based on a mountable electroantennography (EAG) device incorporating silkmoth antennae.
We developed a mountable EAG device including sensing/processing parts with a Wi-Fi module. The device was equipped with a simple sensor enclosure to enhance the sensor directivity. Thus, odor source localization was conducted using the spiral-surge algorithm, which does not assume an upwind direction. The experimental bio-hybrid odor-detecting drone identified real-time odorant-concentration differences in a pseudo-open environment (outside a wind tunnel) and localized the source. The developed drone and associated system can serve as an efficient odorant molecule-detection tool and a suitable flight platform for developing odor source localization algorithms owing to its high programmability.

This study introduces experimental protocols for a bio-hybrid odor-detecting drone based on silkmoth antennae. The operation of an experimental electroantennogram device with silkmoth antennae is presented, in addition to the structure of a bio-hybrid drone designed for odor source localization using the spiral-surge algorithm.

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their ...

Engineering

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

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