M. Tasin was supported by the Swedish Research Council for Sustainable Development (Formas, Grant 2013-934).

1. Preparing Glass Tubes
<ol>
	<li>Prepare the glass tubes (e.g., 2.8 cm diameter, 13 cm long) and close one end with a plastic snap cap.</li>
	<li>Separate 10 insects into the capped glass tubes and cover the remaining end with gauze using a rubber band. Allow the insects to acclimatize to the temperature, light conditions and humidity of the wind tunnel room for at least 2 h. 
	NOTE: The number of insects inside each tube depends on the species and research question.</li>
</ol>
2. Preparing Odor Sources
<ol>
	<li>Sprayer protocol.
	<ol>
		<li>Fill the 1 mL gastight syringe with a diluted headspace collection or a synthetic odor mix. 
		NOTE: The content of the diluted headspace or synthetic odor mix depends on insect species and research question. In general, the concentrations used should correspond to the natural release rates of the authentic odor source.</li>
		<li>Connect the syringe tip to the FEP tubing using tubing adaptors and insert the syringe into the syringe pump.</li>
		<li>Start the syringe pump.</li>
		<li>Start the broadband ultrasonic generator. 
		NOTE: The release of the aerosol from the sprayer can be confirmed by pointing a torch light from below the release point.</li>
		<li>Run the sprayer with 96% ethanol for a minimum of 10 min between the treatments to clean the inside of the tubes and nozzle. Use a separate syringe, dedicated to pure ethanol, for all cleaning.</li>
		<li>Clean the syringes and the tip of the nozzle with 96% ethanol after use.</li>
	</ol>
	</li>
	<li>Authentic odor source protocol.
	<ol>
		<li>Insert the authentic odor sources into the wind tunnel or into a 2 L headspace collection jar.</li>
		<li>Connect the headspace collection jar to the laboratory air flow and release into the wind tunnel. 
		NOTE: Collect plant material as close to the start of the experiments as possible and prevent wilting by inserting the cut end into a small vial with water. The amount and type of plant material, or other authentic odor source, depends on insect species and research question.</li>
	</ol>
	</li>
	<li>Position the glass tube with the insect(s) onto a holder 180 cm downwind from the odor/visual source and 30 cm from the ground. The capped end should point upwind.</li>
</ol>
3. Starting the protocol
<ol>
	<li>Use two cameras to capture two different views. Mount the cameras above the flight arena and angled to capture two different views.</li>
	<li>Open the cap.</li>
	<li>Start the timer. 
	NOTE: Different species require different time frames to respond, e.g., the apple fruit moth (Argyresthia conjugella) will respond within 4 to 5 min11, but the grapewine moth (Lobesia botrana) require up to 20 min to respond18.</li>
	<li>Observe the flight pattern and pay special attention to the flight characteristics and upwind orientation. Score the flight performance according to predefined behavioral categories, e.g., take off, oriented flight over a short distance (minimum 20 cm), oriented flight over longer distance (&#60; 5 cm from the source) and landing. 
	NOTE: Filming and 3D tracking using two cameras can be used to give more detailed information on the flight characteristics. In general, the cameras are mounted above the flight arena and angled to capture two different views.</li>
	<li>Collect the insects landing on the walls of the wind tunnel, outside the odor plume, and replace back onto the holder. 
	NOTE: The insects can be given one chance to respond to odors with upwind flight and not be replaced.</li>
	<li>Change to clean glass hardware between the treatments. 
	NOTE: Frequent control treatments should be run to identify possible contamination sources.</li>
	<li>Euthanize the insects with CO2 or by freezing after the experiments. 
	NOTE: Depending on the research question, females can be scored for egg development or visually inspected to verify the condition of wings or antennae.</li>
</ol>
4. Cleaning
<ol>
	<li>Wash all metal and glass hardware with ethanol and water and leave until dry.</li>
	<li>Heat all metal and glass hardware to 300 &#176;C for 6 h to remove the contaminants.</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigational+strategies+used+by+insects+to+find+distant,+wind-borne+sources+of+odor.">Navigational strategies used by insects to find distant, wind-borne sources of odor.</a> Journal of Chemical Ecology. 34 (7), 854-866 (2008).</li><li>Baker, T. C., Fadamiro, H. Y., Cosse, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moth+uses+fine+tuning+for+odour+resolution.">Moth uses fine tuning for odour resolution.</a> Nature. 393 (6685), 530 (1998).</li><li>Bruce, T. J. A., Wadhams, L. J., Woodcock, C. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+odor+and+visual+stimulus+interaction+during+intratree+host+finding+behavior+of+Rhagoletis+pomonella+flies.">Host odor and visual stimulus interaction during intratree host finding behavior of Rhagoletis pomonella flies.</a> Journal of Chemical Ecology. 19 (11), 2671-2696 (1993).</li><li>Reeves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+should+not+be+overlooked+as+an+important+sensory+modality+for+finding+host+plants.">Vision should not be overlooked as an important sensory modality for finding host plants.</a> Environmental Entomology. 40 (4), 855-861 (2011).</li><li>Knudsen, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrepancy+in+laboratory+and+field+attraction+of+apple+fruit+moth+Argyresthia+conjugella+to+host+plant+volatiles.">Discrepancy in laboratory and field attraction of apple fruit moth Argyresthia conjugella to host plant volatiles.</a> Physiological Entomology. 33 (1), 1-6 (2008).</li><li>Aak, A., Knudsen, G. K., Soleng, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wind+tunnel+behavioural+response+and+field+trapping+of+the+blowfly+Calliphora+vicina.">Wind tunnel behavioural response and field trapping of the blowfly Calliphora vicina.</a> Medical and Veterinary Entomology. 24, 250-257 (2010).</li><li>Montgomery, M. E., Wargo, P. 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The wind tunnel is a helpful tool for identifying both attractive and repellent odors for many insects4,9. With sound knowledge of the ecology, biology and behaviour of the insect studied, its flight characteristics can be easily identified and the environmental conditions, wind speed, visual stimuli and odor application can be tailored to fit. It is recommended when starting out with a new species, to fine tune the wind tunnel parameters using the most attractiv...

The wind tunnel is an important tool in insect chemical ecology studies that allow laboratory testing of insect flight responses to semiochemicals. By releasing odors into a controlled wind stream, the insects&#39; behavioural response to these stimuli can be directly monitored by studying their upwind flight towards the source. Olfaction is the most important sensory mechanism by which many insects interact with their biotic environment1. Insects use odor cues to find suitable partners for mating. Similarly, they use odor bouquets from host resources to find food for themselves, or the offspring. Plants release floral odors in combination with nectar and pollen rewards to secure insect pollination efficiency. All these volatile cues diffuse passively into the environment and insects need to identify and interpret their individual relevance. As volatiles are released into the environment, the molecules travel with the wind as filaments, retaining the initial concentration for long distances downwind, before eventually being broken up and diluted by turbulence and diffusion2. Insects can detect minute changes in the volatile signal and direct their movement upwind, towards the source. Insects display a flight behavior with fast upwind surges when in contact with an attractive odor, and casting sideways upon the loss to relocate the odor plume3,4. The co-localized arrangement of olfactory neurons in the sensilla of the insect antennae can facilitate behavioral responses to the onset and loss of plume contact with remarkable high resolution5 and enable the insects to distinguish between similar odor molecules originating from different sources6. Visual feedback while in flight, termed optomotor anemotaxis, is fundamental to identify wind direction, objects and relative displacement2,7. By the use of sensory interaction and sophisticated behavior, insects can locate point sources in a three-dimensional environment.
The identification of insect attractants and repellents can have several important applied aspects. Sex pheromones (intraspecific signals) of many pest insects can be synthesized and released into the air to disrupt the mating behavior8. Both pheromones and kairomones (interspecific signals) can be used for mass trapping, attract and kill in monitoring traps to give direct information of pest status. Insect repellents, such as for mosquitoes9, can also be studied in wind tunnel bioassays. These methods play an important part of integrated pest management and decision support systems for farmers.
Wind tunnel bioassays, where the odor mediated behavior repertoire of a species can be monitored, is a powerful method to identify potential new tools for pest control to replace or reduce the impact of pesticide use.
The theoretical reasoning behind the wind tunnel design is thoroughly described10. Here, we describe the wind tunnel construction, odor application and flight behaviour that has been used in several experiments to determine the wind tunnel bioassay protocol. The wind tunnel (Figure 1) at Nibio (&#197;s, Norway) is constructed from scratch resistant transparent polycarbonate. The flight arena is 67 cm high, 88 cm wide and 200 cm long. In front of the flight arena, there is an additional polycarbonate section, 30 cm long. This part of the wind tunnel serves as a utility section for the application of odors. If the volatiles get into contact with the polycarbonate housing in the flight arena, they may later on be re-released and contaminate between sessions. On each end of the utility section, there is therefore a perforated metal grid. Both grids restrict the airflow and create a slight overpressure on the upwind side. This results in increased laminar flow on the downwind side. The upwind grid is made from a perforated metal plate with 8 mm holes evenly dispersed across the cross section of the tunnel to provide 54% open area. The downwind grid has holes of 3 mm and a 51% open area. This reduces the turbulence and ensures that the odor plume travel centrally down the length of the flight arena. The odor plume will have the shape of a narrow cone and can be visualized by the use of smoke. On the floor of the flight arena, plastic or paper circles of varying sizes (from 5 to 15 cm in diameter) are laid out to give insects visual feedback during the flight. There is a 25 by 50 cm access door on the upwind end of the flight arena and in the utility section. Between the downwind end of the flight arena and the exhaust filter section, there is a 60 cm open area for insect handling. This access area is covered on the sides with a 0.8 mm meshed fabric to prevent the insects escaping into the room.
Air is drawn into the first filter housing by a fan. The air passes through a dust filter before it is purified by 24 high capacity active charcoal filters and released in the tunnel. The air exiting the tunnel is passed through a similar filter housing before being released back into the room. It might be beneficial to exhaust the air to the outside of the building through a fume hood. The fans on both filter housings are run with equal flow. Both fans have a continuous dimmer switch and are calibrated to different wind speeds using a flowmeter. The air speed is dependent on the species tested. 30 cm s-1 is often a good starting point. For small insects, the ideal air speed may be reduced, and for strong flyers, the airspeed can be higher to increase the relative flight distance.
The wind tunnel room facilitates the control of temperature, humidity and light intensity. LED strips are placed behind a 3 mm opaque poly(methyl methacrylate) pane to create a diffuse light source above and behind the flight arena. Both light sources can be controlled independently.
Odor application can be achieved by several means. Generally, odors are released into the airflow in the center of the upwind end of the flight arena. Depending on the research questions at hand, the release point can be exposed or covered. A glass cylinder (10 cm diameter, 12.5 cm long) with a metal mesh (2 &#215; 2 mm mesh size) on the downwind side can visually block the odor source and at the same time serve as a landing platform for insects. In many experiments, a horizontal glass platform can be used for presenting odor sources, or visual signals close to the release point. There is also the opportunity to release two odors at the same time, side by side, to facilitate choice assays. The release points are then placed 20 cm apart and the odor plumes overlap from halfway down the tunnel. The choice can then be identified by which plume the insect is following upwind.
The wind tunnel design facilitates numerous volatile release methods. For example, a specific odor can be released in front of a background odor such as emitted by a crop plant11,12. Also, different visual stimuli can be tested13,14. The experimental setup must be adapted to each species and research question.
Natural odor sources, such as plant parts and synthetic odors from dispensers can be introduced directly into the flight arena. To isolate odor mediated behaviors from visual, the odor source can be covered, or the volatiles carried into the flight arena via a charcoal filtered laboratory air supply from the outside. The odor source is then confined to a glass jar and the air is pushed through the jar into the wind tunnel via Teflon tubes and glass pipes. The airspeed at the release point should match the wind speed in the arena.
To release odors at specific blend ratios, a sprayer can be used. The sprayer is an ultrasonic nozzle with a conical tip and an inserted microbore to facilitate a liquid flow at 10 &#181;L min-1. The nozzle is connected to a broadband ultrasonic generator and operates at 120 kHz. A syringe pump is pushing the odor sample into the sprayer nozzle. Fluorinated ethylene propylene (FEP) tubing with 0.12 mm inner diameter is connecting the 1 mL gastight syringe and the nozzle. Tubing adaptors that swell in ethanol and shrink in air, facilitate tight fitting with no internal volume. The aerosol droplet size generated from the vibration of the nozzle is frequency dependent and depends on the specific solvent used. The small droplets evaporate and are brought down the wind tunnel as volatiles. Other sprayer designs also exist and a cheaper version utilizing a piezo driven glass capillary provides a similar solution15.
Synthetic mixes or headspace collections can be used with the sprayer. The samples are diluted with pure ethanol to the desired concentrations. With volatile collections, the sample can be diluted to correspond to the collection time. This means that a volatile collection sampled over 3 h should be diluted to 1800 &#181;L, which at a release rate from the sprayer at 10 &#181;L min-1 corresponds to 3 h.
The identification of the flight behavior can be done directly by manual observation or by post hoc video analysis. The oriented flight should be distinguished from random flight. Odor mediated behavior can be recognized by the following characteristics: zig-zag flight across the odor plume, straight upwind flight when inside the plume, and looping back if the contact with the plume is lost. Upon the loss of an attractive plume, the insects can also start to zig-zag with increasing arches to reconnect to the lost plume3,4. This behaviour is fundamental in a field setting where the insects following an attractive odor need to cope with turbulence and shifting wind directions. The flight pattern is not uniform and varies across the insect orders. As an example, strong flyers such as blowflies have a faster upwind orientation with wider casting pattern than moths, and the wind speed should be increased to facilitate a longer relative flight path.
The flight of an insect can also be filmed. With a single camera, simple flight characteristics can be described by plotting the x y coordinates16. By using two cameras with synchronized frame capture, the 3D flight can be reconstructed using an external software17. The flight track can then be analyzed to give information about the flight speed and distance, the flight angles with respect to wind direction and the details about the flight characteristics in relation to the odor plume. There are both custom and commercial equipment and software available which enable automatic frame by frame tracking. The calibration frames should be used to reference real world space, and rectilinear wide-angle lenses should be used to minimize lens distortion. Care should be taken to reduce visual background noise, such as edges and corners in the wind tunnel arena, and to maximize insect to background discrimination. By using an infrared light source, the reflection (e.g., from nocturnal mosquitoes) can be filmed with monochrome CCD cameras17.

<table><tbody><tr><td>Flight arena</td><td>any</td><td>NA</td><td>Construct to fit the filter housing</td></tr><tr><td>Filter housing x 2</td><td>Camfill Farr</td><td></td><td>Contains the dust and charcoal filters</td></tr><tr><td>Fan x 2</td><td>Fischbach</td><td>Model D640/E35</td><td>Silent fan with continous dimmer switch</td></tr><tr><td>Perforated grids</td><td>any</td><td>NA</td><td>Two different open areas are needed, e.g. 54 and 51%</td></tr><tr><td>Flowmeter</td><td>Swema air</td><td>Swema air 300</td><td>Identifying the wind speed</td></tr><tr><td>Ultrasonic sprayer</td><td>SonoTek</td><td></td><td>Sprayer nozzle with conical tip and inserted microbore</td></tr><tr><td>Broadband ultrasonic generator</td><td>SonoTek</td><td></td><td>Function generator</td></tr><tr><td>Syringe pump</td><td>CMA microdialysis</td><td>CMA 102</td><td>Liquid delivery</td></tr><tr><td>FEP tubing</td><td>CMA microdialysis</td><td></td><td>0.12 mm inner diameter</td></tr><tr><td>Tubing adaptors</td><td>CMA microdialysis</td><td></td><td>Connectors for zero internal volume</td></tr><tr><td>Gastight syringe</td><td>any</td><td>NA</td><td>1000 &amp;micro;L syringe for headspace collections and synthetic blends</td></tr><tr><td>Gastight syringe</td><td>any&amp;nbsp;</td><td>NA</td><td>1000 &amp;micro;L syringe for cleaning sprayer</td></tr><tr><td>Torch</td><td>any</td><td>NA</td><td>Small light source for checking sprayer release</td></tr><tr><td>Timer</td><td>any</td><td>NA</td><td>Timer with alarm function&amp;nbsp;</td></tr><tr><td>Holder for insect release</td><td>any</td><td>NA</td><td>Metal construction</td></tr><tr><td>Lighting</td><td>any</td><td>NA</td><td>LED is preferable due to low heat production</td></tr><tr><td>Moisturiser</td><td>any</td><td>NA</td><td>Size depends on volume of wind tunnel room</td></tr><tr><td>Temperature control</td><td>any</td><td>NA</td><td>Temperture range depends on species</td></tr><tr><td>Glass tubes</td><td>any</td><td>NA</td><td>Tubes (2.8 cm diameter, 13 cm long) for&amp;nbsp; insects</td></tr><tr><td>Snap cap</td><td>any</td><td>NA</td><td>Snap cap that fits the glass tube</td></tr><tr><td>Gauze</td><td>any</td><td>NA</td><td>Fabric to close the glass tube</td></tr><tr><td>Rubber band</td><td>any</td><td>NA</td><td>To hold gauze in place</td></tr><tr><td>Glass cylinder</td><td>any</td><td>NA</td><td>Cylinder for odour containment and landing platform (10 cm diameter, 12.5 cm long)</td></tr><tr><td>Glass jars</td><td>any</td><td>NA</td><td>Glass jars for dynamic headspace collection</td></tr><tr><td>Connectors and tubes</td><td>any</td><td>NA</td><td>Tubes and connectors depends on type of glass jars</td></tr><tr><td>Air supply</td><td>any</td><td>NA</td><td>From laboratory air or bottles</td></tr><tr><td>Charcoal filters</td><td>any</td><td>NA</td><td>For cleaning the outside air sypply</td></tr><tr><td>Vial</td><td>any</td><td>NA</td><td>Small vial with water to keep plant material fresh</td></tr><tr><td>Oven</td><td>any</td><td>NA</td><td>Heat metal and glassware to 300 degrees to decontaminate</td></tr></tbody></table>

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.

Blowflies responds strongly to odors from dead animals which represents an ephemeral larval growth substrate19,20. Using dead mice as a natural odor source, we investigated the details of the flight behavior of 15 day old, mated female C. vicina, with or without, a visual stimuli next to the odor release point13. To eliminate the natural visual cue, we used the glass jar system described above. Wit...

Watch this Scientific Journal Video about A Wind Tunnel for Odor Mediated Insect Behavioural Assays at JoVE.com

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

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 ...

a-wind-tunnel-for-odor-mediated-insect-behavioural-assays

Research

JoVE Journal

Behavior

11.6K Views.  Norwegian Institute of Bioeconomy Research (Nibio). 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.

Video: A Wind Tunnel for Odor Mediated Insect Behavioural Assays

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 ...

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, ...

Neuroscience

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

Designing a Flexible, Cost-Effective Olfactometer for Rodent Olfactory Experiments

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These sophisticated devices allow researchers to precisely control and deliver odor stimuli, enabling the investigation of complex olfactory processes in rodents. Although commercially available olfactometers are convenient, they present challenges when technical issues arise, often requiring costly assistance and potentially disrupting research timelines. This article details the construction of a custom olfactometer specifically designed for mouse olfactory behavior experiments, providing a comprehensive list of parts and step-by-step instructions. The olfactometer is controlled through MATLAB, offering a user-friendly interface for researchers. Importantly, the open-source code allows users to modify and adapt the system, tailoring behavioral tasks to meet specific experimental needs. Building a customized olfactometer empowers users with the knowledge and capability to perform custom experimental design and troubleshooting independently, saving both time and resources. This approach not only enhances research flexibility but also fosters a deeper understanding of the equipment's functionality, ultimately leading to more robust and reliable olfactory studies in rodents.

Constructing an Olfactometer for Rodent Olfactory Behavior Studies Near-Infrared Spectroscopy Hyperscanning Study in Psychological Counseling

This protocol describes the construction of an olfactometer for go/no-go olfactory behavior experiments. Step-by-step instructions, along with images, are provided to ensure the successful construction of the olfactometer. Information for troubleshooting issues encountered during the process is also included.

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These ...

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 ...

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.

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 ...

Four-Way Olfactometer Assay: A Method to Assess Odorant-Cued Responses in Drosophila

Source: Lin, C.C., et al. <a href="https://www.jove.com/video/54346/" target="_blank">Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer</a>. J. Vis. Exp. (2016).
Fruit flies have a complex olfactory system, can discriminate hundreds of odorants, and use their sense of smell to direct behavioral decisions. This video describes the four-way olfactometer assay, a method to study olfactory behavioral responses in flies. The featured protocol clip shows how to handle test flies and what are critical steps while performing the assay.

Czterokierunkowy test olfaktometryczny: metoda oceny reakcji zapachowych u Drosophila

Source: Lin, C.C., et al. Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer. J. Vis. Exp. (2016).
Fruit flies ...

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

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Rozdziały w tym wideo

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

Visually Mediated Odor Tracking During Flight in Drosophila

Vertical T-maze Choice Assay for Arthropod Response to Odorants

A Single-fly Assay for Foraging Behavior in Drosophila

Testing Drosophila Olfaction with a Y-maze Assay

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

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

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

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

Czterokierunkowy test olfaktometryczny: metoda oceny reakcji zapachowych u Drosophila