Name: a wind tunnel for odor mediated insect behavioural assays
Uploaded: 2018-11-30T13:30:00
Description: Watch this Scientific Journal Video about A Wind Tunnel for Odor Mediated Insect Behavioural Assays at JoVE.com

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. ...

<ol>
	<li>Hansson, B. S., 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=.">.</a> Insect olfaction. , (1999).</li><li>Murlis, J., Elkinton, J. S., Card&#233;, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+Plumes+and+How+Insects+Use+Them.">Odor Plumes and How Insects Use Them.</a> Annual Review of Entomology. 37, 505-532 (1992).</li><li>Todd, J. L., Baker, T., Hansson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+3.">Ch. 3.</a> Insect olfaction. , 67-96 (1999).</li><li>Carde, R. T., Willis, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+host+location:+a+volatile+situation.">Insect host location: a volatile situation.</a> Trends in Plant Science. 10 (6), 269-274 (2005).</li><li>Srinivasan, M. V., Zhang, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+motor+computations+in+insects.">Visual motor computations in insects.</a> Annual Review of Neuroscience. 27, 679-696 (2004).</li><li>Rhainds, M., Kettela, E. G., Silk, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty-five+years+of+pheromone-based+mating+disruption+studies+with+Choristoneura+fumiferana+(Clemens)+(Lepidoptera:+Tortricidae).">Thirty-five years of pheromone-based mating disruption studies with Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae).</a> Canadian Entomologist. 144 (3), 379-395 (2012).</li><li>Sharpington, P. J., Healy, T. P., Copland, M. J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wind+tunnel+bioassay+system+for+screening+mosquito+repellents.">A wind tunnel bioassay system for screening mosquito repellents.</a> Journal of the American Mosquito Control Association. 16 (3), 234-240 (2000).</li><li>Baker, T. C., Linn, C. E., Hummel, H. E., Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in pheromone research. , 75-110 (1984).</li><li>Knudsen, G. K., Tasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spotting+the+invaders:+A+monitoring+system+based+on+plant+volatiles+to+forecast+apple+fruit+moth+attacks+in+apple+orchards.">Spotting the invaders: A monitoring system based on plant volatiles to forecast apple fruit moth attacks in apple orchards.</a> Basic and Applied Ecology. 16 (4), 354-364 (2015).</li><li>Knudsen, G. K., Norli, H. R., Tasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ratio+between+field+attractive+and+background+volatiles+encodes+host-plant+recognition+in+a+specialist+moth.">The ratio between field attractive and background volatiles encodes host-plant recognition in a specialist moth.</a> Frontiers in Plant Science. 8, (2017).</li><li>Aak, A., Knudsen, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+olfaction-mediated+visual+acuity+in+blowflies+and+its+consequences+for+gender-specific+trapping.">Sex differences in olfaction-mediated visual acuity in blowflies and its consequences for gender-specific trapping.</a> Entomologia Experimentalis et Applicata. 139, 25-34 (2011).</li><li>Th&#246;ming, G., Norli, H. R., Saucke, H., Knudsen, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pea+plant+volatiles+guide+host+location+behaviour+in+the+pea+moth.">Pea plant volatiles guide host location behaviour in the pea moth.</a> Arthropod-Plant Interactions. 8 (2), 109-122 (2014).</li><li>El-Sayed, A., Godde, J., Arn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sprayer+for+quantitative+application+of+odor+stimuli.">Sprayer for quantitative application of odor stimuli.</a> Environmental Entomology. 28 (6), 947-953 (1999).</li><li>Haynes, K. F., Baker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+anemotactic+flight+in+female+moths+stimulated+by+host+odour+and+comparison+with+the+males'+response+to+sex+pheromone.">An analysis of anemotactic flight in female moths stimulated by host odour and comparison with the males' response to sex pheromone.</a> Physiological Entomology. 14 (3), 279-289 (1989).</li><li>Spitzen, J., Takken, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+mosquitoes:+A+review+of+tools+to+track,+record+and+analyse+mosquito+flight.">Keeping track of mosquitoes: A review of tools to track, record and analyse mosquito flight.</a> Parasites and Vectors. 11 (1), (2018).</li><li>Masante-Roca, I., Anton, S., Delbac, L., Dufour, M. -. C., Gadenne, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attraction+of+the+grapevine+moth+to+host+and+non-host+plant+parts+in+the+wind+tunnel:+effects+of+plant+phenology,+sex,+and+mating+status.">Attraction of the grapevine moth to host and non-host plant parts in the wind tunnel: effects of plant phenology, sex, and mating status.</a> Entomologia Experimentalis et Applicata. 122 (3), 239-245 (2007).</li><li>Johansen, H., 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=Blow+fly+responses+to+semiochemicals+produced+by+decaying+carcasses.">Blow fly responses to semiochemicals produced by decaying carcasses.</a> Medical and Veterinary Entomology. 28, 9 (2014).</li><li>Paczkowski, S., Maibaum, F., Paczkowska, M., Schutz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decaying+Mouse+Volatiles+Perceived+by+Calliphora+vicina+Rob.-Desv.">Decaying Mouse Volatiles Perceived by Calliphora vicina Rob.-Desv.</a> Journal of Forensic Sciences. 57 (6), 1497-1506 (2012).</li><li>Aluja, M., Prokopy, R. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+and+other+host-derived+volatiles+as+attractants+to+beetles+that+bore+into+hardwoods.">Ethanol and other host-derived volatiles as attractants to beetles that bore into hardwoods.</a> Journal of Chemical Ecology. 9 (2), 181-190 (1983).</li><li>Skals, N., Anderson, P., Kanneworff, M., L&#246;fstedt, C., Surlykke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Her+odours+make+him+deaf:+Crossmodal+modulation+of+olfaction+and+hearing+in+a+male+moth.">Her odours make him deaf: Crossmodal modulation of olfaction and hearing in a male moth.</a> Journal of Experimental Biology. 208 (4), 595-601 (2005).</li><li>Willis, M. A., Avondet, J. L., Zheng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vision+in+odor-plume+tracking+by+walking+and+flying+insects.">The role of vision in odor-plume tracking by walking and flying insects.</a> Journal of Experimental Biology. 214 (24), 4121-4132 (2011).</li><li>Martel, J. W., Alford, A. R., Dickens, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+and+greenhouse+evaluation+of+a+synthetic+host+volatile+attractant+for+Colorado+potato+beetle,+Leptinotarsa+decemlineata+(Say).">Laboratory and greenhouse evaluation of a synthetic host volatile attractant for Colorado potato beetle, Leptinotarsa decemlineata (Say).</a> Agricultural and Forest Entomology. 7 (1), 71-78 (2005).</li><li>Salvagnin, U., 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=Adjusting+the+scent+ratio:+using+genetically+modified+Vitis+vinifera+plants+to+manipulate+European+grapevine+moth+behaviour.">Adjusting the scent ratio: using genetically modified Vitis vinifera plants to manipulate European grapevine moth behaviour.</a> Plant Biotechnology Journal. 16 (1), 264-271 (2018).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flight arena</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td style="width:489px;">Construct to fit the filter housing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter housing x 2</td>
			<td style="width:152px;">Camfill Farr</td>
			<td style="width:119px;"></td>
			<td>Contains the dust and charcoal filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fan x 2</td>
			<td style="width:152px;">Fischbach</td>
			<td style="width:119px;">Model D640/E35</td>
			<td>Silent fan with continous dimmer switch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Perforated grids</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Two different open areas are needed, e.g. 54 and 51%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flowmeter</td>
			<td style="width:152px;">Swema air</td>
			<td style="width:119px;">Swema air 300</td>
			<td>Identifying the wind speed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrasonic sprayer</td>
			<td style="width:152px;">SonoTek</td>
			<td style="width:119px;"></td>
			<td>Sprayer nozzle with conical tip and inserted microbore</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Broadband ultrasonic generator</td>
			<td style="width:152px;">SonoTek</td>
			<td style="width:119px;"></td>
			<td>Function generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pump</td>
			<td style="width:152px;">CMA microdialysis</td>
			<td style="width:119px;">CMA 102</td>
			<td>Liquid delivery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FEP tubing</td>
			<td style="width:152px;">CMA microdialysis</td>
			<td style="width:119px;"></td>
			<td>0.12 mm inner diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing adaptors</td>
			<td style="width:152px;">CMA microdialysis</td>
			<td style="width:119px;"></td>
			<td>Connectors for zero internal volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gastight syringe</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>1000 &#181;L syringe for headspace collections and synthetic blends</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gastight syringe</td>
			<td style="width:152px;">any&#160;</td>
			<td style="width:119px;">NA</td>
			<td>1000 &#181;L syringe for cleaning sprayer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Torch</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Small light source for checking sprayer release</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Timer</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Timer with alarm function&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Holder for insect release</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Metal construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lighting</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>LED is preferable due to low heat production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moisturiser</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Size depends on volume of wind tunnel room</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Temperature control</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Temperture range depends on species</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass tubes</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Tubes (2.8 cm diameter, 13 cm long) for&#160; insects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Snap cap</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Snap cap that fits the glass tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gauze</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Fabric to close the glass tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rubber band</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>To hold gauze in place</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass cylinder</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Cylinder for odour containment and landing platform (10 cm diameter, 12.5 cm long)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass jars</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Glass jars for dynamic headspace collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Connectors and tubes</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Tubes and connectors depends on type of glass jars</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Air supply</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>From laboratory air or bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Charcoal filters</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>For cleaning the outside air sypply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vial</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">NA</td>
			<td>Small vial with water to keep plant material fresh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oven</td>
			<td style="width:152px;">any</td>
			<td style="width:119px;">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 ...

This method can answer key questions in the field of chemical ecology, such as identifying behaviorally active components. The main advantages of this technique is that we can release odors by many different means, and we can monitor the whole behavioral repertoire of the insect. Prepare the glass tubes with gauze, using a rubber band.Separate 10 insects into the prepared glass tubes, and cover the remaining end with plastic snap caps. Allow the insects to acclimatize to the temperature, light conditions, and humidity of the wind tunnel room for at least two hours. Here the sprayer protocol is demonstrated to prepare odor sources.Fill the one-milliliter, gastight syringe with a synthetic odor mix, and insert the syringe into the syringe pump. Connect the syringe tip to the fluorinated ethylene propylene tubing using tubing adapters. Now, start the syringe pump, followed by the broadband ultrasonic generator.The release of the aerosol from the sprayer can be confirmed by point a light from below the release point. Position the glass tube with the insects onto a holder 180 centimeters downwind from the odor visual source, and 30 centimeters from the ground. The capped end should point upwind.To start the protocol, open the cap and start the timer. Observe the flight pattern, and pay special attention to the flight characteristics and upwind orientation. Shown here, is an oriented flight without landing.Oriented flight with landing is shown here, on a natural odor source, which is included as a control. A closeup of a take off can be seen here, as well as a closeup of a landing on a natural odor source. Score the flight performance according to predefined behavior categories, such as take off, oriented flight over a short distance, oriented flight over a longer distance, and landing.Collect the insects landing on the walls of the wind tunnel, outside the odor plume, and replace them back onto the holder. Use a separate syringe, dedicated to pure ethanol, for all cleaning. Run the sprayer with 96%ethanol for a minimum of 10 minutes between the treatments, to clean the inside of the tubes and nozzle.Clean the syringes and the tip of the nozzle with 96%ethanol, after use. Upon completion, wash all metal and glass hardware with ethanol and water, and leave until dry. Heat all metal and glass hardware to 300 degrees Celsius for six hours, to remove any contaminants.Visually guided odor responses of blow flies are shown here. Details of flight behavior with and without a visual stimuli were studied. There was a significant effect of visual cue on landing response, but not on the other behavioral steps.A comparison of landing responses of pea moths to pea plants and the corresponding volatile collections are presented here. Similar landings were recorded towards both types of cues. A synthetic blend of pea flower volatiles resulted in slightly lower landings, as compared to natural flower volatiles.Here is shown the effect of plant volatile backgrounds on apple fruit moth response to synthetic blends. Odor dispensers with a complex blend and a simple blend resulted in similar upwind attraction, with a clean background. However, when embedded in an apple volatile background, the moths preferred the complex blend.When attempting this procedure, it's important to remember that the flight pattern is not uniform. It varies a lot across insect orders. Blow flies are strong fliers and have a wider casting movements, compared to the moths.The wind tunnel facilitates numerous methods for releasing odors into the wind tunnel, depending on the question at hand. We can release from odor dispensers, from authentic sources, synthetic mixes, and headspace collections. Also, a specific odor can be released on top of a background odor, we can use different visual stimuli, and we can perform two-choice bioassays.Wind tunnel experiments are important tools in identifying behaviorally active compounds and chemicals, both in applied research, as well as in basic research. A good example is if you want to develop tools for pest control.

Research

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Behavior

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