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>
	<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 attractive source possible. With kairomones, this is usually live host plant material or a natural food source and with pheromones, a caged calling female or male (depending on the insect studied). To identify the repellents, there is also a need for an attractive source on which to measure the antagonism9. These initial results will also serve as a baseline for further experimentation.
The wind tunnel dimensions should be considered to give room for free movement and display of innate flight characteristics. With strong flyers and large insects, large flight arenas may be necessary. For smaller insects a portion of the arena may be used. The wind speed may be specific to the insect species studied and should be adjusted to fit the flight capacity. With all odorants, but in particular with pheromones care should be taken to avoid contamination and frequent control treatments should be made to avoid false conclusions.
The activated charcoal filters will supply a blank canvas for the application of the desired odors. But there may be a flip side to clean laboratory conditions, because natural background odors may interact with the developed field dispensers11,23. Translating wind tunnel blends of ubiquitous volatiles to the field conditions may then be less straight forward and options to include background odors in the wind tunnel should be considered. With pheromones, which are distinct volatiles, the translation to field conditions are less of a problem. This also applies to volatiles from rare resources, such as cadaver kairomones utilized by Calliphorid flies24.
The sprayer is an excellent tool for releasing volatiles at known concentrations and blend ratios. The sprayer circumvents the problem of calculating release rates which quickly turns complicated. With sprayed headspace, it is easy to compare the efficiency of a volatile headspace collection by comparing the attraction to living plants and intact plant materials. The content in volatile collections can be identified with a gas chromatograph coupled to a mass spectrometer. The sprayer utilizes a solvent diluted sample. The solvent may interact with insect flight performance and care should be taken to identify the impact. Ethanol is an attractant for some insect species, e.g., woodboring beetles25. As the sprayer functions by vibrating at ultrasonic frequencies, care should also be taken to consider the anti-predatory response on certain species capable of detecting bats26.
Attraction of crawling insects could also be tested in a wind tunnel27. A raised platform, parallel to and in the centre of the odor plume could then be fitted inside the arena28. However, particular care should be taken to handle contamination issues.
The wind tunnel can also be a powerful tool to validate biotechnological solutions, as a study on genetically modified Vitis vinifera plants with altered kairomone emission ratio and corresponding L. botrana attraction has shown29. Transgenic plants, headspace extracts and synthetic blends were tested for L. botrana attraction in the wind tunnel and resulted in reduced attraction compared to control plants of V. vinifera29.
There are, however, some limitations to the use of wind tunnels which should be carefully considered in each particular case. As insects normally are used in the arena only once, species with long generation time is less ideal for wind tunnel experiments. Also red listed species can be controversial to test in laboratory behavioural studies. However, pest insects, for which there is a considerable need for attractive lures, usually have short generation time, and sufficient numbers can be gained from field collection or a rearing protocol.
The wind tunnel certainly has its place in chemical ecology behavioural studies. It can be constructed in various ways, depending on the budget, and functionality can be added to fit with various research questions. The wind tunnel has the benefit of allowing observations and measurements of the flight behaviour of insect responses to odors and other sensory stimuli.

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. With a wind speed of 30 cm s-1 blowflies displayed, more than 80% take-off and oriented flight behavior in the downwind end of the wind tunnel and more than 60% of them advanced with controlled and oriented movement into the upwind part of the tunnel (Figure 2). There was no significant effect of the visual cue on oriented flight behavior. However, to efficiently display the full range of behaviors that under natural conditions will provide blowflies with the opportunity to deposit the eggs on a carcass (take-off, oriented flight and landing), an addition of a visual cue is needed to mark the odor source. The use of a visual cue significantly increased landings at the source from 14% to 40%. This simple extraction of a larger wind tunnel experiment13 illustrates the need for a full account of the sensory modalities used in resource location by insects, and it shows that expanded information regarding behavior may strengthen the results and subsequently allow more knowledge to be extracted from each experiment.
To answer which volatile cues the pea moth is using to find a suitable host plant for oviposition, we studied the upwind orientation of 5 - 7 day old mated females towards pea plants in different phenological stages (leaf, bud, flower, pod) in the wind tunnel14. Wind speed was 30 cm s-1, light intensity 1000 lux, temperature 20 - 22 &#176;C and RH 60 - 70%. We used a combination of visual and olfactory cues by placing the living plants directly into upwind end of the wind tunnel arena and comparing the behavioural response with the corresponding headspace extracts. We also tested a synthetic blend of ten antennaly active pea plant volatiles. The behavioural response of the pea moth (Figure 3) showed that mated females were much more attracted to pea plants with flowers (58%) and buds (52%) than to pea plants in the leaf (10%) or the pod stage (24%). Similar responses were observed using the corresponding headspace extracts. Mated females were most attracted to the headspace extracts obtained from flowering pea plants (56%), followed by pea plants with buds (42%), and low responses were recorded for the pod (28%) and the leaf stage (10%). Testing a sprayed syntheic blend of ten antennaly active pea volatiles as stimuli, resulted in a landing response of 34%.
The results show the link between host plant phenology and the corresponding plant odor, as well as its impact on the behaviour of mated C. nigricana females. Mated females have a clear preference for pea plants during flower development and the associated odor profile is crucial for the host location. Moreover, this experiment is showing that mated C. nigricana females can distinguish between different phenological stages of pea plants when sensing only volatile cues. Sensory integration is important for host location and may increase the ability to percieve minor differences, particularly in females13,21,22. Nevertheless, in this wind tunnel experiment, the landing responses on headspace extracts from flowering pea plants (56%) without the presence of visual cues were the same as the responses to the real plant (58%). The similarity between headspace extracts only and real plants imply that the odor is the fundamental host plant cue for mated C. nigricana females.
One of the main challenges when developing kairomone lures from wind tunnel behavioural assays, is translating the finished blend into the field environment. The wind tunnel has a clean odor background, whereas the field conditions are permeated by odors from the surrounding vegetation which may alter the chemical information.
Experiments in the wind tunnel were&#160;performed with field collected females at the wind speed of 30 cm s-1, light intensity of 5 lux, temperature of 19 - 20 &#176;C and a RH of 55-65%. The experiments with and without background odors aided in the development of a plant volatile based field lure for the apple fruit moth Argyresthia conjugella11. The results show that the odor dispensers with a complex blend (7 components) and a simple blend (2 components) have similar upwind attraction when presented alone on a clean background (Figure 4A). In a choice assay, however, with the field dispensers embedded in an apple volatile background, the apple fruit moth females preferred the complex but not the simple blend (Figure 4B).
The results show that the blend complexity is a key player to overcome the plant background influence and that the background interaction needs to be considered when developing kairomones for field use.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58385/58385fig1.jpg" /> 
Figure 1. Schematic of wind tunnel located as NIBIO, &#197;s - Norway. The wind tunnel is placed in a climate controlled room. The airflow is filtered by activated charcoal filters before and after odor application, and is then circulated back into the room. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58385/58385fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58385/58385fig2.jpg" /> 
Figure 2. Average (&#177;SE) percent behavioural response of Calliphora vicina to natural odor sources with and without visual stimuli. The odor stimuli was confined to a glass jar and introduced into the wind tunnel by a charcoal filtered air stream. This figure has been modified from [Aak, A. &#38; Knudsen, G. K. (2011) Sex differences in olfaction-mediated visual acuity in blowflies and its consequences for gender-specific trapping. Entomologia Experimentalis et Applicata 139 25-34]. Significant differences are identified by t-tests (significance level: p = 0.05) on a total of 50 flies per experimental treatment, with the averages based on the proportion of responders among 10 flies tested on five separate days. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58385/58385fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58385/58385fig3.jpg" /> 
Figure 3. Percent landing response (&#177;SE) of Cydia nigricana to pea plants in different phenological stage, the corresponding headspace collection and a synthetic volatile blend of 10 antennaly active compounds. The headspace and the synthetic blend was released from an ultrasonic sprayer. The plant material was placed directly into the flight arena. This figure has been modified from [Th&#246;ming, G., Norli, H. R., Saucke, H. &#38; Knudsen, G. K. (2014) Pea plant volatiles guide host location behaviour in the pea moth. Arthropod-Plant Interactions. 8 (2), 109-122]. Significant differences are identified by ANOVA (significance level: p = 0.05). For all treatments, at least 50 females were tested and the moth had 6 min to respond to the odor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58385/58385fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58385/58385fig4.jpg" /> 
Figure 4. Percent Argyresthia conjugella approaching complex and simplified attractants (&#60;5 cm). (A). Upwind attraction to field dispensers without the background. (B). Upwind attraction to field dispensers embedded in the plant volatile background. This figure has been modified from [Knudsen, G. K. &#38; Tasin, M. (2015) Spotting the invaders: A monitoring system based on plant volatiles to forecast apple fruit moth attacks in apple orchards. Basic and Applied Ecology 16 (4), 354-364]. Significant differences are identified by ANOVA (significance level: p = 0.05). For all treatments, at least 45 females were tested and the moth had 5 min to respond to the odor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58385/58385fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression

Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the frequency of their negative thoughts by actively suppressing them. We aim to identify the neural correlates underlying thought suppression in at-risk and depressed individuals. Three groups of women were studied; a major depressive disorder group, an at-risk group (having a first degree relative with depression) and controls. Participants performed a mixed block-event fMRI paradigm involving thought suppression, free thought and motor control periods. Participants identified the re-emergence of &#8220;to-be-suppressed&#8221; thoughts (&#8220;popping&#8221; back into conscious awareness) with a button press. During thought suppression the control group showed the greatest activation of the dorsolateral prefrontal cortex, followed by the at-risk, then depressed group. During the re-emergence of intrusive thoughts compared to successful re-suppression of those thoughts, the control group showed the greatest activation of the anterior cingulate cortices, followed by the at-risk, then depressed group. At-risk participants displayed anomalies in the neural regulation of thought suppression resembling the dysregulation found in depressed individuals. The predictive value of these changes in the onset of depression remains to be determined.

We aim to identify the neural correlates underlying sustained and transient thought suppression, and thought re-emergence in controls, at-risk and depressed individuals. Activation was greatest for controls compared to the at-risk and the depressed group in the dorsolateral prefrontal cortex during thought suppression and anterior cingulate cortex during thought re-emergence.

Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the ...

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Event-related Potentials During Target-response Tasks to Study Cognitive Processes of Upper Limb Use in Children with Unilateral Cerebral Palsy

Unilateral Cerebral Palsy (CP) is a neurodevelopmental disorder that is a very common cause of disability in childhood. It is characterized by unilateral motor impairments that are frequently dominated in the upper limb. In addition to a reduced movement capacity of the affected upper limb, several children with unilateral CP show a reduced awareness of the remaining movement capacity of that limb. This phenomenon of disregarding the preserved capacity of the affected upper limb is regularly referred to as Developmental Disregard (DD). Different theories have been postulated to explain DD, each suggesting slightly different guidelines for therapy. Still, cognitive processes that might additionally contribute to DD in children with unilateral CP have never been directly studied. The current protocol was developed to study cognitive aspects involved in upper limb control in children with unilateral CP with and without DD. This was done by recording event-related potentials (ERPs) extracted from the ongoing EEG during target-response tasks asking for a hand-movement response. ERPs consist of several components, each of them associated with a well-defined cognitive process (e.g., the N1 with early attention processes, the N2 with cognitive control and the P3 with cognitive load and mental effort). Due to its excellent temporal resolution, the ERP technique enables to study several covert cognitive processes preceding overt motor responses and thus allows insight into the cognitive processes that might contribute to the phenomenon of DD. Using this protocol adds a new level of explanation to existing behavioral studies and opens new avenues to the broader implementation of research on cognitive aspects of developmental movement restrictions in children.

Several children with unilateral Cerebral Palsy seem to disregard the preserved capacity of their affected upper limb. This Developmental Disregard is extensively described in the literature but the involved cognitive processes have not been studied. To study underlying cognitive factors of upper limb control, an event-related potential protocol was developed.

Unilateral Cerebral Palsy (CP) is a neurodevelopmental disorder that is a very common cause of disability in childhood. It is characterized by unilateral ...

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

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

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

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

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...