The Citrus Research and Development Foundation is acknowledged for providing funding. Thanks to Angel Hoyte and Michael Flores for conducting bioassays.

<ol>
	<li>Assembly of a custom-designed, two-port divided T-olfactometer previously described in Mann et al.10 is shown in Figure 1. The olfactometer consists of a 30 cm glass tube that is bifurcated into two separate ports with a Teflon strip forming a T-maze. Each half is analogous to an arm of a 2-choice style olfactometer enabling the test subject to choose between two potential odor fields.
	<ol>
		<li>Assembly and subsequent assays should take place within a temperature-controlled room or environmental chamber.</li>
		<li>Mount glass T-maze olfactometer vertically as shown in Figure 1 and position it under a fluorescent 900 lux light bulb.</li>
		<li>For optimal light diffusion, the assembly should be mounted within a 1.0 x 0.6 x 0.6 m opaque container to maximize uniform light diffusion.</li>
		<li>Connect the olfactometer arms to odor sources that will be housed in solid-phase micro-extraction holding/odor source chamber (SPMEC) (ARS, Inc., Gainesville, FL) through Teflon;-glass tube connectors. Each SPMEC is comprised of a straight glass tube (17.5 cm long x 3.5 cm wide) supported with an inlet and outlet valve for incoming and outgoing air streams (Figure 1).</li>
		<li>Connect the air delivery system to an external source of humidified and carbon-purified air that is under constant pressure (the air delivery system has both an internal carbon filter for air purification and an air bubbler for humidification if external sources are unavailable).</li>
		<li>Dissolve desired chemical samples within appropriate diluent (solvent) and pipette onto a slow release substrate such as a cotton wick (Petty John Packaging, Inc., Concord, NC). The control treatment should consist of a cotton wick impregnated with solvent only.</li>
		<li>To prevent significant contamination of the glassware, envelop the treated and control cotton wicks in laboratory tissue (Kimwipes, Kimberly-Clark, Roswell, GA) and place into the two SPMEC arms (ARS, Inc., Gainesville, FL). In the case of live plant samples, a guillotine volatile collection chamber can be attached to the olfactometer containing treatments in place of the SPMEC (Figure 1).</li>
		<li>Deliver purified and humidified air through the SPMEC (or guillotine volatile chamber) via two pumps connected to an air delivery system (Figure 1). Maintain airflow rate at 0.1 L/min through both arms of the olfactometer.</li>
	</ol>
	</li>
	<li>Employing olfactometer to test arthropod response to putative repellents. In the present description, a hemipteran insect, Asian citrus psyllid, Diaphorina citri, was used as a test arthropod. Response was tested to citrus volatiles alone or in combination with a repellent (dimethyl disulfide) and clean air as odor sources.
	<ol>
		<li>Conduct all experiments at a standardized temperature and relative humidity.</li>
		<li>Prior to all tests, expose test subjects to clean air versus clean air or solvent versus clean air in the T-maze olfactometer to verify the absence of positional bias or an effect of solvent on behavior of the test subject.</li>
		<li>Assign an odor source randomly to one of the arms of the olfactometer at the beginning of each bioassay. Reverse this position after every 10 test subjects assayed, at minimum, to eliminate potential of positional bias.</li>
		<li>Test the response of a minimum of 30 (and up to 120) test subjects per treatment combination.</li>
		<li>Allow test subjects 100-300 sec to exhibit a behavioral response based on previously established response (latency) interval.</li>
		<li>Record if subject enters the treatment arm, control arm or remains in the release port or below the T-maze division.</li>
		<li>Score a treatment or control arm choice when a test subject crosses the division in the T-maze and moves into either olfactometer arm.</li>
		<li>Score a release arm choice when a test subject remains in the release port or below the T-maze division.</li>
		<li>Clean the olfactometer and connecting tubes thoroughly with 2% soap solution and bake the glass components at 200 &deg;F between the use of different odor treatments.</li>
		<li>For assays in which putative repellent treatments are presented in the T-maze olfactometer with or without a chemical treatment versus clean air, compare the number of test subjects remaining at the release point and not entering the olfactometer between treatments by one way analysis of variance (ANOVA) followed by Tukey's HSD test (&alpha; &lt; 0.05). In cases in which the test subjects leave the release arm, compare the number choosing the control arm versus the treatment arm with Chi square (&chi;2) analysis at &alpha; &lt; 0.05.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Resh, R. H., 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=.">.</a> Encyclopedia of Insects. , (2003).</li><li>Aluja, M., Lesky, T. C., Vincent, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biorational tree-fruit pest management. , (2009).</li><li>Rodriquez-Saona, C., Stelinski, L. L., Peshin, R., Dhawan, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior-modifying+strategies+in+IPM:+Theory+and+Practice.">Behavior-modifying strategies in IPM: Theory and Practice.</a> Integrated Pest Management: Innovation - Development Process. , 261-311 (2009).</li><li>Card&#233;, R. T., Millar, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in insect chemical ecology. , (2004).</li><li>Knight, A. L., Stelinski, L. L., Hebert, V., Gut, L., Light, D., Brunner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dispensers+simultaneously+releasing+pear+ester+and+sex+pheromone+for+disruption+of+codling+moth+(Lepidoptera:+Tortricidae).">Novel dispensers simultaneously releasing pear ester and sex pheromone for disruption of codling moth (Lepidoptera: Tortricidae).</a> Journal of Applied Entomology. 136 (1-2), 79-86 (2012).</li><li>Mann, R. S., Tiwari, S., Smoot, J. M., Rouseff, R. L., Stelinski, L. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+visual+responses+of+flying+mosquitos.">The visual responses of flying mosquitos.</a> Proceedings of the Zoological Society of London A. 109 (4), 221-242 (1940).</li><li>Miller, J. R., Roelofs, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained-flight+tunnel+for+measuring+insect+responses+to+wind-borne+sex+pheromones.">Sustained-flight tunnel for measuring insect responses to wind-borne sex pheromones.</a> Journal of Chemical Ecology. 4 (2), 187-198 (1978).</li><li>Mann, R. S., Rouseff, R. L., Smoot, J. M., Castle, W. S., Stelinski, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfur+volatiles+from+Allium+spp.+affect+Asian+citrus+psyllid,+Diaphorina+citri+Kuwayama+(Hemiptera:+Psyllidae),+response+to+citrus+volatiles.">Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), response to citrus volatiles.</a> Bulletin of Entomological Research. 101 (1), 89-97 (2011).</li><li>Onagbola, E. O., Rouseff, R. L., Smoot, J. M., Stelinski, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guava+leaf+volatiles+and+dimethyl+disulfide+inhibit+response+of+Diaphorina+citri+Kuwayama+to+host+plant+volatiles.">Guava leaf volatiles and dimethyl disulfide inhibit response of Diaphorina citri Kuwayama to host plant volatiles.</a> Journal of Applied Entomology. 135 (6), 404-414 (2011).</li><li>Baker, T. C., Linn, C. E., Hummel, H., 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=Wind+tunnels+in+pheromone+research.">Wind tunnels in pheromone research.</a> Techniques in Pheromone Research. , 75-110 (1984).</li><li>Stelinski, L. L., Rodriguez-Saona, C., Meyer, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+foreign+oviposition+marking+pheromone+in+a+multitrophic+context.">Recognition of foreign oviposition marking pheromone in a multitrophic context.</a> Naturwissenschaften. 96 (5), 585-592 (2009).</li><li>G&#246;k&#231;e, A., Stelinski, L. L., Whalon, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+leafroller+moths+to+selected+plant+extracts.">Behavioral and electrophysiological responses of leafroller moths to selected plant extracts.</a> Environmental Entomology. 34 (6), 1426-1432 (2005).</li><li>Ali, J. G., Alborn, H. T., 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=herbivore-induced+plant+volatile+increases+biological+control+activity+of+multiple+beneficial+nematode+species+in+distinct+habitats.">herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats.</a> PLoS ONE. 7 (6), e38146 (2012).</li><li>Mann, R. S., Ali, J. G., 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=Induced+release+of+a+plant+defense+volatile+'deceptively'+attracts+insect+vectors+to+plants+infected+with+a+bacterial+pathogen.">Induced release of a plant defense volatile 'deceptively' attracts insect vectors to plants infected with a bacterial pathogen.</a> PLoS Pathogens. 8 (3), e1002610 (2012).</li></ol>

The author received funding that was provided by the Citrus Research and Development Foundation.

Herein, an assay device and protocol are described for measuring the response of small arthropods (Insecta: Hemiptera: Psyllidae) to odorants. The method involves a choice test, allowing the insect to make a choice between two odorant fields in the case of assays evaluating a putative attractant. Furthermore, the test subject may display three types of behaviors by leaving the release arm and entering either one of two potential odor fields, or remaining in the release arm, in the case of repellency assays. The olfactometer allows for high throughput data collection because it takes advantage of the negative geotactic (tendency to move upward) and positive phototactic (tendency to move toward light) natural behavioral response of many arthropods. Although the present demonstration uses a psyllid insect test example, the assay can be readily adapted to arthropods broadly, both those that use flight or walking as predominant modes of transportation.
Olfactometers designed for measuring the response of insects were initially developed decades ago and have played a major role in elucidating insect attractants and repellents12. The specific designs of such olfactometers vary widely; however, variations on the general theme of the two-choice assay described here, as well as, similar Y-tube assays have been often used to measure arthropod response to chemicals. Larger flight tunnels that may cause sustained flight of insects9 have also resulted in collection of groundbreaking data elucidating the fundamental mechanisms of insect flight and orientation to semiochemicals, as well as, data informing practical use of pest control tools.
It is often necessary to tailor design an olfactometer and associated instrumentation for the biology of the specific arthropod test subject. Those olfactometers that can be used among a general group of insects are more useful than ones specific to a small group; however, sometimes the economic significance of a small group of insects dictates the need for development of a very specific olfactometer and assay technique. The currently described design builds on previously familiar arthropod techniques. It allows for a more standard two-choice assay than Y-tube bioassays in which the experimental arena or choice test takes the form of a Y-shaped glass device7. Typically, one arm of such a Y-tube will receive a treatment odorant, while the other will be left blank13. Variations on such olfactometers may include addition of multiple radiating arms14 and even addition of a soil medium for assaying the behavior of organisms moving through soil15. In developing such olfactometers and associated assays, it is important to consider how closely natural conditions are replicated and thus the true relevance of the assessed behavioral responses in terms of the test subjects' biology. To a large degree, the data collected will only be as useful as the relevance of the behavioral bioassay with respect to the organism's behavioral ecology16.
The currently described olfactometer and behavioral bioassay is specifically designed for a hemipteran insect that tends to initiate flight as short "jumps"16. The vertical orientation of the olfactometer and the light source arrangement facilitate initiation of insect movement and thus subsequent chemically-mediated orientation behavior can be assayed efficiently and with high throughput. This behavioral assay arrangement can likely be used for a broad array of flying or walking arthropod taxa or could be also easily modified to fit the need of non-arthropod organisms.

Arthropods (including insects and arachnids) are often defined as pests, because they either compete with human consumption in agricultural or urban settings or transmit causal pathogens of disease1. A large amount of investment is made into development of control tools for such pests. These tools can vary widely and significant progress has been made in developing biorational tools that either modify or exploit the natural behavior of pests with the use of behavioral attractants or repellents2,3.
Arthropod pests often rely on olfactory signals or cues for mate and/or host location4. Therefore discovery of effective attractants or repellents for pests can often lead to development of control tools. This process of discovery involves several steps. First, the natural behavior of the test subject must be understood. For example, one must ascertain whether one sex of a given species attracts the opposite sex for mate location. If so, one must determine whether this attraction is mediated by a chemical and where that chemical is produced. Further steps include determining when the chemical is produced and its eventual identification. Throughout this procedure, the test subject's (insect or mite) behavioral response to odorants must be tested. Initially, the behavior of the test subject will be tested to the natural material. For example, a male moth's response may be tested to that of the extract of its female conspecific's pheromone gland. Alternatively, an herbivore's response to the natural odors of its host plant may be tested. While these investigations often have a practical focus on development of management tools5,6, they can also be applied to research that addresses more fundamental questions, such as speciation7. Subsequent steps involve testing the arthropod's response to synthetic versions of identified natural products.
Understanding how arthropods respond to chemicals requires the development of appropriate tools to measure their behavior. The data generated by such tools will depend on how well the investigator is able to measure natural behaviors in a controlled setting. Over six decades ago, sustained flight olfactometers and tunnels were developed to measure insect response to odorants8,9. Olfactometers vary significantly in design and are often tailored to the specific insect or problem being investigated. The goal of this article is to describe the design and use of a vertically oriented, T-maze olfactometer for measuring insect response to odorants (Figure 1). The olfactometer's vertical orientation takes advantage of the negative geotaxis (tendency to move up) exhibited by many insects. A light source positioned directly above the olfactometer takes advantage of the positive phototaxis (tendency to move toward source of light) exhibited by many insects. The olfactometer is particularly useful for hemipteran insects that tend not to exhibit movement readily in horizontally-oriented olfactometers and that are heavily influenced by light intensity with respect to orientation. However, this device can be used for a wide range of flying and crawling insects, as well as arachnids. The olfactometer can be employed to evaluate both attractants and repellents. In the case of attractants, the bifurcated odor field (Figure 1) is established with the putative attractant released on one side versus clean air released on the opposite side. A choice is recorded when the insect released at the base of the olfactometer chooses one of the two odor fields. In the case of repellents, a third behavior is possible. In addition to upward movement into either of the two odor fields, lack of upward movement may be recorded indicating a non-responsive stimulus or repelling stimulus from either odor fields.

<table><tbody><tr><td>Two port divided T-olfactometer</td><td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td><td>None(Custom made)</td><td>If unavailable from ARS, Southern Scientific (Gainesville, FL) also currently builds and distributes such equipment.</td></tr><tr><td>Solid-phase micro-extraction holding/odor source chamber with Teflon;-glass tube connectors.</td><td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td><td>RV-R3</td><td>See above comment</td></tr><tr><td>Air delivery system</td><td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td><td>HADS-2AFM2C.4</td><td>See above comment</td></tr><tr><td>Guillotine chamber</td><td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td><td>L3GP3</td><td>See above comment</td></tr></tbody></table>

vertical t-maze choice assay for arthropod response to odorants

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

Attraction of Asian citrus psyllid (Diaphorina citri) adults to the odors of their natal host plant volatiles (citrus) is depicted in Figure 2A. Significantly (&alpha; &lt; 0.05) more adults chose the arm of the T-maze olfactometer receiving odors from living citrus plants as compared with a blank (clean air) control.
An example of repulsion is shown in Figure 2B. In this case, the insects were exposed to one arm of the T-maze receiving citrus volatiles, while the second arm received volatiles from a citrus plant that was treated with the known repellent, dimethyl disulfide11. In this case, three types of behaviors were observed. The number of psyllids arrested at the release arm and not responding did not differ statistically from the number entering the arm receiving the odors from citrus plants alone (Figure 2B). However, no psyllids entered the arm containing the host plant volatile when co-presented with the repellent (Figure 2B).
<img alt="Figure 1" src="/files/ftp_upload/50229/50229fig1.jpg" /> 
 Figure 1. Schematic diagram of vertically oriented T-maze olfactometer joined to air delivery system and odor treatment release devices. The diagram is an adaptation of Figure 1 in Mann et al.10. A test subject is placed individually into the release chamber of the 2-port divided T-olfactometer. It moves towards the odors through a gate valve which allows controlled upward movement into the olfactometer and prevents the insect from crawling back into the release point area. The tube bifurcates 10 cm after the gate value. A positive response to an odor is recorded once the insect moves 0.5 cm into a specific side of the division. The guillotine volatile collection chamber may be used in place of a solid-phase micro-extraction holding/odor source chamber (SPMEC) as an odor source.
<img alt="Figure 2" fo:content-width="3.5in" fo:src="/files/ftp_upload/50229/50229fig2highres.jpg" src="/files/ftp_upload/50229/50229fig2.jpg" /> 
 Figure 2. Response of Asian citrus psyllid (Diaphorina citri) adults to volatiles from citrus host plants versus clean air control (A) or citrus volatiles versus citrus volatiles co-released with a repellent (dimethyl disulfide) (B) in a vertically oriented T-maze olfactometer. Figure 2A: One-way analysis of variance (ANOVA) followed by Tukey's HSD test (&alpha; &lt; 0.05) was performed to compare the number of psyllid making a choice between either arm (2 treatments, n = 120). Columns indicated by different letters are significantly different from one another (&alpha; &lt; 0.05). Figure 2B: One-way analysis of variance (ANOVA) followed by Tukey's HSD test (&alpha; &lt; 0.05) was performed to compare the number of psyllid making a choice between either arm or remaining in the release point (3 treatments, n = 120). Columns indicated by different letters are significantly different from one another (&alpha; &lt; 0.05).

Watch this Scientific Journal Video about Vertical T-maze Choice Assay for Arthropod Response to Odorants at JoVE.com

Vertical T-maze Choice Assay for Arthropod Response to Odorants

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

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

vertical-t-maze-choice-assay-for-arthropod-response-to-odorants

Research

JoVE Journal

Neuroscience

14.6K Views.  University of Florida. A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Video: Vertical T-maze Choice Assay for Arthropod Response to Odorants

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

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

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

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

Biology

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

Behavior

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

A Single-fly Assay for Foraging Behavior in Drosophila

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

A Single-fly Assay for Foraging Behavior in Drosophila

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

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

Testing Drosophila Olfaction with a Y-maze Assay

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

Testing Drosophila Olfaction with a Y-maze Assay

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

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

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

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

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

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

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

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

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

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

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

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

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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

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

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

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Summary

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Chapters in this video

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

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Drosophila Adult Olfactory Shock Learning

A Single-fly Assay for Foraging Behavior in Drosophila

Testing Drosophila Olfaction with a Y-maze Assay

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

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

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

C. elegans Chemotaxis Assay

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