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 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.
States of hunger promote two types of appetitive behaviors: food search and food consumption1. This simple behavioral assay is useful for the study of chemotactic behaviors associated with foraging2,3. Specifically, it tracks fly position, walking speed and latency to locating a food odor target. Latency of food finding serves as metric for measuring changes in the sensitivity of the fly's odor detection system downstream of changes in its internal appetitive state. A manual version of this assay was previously used to show GABA-B receptor signaling is important for odor localization behavior in adult flies3. The current automated version of the assay was instrumental in the study of how short neuropeptide-F (sNPF) signaling reshapes the olfactory map in Drosophila and influences appetitive behaviors2.
Testing is done in a dark, temperature and humidity controlled room. Digital video cameras set above the clear acrylic testing plates track flies backlit by 660-nm LED illumination. Information from the camera is processed in real time by a computer stationed next to the testing area. We use data acquisition software to record and save the coordinates of fly positions during the testing period.
In this paradigm, the subject is released into an arena that contains a food odor at the center; the odor object creates a food odor gradient within the arena that induces food search behavior in the fly. A similar odor search protocol has been applied toward the study of chemosensation in single Drosophila larvae7. While other behavioral assays such as the four-field olfactometer4,5 or the t-maze6 evaluate odor aversion or attraction behaviors, this paradigm is best suited to evaluate olfactory sensitivity and chemotaxis behaviors.
Several key advantages accompany this assay. First, it permits rapid acquisition of large data sets, because data collection and analysis are mostly automated. Second, this assay isolates and measures the behavior of single flies, thus eliminating social olfactory cues that may influence their behaviors. Third, the simplicity of the protocol and simple experimental design make the assay efficient and easy to teach others.
In addition, this assay may be used to further probe neural circuits underlying food search behavior by combining it with the extensive genetic toolkit available to Drosophila melanogaster8. Targeted expression of transgenes that silence or excite neurons can be achieved with tools such as the GAL4-UAS system as well as the UAS-shibirets1, UAS-tetanus-toxin, and UAS-TrpA1(B) transgenes9-12.
1. Fly Collection and Starvation
2. Preparation of the Food Odor
3. Testing Room and Behavior Chamber Setup
4. Fly Loading into the Testing Plates
Diagrams with specifications for testing plates can be found in the Supplemental Files section. The testing plate is made of clear acrylic and consists of 6 testing arenas. A simple slider contains holding chambers that permit fly loading, temporary containment, and simultaneous release of 6 flies into their respective chambers at the start of the experiment. Cross-hairs etched at the center of each arena in the plate indicate where odorants should be pipetted.
5. Positioning the Testing Plate
6. Record the Fly Position During the Experiment
7. Data Analysis Using Custom Software
“Data Analysis for Fly Tracking—Six Zones” can be found in the Supplemental Files section. During data acquisition, the acquisition software records individual fly position coordinates for each time point in a text file. A single digital camera positioned above the testing plates acquires images at a frame rate of 0.5 Hz. The analysis software program "Data Analysis for Fly Tracking-Six Zones" extracts information from that text file to a) calculate the average speed, b) determine the time point at which a fly successfully located the odor source, and c) build graphical windows that allow the user to view: fly location, distance of the fly from the odor source over time and average fly speed over time. It also formats the data for easy export into a spreadsheet program. In this macro, food search latency is defined as the time point at which flies spend at least 5 sec within a 5 mm radius of the center of the arena.
8. Export Data from the Data Analysis Software to a Spreadsheet Program
The data analysis software and the layout, an example of which can be seen in Figure 1, are used to evaluate each fly's performance during its 10 min trial according to a set of analysis criteria. The following criteria are used to determine whether data from each fly will be used for data analysis and are designed to eliminate those flies that are unable to perform the food search task due to injury, illness, stress, or lack of motivation.
Flies that are inactive for more than 300 sec are considered 'inactive' and are rejected from the data set unless they a) already have succeeded in locating the food source or b) exhibit an average speed >10 mm/sec for at least 100 sec following the inactive period (Figures 2a, 2b, 2c).
Healthy flies exhibit robust search behavior immediately upon release from their holding chambers. Thus, to select only those flies that exhibit healthy speeds during the early stages of active odor search, only flies that move within a certain range of speeds for the first 50 sec of the trial are accepted for data analysis. This criteria is based on our observations that a) as flies near the odor source, their speeds decrease and b) few flies reach the odor target within the first 50 sec of the assay. The speed criteria are determined by evaluating the average speeds of at least 100 control flies at a given experimental temperature. The upper/lower speed limits are set by the average speed +/- standard deviation, respectively. For example, at 21 °C, only flies that move between 3.5-10.5 mm/sec in the first 50 sec of the trial are used for data analysis. Exceptions to this rule are made for flies that successfully located the odor source within the first 50 sec and are thus slower than the lower speed limit.
Flies that do not move through all four quadrants in the arena and head straight for the food source after the trial begins are rejected (Figure 2d).
Flies that weave towards and away from the food source within a 10 mm radius for a minimum of 50 sec are considered to have successfully found the food source. The plot depicting distance of the fly from the odor source over time can be used to evaluate this rare case. This is the only instance of a successful search that is not automatically detected by the current data analysis macro and must be detected manually (Figure 2e)
Arenas with visible artifacts in fly position trace are rejected. Artifacts can be created by any event where the data acquisition software detects an object other than the fly. They often appear as long, straight lines that span across the arena or radiate from its center (Figure 2f).
In Figure 3, adult flies starved 18-24 hr exhibit a higher olfactory sensitivity to food related odors than their fed counterparts1. A graphical plot of the cumulative percentage of flies that successfully locate a food odor source shows 30% of all starved flies succeed within a 10 min window; in contrast, only 7% of all fed flies do so (Figure 3). This heightened olfactory behavioral response has been previously shown to require intact antennae1. Failure to observe a clear difference between fed and starved control flies in this assay can be resolved by examining environmental rearing and testing conditions.
One useful strategy for troubleshooting testing conditions is to examine whether flies are attracted to additional cues other than the odor target by measuring fly attraction to the odor vehicle, agarose. Starved flies should exhibit a significantly greater attraction to vinegar than to the agarose vehicle alone (Figure 4b). Figure 4a exhibits results from a food search experiment that was performed at 32 °C with an environmental humidity of 35% using wild type flies. In this data set, no significant difference between fly attraction to vinegar and the agarose control was detected. This is likely due to an increasing attraction to the water found in the agarose droplet under warmer testing temperatures. By increasing testing humidity to 50-60%, we were able to correct for this behavioral shift and restore the significant difference between attraction to vinegar and the agarose vehicle (Figure 4b, * denotes p-value <0.05).
Figure 1. A typical analysis software data layout illustrates fly position over time, fly distance from odor source over time, and fly speed over time. The 2 column table in the upper left corner of each arena displays average speed (mm/sec) during the first 50 sec (column 1) and the latency of food finding in sec (column 2). In addition, the name of the opened text file is appended to the lower left corner (illustrated as, "OZ120807_ORCODTKRi_1%_S4"). Click here to view larger figure.
Figure 2. Examples of different types of traces addressed in Analysis criteria. A.) Fly that has been inactive for 300+ sec is rejected. B.) Fly that has been inactive after successfully locating food is accepted. C.) Fly that has been inactive for 300+ sec but shows robust activity for at least 100 sec after inactive period is accepted. D.) Fly that heads straight for the food source after release is rejected. E.) Flies that weave towards and away from the food source within a 10 mm radius for a minimum of 50 sec are considered to have successfully found the food source and are accepted. F.) Arenas with visible artifacts in the layout are rejected. Click here to view larger figure.
Figure 3. Graphical plot showing a cumulative percentage of fed and starved flies that find odor source over time using latency of food finding. (n = 88-96 flies; * denotes p-value <0.05, **denotes p-value < 0.01).
Figure 4. Troubleshooting testing conditions A) Graphical plot showing cumulative percentage of flies that find 1% vinegar or agarose vehicle over time. No significant difference was detected in fly attraction to either odor target when testing conditions were at 32 °C and 35% humidity (n = 62-94 flies). B) Graphical plot showing cumulative percentage of flies that find 1% vinegar or agarose vehicle over time. Testing conditions were at 32 °C and 50-60% humidity. Under these conditions, flies are significantly more attracted to 1% vinegar than to the agarose vehicle (n = 55-71 flies; * denotes p-value < 0.05).
In this protocol, we describe a step-by-step procedure for the food search behavior assay. In addition to food related odors, it may also be adapted for the study of the fly's ability to locate other odor objects. For example, it may be applied toward the study of mate localization behavior in male flies3 .There are several additional considerations for this protocol that we will mention here regarding this procedure:
First, the rearing temperature determines how long experimental flies should be aged before testing. It is recommended that a range of ages be examined to determine the most appropriate age for the experiment. For example, in our experience, when reared at 21 °C, differences between fed and starved fly responses are most robust after they have been aged 4-5 days.
Second, the LEDs illuminate a glass diffuser plate which serves to create constant, even illumination beneath the acrylic chambers. Sufficiently even, constant, and bright illumination is critical for automated tracking of fly movement. Uneven lighting or flickering light sources may lead to errors in automated tracking of the fly that either result in intermittent inability to detect the fly's position or cause the software to mistake the light artifacts as the fly. We've found both a commercially available LED backlight or a custom built LED array work equally well in meeting the lighting needs for this assay.
Third, if the software mistakenly detects small changes in lighting or agarose as extra objects, the object detection settings in the "Fly Tracking-Six Zones" acquisition software may be adjusted for threshold as well as object size. Adjusting the detection settings ensures that only one object is detected in each arena. To view the number of objects being tracked in each arena, click the "Threshold" tab of the "Fly Tracking-Six Zones" acquisition software. If more than one object is being tracked, one may adjust the Min Size, Max Size, Min Threshold, or Max Threshold until the detected artifact disappears.
Fourth, fly stocks used in these experiments should be isogenized. Behavioral performances in this paradigm are highly sensitive to differences in genetic backgrounds. Mated females are used to reduce potential behavioral variability associated with mating status or sex. There is no reason to believe this assay would not be equally effective in studying the behavior of males or virgin females.
Fifth, the sieve should be suspended slightly above the light diffuser plate to prevent odor saturation of the odor gradient (the suspension is approximately 2 cm with our commercially purchased model). We use commercially available sieves to create a porous floor beneath the testing plates.
Finally, in order to produce reliable data sets, consistency is required in experimental rearing and testing conditions. Any failure to see significant differences between fed and starved responses in control flies can be resolved by checking to make sure 1) flies are reared under stable temperature and humidity conditions, 2) flies are fed with fresh food and are not reared in crowded conditions, 3) newly eclosed fly exposure to CO2 is minimized, 4) flies experience the same length of starvation, 5) flies are tested under stable temperature and humidity conditions, and 6) the testing environment and chambers are not contaminated with odors from previous trials or experiments. In addition to the aforementioned parameters, isogenization of fly stocks is important as different genetic backgrounds can influence fly performance in this assay. Furthermore, if vinegar is used as the odor source, care must be taken to ensure that it does not lose its potency by keeping it tightly sealed and stored at 4 °C.
In its current form, this odor search assay is best suited for the study of chemotaxis and evaluating differences in olfactory sensitivities across different genotypes or experimental conditions. Chemotaxis inherently requires sensitivity to an odor, attraction to that odor, and the motivation to seek the odor target. Odor attraction or aversion per se, however, are best measured by behavioral assays that present the fly with odor choices such as the t-maze6 or the four-field olfactometer4,5.
The authors have nothing to disclose.
This work was supported by research grants to J.W.W. from the National Institute of Health (R01DK092640) and National Science Foundation (0920668).
Name of Reagent/Material | Company | Catalog Number | コメント |
Apple Cider Vinegar | Spectrum | commercially available | |
Agarose, Type VII | Sigma-Aldrich | A0701 | low gelling temperature agarose |
Acrylic Testing Plate | custom | Plate contains 6 arenas. Each arena is 60 mm in diameter 6 mm in height. See testing plate diagrams for specific measurements. | |
LabVIEW V.8.5 | National Instruments | 776670-09 | platform for programs: PositioningTool.vi, FlyTracking–Six Zones.vi NOTE: "elapsed time.vi", "time into file.vi", and "two object detect.vi" are included subroutines that must be available in order for the main data acquisition program "FlyTracking–Six zones.vi" to run. |
LabVIEW Vision 8.5 | |||
LabVIEW Vision Acquisition Software 8.5 | |||
LabVIEW Vision Builder AI 3.5 | |||
Igor Pro V.6 | Wavemetric, Inc. | platform for macro: Data Analysis for Fly Tracking–Six Zones | |
Basler scA1390-17fm | National Instruments | 779980-01 | Digital Camera NOTE: driver for camera available at Baslerweb.com |
8 mm lens | National Instruments | 780024-01 | Lens for Basler Digital Camera |
Ground Glass Diffuser Plate | Edmund Optics | custom | Diffuses light, 25 cm x 30 cm |
US Std. No. 100 | Fischer Scientific | 04-881X | Sieve with nominal opening of 150 μm |
Lighting Option 1 | |||
LED backlight 660 nm (20 cm x 20 cm) | Spectra West | BL47192 | a simpler but more expensive lighting option. |
Power Supply for LED Backlight | Spectra West | ||
Lighting Option 2 | |||
660 nm LEDs | Superbrightleds | RL5R1330 | Wavelength 660 nm (approximately 7 x 7 LED array for a 14.7 inch x 9.75 inch panel) |
Linear DC Power Supply | GW Instek | GPS-1830D | Power supply for LED Panel |
Solderless Breadboard | Digikey | 922354-ND | Breadboard for LEDs |