The overall goal of this procedure is to quantify behavioral changes triggered by food deprivation in the adult fly Salala Melanogaster. This is accomplished by first starving flies for 18 to 24 hours. The second step is to load individual flies into behavior chambers, each of which contains an attractive odor source at its center.
Next, a computer system with a digital camera tracks the position of each fly over time and records. Its coordinates for later data analysis. The final step is to determine the latency of each fly to reach the odor source using custom software.
Ultimately, this high throughput food search behavior assay can be used to study the relationship between neural circuit function and behavioral responses. The main advantage of this assay over existing method, like the teammates, is that it quantifies chemotactic behavior in single flies. This method can help answer question in neurobiology such as which neuro circuits are important for chemotactic behavior and how these neuro circuits are moderated by the animal's internal physiological state.
The goal of this method is to obtain large behavior data sets in a short time. Although this method has given us insights into foraging behavior, it can also be used to study other behaviors such as make localization. Visual demonstration of this method is critical.
Proper fly handling and execution of the computer software are both essential to the success of this experiment. Generally, individual new to this method may struggle a little bit at the beginning because it takes some time and patience to learn how to handle the flies gently demonstrating the procedure will be arou the needle. Reach a technician for my laboratory Before fly collection.
Rear the experimental flies under controlled temperature and humidity conditions on a 12 hour light dark cycle. On the day of EEC explosion, anesthetize the flies under carbon dioxide before sexing them under a microscope. Several basic criteria can be used to differentiate between males and females.
Males have a patch of black bristles on their four legs called sex combs that females do not have. Also, the tip of the male abdomen is covered by a dark cuticle and displayed genitalia not seen in females, collect female flies and place them along with four to five white-eyed males into new food files with a maximum of 30 flies per vial age. The flies for two to five days prepare the chambers for fly starvation by first pushing a single tissue down to the bottom of an empty plastic vial, completely soak the tissue with distilled water.
Then use an object to push down on the tissue and gently squeeze out excess water. Invert the vial to discard the extra water. There should be enough water to keep the flies hydrated and the starvation chamber moist but not enough to drown the flies.
About 18 to 24 hours before the experiment begins, transfer the flies from the food vial into a starvation chamber and plug the vial. Store the vials under controlled temperature and humidity conditions overnight until the experiment begins. Prepare a 1%agro solution by adding 100 milligrams of low melting temperature aros to 10 milliliters of distilled water.
In a glass flask. Heat the agro solution in a microwave just until it begins to boil, but while before it boils over, stop the microwave and swirl the flask once. Repeat this step twice more until the aros has completely dissolved.
Maintain the Aros solution in a liquid state by keeping the flask warm on a hot plate. Set at 50 degrees Celsius, add 990 microliters of the 1%aro solution and 10 microliters of apple cider vinegar to a 1.5 milliliter upend DF tube. To make a 1%apple cider vinegar solution, vortex the solution until mixed before placing in a dry bath incubator set to 50 degrees Celsius, set the testing room to the desired conditions such as temperature and humidity.
Turn on the 660 nanometer LED panel. Rinse the sieves and testing plates with hot water and heat them in a drying oven until all moisture is evaporated.Cool. The sieves and plates down to the testing room temperature.
Before beginning experiments, position a shallow dish on top of the diffuser plate and fill it with water to increase the local humidity and to mask the water in the aros droplet. Finally, position the sieves over the water dish. The testing plate is made of clear acrylic and consists of six testing arenas.
A slider contains holding chambers that permit fly loading, temporary containment and simultaneous release of six flies into their respective chambers. At the start of the experiment, cross hairs etched at the center of each arena in the plate indicate odorants should be pipetted. Insert the sliders into the acrylic testing plate.
Gently slide the aspirator into the vial past the cotton plug and allow about six flies to walk into the aspirator. It is critical to be as gentle as possible in handling them. One may take advantage of photo tactic, fly behavior to induce flies, to walk towards the aspirator by pointing the vial opening towards a dim light source.
If necessary, gentle suction can be applied to aspirate approximately six female flies. Avoid selecting white-eyed male flies. Insert the tip of the aspirator into the first hole of the testing plate.
Allow a single fly to pass into the holding cell and gently advance the slider forward to load another fly into the next hole. Continue until flies. Occupy all six holding cells of the plate.
Then pipette five microliters of a 1%apple cider vinegar agarro solution directly onto the center of the crosshairs on the inside face of the testing plate. To center the testing plate, open the file named Positioning tool vi. Run the file by clicking on the white arrow in the upper left corner of the screen.
Place the testing plate on top of the sib such that the arena opening faces the SIV floor and the odor target is on the ceiling of the plate. Align the cross hairs of the testing plate with the cross airs on the monitor screen when alignment has been completed. Abort execution by clicking on the red dot located near the upper left corner of the monitor to track and record the coordinates of the fly.
During each food search trial, open the acquisition software file. Fly tracking six vi. Run the file by clicking on the white arrow in the upper left corner of the monitor.
Assign the file a name and then click okay. Advance the sliders in the testing chambers to release the flies into the testing arenas. Be careful not to move the testing chambers as this will lead to improper recording of the coordinates.
Click on start and ensure that the only source of light in the testing chamber is the LED panel. When the trial is finished, remove the sieve and behavior chamber. Lift the testing plate from the sieve and remove the flies by submerging the plate in ice.
Gently clean the plate with hot water and remove any agros debris. Place the testing plates in a drying oven to remove moisture. Ventilate the testing area by turning on a small fan for approximately two minutes.
Turn the fan off and load the next group of flies into the next testing plate. Proceed to perform data analysis using custom software as described in the text protocol. The data analysis software and the layout are used to evaluate each fly's performance during its 10 minute trial according to a set of analysis criteria.
These 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 seconds are considered inactive and are rejected from the data set unless they already have succeeded in locating the food source or exhibit. An average speed greater than 10 millimeters per second for at least 100 seconds following the inactive period 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 seconds of the trial are accepted for data analysis 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.
Flies that weave towards and away from the food source within a 10 millimeter radius for a minimum of 50 seconds 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 arenas with visible artifacts in fly position. Trace are also 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. Adult flies starved 18 to 24 hours exhibit a higher olfactory sensitivity to food related odors than their fed counterparts.
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 minute window. In contrast, only 7%of all fed flies do so. Starved flies should exhibit a significantly greater attraction to vinegar than to an agros vehicle alone, and this observation can be used to troubleshoot testing conditions.
For example, when this food search experiment that was performed at 32 degrees Celsius with an environmental humidity of 35%using wild type flies, no significant difference between fly attraction to vinegar and the aros control was detected, this is likely due to an increasing attraction to the water found in the agros droplet under warmer testing temperatures. By increasing testing humidity to between 50 and 60%this behavioral shift was corrected and the significant difference between attraction to vinegar and the agros vehicle was restored. Once mastered, this technique can be performed in 12, including the 10 minute experimental trial if it is performed properly.
While attempting this procedure, it's important to remember to handle the flies very gently to avoid injuring or disturbing them. Please remember that the testing plates and sieves can be extremely hot when in the drying oven, and therefore they should be removed while wearing oven mitts after its development. This technique paved the way for researchers in the field of neurobiology to explore chemotactic behaviors.
This technique can also be adapted to study the fly's ability to locate other odor objects. For example, it can be applied toward the study of mate localization behavior in male flies. After watching this video, you should have a good understanding of how to prepare for the experiment, adjust the experimental conditions, run the experiment, and analyze the performance of each fly, which will ultimately provide you with a cumulative plot showing the percentage of flies that have successfully located the odor source.
