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

The overall goal of this behavioral bioassay is to investigate odor guided behavior in vinegar flies. Fly Walk is a high throughput system that tracks and analyzes the behavioral responses of multiple individual flies to olfactory stimuli automatically. This new technique can help answer key questions in the area of insect olfaction.

Like what is the hedonic balance of an odor, or what does the addition of additional odorants mean to this balance? The main advantage of this experiment is that we can odor guided behavior in high temporary resolution, and we use odor stimuli that are similar to those that insects experience in nature or to those that we also use in electrophysiological recordings. Generally, individuals who try to use this method might struggle because it really, it needs an airtight system, so you need to know where the air is going through and you need to be sure that there are no leaks at all where the air can leave the system I For these experiments, it is important to rear the flies under a 12 to 12 light dark cycle in low to midden cultures containing a normal food medium.

Keep the room temperature between 23 and 25 degrees Celsius, and the relative humidity should be around 70%in preparation. Using large culture vials made 20 to 30 adults for a week and collect groups of 30 to 40 offspring of mixed sex to make an experimental cohort. When a cohort is two to four days old, transfer it to a new vial containing a moist foam plug or a moist tissue paper.

Let the flies starve in this vial for 24 hours before the test. It is vital that the flies are not anesthetized during this transfer. For this setup, 250 milliliter bottles serve as many humidifiers.

Fill each bottle with 100 milliliters of distilled water. Next, prepare half milliliter working stocks of each odorant as one 1000 dilution of the pure odors and mineral oil. Similarly, prepare one PCR tube with just mineral oil to serve as a negative control.

Now prepare the odor delivery system for each odorant. In the experiment. Connect two ball check valves to each odor vial.

Make sure to connect the odor vials in the right flow direction. A wrong connection will not only compromise the planned experiment, but it may also contaminate the delivery system. Next, check the system for leaks.

First, seal the outlet of the mixing chamber. Then check that all airflow before the stimulus device drops to zero. If there are leaks in the system, listen for them.

They can usually be resolved by exchanging rubber gaskets at crucial connections or exchanging Teflon tubing. For the first step of this procedure, breaking glass is a concern, so put on protective gear. Carefully transfer 15 individual test flies to the individual glass tubes Using an aspirator, close the glass tubes on both sides using the corresponding adapters, they must fit very tightly.

Next, connect the loaded glass tubes to the fly walk set up. Once all the tubes are attached, let the flies habituate to the new environment for at least 15 minutes before starting the experiment. Now switch on the light source.

Make sure to provide enough light for efficient tracking without increasing the temperature inside the glass tubes. The odor vials must all be connected at this point to create a closed system. The habituation period relies on a closed system without leaks, so the airflow through the tubes is at the same speed as during the test periods.

Check the airflow on the computer, connect it to the digital flow meters. The total airflows exiting the system should equal the airflow entering the system. If not, there is a leak present.

The leak will affect airflow in all tubes, but the problematic tube usually is the one that displays the largest drop in airflow. If a new leak is identified, it is usually caused by a glass tube that broke inside the adapter, or it is caused by shards of glass within the adapters from previous experiments. During this process, make certain the humidity of the air exiting the system between 60 and 80%After the flies have acclimated to their tubes, the experiment can be run.

In principle, any sequence of odors can be presented thus far. The best results can be obtained by randomizing the stimuli and separating them by at least 90 seconds. In total.

Present each odor 40 times in 500 millisecond pulses. Next, set up a region of interest by dragging a frame across the area to be monitored. Include all 15 glass tubes in the frame, but exclude the outer five millimeters of the tubes ends.

The next step is to turn off the tracking software and make modifications to the script. That will change the Y-axis position of the 14 parallel lines that separate the individual tubes. When the tracking software is back on, only one fly should be present between any two lines.

Next, make certain the camera parameters are set so that the flies are reliably tracked throughout the glass tubes. If the flies are lost at the edges of the region of interest, increase the brightness or gain of the tracking software. Now that the system is fully prepared, start the stimulus protocol.

Record the flies positions at a rate of 20 frames per second and log these coordinate positions in combination with the odor valve status into text files. Because the experiment is very sensitive to odors, regular cleaning is required to clean the glass tubes, soak them in detergent, and then rinse them off under running distilled water. Following the rinse, dry the tubes under pressurized air and then heat them to 200 degrees Celsius for eight hours.

To clean the odor delivery system first, remove all the odor vials and tubing from the central mixing chamber. Then remove the tubing adapters from the mixing chamber. Clean the emptied mixing chamber by rinsing it with a solvent under a hood like the tubes.

Dry the mixing chamber using pressurized air and heat it at 200 degrees Celsius for eight hours. To clean the odor vials and the valves, remove the steel plugs, discard the rubber gaskets, and remove the check valves, soap check valves and ethanol, and other components in both ethanol and acetone. Next, sonicate the soaked components for 30 minutes at the maximum setting and rinse them off with distilled water.

As before, use pressurized air for drying and heat the components to 200 degrees Celsius for eight hours. Flush them with ethanol When acetone in the direction of the airflow, because acetone attacks rubber parts, immediately dry each flushed check valve with pressurized air to remove all the residual odors from the flushed check valves. Pulse air through them for several days, set them up in a 60 degree Celsius incubator with airflow pulsing on and off on one second intervals.

15 starved female flies were exposed to ethyl butyrate. The yellow bar represents the odor pulse. The blue squares depicts upwind movement within a 100 millisecond timeframe.

Correspondingly red squares depict downwind movement. The same flies also displayed upwind movement in response to stimulation with isop pentyl acetate, ethyl acetate or two three butane d averaged to the population. The fly's response to ethyl acetate had a sharp onset, high amplitude, and a short duration.

In contrast, the response to two three butane Dion had a later average onset, lower amplitude, and longer duration. Ultimately, data from each odorant was resolved in this manner and then further analyzed to show all four odors elicited a higher upwind displacement within four seconds than the solvent. In addition, by opening odorant vials, synchronously flies were exposed to all possible mixtures.

After stimulating the flies with the four individual odors stimulations with blends of the four odors were all at least as attractive as the most attractive single compound in the blend. Once master, you can run this experiment within one hour, and then it runs for eight hours and already 30 minutes later you can visualize your first results. So this method really allows obtaining data from many flies in short time with high temporary resolution.

But be careful. This experiment requires extensive cleaning between the experiments. If your system still is contaminated with the ants from the last experiment, you can forget about your results in the fly walk.

You can of course, also test genetically modified flies you by that you can investigate the impact of individual or factory neural circuits on the OD guided behavior. After the development of this new technique, it paved the way for us doing research in this area, insect faction, to understand coding of these odors in the brain of the insect. And importantly, using the same kind of stimulation for behavior in the fly walk and for physiological experiment means that we can really compare these two different experimental paradigms.

After watching this video, you should have a good idea about how to establish the fly walk, and you should be able to establish this experiment in your own lap and start your own high throughput experiments on insect affection soon. But don't forget, working with glassware and chemicals can be extremely dangerous, so therefore, always wear a lab coat. Use goggles and gloves.