Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

The goal of the following experiment is to train Dsof and Melanogaster using a novel endurance exercise program and to assess the effects of exercise training. This is achieved by using an automated device that lifts and drops several vials of flies repetitively activating the fly's instinctive climbing response, and thus giving the flies a workout session. The flies are trained daily on a ramped schedule from two to three hours over a three week long training period.

Next flies are subjected to various behavioral assays in order to measure the effects of exercise. Results show that exercise flies climb faster as measured by the rapidly induced negative geo axis or ring assay. The main advantage of this technique over existing methods like endurance training other animals is the extensive collection of tools available for manipulation of drosophila genetics.

In the study of endurance exercise, We first had the idea for this method when we were conducting many single negative geo taxis assays. We wondered how long flies could keep climbing and if we could endurance train them in this way. The power tower is built on a rectangular piece of plywood that is clamped to a table.

A 4.5 rotation per minute. Gear motor rests on several boards and is wired to a speed control, a fuse, and a power switch. In a juncture box, a three inch fan held in a metal tube is directed at the motor for cooling.

Connected to the motor is a rotating arm with a roller. On the end, the motor and arm rotate pushing down one side of a bent rod that is attached to the plywood. With a door hinge on the other side of the rod, a wheel is attached so it can roll against a platform.

It pushes up and down as the motor spins, the power of the motor is adjusted, such that a full rotational period is 15 seconds long On the platform, fly vial racks are attached by bungee cords, and the vials placed into these racks are secured with a screen that is also attached to the platform. By bungee cords, the platform is able to slide up and down along standard draw sliders and a styrofoam cushion dampens the shock of its drop. The power tower is operated in a temperature controlled room to match the conditions the animals are reared under.

To run an exercise experiment, a minimum of 240 age matched male flies are required for longitudinal monitoring of endurance and negative geo axis or climbing ability. Maintain the flies in populations of 20 per vial. Divide the vials into two equally sized groups, one of which will be exercised, that group's vials should be plugged with flugs, whereas the control group should be plugged with sponge stoppers.

A sponge is used for immobilization because its adaptive size allows for easy placement near the bottom of the vial. The softer sponge material also reduces injuries caused by the more solid flugs. The food vials should be replaced every other day before testing.

During training, the flies remain in the food vials because if empty vials are used, many more flies suffer. Training related injuries. When loading the vials of control flies on the power tower, push down their sponge stoppers to about three millimeters above the food level, hence, limiting the flies.

Movement for consistent results. Test both groups of flies five consecutive days per week, utilizing a ramped schedule that increases the time per session from two to three hours a day over a three week period. Of course, any regime could be employed.

Consistency in treatment, time of day, and assay methods over a three week time span is essential. Careful scheduling and practice procedures reduce the possibility for error Fatigue. Assays can be performed longitudinally on flies before, after, or during the training regime.

It can also be used to ass safer response to an environmental variable. Run the fatigue assay on a day in which training is not taking place. At the beginning of a day load both groups of flies on the power tower.

Activate the apparatus and observe each vial once every 10 minutes. If on four consecutive drops, five or fewer flies in a vial climb higher than two inches, then that cohort of flies is considered fatigued. Remove the fatigued flies from the power tower while it is still operating and record the time it is removed.

Observe all the vials for a maximum of 10 hours and compile the results. One assay for quantitating. The effects of training is a daily measurement of running speed, which can be performed throughout the training regime and beyond.

To measure the long-term effect of training. To begin the climbing speed or ring assay first transfer flies into new empty vials and place a row of six vials into a vial rack. Rolled pieces of tape are used to secure the vials to the rack.

Then set the rack in front of a light source, like an x-ray view box that will provide even illumination of the vials. Now set up a camera to photograph the rack so the position of each fly will be visible in each vial. Then tap the six vials to knock down all the flies to the bottom of the vials and take a timed photograph.

Two seconds after the flies are all knocked down. If the camera's flash is used, it should be used consistently. Repeat this process four times for each set of vials before proceeding with the power tower.

To analyze the data, score each photo individually using software to find the upper and lower boundaries of the vial. Then measure the climbing height for each fly. Then copy the climbing heights into excel.

Each fly in each vial is given a score from zero to four corresponding to the height of the quadrant. They climb to four for the highest quadrant and zero for not climbing at all. For each vial, calculate the score for each of the 20 flies and take the average score of the cohort.

This is called the climbing index. The average of each cohort's four climbing indices on that day is used for data analysis. If image processing software is not available, quadrant lines can be drawn onto the empty vials, and flies can be manually assigned Quadrant values while type flies responded to an endurance protocol with a diminished age-related decline in climbing ability.

That persisted following the end of training as reflected in longitudinal ring assays across five weeks of age. Thus, induction of exercise by the power tower program can be used as a behavioral input and the ability of genetic or environmental factors to modulate this effect can be assessed. Conversely, the power tower can also be used as an output.

For example, when flies with a varying percentage of sucrose in their diet, were tested for time to fatigue. Increased sucrose content correlated with increased endurance capacity. While attempting this procedure, it's important to remember to make sure that all fly cohorts are age matched, and that methods are performed consistently throughout the course of the experiment.

After watching this video, you should have a good understanding of how to endurance, train, and assess flies of any genotype.