This method can help answer key questions in the field of metabolic research such as determining whether particular lifestyle interventions like exercise can benefit specific individuals. Often individuals new to this method will struggle with identifying larvae in the first instar stage. The key is to collect larvae nearly the same size as the eggs.
The main advantage to this technique is that it can be used to assess the impact of exercise across many experimental conditions and genotypes. First, assemble laying chambers for each experimental group. Apply a dollop of yeast paste onto an apple juice agar plate and secure the plate to a six ounce plastic bottle.
Apple juice agar plates are made by making a 3%agar solution in store-bought apple juice. And then filling 35 by 10 millimeter dishes 3/4 of the way up the wall. Yeast paste is made by mixing store-bought active dry yeast with water at a ratio of about two milliliters of distilled water per gram of yeast.
Keep the paste covered loosely to allow de-gassing and apply about three millimeter dollops to each plate. The bottle, made of polypropylene, must be prepared with small holes for ventilation. After preparing the components for the chamber, proceed with loading in the adults.
Once the adults of the desired genotype are all in the bottle, cap the bottle with the plate and secure the assembly with rubber bands. Ensuring sufficient numbers of adult flies for the laying chambers likely requires one to begin building population numbers several generations prior to the planned experiment. Now, change the apple juice agar plates twice a day, once in the morning and once in the evening.
Then let the eggs develop on the collected plates for 12 to 24 hours into first instar larvae. To collect these larvae, use a thin paintbrush or a pick made from a flexible thin needle and a handle. For each experimental group, transfer 50 first instar larvae to a fresh food vial and let them develop to adulthood.
To make sure that you are able to collect enough larvae for each of your replicates, ensure that there are researchers available that are efficient at collecting larvae in order to get the numbers you need. Upon pupation, collect the pupae by first wetting them using a small damp paintbrush. Then gently scoop pupae off the sides of the vials with a spatula.
Load the pupae into empty vials. Then, upon eclosion, transfer the adults to standard food vials. One to five days after eclosion, separate the flies based on sex and divide each sex into two groups, experimental and controlled.
Maintain the adults at 50 flies per vial. Give the flies fresh vials with live yeast every two days. While it is possible to maintain adults without live yeast supplementation, we have found that adults perform better with the supplement.
Live yeast supplementation is a variable that researchers can modify to suit their specific research goals. The exercise device uses a simple belt and pulley system powered by a variable speed motor. Prior to using the exercise device, remove any vials attached to it and calibrate its motor speed using the power supply such that a full rotational period is 15 seconds.
To time the rotational speed, attach a small paintbrush to the motor cover and position the brush so it touches one of the rotating brackets. Then time the number of rotations made by counting the contacts of the brush and the bracket per minute. Using a calibrated machine, proceed with the first day of exercise, usually a Monday.
Push vial plugs into the vials. Leave the flies one centimeter of space in control vials and six centimeters of space in exercise vials. Now place the vials in the clamps and with 10 minutes to let the flies acclimate.
The exercise device can hold up to 48 vials. The room temperature should remain constant throughout the multi-day procedure or the exercise device can fit into an incubator, if desired. The experimental design consists of five consecutive days of exercise per week.
Each exercise day contains alternating periods of exercise and rest. On each day, five minutes is added to one of the exercise periods so that over a five-day workweek, all four of the exercise periods will increase by five minutes. Once the flies are acclimated, start the first exercise period.
Run the machine for 15 minutes. During the five minute inter-period rest, leave the vials on the clamps. After completing the fourth cycle of exercise, return the food vial plugs to their normal positions and return the flies to their incubator.
To establish whether exercise on the TreadWheel influenced metabolic traits, triglyceride storage in Oregon R and yellow one white flies was measured and normalized against the flies'protein concentration. Statistical analysis revealed significant genotype by exercise interaction affecting triglyceride storage. There was also a significantly sexually dimorphic effect with males storing more triglycerides than females.
Female exercised flies had significantly lower triglyceride levels than their unexercised counterparts. This was not observed in males. Next, flies were raised on variable diets, put through the exercise regime, and then tested on a RING-like negative geotaxis assay.
Females climbed significantly higher when reared on the high fat diet than on any of the other treatments. For the males, exercise only improved climbing when they were raised on a normal diet. A high fat diet had no effect.
Intriguingly, there was a decrease in climbing performance following exercise for females from the DGRP 153 line when raised on a normal diet. Thus, exercise may not be a uniformly positive intervention for all genotypes. Following this procedure, other methods such as hemolymph glucose assays or lifespan analyses can be performed to assess the effect of exercise on Type II diabetes or longevity.
This technique allows researchers in the fields of genetics and metabolism to explore the role of exercise in maintaining metabolic health in the model organism, Drosophila.
