Lifespan Protocol for Drosophila melanogaster: Generating Survival Curves to Identify Differences in Fly Longevity

- Start by cohousing a set number of male and female Drosophila melanogaster fruit flies in a vial containing cornmeal medium. This allows for a consistent density of offspring.

Allow the female flies to lay eggs. Then remove the adult flies. The eggs will become larvae, pupae, and finally adult flies in a span of 10 days. New flies emerging earlier than their cohort should be removed before day 9. Divide the now synchronized flies into groups for the control and experimental conditions. Cohousing male and female flies allows mating, removing a variable that affects lifespan.

After several days, separate the flies by sex into groups. Place an equal number of flies into the corresponding experimental condition. Every three days, transfer the living flies into a new vial and record the number of dead flies. Repeat this process until all the flies have died.

After at least three replicate experiments, plot the mean percentage of surviving flies by day to generate mean survival curves for each condition. In this example, we will see how to generate survival curves for cohorts treated with endocrine-disrupting chemicals.

- In the lifespan protocol, first set up 20 vials with eight females and four males, each filled with 10 milliliters of cornmeal food, and house them at 25 degrees Celsius. After four days, discard the flies and place the vials back into the incubator. In the late afternoon of day 9, remove all newly emerged flies from the vials and return the vials to the incubator.

After 16 to 24 hours, divide at 250 one-day-old adult flies of both sexes into four groups under light carbon dioxide anesthetization and transfer them to 250 milliliter bottles containing adult medium food, three supplemented with different EDC concentrations, and one with the EE2 solvent alone. Maintain the flies at 25 degrees Celsius for two to three days to allow them to mate. Then sort each cohort of flies by sex into two groups. Within each treatment condition for both sexes, randomly subdivide each group into five parallel vials at a density of 20 individuals per vial. Prepare a lifespan spreadsheet in which the number of dead flies is subtracted from the number of surviving flies from the previous transfer so that the number of survivors at each transfer is automatically obtained.

After three days, transfer the flies without anesthesia to new vials containing the corresponding food at the same time and check for death. Record the age of the flies and the number of dead flies. Repeat the transfer every three days until all of the flies die.