To prepare for the experiment, be sure to obtain cultures of Drosophila melanogaster at least two weeks ahead of the planned experiment day. NOTE: Because adult females store sperm after mating, you will need this time to generate virgin females by breeding the flies.
You should get two stock cultures: wildtype, that is homozygous dominant red eyed flies, and white chromosome one mutant, which are homozygous recessive white eyed flies.
Purchase or create Drosophila habitats consisting of a clean vial, 3 cm of prepared food media, and a stopper to close the open end of the vial.
When not being moved or counted, keep the flies in an incubator or room kept at 23 – 25 °C, where the lifecycle will take around 12 days. NOTE: From the egg, Drosophila progress through four main stages of development: embryo, larva, pupa, and adult. The complete lifecycle takes about 10 days at 25 °C, but it can be affected by temperature. For example, at 18 °C, the lifecycle is about 19 days and at 29 °C, the lifecycle is only seven days.
As an overview, to set up for the student experiment, you will generate vials for each student group containing white eyed virgin female flies only, white eyed males only, red eyed virgin females only, and red eyed males only. These will be referred to as the experimental vials.
When your vials from the supplier arrive, referred to as the stock vials, these will contain a mix of male and female flies, as well as larvae, pupae, and eggs.
Beginning with your stock vials, you will take around 3 – 5 males and the same number of females, keeping white eyed and red eyed flies separate at all times, and place these together into a new nursery vial containing food. NOTE: You can sex the flies using a magnifying glass. Look at the posterior of the fly. Males are slightly smaller and have darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
You will leave the flies in these nursery vials for around 3 – 5 days to mate and lay eggs.
After this, you will move them back to their stock vials.
Next, you will observe as the eggs and larvae in the nursery vials mature, checking the vials every 6 – 8 hours. As it takes females around 8 – 10 hours to become sexually mature, this should ensure that any females found in the nursery vials are virgin.
As the new adult flies appear, you should transfer them from the nursery to the appropriate experimental tube. For example, if it is a male, transfer it to the experimental male tube for the appropriate eye color. If it is a female, it should be placed in an experimental vial labeled with eye color and virgin female.
Keep adding flies to the appropriate vials until each experimental vial has at least five flies. NOTE: You will need to make enough experimental vials so that each student group has one of each eye color type and sex, with a few class spares. You should create enough nursery vials to account for this. A good rule of thumb is that it generally takes about two weeks for 25 healthy adults to produce about 50 new adults.
To begin the process, first label empty habitats to make the nursery vials to raise larvae and pupae for each strain of flies: red eyed and white eyed.
To move the adult flies into the nursery vial to lay eggs, use an anesthesia wand according to the manufacturer's instructions, placing it into the stock vial of mature flies. Wait until the animals become unconscious. Now they can be gently tapped onto a piece of white paper and sorted into piles of the same sex using a brush.
Transfer a few male and female flies to the appropriate vial labeled with their eye color phenotype and sex.
Place the nursery vials to incubate at 23 to 25 °C and the flies will lay eggs.
Remove the adults after 3 – 4 days using anesthesia and put them back into the stock vial.
Remove any adult flies that have eclosed in the 6 – 8 hour period using an anesthesia wand.
Then, tip out the adults onto paper and sort them into groups based on their sex before placing each fly into an appropriate experimental vial marked male or virgin female and red or white for the phenotype. NOTE: Each experimental vial should contain five flies. Once this number is reached, then a new vial should be started. If you miss the 6 – 8 hour window, females may not be virgins, so these should be discarded into a morgue tube containing 70% ethanol or returned to the stock vial. Males do not need to be virgins, but do need to be kept in male only containers.
Each lab group will need one tube of five virgin red eyed females, one of five virgin white eyed females, one tube of red eyed males, and one of five white eyed males. In addition, they will need a morgue vial containing ethanol, two anesthesia wands, fly anesthetic, a paintbrush, a magnifying glass, some plain paper, and two empty fly vials for crosses.
