- Fruit Fly I

Procedure -
Our goal for the next several weeks is simple: Isolate a virgin female and one male of known phenotype into a clean, labeled, fly vial for mating. We use these as parents for our F1 generation and the F1's as pareents for an F2 generation. from the phenotypes of the progeny flies we can deduce the genotypes of the parents.

For this week your job is prepare for your fly matings.

We need to develop a stock of flies, from which we can obtain virgin females. Come to class this week take the quiz and get your fly stocks to use for your crosses. Read the below material in preparation for following weeks.

Remember to have the questions at the bottom of this page ready to turn in.

Isolating virgins -

Female Fruit Flies store sperm from all their matings using them randomly. Fro our experiments, we need a controlled mating to determine genetic relationships. Therefore we need to isolate virgin female files (see below). The best way to do this is to obtain very young flies. Eclosure of the flies occurs in a day or two after wings (black bands) become visible on the dorsal surface of the pupa. Release adults NO MORE THAN 10 hours before planned isolation.

We (you) have two options for isolating virgins for the matings .

one option - Isolate virgins before before coming to class. Take the fly vial home. Release ALL adults the morning before lab. The remaining flies, as long as they are less than ~ 12hrs old, should be virgins.

Option two - Release ALL adults after 10pm the night before lab. If new adults eclose, re-isolate virgins before right before class. You may do either or both as time and flies permit.

Transfer newly eclosed adults to our anesthetizer, you need to select the females for mating. Try not to over anesthetize the adult flies. Be careful how hard you tap the vials to remove adults. Tapping too hard will cause a media avalanche, destroying your virgin(s) and the culture they came from.

When the anesthetized files just stop moving empty them onto a quarter sheet of white paper. Use a dissecting microscope to separate males from females, noting the numbers of the different phenotypes. Isolate a few females of mutant phenotype for mating, you can put several female in the same vial. Write in your data book the date, time, phenotype, and number of females. Ask your instructor for the male flies to be used in the matings. Be sure to place the anesthetized flies on the side of horizontal vial. Keep vial horizontal until flies can at least walk. If you don't, the flies will get stuck in water or media since they are too weak to pull free and therefore die. Be sure to label your vials. Store vials in lab until next week.

Take some time to read (and understand) the....

Life Cycle of Drosophila melanogaster: There are four stages in the life cycle of the fruit fly: egg, larva, pupa, and adult. At a typical classroom temperature (21 C), Drosophila melanogaster will produce new adults in two weeks; eight days in the egg and larval stages, and six days in the pupal stage. The total life span of a fruit fly is about 4 weeks. Twenty-four hours after the egg is laid, the larva hatches. The larva has two molting periods, during which the cuticle, mouth, hooks, and spiracles are shed.

The larva is called an instar during the periods of growth before and after molting.

The fruit fly has three instars. The puparium develops from the third instar which becomes hard in texture and dark in color. The puparium is where metamorphosis takes place. Just before the adult fly emerges from the puparium, the pupa darkens.

Approximately 24 hours before the adult fly emerges, one can see the folded wings and the pigment of the eyes if one looks closely through the puparium. Upon completion of metamorphosis, the adult forces its way through the operculum (anterior end) of the puparium. Initially the fruit fly appears light in color with a long abdomen and unexpanded wings. In just a few hours the fly gets darker in color, rounder in the abdomen and extends its wings.

Approximately 48 hours after emerging from the puparium, it is possible for females to start laying eggs. However, there are only 12 hours that a female can be considered virgin after emerging as an adult. After reaching this maturity, the flies are fertile for life. A female fruit fly can store sperm after a single insemination and use it for many reproductions; therefore it is important to use virgin females for genetic crosses.

Egg Stage

• lasts about 24 hours depending on temperature
• egg has appendages which help it stay afloat in liquefied fruit

Larval Stage

• purpose is to eat and grow
• feeds on the organisms decaying the fruit and the liquefied fruit
• primary feeding stage
• outer portion of the exoskeleton is hard and jointed - called a cuticle when the larva has grown enough to fill the exoskeleton it molts molting consists of splitting the cuticle larva crawls out takes on water to stretch the exoskeleton before the new cuticle hardens
• growth is a matter of replacing the water taken in during molting with cells
• divided into three substages called instars
• each instar is internally more complex than the previous one
• egg hatches into the first instar wormlike about 1-2 mm long feeds and grows for about 2 days
• first instar molts into the second instar wormlike about 2-3 mm long feeds and grows for about 2 days
• second instar molts into the third instar wormlike about 4 mm long feeds and grows for about 2 days
• as the third instar draws to a close the larva looks for some solid, dry object upon which to climb out of the food
• once out of the liquefied food it will glue itself to the solid object and become a pupa

Pupal Stage

• the cuticle dries and darken
• no feeding
• metamorphosis takes place
• continues about 6 days before eclosure takes place anterior end splits open
• adults squeeze out of pupal case and harden,
• fly away and mate

Adult Stage

• primary purpose is to reproduce
• live about one month
• in the adult stage flies do not grow immediately after eclosure the wings must be inflated before they dry and harden
• female mates about 10 hours after eclosure female will mate frequently throughout life with whatever male is available
• female stores sperm in special organs for that purpose
• female lays eggs in about 48 hours if conditions are suitable
• sperm are used randomly (not in the order they were obtained) to fertilize the eggs as they are laid.
• female uses ovipositor to poke hole in overripe or bruised fruit in order to deposit egg

During lab next week you will isolate your male flies for the mating, it should be short. You will also have to tend to your cultures during the week. You must have mated files before the penultimate week of our lab.

Questions:

1. What is a dominant allele?

2. Why do you need to isolate Virgin Flies for your crosses?

3. Define the following:

Vestigial

Apterous

Wild type

4. How can you tell the difference between a male and female fly? Circle the male fly in the below image.

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