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Genetic Analysis in Drosophila melanogaster

Introduction

The animal most often used for genetic studies is the fruit fly, Drosophila melanogaster. It is easily
cultured in the laboratory and has many easily distinguishable mutations. In addition, D. melanogaster
has a short life cycle and is highly prolific. Thomas Hunt Morgan, Nobel Prize winner, pioneered the
study of genetics of D. melanogaster in the early part of the 20th century. Morgan first studied at
Columbia University and later moved to the California Institute of Technology. He, and many of those
who worked with him, made genetics a science and in the process developed D. melanogaster as an
unparalleled tool for genetic research. D. melanogaster has a tremendous amount of genetic variability
and a wide range of phenotypic and chromosomal mutations. The Genetic Stock Center at Cal Tech
currently houses over 1675 strains and mutant stocks.

Culture

Laboratory cultures of Drosophila are maintained in clear plastic vials approximately

9.5 cm in height and 3.4 cm in diameter. Foam stoppers are used to plug the vials. Vials must be
washed and disinfected in bleach for two hours before they are reused. Foam stoppers must be washed
and autoclaved before they are reused.

The fruit flies are provided with Formula 4-24 Instant Drosophila Medium from Carolina Biological
Supply Company. One measuring cup of medium is placed in a vial with an equal amount of distilled
water. After two minutes, 6 to 10 grains of yeast are added to the surface of the medium. The yeast will
grow through the culture medium. The larvae and adults will feed on the yeast and the sugars found in
the medium. Putting too much yeast in a vial is harmful to the flies. One of the metabolic by-products
of yeast is carbon dioxide. Excess carbon dioxide may cause sterility and/or death of the flies.

Fly cultures may become infected with bacteria, fungi, or mites. These contaminants will reduce the
hardiness of the flies. If you suspect your cultures are contaminated, immediately consult with your
instructor. Flies transferred from an infected culture to a clean one will carry the contaminant with
them. Cleanliness is the best prevention against contaminated cultures. All utensils and the work area
should be kept clean. Fly cultures should not be kept over a month. Used medium should be discarded
and used vials should be washed and placed in a container away from the work area.

Life Cycle

Drosophila occurs naturally on rotting fruits. Both the adults and the larvae feed on the sugars and the
yeasts that grow on the rotting fruit. The adult female will lay her eggs on the surface of the fruit. D.
melanogaster is a true fly (Order Diptera) and, so, has a complete metamorphosis. The fly passes
through four stages: egg, larva, pupa, and adult. A newly laid egg will emerge as an adult in
approximately 9-10 days at 25°C. Fruit flies exposed to slightly lower temperatures will have a longer
developmental period while those exposed to slightly higher temperatures will have a shorter

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developmental period. Drosophila cultures exposed to temperatures above 30°C or below 10°C will
have greatly increased mortality.

The Drosophila egg is about 0.05 mm long, white, oval, and has two thin stalks emerging from its
anterior end. The day after the egg is laid it hatches into a white, segmented larva or maggot. It has
black mouth parts (jaw hooks) in a narrowed head region. It has no eyes and, so, is blind. Since the
larva has no appendages, it must eat and push its way through the environment. The larva has a pair of
spiracles (air pores) on the sides of its body at both the anterior and posterior ends. The spiracles
connect to tracheae which branch and re-branch for gas exchange.

The larva passes through three instars during which its main purpose is to store energy necessary for
metamorphosis. The larva feeds on yeast growing throughout the culture medium. The different larval
instars are similar in form but differ in size. The first two instars are terminated in molts. Each molt
consists of completely shedding its cuticle, mouth parts, and spiracles. Molting allows the larva to
increase in size. Near completion of the third instar, the larva stops feeding, leaves the medium, and
seeks a dry place (walls of the culture vessel) to pupate. The cuticle hardens and becomes darker to
form the puparium. Third instar larvae are approximately 4.5 mm long. The larval stage is completed
in about 4 days at 25°C.

In insects with complete metamorphosis there is considerable reorganization within the pupal stage.
Structures in the larva (such as - the heart, nervous system, and tracheal system) change very little
during pupation. Other adult structures (such as - eyes, wings, sex organs, and antennae) are present in
a rudimentary form in the larva, and are triggered by changes in hormone levels to develop into their
adult form. Pupation takes approximately four days at 25°C.

The adult (imago) first emerges (ecloses) by forcing its way through the anterior end of the puparium.
Within an hour, the wings will expand, the cuticle will harden and darken, and the body will gradually
become more rounded. The function of the adult is reproduction. Adult females will begin to mate
eight hours after emerging from the puparium. While females require some time to reach sexual
maturity, males will mate as soon as they emerge. The female can store and use the sperm from a
single insemination for the major portion of her reproductive cycle. The sperm are stored by the female
in a sac-like structure (the spermatheca) and are gradually released into the oviduct. As eggs are
produced by the female, they pass into the oviduct to be fertilized and then move into the vagina to be
deposited on the medium. Females start laying eggs two days after emerging and will remain fertile for
as long as they live. The female will initially deposit 50 to 75 eggs in the first two or three days, but
there is a gradual decrease of egg production over time. Under good environmental conditions, the
adults will survive for approximately a month. Generation time is just under two weeks at 25°C.

Anesthetizing

FlyNap is used to anesthetize the fruit flies. It must be stored and used in a fume hood. It is necessary
to keep the lid on the bottle and tightly closed at all times, except when charging anesthetizer. The
principle hazardous component of FlyNap is triethylamine. The MSDS for FlyNap will be kept on top
of the fume hood. Anesthetizing the flies will be demonstrated to you in class. Use approximately 1.0

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mL of Fly Nap to charge the anesthetizer. One to two minutes after the flies have stopped flying
around in the anesthetizer transfer them to a white card for scoring with a dissecting microscope. If the
flies are not anesthetized within five minutes, try recharging your anesthetizer. The flies should remain
immobile for 30 to 45 minutes. Do not be surprised if some of the flies exhibit trembling of the legs or
wings; this is normal. Maneuver the flies on the white card, very gently, with a paint brush.

Determining Sex

The ability to determine the sex of D. melanogaster is crucial for successfully mating and ascertaining
the inheritance pattern of a trait. There are several easily distinguishable anatomical differences.
1. Genitalia. The sex of Drosophila is most reliably determined by examining the genital organs with
magnification. The genitalia are located on the posterior portion of the ventral side of the abdomen. In
the male, they are darkly pigmented; in the female they are non-pigmented. Females have a large
vaginal plate with a longitudinal crease. An anal arch marks the end of the abdomen. Males have a
prominent well scleritized genital arch and a separated anal plate. He also has a pair of claspers
arranged in a circular fashion just ventral to the tip of the abdomen.
2. Sex combs. Males have a sex comb (small tuft of black bristles) on the margin of the basal tarsal
segment of each front leg. The appearance of the sex comb is the only sure way to distinguish male and
female flies less than two hours old because the other adult traits are not immediately recognizable.
3. Color of the abdomen. Alternating dark and light bands occur on the posterior portion of the dorsal
side of the abdomen. The female has six pigmented bands; and, on the male, the four most posterior
bands are fused. On the male, the markings extend completely around the abdomen and meet on the
ventral side.
4. Shape of the abdomen. The abdomen of the female is sharp and protruding; the male's is round and
blunt. The abdomen of the female is distended and appears spherical and ovate; whereas, the male has
a narrow and cylindrical abdomen.
Determining sex by examining genital organs is very time-consuming and subject to error by those
who are not experienced in insect morphology. The differences in the color and shape of the abdomen
between the sexes are variable and so are less reliable for sex determination. The best method for
determining sex is the presence of the sex combs in the males.

Mating

Anesthetized flies should be placed in the center of a white card and examined with a dissecting
microscope. Sort the flies by gently pushing them to one side or the other with a paint brush.

It is necessary to have virgin females for your experimental crosses. Males do not need to be virgins.
Females will not mate until 8 to 10 hours after emerging from the puparium. To collect virgin females,
clear all adult flies from the vial late in the day or evening. Then collect virgin females as they emerge.
Flies tend to emerge in greater numbers during the early part of the day.

To initiate a cross, you should have a minimum of eight virgins. The number of adults per vial should
not exceed 25. Females prefer to have a choice for their partner, so if possible, provide a 2:1 male to
female ratio.

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About 7 to 10 days after a cross has been set up, the parents should be removed from the vial. This will
prevent breeding between generations. Generally, females will emerge first in the new generation and
males will follow a day or two later. The progeny should be scored every other day for at least 10 days,
so as to include flies with slower developmental rates. Counts made beyond 10 days may include flies
from the next generation. All flies not needed for genetic crosses should be disposed of in the morgue
(a bottle containing ethanol, vegetable oil, or detergent).

It is important to keep clear and concise records of all your crosses. Label all of your vials with the
following information: parental phenotypes, date, and your name. You must keep this information in
your notes and on WebCT. Also include the number and sex of the flies used to set up the cross. It is
necessary to do at least 3 repetitions of each cross. This will ensure that there will be enough progeny
for analysis in case a rep must be discarded due to error or contamination. Initially, the data from each
rep will be kept separate; however, the reps of a cross will be combined for the chi square analysis.

Procedure

You will be given two stock cultures of Drosophila melanogaster. One will contain pure breeding
wild type flies and the other will contain pure breeding flies with one or more mutant phenotype(s).
You must set up crosses to determine the inheritance pattern of the unknown trait/traits. Plan and
perform these crosses in conjunction with the other members of your group. The parental generation,
P, would consist of the flies used to set up your first cross. The progeny of the first cross is the first
filial generation, F1. If there was an F1 mated with an F1 then the progeny from that mating would be
the F2 generation.

The two parental stock strains need to be maintained so you will have pure breeding flies for future
crosses. To maintain these stocks, you simply need to transfer the flies from their current vial to a new
vial with food. This technique will be demonstrated to you in lab. (The first day of lab you will need to
save one vial of each stock culture for examining the phenotypes, to practice sexing, and collecting
males for your crosses.) Plan to make new parental stock cultures about every two weeks. The more
stock vials you have the easier it will be to collect pure breeding flies and virgin females.

Anesthetize the remaining vial of wild type flies, and in a separate anesthetizer, the remaining vial of
the mutant flies. Place about 15 flies of both strains on a white index card on the stage of a dissecting
microscope. Examine the wild-type flies and then compare these to the mutant strain. At the same
time, practice sexing the flies. The rest of flies in the anesthetizers should be used to start new stock
cultures or for your reciprocal crosses.

During the first Drosophila lab set up reciprocal parental crosses as follows:
wild type female x mutant male
mutant female x wild type male
The females must be virgins for all of your crosses. These virgin females must be collected from your
parental stock vials.

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After observing the progeny from the first set of crosses, make a hypothesis about the inheritance
pattern of your unknown trait(s). Thereafter, the goal of your crosses should be to provide further
proof of your hypothesis.

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