Teacher Information

Development in

Drosophila melanogaster

Katherine Giunta and Barbara Grosz

Overview

Fruit Flies have been used in classrooms to study the mechanics of genetics for many years. In the last decade they have been "rediscovered" as an important research organism for studying developmental genetics. Fruit fly development is a subject with which our students are much less familiar. In this exercise students have the opportunity to study what is probably the least familiar aspect of development in Drosophila melanogaster, development of the pupa. "First-day" pupae are isolated and cultured. They are dissected daily to study the progressive developmental changes. Particular attention is given to the process of sense organ development.

Biological concepts

• Life cycle of Drosophila melanogaster

• Development and Differentiation

• Metamorphosis

• Development of sense organs

Class Time

This activity will require 45-55 minutes on day one and 10-15 minutes on each of the following four days. A discussion period following the exercise is suggested.

Background Information

Amphibian metamorphosis is characterized by the remodeling of existing tissues. In contrast, the holometabolous insects (flies, beetles, moths, and butterflies) experience a dramatic and sudden transformation that occurs between the larval and adult stages. The juvenile larva (caterpillar, grub, maggot) undergoes a series of molts as it becomes larger. The molts occur because the newly hatched insect larva is covered by a hard cuticle. In order to grow the insect must produce successively larger cuticles and shed or degrade the old ones. This shedding is called a molt. The stages between the molts, characterized by growth and feeding, are called instars. Drosophila undergoes four molts in its life cycle. The transformation of juvenile into adult (the fourth molt) occurs within the pupal cuticle (the puparium). When the third instar larva is ready to pupate, it leaves the culture medium, its body shortens and stops moving, and it attaches to a firm substrate such as the side of the culture bottle. The larval cuticle transforms into a puparium which is initially soft and white. It soon hardens and turns tan. By the end of pupal life it is brown and brittle. Eclosion marks the end of pupation and the beginning of adult life. This usually occurs around dawn in the fly's natural environment, when the leaves of plants would be damp with dew. The insect cracks open the puparium anteriorly and emerges from the pupal case.

During pupal development most of the original body of the larva is degraded as new adult organs develop from undifferentiated groups of cells called imaginal discs. The adult body parts develop from ten pairs of these discs. Pairs of discs form epithelial folds which roll into tubes from which structures such as the antennae, the eyes, the legs, and the wings will develop. Non-epithelial cells migrate into the discs and give rise to the muscles and nerves that serve that particular structure.

Teacher Information

Stages of Metamorphosis in Drosophila

(Table modified from Tyler who used as her source Bainbridge and Bownes,1981)

Hours* Developmental Event (at 25 C)

0 - 24 ** Larva stops wriggling

** White puparium turns brown

** Large gas bubble appears first in the posterior abdomen and then

is seen in the anterior abdomen.

Leg and wing formation begins

24 - 48 Malpighian tubules turn green

Eye cup is visible and outlined in yellow

48-72 ** Eyes become bright yellow, darken to amber, and eventually become bright red

72 - 96 ** Head and thoracic bristles darken

** Wings turn from grey to black

Sex combs (found in males only) darken

96 - 103 ** Legs twitch and fly is able to walk prematurely if puparium is removed

** Eclosion is complete

*Times start on approximately day four of larval life when the larva is still white but is no longer

able to crawl.

** Events your students are more likely to be able to see

Materials

• Drosophila cultures/ one for each team of students

• morgue (mineral oil or alcohol) for disposal of adult flies

• microscope slides • super glue

• empty fly vials with stoppers • dissecting microscope

• microknives (see teacher preparation) • filter paper

• microneedles (see teacher preparation)

Teacher Preparation

Each student team will need a culture. You can produce at least 10 subcultures from each stock culture. Subculture the flies so that you will have 5-day old cultures on a Monday. Eggs take about 24 hours to hatch and the first sign of pupation, the cessation of wriggling, occurs on approximately day four of larval life. Therefore on day five, a culture bottle should have many first-day pupae. Remove the adults from the culture bottles before giving the cultures to the students, or have the students clear the cultures before they begin.

The students will use super glue to attach the pupae to a glass slide. This will keep them stationary for dissection. At least 20 of the same age are chosen. If the students open five each day they can observe the sequential changes. This technique is a modification of one developed by Mary S. Tyler of the University of Maine. Dr. Tyler's exercises and insightful comments can be seen in her lab manual, Developmental Biology, A Guide for Experimental Study.

eacher Information

Brush-On Fingernail Glue, available where manicure supplies are sold, works especially well

because the brush applicator makes it easy to get very small dabs of glue on the slide.

Microknives can be made from chips of double-edge razor blades mounted on dowels. Goggles and leather work gloves must be worn when breaking the razor blades. If a hood is available in the lab it would be best to work behind the protection of glass. Break a razor blade into several small pieces by cutting it with heavy-duty scissors. The ideal shape is like a half-spear. One edge of the blade can be covered with tape, leaving a sharp point exposed for dissection. If a handle is desired, the tape can be omitted and the blade can be glued into a slit made in the end of a six-inch length of quarter-inch dowel. More simply, unbroken double edge razor blades with one side taped for safety can be used.

Microneedles can be made from insect pins (#00) mounted in the ends of quarter-inch dowels. Cut the plastic ball end of the pin off with wire cutters and glue the blunt end of the pin into a hole in the end of the dowel. The hole can be made with a hammer and a fine finishing nail.

Blades and needles can be stored with a piece of drinking straw over the end to protect the tip.

Extensions/ Variations

Using the technique for isolating pupae of the same age students can investigate the effects of various conditions on development and differentiation. Carbon dioxide can be pumped into the development chamber or a small slice of sponge stopper can be soaked in a volatile agent such as alcohol and placed in the bottom of the development chamber. This would allow the student to measure the effects of these agents on rate of development or to look for teratogenic effects. The chambers can be exposed to UV light or changes in temperature as well. Given the opportunity, students will certainly find many variables worthy of further investigation.

Resources

Developmental Biology, fourth edition, Scott Gilbert ,1994 , Sinauer Associates

Developmental Biology: A Guide for Experimental Study, Mary S. Tyler, 1994 Sinauer Associates

About the Authors

Katherine Giunta, St. Ignatius College Prep, 1076 W Roosevelt Road, Chicago, IL 60608

Kathy LG@aol.com

Barbara Grosz, Pine Crest School, 1501 NE 62 Street, Fort Lauderdale, FL 33334

groszb@aol.com

Student Activity

Name:________________________Date:______________Period__

Development in

Drosophila melanogaster

Overview

What happens inside the pupal cuticle of an insect during metamorphosis? In this laboratory activity, you will isolate the pupae of fruit flies and dissect them at each day of pupation in order to study the development and differentiation that occurs from day to day. A review of the basic life cycle of Drosophila should be done before you begin this lab.

Materials

• Drosophila cultures/ one for each team of students

• morgue (mineral oil or alcohol) for disposal of adult flies

• microscope slides • super glue

• empty fly vials with stoppers • dissecting microscopes

• microneedles • filter paper

• microknives

Procedures

I. Review the life cycle of the fruit fly by observing the cultures on display in the lab. For

each stage make a drawing and indicate the length of time the fly spends in that stage:

a. egg c. pupa

b. larva d. adult

II. Study Development

A. The change from larva to pupa involves a process called molting. During a molt the old cuticle is degraded and a new one is produced. Why do you think this is necessary?

B. The stages between molts are called instars. The cuticle of the last instar larva

hardens and darkens to become the puparium. What stage(s) exist in your

culture bottle?

C. Metamorphosis occurs within the puparium. Observe the daily developmental changes that

occur during metamorphosis.

Student Activity

1. Select 20 larvae that are on the sides of the culture bottle and which appear to have stopped wriggling. If they are still white they are probably first-day pupae.

Remove them from your culture bottle and carefully glue each one to a glass slide,

using a tiny drop of super glue to adhere each to the slide. Try to orient each one

in the same direction. You may find it easier to remove about ten at a time and cluster them on one end of your slide. Then space 10 tiny dabs of super glue on the slide and move each one into the glue. Let the glue harden. Prepare a second slide.

Note: A good dissection requires considerable patience and dexterity, but even a

crude dissection will allow you to see some of the most dramatic changes. In fact

you can see some of the changes without even cutting open the puparium.

2. Use a microknife to chip back the pupal case. This will uncover the tender body of

the metamorphosing pupa within. During pupation the larval organs

are self-destructing and the imaginal discs are differentiating to form the adult.

In the first 24 hours of pupal development the heart stops beating and a gas bubble becomes visible in the abdomen.

>Can you see the gas bubble? Is it in the anterior or posterior of the abdomen?

>Why does the interior of the puparium look the way it does at this stage?

3. Store the slides with the remaining pupae in a development chamber made from a culture vial. Take a small slice of sponge (about 1/4 of a sponge

stopper), moisten it slightly, and place it in the bottom of a culture vial. Place your

slide into the chamber and plug it with a sponge stopper.

Student Activity

` 4. Each day cut open 4-5 more pupae and observe the changes.

Between 24 and 48 hours the eye cup develops and you may be able to see it.

>What color outlines the eye cup?

Between 48 and 72 hours eye color develops. If you can, make observations at several times during this time period.

>What color changes does the eye go through as the color develops?

> How can you relate the color changes in eye development to some of

the eye color mutations?

Between 72 and 96 hours bristles develop on the head and thorax and the wings

appear. At first they are gray.

>Can you see the wings folded up? What color are they?

>Where do you see bristles?

Between 96 and 103 hours the legs begin to twitch. In fact the flies should

be able to walk prematurely if the puparium is removed at this stage. By now you

may be skilled enough to remove it.

>Try to remove the pupal covering. Can the prematurely eclosed fly walk?

5. Now that you are familiar with the normal pattern of development, perhaps you

can use the techniques you have learned to investigate the effect of various

environmental conditions on the development of Drosophila melanogaster. Discuss

this possibility with your teacher.

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