FACULTY OF ENGINEERING AND NATURAL SCIENCES

DEPARTMENT OF GENETICS AND BIOENGINEERING

BIO 323: GENETIC ENGINEERING-1

LABORATORY REPORT

Experiments Name: Drosophila Melanogaster

Experiments Date and Submission Date: (16.10.2016)&(19.12.2016)

Students Name / Surname: Necla YÜCEL

Students ID Number: 116201132

Evaluator Name: Asst. Prof. Dr. Özgür GÜL

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CONTENTS

List of Tables……...

……………………………………………………………………………………..3

List of Figures..………………………………………………………………………………………….3

Aim……………………………………………………………………………………………………...4

Introduction…………………………………………………………………………………………...4-6

Materials………………………………………………………………………………………………...6

Method………………………………………………………………………………………………..7-8

Result………………………………………………………………………………………………...9-10

Discussion…………………………………………………………………………………………..10-11

Conclusion…………………………………………………………………………………………..…11

References…………………………………………………………………………………………...…11

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LIST OF TABLES

Table 1: Result of Drosophila’s Numbers

LIST OF FIGURES

Figure-1: Life cycle of Drosophila melanogaster

Figure-2: Body size – female is generally larger than male.

Figure-3: Show Male (left) and Female (right) wild-type Drosophila

Figure-4: Dorsal view of Drosophila

Figure-5 : Drosophila's Medium Culture

Figure-6: Cross of Sepia and Wild Type

Figure-7: The appearance of Drosophila unconscious

Figure -8 :Wild type Drosophilla melanogaster

Figure -9: Sepia Drosophila melanogaster

Figure-10: Showing the poured Medium Culture

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AIM

The purpose of this experiment, in the sex linked cross of Drosophila Melanogaster; a phenotypic ratio

of 1:1 was obtained, to introduced normal "wild type" and various mutant phenotypes, to determine

the ratio of monohybrid cross of Drosophila melanogaster, and to differentiate male and female of

Drosophila melanogaster.

INTRODUCTION

Life cycle of Drosophila melanogaster

D. melanogaster exhibits complete metamorphism, meaning the life cycle includes an egg, larval

(worm-like) form, pupa and finally emergence as a flying adult. This is the same as the well-known

metamorphosis of butterflies and many other insects. The larval stage has three instars, or molts.1

Life cycle by day

Day 0: Female lays eggs

Day 1: Eggs hatch

Day 2: First instar (one day in length)

Day 3: Second instar (one day in length)

Day 5: Third and final instar (two days in length)

Day 7: Larvae begin roaming stage. Pupariation (pupal formation) occurs 120 hours after egg laying

Day 11-12: Eclosion (adults emerge from the pupa case).2

The time from egg to adult is temperature- dependent. The above cycle is for a temperature range of

21-23 degrees C. The higher the temperature, the faster the generation time, whereas a lower (to 18

degrees C) temperature causes a longer generation time. Females can lay up to 100 eggs/day. Virgin

females are able to lay eggs; however, they will be sterile and few in number. After the eggs hatch,

small larvae should be visible in the growing medium. If your media is white, look for the black area

at the head of the larvae. Some dried premixed media is blue to help identify larvae however this is not

a necessity and with a little patience and practice, larvae are easily seen. In addition, as the larvae feed

they disrupt the smooth surface of the media and so by looking only at the surface one can tell if larvae

are present. However, it is always a good idea to double check using a stereomicroscope. After the

third instar, larvae will begin to migrate up the culture vial in order to pupate.3

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Figure-1: Life cycle of Drosophila melanogaster4

Sex difference

Several criteria may be used to distinguish male and female Drosophila melanogaster

Figure-2: Body size – female is generally larger than male.

Abdomen shape – the female abdomen curve to a point, the male abdomen is round and much shorter.

Figure-3: Show Male (left) and Female

(right) wild-type Drosophila

Mark on their abdomen - Alternating dark and light bands can be seen on the entire rear

portion of the female; the last few segments of the male are fused.5

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DORSAL VIEW

Figure-4: Dorsal view of Drosophila

Why use Drosophila?

1. They are small and easily handled.

2. They can be easily anesthetized and manipulated individually with unsophisticated equipment.

3. They are sexually dimorphic (males and females are different), making it is quite easy to

differentiate the sexes.

4. Virgins fruit flies are physically distinctive from mature adults, making it easy to obtain virgin

males and females for genetic crosses.

5. Flies have a short generation time (10-12 days) and do well at room temperature.

6. The care and culture of fruit flies requires little equipment, is low in cost and uses little space

even for large cultures.6

MATERIALS

- Drosophila Melanogaster (Male-Female)

- Drosophila Medium (Each vial contain 10 mL Media and 10 mL Distilled water)

- Anesthefly Solution

- Vial Tube With Sponge Cover

- Soft Paint Brush

- Marker (pen)

- Magnifying Glass

- q-tips

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METHODS

For Anesthetizing system

At the beginning, anesthefly solution was dropped on the cotton which placed under the etherizer cap

and closed the bottle for a few seconds until the ether gas fulfill the entire bottle. Then, the base of the

bottle was stroke lightly on the palm of the hand so that the flies will drop to the bottom. Next, the

bottle cap was removed, quickly replaced it with mouth of etherizer, the bottle was inverted over the

etherizer and shaked the flies into the etherizer. Didn’t invert the bottle over the etherizer because the

ether is heavier than air and it could flow to the culture tube and kill the larvae and pupa. Both

etherizer and culture tube were inverted and stroke slowly until the adult Drosophila drop down in to

the bottom. The flies were then subjected to the ether for a minute or until they ceased moving. After

that, the etherized flies were transferred on the A4 paper. The etherized flies were examined with a

magnifying glass. A soft brush was used for moving the flies about on stage of the magnifying glass.

Finally, after finishing our experiment, the Drosophilas were discarded in a morgue. After this step, a

check was made in 5 hours. Drosophila was separated according to sex. Then, separated flies were put

in different mediums. Meanwhile, medium culture prepare was mixed 10 mL of media and 10 mL of

distilled water. (Drosophila that were not controlled in 5 hours, morgue was thrown. Because maybe

they were not virgin.)

Figure-5 : Drosophila's Medium Culture

Procedures for monohybrid crosses

Until the crosslink (Wild Type x Sepia) step, we were obtained 3 female and 11 male (wild type). For

monohybrid crosses were used 6 wild types (red eyes, spread (normal) drosophila) and 6 sepia (sepia

eyes, normal wings). 6 male (wild type) Drosophila and 6 female (sepia) Drosophila were shifted into

the vial which contains new medium and the vial was closed with the cotton.

Culture Medium

Pupa Formation

Drosophila

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Figure-6: Cross of Sepia and Wild Type

The rest was killed and the traits were observed. The vial was held horizontally, until the Drosophila

woke up.

Figure-7: The appearance of Drosophila unconscious

Drosophila was so held for 2 weeks. Then, Drosophila was kept at 18 oC. So that their development

will slow down.

Temperature cycling

It is possible to maximize the number of virgins by using temperature cycling. When cultures are

maintained at a temperature of 18°C, development is slowed so females will not mate enclosure.

Totally, we were obtained 19 female Drosophila and 20 male Drosophila. Drosophila to until this

stage constitute the F1 phenotype.

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RESULT

Monohybrid Crosses

Figure -8 :Wild type Drosophilla melanogaster7 Figure -9: Sepia Drosophila melanogaster7

The crosses between wild type (female) × sepia (male)

Se+ is dominant allele for wild type

se is recessive allele for sepia

male normal eye (wild type) female sepia eye

Parent se+ste+ × sese

Gamete

F1 se+se

All wild-type

During test Mendel’s Law of Segregation, we were examined the inheritance of eye color by crossing

two pure breeding strains of Drosophila melanogaster that is wild type and sepia 8. We determined

which allele is dominant by setting up the cross se+se+ males × sese females as described above.

Until to crosslinker for F1 (Parents of F1), we were obtained 24 male (wild type) and 16 female (wild

type). F1 crossed result 3 females and 11 males were obtained.

se+ se

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Table 1: Result of Drosophila’s Numbers

DROSOPHILA TOTAL OBTAINED Total VirginParent (Male) 24 11 (virgin)Parent (Female) 16 3 (virgin)F1 (Male) 20F1 (Female) 19Using for Cross F1 (Wild Type) Male

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Using for Cross F1 (Sepia) Female

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DISCUSSION

In this experiment, Parental generation (the parents of F1), 24 male and 16 female Drosophila were

obtained. However, in the F1 crossover phase, there were only 3 females and 11 male Drosophila.

The reasons for the decrease of flies:

1) The flies need to be inspected every 5 hours, against a possible mating. But, me and my group

of friends have not done the checks regularly. At the controls over 5 hours, we put female

Drosophila in morgh and put their male Drosophila in male medium culture (Against a

possible mating). If the controls are not exceeded for 5 hours, we put the females in the female

medium.

2) Drosophila should be careful when taking them to the medium when they are unconscious,

because the Drosophila stick to medium culture and died there. That's why, when the flies are

unconscious and the tube should be on the horizontal side. However, when we were

experimenting, we did not pay attention to this step.

3) Flies on the medium culture may have died from oxygen deficiency.

4) During the anesthesia procedure, when the vial hit the bench, medium culture was poured.

Puppies in the media died. Living Drosophila may have been died the same reason.

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Figure-10: Showing the poured Medium Culture

The reasons I have mentioned above and there may have been a number of decrease in flies due to

many reasons I can not count.

CONCLUSION

In this experiment, I learn on how to conduct a genetic experiment which spans of generation and

learn how to design genetic crosses to illustrate segregation, independent assortment and sex-linkage.

There are four stages of Drosophila melanogaster life cycles that are egg, larva, pupa, and adult. From

study of its life cycle, I'm able to perform this experiment. I can differentiate the male and female of

Drosophila melanogaster based on several characteristic such as size of adult, shape of abdomen,

markings on abdomen or etc.. This making easier for me to differentiate them especially in the

experiment about sex-linkage.

REFERENCES

1) Paul Arnold (2009). Human Genetics and the Fruit Fly Drosophila Melanogaster. Retrieved

March 29, 2010, from http://www.biol.org/DrosPics.html

2) Celeste A. Berg, Ph.D. University of Washington, from http://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

3) Christin E. Arnini (1996). Using Drosophila to Teach Genetics. Retrieved March 29,

2010,http://www.google.com.my/search?hlen&qdrosophila+melanogaster+phenotypes&revid

4) Picture found in http://gfe.uni-muenster.de/Media/FindMediaOutput.php?thema=Genetics

5) Retrieve on 8 April 2010 at ,

http://www.mun.ca/biology/dinnes/B2250/DrosophilaGenetics.PDF

Poured Medium Culture

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6) Celeste A. Berg, Ph.D. University of Washington, from http://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

7) Quoting from this site:

http://www.dreamessays.com/customessays/Science%20Research%20Papers/11452.htm

8) Quoting from this site:

http://www.biologyjunction.com/lab_7_sample_3_fruitflies.htm