Drosophila melanogaster or the fruit fly is a model organism and can be cultured in lab easily for study of genetics and other branches of biology

1. Drosophila Culture
By RUCHIRA AGARWAL
Bsc(H) ZOOLOGY 3rd year
Report On:

2. To know –
1. the morphological characteristics of Drosophila melanogaster.
2.how to culture Drosophila in lab
3.How to handle the flies for observation
Objectives of the Report

3. • Drosophila melanogaster literally means
Black belly dew lover.
• Commonly called fruit fly
• Small in size (2-3mm)
• Weighs around 0.8-1.5 mg
• Exhibits sexual dimorphism.
Drosophila melanogaster
Credits: NASA/Dominic Hart
Characteristics

4. KINGDOM: Animalia
PHYLUM: Arthropoda
CLASS: Insecta
ORDER: Diptera
FAMILY: Drosophilidae
GENUS: Drosophila
SPECIES: melanogaster
Taxonomic Classification
Credits: NASA/Ames Research Center

5. Body consists of-
Head - Possesses a pair of compound eyes, a pair of antennae and proboscis (mouthparts used
for feeding)
Thorax - consists of 3 segments: prothorax, mesothorax and metathorax
• Each segment possesses a pair of legs
• The wings (forewings) attach to the mesothorax.
• Halteres- a pair of small paddle-like organs used to maintain balance in the adult fly attach
to the metathorax. They are the morphological equivalent of the metathoracic/hind wings.
Abdomen - Striped, abdominal segments possess spiracles and genitalia
External Morphology

6. HEAD
THORAX
ABDOMEN
3 PAIRS OF THORACIC LEGS
HALTERE
FOREWING
COMPOUND
EYEPROBOSCIS
Credits: jeb.biologist

7. Mitotic chromosomes of D. melanogaster, showing euchromatic regions,
heterochromatic regions, and centromeres. Arms of the autosomes are
designated 2L, 2R, 3L, 3R, and 4. Credits: Science Magazine
• Entire genome consists of 139.5 million base pairs and contains around 15,682 genes
• contains 4 pairs of chromosomes – an X/Y pair (Males-XY, Females: XX), and three
autosomes labeled 2, 3, and 4.
• More than 60% of the genome appears to be functional non-protein-coding DNA involved in
gene expression control.
• About 75% of human disease genes have similar genes in the fruit fly
Genome of Drosophila melanogaster

8. • Determination of sex occurs by the X:A ratio of X
chromosomes to autosomes.
Gynandromorph of D. melanogaster in which the left side is female
(XX) and the right side is male (XO) Credits:The Journal of Heredity.
Wild type Female (left) and male (right) Drosophila.
Credits: The Exploratorium

9. • Easy to grow in lab.
• High number of progeny produced facilitates statistical analysis.
• Has a short life cycle and is completed in 10-11 days at 25°C temperature.
• Has only 4 pairs of chromosomes which makes it suitable for genetic studies.
• Has high number of known mutants.
• Special chromosomes like Polytene Chromosomes are used to study the function of
genes in transcription
Why do we use Drosophila for Culture?

10. Metcalfe,2013Timeline of the use of Drosophila melanogaster in the history of medical and scientific research
Polytene chromosomes
Ebony mutants (Left) & Dichaete mutant (right) of D. melanogaster
Credits: The Exploratorium
Credits: The Exploratorium

11. • 1933- T.H. Morgan: role played by the chromosome in heredity
• 1946- Herman Muller: production of mutations by means of X-ray irradiation.
• 1958- George Beadle & Edward Tatum: genes act by regulating definite chemical events
• 1995-Edward B. Lewis, Christiane Nüsslein-Volhard & Eric F. Wieschaus: genetic control of
early embryonic development
• 2004- Richard Axel & Linda B. Buck: odorant receptors and the organization of the olfactory
system
• 2011- Bruce A. Beutler and Jules A. Hoffmann: activation of innate immunity
• 2017-Jeffrey C. Hall, Michael Rosbash and Michael W. Young: molecular mechanisms that
control circadian rhythms.
Following Nobel Prizes have been awarded for research using Drosophila :
Model organism: For Research

12. NASA is studying fruit flies in space to help address the effects of long-duration space missions on an
astronaut’s body and the way the body responds to new and stressful environments.
Approximately 75% of human disease genes have similar genes in the fruit fly, so studying them can help
us understand human biology, too.
Food Changeout Platform with fresh fly food (left in blue)
and Fly Cassette (center) containing the biospecimens.
Credit: NASA / Dominic Hart
The fly cassette is a habitat for the fruit fly.
Credits: NASA / Dominic Hart
Model organism: Research in Space

13. • dissection microscope
• Bottles for culture medium
• Etherizer, Re-etherizer, Powder Funnels,Sponge
• Frosted glass plates
• Di- ethyl- ether in dropping bottles Droppers,
• Fine camels hair brush, Marker pens, Dissection needles, Forceps
• Morgue containing 70% Alcohol
Procedure for Culturing Drosophila in lab
Glassware and materials needed :

14. • Weigh 1 gm of agar and dissolve it in 350 ml of
distilled water
• Weigh corn flour, sugar and yeast to the above
• Boil the mixture and then cool it to obtain a semi solid
consistency. Switch off the gas.
• Add a pinch of sodium benzoate.
• Lastly add propionic acid.
Preparation of Culture medium (food for the flies)
Required material and Procedure :

15. • Properly cooked and hot food in semi– fluid condition is dispensed into
culture bottles covered with muslin cloth, cooled, and solidified.
• The moisture condensed on the sides of the bottles is wiped with dry
filter paper and the bottles are plugged with sterilized cotton plugs.
Preparation of culture
Cotton plug
Culture medium
Credits: UPV

16. To ensure maintenance of the culture, the flies need to be given:
 Fresh Food : If the flies are kept at 22-25°C, they need to be moved to fresh vials
approximately once a week. If the flies are kept at 18 °C or lower, they need to be
moved to fresh vials approximately once every three weeks.
 Temperature: At 25 °C, the generation time (egg to adult) is approximately 10 days.
The lower the temperature that the flies are kept in, the longer they will take to
develop.
 Appropriate Lighting: To encourage the flies to eclose (emerge from their pupae) in
the morning, the lighting should be somewhat natural (light during the day, dark
during the night). This can be accomplished by using natural lighting from a window, or
by turning the light on in the morning and off at night.
Maintenance of Culture

17. 1.Place few drops of ether on the absorbent cotton
of the etherizer
2.Tap gently the base of the culture bottle on the
palm so that the flies will drop to the bottom
3.Remove the cotton plug from the culture bottle,
place it on the mouth of the etherizer, invert the
bottle and shake the flies into the etherizer
4.The flies will cease to move after 30 seconds.
5.Avoid over etherization.
Handling the flies
Anaesthetizing flies
Anaesthetized flies
Credits : UPV

18. 1. Transfer the etherized flies into a filter paper/glass plate & observe them
at10X/ 40X in a dissection binocular microscope &manipulate the flies with a
soft brush or a needle
2. If the flies revive, re- etherize them.
3. All the flies after observation, if not needed further can be discarded in the
morgue.
Handling the flies
Observing the flies

19. Life Cycle of Drosophila melanogaster

21. • The eggs hatch about 24 hours after being laid.
• during the next four or five days the larvae moult twice, progressing from first
instar through second instar to third instar stages.
• At the end of this very rapid growth the larvae leave the food medium and
pupate on any available surface.
• Metamorphosis is holometabolous type takes place in the pupae over a period
of four or five days.
• Shortly before emergence or eclosion, the eyes and wings of the adult fly may
be seen within the pupa case.
• In newly emerged flies, the wings are crumpled and soft and the body pale-
colored, soft and elongated
• Within an hour or two the wings expand and the chitinous exoskeleton
hardens and darkens in colour.

22. • Females are not receptive to the male courtship display until they are about 8 hours
old; we can take advantage of this delay to isolate newly emerged females as virgins
for use in crosses. One of the best indicators for a virgin female is the presence of
the meconium in the gut.
• Once inseminated, a female will store sperm in organs called spermathecae, and
these sperm will fertilize all the eggs she lays.
FlyMove
Adult Male and female flies & virgin females (with distinct meconium)

23. •The embryo: Embryos are small, oval
shaped, and have two filaments at one end.
•The larval stage: The larva look like worms.
They use black mouth hooks to eat.
• The pupal stage: A pupa undergoes four
days of metamorphosis. They form a hard
and dark pupal case.
• The adult stage: Adult flies have a head,
thorax, abdomen, six legs, and two wings.
Identify the different stages of Drosophila development
Egg
Larva
Pupa
Credits: UPV
Credits: UPV

24. To identify males from females following characters are observed:
1. Size of the individual
2. Colour and Size of the abdomen
3. Presence of Sex Comb
4. External genitalia
Differences between Male & Female flies

25. 1. Size of the individual
Females (right) are usually bigger in size than males (left).
Credits: UPV

26. 2. Colour & Size of the abdomen
Credits : ScienceDirect
Posterior end of abdomen in males (left) is dark and rounded
while in females (right) it is striped and pointed.
• Alternating dark and light bands can
be seen on the entire rear portion i.e
females are striped at end of
abdomen which is pointed.
• In males the last few segments are
fused and are black in color & tip of
abdomen is more rounded

27. In males there is a tiny tuft of hairs on the front legs called sex combs which are
entirely absent in females.
Sex comb in male:
a) front leg with sex comb marked with black arrow;
b) sex comb bristles
View of first pair of legs
Credits: FlyMove
Black dots are the sex comb
Credits: UPVCredits: Dragana Miličić
3. Presence of Sex comb

28. 4. External genitalia on abdomen
Located at the tip of the abdomen, the ovipositor of the female is pointed. The
claspers of the male are darkly pigmented, arranged in circular form, and
located just ventral to the tip.
Ovipositor of female (laying eggs)Ventral Abdomen of Male (left) & female (right)
credits: ScienceDirect

29. • All Glasswares, vials, bottles etc should be washed in detergent rinsed in ethanol first ,dried
in an hot oven before commencing with the procedure.
• Ether is flammable, has a strong odor and will kill flies if they are over-etherized so care
should be taken while anesthetizing the flies
• It is preferable to keep flies out of direct sunlight or heat sources. These will rapidly dry the
media, necessitating frequent media changes and the potential to dehydrate the flies.
• Flies should be transferred every 10 to 14 days into new vials.
• It is advisable to maintain a backup culture
• virgin females must be collected 8 hours after hatching to prevent them from mating with
males
Precautions

30. • https://www.nasa.gov/ames/fruit-fly-lab
• https://www.nasa.gov/sites/default/files/atoms/files/fruitflyfs_26oct16.pdf
• https://science.sciencemag.org/content/287/5461/2185.full
• https://www.ncbi.nlm.nih.gov/pmc/article
• http://annex.exploratorium.edu
• https://flybase.org/
• https://jeb.biologists.org/
• https://en.wikipedia.org/wiki/Drosophila_melanogaster#Genome
• https://www.sciencedirect.com/topics/neuroscience/drosophila-melanogaster
• https://www.researchgate.net/figure/Sex-comb-in-Drosophila
• https://www2.le.ac.uk/departments/genetics/research/staff-research-interests/tauber-
research-lab/livegene/fly-manual-1
• https://www.nobelprize.org/prizes/medicine/
References