Drosophila Features, Life Cycle & Culture

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 3 rd year Report On:

2. To know – 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 EYE PROBOSCIS 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. H as 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 d o w e use Drosophila for Culture?

10. Metcalfe,2013 Timeline 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. Place few drops of ether on the absorbent cotton of the etherizer Tap gently the base of the culture bottle on the palm so that the flies will drop to the bottom 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 The flies will cease to move after 30 seconds. Avoid over etherization . Handling the flies Anaesthetizing flies Anaesthetized flies Credits : UPV

18. 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 If the flies revive, re- etherize them. 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

20. 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 .

21. 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)

22. 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

23. 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

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

25. 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

26. 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: UPV Credits: Dragana Miličić 3. Presence of Sex comb

27. 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

28. 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

29. 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