Drosophila Life Cycle and culturing technique.pdf

Why drosophila is called as Cindrella of Genetics and why it takes away huge attention within the scietific community, along with its life cycle, all the developmental growth stages, tools for culturing them, what are the requirement and precautions should be taken while maintaing its stock are mentioned very well in this presentation.

1. Culture and handling of Drosophila
and study of its life cycle, stages of
growthand identification of
anatomical structures
Presented By: Praddum Kumar Namdev
Research Scholar
Department of Zoology
School of Biological Sciences
Dr. Harisingh Gour Vishwavidyalaya

2. Introduction:
•Drosophila is a genus of small flies belonging to the
Drosophilidae family, commonly called "fruit flies"
•There are around 1,500 diverse species that can thrive
in many habitats.
•One species, D. melanogaster, is extensively used in
genetics and developmental biology research as a model
organism.
•Basic genetic mechanisms are conserved across
organisms, so studying them in Drosophila provides
insights applicable to other species including humans.
Domain: Eukaryota
Kingdom: Animalia
Phylum: Arthropoda
Class: Insecta
Order: Diptera
Family: Drosophilidae
Subfamily: Drosophilinae
Genus: Drosophila

3. Why Drosophila?
•Hardy and easy to culture with simple nutritional needs like fermenting fruit and very little space
required
•Rapid life cycle of around 12 days at room temp allows quick analysis of genetic crosses
•Females lay hundreds of eggs producing large offspring numbers for statistical significance
•Adults are small (3mm) making examination easy, even under low magnification
•Reduced complexity with only 4 pairs of chromosomes (humans have 23 pairs)
•Special giant polytene chromosomes in larval salivary glands allow detailed visualization
•Thousands of genetically defined mutant strains available covering most biological processes
•75% of human disease genes have a functional homolog in the Drosophila genome
•Genome is relatively small (180 million base pairs) compared to humans (3 billion bp)
•Techniques available to induce mutations in specific genes

4. Life Cycle:
•Holometabolous life cycle with 4
stages - egg, larva, pupa, adult
•At 25°C: egg->larva 5 days, larva-
>pupa 4 days, pupa->adult 4 days (total
10-12 days)
•At 20°C the cycle is extended by
several days in each stage
•Temperatures below 20°C or above
25°C can impair viability and
development

5. The Egg
•Oblong shape around 0.5mm
long with hexagonal chorion and
dorsal filaments.
•Micropyle for sperm entry,
female stores sperm from mating to
fertilize eggs at laying.
•Embryogenesis occurs within the
egg producing the larva.

6. The Larval Stages
•3 instar larval stages separated by
molts/ecdysis of the cuticle.
•Larva is translucent allowing
visualization of organs like gut,
Malpighian tubules, gonads.
•Feeding and growth primarily by
increase in cell size rather than
multiplication.
•Imaginal discs are progenitors of
adult organs and grow by cell division.

7. The Pupa
•Puparium forms from tanned larval
cuticle after contractions.
•Within the pupal case, the animal
separates itself as a prepupa to undergo
metamorphosis.
•Most larval tissues break down and
get replaced by histogenesis from
imaginal discs.
•Wing, leg, eye-antennal and genital
imaginal discs develop into their adult
forms.

8. Adult Stage
•Immature pale adult emerges and
darkens within hours
•Females become receptive to mating
around 10-12 hours post-eclosion
•Adults feed on yeasts and decaying
matter using sponging mouthparts
•Under optimal conditions, adults
can live 4-6 weeks

9. Sex Determination
•Males are slightly smaller than
females.
•Male abdomen is darker and rounded
at the tip, female is elongated.
•Males have thick black sex combs on
fore-legs, absent in females.
•Ovarian and testicular rudiments are
visible through larval cuticle in later
instars.

10. 1. Size
•Females are generally larger (10-20%) than males
•But there is some overlap, so size alone is not fully
reliable
2. Abdominal Shape
•Females have an elongated, cylindrical abdomen with
7 visible segments
•Males have a more rounded/tapered abdomen with 5-6
segments visible at the tip
•This is due to fusion of the last two segments in males

11. 3. Markings on the abdomen
•Females have 7 distinct abdominal segments visible dorsally
•Each segment has alternating dark pigmented bands and light inter-band
regions
•This banding pattern extends along the entire abdomen in females
•In males, only 5-6 segments are clearly visible
•The terminal segments appear fused and uniformly pigmented in males
4. Appearance of sex comb
•The sex comb is a rotating, fringe-like row of 10-12 thick black bristles
•Present only in males, on the first tarsal segment of the forelegs
•Serves to grasp the female during mating
•Visible even in newly eclosed adults, but more prominent in older flies
•Can also be seen in late pupal stage through the pupal case

12. 5. Female External Genitalia:
•The ovipositor is the female egg-laying structure located at the abdominal tip
•It appears as a pointed, elongated structure protruding from the 7th abdominal segment
•The ovipositor is lightly pigmented and has an upward orientation when extended
6. Male External Genitalia:
•The male copulatory organs are darkly pigmented and arranged in a circular pattern
•This includes the claspers used to grasp the female during mating
•The male genital structure is located just ventral (underneath) to the abdominal tip
•It appears as a fused, sclerotized ring at the end of the 5-6 visible abdominal segments

13. Male Larvae:
•In late 3rd instar larvae, the developing testes appear as a large, opaque white
mass
•This testicular tissue is located laterally in the posterior 1/3 region of the larva
•It is embedded within the larval fat body and clearly visible through the
translucent cuticle
Female Larvae:
•The ovarian tissue in females appears as a much smaller, thinner white streak
•It is located more medially compared to the male testes
•The ovaries are often difficult to distinguish, especially in early larval stages

14. Tools for Culturing
•Binocular stereomicroscope
•CO2 pad and anesthesia plate or ether
vapor for immobilizing flies
•Supplies like brushes, morgue, etc. for
transferring and discarding flies
•Humidified incubators allow
maintaining constant temperature

15. Stock Keeping
•Healthy stocks can be maintained by transferring a portion to fresh food every 2-3 weeks
•It's wise to retain the old culture for a couple weeks as a backup
•Optimal density is important - overcrowding can cause poor viability

16. Culture Contaminants
•Mite infestations can overtake
cultures by eating eggs and
medium
•Fungi and bacteria create
unhygienic conditions by
overgrowing medium
•Preventive measures include
discarding old stocks, using
inhibitors, autoclaving

17. Culture Conditions
Timing/Temperature:
•Drosophila are poikilothermic (cold-blooded), so their metabolic rates and
developmental times are temperature-dependent
•The optimal temperature range is 20-25°C for normal development and viability
•Below 18°C, development slows significantly taking over 19 days to go from egg
to adult
•Above 28°C, development rate increases but viability is reduced due to desiccation
•Fluctuating temperatures can disrupt biological cycles like circadian rhythms
•Therefore, maintaining constant temperatures in the 22-25°C range in incubators is
recommended

18. Lighting:
•Drosophila are attracted to lights and use visual cues for key behaviors like mating
•A 12hr light/12hr dark cycle syncs their circadian clocks and stimulates activities
•During courtship, males use visually perceivable traits and dances to attract
females
•Light also regulates rhythmic processes like oscillation in brain neurotransmitters
•Using full spectrum lighting or daylight LED bulbs can better mimic natural
conditions
•However, excessive direct light exposure can cause flies to desiccate faster

19. Breeding Methods
•Standard medium contains agar,
cornmeal, molasses, yeast, acid
preservatives
•Adults mate at 1 male : 3 female
ratio to obtain large progeny
numbers
•Larvae consume the solid media,
adults feed on liquids/microbes on
surface

21. Summary
•- Drosophila melanogaster's attributes like fecundity, short life cycle, easy culturing make it a powerful
genetic model
•- Developmental stages, anatomy, sexing methods are important basics
•- Following best practices for food, environment, contamination control ensures healthy stocks
•- Its conserved genetics combined with vast knowledge base enable profound insights