Drosophila melanogaster : The wonder fly!

Drosophila melanogaster : The wonder fly! 

Being the most common model organism in Biological Sciences, we present to you a comprehensive account of the fruit fly Drosophila melanogaster.

KINGDOM: ANIMALIA 

PHYLUM: ARTHROPODA 

CLASS: INSECTA 

SUBCLASS: HOLOMETABOLA 

ORDER: DIPTERA 

FAMILY: DROSOPHILIDAE 

GENUS: Drosophila (dew lover) 

SPECIES: melanogaster (dark gut)

−The first documented use of Drosophila in the laboratory belongs to William Castle and his group at Harvard in 1901, and more than a century later, Drosophila melanogaster (or the fruit fly / vinegar /wine / pomace fly / gnats)  has touched nearly all fields of biological research – be it Genetics, Molecular Biology, Embryology, Evolution, Behavior, Aging, Microbial pathogenesis, or Physiology; these tiny flies are everywhere! 

− Thomas Hunt Morgan began using Drosphila in his Genetic Studies in 1908, and in 1915 published his ‘Chromosomal Theory of Inheritance’ in a manuscript entitled Mechanism of Mendelian Heredity. His pioneering work on chromosomes & genes to be the carriers of heredity aptly earned him the title of Father of Drosophila Research and the Nobel Prize in Physiology or Medicine in 1933. 

− The Drosphila genome sequence was published in March, 2000 and since then its sequence and fully annotated genome are publicly available at FlyBase, an online database dedicated to Drosophila genes, gene products, mutations and related literature.

Drosophila melanogaster AS A MODEL ORGANISM 

Drosophila serves as an excellent model organism due to several reasons: 

− They are small, easy to handle/culture and relatively inexpensive. Even large cultures could be maintained in relatively smaller spaces. 

− Can be easily anesthetized for examination and manipulated individually with unsophisticated equipment. 

− Due to Sexual Dimorphism (Physically distinct males and females), their sexes could be easily differentiated. 

− A strong distinction also exists between mature adults and virgin flies, hence they could be readily isolated for genetic crosses. 

− Have a short generation time (10-12 days) and do well at room temperature. 

− Have a very high fecundity rate (females can lay ~100 eggs/day for a period of 20 days). Also, they are externally laid embryos which could be easily manipulated. 

− Have only 4 pairs of Chromosomes (3 Autosomes and 1 Sex Chromosome). 

− Males lack meiotic recombination, which facilitates genetic crosses. 

− One of the most remarkable feature of Drosophila genome is that, it is 60% homologous to that of humans, less redundant, and about 75% of the genes responsible for human diseases have homologs in flies. 

− Availability of strong Genetic Modification techniques and many mutant forms. 

All these reasons coupled with the fact that it is the most studied organism till date, with its genetics well documented, completely annotated and publicly accessible, make it an excellent experimental organism of choice. 

HABITAT 

Drosphila melanogaster has been introduced to every continent including islands (except Antarctica). Although it is believed to be initially belonging to Tropical Regions, currently it inhabits almost all Temperate and Tropical Regions around the world. 

Temperature and water availability play a vital role in its development and adults cannot survive in colder environments at high altitudes. In Temperate regions, they could be found in fruit cellars, or other man-made structures with a large supply of food.

FOOD HABIT 

Contrary to popular belief, fruit flies, as the name might suggest, actually eat the yeasts growing on the fruit rather than the fruit itself. Adults thrive on rotting plants while the eggs are laid on unripened fruits so that by the time the larva develop, the fruit too would have ripened and they could utilize it for nutrition. 

MORPHOLOGY 

− Wild type Drosophila sp. are small, about 2–4 mm long, pale yellow (tan) to reddish brown, with brick-red eyes and transverse black rings across the abdomen.

A fly showing the characteristic body divisions, prominent eyes and wings. Image Source: Fruit flies in the laboratory

− Body is covered with chitinous exoskeleton and divided into head, thorax and abdomen. 

   a. Head bears 2 prominent red eyes, short, characteristic antennae above eyes and mouth parts (sapping type.) 

   b. Thorax bears 3 pairs of legs and a pair of wings (functional) attached to it. 

   c.  Females can be distinguished from males by looking at the number of bands and shape of the abdominal tip. 

− Wings are Di-paired (last segment of thorax has a 2nd pair of rudimentary wings, called Haltere which assist in maintaining balance during flight). Wings beat ~220 times per second. 

− The brick red colour of the eye arise due to 2 pigments – Xanthommatin and Drosopterins.

BEHAVIOUR 

− Show simplistic behaviour and are crepuscular (active at dawn or dusk) in nature. 

− Are easily drawn towards the smell of any food. 

− Are sensitive to air currents and differences in light intensity. 

− Fly away when they sense a shadow or any movement. 

− In culture vials, they have a propensity to fly towards the brightest source of light. 

− Mate indiscriminately with any individual of the opposite sex. (Polygamous nature) 

    a. Females exhibit Mate choice copying. Where if virgin females are shown other females copulating with a certain type of male, they tend to copulate more with this type of male afterwards than naive females (which have not observed the copulation of others). 

    b. Males exhibit a strong Reproductive learning curve. That is, with sexual experience, these flies tend to modify their future mating behavior in multiple ways. These changes include increased selectivity for courting only intra-specifically, as well as decreased courtship times.

MATING & REPRODUCTION 

Both males and females show Polygamy. Males perform a sequence of five behavioral patterns to court females. 

A. Orientation, B. Tapping, C. Singing Males orient themselves while playing a courtship song by horizontally extending and vibrating their wings. 

D. Licking 

Next, the male positions himself at the rear of the female's abdomen in a low posture to tap and lick the female genitalia. 

E. Copulation 

Finally, the male curls his abdomen and attempts copulation. At this stage, Females can either reject males by moving away, kicking, and extruding their ovipositor or spread her wings laterally to allow insemination to occur.

In 15–20 minutes of copulation, males transfer a few hundred sperms in the seminal fluid, which are stored by the females in a tubular receptacle and in spermathecae. Sperms from multiple matings compete for fertilization and the last male to mate with a female, fathers about 80% of her offspring. (Source of Image: The New York Times/Illustration by Leif Parsons)

The female lays the eggs on the fruit surface, with the embryos developing in the egg membrane.

LIFE CYCLE OF D. melanogaster 

Have a complete life cycle (Holometabolous) with four distinct life stages – Egg, Larva, Pupa and Adult. 

The Egg: small (0.5mm long), ovoid, shiny white in colour that could be barely seen with naked eyes. Internal fertilization causes the embryonic development to begin before the eggs are laid (oviposition) and hence these are actually Zygotes. Virgin female flies can also lay eggs, but these are sterile and do not develop. 

2 phases of development: 

a. Embryonic phase: from fertilization to hatching of mobile larva 

b. Post-embryonic phase: comprises larval, pupal and adult stages. 

Larva: In normal conditions of 25°C, eggs hatch into small, white first instar maggots (larvae) in about 1 day. The maggots actively consume food like bacteria, microbes, germs, yeasts, vegetable matter and detritus that may be present on the rotten, decaying fruit substrate and the sugar of the fruit itself. Further, they molt twice into 2nd & 3rd instar larva at 24 hour and 48 hours mark (after hatching) respectively. End of each stage (instar) is marked by a molt. At each molt the entire cuticle including many specialized cuticular structures, mouth armature and spiracles is shed and rebuilt again. The final 3rd instar larva is ~4.5mm long. 

1 st instar larva: Hatching to 1st molt 

2 nd instar larva: 1st molt to 2nd molt 

3rd instar larva: 2nd molt to pupal stage 

Pupa: After ~5 days of growth (after hatching), the 3rd instar larva crawls up to the walls of jar and seeks a dry place to form a puparium around itself. The larva initially enters a ‘Prepupal stage’ in which its white cuticle is slowly hardening and acquiring darker pigmentation (completed after 3 and half hours of puparium shaping). After 4 hours, the animal separates from the puparium and becomes a headless individual lacking wings or legs. This stage is called Prepupa. 

A very fine prepupal cuticle is secreted and surrounds the prepupa. After ~12 hours, pupation occurs in which a typical pupa with head, thorax and abdomen is shaped and the animal is enclosed in 3 membranes. During metamorphosis certain larval tissues and organs are lysed and adult structures are formed from Imaginal discs (undifferentiated embryonic cells which were dormant since the embryonic stage). 

Adult: Once metamorphosis is complete (after ~4 days of puparium formation), the adult fly emerges from the pupa (Eclosion). They are pale & fragile and acquire the normal coloration of adult in a few hours. The difference in colour enables one to distinguish young flies from mature adults. They live for a month and then die.

Development in Drosophila melanogaster, is temperature dependent and duration of life cycle increases in both very cold (18°C & 12°C) and very high temperature. (30°C) 

Shortest Life cycle – at 28°C of 7 days.

VIRGIN FLIES 

For Cross Breeding experiments, it is important to know the genotype of both males and females. Since, females are capable of storing a large quantity of sperm in their spermathecae, in controlled breeding experiments, only those females are used which have not mated before. These are called Virgin females. Females become receptive to mating only after 10-14 hours after eclosion (emergence) hence, sexing & segregation of individuals is usually done at 8-10 hours mark. Virgin males and females are larger than mature adults.

CULTURE PREPARATION

Bottles and Vials are generally used according to need and purpose of experiment. The food medium to raise Drosophila could be either prepared or bought from a commercial facility. Normally, even a mashed banana would do, but it becomes difficult to remove flies from it, hence a solidifying agent is used in medium. 

To prepare a medium, following components are added:

                                                       Substance                                  Quantity 

                                                   Distilled Water                                350ml 

                                                       Agar agar                                     3.01g 

                                                      Corn flour                                     20.5g 

                                                        Sugar                                            44g

                                                        Yeast                                             11g 

                                     10% Nipagine (mould inhibitor)                 3.5ml 

                                              Propionic Acid                                     3.5 ml

Water is boiled and the components are added one by one with constant stirring. The fungicide is dispensed when the solution has appropriately cooled to avoid heat inactivation. As soon as the medium is ready, it is poured into vessels after which it quickly solidifies. Presence of yeast in medium improves the nutritional content and flies thrive better on them. Then, the flies are added and the vessel is plugged in.

Drosophila bottle showing larvae and pupa. Image Source: Jennings, B. H. (2011)

GENOME & SEX DETERMINTION 

Drosophila GENOME

Metaphase chromosomes from dividing cells of larval ganglion (brain) of Drosophila melanogaster: (a) chromosomes of female consist of three pairs of autosomes and one pair of rod‐shaped X chromosomes; (b) chromosomes of male consist of three pairs of autosomes, one X chromosome, and one Y chromosome; bar on photograph: 10 μm (Image Source: Farzana Khan Perveen, Introduction to Drosophila, IntechOpen, 2018.) © 2018 The Author(s). Licensee IntechOpen. This image is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

SEX DETERMINATION

BIBLIOGRAPHY