Drosophila melanogaster
Fruit flies
Common Name : Fruit Fly
Scientific Name : Drosophila melanogaster
Eukaryote Insect
Biosafety level : BSL1
Benefits and applications as model organism
Drosophila is a versatile, low maintenance and non-harming model organisms, they can be
easily used in all fields of life sciences like Genetics, Biotechnology, Cancer biology, Genomics,
Reproductive biology, Developmental biology, Micro chemical studies, and ecology.It has turned
out as one of the best metazoan insect model organisms. It is one of the best model organisms
for the biological studies ranging from molecular genetics of diseases to the ecosystems and up
to the evolutionary scales.
Today, visible mutants, chromosome mapping, availability of complex genetic networks by
multiple genome sequences (the 12 genomes project), systematic gene disruption or
knock-down (RNAi stock library), microarray analysis, protein interaction maps and the FlyBase
integrated database have made Drosophila a sought after model system.
We are able to synchronize the life cycle in captivity by seeding new embryos, feeding
larvae/flies and sacrificing the adult flies at precise times. This synchronization of fly population
is particularly useful for developmental studies
Other benefits include
- They are small and easily handled.
- They can be easily anesthetized and manipulated individually with unsophisticated
equipment. - They are sexually dimorphic (males and females are different), making it quite easy to
differentiate the sexes. - Virgins fruit flies are physically distinctive from mature adults, making it easy to obtain virgin
males and females for genetic crosses. - Flies have a short generation time (10-12 days) and do well at room temperature.
- The care and culture of fruit flies requires little equipment, is low in cost and uses little space
even for large cultures.
In terms of developmental biology, Drosophila has been used to model development and aging,
disease and homeostasis, stem cells and differentiation, neurogenesis and neuronal generation.
Drosophila is used in neuroscience because of its genetic tractability, complex behaviors,
well-known and simple neuroanatomy, and many orthologues to human genes.
Drosophila share 75% of genes that cause diseases in humans and are used as a
complementary model system, along with other animal models, to understand disease
progression and immunology.
Maintenance
Drosophila are ectothermic insects whose body temperature changes with the ambient
temperatures. These insects can easily survive between 12–21 degrees Celsius. Temperature
impact on their viability, fertility, developmental period, foraging activity, feeding, and breeding
could easily be seen in laboratory stocks and distribution and population dynamics under field
conditions.The easiest way to grow flies is at room temperature. However, the optimum rearing
condition is a temperature of 25°C and 60% humidity. In these conditions generation time is
shorter (9-10 days from egg to adult).
Large containers known as fly "population cages" are used to cultivate flies in a laboratory
setting . These cages consist of a cylinder made of plastic covered by a net on both sides to
allow the introduction of the food inside the cage without the flies escaping.
Life Cycle of Drosophila
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).
Females become sexually mature 8-10 hours after eclosion
- The generation time of Drosophila melanogaster varies with temperature. The above cycle is
for a temperature of about 22°C (72°F). Flies raised at lower temperature (to 18°C, or 64°F) will
take about twice as long to develop. - Females can lay up to 100 eggs/day.
- Virgin females are able to lay eggs; however they will be sterile and few in number.
Seeding Embryos
The cycle starts with 1.5 g of collected material (a mixture composed of embryos and/or some
first instar larvae) from the previous cycle. This material will be placed in a plastic container.with
an active yeast mixture until the pupal phase. The container will be closed after the introduction
of the biological mixture of embryos and larvae with the lid to avoid allowing the larvae to
escape. It is necessary to make holes into the lid in order to allow air circulation. To avoid having
larvae escape through the holes, foam plugs are used. Finally, It is recommended to wear
gloves and lab coats, not only in this section but also in the whole protocol, to avoid getting dirty
or staining clothes with bleach.
Set up the plastic container.
Make three square holes with a razor blade in the plastic container lid of approximately 2.2 cm.
Add tape (3/4 inch labeling tape) to the four corners of each hole to ensure a tight fit for the
foam plugs (50 x 55 mm, d x l), and then place one foam plug in each hole.
Cover the inside of the plastic container with plastic wrap. Over this film, add a layer of cotton to
cover the bottom of the plastic container. Tear the cotton with your fingers.
Prepare the fly food.
Add 333 ml of deionized water in a 500 ml beaker. While stirring the beaker with a magnetic bar,
add 167 μl of propionic acid and 1.08 ml of phosphoric acid, from the stock solutions (99.96%
and 85%, respectively).
Slowly add 77.5 g of active dry yeast, avoiding big clumps. After dissolving the dry yeast, add
38.8 g of sucrose. NOTE: Sucrose should be the last ingredient to be added because
immediately after adding this component, fermentation will begin.
Immediately after dissolving the sucrose, pour the food over the cotton, and make sure to cover
the cotton evenly. Close the plastic container with the lid to avoid escaped flies within the
laboratory contaminating the food.
Resuspend 1.5 g of the harvested embryos (not dechorionated) from the previous cycle with 5
ml of 70% ethanol. Cut in half two filter papers and distribute the biological mixture evenly over
the 4 pieces using a spatula or a transfer pipet with a wide tip (cut if necessary). Lay the filter
papers on top of the soaked cotton and close the lid. Finally, incubate the plastic container at RT
(24ºC) and humidity (35%) until the pupa stage. NOTE: The plastic container should not be
placed in a humid chamber, otherwise this will encourage the growth of bacteria.
Collecting virgin females
It is important to recognize the time factor involved for obtaining
virgins. Females remain virgins for only 8-10 hours after enclosure and must be collected within
this time frame. Females have the ability to store sperm after a single mating, so if the female
for a cross is not a virgin, you will not know the genotype of the male used for your cross.
It is strongly suggested that you obtain extra virgins in case a mistake is made in identification
or the fly dies before mating and egg laying can occur. In a strong culture, multiple virgin
females should be easily obtained. Although females are able to lay eggs as virgins, they will be
sterile and no larvae will be produced. Below are three ways to obtain virgins, the ‘removal
method’ being most encouraged for beginners.
Anaesthetisation
The least harmful to the flies is either carbon dioxide or cooling anesthetizing. Of these two
choices, cooling is the simplest, requiring only a freezer, ice and petri dishes. In addition, it is the
only method which will not affect fly neurology, therefore behavior studies may begin after the
flies have warmed up sufficiently.Carbon dioxide works very well, keeping flies immobile for long
periods of time with no side effects, however CO2 mats (blocks) are expensive and a CO2
source (usually a bottle) and delivery system (vials and clamps) are necessary, increasing the
costs
Anaesthetising flies
Mutagenesis
The three general techniques for mutagenesis in Drosophila are mutagenesis by irradiation,
chemical mutagenesis, and genetic mutagenesis (i.e., by transposon insertion). The choice
between irradiation and chemical mutagenesis is determined by the objective of the experiment.
Irradiation is done by UV exposure or X-ray source while Ethyl Methanesulfonate or
Ethylnitrosourea are used as effective chemical mutagens for drosophila.
3-5-day-old males are mutagenised. After treatment, the males are mated immediately to
harems of virgin females (usually as 20-pair bottles). These cultures should be transferred daily
for 6 days. They can then be discarded or the males removed and the females further
subcultured; this ensures that only post-meiotic stages are sampled and will avoid recovering
clusters of identical mutations. Bottles should be labeled in such a way that identification of
progeny from the same batch of parents is possible.
Harvesting embryos
Harvesting is two days after eclosion by placing a new molasses tray. Embryos are observed as
white dots.
Use a large paintbrush and distilled water gun to wash embryos and yeast off of molasses.
Discard plates and molasses into a biohazard box.
Pour solution carefully through a three sieve set (coarsest sieve 30 on top, 40 in the middle and
100 on bottom). Wash clumps on top level with a distilled water gun until no clumps remain. The
adult flies and 3rd instar larvae will be retained in the sieve 30.Rinse the autoclave tray with
distilled water and pour the rinse water through the sieves. Remove top sieve and repeat the
process if yeast clumps remain. Most of the 1st and all of the 2nd instar larvae will remain in the
sieve 40.
Remove the second sieve. The yellowish material in the third (bottom) sieve is the mixture of
embryos and small 1st instar larvae. Use a distilled water gun to move all the eggs to one side
of the sieve. Collect them with a spatula and weigh them.
During these cycles the collection yield of embryos/larvae ranges between 7 and 13 g. Use only
1.5 g of this material to start a new cycle (section 1). Use the rest of the embryos for further
processing or can be discarded.
Crossing flies
Once females are deemed virgins, add males. When setting up crosses, a 3:1
ratio of virgin females to males is ideal. Generally, males will mate more efficiently if they have
matured 3 days or longer. Be sure to select robust, healthy males; the older the flies, the lower
the mating efficiency. Mating occurs quickly and the behavior is interesting to watch, but will not
be addressed here. Females begin laying fertile eggs soon after mating. Refer to the life cycle
chart for evidence of F1 larvae. Remove adults once it has been established that enough larvae
are present (typically 7-8 days after the cross) since you may not be able to distinguish parents
from the F1 generation.
Euthanizing
This is an unfortunate necessity when using flies. A bottle or beaker with soapy water, or
mineral oil is generally used. Dump anesthetized flies directly into the soapy water or mineral oil
where they drown. A bottle (beaker, or screw-capped jar) filled with ethanol or isopropanol can
also be used as a morgue.
Maintaining Stocks
Most large fly laboratories maintain stocks that are not in everyday use at 18oC on a 4-5-week
generation cycle. Stocks should be kept as two to four independent cultures, and it may be
convenient to keep these on alternating generations, 2 weeks apart. Stocks are normally
maintained in vials.
Most stocks can be kept by dump-transfer of flies to fresh vials. However, it is important to avoid
overcrowded cultures, and only 20 or so flies should be transferred. It is good practice to inspect
the flies on transfer, to ensure that both sexes are present and that their phenotype is as
expected.
Freezing embryos
The embryos not utilized for continuation of the population can be used immediately for
experiments or alternatively can be frozen at -80 ºC. For both options, the embryos may need to
first be dechorionated.
For dechorionation, wash the embryos for 2 min in 50% bleach solution and rinse thoroughly
with distilled water.
To freeze the embryos, first dry by pressing firmly with a paper towel, and then weigh and place
them, with the help of a spatula, into a 15 or 50 ml conical tube. Finally submerge the conical
tube in liquid N2 for a few seconds.