An introduction to fruit flies

This guide is adapted from the University of Arizona Department of Biochemistry and Molecular Biophysics General Biology Program for Science Teachers: Drosophila Melanogaster and Mendelian Genetics, by Pete Geiger.

An Introduction to Drosophila melanogaster

Drosophila melanogaster is a small, common fly found near unripe and rotted fruit. It has been in use for over a century to study genetics and behavior. Thomas Hunt Morgan was the preeminent biologist studying Drosophila early in the 1900’s. He was the first to discover sex-linkage and genetic recombination, which placed the small fly in the forefront of genetic research. Due to it’s small size, ease of culture and short generation time, geneticists have been using Drosophila ever since.

Fruit flies are easily obtained from the wild and many biological science companies carry a variety of different mutations. In addition these companies sell any equipment needed to culture the flies. Costs are relatively low and most equipment can be used year after year. There are a variety of laboratory exercises one could purchase, although the necessity to do so is questionable.


Why use Drosophila?

Teachers should use fruit flies for high school genetic studies for several reasons:
     1. They are small and easily handled.
     2. They can be easily anesthetized and manipulated individually with unsophisticated equipment.
     3. They are sexually dimorphic (males and females are different), making it is quite easy to differentiate the sexes.
     4. Virgins fruit flies are physically distinctive from mature adults, making it easy to obtain virgin males and females for genetic crosses.
     5. Flies have a short generation time (10-12 days) and do well at room temperature.
     6. The care and culture of fruit flies requires little equipment, is low in cost and uses little space even for large cultures.

By using Drosophila, students will:
     1. Understand Mendelian genetics and inheritance of traits
     2. Draw conclusions of heredity patterns from data obtained
     3. Construct traps to catch wild populations of D. melanogaster
     4. Gain an understanding of the life cycle of D. melanogaster, an insect which exhibits complete metamorphosis
     5. Construct crosses of caught and known wild- type and mutated flies
     6. Learn techniques to manipulate flies, sex them, and keep concise journal notes
     7. Learn culturing techniques to keep the flies healthy
     8. Realize many science experiments cannot be conducted and concluded within one or two lab sessions

National standards covered in these lessons:
     1. Organisms require a set of instructions for specifying traits (heredity)
     2. Hereditary information is located in genes.
     3. Combinations of traits can describe the characteristics of an organism.

Students goals:
     1. Identify questions and concepts that guide scientific investigations
     2. Design and conduct scientific investigations
     3. Formulate and revise scientific explanations and models using logic and evidence
     4. Communicate and defend a scientific argument

The genetics of Drosophila are well documented and several public-domain web sites feature the complete annotated genome. Therefore, those teachers or students wishing to see where their mutations occur have a ready reference available.

Since Drosophila has been so widely used in genetics, there are many different types of mutations available for purchase. In addition, the attentive student may find mutations within their own wild-caught cultures since, due to a short generation time, mutations are relatively common compared to other animal species.

     Domain: Eukarya
     Kingdom: Animalia
     Phylum: Arthropoda
     Class: Insecta
     Order: Diptera
     Family: Drosophilidae
     Genus: Drosophila (“dew lover”)
     Species: melanogaster (“dark gut”)

Life cycle of Drosophila melanogaster
Drosophila melanogaster exhibits complete metamorphism, meaning the life cycle includes an egg, larval (worm-like) form, pupa and finally emergence (eclosure) as a flying adult. This is the same as the well-known metamorphosis of butterflies. The larval stage has three instars, or molts.

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.

After the eggs hatch, small larvae should be visible in the growing medium. If your media is white, look for the small black area (the mouth hooks) at the head of the larvae. Some dried premixed media is blue to help identify larvae however this is not a necessity and with a little patience and practice, larvae are easily seen. In addition, as the larvae feed they disrupt the smooth surface of the media and so by looking only at the surface one can tell if larvae are present. However, it is always a good idea to double check using a stereo microscope. After the third instar, larvae will begin to migrate up the culture vial in order to pupate.


Care, Maintenance and Manipulation of Drosophila

In order to incorporate fruit flies in the classroom, it will be necessary to maintain cultures of flies for manipulation in crosses and as a backup for any mishaps which may occur. Culturing is very easy and it is recommended to have students maintain their own cultures of flies. In that way, each student or group would be directly responsible for the care and long-term maintenance of the flies, including making large culture populations for their crosses. When directly involved, students gain proficiency and a greater understanding of the flies requirements and behavior. The teacher should remain as coach, not lecturer, assisting students in techniques. The instructor needs to maintain stock cultures of all strains and mutants used by students in case the aforementioned unforeseeable incident occurs and student cultures die out or become intermixed. Losing cultures is the exception rather than the rule, and as long as students re-culture their flies on a regular basis and no mass contamination occurs, flies can be maintained for decades.

Bottles and vials
Thomas Hunt Morgan used glass milk bottles for his experiments and, indeed, any container will do, including baby jars and assorted containers. However, for ease of culturing and transferring cultures, uniform bottles and vials are the best approach. Both can be purchased from a biological supply store. Bottles are used mainly for the maintenance of large populations of flies whereas culture vials are useful for maintaining smaller populations and are the preferred container for constructing student crosses. If there is a desire to maintain stock cultures for a long period of time, or to reuse bottles and vials it is important completely clean and sterilize them. This is to prevent outbreaks of pests and diseases.

To clean bottle and vials, first freeze them to kill any flies in them. Remove the food, wash well, then sterilize by autoclaving (for 20 minutes at 121°C and 15 psi; if containers are plastic, be sure they can be autoclaved) or washing in a 10% chlorine bleach solution.

Bottles and vials can be purchased in a variety of sizes and materials. Glass is effective, however if dropped a student could lose 2 weeks of data in a single spill. Autoclaved (sterile) plastic vials are available and are preferable for student use. Vial sizes range from 96 mm by 25 mm to larger sizes, however the smaller size is recommended for making crosses and maintaining small cultures. There are a variety of plugs available from soft cotton to foam plugs. This is a matter of preference and costs, however cotton works fine and can be bought at a local drug store in a pinch.

Where to buy supplies:
Carolina Biological Supply Company, A division of Genesee Scientific

What they look like:

Stereo microscope
Drosophila vial
Drosophila vial
Drosophila bottles

Fly food
The first step in preparing culture vials is adding food media. There are a variety of types of food available for the flies; some require cooking and others are bought already prepared and dehydrated. The latter can be purchased from a biological supply company. This is, of course, much quicker and easier than preparing cooked media, so much so that students can fill their own vials with media. However, it must be completely rehydrated for best results, since this is the only water source for adults and larvae. Therefore, follow the suggestions below to ensure a completely hydrated media:

Dehydrated media
Add dry media to the bottle or vial to about 1/5 to 2/5 volume. Add water until media appears completely moistened. Allow the vial to sit for a few minutes, adding additional water if necessary until the media is completely hydrated. The surface should be moist with a shiny appearance and there should be no spaces in the media. If the media is not completed hydrated, production of vigorous cultures is compromised. Flies may be added minutes after media has been hydrated. Remember to add several grains (but not more) of yeast to the media surface before adding flies.

Cooked media
When dispensing cooked media, it should fill the culture vial, bottle or vial 1/5th to 2/5th full. Keep the media out overnight to cure, keeping the vials covered with cloth to keep wild flies from laying eggs in them. The next day, add yeast and plugs. Refrigerate any unused media vials. Cooked media can be stored in a refrigerator for several weeks. Allow media to warm to room temperature before adding flies. Do not allow media to dry out.

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). Unless equipment is readily available this is unnecessary for successful rearing and crossing of flies. It is preferable to keep flies out of drafts and direct sunlight or heat sources. These will rapidly dry the media, necessitating frequent media changes and the potential to dehydrate the flies.

Anesthetizing flies
The problem with fruit flies is that they fly! Therefore a variety of methods have been developed to anesthetize flies. Include are ether, commercial brands such as Flynap, carbon dioxide, and cooling. Each has its strengths and weaknesses. Ether is flammable, has a strong odor and will kill flies if they are over-etherized (and can anesthetize younger students!). Flynap, from Carolina Biological, is messy and has an odor that some find offensive. Each of these, however, requires low-cost equipment which can be easily purchased. 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. If resourceful, one can use the CO2 emitted from Alka-Seltzer tablets to anesthetize flies for short periods of time. Set up a large test tube with a tube and stopper system. Add water in the tube, then the Alka-Seltzer tablet. Carbon dioxide gas will be emitted.

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.

Anesthetizing flies by cooling
In order to incapacitate the flies, place the culture vial in the freezer until the flies are not moving, generally 8-12 minutes. Dump the flies onto a chilled surface. This can be constructed by using the top of a petri dish, adding crushed ice, then placing the bottom of the petri dish on top. Adding flies to this system will keep them chilled long enough to do each experiment. Simply place the flies back into the culture vial when finished. Flies will “wake up” relatively quickly once off the ice, so keep them cold. There are no long-lasting side effects to this method, although flies left in the refrigerator too long may not recover. Another way to keep flies chilled is adding water to zip-lock type freezer bags, place in the freezer with a petri dish nestled on the bag, and allow to freeze.

Transferring flies from one vial to another
Flies should be transferred every 10 to 14 days. Students should maintain a backup culture of their flies and the instructor should maintain backup stock cultures of all fly strains. There are two basic ways to transfer flies when forming new cultures. One requires no anesthetizing but quick hands.
A) Place a funnel in the mouth of a fresh culture vial that already has media added. In the old vial (the one with flies in it), gently tap the flies down by softly tamping the vial on a soft surface, such as a mouse pad. The flies will fall to the bottom and remain there for a few seconds (no more than that!), enough time to quickly take the plug off the vial, invert it into the funnel, and gently tamp, together, the two vials to force flies down into the new vial.
B) An alternative way is to put the flies in the freezer for about 8 minutes. This will cause the flies to fall into a state of stupor. After placing a funnel on the new vial, invert the vial with motionless flies into the funnel. This is not as much fun but you won’t have any flies flying around the classroom.

Sexing flies
It is quite easy to tell males from females and with a little practice students will become confident of their ability to do so. Notice that males are generally smaller and have a darker and more rounded abdomen. The coloration of the abdomen is the easiest to recognize. In addition, males have tarsal sex combs on their first pair of legs. These are black and very distinctive but can only be seen under relatively high magnification. With a little practice, by looking at the abdomen students will become proficient in accurately sexing flies. Sexing flies is critical when making crosses, so be sure student are confident in identifying the difference between the sexes. In order for students to feel comfortable sexing flies, give or have them obtain 25 or more mixed sex flies and allow them to sort the flies into two piles, male and female. Other students in the group and the instructor should verify the sorting. Each member of the group should be able to sex flies.

Pictures of males and females

Ventral view of a male (top) and female (bottom).

Lateral view of a male (top) and female (bottom).

Note the darker abdomen and more rounded appearance of the male. Females also tend to be larger.

Collecting virgin females
While it’s a simple matter of placing virgin females with males, it is important to recognize the time factor involved for obtaining virgins. Females remain virgins for only 8-10 hours after eclosure and must be collected within this time frame. NOTE: 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 lying 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.

Removal method
Remove all flies 8-10 hours before collecting (generally this is done first thing in the morning). Visually inspect surface of food to ensure complete removal of flies. After 8-10 hours (usually before you leave work) collect all females that are present. All will be virgins. Place in a fresh culture vial and wait 2-3 days look for larvae. Virgin females can lay eggs, but they will be sterile. Since they are photoperiod- sensitive, females tend to eclose early in the morning. Therefore early collections will ensure the greatest number of virgins for experimentation. However, collection is possible later in the day.

Visual method
Being able to recognize virgin females removes the necessity of emptying culture vials on a timely basis and allows students to collect their own without the necessity of coming to class at odd times of the day. Note that virgin females are much larger than older females and do not have the dark coloration of mature females. In addition, in the early hours after eclosure, there will be visible a dark greenish spot (the meconium, the remains of their last meal before pupating) on the underside of the abdomen.

Temperature cycling
It is possible to maximize the number of virgins in a morning collection by using temperature cycling. When cultures are maintained at a temperature of 18°C, development is slowed so females will not mate until 16 hours after enclosure. By removing flies in the afternoon/evening and placing the vials in an 18°C incubator, 98% of flies obtained in the morning will be virgins. Placing virgins in their own vials for 2-3 days will eliminate those 2% that are non-virgins.

Pictures of virgin males and females:

A newly eclosed female. This is the “wet” stage where the fly is sticky to the touch.   The wings and body have a wet appearance.

A newly eclosed female. This is the “wet” stage where the fly is sticky to the touch.
The wings and body have a wet appearance.

Virgin female showing the meconium (arrow). The meconium is a dark green area and is the remains of larval food

Virgin female showing the meconium (arrow).
The meconium is a dark green area and is the remains of larval food

Comparison between a mature (top) and virgin (bottom) female. This is not long after eclosure; after  4+ hours it becomes more difficult to tell the difference between the two. Note the meconium on the virgin female.

Comparison between a mature (top) and virgin (bottom) female. This is not long after eclosure; after 4+ hours it becomes more difficult to tell the difference between the two.
Note the meconium on the virgin female.

Comparison between a mature (top) and virgin (bottom) male. The coloration is similar to virgin females however the genitalia are distinctly different. The meconium is also found in young virgin males as in females.

Comparison between a mature (top) and virgin (bottom) male. The coloration is similar to virgin females however the genitalia are distinctly different. The meconium is also found in young virgin males as in females.

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.

Killing Flies: The Morgue
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.

Basic Drosophila Genetics Nomenclature and Definitions

Drosophila melanogaster flies have 4 chromosomes.
The genotype is written as:

Chromosome or Chromosome / Chromosome

This common nomenclature shows one chromosome on top and its homologue on the bottom, as the chromosomes would appear during meiosis when contributing gametes.

When writing the genotype, in general, chromosomes are separated with a semicolon.

X chromosome; chromosome II; chromosome III; chromosome IV

Wild-type is denoted as “+” or WT

Dominant mutations are written with a capital letter:
For example: Bar or B

Recessive mutations are written with a lower case letter:
For example: white or w

Mutations are alleles (alternative forms of a gene occupying a given locus on a chromosome) that are inherited with chromosomes.

Homozygote – An individual with the same allele at corresponding loci on the homologous chromosomes.

Heterozygote – An individual with different alleles at corresponding loci on the homologous chromosomes.

Genotype – The genes that an organism possesses.

Phenotype – The observable attributes of an organism.

P1 – Parental generation.

F1 – Filial generation, or offspring generation. F1 is the first offspring generation.

F2 – The second offspring generation.

Other great web resources:

Gerard Manning wrote a simple introduction to Drosophila genetics.

Genetics on the Fly: A Primer on the Drosophila Model System by Karen G. Hales et al (2015).

Taking Stock of the Drosophila Research Ecosystem by David Bilder and Kenneth D. Irvine (2017).

FlyBase is an encyclopedic resource for Drosophila researchers, with detailed information on fly stocks, genes, mutants, researchers, publications and much more.