The Fly Manual a Guide to Working with Drosophila

The Fly Manual A guide to working with Drosophila

Produced for

LiveGene!

by the Tauber Lab

www.tinyurl.com/livegene Table of Contents INTRODUCTION ...... 3 THE ORGANISM ...... 3 WHY THE FLY? ...... 3 THE LIFE CYCLE ...... 3 WILD-TYPE AND MUTANT STOCKS ...... 4 SEXING FLIES ...... 5 MUTANTS ...... 7 WHITE ...... 7 CURLY ...... 7 YELLOW ...... 7 EBONY ...... 8 EYES ABSENT ...... 8 DICHAETE ...... 8 PROTOCOLS AND TECHNIQUES ...... 9 VIRGIN COLLECTION ...... 9 TRANSFERRING FLIES ...... 10 ANAESTHETISING FLIES ...... 11 FOOD PREPARATION ...... 12 GENERAL FLY MAINTENANCE ...... 13

2 INTRODUCTION The Organism Drosophila melanogaster, commonly known as the fruit fly, is found through out the world, and its natural habitat is rotting fruit. Raised at 25°C in rich culture medium in the laboratory, flies complete their life cycle in less than 2 weeks, and a single female can lay several hundred eggs. Flies are small enough to be raised in large numbers in a confined space, but large enough for morphological, and more recently, behavioural, mutants to be easily recognisable. Biochemical differences, too, can be identified in single flies.

The haploid chromosomes number is four. In certain larval tissues, notably the salivary glands, the chromosomes are very large (polytene chromosomes) and have been fully described in terms of their easily visible banding patterns. This makes Drosophila an excellent subject for the study of chromosome structure and function, and of gene action & its control.

Why the Fly? The fruit fly was originally regarded as a menace, especially to farmers; however since Nobel Prize winner Thomas Hunt Morgan began to untangle their genetics with the discovery of the first mutant, white, the humble fly has become one of the forerunners in research. Now it is used as a ‘model system’ for animals – studying a range of topics from development, behaviour and even the basis of human disease. The fly has even been used to study alcoholism and as the starting point for drug trials in human medicine!

And many of the reasons the fly is a perfect fit for research can be carried over for simple classroom experiments. Being small (approximately 3mm long) large populations of flies can be stored in minimal space, and their needs are relatively simple resulting in a lower cost maintenance. It’s short life cycle allow for a fast turnover of generations to study. And now it has the backing of many decades of research lending the wealth of knowledge of mutations and methods to draw upon.

The Life Cycle The eggs hatch about 24 hours after being laid, and 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 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-coloured, soft and elongated. Within an hour or two the

3 wings expand and the chitinous exoskeleton hardens and darkens in colour. This life cycle is illustrated in the diagram on the next page.

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. Once inseminated, a female will store sperm in organs called spermathecae, and these sperm will fertilise all the eggs she lays.

Egg 24 hours

Adult Larva

4 - 5 days Pupa 4 - 5 days

Wild-type and mutant stocks A stock of Drosophila is said to be wild-type if the ﬂies are similar to those found in natural populations. Such stocks have been cultured in the laboratory for many years; at regular intervals, a small number of ﬂies are transferred to a fresh culture bottle. Such stocks are usually "true breeding“ for all wild-type characteristics, though they may still carry some "hidden" genetic variation due to recessive mutations. Very rarely, a mutant individual may be found in the stock, but this will be removed from the wild-type stock during subculturing.

Since D. melanogaster was first used for genetic research at the beginning of this century, thousands of different mutant types have been identified and isolated, many of them induced by X-rays or by chemical mutagens. A "mutant stock" is a culture that breeds true for one or more particular mutant characteristic. Each mutant stock has been "constructed" by special selection methods or crosses at some time in the past and it is now carefully maintained by repeated subculturing.

The characteristics of a mutant stock may be due to a mutant form of a single gene or to several different genes, depending on how the stock has been constructed. Genetic analysis of crosses between mutant and wild-type stocks is necessary to determine the number of genes involved.

4 Sexing flies In order to set up crosses, it is important to be able to tell male flies from female flies. With a little practice, it is quite easy to tell the difference between the two Drosophila sexes.

There are several characteristics that differentiate males from females. The most simple characteristic to use to differentiate the two is to look at the genitalia of the flies. Males have dark, rounded genitalia at the tip of their abdomen, whereas females have light, pointed genitalia.

Although the genitalia should serve as the primary sexing characteristic, it can be useful to use others as a guide as well. Male Drosophila are generally smaller than their female counterparts, and have a darker abdomen. The posterior segments of the Drosophila female are only pigmented in their posterior halves, whereas these segments are almost completely pigmented in the males.

The males also have a unique characteristic on their forelegs; the sex comb. The sex comb is a small patch of bristles visible on the forelegs of the male and is perhaps the most reliable method of sexing the flies, but may be too time consuming in practice. These differences are summarised in the table below.

Females Males Body shape Pointed abdomen with a Rounded abdomen “spike” on dorsal surface at rear Colour Each abdominal segment Rearmost abdominal carries a narrow dark band segments almost uniformly dark Genitalia Few structures visible Complex structures visible on ventral surface Sex combs None A short row of thick, closely spaced bristles appearing as a dark mass on the fourth segment of the front legs (often seen best with fly lying on its back)

5 To help demonstrate, presented below is a picture of a female wild type fly (left), and a male wild type fly (right). The differences in pigmentation are clearly visible between the male and female.

Below shows the clear differences in genitalia between male (left) and female (right) flies.

6 Mutants White

Location Sex-linked (X chromosome) Description Mutations in the white gene lead to flies with white eyes instead of the normal wild type red eyes.

Curly

Location Autosomal (chromosome 2L) Description Flies with mutations in the curly gene will have wings that curl upwards and outwards. If kept in 18c, the flies will appear similar to wild type flies. The expression of the curly-winged phenotype will increase with temperature, making this a good trait to demonstrate genetic-environmental interactions. © The Exploratorium, www.exploratorium.edu

Yellow

Location Sex-linked (X chromosome) Description Mutations in the yellow gene produce flies with much paler bodies than wild type flies which look yellowish in colour.

Note: the pictured flies also have a white mutation © The Exploratorium, www.exploratorium.edu

7 Ebony

Location Autosomal (chromosome 3R) Description A mutation in the ebony gene causes the body colour of the flies to be much darker than in the wild type. The dark body phenotype is easily visible upon emergence, but will darken with age.

Eyes absent

Location Autosomal (chromosome 2L) Description Mutations in the eyes absent gene lead to flies with missing eyes - a very easy phenotype to see.

Dichaete

Location Autosomal (chromosome 3L) Description The main phenotype caused by a mutation in the dichaete gene is that the wings of the mutated flies are extended away and above from the body.

8 Protocols and techniques Virgin collection In order to perform crosses, all of the females used must be virgins. Virgin females (denoted by the symbol ) have several defining characteristics that separate them from mated females: 1. Very pale 2. Dark spot seen in abdomen (the meconium, waste products remaining from pupation). 3. Distended, large abdomen Note that very young, newly eclosed, females can have their wings folded and the abdomen may not have inflated yet. One of the best indicators for a virgin female is the presence of the meconium in the gut.

The flies eclose early in the morning, therefore this is the best time to collect virgins however it is possible later in the day. If this is an issue, the safest method to collect virgins is to empty the vial of flies a few hours prior to collecting virgins. After eclosion, the flies present are likely to be virgins for approximately 8-10 hours, although they will be more difficult to identify as they mature.

As newly eclosed males are also very pale, and can also have visible meconium be sure to check the genitalia of the fly!

9 Transferring flies Transferring flies from one vial to another is a simple process that becomes very easy with practice. Transferring flies simply requires a vial of flies and a new, empty vial (with food already at the bottom).

Protocol 1. Softly but firmly tap the bottom of the vial of flies onto a surface such as a mouse pad; this will cause the flies to fall to the bottom of the vial and become momentarily stunned. 2. Remove the plug from both vials and put the empty vial on top of the vial containing your flies. 3. Holding the vials together, invert them and again tap them onto a surface so the flies will fall into the new vial. 4. Insert plugs back into both of your vials.

10 Anaesthetising flies To anaesthetise the flies using the cooling method, you will need the following equipment:

1. Ice container (box, bucket, etc) 2. Cool packs (ideally with a flat surface to support a petri dish) 3. 50ml falcon tubes. 4. A plastic funnel compatible with the 30ml centrifuge tubes.

Protocol 1. Funnel the flies into a 30ml centrifuge tube 2. Place the tube into the ice container for around 10 minutes (until the flies are not moving) 3. Place a petri dish on top of a cool pack (to create a cooled surface upon which to work with the flies) 4. Tap the flies out onto the cooled petri dish

Notes The flies will generally stay anaesthetised for a period of 20-30 minutes, but will wake up rather quickly once they warm up.

11 Food Preparation To make 200ml of fly food (approximately 25-30 vials), you will need: • 200ml water • 9.2g sugar • 9.2g yeast • 2.5g bio-agar • 2ml 20% nipagen in ethanol

Fly food protocol: 1. Create a 50ml master mix of nipagen dissolved in ethanol; 50ml 100% ethanol and 10g nipagen. Wait until dissolved. 2. Mix together 9.2g sugar, 9.2g yeast, 2.5g bio-agar, and 200ml of water. Do not add the nipagen solution yet. Mix by swirling. 3. Once mixed, put into microwave without a lid on full power for around 2 minutes, until the mixture begins to boil. Note: if the mixture is left to boil in the microwave for too long, it will rapidly overflow so supervision is required whilst microwaving. 4. Cool the microwaved solution to room temperature. 5. Add the nipagen-ethanol solution. 6. Pour into empty Drosophila vials before it sets.

12 General fly maintenance Keeping your own stocks is simple; the flies need to be given: • Fresh food • A reasonable temperature • Sensible lighting

Food If the flies are kept at 22-25c, they need to be moved to fresh vials approximately once a week. If the flies are kept at 18c or lower, they need to be moved to fresh vials approximately once every three weeks.

Temperature At 25c, 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.

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.