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 sub- culturing. 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. © The Exploratorium, www.exploratorium.edu Below shows the clear differences in genitalia between male (left) and female (right) flies. © FlyMove 6 Mutants White Location Sex-linked (X chromosome)