rane {An introduction to Drosophila melanogaster — biology arizona edu — Readabilty Obiotogy.arizona.edu An introduction to Drosophila melanogaster 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 lends itself well to behavioral studies. Thomas Hunt Morgan was the preeminent biologist studying Drosophila early in the 1900's Morgan was the first to discover sex-linkage and genetic recombination, which placed the small fly in the forefront of genetic research. Duc to it's small size, ease of culture and short generation time, geneticists have been using Drosophila ever since. It is one of the few organisms whose entire genome is known and many genes have been identified. Fruit flies are easily obtained from the wild and most 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 area 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. You can anesthetize them easily and manipulated individuals with very unsophisticated equipment. 3. Drosophila are sexually dimorphic (males and females are different), making it is quite easy to differentiate the sexes. 4. It is easy to obtain virgin males and females, as virgins are physically distinctive from mature adults. 5. Flies havea short generation time (10-12 days) and do well at room temperature. 6. The care and culture 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 ey cle 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 twolab www readabilly.comvartclesinaklwr wrane {An introduction to Drosophila melanogaster — biology arizona edu — Readabilty National standards covered in these lessons: Conten 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, Inquiry: Students will 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 known and several web sites feature the complete genome, In additions, many gene loci are known and these, too, are in the public-domain web sites. Therefore, those teachers or students wishing tosee where their mutations occur have a ready reference available. Since Drosophila has been so widely used in genetics, there are many different ty pes of mutations available for purchase, In addition, the attentive student may find mutations within their own cultures since, due toa short generation time, mutations are relatively common compared to other animal species. Classification: Domain: Eukarya Kingdom: Animalia Phylum: Arthropoda Class: Insecta Order: Diptera Family: Drosophilidae Genus: Drosophila ("dew lover") Species: melanogaster ("dark gut") Life cycle of D. melanogaster D. melanogaster exhibits complete metamorphism, meaning the life ey cle includes an egg, larval (worm like) form, pupa and finally emergence (eclosure) asa flying adult. This is the same as the well-known metamorphosis of butterflies and many other insects. The larval stage has three instars, or malts. Life eycle by day 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 time from egg to adult is temperature- dependent. The above cy le is for a temperature range of 21-23 degrees ©. The higher the temperature, the faster the generation time, whereas a lower (to 18 degrees C) temperature causes a longer generation time, www readabilly.comvartclesinaklwr anrane {An introduction to Drosophila melanogaster — biology arizona edu — Readabilty Females can lay up to100 eggs/day. ‘Virgin females are able tolay eggs; however they will be sterile and few in number. After the eggs hatch, small larvae should be visible in the growing medium. Ify our media is white, look for the black area at the head of the larvae. Some dried premixed media is blue to help identify larvae however this isnot 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 iflarvae are present. However, it is always a good idea to double check using a stereomicroscope. After the third instar, larvae will begin to migrate up the culture vial in order to pupate. Next Care, maintenance and manipulation of Drosophila ‘This section contains the following information: 1, Culturing techniques a. Introduetion ». Bottles and vials ©. Media, 4. Environment ¢. Anesthetizing flies £ Transferring flies from one vial to another 2. Sexing flies 3. Collecting virgins 4. Crossing flies 5. Diseases 6. The morgue Culturing techniques Introduction In order to incorporate D. melanogaster in the classroom, it will be necessary to maintain cultures of flies for manipulation in crosses and asa backup for any mishaps which may occur. Culturing is very easy andit 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 ofthe 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 ofall strains and mutants used by students in case the afore-mentioned 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 reculture their flies on a regular basis and no mass contamination ‘occurs (see pests and diseases section), flies can be maintained for decades. Bottles and vials ‘TH 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 www readabilly.comvartclesinaklwr arane {An introduction to Drosophila melanogaster — biology arizona edu — Readabilty container for constructing student crosses. If there is a desire to maintain stock cultures for a long period of time, or toreuse 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 freeze them if there are flies in them. Remove any food, wash well, then either autoclave for 20 minutes at 121 degrees C and 15 psi (if plastic be sure it can be autoclaved) or wash vials in a 10% chlorine bleach solution, Bottles and vials ean 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 tolarger 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 isa matter of preference and costs, however cotton works fine and can be bought at a local drug store in a pinch Where tobuy What they look like Medi ‘The first step in preparing culture vials is adding food media. There are a variety of ty pes 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, somuch sothat students can fill their own vials with media. However, it must be completely rehy drated 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: Add dry media to the bottle or vial toabout 1/5 to2/5 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. Ifthe 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 tothe media surface before adding flies, Cooked media can be stored in a refrigerator for several weeks. Be sure to allow media towarm toroom temperature before adding flies. Do not allow media to dry out, Media should fill the culture vial, bottle or vial 1/sth to2/5th full. Keep the media out overnight to cure, being sure to completely cover the vials with cloth to keep flies from laying eggs in them. The next day, add yceast and plugs. Refrigerate any unused medai vials. Unused media can last up to twoweeks. Where tobuy Recipes to use Environment The easiest way to grow flies is at room temperature. However, the optimum rearing condition isa temperature of 25C and 60% humidity. In these conditions generation time is shorter (9-10 days from egg toadult). Unless equipment is readily available this is unnecessary for successful rearing and crossing of flies. It is preferable to keep out of drafts and direct sunlight or heat sources. These will rapidly dry the media, necessitating frequent media changes and the potential todehy drate the flies. Anesthetizing flies ‘The problem with fruit flies is that they fly ! Therefore a variety of methods have been developed to anesthetize flies. ww readabilly.comvartclesinaklwr anarin ‘An ntoducton to Drosophila melanogaster — biology arizona edu — Readabilty Include are ether, commercial brands such as Fly nap, carbon dioxide, and cooling. Each has its strengths and weaknesses. Ether is flammable, has strong odor and will kill fies if they are ov er-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 002 source (usually a bottle) and delivery system (vialsand 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 ontoa 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 sy stem will keep them chilled long, ‘enough to doeach 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 ziplock type freezer bags, place in the freezer with a petri dish nestled on the bag, and allow to freeze. Where tobuy ransferring flies from one vial to another Flies should be transferred every 10 to14 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 asa 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 isto put the flies in the freever for about 8 minutes. This will cause the flies to fall intoa 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 totell males from females and with a little practice students will become confident of their ability to do so, Notice that malesare generally smaller and havea 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 tofeel comfortable sexing flies, give or have them obtain 25 or more mixed sex flies and allow them tosort 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. www readabilly.comvartclesinaklwr srrane {An introduction to Drosophila melanogaster — biology arizona edu — Readabilty ictures of mal nd females Collecting virgin females While it's 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. Alternatively, it is quite easy to distinguish virgins from mature flies visually. 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. What virgins look like Removal method Remove all flies 8-10 hours before collecting. Visually inspect surface of food to ensure complete removal of flies. After 8-10 hours collect all females that are present. All will be 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 the day gins, Place in a fresh culture vial and wait 2-3, for exper mentation. However, collection is possible later Visual method Being able torecognize virgin females removes the necessity of empty ing 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 degrees 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 degrees incubator, 98% of fies 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, Crossing flies Once females are deemed virgins, add males. 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 lay ing fertile ‘eggs soon after mating. Refer tothe life cycle chart for evidence of Fi larvae. Remove adults once it has been established that enough larvae are present since you may not be able to distinguish parents from the Fi generation. Diseases Purchased cultures are almost always disease-free. However ifyou are collecting wild populations, mites and bacteria may be present. In addition, food may develop molds or bacterial growth. These are generally not a major problem for small-scale use, as in a classroom. However, mites are a major concern. Mites ‘There are two ty pes of mites tobe wary of: food mites and parasitic mites. For both, immediately quarantine the cultures from the rest of the stocks (put them in a separate room) to prevent continued contamination. Mites ean be persistent and debilatory on the health of the flies and success of crosses. www readabilly.comvartclesinaklwr erarin ‘An ntoducton to Drosophila melanogaster — biology arizona edu — Readabilty Food mites Food mites, which donoharm to the flies, need tobe eradicated none+the less. These small mites will be seen crawling on the surface of the food and, toa lesser extent, on the sides of the vial. First, isolate these cultures and destroy them ifyou donot need the flies. Ifyou need the flies, follow this procedure. 1. In an empty vial, add moist paper towels to cover the bottom 2. Add the flies with the mites 3. After an hour, transfer the flies intoa new vial. Invert the new vial over the old one. Fies will climb into the new vial faster then the mites. You may need torepeat this procedure. Remember to keep the affected flies in quarantine until you are positive they are mite-free. rasitic mites ‘The second type of mite is parasitic on the flies themselves. These are evident by looking at the flies and seeing mites on most any body part, including the legs, abdomen and head. The method of removal isto destroy the afflicted flies. First, be sure to immediately quarantine any infected flies from other stock cultures, as with food mites. Then, freeze the vial and use sterilizing techniques or throw the vial away. jcture of parasitic mites on a fly Bacteria ‘There is little effect of bacteria on the flies themselves, however food media may become infected and inhibit growth and development of larvae. White food media take on a reddish-brown color, indicating bacterial contamination. Bacteria may be transferred via flies, airborne particles, yeast contamination or equipment. Treatment is to remove from contaminated culture vials and, of course, not use discolored vials to maintain flies. Keeping food in a refrigerator until ready touse will help keep infection at a minimum. Be sure toallow time for the food to warm to room temperature before adding flies. Molds Much like bacteria, fungal infections are mainly isolated to the media. Most are inhibitory towards larvae. Treatment is the same as with bacteria - removal of the flies from the infected culture vial. All media should contain a fungicide (see media recipes), including dehydrated media. Picture of blue mold on food Killing Flies: The Morgue ‘This is an unfortunate necessity when using flies. A bottle or beaker with mineral oil added is generally used. Dump anesthetized flies directly into the mineral oil where they drown. A bottle (beaker, ot screw, capped jar filled) with ethanol or isopropanol can also be used as a morgue. Previous Original URL ip: biology arizona,edu/sciconn/lessons2/Geigerintro.him, www readabilly.comvartclesinaklwrrane Maintenance of a Drosophila Laboratory: General Procedures — cshprotocols.cship.org — Readabi ‘Dreshprotocols.eshlp.org Maintenance of a Drosophila Laboratory: General Procedures Keeping Stocks ‘Most large fly laboratories maintain stocks that are not in every day use at 18°C 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 too overcrowded cultures, and only 20 of 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. Fly laboratories may keep some stocks that require selection of each generation, and it is important that the stock keeper knows of any special requirements to keep any stock (these should be entered on the stock database, see below). The “sick tray” is an inevitable part of any stock room, a place where sickly stocks, or stocks going through a crisis, are kept under special attention. It is very good practice tokeep the old cultures for 2 weeks (at 18°C) after transfer, so that they can be used as a backup should the new stocks fail for any reason. Collecting Virgins ‘Twogeneral methods ensure that female D. melanogaster are virgin when used to set up a cross. These can be called the “biological” and “genetic” methods. Only the former is considered here; for genetic tricks useful for virgin collecting, see Chapter 12 of Ashburner (1989). Although some variation between stocks exists, the general rule is that females will not accept a male mate until they are 10-12 hours old (i.e., after eclosion from the pupa). Thus, flies can be collected during this window (or, better, between 8 and 10 hr after eclosion), anesthetized, separated into males and females, and stored until needed in yeasted vials. The females will then usually be virgin when used. Asa preliminary check, the vials that were used for storing the virgin females should be kept and inspected 3 or 4 days later for any signs of larvae. Iflarvae are present, it is clear that at least one female in that vial was not virgin. Of course it does not matter toomuch ifa single female is incorrectly stored with the males (as long as she is discarded); but a single male in the tube of females will play havoc. The rule for sexing for virgin collecting, especially when tired or rushed is: Ifin doubt, it isa male. The following is a convenient schedule for virgin collection. Day 0: Clear all flies from emerging cultures in the late afternoon or early evening (¢.g., 5:00 p.m. to.6:00 p.m.). Discard these flies. Store emerging cultures at 18°C in the dark. Day 1; Put cultures at 25°C in the light, first thing in the morning. Clear all flies from the cultures ~1 hour later, anesthetize, separate into males and females, and store these in separate vials at 18°C until required. The young females, i.e., those that are relatively unpigmented and/or have unexpanded wings, will almost certainly be virgin. Check that the emerging cultures have no adult flies. Return the emerging cultures to 25°C in the light and possibly collect virgins last thing in the evening, Keep the “female” vials after using the virgins and inspect 3-4 days later for larvae. I'present, presume that any females from that vial were nonvirgin at the time of use. (Note that the presence of eggs in the female-holding vials is not evidence of nonvirginity, even virgin females will lay eggs, albeit at a low rate in comparison with mated females.) ww readabilly.comvartclesizbrects 1rane Maintenance of a Drosophila Laboratory: General Procedures — cshprotocols.cship.org — Readabi In practice, fly workers develop their own protocols for virgin collection that suit not only the flies, but also social and other activities. But please bear in mind, nonvirginity is by far the most common reason for an “unexpected” result from a cross. It is therefore very good practice to design crosses so that nonvirgin progeny will be evident by their phenotype, especially if unexpected nonvirgin progeny could confuse the analy sis of an experiment. Mutagenesis ‘The three general techniques for mutagenesis in Drosophila are mutagenesis by irradiation, chemical mutagenesis, and genetic mutagenesis (i.e., by transposon insertion). Only the former twoare discussed here. The choice between irradiation and chemical mutagenesis is determined by the objective of the experiment. In very general terms, only about one third of irradiation-induced mutations will be associated with chromosome aberrations, whereas most mutations induced with either of the two chemicals discussed here will be “point” mutations (usually due to single- base-pair changes; see Chapter 9 of Ashburner [1989] for a more systematic treatment, and Grigliatti [1998] for detailed protocols). In this section, we discuss only the general protocols for the mutagenesis itself, not the genetic schemes required to detect the desired mutations. For all routine purposes, only 3-5-day-old males are mutagenized. 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 postmeiotic stages are sampled and will avoid recov ering clusters of identical mutations. Bottles should be labeled in such a way that identification of progeny from the same batch of parents is possible. Irradiation Flies may be irradiated with either y or X rays, Many small laboratories use whatever source is conveniently available (e.g., a machine otherwise used for therapy); for purchase, a small industrial X-ray machine is strongly recommended. X-ray equipment, such as the Torrex TRX2800 and Torrex 120/150D, 24-inch cabinet, is available from Faxitron Corporation. y-ray equipment is available from AEA Technology ‘An X-ray machine has three advantages over a y-ray source: (1) the relative biological effectiveness of X rays is higher than that of y rays; (2) X-ray machines are safe when switched off, whereas y-ray sources require expensive shielding that must be maintained; and (3) for y-ray sources, the exposure time will need recalibration over time, since the source will be decaying (see Ashburner 1989 [p. 307]). Self-contained X-ray machines suitable for irradiation of flies, ic., with an operating voltage of 100 kV or more and 5 mA, are available. These machines, designed for industrial use, need to be modified to remove damaging low-energy X rays for irradiating flies. This is done by inserting a 1-mm filter of aluminum or Perspex between the source and the flies. For irradiation, male flies are placed either in small plastic vials or gelatin capsules (use capsules with a 3-mm-thick wall for Coy rays). For the routine induction of mutations, and chromosome aberrations, use a dose of 4 KR with X raysand 5 kRwith Coy rays. The dose rate of X-ray machines needs to be calibrated, which may be performed by the supplier, or a dosimeter can be used, for example, the Farmer Dosimeter 257 from NETechnology. A monitor should be used to test the machine for X-ray leakage, e-g., the Mini-monitor from Mini Instruments. Chemical Mutagenesis Bthyimethanesulfonate (EMS): The most convenient chemical for routine mutagenesis is EMS, administered by feeding toadult males. The standard dose for EMS (available from Sigma BioSciences) is 25 mM (0.24 ml of EMS in 100 ml of 1% aqueous sucrose, dispersed by repeated aspiration with a 10-ml syringe or P1 000 micropipettor). Males are placed in an empty bottle and starved for 12-24 hours before being allowed to feed for 12-24 hours on the EMS solution. A piece of tissue paper (or a paper towel) is fitted tightly to the floor and sides of the bottle, so that the flies do not get trapped in nooks and crannies. Freshly made up EMS solutions can then be conveniently dispensed on the tissue paper with a P5000 micropipettor. (Starvation of males can lead toa high death rate, and consequent loss of ww readabilly.comvartclesizbrects 218rane Maintenance of a Drosophila Laboratory: General Procedures — cshprotocols.cship.org — Readabi eld; this is especially true ifthe males are already genetically weak. Ifso, keep them overnight in a food vial without any additional yeast.) Ethylnitrosourea (ENU): ENU is, like EMS, a very effective mutagen for Drosophila. Although EMS may induce chromosome aberrations, EN Vis far less effective in this respect. ENU can be administered to flies in the same way as EMS. It can be purchased from Sigma as “Isopac” vials and should be freshly made up before use. To dissolve the ENU, make up a 0.01 M solution of sodium acetate buffer (pH 4.5), and inject it into the “Isopac” vial containing the U. Then dilute the ENU in a 1% sucrose solution until the final concentration of ENU is 5 mM (Grigliatti 1998). Controlling Plagues and Diseases ‘One reason for the success of D. melanogaster as a laboratory organism is that it is relatively resistant to plagues and diseases. The two most common problems are molds overgrowing the medium and mites, There have also been disturbing reports recently of viral infections in some laboratories. The culprit is apparently a picornavirus (Drosophila C virus, DCV) and the sy mptoms of infection are black, dying pupae. The extent towhich fy laboratories suffer from molds and mites varies greatly, but there is some general advice that can be given. The ‘most important advice is that prevention is better (far better) than cure; two main guidelines for preventing plagues and diseases are below. Cleanliness. Keep a clean fly room, fly kitchen, and culture environment. For cleaning surfaces in fly rooms, use alcohol or a spray disinfectant, e.g., “Astell D.” Isolation of new stocks. Quarantine all incoming stocks, no matter from what source, even if the distributor swears that they are free of mold, mites, or viruses. A quarantine facility (e.g., a dedicated incubator) should be situated as distant from the normal fly and culture rooms as possible, and all materials, especially discarded vials, must be segregated from those in regular use. It is probably sufficient to quarantine for two generations, and only transfer the stocks to the regular facility when close inspection shows them to be free of infection or infestation. For flies brought tothe laboratory straight from the wild, four generations of quarantine are recommended, fan infection or infestation occurs, the first rule is to isolate all affected cultures toa quarantine facility. What is done next depends very much on the nature and extent of the problem Bacteria ‘The most common bacterial problem is mucus-producing bacteria on the food, which often produce @ reddish-brown pigment (.g., Acinebacter sp.). The addition of antibioties (streptomycin, tetracy cline, oF ampicillin) tothe food at a concentration of 250 mg/liter is usually sufficient to cure the problem within one generation, Ifthe problem is recurrent, then investigate the possible sources of the contamination (e.g., the yeast). The use of dextrose, rather than sucrose, in the fly medium should prevent most bacterial growth. The routine use of antibioties in the food ‘medium is not recommended, as this will inevitably lead to resistance Molds Molds, usually species of Penicilum or Aspergillus, are a common problem, as fly medium isan ideal substrate for their growth. With healthy cultures, the flies normally out-compete these fungi, but they can prove tobe serious for weak stocks or for cultures at low density. It is now routine practice to include mold inhibitors in medium. Those most commonly used are Nipagin M or propionic acid. For both bacteria and molds, persistent infections that are refractory to treatment can best be overcome by washing eggs in 70% alcohol. Mites Several species of mites ean infect cultures of Drosophila (see Chapter 38 of Ashburner 1989). Broadly speaking, the ww readabilly.comvartclesizbrects siaarin Maintenance ofa Drosophila Laboratory: General Procedures — eshorotocols.ship.org — Rada mites may be interested either in the flies'food (food mites) or in the flies themselves. The fly mites are rarer than the food mites, but far more dangerous. Food mites often come in with the raw materials of ly medium (e.g., corn meal). For this reason, it is good practice to store bulk meal at -20°C and tobe scrupulous about cleaning up any spills, One of the commonest causes ofa serious mite infestation is allowing old fly cultures to fester in culture rooms or the fly room. These rooms must be inspected regularly by someone with the authority to autoclave old cultures without question. This is not an issue where the liberal social attitude so characteristic of fly labs can be allowed to constrain effective management. If mites are found, then the affected cultures must be immediately quarantined (even better, autoclaved, but this is not always acceptable). If foam or muslin cotton-wool bungs are used, then replace these immediately with bungs of nonabsorbent, tightly balled cotton wool; these should prevent the mites spreading further. Rapid (i.e., daily) transfer of stocks or cultures can rid them of mites, but this can be dangerous for weak stocks. An alternative is to collect eggs and wash them free of any mite eggs before transfer to clean vials, or to collect pupae on paper inserts and wash them free of mites and mite eggs in 70% ethanol, again before transfer to clean vials. Tedion has been found tobe effective against some common species of food mite. Tedion is available from the Sigma Aldrich Library of Rare Chemicals and also from local suppliers of agrochemicals. Dilute the commereial product (usually 8% active compound) to 5000 ppm in acetone and soak 7-em filter papers in this solution. Allow filters to dry completely and introduce one into each culture. Serious endemic mite infestations should never be allowed to build up. Iftthey do, then seek professional advice to combat them, since they will require complete fumigation of all fly-handling rooms and equipment (see Ashburner 1989 [p. 1218]). Viruses In contradiction to the statements in Ashburner (1989, p. 1192), infection with the double-stranded RNA virus DCV hasbeen found tobe a serious problem in a few fly laboratories in recent years. Its symptoms are the presence of dying black pupae, particularly in old cultures. In addition, DCV infection seems to block the induction of transgenes under the Hsp70 heat shock promoter (T. Tully, pers. comm.). T. Tully (pers. comm.) has developed protocols for eradicating viral infection. Original URL: http://eshprotocols.eshlp.org/content/2007 /3/pdb.ipg5.full?sid=bdedbgad-B8ec6-412b-9d1e-e0315cb6666e ww readabilly.comvartclesizbrects a8e2it2 Comparisons betweem male D — biology arizona edu — Readability Obiotogy arizona.cau Comparisons betweem male D Comparisons between male D, melanogaster and D, simulans Pictures of male D. simulans.Note the small orange-red protuberance (arrow) on the D. simulans genitalia ‘Comparison between D. melanogaster (left) and D. simulans.Again, notice the absence of a large genitial arch in D. melanogaster compared to D. simulans. ww readabilly.comvartclesidkit2h wee2it2 Comparisons betweem male 0 — biology arizona edu — Readabilty - Drawing of D. simulans (left) and D. melanogaster males (fom Ashburner, 1989). Original URL: ‘utp biology arizona.edu/sciconn/lessons2/Geiger/Picpages/male_%20melanogaster_vs_sims.htm ww readabilly.comvartclesidkit2hrite Differences between males and females — biology arizona ed — Readability Obiotogy.arizona.edu Differences between males and females Differences between males and females Ventral view of a female (left) and male D. melanogaster. Note the darker abdomen and more rounded appearance of the male. Females also tend tobe larger. melanogaster. Arrow points to sex comb on the male, y Lh r \ Original URL |htp:biology_arizona.edwsciconn/lessons2/Geiger;Piepages/males_vs_females.htm www readabilly.comiartclosirjg38x0 Lateral view of a male (left) and female D. a"e2it2 Construction fy raps — biology arzona.edu — Readability Obiotogy.arizona.edu Construction fly traps How to construct fly traps Materials are a vial, a bit of banana, a funnel and yeast. Original URL Inu: biology arizona.edwsciconn/lessons2/GeigerPicpages/Fly_trap_picture_page-htm www readabilly.comiartcles/4zyiw) ‘The completed setup look like this: werane www readabilly.comiartcles/4zyiw) Construction fly traps — biology arizona edu — Readability 22rite [Materials necessary for working with fies — biology sizone.edu— Readabiliy Obiotogy.arizona.edu oRIGIN Materials necessary for working with flies Materials necessary for working with flies. Stereomicroscope, fine paint brush, tamping pad and funnel (for transferring flies) Glass vial with cotton plug. There are plastic alternatives to glass vials, which are recommended when working with students. ‘The media is not from adehy drated mix but is a cornmeal-sugar recipe. Bottles are useful for culturing large populations of flies ww readabilly.comvartcles/zuybnt73 werite [Materials necessary for working with fies — biology sizone.edu— Readabiliy utp: /biology arizona.edu/sciconn/lessons2/GeigerPicpages/Materials_for_working_with_fles_pictures.htm ww readabilly.comvartcles/zuybnt73e2it2 Pictoral alas of selected mutant fies — biology arizona edu — Readabilty Obiotogy arizona.cau Pictoral atlas of selected mutant flies Pictures of Selected Mutant Fly Strains Antennapedia: Instead of regular antenna, these flies have legs for antenna. Antennapedia (bottom) with wild ty pe (top) Dumpy wing: while wings are fully formed, they do not take on the fullness as wild ty pe flies. Dumpy female on top, wild type male on bottom. Heldout: wings are at a 90° angle from the body in their normal position. www readabilly.comiartles/t7etscht 18e2it2 Pictoral alas of selected mutant fies — biology arizona edu — Readabilty Sepia eyes: eyes are sepia colored, not the typical bright red exhibited in wild types. Sepia female on the right and wild ty pe female on the left. Closer view of sepia eye color. www readabilly.comiartles/t7etscht 218rane Pictoral alas of selected mutant fies — biology arizona edu — Readabilty White eye mutants exhibit sex-linkage when crossed with wild types. White eyed on right, wild ty pe on left Wingless: Flies have no wings. Wingless female on the top, wild type female on the bottom, www readabilly.comiartles/t7etscht 38rane Pictoral alas of selected mutant fos — biology arizona edu — Readability \Wrinkled wing: Wings appear as if the flies have just eclosed and have not expanded their wings yet. Wrinkled female on the left, wild ty pe on the right. More wrinkle-winged flies. Vestigial/ebony: This mutant is an example of two phenoty pes. Wings are greatly reduced and body type is noticeably darker than the wild type. Vestigial/ebony in comparison to wild ty pe. www readabilly.comiartles/t7etscht 48rite Pictoral alas of selected mutant fies — biology arizona edu — Readabilty www readabilly.comiartles/t7etschtrite Pictoral alas of selected mutant fies — biology arizona edu — Readabilty Original URL: |hup biology arizona,edu/sciconn/lessons2/Geiger/Picpages/SelectedM utant_Fly_Strains.hem www readabilly.comiartles/t7etschtrite Pictures of parasitic mites and blue mold — biology arizona.edu — Readability Obiotogy.arizona.edu Pictures of parasitic mites and blue mold Pictures of parasitic mites and blue mold ‘An extreme case of parasitic mites. They will cover all exposed surfaces is allowed.Once established they may be difficult to eradicate. Flies must be immediately quarantined then destroyed being careful not tocontaminate other cultures. Blue mold in a culture tube.This is q generally not a problem, however steps should be taken toremove any vials showing mold immediately See text for det Original URL: ww readabilly.comvartclesmbkdegna werane Pictures of parasitic mites and blue mold — biology arizona.edu — Readability up: biology arizona.edu/sciconn/lessons2/Geiger/Piepages/parasitie_mites_and_blue_mold.htm ww readabilly.comvartclesmbkdegna 22rite Pictures of viegin male and female fles — biology arizona edu — Readabilty Qbiotogy. arizona. Pictures of virgin male and female flies Pictures of virgin male and female flies A newly eclosed female. This is the “wet” stage where the fly is sticky tothe touch. ‘The wings and body have a wet appearance. ww readabilly.comiartclesikeypuszn Virgin female showing the meconium, (arrow). ‘The meconium isa dark green area and is, theremains of larval food. ‘Comparison between a mature (left) and virgin (right) female. This is not long after closure; after a 4+ hours it becomes more difficult totell the difference between the two, Note the meconium on the virgin female. ‘Comparison between a mature (bottom) and virgin (top) male. The coloration is similar to virgin females however the genitalia are distinetly different. ‘The meconium is also found in young, virgin males asin females. werite nd female fies — biology arizona.edu — Readabilty Original URL: tp :/biology arizona.edu/sciconn/lessons2/Geiger/Piepages/virgin_male_and_female_flies.htm ww readabilly.comiartclesikeypusznrite References — biology. srizona.edu —Readabilty Obiotogy.arizona.edu References Drosophila Melanogaster By Pete Geiger email: pgeiger@mail-hockaday.org Selected References and Websites Glossary Access Excellence. This is very complete and includes terminology related to molecular genetics as well as Medelian. Where to buy equipment 1. VWR, Vials, bottles, anesthetizing equipment including CO2 pads 2. Carolina Biological. Basic fly equipment a rds, Basie fly equipment Where to obtain flies General Biological Supply Companies: For basic mutant types good for any High School Class 1. Carolina Biological. Wild type anda variety of mutants rds, Wild type and a variety of mutants University Stock Centers: For more obscure strains, generally for researchers. 1, Tucson Species Center 2, Bloomington Drosophila Stock Center Indiana University, Alsohas great links to fly genomics. Fly genome sites 1, The National Center for Biotechnology Information, From the US government 2. FlyBase: A Database of the Drosophila Genome. An oft-used site for researchers. 3. Berklev Drosophila Genome Project Food sites Cooked food sources 1, The Carolina Drosophila Manual, page 6, has different recipes. This is free with an order of flies or $4.55 per ‘manual from their on-line catalog (July 2002) 2, Media recipes from Bloomington. Bight different recipes to choose from. Dried food sources 1. YWR 2. Carolina Biological. 3. Wards, Selected Web References 1, Great resources from Access Excellence. There are a variety of sections: Section II, "Drosophila Unit: from Capture to Chemistry "is full of great ideas and techniques ‘ww eadabily.comfatlestobsute0 tnrane References —blogy.arizona.edu—Readabilty 2, Aweb site devoted to mutant strains of flies. From the Exploratorium, a readable for students and contains cartoon drawings of selected mutants. 3. From the University of Arizona's Biotech Project, "Analy zing the Products of DNA:Chromatography of Fruit Fly Eye Color” shows the pathway of eye coloration. 4. The Interactive Fly: and microphotographs of developmental stages and adult Drosophila cyberspace guide to Drosophila genes and their roles in development. Great information 5. Using Punnett Squares with Drosophila. An access excellent site. 7. MendelWeb: ‘an educational resource for teachers and students interested in the origins of classical genetics, introductory data analy sis, elementary plant science, and the history and literature of science." 8, Mendel’s Original Paper, "Experiments in Plant Hy bridization" (1865) mendels original paper 9. The Monk in the Garden web site. Information on the book and links to Medelian geneties sites. Books 1, Roberts, D.B Drosophila, A Practical Approach,.. (editor): Good general reference 2, Greenspan, R.J., 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: Good general reference 3. Hartenstein, V., 1993 Atlas of Drosophila Development. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: For more advanced study on larval development into adults. 4. Demerec, M., 1994 Biology of Drosophila. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: A complete work suitable for research purposes Henig, R.M., 2000 5. The Monk in the Garden. Houghton Mifflin Company, New York. A nice, readable book on the life of Mendel and the resurrection of his experiments. hburner http://biology .arizona.edu /sciconn /lessons2 /lessons. html All contents copyright © 2002. All rights reserved. Original URL lip :?biology arizona, edu/sciconn/lessons2iGeigerteferences. htm, ww readabilly.comiartclesiroSsuhr0 22