Information Panel

Maintenance of a Drosophila Laboratory: General Procedures

Adapted from "Laboratory Culture of Drosophila," Chapter 35, in Drosophila Protocols (eds. Sullivan et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.

INTRODUCTION

This article provides a general introduction to keeping Drosophila stocks, making and scoring crosses, mutagenesis, and controlling diseases in the laboratory.

RELATED INFORMATION

General information on setting up a Drosophila laboratory can be found in Culture of Drosophila: The Laboratory Setup.

PROCEDURES

Keeping 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 too 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. 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 to keep the old cultures for 2 weeks (at 18ºC) after transfer, so that they can be used as a backup should the new stocks fail for any reason.

Collecting Virgins

Two general 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. As a 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. If larvae are present, it is clear that at least one female in that vial was not virgin. Of course it does not matter too much if a 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: If in doubt, it is a 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 (e.g., 5:00 p.m. to 6:00 p.m.). Discard these flies. Store emerging cultures at 18ºC in the dark.

Day 1: Put cultures at 25ºC 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 18ºC 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 25ºC 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. If 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.)

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 analysis of an experiment.

Setting Up and Scoring Crosses

It is impossible to give any universal rules for setting up or scoring crosses, since the precise protocol will vary from experiment to experiment. Crosses can be set up readily with a single pair of parents, although the failure rate can be quite high; normally one would use four pairs for crosses in vials and between five and ten pairs for crosses in bottles. Crosses can be transferred to new vials or bottles after 2 days. When scoring crosses, it is usually important to score at least once a day and to continue scoring for 9 days after the first progeny have emerged, since many genotypes have a delayed development (and/or a short life span). (Scoring for >9 days will cause confusion because F₂ flies may be emerging.)

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 two are 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 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.

Irradiation

Flies may be irradiated with either 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. -ray equipment is available from AEA Technology.

An X-ray machine has three advantages over a -ray source: (1) the relative biological effectiveness of X rays is higher than that of rays; (2) X-ray machines are safe when switched off, whereas -ray sources require expensive shielding that must be maintained; and (3) for -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, i.e., 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 ⁶⁰Co rays). For the routine induction of mutations, and chromosome aberrations, use a dose of 4 kR with X rays and 5 kR with ⁶⁰Co 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 257D 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

Ethylmethanesulfonate (EMS): The most convenient chemical for routine mutagenesis is EMS, administered by feeding to adult 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 P1000 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 to a high death rate, and consequent loss of yield; this is especially true if the males are already genetically weak. If so, 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, ENU is 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 ENU. 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 symptoms of infection are black, dying pupae. The extent to which fly 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 to the laboratory straight from the wild, four generations of quarantine are recommended.

If an infection or infestation occurs, the first rule is to isolate all affected cultures to a 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 a reddish-brown pigment (e.g., Acinebacter sp.). The addition of antibiotics (streptomycin, tetracycline, or ampicillin) to the food at a concentration of 250 mg/liter is usually sufficient to cure the problem within one generation. If the 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 antibiotics in the food medium is not recommended, as this will inevitably lead to resistance.

Molds

Molds, usually species of Penicillium or Aspergillus, are a common problem, as fly medium is an ideal substrate for their growth. With healthy cultures, the flies normally out-compete these fungi, but they can prove to be 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 can infect cultures of Drosophila (see Chapter 38 of Ashburner 1989). Broadly speaking, the 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 fly medium (e.g., corn meal). For this reason, it is good practice to store bulk meal at -20ºC and to be scrupulous about cleaning up any spills. One of the commonest causes of a 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 to be 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 commercial product (usually 8% active compound) to 5000 ppm in acetone and soak 7-cm 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. If they 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 has been found to be 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.

REFERENCES

Ashburner, M. 1989. Drosophila. A laboratory handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Grigliatti, T. 1998. Mutagenesis. In Drosophila. A practical approach, 2nd ed, (ed. D. Roberts), pp. 55–83. IRL Press at Oxford University Press, UK.