CHAPTER 36

The Drosophila melanogaster Assay

Wendy J. Davies and Stuart J. Freeman

1. Introduction

The method described is based on that developed by Schuler and col- leagues (1,2) and recently refined (3).

2. Materials 1. Drosophila melanogaster: Oregon-R wild type. 2. Drosophila medium. 3. Yeast: Baker’s live. 4. Flint-glass shell vials (30 mL). 5. Refined cotton. 6. Cheesecloth. 7. Dissectmg microscope.

3. Methods 3.1. Maintenance

1. Instant plain Drosophila medium is reduced to a powder using a mortar and pestle. One gram is placed m a flint-glass vral and dissolved in 5 mL distilled deionized water. A small amount (~1 mg) of yeast is sprinkled onto the surface of the setting medium. Vials are stoppered with refined cotton and covered with cheesecloth.

2. Drosophila cultures in vials are incubated at 25 f 1 “C, at 60-70% relative humidity, and with a 12 h light/dark diurnal cycle.

3.2. Mating 1. Newly enclosed males and females (l-24-h-old) from stock cultures are

CO:! anesthetized before collection into holding vials (single sex). Virgin flies are collected, usually for 3 d (Wednesday to Friday).

From Methods m Molecular Srology, Vol 43. In Wro Toxdty Testing Protocols Edited by: S O’Hare and G K. Attetwlll Copyright Humana Press Inc , Totowa, NJ

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318 Davies and Freeman

2. On the 3rd d or when sufficient flies have been collected, they are anesthe- tized and transferred to culture flasks (about 20 of each sex, from different stocks) for mass-mating.

3. After 3-5 d (usually over the weekend), the flies are anesthetized again, and inseminated females are removed and introduced singly mto each assay vial (freshly prepared). They are also introduced mto stock vials for the maintenance of stocks.

3.3. The Assay

1. Assay vials are prepared as descrtbed in step 1 above, but using a solutron of test chemical (or a dilution of) instead of water.

2. Following 20 h each female is removed from the assay vial and discarded. Deposited eggs are counted using a dissectmg microscope.

3. After about 9 d (thus can vary with test compound, and is carefully checked for each vial), adult flies begin to emerge from puparmm (eclosure).

4. Adult flies are harvested daily for 10 d following the first day of eclosure and counted. Flies are not counted after 10 d, in case a subsequent genera- tion of flies are counted.

5. On the 10th d for each vial, survival data is totaled (number of harvested flies/number of eggs deposited).

6. Initial range-finding tests are carried out. Three vials at each of 14 concen- trations from O-80 mg test compound/vial are used.

7. A closer range and fewer concentrations are then used to produce a gradi- ent of mortality ranging from 100 to 0%. This can be analyzed by probit analysts (4) to establish concentrations for further tests.

8. These next screenmg test(s) use half-logarithmic mterval concentrations including and below the LC& concentration (concentration lethal to 50% of the developing flies), and controls.

9. Flies used for screenmg tests are selected usmg a dissectmg micro- scope: males before mass-mating and females before egg deposition. Vig- orous and morphologically normal males and the most fecund females are selected.

10. The number of vials is adjusted to yield approx 200 offspring for every concentration tested: The number of vials can be estimated from data obtained in the range-finding tests.

11. If insufficient flies are produced at a concentration, more vials are used m a subsequent repeat. Concentrations are adjusted upward if necessary so that a 50% mortality is always achieved at the highest concentration.

12. Flies are exposed to a continuous flow of CO2 gas after harvestmg. They are scored on the day of eclosure if possible, and not later than 1 wk afterward.

The Drosophila melanogaster Assay 319

nna orbltals

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Fig. 1. Drosphila melanogaster morphology. Adapted from Schuler et al. (2).

13. Each fly is exammed m several orientations for external morphological abnormalities (Fig. 1). All major bristles, legs, segmentation halteres, wings, antennae, eyes, and mouth parts are examined; also, the general appearance of the thorax, abdomen, small bristles, and entire body (includ- mg size and color). A check for body alignment, excess tissue growth, and extra or absent body parts is also carried out.

3.4. Evaluation

1. Incidence data for the same concentrations from different runs are pooled. 2. If a stattstically stgnlficant concentration-dependent mcidence of any mal-

formation is observed (chi-square, p < 0.05) (4) repeat testing is terminated. The result 1s then considered to be positive: The compound is predicted to have teratogenic potential. Note: Concentrations tested are less than the LCso.

3. If this criterion is not met after challenging with increasing concentrations and obtaining data from a minimum of 500 pooled flies, the result is con- sidered to be negative and testing stops.

4. Notes

1. A simpler method is to allow 3 pairs of adult flies to oviposit in a series of vials (2 d/vial) containing different concentrations of test compound (5).

Davies and Freeman

Emerging flies are examined as usual. Larvae produced in this way in untreated vials can also be collected at selected developmental stages for subsequent exposure to compound.

2. Combined adult and developmental toxicity assessments can be made by allowing an adult pair to remain in a vial for 7 d before scormg (6).

3. An important discovery recently has demonstrated that it may only be nec- essary to assess bent bristles and wing notches for an improved screenmg test for developmental toxicity (3).

4. Drosophila embryonic cell cultures have also been used to detect terato- gens (7). These shall not be discussed except to report that they have been used to demonstrate the presence of a metaboltc activating system m Drosophila (8).

References 1 Schuler, R. L , Hardm, B. D , and Niemerer, R. W. (1982) Drosophzlu as a tool for

the rapid assessment of chemicals for teratogenicity Terat. Cart. Mutat. 2,293-301 2. Schuler, R. L., Radike, M. A., Hardm, B. D., and Nremeier, R. W. (1985) Pattern

of response of intact Drosophila to known teratogens. J Am. Co11 Toxic01 4(4), 291-303.

3. Lynch, D. W., Schuler, R. L , Hood, R. D., and Davis, D G. (1991) Evaluation of Drosophila for screening developmental toxicants: test results with eighteen chemi- cals and presentation of a new Drosophila bioassay. Terat. Cart. Mutat. 11,147-173

4. Sachs, L. (1984) Applied Statutics: A Handbook of Techniques, 2nd ed., Sprmger- Verlag, New York.

5. Ranganathan, S., Davis, D. G., and Hood, R. D. (1987). Developmental toxicity of ethanol in Drosophila melanogaster. Teratology 36,45-49.

6. Goldstem, S. H. and Babrch, H. (1989) Drfferential effects of arsemte and arsenate to DrosophiZu melanogaster in a combined adult/developmental toxicity assay. Bull. Environ. Contam. Toxicol. 42,276-282.

7 Bournias-Vardiabasis, N., Teplitz, R. L., Chernoff, G. F , and Seecof, R. L. (1983) Detection of teratogens in the Drosophila embryonic cell culture test: assay of 100 chemicals. Teratology 28,109-122

8. Bournias-Vardiabasis, N. and Flores, J. (1983) Drug metabolising enzymes in Drosophila melanogaster teratogenicity of cyclophosphamide in vitro. Terat. Care. Mutat. 3,255-262