In general, Drosophila are cultured at 25°C on a 12-hour light:dark cycle with 60-70% humidity. Flies may be kept in bottles or vials that have food on the bottom. Appropriately sized foam stoppers may be convenient to seal the top; however, a chunk of cotton wool may also be used. As has been mentioned, temperature has a significant effect on lifespan in flies. The minimum permissive temperature is 18° C, and the upper restrictive temperature is 29°C. If there are only a few hundred flies of a few stocks, one or two 25° C incubators are sufficient. However, if there are going to be large numbers of flies, a 25°C room will be a preferred choice.
Fly stocks can be successfully cultured by regular mass transfer to fresh food. The frequency of the transfer needs to be established depending on the fly strains, the temperature and the density of the cultures. Stocks kept at 25°C (200-300 adults per half-pint bottle) should be transferred to fresh food at least once every 2 weeks. In older stocks, mites and mold may appear and cause problems, so the stocks should be monitored from time to time regardless of the intervals. This period can be extended by keeping the stocks at a lower temperature (e.g., 18°C); however, not all strains survive well at 18°C, and mold can be a serious problem for certain strains.
To obtain healthy adults, optimum numbers of parental flies need to be established. In most cases, 300600 flies are obtained from a bottle, in which around 100 females (and a few tens of males) are left for a day and allowed to lay eggs. All the parental flies need to be emptied afterwards. If larvae become overcrowded, baker's yeast should be added to provide additional nutrition.
There are many types of fly food used by different laboratories. Food is commercially available, but can also be cooked in laboratories (for recipes, see the ''Recommended Resources'' section: Roberts and Standen (1998), website for Bloomington stock center). The food may be kept at 4°C for about a month. Nipagin M ( p-hydroxy benzoic acid methyl ester) is an antioxidant that is often included in food. In vitro, it is able to scavenge hydrogen peroxide production by mitochondria effectively at relatively high concentrations, although there is no report of the effects of Nipagin on lifespan.
Dietary restriction in flies has been applied by food dilution, and this method can extend lifespan in flies. However, there have been concerns about how much flies eat, since flies are cultured on food in a bottle and have access to food all the time; the flies on diluted food may eat more to make up the calories. Indeed, a recent study measuring food intake using isotope labeling reported that this is the case (Carvalho et al., 2005). Further work will be needed to verify the dietary restriction effect of the dilution method if any, as well as to develop alternative methods for dietary restriction in flies.
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