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, 11 (1), 100-5

A Holidic Medium for Drosophila Melanogaster

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A Holidic Medium for Drosophila Melanogaster

Matthew D W Piper et al. Nat Methods.

Erratum in

  • Nat Methods. 2014 Sep;11(9):971
  • Nat Methods. 2015 Nov;12(11):1098

Abstract

A critical requirement for research using model organisms is a well-defined and consistent diet. There is currently no complete chemically defined (holidic) diet available for Drosophila melanogaster. We describe a holidic medium that is equal in performance to an oligidic diet optimized for adult fecundity and lifespan. This holidic diet supports development over multiple generations but at a reduced rate. Over 7 years of experiments, the holidic diet yielded more consistent experimental outcomes than did oligidic food for egg laying by females. Nutrients and drugs were more available to flies in holidic medium and, similar to dietary restriction on oligidic food, amino acid dilution increased fly lifespan. We used this holidic medium to investigate amino acid-specific effects on food-choice behavior and report that folic acid from the microbiota is sufficient for Drosophila development.

Figures

Figure 1
Figure 1
A holidic diet for adult Drosophila. (a) Drosophila median lifespans. Under the same experimental conditions using an oligidic diet, fly lifespan varied over a 25-day range during the period September 2005 – July 2012; n = 65. (b) Adult lifespan of flies on holidic and oligidic diets. This trial, flies on the holidic diet were longer lived than on 1SY (P < 0.001, n = 100, Log-rank test). (c) Fitness traits of flies on holidic and oligidic diets. While average median lifespan on holidic and oligidic diets were no different (P = 0.52, n = 7, Wilcoxon rank-sum test), inter-trial variance tended to be lower, but was not significant (P = 0.059, n = 7, F-test). There was significantly less variance in egg-laying on the holidic diet (P = 0.03, n = 17, F-test), but at a ~25% lower level (P = 0.008, n = 17, Wilcoxon rank-sum test). Data shown collected May 2006 – November 2011. Lines connect cohorts in the same experiment. (d) Lifespan of insulin-mutant (wDah; dilp2-31,5) and control (wDah) flies on holidic and oligidic diets. Insulin mutants were longer lived than controls (P < 0.0001, n = 100, Cox proportional hazards) on both food types (effect of diet: P = 0.53, n = 100, Cox proportional hazards). There was a significant genotype x diet interaction (P = 0.004, n = 100, Cox proportional hazards) due to the enhanced lifespan of control flies on holidic diet.
Figure 2
Figure 2
Nutrient-specific effects on Drosophila fitness traits. (a) From the holidic diet, omitting cholesterol (– sterol), sugar (– S), amino acids (– N), arginine alone (– arg), isoleucine alone (– ile) or vitamins (– vit) shortened lifespan (P < 0.001, n = 100, for all comparisons, Log-rank test). Cumulative egg-laying was reduced by omission of either cholesterol, amino acids, arginine alone, isoleucine alone or vitamins (P < 0.016, n = 10 for all comparisons, Wilcoxon rank-sum test). Omission of the non-essential amino acids (– NEAA) had no effect on longevity (P = 0.28, n = 100, Log-rank test) or egg-laying (P = 0.71, n = 10, Wilcoxon rank-sum test) and removal of Ca, Cu, Fe, Mn and Zn (– metal) resulted in reduced egg-laying (P = 0.009, n = 5, Wilcoxon rank-sum test), and a small, but significant increase in lifespan (P = 0.002, n = 100, Log-rank test). Asterisks indicate significance (P < 0.05) versus complete holidic diet control. Bars represent mean ± s.e.m. (b) Lifespan and egg laying for flies on media without folic acid. Omission of dietary folic acid significantly shortened lifespan (P < 0.016, n = 100, in both comparisons, Log-rank test) and reduced egg laying (P < 0.002, n = 10, in both comparisons, Wilcoxon rank-sum test). This was not rescued by addition of para-amino benzoic acid that microbes but not flies can convert to folic acid. Unique letters indicate significant differences. Bars represent mean ± s.e.m.
Figure 3
Figure 3
Holidic diet as a tool to study behavior (a, b) Activity and sleep profiles of flies on holidic and oligidic diets (a) (summed into 30 minute bins) for female and male (Supplementary Fig. 4) flies recorded for 72 h following a 24 h acclimation period. Black and white boxes represent light and dark periods. (b) Average sleep amount and total waking activity were summed during the 72 h recording. They did not significantly differ between diets (P > 0.35, n = 16, for both comparisons, Wilcoxon rank-sum test). Plotted data represent mean ± s.e.m. (c) Diet preference test. After maintenance on holidic diets lacking either sucrose or amino acids for a period of 3 or 6 days, flies were then assayed for their choice of sugar alone or yeast alone. Flies deprived of amino acids showed a significantly greater yeast preference (Yeast Preference Index; YPI) than those deprived of sugar (P < 0.0001, n = 13 for 3-day deprived and n = 14 for 6-day deprived flies, Wilcoxon rank-sum test).
Figure 4
Figure 4
Drug bioavailability is increased on holidic medium. (a) RU486-induction of transgenic Aβ42 expression. Only 25 μM RU486 was required on holidic medium to match Aβ42 peptide expression found for flies feeding on oligidic diet with 200 μM RU486 (P = 0.05, n = 3, Student’s t-test). Climbing ability was similarly affected over time for flies on both media when Aβ42 was induced to the same level (diet by age interaction, P = 0.47, n = 3, 2-way ANOVA). There was a significant effect of diet (P = 0.0006, n = 3, 2-way ANOVA) due to the flies on holidic diet starting with a higher performance index, and age (P < 0.0001, n = 3, 2-way ANOVA). Supplementary fig. 5 shows a wider range of RU486 concentrations. Bars represent mean ± s.e.m. Unique letters indicate significant differences. (b) Effect of rapamycin on egg laying. Only 10 μM rapamycin was required to completely block egg-laying on holidic food (P < 0.0001, n = 5, Wilcoxon rank-sum test), whereas a dose of 200 μM was required in SY food (P = 0.001, n = 5, Wilcoxon rank-sum test). Unique letters indicate significant differences. Bars represent mean ± s.e.m.
Figure 5
Figure 5
Development on holidic media. (a) Development was delayed on holidic medium (P < 0.0001, n = 13 oligidic diet (SY) and n = 20 holidic diet, Wilcoxon rank-sum test). The delay was significantly reduced by addition of water-soluble yeast extract (YE) (P < 0.0001, n = 29, Wilcoxon rank-sum test). (b) Adding 50% more vitamins to holidic food improved eclosion success (P < 0.05, n = 3, Student’s t-test) to match oligidic food. (c) Rearing conditions affect adult fitness. Wing area was smaller for flies reared on holidic medium (P < 0.001, n = 22 SY, n = 16 holidic diet, Wilcoxon rank-sum test). Holidic and oligidic diets supported egg laying equally although the larger flies reared on oligidic food laid more eggs than those from holidic food (effect of rearing diet P < 0.0001, n = 8; effect of adult diet, P = 0.31, n = 8; interaction P = 0.85, n = 8, Linear Model). (d) Folic acid requirement for development. Untreated or bleach treated eggs were transferred to sterile holidic media either with or without folic acid. Removing folic acid delayed development of untreated eggs (P < 0.0001, n = 40, Wilcoxon rank-sum test) and abolished development of bleach-treated eggs. Bleach treatment slowed development in the presence of folate (P = 0.015, n = 40, Wilcoxon rank-sum test), which was rescued by pre-exposing developmental medium to male flies. Within panels, unique letters indicate significant differences. Data expressed as means ± s.e.m.

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