PMC

Triglyceride and lactate content of L3 larvae grown on different media. Relative amounts of (a) triglycerides and (b) lactate in L3 wandering‐stage larvae from the indicated crosses, after normalization to values from control larvae (transgenic recipient strain w ¹¹¹⁸, cultured on complete medium); means + SD of ≥ 6 batches of 10 larvae of each genotype and culture medium. Two‐way ANOVA (Figure S4B, all controls vs. AOX‐expressing larvae, i.e., UAS‐AOX ^F6 × daGAL4 cross) found no significant differences based on genotype, but significance for diet as determinant of both lactate (p < .001) and triglyceride (p < .01) levels, as well as for pyruvate (see Figure S4A). ANOVA, analysis of variance; AOX, alternative oxidase; AU, arbitrary units; SD, standard deviation

Treacle aqueous fraction does not contain an AOX inhibitor. (a) Respirometry trace for 5 × 10⁶ AOX‐expressing (doxycycline‐induced) cells treated with substrates and inhibitors as indicated: Dig 15 (15 ng/ml), PGM (20 mM each), Succ (20 mM), ADP (5 mM), Trcl 1/100, 1/10, and 1/1 (treacle aqueous fraction equivalent to 1%, 10%, and 100% of the standard concentration in fly food), Aa (60 ng/ml), and nPG (0.2 mM). Numbers opposite the trace represent oxygen consumption rate before and after treacle addition (nmol/min per 5 × 10⁶ cells), extrapolated from slope. (b) Respirometry data on permeabilized cells (averaged data from two experiments), with and without induction by doxycycline as shown. Extent of antimycin resistance with and without the addition of treacle (aqueous fraction, added to equivalent level as in fly food). See also Figure S3. Aa, antimycin A; ADP, adenosine diphosphate; AOX, alternative oxidase; Dig, digitonin; nPG, n‐propyl gallate; PGM, sodium pyruvate, glutamate, and malate; Succ, sodium succinate [Color figure can be viewed at wileyonlinelibrary.com]

Doxycycline does not influence the development of AOX‐expressing flies. Proportion of pupae eclosing on the indicated media, of the genotypes or crosses (female × male) shown (means + SD, n ≥ 4). (a) Complete medium or low‐nutrient medium with and without 100 μg/ml doxycycline. (b) Low‐nutrient medium with and without supplementation by treacle and/or doxycycline at the indicated doses, alongside daGAL4 control on low‐nutrient medium. (c) Low‐nutrient medium with and without supplementation by maize flour and/or 100 μg/ml doxycycline. In each panel, horizontal lines annotated with asterisks (*, **, ***) denote significant differences between groups within a genotype or doxycycline concentration (one‐way ANOVA with Tukey post hoc HSD test, p < .05, .01, and .001, respectively). Symbols above individual bars represent significant differences in pairwise comparisons between (a, c) genotypes or (b) doxycycline concentrations (Student’s t test, ^¤ and ^#: p < .05 and .001, respectively). ANOVA, analysis of variance; AOX, alternative oxidase; HSD, honestly significant difference; SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Citrate addition does not rescue the developmental failure of AOX‐expressing flies. Proportion of pupae of the genotype or cross (female × male) shown, eclosing on low‐nutrient medium supplemented with the indicated levels of citrate (means + SD, n ≥ 4). Horizontal lines annotated with asterisks (* and **) denote significant differences between groups within a genotype (one‐way ANOVA with Tukey post hoc HSD test, p < .05 and .01, respectively). Symbols “#” above individual bars represent significant differences in pairwise comparisons between genotypes for a given citrate concentration (Student’s t test, p < .001). Note that this experiment was conducted at 26°C for technical reasons, also giving a more robust phenotype (Saari et al., ). ANOVA, analysis of variance; AOX, alternative oxidase; HSD, honestly significant difference; SD, standard deviation

Developmental failure in AOX‐expressing flies occurs during metamorphosis. Strains are denoted as w¹¹¹⁸ (transgenic recipient strain), AOX^F6 (UAS‐AOX ^F6, AOX transgenic strain dependent on GAL4 for expression), GFP (UAS‐GFP ^Stinger, control strain transgenic for nuclear‐localized GFP, also GAL4‐dependent), daGAL4 (ubiquitously expressing GAL4 driver). (a) A number of eggs laid in the indicated crosses, on low‐nutrient medium containing 3.5% yeast and 5% glucose (means + SD of 3–6 individual vials in each case). There were no significant differences between strains or crosses (one‐way ANOVA). (b) Percentage of eggs laid from the different crosses on low‐nutrient medium (3.5% yeast and 5% glucose), reaching the pupal stage (means + SD of 3–6 individual vials in each case). There were no significant differences between strains or crosses (one‐way ANOVA). (c) Proportion (%) of eclosing adult flies in three replicate crosses (3–6 vials per strain in each cross) on low‐nutrient medium (3.5% yeast and 5% glucose). Means + SD; **Significant difference (p < .01) from all other classes (one‐way ANOVA followed by Tukey’s post hoc HSD test). See also Figure S1. ANOVA, analysis of variance; AOX, alternative oxidase; GFP, green fluorescent protein; HSD, honestly significant difference; SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Specific treacle fractions support development of AOX‐expressing flies. Proportion of pupae from UAS‐AOX ^F6 × daGAL4 cross (female × male, means + SD, n ≥ 4 except where indicated), eclosing on low‐nutrient medium supplemented with the indicated treacle fractions. (a) Aqueous and ether fractions following ether extraction, and a 50/50 mixture of the two fractions. Horizontal lines annotated with asterisks (**) denote significant differences between groups (one‐way ANOVA with Tukey post hoc HSD test, p < .01). (b) Pellet and supernatant fractions following precipitation of the ether‐extracted aqueous fraction, using the indicated ethanol concentrations. Vertical bars denote separately executed series of experiments. The set of precipitations at 40%, 60%, and 75% ethanol are the combined data from all replicate vials in two separate experiments using the same extracts, n > 5. The symbol “**” above individual bars represent significant differences in pairwise comparisons between a given pair of supernatant and pellet fractions (Student’s t test, p < .01). ANOVA, analysis of variance; AOX, alternative oxidase; HSD, honestly significant difference; SD, standard deviation

Developmental failure of AOX‐expressing flies is not corrected by many specific dietary supplements. Proportion of pupae eclosing on the indicated media, of the genotypes or crosses (female × male) as shown (means + SD, n ≥ 4). (a) Complete (high‐sugar) medium or low‐nutrient (3.5% yeast and 3% glucose) medium to which the indicated component was added at the concentrations shown, or as given in Section 2. Separated groups of bars represent trials conducted in separate series of experiments (UAS‐AOX ^F6 × daGAL4 cross and daGAL4 controls always studied in parallel in each case). Horizontal lines annotated with symbols denote significant differences between groups within a genotype and a given experiment (one‐way ANOVA with Tukey post hoc HSD test). Symbols above individual bars represent significant differences in pairwise comparisons between genotypes, for a given additive in a given experiment (Student’s t test). (b) Low‐nutrient (3.5% yeast and 3% glucose) medium to which multivitamins were added at the indicated dilutions from the standard amount of one tablet per 300 ml of fly food, in combination with 3% fructose and 1.5% sucrose, for the UAS‐AOX ^F6 × daGAL4 cross only. There were no significant differences between the groups (one‐way ANOVA). (c) Low‐nutrient medium plus CuSO₄ at the indicated concentrations. Within each genotype, there were no significant differences between the groups (one‐way ANOVA). Symbols denote significant differences between the genotypes in pairwise comparisons, at each CuSO₄ concentration tested (Student’s t test). (d) Low‐nutrient medium plus DMEM. Note that preliminary trials were conducted to determine a concentration of DMEM that was completely nontoxic to control flies. As for other mixed additives that had no measurable effect, it cannot be excluded that DMEM contains both positively and negatively acting components that cancel each other out. Within each genotype, there were no significant differences between the groups (Student’s t test). Symbols denote significant differences between the genotypes in pairwise comparisons, with and without DMEM (Student’s t test). Statistical significance in all panels is denoted by ^¤, ^#, and *: p < .05, .01, and .001, respectively. ANOVA, analysis of variance; AOX, alternative oxidase; DMEM, Dulbecco’s modified Eagle medium; HSD, honestly significant difference; SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Developmental failure of AOX‐expressing flies is corrected by complex dietary supplements. The proportion of pupae eclosing on the indicated media, of the genotypes or crosses (female × male) as shown (means + SD, n ≥ 4). (a) Standard high‐sugar medium lacking each of the indicated ingredients. Statistical analysis comparing eclosion frequency of flies of each given genotype on different media (by one‐way ANOVA) and also comparing flies of the two different genotypes on each medium with each other (using Student’s t test), revealed no significant differences. (b) Low‐nutrient (3.5% yeast and 3% glucose) medium to which the indicated component from the standard high‐sugar medium was added, at the concentrations indicated in Section 2. * and **Significant differences (p < .05 and .01, respectively) in eclosion frequencies of flies of each given genotype on different media (by one‐way ANOVA followed by Tukey’s post hoc HSD test). Pairwise comparisons of eclosion frequencies of flies of the two different genotypes on each given medium (using Bonferroni‐corrected Student’s t test), also revealed a significantly decreased eclosion rate in AOX‐expressing flies for most individual added ingredients (for clarity, not shown on the figure): p < .001 for sucrose (or for minimal medium with no additions), p < .05 for wheat‐germ, treacle, and soy flour. (c) Dose‐response to different amounts of yeast or glucose added to, or subtracted from, the low‐nutrient medium. Note that a single data set was used for the “3% glucose” and “3.5% yeast” conditions, as denoted by the boxes, this also being the standard composition of the low‐nutrient medium used elsewhere in the study. *Statistically significant differences between different nutrient conditions within a genotype (one‐way ANOVA followed post hoc by Tukey’s HSD test, p < .05); ^#Statistically significant differences in a pairwise comparison of genotypes at each given nutrient condition (Student’s t test, p < .001). (d) Dose‐response to different amounts of two active ingredients from the standard medium, treacle, and maize flour (% w/v as shown), for the UAS‐AOX ^F6 × daGAL4 cross only. * and **Significant differences between concentrations of a given nutrient (one‐way ANOVA followed post hoc by Tukey’s HSD test, p < .05 and .01, respectively). All flies were cultured at 25°C, except for the experiment of the panel (c), where 26°C was used, for technical reasons. ANOVA, analysis of variance; AOX, alternative oxidase; HSD, honestly significant difference; SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]