Alternative oxidase confers nutritional limitation on Drosophila development

© 2019 The Authors. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology Published by Wiley Periodicals, Inc. J. Exp. Zool. 2019;331:341–356. wileyonlinelibrary.com/journal/jez | 341 Received: 23 December 2018 | Revised: 12 April 2019 | Accepted: 17 May 2019 DOI: 10.1002/jez.2274 RESEARCH PAPER Alternative oxidase confers nutritional limitation on Drosophila development Sina Saari 1 | Esko Kemppainen 2 | Tea Tuomela 1 | Marcos T. Oliveira 3 | Eric Dufour 1 * | Howard T. Jacobs 1,4 * 1 Faculty of Medicine and Health Technology and Tampere University Hospital, Tampere University, Tampere, Finland 2 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland 3 Departamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Jaboticabal, SP, Brazil 4 Institute of Biotechnology, University of Helsinki, Helsinki, Finland Correspondence Howard T. Jacobs, Faculty of Medicine and Health Technology, FI‐33014 Tampere University, Finland. Email: [email protected] Funding information FP7 Ideas: European Research Council, Grant/ Award Number: 232738; Tampere University Hospital Medical Research Fund; Suomen Akatemia, Grant/Award Numbers: 272376, 283157; Sigrid Juselius Foundation; Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/Award Number: 2014/02253‐6; Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: 424562/2018‐9 Abstract The mitochondrial alternative oxidase, AOX, present in most eukaryotes apart from vertebrates and insects, catalyzes the direct oxidation of ubiquinol by oxygen, by‐ passing the terminal proton‐motive steps of the respiratory chain. Its physiological role is not fully understood, but it is proposed to buffer stresses in the respiratory chain similar to those encountered in mitochondrial diseases in humans. Previously, we found that the ubiquitous expression of AOX from Ciona intestinalis in Drosophila perturbs the development of flies cultured under low‐nutrient conditions (media containing only glucose and yeast). Here we tested the effects of a wide range of nutritional supplements on Drosophila development, to gain insight into the physiological mechanism underlying this developmental failure. On low‐nutrient medium, larvae contained decreased amounts of triglycerides, lactate, and pyruvate, irrespective of AOX expression. Complex food supplements, including treacle (molasses), restored normal development to AOX‐expressing flies, but many individual additives did not. Inhibition of AOX by treacle extract was excluded as a mechanism, since the supplement did not alter the enzymatic activity of AOX in vitro. Furthermore, antibiotics did not influence the organismal phenotype, indicating that commensal microbes were not involved. Fractionation of treacle identified a water‐ soluble fraction with low solubility in ethanol, rich in lactate and tricarboxylic acid cycle intermediates, which contained the critical activity. We propose that the partial activation of AOX during metamorphosis impairs the efficient use of stored metabolites, resulting in developmental failure. KEYWORDS AOX, cataplerosis, mitochondria, nutrition, TCA cycle 1 | INTRODUCTION Animals have evolved a wide variety of physiological mechanisms to tailor their development and feeding behavior to the nature and availability of food resources. Most holometabolous insects, for --------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. *Eric Dufour and Howard T. Jacobs contributed equally to the supervision of this work. example, lay their eggs on rich nutrient sources, which sustain rapid growth during larval development. Metabolites that are synthesized and stored during the larval stages are then used to drive the subsequent processes of cellular proliferation, migration, and differentiation. The program of metamorphosis during pupal stage is then executed in an environment where further feeding is not possible. The metabolic processes that characterize the larval and pupal stages may be crudely described as anabolic and catabolic, respectively. Nevertheless, the biosynthesis that occurs during larval development depends on a supply of biological energy, while pupae rely on converting some of their stored resources into new biomolecules, as well as fueling and regulating the morphogenetic program. Importantly, most of the energy generation, as well as the production of relevant metabolites for biosynthesis and signaling, originate within the mitochondria. The tricarboxylic acid (TCA) cycle, in particular, is central to catabolism, anabolism, and signaling, and is in turn reliant on the mitochondrial respiratory chain. The model organism Drosophila melanogaster has been extensively studied with regard to the molecular and cellular processes that underpin this developmental program. However, the metabolic events which accompany development have received less attention. D. melanogaster is considered a cosmopolitan species (Markow, Nazario‐Yepiz, & Ramirez Loustalot‐Laclette, 2017; Matzkin, Johnson, Paight, Bozinovic, & Markow, 2011), able to grow on a wide variety of food sources. These commonly include glucose and other sugars from decaying fruit, as well as yeast, which represents a rich source of amino acids and other nutrients. The major metabolic fuel for the pupal stage, accumulated during larval development, is triglycerides (Church & Robertson, 1966; Kühnlein, 2012; Merkey, Wong, Hoshizaki, & Gibbs, 2011). Lactate, the major glycolytic end‐product, may also be important as a fuel, at least during the onset of metamorphosis. Lactate dehydrogenase (LDH) is required for both the synthesis and remobilization of lactate. It is highly expressed in larvae and is also induced by steroid signaling (Abu‐Shumays & Fristrom, 1997). LDH activity declines during metamorphosis (Rechsteiner, 1970), reflecting the drop in its messenger RNA late in larval development (Graveley et al., 2011), with the accumulated lactate being mostly used up during the prepupal stage (Li et al., 2017). In larvae, glycolysis serves the needs of adenosine triphosphate (ATP) production and supplies carbon skeletons for biosynthesis via the TCA cycle (Tennessen, Baker, Lam, Evans, & Thummel, 2011), while in the pupa, triglycerides are catabolized mainly in mitochondria. Efficient mitochondrial respiration is therefore crucial at both stages. In an earlier series of experiments, we found that flies expressing the alternative oxidase AOX from the tunicate Ciona intestinalis failed to complete development when reared on a low‐nutrient agar medium containing only yeast and glucose (Saari et al., 2018). AOX branches the mitochondrial respiratory chain, bypassing complexes III and IV in a non‐proton‐motive reaction that oxidizes ubiquinol directly by molecular oxygen (Rogov, Sukhanova, Uralskaya, Aliverdieva, & Zvyagilskaya, 2014). The gene for AOX is present in most groups of eukaryotes, including animals (McDonald & Gospodaryov, 2018), but has been lost from specific lineages during the course of evolution, notably from vertebrates and advanced insects. The reasons for its evolutionary loss or retention are unclear. In lower eukaryotes and plants it confers resistance against stresses or metabolic disruption resulting from overload, inhibition, or damage to the standard mitochondrial respiratory chain (Dahal, Martyn, Alber, & Vanlerberghe, 2017; Dufour, Boulay, Rincheval, & Sainsard‐ Chanet, 2000). Such stresses include the excess production of reactive oxygen species (ROS), limitations on ATP synthesis, restraints on metabolic flux, and disturbances to cellular redox and ionic homeostasis. AOX is believed to play a similar protective role in animals (McDonald & Gospodaryov, 2018; Saari et al., 2018). Since similar metabolic stresses arise in humans experiencing pathological dysfunction of mitochondria, we reasoned that AOX could be developed as a potential wide‐spectrum therapeutic (El‐Khoury et al., 2014). As a first step, we have established the transgenic expression of Ciona AOX in model organisms, including both Drosophila (Fernandez‐ Ayala et al., 2009) and the mouse (El‐Khoury et al., 2013; Szibor et al., 2017), to evaluate its effects on development, physiology, and pathology. In plants, AOX is enzymatically active only under conditions where the quinone pool becomes highly reduced (Castro‐Guerrero, Krab, & Moreno‐Sanchez, 2004; Hoefnagel & Wiskich, 1998). Thus, it contributes negligibly to electron flow under standard physiological conditions, while being available as a stress buffer whenever required. The same appears to be so for Ciona AOX expressed in the mouse (Dogan et al., 2018). In accordance with this, the ubiquitous expression of Ciona AOX in both flies (Fernandez‐ Ayala et al., 2009) and mammals (Szibor et al., 2017) has almost no detectable physiological effect under nonstressed conditions. However, if the standard respiratory chain is dysfunctional, for example, due to toxic inhibition (El‐Khoury et al., 2013; Fernandez‐Ayala et al., 2009; Szibor et al., 2017), overload (Mills et al., 2016), or genetic damage (Kemppainen et al., 2014; Rajendran et al., 2018), AOX is able to compensate the resulting phenotypes to a significant degree. However, since AOX bypasses two of the proton‐pumping steps of the standard respiratory chain, it is not able to fully restore ATP production. Thus, it cannot compensate null mutations or even profound knockdown of core subunits of cytochrome coxidase (complex IV), nor mutations that abolish the synthesis of a vital prosthetic group of the enzyme (Dogan et al., 2018; Fernandez‐Ayala et al., 2009; Kemppainen et al., 2014). The developmental failure of AOX‐expressing flies under nutritional stress (Saari et al., 2018) implies that transgenic AOX becomes enzymatically activated during at least part of the life‐cycle and/or in some crucial tissue(s), under low‐nutrient conditions. In addition, since activated AOX is known to facilitate TCA cycle reactions and decrease ROS production, but also to result in less ATP synthesis, developmental failure could result from multiple, nonexclusive causes. This might explain why the addition of either glucose or yeast to the low‐nutrient medium failed to improve the developmental outcome for AOX‐expressing flies. 342 | SAARI ET AL. To investigate the phenomenon further, we embarked on a study to identify the missing dietary components that would enable AOX‐ expressing flies to complete development, as well as to pin down more accurately the timing of developmental failure and its correlates in terms of stored nutrient accumulation. Our findings unambiguously ascribe AOX‐dependent developmental failure to the pupal stage. We also found that L3 (wandering‐ stage) larvae reared on the low‐nutrient medium are deficient in specific stored nutrients, regardless of genotype. Together, these results imply that AOX‐expressing flies are unable to make proper use of this depleted metabolic store, so as to complete development. Finally, we identified a specific set of metabolites as the most likely candidates to rectify this deficiency, thus enabling AOX‐expressing flies to fully undergo metamorphosis. 2 | MATERIALS AND METHODS 2.1 | Drosophila strains and culture Drosophila strains used in the study were the transgenic line UAS‐ AOX F6 for C. intestinalis AOX (on chromosome 2), constructed in‐ house and described previously (Fernandez‐Ayala et al., 2009), and the standard ubiquitous driver line daGAL4 (insertion on chromosome 3). Both lines were maintained as homozygotes and crossed together as indicated in figures. In addition, RNAi lines 30282 (Vienna Drosophila Resource Centre, GD library, chromosome 3, maintained over the TM3Sb balancer) and 65175 (Bloomington, TRiP line, chromosome 2, maintained over the CyO balancer), both for ATPCL, were crossed with daGAL4 to test the effects of ATPCL knockdown, using an internal control. Flies weremaintainedandculturedonstandardhigh‐sugar medium (Fernandez‐Ayala et al., 2009), described here as a complete medium, with 12 hr cycles of light and darkness at 25°C, except where indicated in specific experiments. Low‐nutrient medium consisted of (w/v) 1% agar, 3% glucose, and 3.5% yeast (Instant SD; Algist‐Bruggerman NV, Gent, Belgium), which were boiled together then cooled to 65°C before addition of standard antibiotics nipagin (to 0.1%) and propionic acid (to 0.5%), before dispensing into food vials. All other food supplements were added to the mixture during boiling, except for doxycycline which was dispensed as a stock solution onto food plugs into which it was allowed to penetrate and dry in a fume hood (final concentrations as shown in figure legends). Food supplements that were added to the low‐nutrient medium in specific experiments, were as follows: sucrose (VWR, 1.5% w/v), with or without fructose (Sigma, 3% w/v), soya flour (Soyolk; Oriola Oyj, Espoo, Finland; 1% w/v), maize flour (Risenta; Paulig Group, Helsinki, Finland; 1.5% w/v except where stated), wheat germ (Elovena Plus Vehnänalkio; Raisio plc, Raisio, Finland; 1% w/v), treacle (Lyle’s Black Treacle; Tate and Lyle Sugars, London, UK; 3% w/v except where stated), multivitamin tablets (Multitabs Family; Pfizer Consumer Healthcare, Helsinki, Finland; one tablet per 300 ml of fly food or diluted as indicated in figures), B‐vitamin supplement (Beko Strong; Orion Oyj, one tablet per 300 ml of fly food), Dulbecco’s modified Eagle medium (DMEM) powder, containing high‐glucose and glutamine but no pyruvate (Gibco; catalog #52100039), 1.24 g per 120 ml fly food, trisodium citrate dihydrate (Sigma), iron (ammonium iron(III)citrate; Sigma) or CuSO 4 (Sigma), used at concentrations indicated in the figures. The effects of different media on eclosion were tested by mating batches of 10 virgin females and five males overnight in food vials containing complete medium, then transferring them daily to fresh food vials containing the medium under test, for egg laying. Pupae per vial and the number of eclosed adults were recorded. 2.2 | Mammalian cells and culture Flp‐In™T‐REx™293 cells transformed with C. intestinalis AOX and their parental cell‐line were cultured as previously (Hakkaart, Dassa, Jacobs, & Rustin, 2006). To induce transgene expression 1.5 × 10 6 cells were seeded in 10 cm 2 plates and cultured for 72 hr in medium containing 1 μg/ml doxycycline (Sigma). The medium containing doxycycline was replaced after 48 hr. AOX transgene expression was verified by western blot analysis using a customized antibody, as previously (Dassa et al., 2009). 2.3 | Respirometry Respirometry on permeabilized cells was conducted essentially as described previously (Cannino et al., 2012), with the sequential addition of substrates and inhibitors as indicated in figure legends. 2.4 | Fractionation of treacle Treacle was fractionated into aqueous and nonaqueous components as follows. In a fume hood, 1.8 g treacle was diluted in water to 40 ml by gentle heating. After cooling, the solution was poured into a separation funnel, mixed with 40 ml of diethyl ether (Sigma), and extracted by gentle inversion approximately 10 times. After the phases had fully separated they were isolated and each extracted twice more with the opposite solvent. A further 40 ml of water was added to the ether fraction and the two fractions were then left overnight in open beakers for all ether to evaporate. Each fraction was then made up into 40 ml fly food by adding the low‐nutrient medium ingredients, for testing in the eclosion assay, or else analyzed further by mass spectrometry, as described below, or used in respirometry. For further fractionation by ethanol precipitation, 10 ml of the aqueous fraction were decanted into 50 ml centrifuge tubes, and 99% ethanol added to bring the final ethanol concentration to the desired level (between 40% and 75%). After overnight precipitation at −20°C and centrifugation at 14,000g max for 30 min at 4°C, supernatants were decanted, and the ethanol evaporated, while pellets were air‐dried and resuspended in 10 ml of water. Individual fractions were then made up into fly food for testing or analyzed by mass spectrometry, as described below. SAARI ET AL. | 343 2.5 | Metabolite assays To assay triglycerides, batches of 10 L3 (wandering‐stage) larvae were homogenized in 100 μl phosphate buffered saline (PBS)‐0.05% Tween (Medicago, Uppsala, Sweden), using a disposable plastic pestle. Samples were heated at 70°C for 5 min, cooled to room temperature, and vortexed. Aliquots of the homogenate (5 μl) were added to 100 μl of Triglyceride Reagent (Thermo Fisher Scientific) in transparent 96‐well plates. After incubation at 37°C for 30 min, absorbance at 540 nm was measured using a plate reader (Plate Chameleon™V; Hidex) and normalized for protein content based on the Bradford assay (Bradford Reagent; Sigma). For lactate and pyruvate assays, batches of 10 L3 larvae were homogenized similarly, in 6M guanidine hydrochloride on ice, then incubated at 95°C for 5 min. Supernatants were transferred to fresh vials and stored at −80°C, then thawed on ice and diluted 1:10 with water. Standards and reaction master mix were prepared according to the manufacturer’s protocol (L‐Lactate assay kit/Pyruvate assay kit; Sigma). The reactions were performed in a black 96‐well microplate by mixing samples 1:5 with the reagent master mix and incubation at room temperature for 30 min. Fluorescence (excitation 535 nm and emission 590 nm) was measured at 1 s intervals using the same plate reader and normalized for protein content. 2.6 | Mass spectrometry Ten microliter aliquots of treacle fractions were mixed vigorously with 300 µl of methanol containing internal standards (0.5 ppm d8‐ valine, 0.5 ppm d4‐succinic acid, 0.5 ppm d5‐glutamic acid, 2.4 ppm heptadecanoic acid) and dried under constant nitrogen flow at room temperature. The resulting residual metabolites were converted into methoxime and trimethylsilyl derivatives by a two‐step procedure. First, dried samples were dissolved in 25 µl of methoxyamine hydrochloride solution (20 mg/ml in pyridine; Sigma‐Aldrich), and incubated for 1 hr at 45°C. After the addition of 25 µl of N‐methyl‐N‐ (trimethylsilyl)trifluoroacetamide (Sigma‐Aldrich) and a further 1 hr incubation at 45°C, samples were spiked with 25 µl of an alkane‐ standard mixture (C10–C30, 10 mg/L; Sigma‐Aldrich). The analysis was performed using a gas chromatograph (Agilent 7890; Agilent Technologies, Santa Clara, CA) combined with a time‐of‐flight mass spectrometer (Pegasus BT; Leco Corp., St. Joseph, MI). Peak identification and data analysis used ChromaTOF software (Leco Corp.), NIST 2014 Mass Spectral Library and open‐source software Guineu v2. See Supporting Information for further details. 2.7 | Statistical analysis Student’sttest (Microsoft Excel) was used to assess significance when performing pairwise comparisons, implementing the Bonferroni correction where more than two groups were compared. When comparing multiple levels of a single factor to each other, one‐way analysis of variance (ANOVA) followed post hoc by the Tukey honestly significant difference (HSD) test (http://astatsa.com/) was applied. To test which of two factors was a significant determinant of a numerical outcome, two‐way ANOVA (GraphPad Prism) was used. Note that post hoc analysis (e.g., via the Tukey HSD test) is only appropriate in cases where a significant interaction between the factors is detected by ANOVA, or where more than two levels of a given significant factor are compared. χ 2 tests to compare observed and expected outcomes were performed online (https://www. graphpad.com/). Details are given in figure legends, as appropriate. 3 | RESULTS 3.1 | Developmental failure of AOX‐expressing flies occurs during the pupal stage We first established that the developmental failure of AOX‐ expressing flies grown on low‐nutrient medium occurs during metamorphosis, rather than during larval development or formation of the pupa (Figure 1). Matings that combined the AOX transgene with the ubiquitous daGAL4 driver produced a similar number of eggs as control strain matings (Figure 1a). Altering the glucose content of the medium had no significant effect on the number of eggs laid, regardless of genotype (Figure S1A). AOX expression also had no effect on the proportion of eggs that developed on low‐ nutrient medium as far as the pupal stage (Figure 1b and S1B). In contrast, the proportion of pupae that finally eclosed was dramatically decreased in AOX‐expressing flies when cultured on the low‐ nutrient medium (Figure 1c). 3.2 | Complex food supplements compensate for the developmental defect of AOX‐expressing flies To identify the dietary component(s) which enables AOX‐expressing flies to complete development on an otherwise low‐nutrient medium, we undertook a series of experiments in which we removed individual components from the standard medium, or added individual components to the low‐nutrient medium. Noting the exquisite temperature‐sensitivity of the phenotype (see Saari et al., 2018), we performed most assays at 25°C, a temperature at which approximately 10–20% of AOX‐expressing flies complete development on low‐nutrient medium. However, as indicated in figure legends, a limited number of assays were performed at 26°C for technical reasons. The omission of any one of the complex food additives, that is, maize flour, soya flour, wheat‐germ or treacle, or of sucrose, from the standard medium, had no effect on the eclosion frequency of AOX‐expressing (or control) flies (Figure 2a). Conversely, the addition to the low‐nutrient medium of any of the complex food additives but not of sucrose was sufficient to restore eclosion almost to control levels (Figure 2b), although wheat‐germ was consistently less effective than other additives. Supplementation with glucose or yeast to varying amounts produced no rescue (Figure 2c), while the effects of both treacle and maize flour were clearly dose‐dependent (Figure 2d). The published nutritional composition of 344 | SAARI ET AL. the complex food additives shows only modest overlap (Tables S1–S4). 3.3 | Specific food additives do not compensate for the developmental defect conferred by AOX Next, we tested whether the addition of specific vitamins, minerals or sugars was able to restore eclosion competence to AOX‐expressing flies. We selected concentrations of the various additives that have either been revealed previously to impact the phenotype of flies with relevant auxotrophies, or that are deemed effective in delivery to humans suffering an equivalent deficiency. In several cases, we tested a range of potentially effective concentrations, as well as combinations of additives. All additives and combinations of additives were found to be ineffective, including iron supplementation using two concentrations of ammonium iron(III)citrate (Figure 3a), multivitamin and B‐vitamin mixes either alone (Figure 3a) or in combination with iron supplements (Figure 3a) or sugars (fructose and sucrose, Figure 3b), various amounts of copper, supplied as CuSO 4 (Figure 3c) and even a complex mix of metabolites used as a medium in mammalian cell culture (DMEM; Figure 3d). 3.4 | Antibiotic treatment does not modify the developmental phenotype of AOX‐expressing flies Next, we tested whether the developmental failure of AOX‐expressing flies on low‐nutrient medium could be related to the growth of commensal bacteria. To address this issue, we tested AOX‐expressing and control flies on complete and low‐nutrient medium supplemented with a wide‐spectrum antibiotic, doxycycline. We used two concentrations of the drug, a stringent dose (100 μg/ml) that had a significant, (a) 100 80 60 40 20 0 % egg-to-pupa 100 80 60 40 20 0 %pupa-to-adult AOX F6 daGAL4 w 1118 GFP AOX F6 GFP w 1118 AOX F6 GFP x daGAL4 x w 1118 ** Eggs per vial 100 80 60 40 20 0 (b) (c) eggs pupae adults FIGURE 1 Developmental failure in AOX‐expressing flies occurs during metamorphosis. Strains are denoted as w 1118 (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’spost 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 SAARI ET AL. | 345 detrimental effect on larval development, but nevertheless allows us to measure relative effects on the completion of metamorphosis, and a much lower dose (15 μg/ml), typically used to eliminate intracellular bacteria such as Wolbachia (Koukou et al., 2006). Neither of these doses of doxycycline compromised eclosion on the complete medium (Figure 4a–c). On the low‐nutrient medium doxycycline also did not improve the eclosion frequency of AOX‐expressing flies, while having no impact on control flies. Doxycycline also did not block the rescue brought about by supplementation with treacle (Figure 4b) or maize flour (Figure 4c). 3.5 | Specific treacle fractions contain the active components rescuing developmental failure Since the addition of specific dietary supplements to low‐nutrient medium failed to rescue the AOX‐associated developmental failure, we attempted to isolate, or at least enrich for, the active material present in the complex food additives. This approach was simplest for treacle, because it contained only very low levels of particulates. The published composition of treacle (Table S1) gives only an approximate description of its biologically active ingredients, insufficient for % eclosion AOX F6 x daGAL4 daGAL4 100 80 60 40 20 0 (a) % eclosion AOX F6 x daGAL4 daGAL4 100 80 60 40 20 0 (b) wheat germ treacle soy flour maize flour % eclosion 0 2 4 6 8 10 12 14 treacle (%) 100 80 60 40 20 0 % eclosion 0 1 2 3 4 maize flour (%) 100 80 60 40 20 0 (d) % eclosion (c) 100 80 60 40 20 0 yeast % 1.5 3.5 7 10 3.5 3.5 3.5 3.5 glucose % 3 3 3 3 1.5 3 6 9 daGAL4 AOX F6 x daGAL4 sucrose complete minimal ** * * ## #### # ** *** * # FIGURE 2 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’sttest), 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’spost hoc HSD test). Pairwise comparisons of eclosion frequencies of flies of the two different genotypes on each given medium (using Bonferroni‐corrected Student’sttest), 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’sttest, 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 346 | SAARI ET AL. the present study. Therefore we used simple chemical fractionation to focus attention on the particular, active components. Ether extraction was used to separate treacle into aqueous and nonaqueous fractions which were then tested for their ability to support the development of AOX‐expressing flies. We reproducibly found the complementing activity only in the aqueous fraction (Figure 5a). It was still active after being combined with the nonaqueous fraction, showing that the latter did not contain an inhibitor of the former. Similar fractionation of wheat‐germ or maize flour was not considered feasible, due to the high level of particulates, but water/ether extraction of soya flour did produce a similar outcome, with the activity mainly in the aqueous fraction (Figure S2). To narrow down further the nature of the active component(s), the aqueous fraction of treacle, following ether extraction, was subjected to precipitation with varying concentrations of ethanol, generating supernatant and pellet fractions which were tested for effects on fly complete iron 80 μg/ml minimal iron 0.8 mg/ml multivitamin mix iron 0.8 mg/ml + multivitamin mix B-vitamin mix iron 0.8 mg/ml + B-vitamin mix % eclosion 100 80 60 40 20 0 AOX F6 x daGAL4 (b) % eclosion AOX F6 x daGAL4 AOX F6 100 80 60 40 20 0 (c) 0 20 50 70100 0 20 50 70100 [CuSO 4 ] (μM) vitamins 0 1/8 1/4 1/2 1 (d) % eclosion 100 80 60 40 20 0 minimal DMEM AOX F6 x daGAL4 daGAL4 minimal sugars + vitamins * ¤ ¤ ## ¤ * % eclosion AOX F6 x daGAL4 daGAL4 100 80 60 40 20 0 (a) * # # # # ## # ## * FIGURE 3 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’sttest). (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 CuSO 4 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 CuSO 4 concentration tested (Student’sttest). (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’sttest). Symbols denote significant differences between the genotypes in pairwise comparisons, with and without DMEM (Student’sttest). 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 SAARI ET AL. | 347 development (Figure 5b). When precipitated with 75% ethanol, the active component(s) were recovered in the pellet fraction, whereas at 40% or 60% ethanol both the pellet and supernatant fractions showed a partial activity, albeit with high variance (Figure 5b). A 65% ethanol precipitation gave an intermediate result, with more activity in the pellet than in the supernatant (Figure 5b). 3.6 | Treacle does not contain an inhibitor of AOX To explain the effect of treacle and other complex supplements, we considered the possibility that it contained an inhibitor of AOX, thus negating the effects of AOX expression on development. To test this possibility, we conducted respirometry on permeabilized HEK293‐ derived cells expressing Ciona AOX under the control of a doxycycline‐ inducible promoter (Hakkaart et al., 2006). After confirming AOX expression by western blot analysis (Figure S3A), we conducted respirometry according to a standard protocol (see Figure 6a), in which we compared oxygen consumption before and after the inhibition of complex III with antimycin. The role of AOX in antimycin‐resistant respiration was confirmed by subsequent treatment with n‐propyl gallate, a specific inhibitor of alternative oxidases. The addition of the water‐ soluble fraction of treacle to the permeabilized cells at a concentration equivalent to that in fly food, as well as 10% and 1% of this amount, had no effect on the AOX‐driven respiration (Figure 6a,b). Instead, treacle addition modestly stimulated respiration in both AOX‐expressing and control cells (Figure S3B). 3.7 | Flies reared on low‐nutrient medium show decreased levels of triglycerides and lactate The above findings strongly suggest one (or both) of two scenarios, relating nutrient storage and AOX. Either (a) accumulated nutritional reserves are lower in AOX‐expressing larvae cultured on low‐nutrient medium, compared with control larvae or with AOX larvae grown on complete medium; or (b) nutritional reserves accumulated on the low‐nutrient medium are similar, regardless of % eclosion AOX F6 x daGAL4 daGAL4 100 80 60 40 20 0 (a)  + +  + + doxycycline, 100 μg/ml complete minimal % eclosion 100 80 60 40 20 0 (b) 15 μg/ml 100 μg/ml daGAL4 control minimal minimal + treacle minimal + dox minimal + treacle + dox (c) % eclosion 100 80 60 40 20 0 complete complete + dox minimal minimal + dox minimal + maize minimal + maize + dox AOX F6 x daGAL4 daGAL4 * ** *** ¤ ** # # ** * ** FIGURE 4 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’sttest, ¤ and # :p< .05 and .001, respectively). ANOVA, analysis of variance; AOX, alternative oxidase; HSD, honestly significant difference; SD, standard deviation 348 | SAARI ET AL. genotype, but those resources are insufficient to enable AOX pupae to complete development, due to AOX activation during metamorphosis. To test these possibilities, we assayed the relative levels of triglycerides (Figure 7a) and lactate (Figure 7b) in wandering‐stage AOX‐expressing L3 larvae and controls grown on complete versus low‐nutrient medium. Growth on the low‐ nutrient medium significantly decreased the relative concentration of triglycerides (two‐way ANOVA, p< .01) and lactate (two‐way ANOVA; p< .001), while AOX expression had no significant effect on these metabolites. Pyruvate, being interconvertible with lactate via LDH was also decreased in larvae grown on low‐nutrient medium (Figure S4, p< .01), but not by AOX expression. These findings support the hypothesis that the low‐nutrientdietrestricts the accumulation of stored nutritional resources and that it is the inability to mobilize these diminished resources that causes developmental failure in AOX‐expressing pupae. 3.8 | Treacle fractionation reveals a list of candidate nutrients for compensating AOX Treacle and its various fractions that were tested earlier in the developmental assay were analyzed further by mass spectrometry, alongside standards for 48 common metabolites. Of these, the majority was detectable in treacle, and most of these were present in the aqueous fraction thereof (Table 1). However, only four were clearly enriched in the pellet after precipitation in 75% ethanol. Since fructose, by far the most abundant of them, had already been tested in the developmental assay (Figure 3b), we proceeded to test the next most enriched, citrate, because of its metabolic role in the generation of cytosolic acetyl‐CoA, the main precursor for fatty acid synthesis. However, citrate was unable to alleviate the developmental failure of AOX‐expressing flies on low‐nutrient medium (Figure 8), even though its conversion to acetyl‐CoA by ATP citrate lyase appears to be essential for Drosophila development (Figure S5). 4 | DISCUSSION In this study, we established that the developmental failure of AOX‐expressing flies on low‐nutrient medium occurs specifically during the pupal stage (Figure 1). Several key metabolites regarded as nutrient stores for metamorphosis were at diminished levels in flies cultured on low‐nutrient medium, regardless minimal + aqueous + ether + combined fractions % eclosion 100 80 60 40 20 0 (b) pellet supernatant 40% 60% 75% 65% 75% EtOH concentration ** ** ** % eclosion 100 80 60 40 20 0 (a) ** FIGURE 5 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. 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International Journal of Biological Sciences,6, 252–267. https://doi.org/10.7150/ijbs.6.252 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Saari S, Kemppainen E, Tuomela T, Oliveira MT, Dufour E Jacobs HT. Alternative oxidase confers nutritional limitation on Drosophila development. J. Exp. Zool. 2019;331:341–356. https://doi.org/10.1002/jez.2274 356 | SAARI ET AL.