Mitochondrion xxx (2013) xxx–xxx

MITOCH-00829; No of Pages 6

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Mitochondrion

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Diet influences the intake target and mitochondrial functions of Drosophilamelanogaster males

Nicolas Pichaud a,b,⁎, Marie Messmer a, Carolina C. Correa a, J. William O. Ballard a

a School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australiab Département de Biologie Intégrative, Université du Québec à Rimouski, Rimouski, QC, Canada

⁎ Corresponding author at: Laboratoire de biologie intégUniversité du Québec à Rimouski, 300 allée des UrsulinCanada. Tel.: +1 418 723 1986x1634.

E-mail address: pichaud.nicolas@wanadoo.fr (N. Pic

1567-7249/$ – see front matter © 2013 Elsevier B.V. anhttp://dx.doi.org/10.1016/j.mito.2013.05.008

Please cite this article as: Pichaud, N., et al., Dtochondrion (2013), http://dx.doi.org/10.10

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 February 2013Received in revised form 26 April 2013Accepted 14 May 2013Available online xxxx

Keywords:HaplotypeMitochondrial functionsDietFeeding rateEnzymatic activitiesP:C ratio

In this study, we examine the dietary protein to carbohydrate ratio (P:C) on the mitochondrial functions oftwo Drosophila melanogaster mtDNA haplotypes. We investigated multiple physiological parameters onflies fed with either 1:12 P:C or 1:3 P:C diets. Our results provide experimental evidence that a specific hap-lotype has a reduction of complex I activity when the flies are fed with the 1:12 P:C diet. This study is of par-ticular importance to understand the influence of diet on mitochondrial evolution in invasive and broadlydistributed species including humans.

© 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction

The goal of this study is to test whether diet can differentially affectmitochondrial functions of organisms harbouring knownmitochondrialDNA differences in a constant nuclear genetic background. Differentialexpression of genes mediated by diet has the potential to influencethe organismal phenotype. To date, however, few examples of such in-teractions have been described (Luan et al., 2001; Zhang et al., 2012)and we still have to determine which suite of genes and genetic path-ways are most affected by diet. We hypothesized that diet compositionis likely to be one parameter that impacts the functional properties ofmitochondria. Nutrients from the diet provide the substrates for mito-chondrial function to produce energy in the form of ATP (the oxidativephosphorylation, OXPHOS). Therefore, diet composition will likely in-fluence mitochondrial functions through the availability of metabolicsubstrates provided to mitochondria.

To critically examine the effect of nutrients present in the diet on mi-tochondrial functions we employed the Geometric Framework (Simpsonand Raubenheimer, 2012). This framework is a state-spacemodelling ap-proach that explores how an animal solves the problemof balancingmul-tiple and changing nutrient needs in a multidimensional and variablenutritional environment. There is a mixture of nutrients that is optimal,which the Geometric Framework calls a nutritional target. The Geometric

rative, Département de biologie,es, Rimouski, Québec G5L 3A1,

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iet influences the intake targ16/j.mito.2013.05.008

Framework has two additional targets. Thefirst is the intake target,whichrepresents the amount of nutrients that an animal needs to ingest in orderto reach its nutritional target. Animals also have a growth target, whichreflects the level of nutrients incorporated into growth and storagetissues.

There is compelling evidence that diet particularly the dietary pro-tein to carbohydrate (P:C) ratio is a key determinant for multiple or-ganismal physiological traits such as reproduction and longevity(Fanson et al., 2009; Lee et al., 2008; Maklakov et al., 2008; Simpsonand Raubenheimer, 2012). However, the specific influence of P:Cratio on mitochondrial functions of flies is currently unknown. Inthis study, we fed flies with diets differing in the P:C ratio. We thenmeasured feeding rate using the capillary feeder (CAFE) assay (Ja etal., 2007, 2009; Lee et al., 2008). Foods varied in sucrose andhydrolysed yeast content, yielding P:C ratios of 1:12 and 1:3. The1:12 diet is expected to result in an increased survival while the 1:3diet is known to result in higher fecundity in D. melanogaster females(Lee et al., 2008).

Moreover, we critically examine these feeding rates in twoD. melanogaster fly lines that harbour a known set of mtDNAmutations in a highly standardised nuclear genetic background. Recentstudies strongly suggest that environmental conditions can differential-ly affect mitochondrial functions of fly lines harbouring distinct mtDNAhaplotypes (Pichaud et al., 2012a, 2012b). The two lines we includedwere created by chromosome replacement using balancer chromo-somes and have highly similar nuclear DNA genomes but retain theiroriginal mtDNA genomes from Japan and Alstonville (Clancy, 2008).The primary difference between these lines occurs at the ND2 subunit

ll rights reserved.

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of complex I (histidine to tyrosinemutation at position 182), though ad-ditional synonymous, tRNASer

AGY and 12S rRNA differences are known.TheND2 subunit is believed to belong to theminimal assembly requiredfor catalysis and may function for the quinone-binding (Amarneh et al.,2003; Ohnishi et al., 2012) and mutations in tRNA or rRNA genes havebeen shown to cause abnormalities which result in decreased mito-chondrial functions (Akashi, 1994; Moreno-Loshuertos et al., 2011).

To assay mitochondrial function of the D. melanogastermales fed ontwo P:C diets we quantified mitochondrial respiration at different stepsof the ETS. Furthermore, we determined citrate synthase activity (CS),as a biomarker of mitochondrial density, and NADH-cytochrome c oxi-doreductase (CI + CIII) activity. Using this integrative approach, wefound that the fly lines harbouring specific mtDNA mutations exhibitmetabolic differences related to the P:C ratio.

2. Materials and methods

2.1. Flies and fly feeding

2.1.1. FliesJapan and Alstonville fly lines used in this studywere created by chro-

mosome replacement using balancer chromosomes (Clancy, 2008). Tominimise the potential influence of mutations accumulating in eachline, females were backcrossed to males from an inbred w1118 line forsix generations immediately prior to the commencement of this study.Resulting flies were sequenced for a diagnostic region of the mtDNA ge-nome to confirm the mtDNA haplotype at the beginning and end of thestudy (Aw et al., 2011).

All lines were maintained at low density at 23 °C under a 12:12light:dark diurnal cycle with 50% humidity on a standard semolina me-dium containing a mixture of 24 g agar–agar, 500 g treacle, 170 g offresh yeast and 250 g of semolina flour dissolved in 2.1 L of water,complemented with 10% methyl-4-hydroxybenzoate. Newly hatchedflies from the last generation of backcrossing were kept for one day onstandard semolina medium and were then housed in groups of sixflies in each vial. Male flies were studied because mitochondrial dys-function is hypothesized to be more pronounced in males than females(Camus et al., 2012; Gemmell et al., 2004; Innocenti et al., 2011).

2.1.2. Food consumptionFood consumption of flies was measured using a modified CAFE

assay (Ja et al., 2007, 2009; Lee et al., 2008). Briefly, two differentdiets were prepared in sterile distilled water. Foods varied in sucroseand hydrolysed yeast content, yielding P:C ratios of 1:12 and 1:3. The1:12 diet is expected to result in an increased survival while the 1:3diet is known to result in higher fecundity in D. melanogaster females(Lee et al., 2008). Macronutrient compositions are based on autolyzedyeast (MP Biomedicals) containing 45% protein, 24% carbohydrate (asglucose equivalent), 21% indigestible fibre, 8%water, and the remaining2% is composed of fatty acids, minerals, and vitamins. Concentration ofnutrients was 180 g.L−1 and 0.01% phosphoric acid and 0.1% propionicacid was added to each diet as antimold agents (Ballard et al.,2010). Calibrated glass micropipettes (5 μL) filled with liquid dietcomplemented with 0.145% agar to avoid dripping and cappedwith a mineral oil overlay to minimise evaporation were providedto the flies to measure food intake. Capillaries were replaced dailyand intake was measured. To ensure flies had access to ad libitumwater each vial was filled with 20 mL of 1% agar–agar sterile dis-tilled water (Ja et al., 2009). Each treatment included three identi-cal vials without flies to determine evaporative loss, which wassubtracted from experimental readings.

Different vials containing six flies of the same haplotype were usedfor all the experiments (n = 5 vials for Alstonville for 1:3 P:C ratio;n = 6 Japan for 1:3; n = 6 Alstonville for 1:12; n = 7 Japan for1:12). Food consumption (μL)wasmeasureddaily for 10 days and aver-aged per fly per day. At 11 days-old, four flies from each vial were

Please cite this article as: Pichaud, N., et al., Diet influences the intake targtochondrion (2013), http://dx.doi.org/10.1016/j.mito.2013.05.008

collected for high-resolution respirometry studies. All remaining flieswere immediately stored at −80 °C for enzymatic analysis.

2.2. Preparation of permeabilized muscle fibres

Fibre permeabilizations of thorax muscle were conducted at 4 °C aspreviously described for Drosophila using a BIOPS relaxing solutioncomplemented with saponin (Pichaud et al., 2011, 2012a, 2012b). Tho-raxes were permeabilized, blotted and weighed using a Sartorius CP2PElectronic Micro Precision Balance (Sartorius, Gottingen, Germany).Each experiment was conducted using two groups of flight muscle fi-bres from 2 thoraxes (e.g. pseudo-replicates). Fibres were immediatelytransferred after permeabilization into each of the two chambers of anOxygraph-2 K respirometer (Oroboros Instruments, Innsbruck, Austria)filled with air-saturated respiration medium containing 115 mM KCl,10 mM KH2PO4, 2 mM MgCl2, 3 mM HEPES, 1 mM EGTA, 0.2% BSA,and pH 7.2.

2.3. High-resolution respirometry

Zero oxygen measurements were taken after sodium dithionite ad-dition and oxygen electrodes of the respirometer were calibrated withair-saturated respiration medium at 23 °C. O2 solubility for the respira-tion medium was calculated at 23 °C according to Rasmussen andRasmussen (2003). Pyruvate, L-proline and malate (10 mM each)were injected into the oxygraph chambers and the samples were trans-ferred shortly thereafter. To enable oxygen diffusion in permeabilizedfibres, oxygenwas injected into the chambers (Gnaiger, 2009). All mea-surements were expressed as means of respiration rates expressed inpmol of oxygen consumed per second per mg of permeabilized fi-bres ± s.e.m.

The different respiration rates measured are presented with the ab-breviation(s) of the complex(es) involved followed by the state of res-piration (complex-STATE). After stabilization of the signal, the LEAKstate (or state 2′) for complex I (CI-LEAK) was monitored (Gnaiger,2009) with pyruvate, L-proline and malate. This state is a resting stateof non-phosphorylating respiration when oxygen flux is maintainedmainly to compensate for the proton leak (Gnaiger, 2009). Injection ofexcess ADP (5 mM) allowed to reach a state of OXPHOS (or state 3)for complex I (CI-OXPHOS). This enables measurement of the maxi-mum oxygen flux when the transport of electrons from complex I iscoupled to the production of ATP. Subsequent injection of cytochromec from equine heart (15 μM) allowed evaluation of the functional integ-rity of the outer mitochondrial membrane (CIc-OXPHOS). Respiratorycontrol ratios (RCR = CI-OXPHOS/CI-LEAK) and increase of oxygenflux after injection of cytochrome c (CIc-OXPHOS/CI-OXPHOS) wereused as quality control of mitochondria in the permeabilized fibres.

Additional O2 fluxes were then determined by sequentially injectingdifferent compounds: sn glycerol-3-phosphate (20 mM) tomonitormax-imumOXPHOS (or state 3)with convergent electronflow from complex Iand glycerol-3-phosphate dehydrogenase (CIc + G3Pdh-OXPHOS); FCCP(optimum concentration reached between 0.75 and 1.25 μM) to stimu-late uncoupled respiration for complex I and glycerol-3-phosphate dehy-drogenase as a measure of ETS capacity (CI + G3Pdh-ETS); rotenone(1 μM)which is an inhibitor of complex I allowing tomeasure the oxygenflux due to the electron input through G3Pdh in the uncoupled state(G3Pdh-ETS), and antimycin A (2.5 μM) inhibitor of complex III to mea-sure residual oxygen consumption. The inhibition of complex III byantimycin A allowed us to measure the residual oxygen consumptiondue to residual oxidative side reactions occurring in permeabilized fibresand correct all the O2 fluxes (Gnaiger, 2009).

Cytochrome c oxidase (complex IV, Cox) is the last complex of theETS and reduces O2 to H2O. Its activity represents the maximalefficiency of complex IV to consume oxygen. Cox activity wasmeasured after inhibition of complexes I and III, by injecting N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and ascorbate (0.5 μM

et and mitochondrial functions of Drosophila melanogaster males, Mi-

Table 1Mean values (± s.e.m) of food consumption, respiratory control ratios for complex I (RCR = CI-OXPHOS/CI-LEAK), and cytochrome c effect (CIc-OXPHOS/CI-OXPHOS). * denotesdifferences (LSD test) between Japan and Alstonville fed with the same diet and † denotes differences (LSD test) between diets in one of the two haplotypes. Significance was set atP b 0.05.

Japan Alstonville

1:3 1:12 1:3 1:12

Food consumption (μL per day per fly) 0.44 ± 0.02† 0.61 ± 0.02⁎ 0.43 ± 0.04† 0.53 ± 0.01Respiratory control ratio for complex I 13.14 ± 0.78 11.95 ± 1.02 15.27 ± 1.35 14.68 ± 1.56Cytochrome c effect 1.03 ± 0.02 0.98 ± 0.02 1.03 ± 0.01 1.01 ± 0.01

Table 2Analyses of variance results showing F ratios obtained with the GLM procedure.

Error df Haplotypedf = 1

Dietdf = 1

Haplotype × Dietdf = 1

Feeding rate 23 4.52⁎ 33.63⁎⁎⁎ 2.13RCR for complex I 23 4.2 0.59 0.06Mass-specific O2 fluxes

CI-LEAK 23 1.99 0.11 0.93CI-OXPHOS 23 6.09⁎ 0.44 1.24CIc-OXPHOS 23 6.43⁎ 0.75 1.37CIc + G3Pdh-OXPHOS 23 3.89 0.41 0.69CIc + G3Pdh-ETS 23 4.25 0.48 0.6G3Pdh-ETS 23 7.59⁎ 1.93 1.73Cox 23 5.48⁎ 0.02 0.08

Enzymatic activitiesCS 23 6.38⁎ 35.45⁎⁎⁎ 0.23CI + CIII 23 6.86⁎ 0.01 0.05

⁎ P b 0.05.⁎⁎⁎ P b 0.001.

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and 2 mM respectively) into the chambers. Due to autoxidation ofTMPD, ascorbate and cytochrome c, chemical backgrounds were eval-uated at the end of each experiment after inhibition of Cox and weresubtracted from the activity (Gnaiger et al., 1998).

2.4. Enzymatic analysis

We included two complimentary enzymatic activity assays of CS andCI + CIII. CS is a key enzyme of the tricarboxylic acid cycle and is consid-ered a good biomarker of mitochondrial content (Larsen et al., 2012).CI + CIII activity represents the conjoint capacity of NADH:ubiquinoneoxidoreductase (complex I) and ubiquinone:cytochrome c oxidoreduc-tase (complex III). CS and CI + CIII activities were measured on a singlethorax homogenised in 200 μL of ice-cold buffer (100 mM KH2PO4,1 mM EDTA, 0.1% Triton X-100, pH 7.5) according to Hébert Chatelainet al. (2011), using a microplate reader Spectramax M3 (Molecular De-vices, CA, USA) set at 23 °C.

For CS activity, homogenates were incubated with a 100 mM imidaz-ole HCl buffer complemented with 0.1 mM 5,5′-Dithiobis(2-nitrobenzoicacid), 0.1 mM acetylCoA (pH 8) and 0.15 mM oxaloacetate. Activity wasmeasured at 412 nm (ε412 = 13.6 mL.cm−1.μmol−1). CI + CIII activitywas measured by incubating homogenates with 0.1 mM sodium phos-phate (pH 8.5), 0.85 mM NADH, 2 mM iodonitrotetrazolium chlorideand 0.03% Triton X-100. Activity was measured at 490 nm (ε490 =15.9 mL.cm−1.μmol−1). Protein concentration was measured using a DCProtein Assay (Bio-Rad, CA, USA) and was used to express enzymatic ac-tivities in U.mg−1 proteins, where U is 1 μmol of substrate transformedper minute.

2.5. Statistical analyses

All statistical analyses were performed with SAS software (9.1.3,SAS Institute, Cary, IN, USA). O'Brien's test was used to verify homo-geneity of the data. Analyses of variance (ANOVA) with two indepen-dent variables (haplotype and diet composition) were performedusing a general linear model (GLM) procedure. When an effect wasdetected, the GLM procedure was followed by a post-hoc Least SquareDifference (LSD) test. Significance was defined at P b 0.05.

3. Results

3.1. Flies and fly feeding

To test our hypotheses that diet could differentially influence the in-take target of flies harbouring different mtDNA haplotypes, the feedingrates of males were measured using the dietary P:C ratios of 1:12 and1:3 (Table 1). The GLM procedure showed that the haplotype and thediet composition but not their interaction had a significant effect onfeeding rate (Table 2). As determined by the post-hoc test, fliesharbouring Japan mtDNA consumed more food than those harbouringAlstonville mtDNA on the 1:12 diet (LSD: P = 0.003) but not the 1:3diet (LSD: P = 0.78). As expected, flies ingested more food when fedwith the 1:12 diet compared to the 1:3 diet (LSD: P = 0.0002 andP = 0.025for Japan and Alstonville respectively).

Please cite this article as: Pichaud, N., et al., Diet influences the intake targtochondrion (2013), http://dx.doi.org/10.1016/j.mito.2013.05.008

3.2. High-resolution respirometry

We used RCR and cytochrome c to determine the quality and func-tionality of mitochondria. RCRs above 3 indicate well-coupled respira-tion (Gnaiger, 2009) while cytochrome c addition tests for intactnessof the mitochondrial outer membrane (Kuznetsov et al., 2008). RCRswere calculated for complex I as CI-OXPHOS/CI-LEAK. In all experi-ments, RCRs obtained using substrates for complex I were above 10.0for each treatment (Table 1), indicating well-coupled respiration inevery preparation. No significant effect of haplotype, diet compositionor their interaction was detected on RCR values (Table 2). As expected,respiration rates before and after cytochrome c additions were verysimilar in all preparations attesting the integrity of mitochondria inthe permeabilized thoraxes (Table 1).

Mass-specific O2 fluxes were measured in permeabilized fibresat different steps of the ETS (CI-LEAK, CI-OXPHOS, CIc-OXPHOS,CIc + G3Pdh-OXPHOS, CIc + G3Pdh-ETS, G3Pdh-ETS). CI-OXPHOS,CIc-OXPHOS and G3Pdh-OXPHOS were influenced by the haplotype(Table 2). These measures are directly related to complex I where thereis a known difference between the Japan and Alstonville mtDNA. Therewere no significant diet or interaction terms (Table 2). Post-hoc LSDtests were used to determine the specific differences between the fliesharbouring different haplotypes but fed with the same diet. Significantlylower fluxes were measured for Japan flies under the 1:12 diet (LSD:P = 0.013 and P = 0.010 for CI-OXPHOS and CIc-OXPHOS respectively)but not when the flies were fed with the 1:3 diet (LSD: P = 0.48 andP = 0.48for CI-OXPHOS and CIc-OXPHOS respectively) (Fig. 1). Further-more, somewhat surprisingly Japan flies fed with the 1:12 diet presenteda higher flux than Alstonville flies when the complex I was inhibited(G3Pdh-ETS) (LSD: P = 0.013; Fig. 1). For G3Pdh-ETS, no differenceswere detected between haplotypes when the flies were fed with the 1:3diet (LSD: P = 0.40).

Cox activity was measured to determine the maximal efficiency ofcomplex IV to consume oxygen. According to the GLM procedure, Coxactivity was influenced by the haplotype but neither by the diet norby the haplotype by diet interaction (Table 2). However, post-hoc

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analyses did not identify significant differences between haplotypeswhen fed with the different diets (LSD: P = 0.06 and P = 0.24 for1:12 and 1:3 diets respectively).

3.3. Enzymatic activities

We assayed CS as a measure of mitochondrial content (Larsen etal., 2012). The GLM procedure indicated that CS activity assay wassignificantly influenced by the mtDNA haplotype and the diet butthere was not a significant interaction between them (Table 2).Post-hoc tests showed that flies fed with the 1:12 diet had lower CSactivity than those fed with the 1:3 diet in both haplotypes (LSD:P = 0.005 and P = 0.0002 for Japan and Alstonville respectively;Fig. 2). No significant differences were detected between haplotypesfed with the same diets (LSD: P = 0.06 and P = 0.13 for 1:12 and1:3 respectively; Fig. 2).

CI + CIII represented the combined activity of complexes I and III.The GLM procedure showed that CI + CIII activity was significantlyinfluenced by the haplotype but not by the diet or by the haplotypeby diet interaction (Table 2). According to the post-hoc test, Japanflies had lower CI + CIII activity than Alstonville flies when fed withthe 1:12 diet (LSD: P = 0.03) but not the 1:3 diet (LSD: P = 0.17).This supports the results obtained for O2 fluxes suggesting that thecomplex I of Japan flies has a lower capacity than Alstonville flieswhen they are feeding on the 1:12 diet but not the 1:3 diet.

4. Discussion

Extensive evidence supports the hypothesis that diet influenceslifespan and fecundity in a variety of organisms (Fanson et al., 2009;Lee et al., 2008; Maklakov et al., 2008; Simpson and Raubenheimer,2012). The current study extends the previous work and asks wheth-er diet differentially influences the mitochondrial functions of organ-isms harbouring specific and known mtDNA differences. If diet candifferentially influence mitochondrial functions and the organismalphenotype then this cornerstone environmental parameter has the

Fig. 1.O2 fluxesmeasured in permeabilized thoraxes ofDrosophilamelanogastermales harbouringare expressed as pmolO2 consumed per secondpermg offibres.Mitochondrial functions are prese(complex-STATE) and were measured in presence of pyruvate + malate + L-proline (CI-LE(CIc + G3Pdh-OXPHOS), + FCCP (CIc + G3Pdh-ETS), + rotenone (G3Pdh-ETS), + antimycinmeans ± s.e.m. For the 1:12 diet, 7 different preparations were tested for Japan and 6 for Alstonvferences (LSD test). Significance was set at P b 0.05.

Please cite this article as: Pichaud, N., et al., Diet influences the intake targtochondrion (2013), http://dx.doi.org/10.1016/j.mito.2013.05.008

potential to influence the fate of specific mutations in populationsand potentially species. If true, this is likely to be of utmost impor-tance in invasive species that colonise new habitats and broadly dis-tributed opportunistic species that feed on a variety of nutrientsincluding humans.

We tested the influence of two P:C diets on the mitochondrialfunctions of two D. melanogaster mtDNA haplotypes expressed in ahighly standardised nuclear background (Clancy, 2008). The resultssuggest that the 1:12 P:C diet but not the 1:3 P:C diet differentiallytriggers the expression of a particular mitochondrial phenotype inJapan but not in Alstonville males. We suggest that this result ismost likely caused by the single non-synonymous mutation in themtDNA encoded ND2 locus (complex I). However, it is possible thatthe known tRNA and 12S rRNA mutations may affect transcriptionalor translational accuracy (Akashi, 1994), as well as mitochondrial bio-genesis (Moreno-Loshuertos et al., 2011). Moreover, unaccounteddifferences in the A + T rich region of the mtDNA and/or in chromo-some 4 (Clancy, 2008) may also influence the mitochondrial func-tions measured (Correa et al., 2012).

A possible explanation for our results is that flies harbouring mito-chondria from the Japan haplotype have a reduced efficiency to reduceNADH at the level of complex I and this competence is exposed on the1:12 diet but not the 1:3 diet. Thismight be due to an impairment ofmi-tochondrial function that changed the nutritional intake target and po-tential aspects of organismal physiology (Simpson and Raubenheimer,2012). The nutritional target is known to change depending on thestage of development and the environmental circumstances. Japanflies consumed more than Alstonville flies under the 1:12 diet but notunder the 1:3 diet and flies harbouring both haplotypes consumedmore food under the 1:12 diet than under the 1:3 diet (Table 1).These results support previous studies showing that carbohydrates en-hanced consumption in flies but proteins induced satiation (Lee et al.,2008; Skorupa et al., 2008). One possible candidate thatmaydetect pro-tein to carbohydrate levels in flies is Hifα. Hifα senses the primary sub-strates reduced by the mitochondria, controls glycolytic substrate fluxvia the transcriptional control of glycolysis, and controls the expression

the Japan or AlstonvillemtDNAhaplotype and fedwith either the 1:12 or the 1:3 diet. Resultsntedwith the abbreviation(s) of the complex(es) involved followedby the state of respirationAK), + ADP (CI-OXPHOS), + cytochrome c (CIc-OXPHOS), + sn glycerol-3-phosphateA used to correct for residual O2 consumption, + TMPD + ascorbate (Cox). Results areille. For the 1:3 diet, 6 preparations were tested for Japan and 5 for Alstonville.* denotes dif-

et and mitochondrial functions of Drosophila melanogaster males, Mi-

Fig. 2. Enzymatic activities of citrate synthase (CS) and NADH-cytochrome c oxidore-ductase (CI + CIII) measured in the two haplotypes of Drosophila melanogaster, Japanand Alstonville, fed with either the 1:12 or the 1:3 diet. Results are means ± s.e.m.For the 1:12 diet, 7 different homogenates were tested for Japan and 6 for Alstonville.For the 1:3 diet, 6 homogenates were tested for Japan and 5 for Alstonville.* denotesdifferences (LSD test) between haplotypes; letters denote differences (LSD test) be-tween flies of the same haplotype fed with different diets with a statistically differentfrom b. Significance was set at P b 0.05.

5N. Pichaud et al. / Mitochondrion xxx (2013) xxx–xxx

of the cytochrome c oxidase genes (Freije et al., 2012). Currently, how-ever,much remains unknown about the specific pathways involved andthe mechanisms that elicit specific responses.

The mtDNAmutations also changed mitochondrial functions. At thelevel of complex I, flies with Japan mtDNA had lower mass-specific O2

fluxes when fed with the 1:12 diet (CI-OXPHOS, CIc-OXPHOS) but hadelevated fluxes when rotenone was included as a blocker (G3Pdh-ETS).These results support the enzymatic activity results of CI + CIII whichshow a significant decrease of activity in Japan flies under the 1:12diet when compared to Alstonville flies under the same diet. These re-sults suggest that the sensitivity of the quinone-binding (which is de-pendant of ND2) to rotenone was also affected in the complex I ofJapan flies. An alternate explanation is that the observed differences inboth O2 fluxes and CI + CIII activity are due to a compensatory mecha-nism (Benard et al., 2010). One possible compensatory mechanism is achange in mitochondrial density. Specifically, Japan flies tend to have alowermitochondrial density thanAlstonville as determined by CS activ-ity (Table 2, Fig. 2). This could reflect a reduction of the number of ETScomplexes and plausibly a decrease in both mitochondrial respirationand enzymatic activity. However, this does not appear to be the case.Somewhat surprisingly, a real time PCR assay indicated that mtDNAdensity was higher in Japan flies suggesting that the number of ETScomplexes was not reduced in Japan flies (Correa et al., 2012). This re-sult also suggests that mtDNA copy can vary independently of mito-chondrial density (Larsen et al., 2012). A second alternate explanationis that Japan flies have reduced mitochondrial efficiency (especially at

Please cite this article as: Pichaud, N., et al., Diet influences the intake targtochondrion (2013), http://dx.doi.org/10.1016/j.mito.2013.05.008

the level of complex I) when the protein: carbohydrate ratio is de-creased (i.e. under the 1:12 P:C diet). Reduced mitochondrial efficiencyin Japan males could lead to an increase in reactive oxygen species(ROS) production and an increase in the rate of ageing (Harman,1956) when compared with Alstonville males (as shown by Clancy,2008). While plausible, this explanation seems less likely becausehigh-protein:low-carbohydrate ratio has been shown to decreaselifespan in Drosophila (Lee et al., 2008; Skorupa et al., 2008) and istherefore thought to bemore challenging in terms ofmitochondrialme-tabolism, ageing, or both.

Our results showed that a low P:C diet is associated with lower CSactivity and higher food consumption. It has been shown that Indy,which is a transporter protein of Krebs cycle intermediates such ascitrate can interact with diet in D. melanogaster to influence food con-sumption (Wang et al., 2009). Since CS is one of the major enzymes ofthe Krebs cycle, it is possible that under the 1:12 diet Indy expressionis decreased. Assaying mRNA expression of Indy would test thishypothesis.

There are two major limitations to this study. First a single cohortof flies was included. Correa et al. (2012) analysed the same fly lines ayear before this study and they observed very similar results for com-plex I substrates and respiration rates measured even though slightlydifferent environmental conditions were assessed (1:3 diet vs 1:3.2diet and liquid diet vs bottom food). Second, only two P:C ratioswere tested. Inclusion of additional diets may show that differentdiets are optimal for specific genotypes.

5. Conclusions

Overall, our results show that diet composition influences the ex-pression of organismal phenotype and mitochondrial functions inmale flies harbouring a small set of mtDNA differences. The ND2 mu-tation may affect the structure of complex I, the tRNASer

AGY mutationmay affect transcriptional accuracy and efficiency (Akashi, 1994) andthe mutation in 12 s rRNA gene may destabilize the structure of the12 s rRNA (Correa et al., 2012). Here, we included males becausethe “mothers curse” hypothesis predicts that mtDNA effects are great-er in males than in females (Camus et al., 2012; Gemmell et al., 2004;Innocenti et al., 2011). To further examine the evolutionary conse-quences of diet on genotype future studies need to examine the influ-ence of diet in females and test important life history traits such asrates of egg production.

Acknowledgements

We would like to thank William Ja for the helpful comments on theCAFE assay. We acknowledge Australian Research Grant DP110104542for funding.

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