Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry

Assessment of MitochondrialFunctions in Daphnia pulexClones Using High-ResolutionRespirometrySANDRINE A. KAKE-GUENA1,KAMAL TOUISSE1, ROLAND VERGILINO2,FRANCE DUFRESNE2, PIERRE U. BLIER2,AND H�EL�ENE LEMIEUX1*1Campus Saint-Jean, University of Alberta, Edmonton, Alberta, Canada2D�epartement de biologie, Laboratoire de Physiologie Animale Int�egrative, Universit�e duQu�ebec�a, Rimouski, Rimouski, Qu�ebec, Canada

ABSTRACT The objectives of our study were to adapt a method to measure mitochondrial function in intactmitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitiveenough to characterize mitochondrial metabolism in clones of the pulex complex differing inploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging tothe Daphnia pulex complex represent a powerful model to delineate the link betweenmitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergentmtDNA can be grown under various experimental conditions. Our study included two diploidclones from temperate environments and two triploid clones from subarctic environments. Thewhole animal permeabilization and measurement of respiration with high-resolution respirometryenabled the measurement of the functional capacity of specific mitochondrial complexes in fourclones. When expressing the activity as ratios, our method detected significant interclonalvariations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximalphysiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported bycomplex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill(Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II.Additional studies are required to determine if these differences in mitochondrial functions arerelated to differences in mitochondrial haplotypes or ploidy level and if they might be associatedwith fitness divergences and therefore selective value. J. Exp. Zool. 9999A: XX–XX, 2015. © 2015Wiley Periodicals, Inc.

How to cite this article: Kake-Guena SA, Touisse K, Vergilino R, Dufresne F, Blier PU, Lemieux H.2015. Assessment of mitochondrial functions in Daphnia pulex clones using high-resolutionrespirometry. J. Exp. Zool. 9999:1–9.

J. Exp. Zool.9999A:1–9, 2015

Grant sponsor: The National Science and Engineering Research Council; grant numbers: RGPIN 402636, RGPIN 155926, RGPIN 222948.Present address of Roland Vergilino is D�epartement des Sciences Biologiques, Universit�e de Montr�eal, Montr�eal, Qu�ebec, Canada.�Correspondence to: H�el�ene Lemieux, Campus Saint-Jean, University of Alberta, 8406 Marie-Anne-Gaboury Street, Edmonton, Alberta, T6C 4G9, Canada.

E-mail: [email protected] 13 August 2014; Revised 12 December 2014; Accepted 15 December 2014DOI: 10.1002/jez.1913Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com)..

RESEARCH ARTICLE

© 2015 WILEY PERIODICALS, INC.

INTRODUCTIONThe water flea Daphnia has been widely studied in ecology andevolution over the past decades (Hebert, '87) and is nowrecognized as an important model system in ecological genomicssince its full genome sequence became available in 2011(Colbourne et al., 2011). The advantages of this model arenumerous and include a short generation time (5–10 days), largebrood size, a high range of phenotypic traits, ease of culture in thelaboratory, and reproduction by facultative parthenogenesis.These characteristics make it ideal to investigate reaction norms(the scope of phenotypic responses to environmental conditionsof a given genome) as a single genotype can be propagated underdifferent environmental conditions. Daphnia is also a popularanimal model in ecotoxicology because it is extremely sensitiveto changes in the chemical composition of its environment and iswidely used for testing chemical safety and for monitoring waterquality (Kiss et al., 2003; Martins et al., 2007).The mitochondrial phylogeny of Daphnia pulex complex hasbeen well studied and comprises at least seven lineages(Colbourne and Hebert, '96; Colbourne et al., '98; Mergeayet al., 2008). These lineages are morphologically very similar butinhabit different environments. D. pulex inhabits ponds whereasthe closely related species,D. pulicaria is typically found in lakes.In the natural environment, these two sexually reproducingspecies form hybrids that are known to reproduce, followinghybridization, by obligate parthenogenesis (Hebert et al., '93) andhave the potential to expand to large numbers as a result of theirasexual nature (Taylor and Hebert, '92; Spaak, '97; Weider et al.,'99). In temperate North America, the hybrids are diploids andreproduce by obligate parthenogenesis (with no meiosis) andalmost invariably have the mitochondrial genome of the pondspecies (D. pulex) whereas in subarctic and arctic areas, triploidsgenerally have the mitochondrial genome of the lake species (D.pulicaria) (Dufresne and Hebert, '94; Hebert and Finston, 2001;Weider and Hobæk, 2003; Vergilino et al., 2009; Vergilino et al.,2011) although some triploid clones with D. pulex mitochondriahave been found in North-Eastern Canada (Vergilino et al., 2009;Vergilino et al., 2011). The asymmetry in mitochondrial genomeof the temperate hybrids is intriguing and might result fromselection on the mitochondrial genome or from D. pulicariamales preferentially mating with D. pulex females in temperateareas. A previous study failed to detect differences in citratesynthase, lactate dehydrogenase, and electron transport system(ETS) activities between Daphnia clones with different mtDNAand ploidy levels after a short term exposure to low and hightemperature (Jose et al., 2009). It could be that qualitativeadjustments of mitochondria to temperature exist despite noquantitative adjustments to temperature.It is well known that metabolic adjustments could be keybioenergetics adaptations to successfully colonize or occupyspecific environments (Hochachka and Somero, 2002; P€ortneret al., 2012). Among these adjustments, it appears that

modulations or adaptations of mitochondrial functions couldpartly dictate the extent to which animals, and particularlyectotherms, could fulfill all their life history requirements inspecific environments (Blier et al., 2001; Wallace, 2007;Parmakelis et al., 2013; Blier et al., 2014).In this context, clones belonging to different lineages of the D.pulex complex (as previously described) could be a powerfulmodel to delineate the link between mitochondrial DNAevolution and mitochondrial phenotypes (see Blier et al., 2001)and to investigate the potential metabolic adaptation associatedwith these mitochondrial phenotypes and specific environments.As a first step, we document mitochondrial functional propertiesamong lineages that differ in mitochondrial haplotypes and inploidy and open the door to future experimental studies that willassess if these different properties have divergent fitness impactand could therefore result from positive selective pressure. Theactual challenge is accordingly to characterize mitochondrialphenotypes of these microcrustaceans with enough precision andresolution to identify functional divergences that could becorrelated to environment or life-history and therefore suggestmitochondrial phenotypic adaptation hypothesis. Since theseorganisms are quite small, it is difficult to consider classicalapproach of mitochondrial isolation and purification. In recentstudies, we have been able to work directly on permeabilizedtissues instead of mitochondrial preparation to investigatemitochondrial functions in invertebrates (Pichaud et al., 2011;Lemieux and Warren, 2012; Pichaud et al., 2012).Our study aimed at developing an accurate method to measuremitochondrial function using a small number of Daphniaindividuals in order to delineate the scope of metabolicphenotypes potentially associated to the evolution of differentlineages. Instead of proceedingwith an isolation ofmitochondria,which would require hundreds of individuals, animal permeabi-lization is used to measure mitochondrial function in differentclones belonging to the D. pulex complex from differentenvironment, ploidy levels (diploid and triploid), and mitochon-drial DNA haplotypes (D. pulex and D. pulicaria).

MATERIALS AND METHODS

AnimalsFour clones belonging to different lineages of the D. pulexcomplex from different latitudes were used in order to comparethe mitochondrial functions. To identify Daphnia clones atmitochondrial level we have amplified and sequenced a fragmentof themitochondrial encoded geneNADH dehydrogenase 5 (ND5)(see Table 1 for primers and protocol; Vergilino et al., 2009). Thediploid clones Fence and Hawrelak are from temperate environ-ments and possesses mitochondrial DNA from D. pulex lineage[ND5 sequence accession numbers HQ434649 and KM983396,respectively]. The triploid clones A24 and K154 are fromsubarctic environments but differ their mitochondrial DNA;

2 KAKE-GUENA ET AL.

J. Exp. Zool.

A24 mtDNA [KC536614] is from D. pulicaria lineage and K154mtDNA [FJ591097] is from D. pulex lineage. At the nuclear level,the Lactate dehydrogenase A gene (LDH-A) was amplified todifferentiate D. pulex (homozygous for the S allele) from D.pulicaria (homozygous for the F allele) and their hybrids(heterozygotes SF) (Crease et al., 2011). Specific primers were usedto amplify separately two different amplicons of 248 nt of theLDH-A gene (Vergilino et al., 2011). Two sets of primer pairs(LDH-A-SF/LDH-A-SR and LDH-A-FF/LDH-A-FR, see Table 1)were used to amplify F and S alleles known to be diagnostic to D.pulicaria and to D. pulex, respectively. All the clones wereconsidered as D. pulex�D. pulicaria hybrids as we were able toamplify both amplicons for each clone (see Table 2).Clones were cultured in environmental growth chambers(Thermo FormaDiurnal Growth Chamber) at pH 7, 20°C, and 16 hrlight: 8 hr dark diurnal cycle for several months prior to theexperiments. They were fed with a suspension of Selenastrum sp.every other day. The water was changed every week withsynthetic pond water containing (per L): 5 g of KCl, 4 g of MgSO4,2.65 g of CaCl2, 0.6 g of K2HPO4, 0.6 g of KH2PO4, 5 g of NaNO3,0.44 g of FeCl3. The water was aerated daily. Clones (Fence, A24,and K154) were sent to Edmonton and kept at room temperaturewith the same feeding for at least two weeks prior to theexperiments.The D. pulex�D. pulicaria hybrid from Edmonton area(Hawrelak) was raised in the laboratory at the Campus Saint-Jean(University of Alberta) for a year. This clonewas also used in orderto perform the tests on the methods (number of animals per

chamber and saponin treatment). The population was kept atroom temperature, with a 12 hr light: 12 hr dark diurnal cycle andwere fed with the algae Chlamydomas sp. The water was aerateddaily.

Permeabilization of the AnimalsThe Daphnia were rinsed twice with 0.5mL of ice-cold relaxingsolution (BIOPS) containing 2.77mM CaK2EGTA, 7.23mMK2EGTA, 20mM imidazole, 20mM taurine, 6.56mM MgCl2-6H2O, 5.77mM ATP, 15mM phosphocreatine, 0.5mM dithio-threitol, 50mM K-MES (pH 7.1 at 0°C). The whole animals werepermeabilized with sharp forceps by piercing holes in the bodyand then agitating the animal for 30min on ice in the BIOPSsupplemented with 50mg.mL� 1 saponin (Veksler et al., '87;Kuznetsov et al., 2004). In additional experiments with Daphniafrom Hawrelak (Edmonton), two saponin concentrations (50 and25mg.mL� 1) and two duration of exposure to saponin (10 and30min) were used. The permeabilized animals (10 individuals forall experiments with K154, A24 and Fence clones, and 10 or 20 forthe experiments with the clone fromHawrelak) were immediatelytransferred into the respiration chamber (OROBOROS Oxygraph2 k, Innsbruck, Austria) containing 2mL of respiration mediumMir05 [110mM sucrose, 60mM K-lactobionate, 0.5mM EGTA,1 g.L� 1 BSA fatty acid free, 3mM MgCl2-6H2O, 20mM taurine,10mM KH2PO4, 20mM K-HEPES, pH 7.1, osmolarity 330 mOsm;(Gnaiger et al., 2000)]. Rinsing the saponin before the measure-ment in the chamber did not show any effect on respiration rateor coupling (results not shown). Respiration was measured at 20

Table 2. Characterization of the clones of Daphnia pulex and Daphnia pulicaria.

Name Hybrids Ploidy Reproduction Mitochondria from Provenance

Hawrelak Pulex� Pulicaria Diploid Asexual D. pulex Edmonton, AlbertaFence Pulex�Pulicaria Diploid Asexual D. pulex Windsor, OntarioA24 Pulex� Pulicaria Triploid Asexual D. pulicaria Churchill, ManitobaK154 Pulex� Pulicaria Triploid Asexual D. pulex Kuujjurapik

Table 1. Primers table.

PurposeAnnealingtemperature

Primername Sequence Amplicon size

ND5 amplification 50°C DpuND5b DpuND5b 50-GGGGTGTATCTATTAATTCG50-ATAAAACTCCAATCAACCTTG

850

ND5 amplification 50°C DpuND5b ND5ColbF 50-GGGGTGTATCTATTAATTCG50-AACTTAGTATCACCAGCAGG

700

LDHA-pulex diagnostic allele 50°C LDH-A-SF LDH-A-SR 50-GAGCGATTTAACGTTGCGCCC50-GGACGACTTGTGTGTGAATTTG

248

LDHA-pulicaria diagnostic allele 50°C LDH-A-FF LDH-A-FR 50-GAGCGATTTAACGTTGCGCCT50-GGACGACTTGTGTGTGAATTTC

248

J. Exp. Zool.

FUNCTIONS IN Daphnia pulex CLONES 3

or 25°C, depending on the experiment. Datlab software (ORO-BOROS Instruments) was used for data acquisition and analysis.

High-Resolution RespirometryThe protocol for evaluation of mitochondrial function ispresented in Figure 1. The final concentration of substratesand inhibitors added to the chambers were: 5mM pyruvate, 5mMmalate, 2.5mM ADP, 10mM cytochrome c (cyt. c), 10mMsuccinate, 0.5mM rotenone, 2.5mM antimycin A, 2mMascorbate, 0.5mM tetramethylphenylenediamine (TMPD),15mM azide. Mitochondrial respiration was corrected for oxygenflux due to instrumental background, and for residual oxygenconsumption (ROX) after inhibition of complexes I and III withrotenone and antimycin A. For complex IV respiration (ascorbateþ TMPD), the chemical background measured in the presence ofazide was subtracted. Respiratory flux in Daphnia is expressed inpmol O2 per s per animal or in flux control ratios (FCR)normalized for maximal OXPHOS capacity with complexes IþIIsubstrates.

Since our objective was to document divergences in mito-chondrial functions, and not in aerobic capacity of organisms, weavoided expressing respiration rate per weight of daphnia or perprotein content to bypass the bias that could have been inducedby weight measurements or protein assays. By focusing onoxygen consumption normalized for oxygen capacity withcomplexes IþII substrates, it ensures a more precise character-ization of mitochondrial functions, and improve possibilities todetect divergences dictated by mitochondrial organization thatcould further be associated to mitochondrial DNA evolution.

Data AnalysisStatistical analyses were performed using SigmaStat 4 (AspireSoftware International, Ashburn, VA). The criteria of normalityand the homogeneity of variance for analysis of variance weretested for each variable with Shapiro–Wilk tests and Spearmantests, respectively. For variables meeting these criteria (fluxcontrol ratio for Complex I-OXPHOS, Complexes IV-OXPHOS,ratios complex I/complex II, respiratory control ratios, andcytochrome c effect ratios), differences between clones weretested with a one factor ANOVA, followed by a pairwisecomparison with Holm–Sidak test. For variables that did notmeet the criteria (respiration in flux per daphnia for all states, fluxcontrol ratios for Complex I-LEAK and Complex II-OXPHOS),differences among clones were tested with Kruskal–Wallis testsfollowed by a posteriori Dunn comparisons.The impact of different permeabilization treatments (differentsaponin concentrations or different duration of exposure tosaponin) was tested using the same tests described above; all thedata were meeting the criteria of normality and homogeneity ofvariance. A t-test for independent samples was used to determinethe effects of the additions of 10 or 20 animals in the chamber.P< 0.05 was considered significant. Results are presented asmeans� standard error of the mean (SEM).

RESULTS

Clones Divergences of Respiratory ParametersRespiratory states are defined as LEAK respiration measured inthe presence of pyruvateþmalate before the addition of ADP andOXPHOS respiration measured after the addition of saturatingADP (Fig. 1). None of the respiratory states in pmol.s� 1 perDaphnia (LEAK or OXPHOS) showed significant differencebetween the four clones (Fig. 2A). In order to correct for thecapacity of the mitochondria, the results were expressed as fluxcontrol ratio (FCR), normalized for maximal OXPHOS capacitywith parallel electron input from complexes IþII substrates. Thisallowed the characterization of the relative capacity of thedifferent complexes (CI, CII and CIV) over the maximalrespiratory capacity of mitochondria (which is a characterdictated by mitochondrial properties and not by mitochondrialcontent) and to compare these capacities among the four

Figure 1. Representative trace for evaluation of mitochondrialrespiratory capacities in permeabilized Daphnia pulex (cloneK154) with a multiple substrate-inhibitors titration protocol. Thetrace represents the oxygen consumption as a function of time.Mitochondrial coupling states are distinguished as LEAK (withoutADP) and OXPHOS (saturating ADP). Themultiple titration protocolcomprised the following steps: (1) LEAK respiration in the presenceof complex I (CI) substrates pyruvateþmalate, OXPHOS respirationin the presence CI substrates and saturating ADP, addition ofcytochrome c to test for integrity of outer mitochondrialmembrane, succinate to measure respiration in the presence ofcomplexes IþII substrates pyruvateþmalateþsuccinate, succi-nate-supported respiration (complex II, CII) after inhibition ofcomplex I with rotenone, residual oxygen consumption afterinhibition of complex III with antimycin A, complex IV respirationin the presence of ascorbateþTMPD, inhibition of complex IV withazide. Arrows indicate times of titrations of the substrates andinhibitors.

J. Exp. Zool.

4 KAKE-GUENA ET AL.

different lineages. The FCR showed lower OXPHOS in thepresence of complex I substrates (P< 0.001, N¼ 6) and higherOXPHOS in the presence of complex II substrates (P< 0.001,N¼6) in the clone K154 compared to Hawrelak, Fence, and A24(Fig. 2B). The difference is also expressed as a ratio of complex I/IIin the four clones, showing in the clone K154 (Fig. 3A; P< 0.001,N¼ 6) a lower capacity to oxidize substrates of complex I relativeto oxidative capacity when the ETS is fed into complex II. Theclone A24 showed significantly lower complex II OXPHOScompared to Hawrelak (P¼ 0.026; N¼ 19), Fence (P¼ 0.005,N¼ 6), and K154 (P< 0.001, N¼ 6) clones (Fig. 2B). The FCR forcomplex IV did not vary among the four clones (Fig. 2A and B).

OXPHOS Coupling and Mitochondrial Membrane IntegrityThe coupling of the OXPHOS process in the Daphnia mitochon-dria was evaluated with the respiratory control ratio (RCR;OXPHOS/LEAK) and the flux control ratio (FCR; LEAK/maximal

OXPHOS with complexes IþII). LEAK respiration is the oxygenconsumption that represents proton leak, electron slip, andproton cycling (Brand et al., '94). LEAK flux normalized for ETScapacity provides an expression of mitochondrial uncoupling(Gnaiger, 2009). In Daphnia, maximal OXPHOS with complex

Figure 2. Mitochondrial respiratory capacity (panel A) and fluxcontrol ratios (panel B) measured in permeabilized D. pulex of fourdifferent clones (Hawrelak, Fence, A24, K154). Mitochondrialcoupling states are distinguished as LEAK (without ADP) andOXPHOS (saturating ADP). Respiration is measured in the LEAKstate (before addition of ADP) or in the OXPHOS state (afteraddition of ADP and cytochrome c) in the presence of complex I(CI) substrates pyruvateþmalate, complexes IþII (CIþII) sub-strates pyruvateþmalateþsuccinate, complex II (CII) substrates(after inhibition of CI with rotenone) succinate-supportedrespiration (complex II, CII), and complex IV substrates (ascorba-teþTMPD; with substraction of the rate after inhibition withazide). The Flux control ratios are normalized for maximal OXPHOScapacity with CIþII substrates. Data are means� SEM. Columnswith different letters are significantly different within a specificstate (P< 0.05). N¼ 6 measurements per clone.

Figure 3. Ratios complex I/complex II (CI/CII, panel A), respiratorycontrol ratio (RCR; panel B), and cytochrome c effect representedby the ratio of complex I without cytochrome c added overcomplex I with cytochrome c added (CI/CIc; panel C) of fourdifferent clones (Hawrelak, Fence, A24, K154). Data are means� SEM. Columns with different letters are significantly differentwithin a specific state (P< 0.05). N¼ 6 measurements per clone.

J. Exp. Zool.

FUNCTIONS IN Daphnia pulex CLONES 5

IþII substrates is equivalent to maximal ETS capacity becausethere is no limitation of OXPHOS by the phosphorylation system(Lemieux and Warren, 2012). FCR (LEAK/maximal OXPHOS,Fig. 2B) were low and similar among the four clones, ruling outdifferences in mitochondrial uncoupling related to the procedurefor permeabilization. The RCR around 3–4 (Fig. 3B) arecomparable to other studies performed on mitochondrialisolation or perfused tissues from aquatic invertebrates(Tschischka et al., 2000; Abele et al., 2002; Pichaud et al.,2012). In the clone Hawrelak, the RCR is significantly highercompared to the clone Fence (P¼ 0.037). The integrity of theouter mitochondrial membrane in the permeabilized animal wasmeasured by the addition of exogenous cytochrome c tomitochondria in the presence of complex I substrates andsaturating ADP (Fig. 1). There was an increase in OXPHOS afteraddition of cytochrome c in the four clones (Fig. 1). The increase isexpressed as the ratio of respiration without cytochrome c overthe respiration after the addition of cytochrome c (Fig. 3C). Aratio of 1.0 means full integrity of the mitochondrial outermembrane (Fig. 3C). The ratios of 0.78 in the Hawrelak, K154, andA24 clones and lower ratio in the Fence clone (0.70) could suggestslight damage to the mitochondrial outer membrane (P¼ 0.005for Fence vs. either Hawrelak or K154 and P¼ 0.015 for Fence vs.A24, N¼ 19 for Hawrelak and N¼ 6 for the other clones). Thedata of OXPHOS used for comparison between the clones werethe data with exogenous cytochrome c added, in order to avoidany bias induced by limitation of cytochrome c availabilityresulting from the permeabilization or manipulation.

Effect of the Number of Animals in the Chamber on theRespiratory RatiosPreliminary test with the clone Hawrelak showed similar FCR forOXPHOS with 10 (N¼ 19 measurements) or 20 animals (N¼ 10measurements) per chamber. This showed that once corrected forthe mitochondrial content in the chamber, similar data wereobtained in both experiments. The FCR LEAK/ETS was, however,

higher in the experiments with 20 animals (Fig. 4A) and the RCRwas lower (Fig. 4B) in the experiments with 20 animals. Thecoupling of the mitochondria might be reduced by thepermeabilization of more individuals in the same volume ofBIOPS. The integrity of outer mitochondrial membrane shownwith the cytochrome c test (Fig. 4C) was similar with 10 or 20animals per chamber (Fig. 4C). Because of the reduced coupling inexperiments with 20 animals, 10 animals per chamber were usedin all other experiments. Further decrease in the number ofanimals per chamber (2, 3, or 5 animals) did not modify the RCRor the impact of cytochrome c addition (results not shown).

Effect of the Saponin Treatment on the Respiratory RatiosSaponin treatment was used in order to ensure that the cellmembranes were well permeabilized. The concentration and timeof incubation were modified (Fig. 5) in order to see if the FCR, theRCR, and the cytochrome c effect would be affected. There was nodifference in any of the ratios with the three saponin treatments(N¼ 19 for 30min with 50mg.mL� 1 saponin; N¼ 6 for 30minwith 25mg.mL� 1 saponin; and N¼ 6 for 10minwith 25mg.mL� 1

saponin). A saponin concentration of 50mg.mL� 1 for 30min wasthen used in all experiments with the four clones, because the FCRLEAK/OXPHOSwere slightly (but not significantly) lower and theRCR was slightly (but not significantly) higher with this specificsaponin treatment.

DISCUSSIONOur study showed that mitochondrial respiration can adequatelybe measured with high-resolution respirometry in a smallcrustacean such as Daphnia and opens new possibilities forusing this well-recognized animal model in different areas ofenvironmental and evolutionary biology. The respiratory ca-pacity of functional and coupled mitochondria can be measuredwhen keeping the mitochondria in their cellular environment.With only 10 individuals, OXPHOS capacities of complexes I, II,IþII, and IV can be measured. The method is very sensitive when

Figure 4. Effects of the number of D. pulex in the respiratory chamber on the flux control ratios (FCR; panel A), the respiratory control ratio(RCR; panel B) and the cytochrome c effect (panel C). The measurements were performed with 10 or 20 animals per chamber at 20°C. N¼ 9with 20 animals and N¼ 19 with 10 animals. Data are means� SEM. N¼ 19 measurements with 10 animals and N¼ 9 measurements with20 animals.

J. Exp. Zool.

6 KAKE-GUENA ET AL.

comparing ratios such as FCR, and slight changes can be detected.The measurement of mitochondrial respiration in Daphnia is ofgreat interest to delineate to which extent perturbation ofmitochondrial metabolism can set the ability of organism tocolonize habitats. This becomes even more important sincemitochondria are the major source of reactive oxygen speciesproduction which lead to oxidative damages and have potentialimpacts on senescence, adaptation to temperature, tolerance topollution, etc. (Blier et al., 2014). TheD. pulex complex model canbe a powerful tool to point out key metabolic adaptation to localconditions.Our study focused on the detection of these specific changesin the OXPHOS complexes function. Our method allowed themeasurement of specific activity of complexes I, IþII, II, and IV.Interestingly, our results showed that the contribution of complexI and complex II to the maximal physiological OXPHOS rate(complex IþII) vary between the clones. In the triploid subarcticclone K154, that has mitochondrial DNA fromD. pulex, there wasa lower contribution of complex I and a higher contribution ofcomplex II to the maximal OXPHOS rate. In contrast, in thetriploid A24, that possess mitochondrial DNA from D. pulicaria,the contribution of complex II to maximal OXPHOS was lower,when compared to the three other clones. Two diploid clonesHawrelak and Fence, with mitochondria from D. pulex, showedhowever no significant differences. These results indicate asignificant reorganization of the mitochondrial function depend-ing on a combination of mitochondrial haplotypes and ploidylevels. According to Blier et al. (2014) the qualitative adjustmentsof mitochondria could be even more important than quantitativeadjustments to latitude and temperature because it wouldensure proper kinetic equilibrium among reactions and adequateregulation process. In this context, the measured divergencesin flux control ratio as well as the ratios of CI/CII could bephysiologically highly significant.Hybridization and polyploidization may lead to genomereorganization and gene expression change (Jackson and Chen,2010; Buggs, 2013; Coate et al., 2013). The interaction between

proteins expressed from different nuclear and/or mitochondrialgenomes may have various outcome and may lead to non-additive genetic processes, e.g., epistasis (reviewed in Chen andYu, 2013), producing different phenotypes (including physio-logical phenotypes) on which natural selection can act. Due to theinteractions between the peptides coded by nuclear genomes as inthe complex II or coded bymitochondrial and nuclear genomes asin complex I, III, and IV, each complex activity outcome may bedifferent depending on the divergence between each nucleargenome and between nuclear and mitochondrial genomes inhybrids (McKenzie et al., 2003). For example, (Ellison and Burton,2006) have shown that a disruption of the activity of complexes I,III, and IV, but not of complex II, occurs in inbred hybrid linespresenting mitochondrial haplotype from one population andnuclear background from another divergent population. Thisresult is likely due to a breakdown of co-adapted cyto-nucleargene complexes in inbred hybrid lines. The diploid and polyploidhybrid clones of the D. pulex complex have different origins(Vergilino et al., 2009, 2011). Polyploid isolates have differentmitochondrial haplotype and have been sampled in subarcticregions whereas the diploid hybrids have been sampled intemperate region (see Table 2). The interactions of the proteinsproduced by the different nuclear and mitochondrial genomesmay have different impacts on the different complex activitiesand then produce various physiological phenotypes as we see forthe complexes I and II.In a previous study, no differences were detected inmitochondrial enzyme activities and in thermal sensitivitybetween four Daphnia clones from subarctic and temperateenvironments (Jose et al., 2009). This previous study, however,measured enzyme activities of citrate synthase and ETS insolubilized mitochondria. The measurement of ETS activityrepresents an estimation of the aerobic potential of the organism(Toth et al., '95), but does not allow to detect specific changesin complexes capacities. Furthermore, the ETS is performed insolubilized mitochondrial whereas our OXPHOS measurementis performed in intact and functional mitochondria. The

Figure 5. Effect of the saponin concentration and the time of treatment on the measurement of the flux control ratios (FCR; panel A), therespiratory control ratio (RCR; panel B), and the cytochrome c effect (panel C). The measurements were performed at 25°C. Data aremeans� SEM. N¼ 19 measurements for 30min saponin and 50mg.mL� 1. N¼ 6 measurements for each of the two other treatments.

J. Exp. Zool.

FUNCTIONS IN Daphnia pulex CLONES 7

measurement of enzyme activities is also often associated withhigh variability and does not always allow the detection of slightdifferences between the groups. These differences in the methodmight explain why our study showed differences in mitochon-drial function between the clones, and emphasizes the impor-tance of measurement of mitochondrial function in intactmitochondria with a method that allow detection of slightdifferences between the organisms.The comparison of mitochondrial OXPHOS between the cloneswas performed at 20°C. This temperature is within the rangetolerated by all the clones including the subarctic one (DufresneandHebert, '98).We do not think that this temperature could havebeen detrimental to the mitochondria of the subarctic clones. Inthe heart mitochondria from a cold adapted fish species,Anarhichas lupus, the sensitivity of complex I at high temper-ature was much more pronounced compared to the sensitivity ofcomplex II (Lemieux et al., 2010). But the decrease in complex Iactivity inA. lupus occurred at a temperature way over the rangeof temperature experienced by the species, i.e., between 25 and 35whereas the natural habitat range from 0 to 16°C (Moksness andPavlov, '96). Furthermore, when the drop in complex I wasobserved in A. lupus, the RCR value with complex I substratesalso dropped dramatically (4-fold) (Lemieux et al., 2010).A. lupusis taxonomically quite distant from Daphnia and we do notpretend that mitochondrial organisation of these two taxonomicgroups is similar but we want to illustrate (with one of the fewstudy available on thermal sensitivity of mitochondrial functionin a strict cold stenothermal species) thatmitochondrial functionscan in some occasion support temperature increase that are out ofthe range of temperature encountered by the species. Further-more, our data do not show significant differences in RCRbetween the clones Fence, A24, and K154. The integrity of theouter mitochondrial membrane, shown with the cytochrome ceffect, was even better in the two subarctic clones than in thetemperate Fence clone. These results indicate that the measure-ment temperature of 20°C did not cause damage to themitochondria of the subarctic clones.The question remains if the changes in specific mitochondrialfunction seen in Daphnia clones with the different mitochon-drial haplotypes and ploidy level could confer a selectiveadvantage. Can an increase in the contribution of complex IIand a decrease in the contribution of complex I to the maximalphysiological OXPHOS capacity translate into an advantage ina low temperature environment? As thermal sensitivity ofcomplex II is more pronounced at low temperature whencompared to thermal sensitivity of complex I (Lemieux et al.,2010), cold adapted populations might require higher propor-tion of complex II activity to maintain normal functions at lowtemperature. The increase in complex II respiration observed inthe K154 clone at 20°C would then compensate for a moresevere loss in activity at low temperature. Interestingly, the twosubarctic clones showed significant divergences in mitochon-

drial function. In order to see if these divergences are associateddirectly with changes in mitochondrial DNA from D. pulex to D.pulicaria, further studies with additional clones with D. pulexand D. pulicaria mitochondrial genome and from differentgeographical locations need to be performed under differenttemperatures.

ACKNOWLEDGMENTSThis study was supported by a discovery grants from the NaturalSciences and Engineering Research Council to HL, FD and PB, aresearch grant and a startup grant from Campus Saint-Jean, aswell as an equipment grant from the Canadian Foundation forInnovation to HL. We are grateful to Micheline Forgues for herhelp with the Daphnia culture.

REFERENCESAbele D, Heise K, Portner HO, Puntarulo S. 2002. Temperature-dependence of mitochondrial function and production of reactiveoxygen species in the intertidal mud clam Mya arenaria. J Exp Biol205:1831–1841.Blier PU, Dufresne F, Burton RS. 2001. Natural selection and theevolution of mtDNA-encoded peptides: evidence for intergenomicco-adaptation. Trends Genet 17:400–406.Blier PU, Lemieux H, Pichaud N. 2014. Holding our breath in ourmodern world: will mitochondria keep the pace with globalchanges? Can J Zool 92:591–601.BrandMD, Chien LF, Ainscow EK, Rolfe DF, Porter RK. 1994. The causesand functions of mitochondrial proton leak. Biochim Biophys Acta1187:132–139.Buggs RJ. 2013. The consequences of polyploidy and hybridisation fortranscriptome dynamics. Unravelling gene expression of complexcrop genomes. Heredity 110:97–98.Chen ZJ, Yu HH. 2013. Genetic and epigenetic mechanisms forpolyploidy and hybridity. In: Chen ZJ, Birchler JA, editors. Polyploidand hybrid genomics. Oxford: John Wiley & Sons, Inc. p 335–354.Coate JE, Powell AF, Owens TG, Doyle JJ. 2013. Transgressivephysiological and transcriptomic responses to light stress inallopolyploid Glycine dolichocarpa (Leguminosae). Heredity110:160–170.Colbourne JK, Crease TJ, Weider LJ, et al. 1998. Phylogenetics andevolution of a circumarctic species complex (Cladocera: Daphniapulex). Biol J Linn Soc 65:347–365.Colbourne JK, Hebert PD. 1996. The systematics of North AmericanDaphnia (Crustacea: Anomopoda): a molecular phylogeneticapproach. Philos Trans R Soc Lond B Biol Sci 351:349–360.Colbourne JK, Pfrender ME, Gilbert D, et al. 2011. The ecoresponsivegenome of Daphnia pulex. Science 331:555–561.Crease TJ, Floyd R, Cristescu ME, Innes D. 2011. Evolutionary factorsaffecting lactate dehydrogenase A and B variation in the Daphniapulex species complex. BMC Evol Biol 11:212.Dufresne F, Hebert PDN. 1994. Hybridization and origins of polyploidy.Proc Royal Soc B-Biol Sci 258:141–146.

J. Exp. Zool.

8 KAKE-GUENA ET AL.

Dufresne F, Hebert PDN. 1998. Temperature-related differences inlife-history characteristics between diploid and polyploid clones ofthe Daphnia pulex complex. �Ecoscience 5:433–437.Ellison CK, Burton RS. 2006. Disruption of mitochondrial function ininterpopulation hybrids of Tigriopus californicus. Evolution60:1382–1391.Gnaiger E. 2009. Capacity of oxidative phosphorylation in humanskeletal muscle. New perspectives of mitochondrial physiology. Int JBiochem Cell Biol 41:1837–1845.Gnaiger E, Kuznetsov AV, Schneeberger S, et al. 2000. Mitochondria inthe cold. In: Heldmaier G, Klingenspor M, editors. Life in the cold.New York: Springer Berlin Heidelberg. p 431–442.Hebert PDN. 1987. Genetics of Daphnia. In: Peters RH, De Bernardi R,editors. Daphnia. Pallanza: Mem Ist ital Idrobiol. p 439–460.Hebert PDN, Finston TL. 2001. Macrogeographic patterns of breedingsystem diversity in the Daphnia pulex group from the United Statesand Mexico. Heredity 87:153–161.Hebert PDN, Schwartz SS,Ward RD, Finston TL. 1993.Macrogeographicpatterns of breeding system diversity in the Daphnia pulex group. I.Breeding systems of Canadian populations. Heredity 70:148–161.Hochachka PW, Somero GN. 2002. Biochemical adaptation. Mech-anism and process in physiological evolution. New York: OxfordUniversity Press.p 466.Jackson S, Chen ZJ. 2010. Genomic and expression plasticity ofpolyploidy. Curr Opin Plant Biol 13:153–159.Jose C, H�ebert Chatelain �E, Dufresne F. 2009. Low flexibility ofmetabolic capacity in subarctic and temperate cytotypes ofDaphnia pulex. J Therm Biol 34:70–75.Kiss I, Kov�ats N, Szalay T. 2003. Evaluation of some alternativeguidelines for risk assessment of various habitats. Toxicol Lett 140–141: 411–417.Kuznetsov AV, Schneeberger S, Seiler R, et al. 2004.Mitochondrial defectsand heterogeneous cytochrome c release after cardiac cold ischemiaand reperfusion. Am J Physiol-Heart Circul Physiol 286:H1633–H1641.Lemieux H, Tardif JC, Dutil JD, Blier PU. 2010. Thermal sensitivity ofcardiac mitochondrial metabolism in an ectothermic species from acold environment, Atlantic wolffish (Anarhichas lupus). J Exp MarBiol Ecol 384:113–118.Lemieux H, Warren B. 2012. An animal model to study humanmuscular diseases involving mitochondrial oxidative phosphor-ylation. J Bioenerg Biomembr 44:503–512.Martins J, Oliva Teles L, Vasconcelos V. 2007. Assays with Daphniamagna and Danio rerio as alert systems in aquatic toxicology.Environ Int 33:414–425.McKenzie M, Chiotis M, Pinkert CA, Trounce IA. 2003. Functionalrespiratory chain analyses in murid xenomitochondrial cybridsexpose coevolutionary constraints of cytochrome b and nuclearsubunits of complex lll. Mol Biol Evol 20:1117–1124.Mergeay J, Aguilera X, Declerck S, et al. 2008. The genetic legacy ofpolyploid Bolivian Daphnia: the tropical Andes as a source for theNorth and South American D. pulicaria complex. Mol Ecol 17:1789–1800.

Moksness E, Pavlov DA. 1996. Management by life cycle of wolffish,Anarhichas lupus L., a new species for cold-water aquaculture: atechnical paper. Aquacult Res 27:865–883.Parmakelis A, Kotsakiozi P, Rand D. 2013. Animal mitochondria,positive selection and cyto-nuclear coevolution: insights frompulmonates. PLoS One 8:e61970.Pichaud N, Ballard JW, Tanguay RM, Blier PU. 2011. Thermal sensitivityof mitochondrial functions in permeabilized muscle fibers fromtwo populations of Drosophila simulans with divergent mitotypes.Am J Physiol Regul Integr Comp Physiol 301:R48–R59.Pichaud N, Rioux P, Blier PU. 2012. In situ quantification ofmitochondrial respiration in permeabilized fibers of a marineinvertebrate with low aerobic capacity. Comp Biochem Physiol AMol Integr Physiol 161:429–435.P€ortner HO, Peck LS, Somero GN. 2012. Mechanisms defining thermallimits and adaptation in marine ectotherms: an integrative view.In: Rogers A, Johnston N, Murphy E, Clarke A, editors. AntarcticEcosystems: An Extreme Environment in a Changing World.Chichester, UK: John Wiley & Sons, Ltd. p 379–416.Spaak P. 1997. Hybridization in the Daphnia galeata complex: arehybrids locally produced?. Hydrobiologia 360:127–133.Taylor DJ, Hebert PDN. 1992. Daphnia galeata mendotae as a crypticspecies complex with interspecific hybrids. Limnol Oceanogr37:658–665.Toth LG, Szabo M, Webb DJ. 1995. Adaptation of the tetrazolimreduction test for the measurement of the electron-transportsystem (ETS) activity during embryonic-development of Medaka. JFish Biol 46:835–844.Tschischka K, Abele D, Portner HO. 2000. Mitochondrial oxy-conformity and cold adaptation in the polychaete Nereis pelagicaand the bivalve Arctica islandica from the Baltic and White Seas. JExp Biol 203:3355–3368.Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA. 1987.Mitochondrial respiratory parameters in cardiac tissue: a novelmethod of assessment by using saponin-skinned fibers. BiochimBiophys Acta 892:191–196.Vergilino R, Belzile C, Dufresne F. 2009. Genome size evolution andpolyploidy in the Daphnia pulex complex (Cladocera: Daphniidae).Biol J Linn Soc 97:68–79.Vergilino R, Markova S, Ventura M, Manca M, Dufresne F. 2011.Reticulate evolution of the Daphnia pulex complex as revealed bynuclear markers. Mol Ecol 20:1191–1207.Wallace DC. 2007. Why do we still have a maternally inheritedmitochondrial DNA? Insights from evolutionary medicine. Annu RevBiochem 76:781–821.Weider LJ, Anders Hobæk A, Hebert PDN, Crease TJ. 1999.Holarctic phylogeography of an asexual species complex - II.Allozymic variation and clonal structure in Arctic Daphnia. MolEcol 8:1–13.Weider LJ, Hobæk A. 2003. Glacial refugia, haplotype distributions,and clonal richness of the Daphnia pulex complex in Arctic Canada.Mol Ecol 12:463–473.

J. Exp. Zool.

FUNCTIONS IN Daphnia pulex CLONES 9