Temperature-related differences in mitochondrial function among clones of the cladoceran Daphnia pulex

Author’s Accepted Manuscript Temperature-related differences in mitochondrial function among clones of the cladoceran Daphnia pulex S.A. Kake Guena, K. Touisse, B.E. Warren, K.Y. Scott, F. Dufresne, P.U. Blier, H. Lemieux www.elsevier.com/locate/jtherbio

PII: DOI: Reference:

S0306-4565(17)30085-2 http://dx.doi.org/10.1016/j.jtherbio.2017.05.005 TB1937

To appear in: Journal of Thermal Biology Received date: 3 March 2017 Revised date: 10 May 2017 Accepted date: 20 May 2017 Cite this article as: S.A. Kake Guena, K. Touisse, B.E. Warren, K.Y. Scott, F. Dufresne, P.U. Blier and H. Lemieux, Temperature-related differences in mitochondrial function among clones of the cladoceran Daphnia pulex, Journal of Thermal Biology, http://dx.doi.org/10.1016/j.jtherbio.2017.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Temperature-related differences in mitochondrial function among clones of the cladoceran Daphnia pulex

Running page head: Mitochondrial function in Daphnia pulex clones

Kake Guenaa SA, Touissea K, Warrena BE, Scotta KY, Dufresneb F, Blierb PU, Lemieuxa* H

a

Faculty Saint-Jean, University of Alberta, Edmonton, Alberta, Canada, T6C 4G9

b

Dept. de Biologie, Laboratoire de Physiologie Animal Integrative, Université du Québec à Rimouski, Rimouski, Quebec,

Canada, G5L 3A1

*Corresponding author: Faculty Saint-Jean, University of Alberta, 8406 Marie-Anne-Gaboury Street, Edmonton, Alberta, T6C 4G9, Canada Email: [email protected] (H. Lemieux).

Abbreviations used: FCFc, Flux control factor; FCR, Flux control ratio; OXPHOS, oxidative phosphorylation; RCR, Respiratory control ratio; ROS, reactive oxygen species; ROX, Residual oxygen consumption.

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Abstract This study assessed the thermal sensitivity of mitochondrial respiration in the small crustacean Daphnia pulex. More specifically, we wanted to determine if clones that inhabit different latitudes and habitats showed differences in the thermal sensitivity of their mitochondrial function. The experimental design included two clones from temperate environments (Fence from Ontario and Hawrelak from Alberta) and two clones from subarctic environments (A24 from Manitoba and K154 from Quebec). The integrated mitochondrial function was measured with high-resolution respirometry following whole-animal permeabilization. Mitochondrial respiration was performed under six different temperatures (10, 15, 20, 25, 30, and 35°C) in the clone Hawrelak and at two temperatures (10 and 20°C) in the three other clones. In the clone Hawrelak, complexes I and II respiration showed higher sensitivity to temperature variation compared to complex IV respiration. Interestingly, the threshold plot showed no excess capacity of complex IV at 20 °C in this clone. The clones showed significant divergence in the ability to oxidize the complex I and complex IV substrates relative to the maximal oxidative capacity of mitochondria. More importantly, some of the clone divergences were only detected under low assay temperatures, pointing toward the importance of this parameter in comparative studies. Future and more complex studies on clones from wider environmental gradients will help to resolve the link between mitochondrial function and adaptations of organisms to particular conditions, principally temperature.

Keywords: Daphnia pulex; Daphnia pulicaria, clones; mitochondrial respiration; thermal sensitivity; mitochondrial haplotypes.

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1. Introduction Temperature has a significant impact on most biochemical reactions of ectothermic organisms (Hochachka and Somero, 2002) and is therefore a major evolutionary driving factor for these animals since it influences their metabolic rates (Angilletta et al., 2002; Brown et al., 2004; Gillooly et al., 2001) as well as their microgeographic distribution, growth, and life history traits (Bozinovic et al., 2011; Pörtner, 2002; Prosser, 1991). One key function of animal metabolism, that is particularly sensitive to temperature variation is the mitochondrial oxidative phosphorylation (OXPHOS). When the ATP generated by mitochondria cannot sustain cell functions, whole homeostasis is threatened , unlessthe organism can escape this bioenergetics bottleneck through either short period of anaerobiosis or metabolic torpor.

In aquatic ectotherms, it has been suggested that mitochondrial function limits aerobic scope at low temperatures whereas excessive demands induced by increased metabolism at high temperatures may result in inadequate ventilation and transport capacities to support oxygen demand (Pörtner, 2002). And in fact, the acclimation at low temperatures in ectothermic animals often results in mitochondrial adjustments that maintain ATP homeostasis (Dos Santos et al., 2013; Fangue et al., 2009; Kraffe et al., 2007; O'Brien, 2011). Several modifications have been observed in the tissues of aquatic ectotherms acclimated to cold temperatures, including changes in mitochondrial content or volume (Chung and Schulte, 2015; Fangue et al., 2009; Guderley, 2004; Guderley and St-Pierre, 2002; Johnston et al., 1998; O'Brien, 2011; Pörtner et al., 2007; St-Pierre et al., 1998), enzymatic activities or substrate affinity (Das, 2006; Fangue et al., 2009; Guderley, 2004; Hochachka and Somero, 2002; Kraffe et al., 2007; Oellermann et al., 2012), membrane composition (Clarke, 1987; Grim et al., 2010; Guderley, 2004; Kraffe et al., 2007), and cristae organization (reviewed by (Guderley, 2004). Implicitly, all of the different reactions involved in mitochondrial metabolism must be tightly adjusted to each other but are differently affected by temperature shifts. The OXPHOS process requires the appropriate activity of the systems responsible for the transport and oxidation of the substrates donating electrons, the transport of electrons in the electron transport system (ETS), the formation of the proton gradient within the mitochondrial intermembrane space, and the phosphorylation process allowing production of ATP from ADP and Pi. The preservation of mitochondrial metabolism in this wide temperature range is

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crucial for maintaining not only the energetic balance, but also the redox balance (Blier et al., 2014). When studying mitochondrial OXPHOS adaptation, it is therefore critical to determine at which specific step the adjustments are required and, in parallel, which molecular and genetic regulation ensure the appropriate metabolic response.

Despite the central bioenergetic function of mitochondria and their close association with most life-history traits, little is known about the specific adaptations of mitochondrial function that are required to ensure proper cellular homeostasis in different environmental and thermal conditions. The aquatic water flea Daphnia pulex could represent a great model to examine metabolic adjustments in natural populations from different thermal environments as the mode of reproduction by facultative (or obligate) parthenogenesis allows the decoupling of genetics and environmental effects by allowing the physiological characterization of individuals with identical genotypes (Kiss et al., 2003; Stollewerk, 2010; Vergilino et al., 2011). Secondly, Daphnia is an active eurythermal organism with a large geographical distribution; as such, it needs to be able to adjust metabolic functions to a wide range of temperatures (Chopelet et al., 2008; Pennak, 1989). The Daphnia pulex species complex includes at least seven morphologically similar lineages (Colbourne et al., 1998; Colbourne and Hebert, 1996; Mergeay et al., 2008) that inhabit different environments, from ponds (D. pulex) to lakes (D. pulicaria). These organisms face not only long-term climate changes, but also the challenge of seasonal and daily fluctuations in temperature. In the laboratory, the species D. pulex can be grown at temperatures ranging from 2 to 30°C (Goss and Bunting, 1983). Due to their small size and high surface-to-volume ratio, the effects of temperature shifts on their metabolism are pronounced and immediate (Stollewerk, 2010), rendering the efficiency of the thermal adjustments even more critical. Clonal differences in thermal tolerance have been reported in numerous Daphnia studies (e.g., Palaima and Spitze, 2004). Clones from lower latitudes were found to have a higher heat tolerance than clones from higher latitudes when tested at low and intermediate but not at high acclimation temperatures (Williams et al., 2012; Yampolsky et al., 2014). Some studies have also observed divergences in metabolic rates among daphnia from different geographical areas (Chopelet et al., 2008).

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While the wide distribution and successful colonization of highly contrasted habitats make this species complex a potentially powerful animal model to explore mitochondrial adaptations to different environmental constraints, the size of these organisms has prevented the precise characterization of mitochondrial function. Fine characterizations of mitochondrial function must be done through respiration measurements because measurements of enzyme activities (i.e., citrate synthase, electron transport system) in daphnia homogenates have failed to reveal differences between D. pulex clones (Jose et al., 2009). A lack of divergence in key enzyme activities (cytochrome c oxidase or citrate synthase activities) has also been noted in fish species and their hybrids (arctic charr, Salvelinus alpinus, and brook charr, S. fontinalis) (Blier et al., 2006). Divergence in key enzymes activities can be a first step to document adjustment or variation of mitochondrial phenotype, but the absence of these divergences does not advocate for identical mitochondrial phenotype. Changes in proportions of different enzymes or adjustments of membranes lipids composition could certainly impact functional properties of mitochondria even without revealing clear divergences when essaying only few key enzymes. A newly developed assay (Kake-Guena et al., 2015) now makes it possible to measure integrated mitochondrial function in Daphnia lineages. This assay has enabled the detection of differences in mitochondrial OXPHOS measured at 20°C between D. pulex clones (Kake-Guena et al., 2015).

The next step is to use this new protocol to determine if there is variation in mitochondrial functions in Daphnia pulex and to identify if there is specific mitochondrial characters associated with particular environmental conditions. To answer this question, we measured mitochondrial respiration at different temperatures and compared key functions of mitochondrial OXPHOS in the D. pulex clones. We included four clones belonging to different lineages of D. pulex complex from different latitudes—two clones from temperate environments and two clones from subarctic environments.

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2. Material and methods

2.1 Animals Clones were sampled from the field and cultured in environmental growth chambers (Thermo Forma Diurnal Growth Chamber) at 20°C under a 16h:8h light:dark regime for several months prior to the experiments. The temperate clones were from Ontario (Fence) and Alberta (Hawrelak) and the subarctic clones from Churchill, Manitoba, (A24) and Kuujjuarapik, Quebec, (K154) (all in Canada). The clone A24 comes from a pond with high conductivity (960uS), K154 inhabits a pond with lower conductivity (150 uS). The conductivities of ponds where Fence and Hawrelak were sampled are not known but are most likely less than 100 uS. The Fence, Hawrelak, and K154 clones had mitochondrial DNA typical of Daphnia pulex whereas A24 had mitochondrial DNA typical of D. pulicaria. At the nuclear level, K154, A24, and Fence clones can be classified as pulex-pulicaria hybrids whereas Hawrelak is a pure D. pulex (Vergilino et al. 2011). Culture water was synthetic pond water containing (per L): 50 mg of KCl, 40 mg of MgSO4, 26.5 mg of CaCl2, 6 mg of K2HPO4, 6 mg of KH2PO4, 50 mg of NaNO3, and 1.1 mg of FeCl3 (modified from Lynch et al., 1986); water was aerated daily and changed every week. All clones were kept under the same conditions for at least two weeks prior to experiments. All clones were fed with a suspension of Selenastrum spp. every other day.

2.2 Permeabilization of the animals Daphnia were rinsed twice with 0.5 ml of ice-cold relaxing solution (BIOPS) containing the following: 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 20 mM imidazole, 20 mM taurine, 6.56 mM MgCl2-6H2O, 5.77 mM ATP, 15 mM phosphocreatine, 0.5 mM dithiothreitol, and 50 mM K-MES (pH 7.1 at 0°C; (Gnaiger, 2014). Whole animals were permeabilized with sharp forceps by piercing holes through the whole surface of the body. In order to ensure complete permeabilization of the cell membrane, the sample was thengitated for 30 min on ice in BIOPS supplemented with 50 μg ml−1 saponin (Kake-Guena et al., 2015). For each measurement, a pool of 10 (K154, A24, Fence) or 20 (Hawrelak) permeabilized animals were immediately transferred into respiration chambers (OROBOROS Oxygraph 2 k, Innsbruck,

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Austria) containing 2 ml of MiR05 respiration medium (110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 1 g L−1 fatty-acid free BSA, 3 mM MgCl2-6H2O, 20 mM taurine, 10 mM KH2PO4, and 20 mM K-HEPES; pH 7.1, osmolarity 330 mOsm ). Respiration was measured at 10, 15, 20, 25, 30, and 35°C for the Hawrelak and at 10 and 20°C for the three others. Datlab software (OROBOROS Instruments) was used for data acquisition and analysis.

2.3 High-resolution respirometry The protocol for the evaluation of mitochondrial function is shown in Fig. 1. The final concentration of substrates and inhibitors added to the chambers were 5 mM pyruvate, 5 mM malate, 2.5 mM ADP, 10 μM cytochrome c 10 mM succinate, 0.5 μM rotenone, 2.5 μM antimycin A, 2 mM ascorbate, 0.5 mM tetramethylphenylenediamine (TMPD), and 15 mM azide. Respiratory states are defined as follows: (1) LEAK respiration is measured in the presence of pyruvate&malate before the addition of ADP and represents the respiration compensating for proton leak and slip, cation cycling, and electron leak (Gnaiger 2009), and (2) OXPHOS respiration is measured after the addition of saturating ADP (coupled oxidative phosphorylation) and substrates feeding electrons into complex I (pyruvate&malate), complex I&II (pyruvate&malate&succinate), complex II (succinate&rotenone), or complex IV (TMPD&ascorbate) of the ETS. The respiratory control ratio (RCR) was calculated as OXPHOS with complex I substrates over LEAK capacity. The integrity of the outer mitochondrial membrane in the permeabilized animals was measured by the addition of exogenous cytochrome c to mitochondria in the presence of complex I substrates and saturating ADP. Mitochondrial respiration was corrected for oxygen flux due to instrumental background and for residual oxygen consumption (ROX) after inhibition of complexes I and III with rotenone and antimycin A. For complex IV respiration (ascorbate&TMPD), the chemical background measured in the presence of azide was subtracted. Respiratory flux in daphnia is expressed as pmol O2 per second per animal.

Different ratios were calculated from the respiratory flux data per animal. The flux control ratio (FCR) is expressed on a common reference state of maximal OXPHOS capacity with parallel electron input from complexes I&II. FCRs represent

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the proportional contribution of the various complexes in relation to maximal OXPHOS capacity, and therefore are dictated by the mitochondrial properties rather than the mitochondrial content. In daphnia, uncoupling with dinitrophenol does not affect mitochondrial respiration, and OXPHOS capacity is equivalent to the ETS capacity (Kake-Guena et al., 2015; Lemieux and Warren, 2012). The coupling of the ETS and the phosphorylation of ADP was evaluated by FCR for LEAK and the conventional respiratory control ratio (RCR; respiration with pyruvate&malate&ADP / respiration with pyruvate&malate). The integrity of the outer mitochondrial membrane was estimated with the flux control factor for cytochrome c (FCFc), which is calculated as (flux with pyruvate&malate&ADP&cytochrome c – flux with pyruvate&malate&ADP) / Flux with pyruvate&malate&ADP&cytochrome c. A ratio of 0.00 means full integrity of the mitochondrial outer membrane while a ratio of 1.00 means that the mitochondrial outer membrane is fully damaged.

2.4 Data analysis Statistical analyses were performed using SigmaStat 4 (Aspire Software International, Ashburn, VA). Criteria of normality and homogeneity of variance were tested for each variable with Shapiro-Wilk and Brown-Forsythe tests, respectively. FCR for LEAK and RCR met these criteria. These variables were compared at six temperatures for the Hawrelak clone with a one-factor (temperature) analysis of variance (ANOVA) followed by a pairwise comparison with a Tukey test. For variables not meeting these criteria (all of the fluxes per daphnia, the FCRs for OXPHOS, and the cytochrome c effect), a Kruskal-Wallis test followed by an a posteriori Dunn comparison were performed. Differences between clones for flux control ratios and cytochrome c effect were tested with a two-way ANOVA, with temperature and clones as the two factors (normality and homogeneity of variance conditions were met). For the respiration rate per daphnia and RCR, nonparametric analyses were used (Kruskal-Wallis and Dunn comparison tests). P<0.05 was considered significant. Results are presented as means ± standard error of the mean (SEM).

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3. Results 3.1 Mitochondrial respiration at six temperatures in the Hawrelak clone Mitochondrial OXPHOS with all substrates (in pmol O2 s-1 daphnia-1) increased with temperature from 10 to 30°C and dropped at 35°C (Fig. 2a). The Q10 values represent the change in rate with an increase of 10°C. Q10 values for all complexes (I, II, I&II, and IV) and states (OXPHOS, complex IV activity, and LEAK) were very similar in the 15–20°C and 20–25°C temperature ranges (Table 1). In the lower temperature range (10–15°C), there was a strong variation in Q10 among the complexes and states: OXPHOS for complexes I, II, and I&II showed a higher sensitivity to temperature (Q10 of 4.2, 4.1, and 3.6, respectively) whereas complex IV showed a much lower thermal sensitivity (Q10 of 2.0). A lower thermal sensitivity of complex IV compared to other complexes was also observed in the 25–30°C temperature range (Table 1). All mitochondrial functions dropped when temperature exceeded 30°C, as shown by the Q10 values under 1.0. The decrease was more pronounced for complexes I, II, and I&II OXPHOS (Q10 of 0.3) compared to complex IV activity (Q10 of 0.7) and complex I LEAK (Q10 of 0.8; Table 1).

To measure how temperature affect the way different complexes vary with respect to each other, respiration rates were expressed as FCR over the maximal OXPHOS capacity with the complex I&II substrates (Fig. 2B), indicative of a qualitative change in the OXPHOS system independent of the mitochondrial content. FCRs show minor variations for complex I and complex II within the full range of temperature (Fig. 2B). The contribution of complex I to the maximal OXPHOS capacity is at a minimum at 10°C and maximum in the 20–30°C temperature range, whereas the contribution of complex II is minimal at 25°C and maximal at 15 or 35°C. In contrast to the minor changes observed with complexes I and II, the FCR for complex IV activity showed a strong temperature effect, with a drastic increase in the FCR at low (10°C) and at high (35°C) temperatures (Fig. 2B).

We then examined the apparent excess capacity of complex IV at 20°C in a physiological state with simultaneous electron flow from complexes I&II. Azide titrations resulted in a parallel hyperbolic inhibition of complex IV and pathway flux

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through complexes I&II (Fig. 3A). The threshold plot display pathway flux as a function of complex IV activity is linear with an intercept of 1(Fig. 3B). The threshold plot means that the inhibition of complex IV activity results in the same inhibition in the pathway flux, so there is no excess capacity of complex IV at 20°C. The FCRs for complex IV showed that the proportion of complex IV over maximal OXPHOS capacity increased in the Hawrelak clone at 10°C and 35°C (Fig. 2B). This suggests that there is an excess capacity of complex IV at these extreme temperatures.

3.2 Clonal comparisons under two temperatures Measurements at 10°C revealed differences in mitochondrial function among clones that were not seen at 20°C. While none of the OXPHOS rates in pmol s-1 per daphnia showed significant differences among the four clones at 20°C (Fig. 4A– D), measurements 10°C revealed differences in complex I OXPHOS, with a significantly higher flux in the Fence clone compared to the Hawrelak and A24 clones (Fig. 4A). When rates were expressed as FCRs, the variability of the data due to differences in body composition and mitochondrial content was reduced, allowing the detection of divergences associated with mitochondrial function. FCRs for complex I OXPHOS showed significant variation among clones: at 10°C, the Fence clone had significantly higher FCR values than did the K154 and A24 clones while the Fence and Hawrelak clones showed significantly higher FCR values when compared to the K154 clone at 20°C, (Fig. 4E). In contrast, the FCR for complex II showed only one significant difference, i.e., between the K154 and A24 clones at 20°C (Fig. 4F). The FCR for Complexes I&II is equal to 1.00 as this state is used as the reference for the FCR calculation (Fig. 4G). The FCR for complex IV showed pronounced differences at the low temperature, with higher values in the Hawrelak and A24 clones (Fig. 4H).

The thermal sensitivity (Q10 for the temperature range 10–20°C) of mitochondrial function for each state and clone are shown in Table 2. For each clone, the thermal sensitivity of the LEAK state was lower compared to the thermal sensitivity of the OXPHOS states with substrates for complexes I and/or II. In the temperate Fence clone, all OXPHOS states (for all complexes) had low and similar thermal sensitivities. In contrast, Q10 values in the three other clones were higher and more variable. The thermal sensitivity of mitochondrial respiration in the K154 clone showed similar values for complexes I, II,

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and I&II (1.98, 1.92, and 1.91, respectively), and a slightly lower value for complex IV (1.71; Table 2). In the Hawrelak and A24 clones, complex IV showed a lower thermal sensitivity compared to the other mitochondrial complexes. In addition, the A24 clone showed a stronger thermal sensitivity of complex I compared to complex II (Q10 of 2.72 and 1.99, respectively); in the three other clones, the thermal sensitivities of complex I OXPHOS and complex II OXPHOS were much closer. The difference between the thermal sensitivity of complex IV and complexes I and II in the Hawrelak and A24 clones explains the high FCR of complex IV in these clones compared to the Fence and K154 clones. A stronger decrease in the function of complexes I and II when temperature drops, coupled with a better preservation of complex IV function at low temperature (Table 2), necessarily leads to a complex IV excess at the low temperature in these two clones.

3.3 Integrity of the mitochondrial membrane and coupling in D. pulex The increase in respiration after the addition of exogenous cytochrome c was expressed as the ratio FCFc and was similar between 10°C and 30°C in the Hawrelak clone (0.24±0.02 at 10°C, 0.19±0.02 at 15°C, 0.22±0.01 at 20°C, 0.21±0.02 at 25°C, and 0.19±0.01 at 30°C). At 35°C, FCFc decreased to 0.14±0.04. This is significantly different from the values at 10, 20, and 25°C, indicating either a better integrity of the outer mitochondrial membrane or higher affinity of cytochrome c for associated membrane components at higher temperatures. When comparing the clones, the cytochrome c effect was higher in the Fence clone, whereas the three other clones showed similar cytochrome c effects (Fig. 5A). This could suggest greater damage to the outer mitochondrial membrane in the Fence clone, which can hypothetically be associated to the membrane composition of mitochondria. Since this clone is the one suspected to live at the highest temperatures, the fatty acid profile of mitochondrial membranes could diverge from composition in the other species. This could affect both the resistance of mitochondria to manipulation as well as the affinity of cytochrome c to membranes and cytochrome c oxidase. To compensate for this divergence, the OXPHOS data used for the comparison between the clones were the data with exogenous cytochrome c added. This allowed us to avoid any bias induced by the limitation of cytochrome c availability resulting from damage to the outer mitochondrial membrane.

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The coupling of the OXPHOS process in daphnia mitochondria was evaluated with the FCR of LEAK and the conventional RCR. The FCRs allowed the evaluation of coupling with reference to a high maximal capacity under conditions that were not limited by substrates or by the phosphorylation system (Gnaiger, 2009; Gnaiger et al., 2015). In the Hawrelak clone, the FCR of LEAK did not vary with temperatures in the10–30°C range (Fig. 2B, full circles). The low value of 0.2 within the 10–30°C temperature range also rules out differences in mitochondrial uncoupling related to the procedure for permeabilization or the assay temperature. The FCR for LEAK was low in all clones (Fig. 5B). No significant variations in the FCR for LEAK were measured between clones at 10°C, and only the Hawrelak clone showed FCR values higher than those in the other three clones at 20°C (Fig. 5B).

In our study, the conventional RCR values are comparable to other studies performed on isolated mitochondria or permeabilized tissues from aquatic invertebrates (Abele et al., 2002; Kake-Guena et al., 2015; Munro et al., 2013; Paital and Chainy, 2012; Pichaud et al., 2012; Tschischka et al., 2000). In the Hawrelak clone, similar RCRs were obtained at temperatures between 10 and 30°C (2.7±0.2 at 10°C, 2.8±0.2 at 15°C, 3.1±0.1 at 20°C, 2.8±0.2 at 25°C, 3.3±0.2 at 30°C; Fig. 5C). At 35°C, the uncoupling of mitochondrial OXPHOS was measured in the Hawrelak clone, with a decrease in RCR (1.9±0.1; data not shown) and an increase in LEAK FCR (0.3; Fig. 2B; filled circles). The RCR values did not vary among the clones at 10°C (Fig. 5C), while a lower RCR was observed in the Hawrelak clone at 20°C, which is indicative of a lower coupling, as measured with the FCR of LEAK (Fig. 5C).

4. Discussion The characterization of mitochondrial functions and their thermal sensitivity in four Daphnia clones has revealed that the main differences occur at the level of complex I and IV respiration and under low assay temperature. The FCR for complex IV varied among clones at 10°C but not at 20°C. Our results at 20°C are in accordance with our previous study with the same clones (Kake-Guena et al., 2015), showing only minor changes in the FCR for complex I and complex II between

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clones and no changes in complex IV. Other studies on fishes (Kraffe et al., 2007) and cephalopods (Oellermann et al., 2012) have detected differences in mitochondrial function at low assay temperatures even larger than those that we found. Also in accordance with our results, populations of cuttlefish (Sepia officinalis) from different thermal environments or acclimated to various temperatures showed variations in mitochondrial function mostly localized at complex I and IV (Oellermann et al., 2012). Similarly, metabolic adjustments following temperature acclimation in the killifish has been shown to specifically target complex I of the ETS (Chung and Schulte, 2015).

Do divergences in mitochondrial function translate into metabolic adaptations? The first step towards answering this question is to relate the specificity in mitochondrial function with the environment as well as the life history of the clones. We would expect, for example, that key adjustments should be correlated with the thermal gradient of the clones, with the clones from colder locations having higher mitochondrial catalytic capacity or lower thermal sensitivity at lower temperature. The Fence and Hawrelak clones are from temperate areas whereas the K154 and A24 clones are from subarctic areas. Surprisingly, the clone from the most southern location (Fence; Windsor, Ontario) had a significantly higher complex I OXPHOS capacity per daphnia and a trend for higher OXPHOS capacity with substrates from complex II and complexes I&II. As this is a respiratory capacity per Daphnia, we cannot exclude the fact that the difference is related to size of the animal. In addition, the temperate Fence clone also showed a smaller effect of temperature on mitochondrial respiration (lower Q10) and a more consistent thermal sensitivity of the mitochondrial complexes at different temperature ranges. The Hawrelak clone (Alberta; temperate environment) had mitochondrial function similar to the A24 clone from Churchill: a low complex I respiration per daphnia and a high FCR for complex IV at 10°C. Clearly clonal variations in mitochondrial function, even at low temperatures, do not correlate with geographical location. Further analyses on more clones from a wider environmental gradient are needed to better understand the relationship between thermal tolerance and mitochondrial functions in Daphnia.

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It should also be noted that mitochondrial characters other than maximal catalytic capacities could be critical for survival and fitness at different thermal regimes or when facing temperature variations. Divergences among clones adapted to different temperatures could reflect adjustments that ensure the proper regulation of mitochondrial function rather than different maximal respiratory capacities. This might help organisms respond to the variability of dissolved oxygen in different environments and at different temperatures, or ensure the precise regulation of reactive oxygen species (ROS) production (see Blier et al. 2014). Daphnia inhabiting ponds have the mtDNA haplotypes of D. pulex whereas daphnia inhabiting lakes have the mtDNA haplotypes of D. pulicaria. In our study, the A24 clone was the only one with an mtDNA typical of D. pulicaria and the only clone that showed a stronger thermal sensitivity of complex I compared to complex II. Additional studies are needed on a larger number of clones differing in mtDNA haplotypes to determine if these can be related to mitochondrial phenotypes.

We observed a striking similarity between the Hawrelak clone from Edmonton and the A24 clone from the coldest location (Churchill, Manitoba): a large increase in the complex IV activity over maximal OXPHOS at 10°C and low apparent excess capacity of complex IV at 20°C. This low excess of complex IV capacity at 20°C, which seems at odds with the literature, is revealed by linear inhibition of maximal OXPHOS starting at low inhibition of Cytochrome oxidase activity. Many previous studies alluded to the appearance of complex IV in excess capacity in different species, i.e., red muscle of brook trout (Salvelinus fontinalis; (Blier and Lemieux, 2001), cardiac muscle of triplefin fish (Hilton et al., 2010), Drosophila species (Farge et al., 2002; Pichaud et al., 2011; Pichaud et al., 2010), and in endotherm species (Lemieux et al., 2017; Letellier et al., 1994; Rossignol et al., 2003; Rossignol et al., 2000; Rossignol et al., 1999) usually based on higher complex IV catalytic capacity compared to maximum OXPHOS flux . It has been proposed that this high catalytic capacity of complex IV compared to OXPHOS respiration rate would ensure the maintenance of ETS in an adequate oxidized state that would allow proper functioning and regulation of mitochondrial respiration (see review by (Blier et al., 2014). Gnaiger and colleagues (Gnaiger et al., 1998) also suggested that an apparent excess capacity of complex IV sustains the high affinity for oxygen in mitochondria and might be especially important when the oxygen level is low, such as under hypoxic

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conditions or in the active state when oxygen affinity decreases. The low apparent excess in complex IV at 20°C, revealed by the titration by sodium azide, could suggest that complex IV and mitochondria are not limited by oxygen access in these micro-crustaceans at temperature close to optimality. It could also suggest that fine regulation of reduction state ETS, by high content of the complex responsible of final reduction of molecular oxygen, is not required by these organisms. The increase in apparent excess of complex IV at 10°C for the Hawrelaak and A24 clones (Fig. 4F) are however in line with a previous study in Drosophila (Pichaud et al., 2013), where the authors found complex IV in excess in muscle mitochondria only at the lower temperature (12°C compared to 18°C). In the mouse heart, it was also recently shown (Lemieux et al., 2017) that at low temperature (4°C compared to 25°C), the FCR for complex IV increased drastically by 3-fold, and that this is associated with a 4-fold increase in excess of complex IV measured with a threshold plot. These data support the utilization of the FCR for complex IV as a good indication of drastic changes in excess capacity at low temperature in our daphnia experiments. Considering the divergences among clones of the temperature impact on apparent excess of complex IV, these clones should be relevant system to explore the potential adaptive significance of this apparent excess. These results reflect the higher thermal sensitivity at low temperature of processes upstream of complex IV, e.g., our observation of the higher Q10 of complex I, complex II, or complex I&II respiration when compared to complex IV Q10 at low temperature for three clones (Hawrelak, K154, A24). This higher sensitivity of processes upstream of complex IV at low temperature can only result in a buildup of excess complex IV. It also suggests a stronger limitation of mitochondrial capacity at low temperature, either at the entrance of electrons into the ETS or at the level of dehydrogenase pathways that provide reducing equivalents (NADH and FADH2) to the ETS. This interpretation is supported by comparative studies on fish and mammals that show a higher relative capacity in ectotherm fish heart mitochondria to oxidize pyruvate (compared to other components of the mitochondrial pathway) in comparison to the mammalian heart mitochondria at low temperature (see (Blier et al., 2014; Lemieux et al., 2010a; Lemieux et al., 2010b). These data suggested that the processes upstream of complex IV are important determinants in thermal adaptation to low temperature and that a putative a key adaptation of ectotherms to function at low temperature, could be an increased ability to supply electrons to ETS through higher activity of the pyruvate dehydrogenase complex.

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One could argue that excess complex IV at extreme temperatures (high and low, as observed in the present study for Hawrelack) might be of adaptive significance. The high relative complex IV activity could ensure higher mitochondrial affinity for oxygen close to lower or higher temperature limits, over which oxygen supply could no longer support aerobic capacity and induce hypoxic cellular conditions (Pörtner et al., 2005; Pörtner et al., 2007). An increase in complex IV following warm acclimation has been observed in the cuttlefish heart (Oellermann et al., 2012), carp heart (Cai and Adelman, 1990), Atlantic cod white muscle (Foster et al., 1993), and Antarctic eelpout liver (Windisch et al., 2011). This increased complex IV capacity was expected to support higher oxygen diffusion to mitochondria in hypoxic conditions and delay heat-formation of ROS. An increase in ROS formation in response to heat stress and environmental hypoxia is common among marine ectotherms (Abele et al., 2007). An increase in complex IV following cold temperature acclimation has also been recurrently observed at least in some fish tissues. However, the apparent excess capacities (at low or high temperature) can simply reflect differences in thermal sensitivity (Q10) among the components of mitochondrial pathways, and the relevance of high relative complex IV activities for restricting ROS upsurge at extreme temperatures will have to be further tested. Our Daphnia system is perfectly suited to delineate the role of complex IV on ROS management and its relation with thermal tolerance or adaptability.

5. Conclusion Our results show important divergences in mitochondrial phenotypes among Daphnia clones. The flux control ratio of complex I, complex II, and complexes I&II, and the relative activity of complex IV as well as their respective thermal sensitivities (Q10) are quite divergent among clones. We show that differences in mitochondrial function among clones are more likely to be detected at low assay temperatures, pointing towards the importance of choosing the proper assay temperature when the objective is to detect variations between close groups such as populations, clones, or strains. Complex IV capacity relative to maximal OXPHOS capacity showed large variations between D. pulex clones, with a large apparent excess capacity of complex IV observed in the clones from Edmonton and Churchill, whereas the two other clones

17

(from Windsor and Kuujjurapik) did not show such an excess capacity of complex IV. Importantly, complex IV apparent excess is evident at temperature extremes and particularly at lower temperatures, which supports the hypothesis that the thermal sensitivity of mitochondrial function at lower temperatures is dictated by processes upstream of the phosphorylation system and cytochrome c oxidase activity (Blier et al. 2014). A question remains to be tested: do these changes in complex IV excess capacity at extreme temperatures confer an advantage for species encountering rapid and extreme changes in temperatures? D. pulex appears to be a model of choice to answer this question, considering the diversity of environmental conditions and habitats where they are found, the scale of their distribution over a latitudinal gradient, and the ease of rearing them in controlled conditions. Our capacity to discriminate different mitochondrial phenotypes makes Daphnia a highly valuable model organism when attempting to correlate mitochondrial function with the phenotype of the organism and its ability to successfully exploit different habitats.

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Acknowledgements This study was supported by a discovery grants from the Natural Sciences and Engineering Research Council (NSERC) to HL, FD, and PB (RGPIN 402636, RGPIN 155926, RGPIN 222948, respectively), an undergraduate student research award from NSERC to K. Touisse, a research grant and startup grant from Faculty Saint-Jean, and an equipment grant from the Canadian Foundation for Innovation to HL. We are grateful to Micheline Forgues for her help with animal care.

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Tables

Table 1: Q10 values for mitochondrial respiratory capacity in permeabilized D. pulex of the clone Hawrelak (Edmonton, AB) for each temperature range.

State

Complex

10–15°C

15–20°C

20–25°C

25–30°C

30–35°C

LEAK

Complex I

2.9

1.5

1.1

2.9

0.8

OXPHOS

Complex I

4.2

1.7

1.0

3.5

0.3

OXPHOS

Complexes I&II

3.6

1.6

1.0

3.2

0.3

OXPHOS

Complex II

4.1

1.4

0.9

3.6

0.3

OXPHOS

Complex IV

2.0

1.5

1.0

2.1

0.7

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Table 2: Q10 values for the 10–20°C temperature range for mitochondrial respiratory capacity in four D. pulex clones.

State

Complex

Fence

Hawrelak

K154

A24

LEAK

Complex I

0.98

2.11

1.55

1.82

OXPHOS

Complex I

1.37

2.69

1.98

2.72

OXPHOS

Complexes I&II

1.33

2.42

1.91

2.29

OXPHOS

Complex II

1.30

2.42

1.92

1.99

OXPHOS

Complex IV

1.37

1.72

1.71

1.39

27

Figure legends

Fig. 1. Representative trace for the evaluation of mitochondrial respiratory capacities in permeabilized Daphnia with a multiple substrate–inhibitors titration protocol. The trace represents the oxygen consumption as a function of time. The measurement was performed at 20°C in the Hawrelak clone. Mitochondrial coupling states are distinguished as LEAK (without ADP) and OXPHOS (saturating ADP). The multiple titration protocol comprised the following steps: (1) LEAK respiration in the presence of the complex I substrates pyruvate&malate, (2) OXPHOS respiration in the presence of complex I substrate, (3) addition of cytochrome c to test for integrity of the outer mitochondrial membrane, (4) addition of succinate to measure respiration in the presence of the substrates of complexes I&II, pyruvate&malate&succinate, (5) succinate-supported respiration (complex II) after inhibition of complex I with rotenone, (6) residual oxygen consumption after inhibition of complex III with antimycin A, (7) complex IV respiration in the presence of ascorbate&TMPD, (8) inhibition of complex IV with azide. Arrows indicate times of titrations of the substrates and inhibitors.

Fig. 2: Mitochondrial respiratory capacity (A) and flux control ratios (B) measured at six different temperatures in permeabilized D. pulex of the Hawrelak clone (Edmonton, Alberta). (A) Respiration is measured in the LEAK and/or OXPHOS state in the presence of complex I, complexes I&II, complex II, and complex IV substrates (see details in Fig.1). (B) The flux control ratios are normalized for maximal OXPHOS capacity with the substrates of complexes I&II. Data are means ± SEM. Within a specific state, symbols with one similar letter are not significantly different (p˃0.05). N = 10, 10, 14, 22, 10, and 14 pools of 20 animals for 10, 15, 20, 25, 30, and 35°C, respectively..

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Fig. 3: Azide titration and complex IV threshold in permeabilized D. pulex from the Hawrelak clone. The measurement was performed at 20°C. (A) The azide titration was performed either on the flux through the OXPHOS pathway in the presence of complex I&II substrates (titration after the addition of succinate; open symbols) or on cytochrome c oxidase (complex IV) activity (after the addition of ascorbate&TMPD; filled symbols). Results are means ± SEM (N = 4 trials). (B) Complex IV excess capacity is illustrated by the threshold plot, which shows the relative flux through the respiratory system (complexes I&II OXPHOS) as a function of relative inhibition of complex IV at identical azide concentrations. The data show a linear regression (R2≥0.92) for the four replicate experiments, with an intercept of 0.98.

Fig. 4: Oxidative phosphorylation (OXPHOS) capacity per daphnia (A–D) and expressed as flux control ratios (E–H) in four different clones of D. pulex at 10 and 20°C. At 10°C, N = 5, 10, 4, and 4 pools of 20 animals for the Fence, Hawerelak, K154, and A24 clones, respectively. At 20°C, N = 6, 14, 6, and 6 pools of 20 animals for the Fence, Hawerelak, K154, and A24, clones, respectively. Data are means ± SEM. Within a specific state, significant differences are indicated with asterisks (*p<0.05; ** p<0.01; ***p<0.001).

Fig. 5: Mitochondrial membrane integrity and mitochondrial coupling in the four clones of D. pulex at two assay temperatures. (A) Integrity of the mitochondrial outer membrane calculated as the flux control factor for cytochrome c (FCFc) calculated from respiratory values in the presence of complex I substrates; 0 indicates full integrity of the outer mitochondrial membrane and 1 indicates a fully damaged outer mitochondrial membrane. (B) and (C) indicate the coupling of the mitochondria. (B) Flux control ratios (FCR) of LEAK over OXPHOS with complexes I&II. (C) Respiratory control ratios (RCR) calculated as OXPHOS with complex I substrates (with cytochrome c) over LEAK state with complex I substrates. Data are means ± SEM. Within a specific temperature, significant differences are indicated with asterisks (*p<0.05; ** p<0.01; ***p<0.001). N are the same as in Figure 4.

29

30

31

32

33

Highlights

34



The present study applies our previously developed method in order to study the mitochondrial functions and their thermal sensitivity in D. pulex clones. Weclearly show that divergences in mitochondrial function among clones are more important at a low temperature assay (10 °C) and mainly target Complexes I and IV capacities.



This measurable phenotypic variability in mitochondrial function in D. pulex makes it an ideal model to study how the genetic background is linked to bioenergetics of organisms and their ability to exploit different thermal environments.