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Laboratoire de Génétique Cellulaire et développementale, Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Institut de Biologie intégrative et des systèmes, 1030 ave de la Médecine, Université Laval, Québec, Canada G1V 0A6
Linking the mitochondrial genotype and the organismal phenotype is of paramount importance in evolution of mitochondria. In this study, we determined the differences in catalytic properties of mitochondria dictated by divergences in the siII and siIII haplogroups of Drosophila simulans using introgressions of siII mtDNA type into the siIII nuclear background. We used a novel in situ method (permeabilized fibers) that allowed us to accurately measure the consumption of oxygen by mitochondria in constructed siII-introgressed flies and in siIII-control flies. Our results showed that the catalytic capacity of the electron transport system is not impaired by introgressions, suggesting that the functional properties of mitochondria are tightly related to the mtDNA haplogroup and not to the nuclear DNA or to the mito-nuclear interactions. This is the first study, to our knowledge, that demonstrates a naturally occurring haplogroup can confer specific functional differences in aspects of mitochondrial metabolism. This study illustrates the importance of mtDNA changes on organelle evolution and highlights the potential bioenergetic and metabolic impacts that divergent mitochondrial haplogroups may have upon a wide variety of species including humans.
It is now well-known that nuclear and mitochondrial genomes evolve at dissimilar rates due to differences in modes of inheritance, recombination, and patterns of mutation (Rand et al. 2004; Burton et al. 2007; Wallace 2007). Specifically, mitochondrial DNA (mtDNA) genes are most often maternally inherited, rarely recombine, and undergo higher rates of nucleotide substitution compared with nuclear DNA (Avise and Vrijenhoek 1987; Avise 1991; Pesole et al. 1999; Blier et al. 2001; Johnson et al. 2003; Burton et al. 2007). These properties have placed the mtDNA at the center of a broad array of evolutionary studies and have led to the suggestion that selection on mtDNA might lead to haplotype-specific adaptation to specific environments (Blier et al. 2001; Fontanillas et al. 2005; Wallace 2007). However, the role of natural selection on the evolution of mtDNA remains elusive, principally because the specific relationships between mtDNA gene variation and potentially adapted function can be extremely complex to establish with traditional tools of molecular evolution. In this context, the direct study of functional properties of mitochondria with different mitochondrial haplotypes may help us to provide a robust link between the genotype and the phenotype.
Drosophila simulans could be an appropriate model to study functional specificity of mtDNA. This species exhibits three different mitochondrial haplotypes (siI, siII, and siIII) with nearly 3% interhaplogroup divergence (Ballard 2000a,b, 2005) but lacks subdivision at any nuclear-encoded loci tested to date (Ballard 2000a; Katewa and Ballard 2007; Melvin et al. 2008). The siII and siIII populations live in sympatry in Kenya where the frequency of the siIII haplogroup is about 40% (Ballard 2004), suggesting that there is the potential for random nuclear gene flow. In four wild-caught siII and four siIII fly lines, low amino acid variation was observed within haplogroups in a 4.5-kb region spanning from position 1450 to position 5983 of the mtDNA genome. In contrast, large divergences were noticed between the haplogroups (0.02% nucleotide divergence, 0.012% amino acid divergence in the same region) (Ballard et al. 2007).
In a previous study, we used an in situ approach to evaluate the mitochondrial respiration of D. simulans siII and siIII in permeabilized fibers (Pichaud et al. 2011). We measured higher catalytic capacities of ETS at 24°C for the siII haplotype when compared to the siIII haplotype. However, we could not conclude that the observed difference resided entirely at the level of mtDNA as mito-nuclear interactions may also be involved. Indeed, different components of the ETS, either encoded by nuclear genome or mtDNA, should be coadapted to efficiently fulfill their task (Blier et al. 2001; Rand et al., 2004). To investigate if divergences in mtDNA are associated with differences in mitochondrial properties, we employ backcrossing of two populations harboring divergent mitochondrial haplotypes but sharing the same nuclear environment.
In this study, we compared mitochondrial functions between D. simulans harboring two mitochondrial haplogroups in the same nuclear background. If the mtDNA dictates the metabolic differences between the two sets of fly lines, we should observe the same set differences in the functional properties of mitochondria when siII-introgressed and siIII-controls are compared with native siII and native siIII flies. Specifically, we predict higher catalytic capacities of ETS for the siII-harboring flies (Pichaud et al. 2011). If, on the other hand, the siII and siIII mtDNA are specifically coadapted with their native nuclear genomes, we should detect a disruption of the catalytic capacities in siII-introgressed flies due to alteration of mito-nuclear coadaptations.
Eight native lines of D. simulans (having siII and siIII haplogroups) collected in Kenya were used for experiments. For each haplogroup, four isofemale lines (namely 2KY0412, 2KY0415, 2KY0418, and 2KY0421 for siII; 3KY0410, 3KY0412, 3KY0414, and 3KY0420 for siIII) were reared from flies collected in Nairobi (Kenya) during November 2004 (Ballard et al. 2007). No nuclear subdivision was detected at any nuclear-encoded loci tested among these lines (Ballard 2000a; Dean et al. 2003; Katewa and Ballard 2007; Melvin et al. 2008).
This study aims to determine whether the previously observed differences in mitochondrial functions between D. simulans harboring two mitochondrial haplogroups result from differences in the mtDNA (siII > siIII) or specific mito-nuclear interactions (Pichaud et al. 2011). The goal of the genetic crosses was to place siII and siIII mtDNA in the nuclear genetic background from flies that natively harbored siIII mtDNA. This strategy was considered conservative because it would disrupt any coevolved interactions between siII mtDNA and its wild-type nuclear genetic background should they exist (siII-introgressed). It would not, however, disturb coevolved genetic interactions between siIII mtDNA and its native nuclear genetic background (siIII-control). To examine the possible influence of haplogroup on mitochondrial metabolism, four crossing pairs (CP1, CP2, CP3, and CP4) were constructed in parallel (Fig. 1). Each pair includes a siII-introgressed line and a siIII-control line. To decrease the potential for interindividual variation between the nuclear DNA of the different isofemale lines, F8 individuals resulting from the four independently disrupted siII-introgressed lines and F’8 flies from the four siIII-controls were pooled for subsequent experiments (Fig. 1).
The crossing strategy was performed to minimize inbreeding depression. Within a crossing pair, siII-introgressed lines were constructed by mating 15 siII virgin females of the four isofemale lines (2KY0415, 2KY0418, 2KY0421, and 2KY0412 for CP1, CP2, CP3, and CP4, respectively) with 30 siIII males for each of the four isofemale lines (3KY0412, 3KY0414, 3KY0420, and 3KY0410 for CP1, CP2, CP3, and CP4, respectively). After three days, adult flies were removed and virgin female progeny (F1) was backcrossed with siIII males (Fig. 1). This was repeated for eight generations using an alternate paternal line at each generation (each of the four isofemale line was used twice), and F8 males were collected for experiments. This backcrossing allows maintenance of maternally inherited mtDNA while increasing the proportion of paternal nuclear DNA. Theoretically, this procedure enables a replacement of more than 99.6% of nuclear DNA to obtain siII mtDNA within siIII nuclear DNA environment. Moreover, it decreases the possible inbreeding effect while minimizing the line-specific effect. The siIII-control lines were developed using a parallel procedure (Fig. 1). A limitation of the crossing strategy is that it reintroduced the nuclear DNA of the parental siIII female in F’8 and may bias the functional properties of mitochondria in favor of siIII phenotype. Again, this crossing strategy is conservative as our hypothesis is the properties of mitochondria harboring siII mtDNA are divergent from that harboring siIII mtDNA.
To avoid fitness problems associated with aging, each backcross was performed with flies less than 14 days old. All lines were maintained at 24°C in two different incubators (A and B) to allow replicates of experimental lines and to avoid any “incubator” effect. The pools of siII-introgressed and of siIII-control individuals were weighted before fiber permeabilizations.
At the beginning and at the end of the study, the mtDNA type of each constructed line was determined using allele-specific PCR (Dean et al. 2003). The males used for each backcross were also verified for mtDNA type during the eight generations. The absence of Wolbachia was verified using conserved 16S rDNA primers (James and Ballard 2000).
PREPARATION OF PERMEABILIZED MUSCLE FIBERS
Fiber permeabilizations were conducted at 4°C with flight muscles from four thoraxes of 10-day-old males D. simulans as previously described (Pichaud et al. 2011). Briefly, fibers were permeabilized using a BIOPS-relaxing solution (Veksler et al. 1987; Letellier et al. 1992) complemented with 81.25 μg.mL−1 saponin (Pichaud et al. 2011) and were transferred into a respirometer (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) filled with air-saturated respiration medium.
Respiration was measured at 24°C, and data acquisition and analysis were performed using the software DatLab (Oroboros Instruments). State 2 was reached after the transfer of the fibers bundles into the respiration chambers filled with respiration medium in presence of pyruvate (10 mM), malate (10 mM), and L-proline (10 mM). The following substrates or uncouplers were then sequentially added to the chamber: ADP (5 mM, to achieve state 3 respiration for complex I, CI), cytochrome c from equine heart (10 μM, as an index of functional integrity of the outer mitochondrial membrane, CIc), succinate (10 mM, to reach state 3 respiration for complex I + complex II, CIc + CII), sn glycerol-3-phosphate (20 mM, to achieve state 3 respiration for complex I + complex II + glycerol-3-phosphate dehydrogenase, Cic + CII + G3Pdh), 2,4-dinitrophenol (uncoupler, optimum concentration between 5 to 15 μM to reach maximum respiration rate, CIc + CII + G3Pdh + u), rotenone (0.5 μM, inhibitor of complex I, CII + G3Pdh + u), malonate (5 mM, inhibitor of complex II, G3Pdh + u), antimycin A (2.5 μM, inhibitor of complex III, residual oxygen consumption reached after inhibition of complexes I, II, and III), TMPD + ascorbate (0.5 mM and 2 mM, respectively, to measure complex IV activity, COX). This protocol allowed us to calculate the respiratory control ratios (RCR = CI/state 2) for complex I, the cytochrome c effect (CIc/CI), as well as the uncoupling control ratios (UCR = CIc + CII + G3Pdh + u/CIc + CII + G3Pdh) with seven fibers preparations for siII-introgressed from each incubator and five for siIII-controls from each incubator. We also measured the respiration rates for CI, CIc, CIc + CII, CIc + CII + G3Pdh, CIc + CII + G3Pdh + u, CII + G3Pdh + u, G3Pdh + u, and for COX with four fibers preparations for siII-introgressed from each incubator and three for siIII-controls from each incubator (Pichaud et al. 2011). Data were expressed as mean respiration rates in pmol of oxygen consumed per second per mg proteins ± SEM corrected with residual oxygen consumption (when complexes I, II, and III were inhibited).
At the end of mitochondrial respiration measurements, fiber bundles were removed and homogenized with a Tekmar homogenizer (Tekmar Company, Cincinnati, OH) and the homogenates were immediately frozen at –80°C for further analyses. Total protein content was determined for homogenates from fiber bundles in duplicate by the bicinchoninic acid method (Smith et al. 1985). Due to addition of cytochrome c during experiments as well as the presence of bovine serum albumin in the ice-cold buffer and in the respiration medium, the protein content of the buffer was subtracted from the fibers preparations.
Chemicals were purchased from Sigma-Aldrich (St Louis, MO).
Statistical analyses were performed with SAS software (9.1.3, SAS Institute, Cary, IN). O’Brien's test was used to verify homogeneity of data. Analyses of variance (ANOVAs) with two independent variables (haplogroup, incubator) and their interaction were performed using a general linear model (GLM) procedure with the Least Square Means method. When an effect was detected, multiple comparisons (post-hoc Tukey's HSD) test was performed. Significance was defined at P < 0.05.
First, we tested whether the flies from the two groups (siII-introgressed and siIII-controls) differed in mass or whether there was any incubator effect. The resulting pools were not different for means mass ± SD between haplogroup or between incubators (0.67 ± 0.1 mg for siII-introgressed incubator A; 0.66 ± 0.08 mg for siII-introgressed incubator B; 0.65 ± 0.09 mg for siIII-controls incubator A; 0.65 ± 0.08 mg for siIII-controls incubator B).
RCRs from 14 and 10 different preparations for siII-introgressed and siIII-control, respectively (seven and five preparations per incubator), were used as an index of functional integrity of mitochondria and showed well-coupled respiration (Table 1). Nested ANOVA showed that neither haplogroup (F1, 20= 0.02, P= 0.90) nor incubator (F1, 20= 0.03, P= 0.86) or their interaction (F1, 20= 0.25, P= 0.62; for haplogroup × incubator) influenced the RCR. No significant differences between haplogroups or between incubators were detected with the Least Square Means method.
Table 1. Values of respiratory control ratio, uncoupling control ratio, and cytochrome c effect in permeabilized fibers of Drosophila simulans (± SEM).
Respiratory control ratio
Uncoupling control ratio
Cytochrome c effect
To assess mitochondrial integrity, O2 flux was compared before and after cytochrome c injection. Consistent with functional integrity of outer mitochondrial membrane, ratios of CIc/CI showed little effect of cytochrome c addition on the mitochondrial respiration (Table 1).
O2 fluxes were also compared before and after the addition of an uncoupler to see if the ATP synthase and the adenine nucleotide translocator (ANT) exerted a limitation on mitochondrial respiration. As previously reported (Pichaud et al 2011), all UCRs were close to 1.0 (Table 1) showing that ATP synthesis and ANT capacity can support the maximum respiration rates induced by experimental substrates. This result suggests that when the ETS is “nearly saturated” with electrons from the three branches (CI, CII, and G3Pdh), both ATP synthesis and ANT capacity can support the maximum electron flux through ETS and consequently that the phosphorylation of ADP and the transport of ADP and/or ATP do not appear to be limiting steps.
A typical graph of O2 concentration and uncorrected O2 consumption (in pmol.sec−1.mL−1) measurements is presented in Figure 2. ANOVAs results showing F ratios are presented in Table 2. No parameter for the O2 fluxes measured at the different steps of the ETS (CI, CIc, CIc + CII, CIc + CII + G3Pdh, CIc + CII + G3Pdh + u, CII + G3Pdh + u, G3Pdh + u, and COX) was influenced by incubator or by the interaction haplogroup × incubator (Table 2). However, the variable haplogroup influenced CIc + CII, CIc + CII + G3Pdh, and CIc + CII + G3Pdh + u (Table 2). Multiple comparisons (post-hoc Tukey's HSD test with eight and six different preparations for siII-introgressed and siIII-controls, respectively) showed significant differences between haplogroups for CIc + CII, CIc + CII + G3Pdh, and CIc + CII + G3Pdh + u (P= 0.04, P= 0.04, and P= 0.03, respectively), with siII-introgressed having higher oxygen consumption than siIII-controls (Fig. 3B).
Table 2. Analyses of variance results—F ratios.
Haplogroup× incubator df=1
*P < 0.05. CI and CIc, complex I (without and with cytochrome c, respectively); CII, complex II; G3Pdh: glycerol-3-phosphate dehydrogenase; u: uncoupled respiration; COX, cytochrome c oxidase.
The results we obtained in this study are broadly consistent with a previous study (Pichaud et al. 2011) performed at the same temperature on native siII and siIII haplogroups (Fig. 3A). We therefore compared the results from the present study with those from the study on native haplogroups (Pichaud et al. 2011) using the Least Square Means method (Table S1). No significant differences were denoted between siIII and siIII-controls for all the respiration rates, and only G3Pdh + u was different between siII and siII-introgressed (P= 0.049).
It is well established that mtDNA mutations may cause disease in humans (review in Taylor and Turnbull 2005). It is less well established how naturally occurring mutations could influence the phenotype and the biochemistry of mitochondrial bioenergetics. In this study, we investigated the influence of naturally occurring mutations in sympatric populations of D. simulans to shed light on the role of natural selection on the evolution of mtDNA. Globally, we observed similar patterns in the functional properties of mitochondria when siII-introgressed and siIII-controls are compared with native siII and native siIII flies. We did not detect any evidence that the disruption of coevolved mito-nuclear genotypes impaired the catalytic capacities of mitochondria in the experimental flies, when measured in the conditions described in the present study. Therefore, the most parsimonious explanation is that the measured mitochondrial functions divergences are mainly conferred by the mtDNA and not by the nuclear DNA or mito-nuclear interactions. To our knowledge, this is the first study that tests the effect of mtDNA differences on the catalytic capacities of the distinct complexes of the ETS with introgressed and wild-type mitochondrial genomes from closely related populations of the same species.
In this study, we used an in situ approach (on permeabilized fibers) to quantify O2 consumption in thorax tissue (Pichaud et al. 2011). O2 fluxes were measured at maximal state 3 with saturating concentrations of substrates to first identify the locus of divergences in terms of catalytic capacities of ETS enzymes in siII-introgressed and siIII-controls populations. Significant differences in O2 fluxes for CIc + CII, CIc + CII + G3Pdh, and CIc + CII + G3Pdh + u (as well as a clear overall pattern for the other parameters) were detected between introgressed and control organisms, with siII-introgressed having higher catalytic capacities than siIII-controls (Fig. 3B). In a previous study, we detected similar differences between native siII and siIII haplogroups (Pichaud et al. 2011) for CIc + CII + G3Pdh, CIc + CII + G3Pdh + u, CII + G3Pdh + u, G3Pdh + u, and COX (Fig. 3A).
Backcrossing has previously been used to study mito-nuclear interactions among different geographically isolated populations of D. simulans and of the marine copepod Tigriopus californicus (Edmans and Burton 1998; Willett and Burton 2001; Sackton et al. 2003; Willett and Burton 2004; Ellison and Burton 2006, 2007, 2008). Globally, these studies showed that in allopatric populations the disruption of mito-nuclear interactions led to a decrease in COX activity levels (Edmans and Burton 1998; Sackton et al. 2003), as well as in the activity of complexes I and III, and ATP production (Ellison and Burton 2006). In contrast, not only did we not detect any decrease in the activity of the ETS complexes in introgressed D. simulans but we measured higher activity in that population. The siII and siIII isofemale lines used for this study were collected in sympatry from Kenya and it is possible that gene flow between the populations may limit the fixation of nuclear-encoded mutations and prevent the establishment of coadapted mito-nuclear complexes. Another possibility would be that if there is a strong effect of coadaptation, then selection would act to keep the specific nuclear genes with coadapted mitochondria (Ellison and Burton 2010). When Ellison and Burton (2010) made recombinant inbred lines in Tigriopus, they found far fewer mismatches between mtDNA and the mitochondrial RNA polymerase than expected by chance, with mtDNA and mitochondrial RNA polymerase from the same line overrepresented in hybrid F2 populations. In the present work, hitchhiking of nuclear genes with mitochondrial DNA introgression would have lead to the observed phenotype modification in the introgressed population. No evidences of subdivision in nuclear genes has, however, been detected between siII and siIII.
The different isofemale lines used could also have accumulated unknown deleterious nuclear mutations that could influence the functional properties of mitochondria. However, this seems quite unlikely to have influenced our results. Within each pair, the siII-introgressed and siIII-control lines were crossed with the four paternal lines in the same order (Fig. 1). Between pairs, siII-introgressed and siIII-control lines were crossed with paternal lines in a different order such that the composition of the nuclear genetic background in the flies differed (Fig. 1). To balance the nuclear contribution, equal numbers of males from the four F8 siII-introgressed lines were pooled and equal numbers of males from the F’8 siIII-controls were pooled in each experiment (Fig. 1).
The overall higher catalytic capacities of siII-introgressed can better be explained by a higher catalytic efficiency of the ETS complexes encoded by siII mitochondrial DNA. This could give an advantage to siII either in terms of intensity of aerobic activity, endurance, and/or lower production of reactive oxygen species (Pichaud et al. 2011) under laboratory conditions (i.e., when the resources are unlimited and when the temperature, the humidity, and the fly density are controlled). Indeed, it has been shown that siII flies outperform siIII when resources are continuous and abundant whereas siIII is favored when resources are discontinuous (Ballard et al. 2007), explaining the persistence of these two haplotypes in Kenya. Previously, we have shown that the different parts of ETS are differently affected by temperature change (Pichaud et al. 2010, 2011) supporting divergent functional properties of ETS between siII and siIII. Further studies on the catalytic properties of ETS and mitochondrial metabolism at different temperatures are required to determine if the functional divergences have any adaptive value according to optimal temperature range of the flies.
Drosophila simulans is a well-developed model to study the influence of distinct mtDNA types on mitochondrial bioenergetics. The next development in our understanding of the influence of mtDNA on bioenergetics may be to relate these differences to the specific fitness associated to haplotypes. These studies have the potential to illustrate the adaptive value of mtDNA or of the coadapted mito-nuclear DNA complexes in a wide variety of species including humans.
Associate Editor: E. Abouheif
Authors would like to thank C. Jutras for animal maintenance and help with virgin females. RMT work is supported by a grant from the Canadian Institute of Health Research. This study was supported by research grants from the Natural Sciences and Engineering Research Council (NSERC) to PUB.