Nuclear–Mitochondrial Epistasis and Drosophila Aging: Introgression of Drosophila simulans mtDNA Modifies Longevity in D. melanogaster Nucle
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遗传学杂志 2006年第1期
Manuscript received June 9, 2005. Accepted for publication September 28, 2005.
ABSTRACT
Under the mitochondrial theory of aging, physiological decline with age results from the accumulated cellular damage produced by reactive oxygen species generated during electron transport in the mitochondrion. A large body of literature has documented age-specific declines in mitochondrial function that are consistent with this theory, but relatively few studies have been able to distinguish cause from consequence in the association between mitochondrial function and aging. Since mitochondrial function is jointly encoded by mitochondrial (mtDNA) and nuclear genes, the mitochondrial genetics of aging should be controlled by variation in (1) mtDNA, (2) nuclear genes, or (3) nuclear–mtDNA interactions. The goal of this study was to assess the relative contributions of these factors in causing variation in Drosophila longevity. We compared strains of flies carrying mtDNAs with varying levels of divergence: two strains from Zimbabwe (<20 bp substitutions between mtDNAs), strains from Crete and the United States (20–40 bp substitutions between mtDNAs), and introgression strains of Drosophila melanogaster carrying mtDNA from Drosophila simulans in a D. melanogaster Oregon-R chromosomal background (>500 silent and 80 amino acid substitutions between these mtDNAs). Longevity was studied in reciprocal cross genotypes between pairs of these strains to test for cytoplasmic (mtDNA) factors affecting aging. The intrapopulation crosses between Zimbabwe strains show no difference in longevity between mtDNAs; the interpopulation crosses between Crete and the United States show subtle but significant differences in longevity; and the interspecific introgression lines showed very significant differences between mtDNAs. However, the genotypes carrying the D. simulans mtDNA were not consistently short-lived, as might be predicted from the disruption of nuclear–mitochondrial coadaptation. Rather, the interspecific mtDNA strains showed a wide range of variation that flanked the longevities seen between intraspecific mtDNAs, resulting in very significant nuclear x mtDNA epistatic interaction effects. These results suggest that even "defective" mtDNA haplotypes could extend longevity in different nuclear allelic backgrounds, which could account for the variable effects attributable to mtDNA haplogroups in human aging.
THE central role of mitochondria in metabolism makes mitochondrial genetics fundamentally important for understanding both the genetic basis and the evolution of aging. Mitochondrial function requires coordinated expression of hundreds of nuclear genes and a few dozen mitochondrial genes, many of which have been associated with either extended or shortened life span. Clearly, it is the alleles of genes and not just the genes themselves that extend or reduce longevity. The complex genetic architecture of mitochondrial function suggests that there should be an equally complex set of gene interactions (epistases) involving genetic variation in the nuclear and mitochondrial genomes that warrants further scrutiny in the context of the various mitochondrial theories of aging.
The mitochondrial free radical theory of aging states that physiological decline with age is a result of the cumulative effects of damage from oxygen radical production (HARMAN 1957; BECKMAN and AMES 1998). Mitochondria are the primary source of reactive oxygen species (ROS) within the cell. During electron transport in the inner mitochondrial membrane, enzyme complexes I and III generate the majority of ROS as byproducts of the coenzyme Q cycle (BALABAN et al. 2005). Mitochondria are also particularly sensitive targets for ROS damage during aging. The mtDNA molecule is not associated with histones and mtDNA has limited DNA repair mechanisms (BOGENHAGEN 1999). Consistent with these observations, mtDNA experiences greater damage from ROS than do nuclear proteins and DNA (RICHTER et al. 1988; WEI et al. 1998).
The basic principles of the mitochondrial theories of aging have gained wide support from biochemical, molecular genetic, and evolutionary studies (AGARWAL and SOHAL 1996; BECKMAN and AMES 1998; FINKEL and HOLBROOK 2000; BARJA 2004). An extensive literature has documented age-specific increases in damage to mitochondrial proteins, membranes, and mtDNA (SOHAL and WEINDRUCH 1996; WALLACE 1997; YAN et al. 1997), age-specific declines in the activity of mitochondrial enzyme complexes (SCHWARZE et al. 1998; FERGUSON et al. 2005), and the accumulation of mutant mtDNAs within cells (heteroplasmy) (CORTOPASSI et al. 1992; WALLACE 1995; WANG et al. 2001). But there is little direct experimental evidence that mtDNA mutations play a causal role in whole animal aging. The notable exception to this trend is the study by (TRIFUNOVIC et al. 2004) where knock-in mice with defective proofreading activity of mitochondrial polymerase lead to elevated mtDNA mutation rates and premature aging. Nevertheless, the distinction between the causes and consequences of mitochondrial decline in aging has been addressed by relatively few genetic experiments that manipulate mtDNA (DE GRAY 1999; LIGHTOWLERS et al. 1999; GOLDEN et al. 2002; JAMES and BALLARD 2003).
While mitochondria are the primary source of ROS, nuclear-encoded antioxidant proteins defend against this damage. The primary antioxidant proteins are the mitochondrial Mn-superoxide dismutase (SOD), and the cytoplasmic CuZn-SOD, catalase, and glutathione peroxidase. This redox balance plays an important role in both cellular signaling and aging, and is determined by the relative levels of oxidant production vs. the activities of antioxidant pathways (FINKEL and HOLBROOK 2000; BARJA 2004). Thus, the free radical theory of aging predicts that longevity should be extended by (1) increasing the level of ROS defenses, or (2) decreasing the levels of ROS production. Overexpressing SOD has been shown to extend life span of Drosophila (PARKES et al. 1998; SUN and TOWER 1999; SUN et al. 2002;), but this effect depends on genetic background (SUN and TOWER 1999; ORR et al. 2003; SPENCER et al. 2003). Overexpression of catalase in the mitochondria of mice also extends longevity, and shows significant variation between replicate overexpression constructs (SCHRINER et al. 2005). These studies support the biochemical argument that reducing ROS levels extends longevity, but reveal how sensitive these manipulations are to genetic background.
The extension of longevity by reducing ROS production is suggestive but remains indirect. Calorically restricted (CR) mice live longer and produce lower levels of ROS (SOHAL et al. 1994; MASORO 2000), but CR increases production of stress response proteins and ROS scavenging enzymes (SOHAL and WEINDRUCH 1996; WEINDRUCH et al. 2001). Flies selected for longevity show lower levels of hydrogen peroxide than control flies (ROSS 2000). But again, the long-lived strain also shows increased expression of antioxidant defenses (DUDAS and ARKING 1995; FORCE et al. 1995), making it difficult to distinguish cause and effect.
Several studies in Drosophila have failed to support predictions that appear logical from these observations, suggesting additional complexity to nuclear–mitochondrial signaling. If mitochondria are responsible for aging, it would follow that some change in metabolic rate or ROS levels in mitochondria should be detected with increasing age. However, metabolic rates do not change with age in most Drosophila species (PROMISLOW and HASELKORN 2002). ROS levels do not change with age in D. melanogaster, and neither CR nor overexpression of adenine nucleotide translocase (important in regulating ADP transport in mitochondria) decreases ROS production (MIWA et al. 2004). Metabolic rate is reduced neither by CR nor by reduced insulin signaling, both of which extend longevity (HULBERT et al. 2004). These observations do not falsify the mitochondrial theories of aging, but suggest that a diversity of pathways may be involved that influence the way nuclear–mitochondrial interactions modulate longevity.
Some likely candidate pathways are oxidative phosphorylation (OXPHOS), insulin signaling, apoptosis, and uncoupling. Down-regulation of a number of nuclear- and mtDNA-encoded genes involved in OXPHOS extended longevity in Caenorhabditis elegans (DILLIN et al. 2002). The insulin-signaling pathway is an evolutionary conserved modulator of longevity (TATAR et al. 2003), with a myriad of downstream metabolic effects that may intersect with mitochondrial function (LARSEN and CLARKE 2002; TATAR and RAND 2002). The role of apoptosis in aging warrants further dissection as it may be a sensitive response pathway to caloric restriction (COHEN et al. 2004) and oxidative stress (KUJOTH et al. 2005). Uncoupling proteins represent an additional class of nuclear-encoded proteins that may modulate mitochondrial aging by permitting the leakage of protons across the inner mitochondrial membrane and reducing ROS production (BRAND 2000; SPEAKMAN et al. 2004). Thus, mitonuclear cross talk has many pathways that can be modulated to reduce or extend longevity.
The molecular genetics of aging has been dissected using a variety of tools available in Drosophila such as transgenic and overexpression constructs (PARKES et al. 1998; SUN et al. 2002; ORR et al. 2005), and hypomorphic or knock-out alleles (LIN et al. 1998; ROGINA et al. 2000; CLANCY et al. 2001; TATAR et al. 2001). But selection for delayed aging has robust responses, so ample genetic variation exists for longevity (LUCKINBILL et al. 1984; ROSE 1984; SGRO et al. 2000). Since variation in rates of aging is inherently a population-level problem, the role of genetic polymorphisms in modulating aging is an important challenge. As the number of candidate genes for aging grows, the role of polymorphism at these genes becomes increasingly important to the genetics of aging, and needs to be incorporated into genetic experiments.
If we consider mtDNA as a candidate gene (or genome) for aging, the high levels of polymorphism in mtDNA provide a large pool of potential variants affecting aging. It is well established that mildly deleterious mtDNA polymorphisms are a general property of animal populations (NACHMAN 1998; RAND and KANN 1998; WEINREICH and RAND 2000). In humans, there is statistical evidence that alternative mtDNA haplotypes are associated with variation in longevity (IVANOVA et al. 1998; TANAKA et al. 1998; DE BENEDICTIS et al. 1999). However, some long-lived haplotypes carry mutations associated with disease suggesting that genetic background is an important modulator of mtDNA effects on aging (ROSE et al. 2001). Studies in Drosophila have established that nuclear–mitochondrial interactions (epistases) are important in the fitness effects of mtDNA variation (CLARK and LYCKEGAARD 1988; KILPATRICK and RAND 1995; RAND et al. 2001; JAMES and BALLARD 2003). While there has been an explosion of research on mitochondrial aspects of aging, there has been a general lack of studies that manipulate nuclear and mitochondrial genetic variation to explore nuclear–mitochondrial epistatic effects on longevity.
Here we present the results of such a study, using strains of Drosophila carrying mtDNAs on different nuclear backgrounds. We compare the longevities of wild-type strains of D. melanogaster carrying alternative mtDNAs from (1) within a Zimbabwe population, (2) between an Old World (Crete) and a New World (Rhode Island) strain, and (3) between mtDNA introgression strains carrying D. simulans mtDNA on D. melanogaster nuclear backgrounds. We test the predictions that variation in longevity is due to (1) mtDNA, (2) nuclear genetic background, and (3) an interaction between nuclear and mtDNA. A further prediction is that the effect of mtDNA on aging should increase in parallel with increasing levels of mtDNA divergence. While the results are consistent with this latter prediction, mtDNA haplotypes affect aging in a nuclear-background dependent manner. These mitonuclear epistases suggest that the main effects of mtDNA on aging could be masked, or even exaggerated, by significant interactions with ubiquitous nuclear allelic variation.
MATERIALS AND METHODS
Fly strains:
Two D. melanogaster strains from Zimbabwe (Zim 2 and Zim 53), one strain each from Crete and Rhode Island, and two mtDNA introgression strains with D. melanogaster Oregon-R chromosomes and D. simulans siII mtDNA were used for longevity analyses. The strain from Iraklion, Crete (Crete 10) was provided by E. Zouros, and the strain from Rhode Island (Four Town Farm 100, FTF 100) was collected by DMR. All strains had been in laboratory culture for >5 years prior to analyses. Some data on longevity for Zim 2, Zim 53, and FTF 100 have been reported previously (FRY and RAND 2002; FRY et al. 2004).
The interspecific mtDNA introgression strains were obtained from K. Sawamura (SAWAMURA et al. 2000). In a search for D. simulans lines (hereafter Dsim) that would rescue the sterility of hybrids from crosses with D. melanogaster (hereafter Dmel), a Dsim line was discovered (C167.4) that produced fertile F1 females in a Dsim female x Dmel male cross (DAVIS et al. 1996). Due to the maternal inheritance of mtDNA, these lines should have the Dsim mtDNA, and we confirmed this by DNA sequencing (see below). The Dmel male used in the initial cross was In(1)AB, while subsequent generations of backcrossing used Dmel Oregon-R males (DAVIS et al. 1996; SAWAMURA et al. 2000). The crossing scheme was as follows (K. SAWAMURA, personal communication): Dsim C167.4 females x Dmel In(1)AB, F1 females x Dmel Ore-R males (F1 cross), F2 female (single) x Dmel Ore-R males (F2 crosses). F2 females were numbered, and the line number represents these females: no. 12 and no. 33 were from the same F1 cross, no. 14 and no. 21 were from the same F1 cross, no. 22 and no. 38 were from the same F1 cross. In the F3 generation, sib-mating was done to establish isofemale lines. In the current study we used descendants of only two of these lines: no. 21 and no. 38. Two sib-matings and four isofemale lines were set up from no. 21 female (on April 11, 2000) and only one isofemale was fertile. Four sib-matings were set from no. 38 female (April 14, 2000) and all four were fertile (one of which was obtained from K. Sawamura). We refer to these lines as "sim-mel 21," and "sim-mel 38" (hereafter sm21 and sm38) to indicate that they carry Dsim mtDNA on a Dmel nuclear background (see below).
When we obtained the lines from K. Sawamura, they had been backcrossed for 4–5 additional generations to Dmel Ore-R males, followed by maintenance in vial cultures for 2 years. On the basis of the conclusions from SAWAMURA et al. (2000), this should select against Dsim nuclear genes linked to fertility factors. Before conducting our demography assays, we treated all lines with tetracycline to remove Wolbachia. PCR assays confirmed that this was successful (data not shown). In addition, we subjected all lines to three additional generations of single-pair backcrossing to Dmel Ore-R males. With free recombination and no selection, these lines should have >99.2% Ore-R nuclear alleles (assuming seven generations of backcrossing, not including the 2 years of culture which should select against Dsim alleles). When not undergoing single-pair backcrossing, all lines were maintained in vial culture at 50 pairs per generation prior to expansion of lines to collect sufficient adults for demography.
mtDNA haplotypes:
The mtDNA of the Zim 53 strain has been completely sequenced (BALLARD 2000). Nucleotide polymorphism surveys for 1500 bp of the NADH dehydrogenase subunit 5 (ND5) gene (RAND et al. 1994; RAND and KANN 1996) indicate that Zim 2 should differ from Zim 53 at <20 nucleotide sites across the coding sequence of the mtDNA (the ND5 data represent about 10% of the entire coding region of Drosophila mtDNA). The Crete 10 and FTF 100 differ by 4 bp in this ND5 region, implying 40 total base pair differences between the coding regions of these two mtDNAs (considerably more than between Zim 2 and Zim 53). Nucleotide polymorphism for pooled samples of Crete and FTF (or either sample and Zimbabwe) are >10-fold higher than polymorphism within either of the samples (A. FRY and D. M. RAND, unpublished results), confirming that between-population variation in Dmel mtDNA (e.g., Crete and FTF) is much greater than intrapopulation variation (between Zim 2 and Zim 53).
We sequenced >10 kb of the mtDNA for each of the sim-mel introgression strains (sm21 and sm38), and as expected they carry the Dsim siII mtDNA haplotype (SOLIGNAC et al. 1986; BALLARD 2000). The Dsim siII mtDNA differs from Dmel Ore-R mtDNA by 539 synonymous mutations and 86 amino acid mutations (BALLARD 2000). This should provide a strong set of mtDNA "alleles" with which to probe mitonuclear interactions in D. melanogaster.
Demography assays:
All longevity assays were done on F1 flies from reciprocal crosses between specific strains carrying the different mtDNAs. While backcrossing is commonly used to remove nuclear genetic variation, subtle allelic variation can be retained, and single male backcrossing leads to inbreeding. Moreover, the interspecific introgression lines could retain nuclear alleles of Dsim that were selected for in backcrosses due to coadaptation with the Dsim mtDNA. These potential nuclear allelic effects were controlled using reciprocal crosses. Reciprocal F1 females (e.g. sm21 female x Crete 10 male vs. Crete 10 female x sm21 male) will be identically heterozygous for any segregating alleles between the two parental strains, but will carry alternative mtDNAs, due to the maternal inheritance of mtDNA. Reciprocal F1 males will be identical for autosomes, but will differ in mtDNA, X, and Y chromosomes. We compared longevities of F1 flies from the following crosses (female x male), which generated different combinations of nuclear and mitochondrial genes (hereafter mitonuclear genotypes):
All crosses were performed at 25° on a 12:12 light:dark cycle using 25 pairs of parents for each cross. Parental strains had been acclimated under the same conditions at controlled density for at least one generation prior to crossing.
Longevity was measured by placing 100 males and 100 females (F1 offspring of a single genotype listed above) into each of three replicate demography cages. Approximately 8400 deaths were recorded in the study (14 genotypes x 3 cages x 2 sexes x 100 flies of each sex). The demography cages were made from one-quart plastic containers with a screened lid and a side coupling for attaching a food vial. The cages were kept in a walk-in incubator on a 12:12 light:dark photoperiod at 25° and 40% relative humidity. The food vials were replaced every other day and dead flies were removed with an aspirator, sexed, and counted. Survival was analyzed with a semi-parametric proportional hazards statistical model using day of death as the dependent variable, and cross (reciprocals above), mtDNA, and their interactions as predictors. Mortality analyses were done on pooled data across the three replicate demography cages for each genotype. Survival curves were compared with log-rank tests, and tested for significance using JMP statistical software.
Age-specific mortality (μx) was reported as μx = –ln(1 – qx) where qx is the proportion of individuals that die in age class x (number of individuals that die, divided by the number of individuals that enter, age class x). We estimated age-specific mortality models for various lines using maximum likelihood (PLETCHER and GEYER 1999; PLETCHER et al. 2000). The Gompertz (G) model describes an exponential increase in mortality rate with age and is given by μx = e–?x, where μ(x) is the mortality rate at age x, is the initial mortality rate or intercept, and ? is the slope or age-dependent increase in mortality. The Gompertz–Makeham (GM) model is the Gompertz plus a parameter, C, that describes age-independent mortality. The Logistic (L) model is the Gompertz plus a frailty parameter, s, to describe late-life mortality rate deceleration. The Makeham and Logistic models can be combined with the Gompertz to produce a four-parameter Logistic–Makeham (LM) model. These models are nested, so likelihood ratio tests can be used to compare model parameters between different lines by allowing parameters to vary, or constraining them to be the same. Significance is determined using twice the difference in likelihood scores, distributed as a chi-square with degrees of freedom equal to the difference in the number of parameters. All such analyses were done with the Winmodest program (PLETCHER et al. 2000).
RESULTS
Longevities of intrapopulation crosses:
The longevity patterns for strains from within the Zimbabwe population (Zim 2, Zim 53) are shown in Figure 1. There are very significant differences between the inbred parental strains (Zim 2, Zim 53), but the longevities of the reciprocal F1 genotypes are very similar. The survivorship curves reveal a clear effect of heterosis, as the two F1 genotypes are significantly longer lived than the two parental strains. Reciprocal F1 females are not significantly different from one another (P > 0.1, log-rank tests), while males are (P < 0.001; reciprocal cross males also differ in any X-linked or Y-linked polymorphisms). The small mtDNA difference between these two strains (<20 bp substitutions; see MATERIALS AND METHODS) suggests that mtDNA haplotype effects cannot override the clear heterosis effects that result from reciprocal crosses between these two strains, which may have accumulated deleterious recessive mutations during laboratory culture (PROMISLOW and TATAR 1998; MACK et al. 2000; YAMPOLSKY et al. 2000).
Longevities of interpopulation crosses:
Survivorship and mortality plots for the interpopulation comparison of the two D. melanogaster strains from Crete and Four Town Farm, Rhode Island (Crete 10 and FTF 100) are shown in the top panels of Figures 2 (females) and Figures 3 (males). Similar data for the reciprocal crosses of these two strains to each of the two sim-mel interspecies introgression strains (sm21 and sm38) are shown in the lower panels of Figures 2 and 3, and will be discussed below. These data are presented in the same figure to facilitate comparison among the interpopulation (Crete 10 and FTF 100) and interspecies crosses (sm21 or sm38 crossed to either Crete 10 or FTF 100). The descriptive statistics of survival and mortality for all strains are presented in Table 1.
Significance tests of survivorship curves were done using log-rank tests, while significance tests of different mortality parameters were done using likelihood ratio tests (see MATERIALS AND METHODS). These tests can have different outcomes that reflect basic differences in survival vs. age-specific mortality. Survivorship curves display cumulative data so differences in survivorship between genotypes in one age interval are carried forward to other ages, which can inflate the effects of log-rank tests. Mortality parameters are a more accurate description of true aging effects since they focus on differences in the slope, intercept, or linearity of the mortality rate between genotypes. The data for all genotypes were contrasted using four mortality models [G, GM, L, and LM (see MATERIALS AND METHODS)]. Comparisons of alternative mortality models using the Winmodest package (PLETCHER et al. 2000) established that the four-parameter LM model was preferred over others for all genotypes presented in Table 1. We then compared each genotype to its reciprocal genotype to determine if any of the four parameters of the LM model differed between flies carrying alternative mtDNAs. This was done by determining the likelihood of a model that allowed each genotype to have a unique set of parameters (eight independent parameters; see Table 1, "unique" column) vs. a model that constrained one of the parameters to be the same for the two genotypes and allowed the remaining six parameters to vary (Table 1, "fixed ," "fixed ?," "fixed s," and "fixed C" columns).
Reciprocal F1 genotypes from crosses between two wild strains of Dmel, Crete 10, and FTF 100 show subtle but significant differences (Figures 2, A and B and 3, A and B). Log-rank tests show a significant difference between these two genotypes (P < 0.001), but likelihood ratio tests of mortality parameters do not show any significant differences. For Crete 10 and FTF 100 females, these likelihoods could not be distinguished (Table 1). In males there is a marginally significant difference in survivorship using a log-rank test (P < 0.02), and additional differences in mortality parameters (P < 0.01), neither of which is significant after correcting for multiple tests (see Table 1). In addition, it is not certain that this reciprocal cross effect in males can be attributed to mtDNA vs. X, or Y-chromosomal factors.
Comparisons between Dmel and Dsim mtDNAs:
Genotypes carrying either a Dmel or a Dsim mtDNA are shown in Figure 2, C–J (females), and Figure 3, C–J (males), representing reciprocal crosses between either Crete 10 or FTF 100, and the two "sim-mel" mtDNA introgression lines, sm21 and sm38. In general, the differences in longevity among flies carrying Dmel vs. Dsim mtDNA are much more pronounced than those carrying alternative Dmel mtDNAs (compare survivorship curves in Figure 2A to those in Figure 2, C, E, G, and I). However, the data clearly show that genotypes carrying the Dsim mtDNA are not consistently shorter lived than those carrying the Dmel mtDNA, as might be expected if Dsim mtDNA disrupts coadapted nuclear–mtDNA interactions in a Dmel nuclear background. Reciprocal crosses involving the "disrupted" mitonuclear sim-mel strains sm21 and sm38 are either shorter or longer lived than the reciprocal crosses between the two "native" mitonuclear Dmel strains FTF 100 and Crete 10 (see Figure 4). When the "sim-mel" introgression strains are crossed to the Dmel strain Crete 10, the sm38 (Dsim) mtDNA shows extended longevity, and sm21 mtDNA shows reduced longevity, relative to the Crete 10 (Dmel) mtDNA. The reverse is true when Dmel FTF 100 is used: sm21 mtDNA lives longer, and the sm38 mtDNA shows reduced longevity, relative to the FTF 100 mtDNA. The direction of these effects was the same in both sexes, although the magnitude of the effects was generally reduced in males (compare Figures 2 and 3; summarized in Figures 4 and 5).
Among the four pairs of reciprocal crosses involving Crete 10, FTF 100, sm38, and sm21, the effect of mtDNA haplotype on longevity is entirely epistatic, i.e., it depends on the allelic constitution of the F1 heterozygous nuclear background (Figure 5). This was quantified in a two-way ANOVA with nuclear background (reciprocal crosses) and mtDNA (Dmel vs. Dsim) as main effects, plus the interaction term. The nuclear x mtDNA interaction terms of these ANOVAs are all highly significant (see Table 2). Table 2 presents three different two-way ANOVAs: one involving the two pairs of reciprocal crosses of Crete to sm21 or to sm38, a second ANOVA involving the two pairs of reciprocal crosses of FTF to sm21 or sm38, and a third involving all four pairs of reciprocal crosses involving Crete, FTF, sm21, and sm38. Data from the three replicate demography cages were pooled for each genotype and a Cox proportional hazard model was used with day of death as the dependent variable and nuclear background and mtDNA plus interaction as the predictors. This treats the two reciprocal crosses with Dsim mtDNA as replicates (e.g., sm21 female x Crete male and sm38 female x Crete male). Thus, there is one degree of freedom for each factor in Crete or FTF ANOVAs, and three degrees of freedom for the nuclear reciprocal factors with all four reciprocals. The three replicate demography cages were generally in close agreement; ANOVA keeping them separate produced the same highly significant nuclear x mtDNA interaction terms (data not shown). To confirm the results of the ANOVAs, the analyses were done ignoring (1) the first 10 days, (2) the last 10 days, and (3) both the first and last 10 days of death events, and the results were qualitatively identical to the data reported in Table 2.
In females, the ANOVA shows strong variation among nuclear genotypes and in the nuclear x mtDNA interaction term, but no main effect of mtDNA. In males, the design violates a basic assumption of ANOVA since reciprocal crosses cannot really be considered replicated effects, so the results are presented for comparison only. Note that in males there are significant main effects of mtDNA, but this incorporates X- and Y-chromosome variation as well. As is evident in the figures, there is more nuclear and nuclear x mtDNA variation among genotypes in females than in males (female r2 = 13.1%, male r2 = 6.6%), suggesting that X- or Y-linked factors are reducing apparent effects of cytoplasmic term even if they are confounding it.
Demographic patterns:
Some additional comments are warranted about differences in mortality patterns among genotypes. Table 1 shows descriptive statistics for each of the reciprocal crosses involving the sim-mel introgression strains, with estimates of the LM terms that characterize mortality. The right side of the table presents the likelihood ratio tests of mortality parameters between reciprocal genotypes that quantify the differences shown in the right-hand panels of Figures 2 and 3 (mortality plots). In clear contrast to the results from the Crete x FTF cross (Dmel mtDNA only), the crosses with Dsim mtDNA strains show many significant differences in mortality parameters. The intercept (), slope (?), and late-life mortality deceleration terms (s) are all different for crosses to Crete 10. The effect of the latter term (s) can be seen in the Crete 10 crosses where a reduction of mortality occurs in mid to late life (Figure 2, C–F). It is also clear that crosses to FTF show fewer significant effects. This may be due to the fact that the sim-mel strains have largely Ore-R nuclear chromosomes, and these should be less differentiated from other North American strains such as FTF. Females show more significant differences than males.
Likelihood ratio tests between other pairs of genotypes further support the notion of complex nuclear–mtDNA interactions. For example, contrasts between the reciprocal crosses with sm38 females show no significant differences in , ?, or s (sm38 x Crete vs. sm38 x FTF), but contrasts between the reciprocal crosses with sm21 females show highly significant differences in , ?, and s (sm21 x Crete vs. sm21 XFTF; data available on request). Similarly, reciprocal crosses where FTF provided the mtDNA are significantly different for and C, but reciprocal crosses where Crete provided mtDNA are highly significantly different for all four parameters , ?, s, and C. Thus, the same Dsim mtDNA haplotype can have very different mortality patterns when crossed to alternative Dmel wild lines, and the same wild Dmel mtDNA has different mortality patterns when crossed to different sim-mel mtDNA introgression strains. These analyses show that nuclear–mitochondrial epistatic interactions can modulate age-specific survival in a variety of ways.
DISCUSSION
More than 90% of the functional mitochondrial genome is encoded in the nucleus. These nuclear-encoded mitochondrial genes (mitonuclear genes) arise either by transfer events from the mitochondrial to the nuclear genome or by recruitment of nuclear genes to a novel mitochondrial function through the acquisition of mitochondrial targeting sequences (RAND et al. 2004). This genetic architecture suggests that the mitochondrial phenotypes should be governed by (1) mitochondrial genes (mtDNA), (2) mitonuclear genes, or (3) genetic interactions between mtDNA and mitonuclear genes. Moreover, coadaptation among mitochondrial and mitonuclear genes predicts that the phenotypic effects of alternative mtDNAs should increase with increasing levels of DNA sequence divergence between native and foreign mtDNAs (e.g., mtDNA from different populations or species; RAND et al. 2004). The current study seeks to test these alternatives in the context of Drosophila aging by studying longevity among reciprocal crosses of strains carrying alternative mtDNAs.
Our results are qualitatively consistent with the coadaptation hypothesis: the inferred effects of alternative mtDNAs on Drosophila longevity increase in parallel with the degree of DNA sequence divergence. Differences in longevity were weak or absent between reciprocal F1 flies carrying alternative mtDNAs from within a Zimbabwe population, a subtle but significant difference was observed between an Old World and New World strain, and highly significant differences were observed among genotypes carrying mtDNAs from either D. melanogaster (Dmel) or D. simulans (Dsim). However, the results are not consistent with the hypothesis that flies carrying foreign mtDNA from a different species are generally disrupted and show reduced longevity. Our results clearly show that the effects of the foreign Dsim mtDNA on longevity depend strongly on the nuclear genetic background, i.e., there is nuclear–mtDNA epistasis for longevity.
Nuclear–mitochondrial epistasis and longevity:
Why are nuclear–mitochondrial epistatic effects stronger than mtDNA main effects on Drosophila longevity? Presumably main effects of mtDNA would be seen if a highly divergent mtDNA (say, from D. pseudoobscura) were carried on a Dmel nuclear background. One possibility is that the roughly 500 silent and 80 amino acid changes that exist between the Dsim and Dmel mtDNAs used in this study represent neutral or nearly neutral substitutions of nucleotide positions that are free to vary, rendering the two haplotypes nearly neutral variants with respect to longevity. Several lines of evidence argue against this. First, deleterious evolution of mtDNA is well established (NACHMAN 1998; RAND and KANN 1998), and non-neutral patterns of evolution have been documented for divergence of Dmel and Dsim mtDNAs (BALLARD and KREITMAN 1994; RAND et al. 1994; BALLARD 2000). Second, population cage studies with these mtDNAs indicate that Dsim mtDNA is indeed selected against relative to Dmel mtDNA (D. RAND, unpublished results). Moreover, less-diverged mtDNA haplotypes within D. simulans show repeatable differences in fitness effects of mtDNA (JAMES and BALLARD 2003; BALLARD and JAMES 2004). While these arguments do not establish that Dmel and Dsim mtDNA are non-neutral for longevity, it seems plausible that if mtDNA effects are detectable at an early age, there should be some additive mtDNA effects for these haplotypes at a later age when selection is less likely to have influenced their evolution.
A second possible explanation for the inconsistent main effects of mtDNA on longevity could stem from genomic imprinting of nuclear chromosomes. To infer an mtDNA effect on longevity, we compared reciprocal F1 genotypes carrying alternative mtDNAs. Reciprocal F1 females will be identically heterozygous and carry alternative mtDNAs, but could also differ in parent-of-origin effects on the nuclear alleles transmitted into the F1 flies studied. While imprinting has been reported for the Drosophila Y chromosome (MAGGERT and GOLIC 2002), this should not affect females. Notably, our results show that the differences in longevity among reciprocal F1 males was less than that for sibling females, so if imprinting was active in our lines it appears to have reduced, not increased, reciprocal cross effects (i.e., inferred mtDNA effects).
A third possible source of the strong epistatic effects is the retention of Dsim nuclear alleles in the mtDNA introgression lines carrying Dsim mtDNA. Introgression studies for a region of the second chromosome reveal that fertility and viability factors accounting for the isolation between Dmel and Dsim are densely distributed (SAWAMURA et al. 2000). During the initial generation of backcrossing between the Dsim C167.4 and Dmel In(1)AB, there may have been Dsim segments of the nuclear chromosomes that escaped the purging effects of hybrid unfitness in the increasingly Dmel nuclear background. Perhaps the presence of a Dsim mtDNA preferentially selected for individual Dsim alleles that are retained in some, but not all, of our sim-mel introgression strains (e.g., in sm21 but not sm38). While spurious selection effects during the backcrossing may also have contributed to the results, the strong reciprocal cross effects in females would require that the selection altered dominance patterns of certain nuclear alleles for a strictly nuclear effect to explain our results. The alternative mtDNAs carried in reciprocal cross females seems like a more parsimonious explanation.
Sequence analyses of 70 nuclear encoded subunits of mitochondrial OXPHOS subunits in all six of our sim-mel introgressions strains show them to have the Dmel Oregon-R allele (as expected from Oregon-R male backcrossing). However, there are hundreds of known mitonuclear genes that remain to be sequenced for these strains, plus many genes of unknown mitochondrial function that could retain putative Dsim alleles that alter longevity. Such loci could have epistatic interactions with the FTF 100 or Crete 10 alleles used in our hybrid crosses. Alternatively, the epistatic effects of these putative nuclear x nuclear interactions could be sensitized by the presence of the alternative mtDNAs in their respective F1 genotypes (ZEYL et al. 2005; WADE and GOODNIGHT 2006).
A further source of the nuclear–mtDNA epistatic effects we observed may stem from alterations of gene expression that result from the crossing of wild strains. Transcript profiling of reciprocal F1 genotypes from crosses between strains of D. melanogaster revealed that up to 33% of genes had significantly different expression patterns from the parental strains, and most of these effects were non-additive (GIBSON et al. 2004). This misregulation effect is also pronounced in interspecific hybrids (RANZ et al. 2004), with some genes showing expression outside the range of either parent.
If the two introgression strains (sm21, sm38) have retained distinct Dsim alleles during the backcrossing process, this could add further variation to the patterns of mis-regulated genes in reciprocal crosses of these strains to the two wild Dmel strains (Crete 10 and FTF 100). For this to account for our longevity data, these misregulation effects would have to be altered by the mtDNA haplotype present in the reciprocal F1 flies. Indeed, for this to occur in reciprocal F1 females, it suggests that mtDNA haplotype might alter the dominance of alternative alleles present in these hybrids. Mitochondrial-to-nuclear (retrograde) signaling modulates gene expression (BUTOW and AVADHANI 2004) and affects longevity in yeast (KIRCHMAN et al. 1999). In this context mitochondrial genotypes should be considered as novel "environments" for alleles and genotypes of nuclear loci. Since both nuclear x nuclear epistatic interactions and genotype x environment interactions affect Drosophila longevity (LEIPS and MACKAY 2000; VIEIRA et al. 2000), the effects of alternative mtDNA haplotype on the misregulation of genes in hybrid crosses is a potentially important component of variation in longevity among individuals in outbred populations, and warrants further study.
Nuclear–mitochondrial coadaptation:
The coadaptation hypothesis predicts that disrupted mitonuclear genotypes (e.g., Dmel nuclear chromosomes with Dsim mtDNA) should have reduced performance, and there is compelling evidence for this in a variety of systems (EDMANDS and BURTON 1999; RAWSON and BURTON 2002; MCKENZIE et al. 2003; SACKTON et al. 2003). It might follow that such genotypes would have reduced longevity due to disrupted OXPHOS functions, possibly resulting in elevated ROS production. However, the relationship between metabolic rates, electron transport and ROS production are not at all clear (SPEAKMAN et al. 2004).
In most species of Drosophila, metabolic rates do not change with age (PROMISLOW and HASELKORN 2002). There is no correlation between age and ROS production, and over-expression of the adenine nucleotide translocase (ANT) decreases ROS levels but shortens longevity (MIWA et al. 2004). Moreover, caloric restriction and reduced insulin signaling have no effect on metabolic rates or ROS production. It remains possible that the disruption of mitonuclear coadaptation could generate cellular states or gene expression patterns that extend longevity. An RNAi screen in C. elegans showed that extended longevity was associated with down-regulation of a number of mitochondrial genes, both nuclear and mtDNA encoded (DILLIN et al. 2002). In mice, extended longevity was associated with higher metabolism due to higher levels of uncoupling (SPEAKMAN et al. 2004).
Thus, it may be simplistic to assume that different sets of heterozygous flies carrying the same divergent mtDNA should show reduced longevity simply because an interspecific coadapted gene complex is disrupted. These genotypes may vary in the levels of expression of OXPHOS or uncoupling proteins that result in reduced or extended longevity in an mtDNA-dependent manner. For example, one pair of reciprocal crosses (sm38 x Crete 10) showed the greatest longevity extension, while a different pair showed the most reduced longevity (FTF 100 x sm21). These kinds of reciprocal crosses among mtDNA introgression lines provide excellent material for dissecting the mechanistic bases of altered longevity as they generate strong genetic effects that should have biochemical and cellular explanations.
Our results demonstrate that even with relatively divergent mtDNA alleles (mtDNA differing at over 700 point mutations including >80 amino acid altering sites), epistatic interactions with nuclear genetic background are a significant component of the mitochondrial genetics of aging. This kind of epistasis could explain why some mtDNA mutations can have very different phenotypic effects in different individuals, possibly obscuring the mtDNA effects in human aging and disease (JACOBS and HOLT 2000; ROSE et al. 2001). Studies that seek to identify mitochondrial genotype effects in aging should not seek to remove nuclear genetic variation, but should embrace it by replicating mitochondrial genotypes across several different nuclear genetic backgrounds. While the human species has relatively low nuclear genetic variation relative to Drosophila, two average people differ by 3 million nuclear base pairs, and 50 mtDNA point mutations. The significance of this vast array of possible mitonuclear genotypes should be considered in the context of the unpredictable nature of mitonuclear epistasis.
ACKNOWLEDGEMENTS
Larry Harshman, Jeff Hofmann, Chester McFly, Colin Meiklejohn, Kristi Montooth, Marc Tatar and Rebecca Wagaman and an anonymous reviewer provided helpful comments. The authors were supported by grants from the National Institutes of Health (GM-67862, AG-16632) and the National Science Foundation (DEB 0343464, DEB 9981497).
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Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Island 02912(David M. Rand, Adam Fry a)
ABSTRACT
Under the mitochondrial theory of aging, physiological decline with age results from the accumulated cellular damage produced by reactive oxygen species generated during electron transport in the mitochondrion. A large body of literature has documented age-specific declines in mitochondrial function that are consistent with this theory, but relatively few studies have been able to distinguish cause from consequence in the association between mitochondrial function and aging. Since mitochondrial function is jointly encoded by mitochondrial (mtDNA) and nuclear genes, the mitochondrial genetics of aging should be controlled by variation in (1) mtDNA, (2) nuclear genes, or (3) nuclear–mtDNA interactions. The goal of this study was to assess the relative contributions of these factors in causing variation in Drosophila longevity. We compared strains of flies carrying mtDNAs with varying levels of divergence: two strains from Zimbabwe (<20 bp substitutions between mtDNAs), strains from Crete and the United States (20–40 bp substitutions between mtDNAs), and introgression strains of Drosophila melanogaster carrying mtDNA from Drosophila simulans in a D. melanogaster Oregon-R chromosomal background (>500 silent and 80 amino acid substitutions between these mtDNAs). Longevity was studied in reciprocal cross genotypes between pairs of these strains to test for cytoplasmic (mtDNA) factors affecting aging. The intrapopulation crosses between Zimbabwe strains show no difference in longevity between mtDNAs; the interpopulation crosses between Crete and the United States show subtle but significant differences in longevity; and the interspecific introgression lines showed very significant differences between mtDNAs. However, the genotypes carrying the D. simulans mtDNA were not consistently short-lived, as might be predicted from the disruption of nuclear–mitochondrial coadaptation. Rather, the interspecific mtDNA strains showed a wide range of variation that flanked the longevities seen between intraspecific mtDNAs, resulting in very significant nuclear x mtDNA epistatic interaction effects. These results suggest that even "defective" mtDNA haplotypes could extend longevity in different nuclear allelic backgrounds, which could account for the variable effects attributable to mtDNA haplogroups in human aging.
THE central role of mitochondria in metabolism makes mitochondrial genetics fundamentally important for understanding both the genetic basis and the evolution of aging. Mitochondrial function requires coordinated expression of hundreds of nuclear genes and a few dozen mitochondrial genes, many of which have been associated with either extended or shortened life span. Clearly, it is the alleles of genes and not just the genes themselves that extend or reduce longevity. The complex genetic architecture of mitochondrial function suggests that there should be an equally complex set of gene interactions (epistases) involving genetic variation in the nuclear and mitochondrial genomes that warrants further scrutiny in the context of the various mitochondrial theories of aging.
The mitochondrial free radical theory of aging states that physiological decline with age is a result of the cumulative effects of damage from oxygen radical production (HARMAN 1957; BECKMAN and AMES 1998). Mitochondria are the primary source of reactive oxygen species (ROS) within the cell. During electron transport in the inner mitochondrial membrane, enzyme complexes I and III generate the majority of ROS as byproducts of the coenzyme Q cycle (BALABAN et al. 2005). Mitochondria are also particularly sensitive targets for ROS damage during aging. The mtDNA molecule is not associated with histones and mtDNA has limited DNA repair mechanisms (BOGENHAGEN 1999). Consistent with these observations, mtDNA experiences greater damage from ROS than do nuclear proteins and DNA (RICHTER et al. 1988; WEI et al. 1998).
The basic principles of the mitochondrial theories of aging have gained wide support from biochemical, molecular genetic, and evolutionary studies (AGARWAL and SOHAL 1996; BECKMAN and AMES 1998; FINKEL and HOLBROOK 2000; BARJA 2004). An extensive literature has documented age-specific increases in damage to mitochondrial proteins, membranes, and mtDNA (SOHAL and WEINDRUCH 1996; WALLACE 1997; YAN et al. 1997), age-specific declines in the activity of mitochondrial enzyme complexes (SCHWARZE et al. 1998; FERGUSON et al. 2005), and the accumulation of mutant mtDNAs within cells (heteroplasmy) (CORTOPASSI et al. 1992; WALLACE 1995; WANG et al. 2001). But there is little direct experimental evidence that mtDNA mutations play a causal role in whole animal aging. The notable exception to this trend is the study by (TRIFUNOVIC et al. 2004) where knock-in mice with defective proofreading activity of mitochondrial polymerase lead to elevated mtDNA mutation rates and premature aging. Nevertheless, the distinction between the causes and consequences of mitochondrial decline in aging has been addressed by relatively few genetic experiments that manipulate mtDNA (DE GRAY 1999; LIGHTOWLERS et al. 1999; GOLDEN et al. 2002; JAMES and BALLARD 2003).
While mitochondria are the primary source of ROS, nuclear-encoded antioxidant proteins defend against this damage. The primary antioxidant proteins are the mitochondrial Mn-superoxide dismutase (SOD), and the cytoplasmic CuZn-SOD, catalase, and glutathione peroxidase. This redox balance plays an important role in both cellular signaling and aging, and is determined by the relative levels of oxidant production vs. the activities of antioxidant pathways (FINKEL and HOLBROOK 2000; BARJA 2004). Thus, the free radical theory of aging predicts that longevity should be extended by (1) increasing the level of ROS defenses, or (2) decreasing the levels of ROS production. Overexpressing SOD has been shown to extend life span of Drosophila (PARKES et al. 1998; SUN and TOWER 1999; SUN et al. 2002;), but this effect depends on genetic background (SUN and TOWER 1999; ORR et al. 2003; SPENCER et al. 2003). Overexpression of catalase in the mitochondria of mice also extends longevity, and shows significant variation between replicate overexpression constructs (SCHRINER et al. 2005). These studies support the biochemical argument that reducing ROS levels extends longevity, but reveal how sensitive these manipulations are to genetic background.
The extension of longevity by reducing ROS production is suggestive but remains indirect. Calorically restricted (CR) mice live longer and produce lower levels of ROS (SOHAL et al. 1994; MASORO 2000), but CR increases production of stress response proteins and ROS scavenging enzymes (SOHAL and WEINDRUCH 1996; WEINDRUCH et al. 2001). Flies selected for longevity show lower levels of hydrogen peroxide than control flies (ROSS 2000). But again, the long-lived strain also shows increased expression of antioxidant defenses (DUDAS and ARKING 1995; FORCE et al. 1995), making it difficult to distinguish cause and effect.
Several studies in Drosophila have failed to support predictions that appear logical from these observations, suggesting additional complexity to nuclear–mitochondrial signaling. If mitochondria are responsible for aging, it would follow that some change in metabolic rate or ROS levels in mitochondria should be detected with increasing age. However, metabolic rates do not change with age in most Drosophila species (PROMISLOW and HASELKORN 2002). ROS levels do not change with age in D. melanogaster, and neither CR nor overexpression of adenine nucleotide translocase (important in regulating ADP transport in mitochondria) decreases ROS production (MIWA et al. 2004). Metabolic rate is reduced neither by CR nor by reduced insulin signaling, both of which extend longevity (HULBERT et al. 2004). These observations do not falsify the mitochondrial theories of aging, but suggest that a diversity of pathways may be involved that influence the way nuclear–mitochondrial interactions modulate longevity.
Some likely candidate pathways are oxidative phosphorylation (OXPHOS), insulin signaling, apoptosis, and uncoupling. Down-regulation of a number of nuclear- and mtDNA-encoded genes involved in OXPHOS extended longevity in Caenorhabditis elegans (DILLIN et al. 2002). The insulin-signaling pathway is an evolutionary conserved modulator of longevity (TATAR et al. 2003), with a myriad of downstream metabolic effects that may intersect with mitochondrial function (LARSEN and CLARKE 2002; TATAR and RAND 2002). The role of apoptosis in aging warrants further dissection as it may be a sensitive response pathway to caloric restriction (COHEN et al. 2004) and oxidative stress (KUJOTH et al. 2005). Uncoupling proteins represent an additional class of nuclear-encoded proteins that may modulate mitochondrial aging by permitting the leakage of protons across the inner mitochondrial membrane and reducing ROS production (BRAND 2000; SPEAKMAN et al. 2004). Thus, mitonuclear cross talk has many pathways that can be modulated to reduce or extend longevity.
The molecular genetics of aging has been dissected using a variety of tools available in Drosophila such as transgenic and overexpression constructs (PARKES et al. 1998; SUN et al. 2002; ORR et al. 2005), and hypomorphic or knock-out alleles (LIN et al. 1998; ROGINA et al. 2000; CLANCY et al. 2001; TATAR et al. 2001). But selection for delayed aging has robust responses, so ample genetic variation exists for longevity (LUCKINBILL et al. 1984; ROSE 1984; SGRO et al. 2000). Since variation in rates of aging is inherently a population-level problem, the role of genetic polymorphisms in modulating aging is an important challenge. As the number of candidate genes for aging grows, the role of polymorphism at these genes becomes increasingly important to the genetics of aging, and needs to be incorporated into genetic experiments.
If we consider mtDNA as a candidate gene (or genome) for aging, the high levels of polymorphism in mtDNA provide a large pool of potential variants affecting aging. It is well established that mildly deleterious mtDNA polymorphisms are a general property of animal populations (NACHMAN 1998; RAND and KANN 1998; WEINREICH and RAND 2000). In humans, there is statistical evidence that alternative mtDNA haplotypes are associated with variation in longevity (IVANOVA et al. 1998; TANAKA et al. 1998; DE BENEDICTIS et al. 1999). However, some long-lived haplotypes carry mutations associated with disease suggesting that genetic background is an important modulator of mtDNA effects on aging (ROSE et al. 2001). Studies in Drosophila have established that nuclear–mitochondrial interactions (epistases) are important in the fitness effects of mtDNA variation (CLARK and LYCKEGAARD 1988; KILPATRICK and RAND 1995; RAND et al. 2001; JAMES and BALLARD 2003). While there has been an explosion of research on mitochondrial aspects of aging, there has been a general lack of studies that manipulate nuclear and mitochondrial genetic variation to explore nuclear–mitochondrial epistatic effects on longevity.
Here we present the results of such a study, using strains of Drosophila carrying mtDNAs on different nuclear backgrounds. We compare the longevities of wild-type strains of D. melanogaster carrying alternative mtDNAs from (1) within a Zimbabwe population, (2) between an Old World (Crete) and a New World (Rhode Island) strain, and (3) between mtDNA introgression strains carrying D. simulans mtDNA on D. melanogaster nuclear backgrounds. We test the predictions that variation in longevity is due to (1) mtDNA, (2) nuclear genetic background, and (3) an interaction between nuclear and mtDNA. A further prediction is that the effect of mtDNA on aging should increase in parallel with increasing levels of mtDNA divergence. While the results are consistent with this latter prediction, mtDNA haplotypes affect aging in a nuclear-background dependent manner. These mitonuclear epistases suggest that the main effects of mtDNA on aging could be masked, or even exaggerated, by significant interactions with ubiquitous nuclear allelic variation.
MATERIALS AND METHODS
Fly strains:
Two D. melanogaster strains from Zimbabwe (Zim 2 and Zim 53), one strain each from Crete and Rhode Island, and two mtDNA introgression strains with D. melanogaster Oregon-R chromosomes and D. simulans siII mtDNA were used for longevity analyses. The strain from Iraklion, Crete (Crete 10) was provided by E. Zouros, and the strain from Rhode Island (Four Town Farm 100, FTF 100) was collected by DMR. All strains had been in laboratory culture for >5 years prior to analyses. Some data on longevity for Zim 2, Zim 53, and FTF 100 have been reported previously (FRY and RAND 2002; FRY et al. 2004).
The interspecific mtDNA introgression strains were obtained from K. Sawamura (SAWAMURA et al. 2000). In a search for D. simulans lines (hereafter Dsim) that would rescue the sterility of hybrids from crosses with D. melanogaster (hereafter Dmel), a Dsim line was discovered (C167.4) that produced fertile F1 females in a Dsim female x Dmel male cross (DAVIS et al. 1996). Due to the maternal inheritance of mtDNA, these lines should have the Dsim mtDNA, and we confirmed this by DNA sequencing (see below). The Dmel male used in the initial cross was In(1)AB, while subsequent generations of backcrossing used Dmel Oregon-R males (DAVIS et al. 1996; SAWAMURA et al. 2000). The crossing scheme was as follows (K. SAWAMURA, personal communication): Dsim C167.4 females x Dmel In(1)AB, F1 females x Dmel Ore-R males (F1 cross), F2 female (single) x Dmel Ore-R males (F2 crosses). F2 females were numbered, and the line number represents these females: no. 12 and no. 33 were from the same F1 cross, no. 14 and no. 21 were from the same F1 cross, no. 22 and no. 38 were from the same F1 cross. In the F3 generation, sib-mating was done to establish isofemale lines. In the current study we used descendants of only two of these lines: no. 21 and no. 38. Two sib-matings and four isofemale lines were set up from no. 21 female (on April 11, 2000) and only one isofemale was fertile. Four sib-matings were set from no. 38 female (April 14, 2000) and all four were fertile (one of which was obtained from K. Sawamura). We refer to these lines as "sim-mel 21," and "sim-mel 38" (hereafter sm21 and sm38) to indicate that they carry Dsim mtDNA on a Dmel nuclear background (see below).
When we obtained the lines from K. Sawamura, they had been backcrossed for 4–5 additional generations to Dmel Ore-R males, followed by maintenance in vial cultures for 2 years. On the basis of the conclusions from SAWAMURA et al. (2000), this should select against Dsim nuclear genes linked to fertility factors. Before conducting our demography assays, we treated all lines with tetracycline to remove Wolbachia. PCR assays confirmed that this was successful (data not shown). In addition, we subjected all lines to three additional generations of single-pair backcrossing to Dmel Ore-R males. With free recombination and no selection, these lines should have >99.2% Ore-R nuclear alleles (assuming seven generations of backcrossing, not including the 2 years of culture which should select against Dsim alleles). When not undergoing single-pair backcrossing, all lines were maintained in vial culture at 50 pairs per generation prior to expansion of lines to collect sufficient adults for demography.
mtDNA haplotypes:
The mtDNA of the Zim 53 strain has been completely sequenced (BALLARD 2000). Nucleotide polymorphism surveys for 1500 bp of the NADH dehydrogenase subunit 5 (ND5) gene (RAND et al. 1994; RAND and KANN 1996) indicate that Zim 2 should differ from Zim 53 at <20 nucleotide sites across the coding sequence of the mtDNA (the ND5 data represent about 10% of the entire coding region of Drosophila mtDNA). The Crete 10 and FTF 100 differ by 4 bp in this ND5 region, implying 40 total base pair differences between the coding regions of these two mtDNAs (considerably more than between Zim 2 and Zim 53). Nucleotide polymorphism for pooled samples of Crete and FTF (or either sample and Zimbabwe) are >10-fold higher than polymorphism within either of the samples (A. FRY and D. M. RAND, unpublished results), confirming that between-population variation in Dmel mtDNA (e.g., Crete and FTF) is much greater than intrapopulation variation (between Zim 2 and Zim 53).
We sequenced >10 kb of the mtDNA for each of the sim-mel introgression strains (sm21 and sm38), and as expected they carry the Dsim siII mtDNA haplotype (SOLIGNAC et al. 1986; BALLARD 2000). The Dsim siII mtDNA differs from Dmel Ore-R mtDNA by 539 synonymous mutations and 86 amino acid mutations (BALLARD 2000). This should provide a strong set of mtDNA "alleles" with which to probe mitonuclear interactions in D. melanogaster.
Demography assays:
All longevity assays were done on F1 flies from reciprocal crosses between specific strains carrying the different mtDNAs. While backcrossing is commonly used to remove nuclear genetic variation, subtle allelic variation can be retained, and single male backcrossing leads to inbreeding. Moreover, the interspecific introgression lines could retain nuclear alleles of Dsim that were selected for in backcrosses due to coadaptation with the Dsim mtDNA. These potential nuclear allelic effects were controlled using reciprocal crosses. Reciprocal F1 females (e.g. sm21 female x Crete 10 male vs. Crete 10 female x sm21 male) will be identically heterozygous for any segregating alleles between the two parental strains, but will carry alternative mtDNAs, due to the maternal inheritance of mtDNA. Reciprocal F1 males will be identical for autosomes, but will differ in mtDNA, X, and Y chromosomes. We compared longevities of F1 flies from the following crosses (female x male), which generated different combinations of nuclear and mitochondrial genes (hereafter mitonuclear genotypes):
All crosses were performed at 25° on a 12:12 light:dark cycle using 25 pairs of parents for each cross. Parental strains had been acclimated under the same conditions at controlled density for at least one generation prior to crossing.
Longevity was measured by placing 100 males and 100 females (F1 offspring of a single genotype listed above) into each of three replicate demography cages. Approximately 8400 deaths were recorded in the study (14 genotypes x 3 cages x 2 sexes x 100 flies of each sex). The demography cages were made from one-quart plastic containers with a screened lid and a side coupling for attaching a food vial. The cages were kept in a walk-in incubator on a 12:12 light:dark photoperiod at 25° and 40% relative humidity. The food vials were replaced every other day and dead flies were removed with an aspirator, sexed, and counted. Survival was analyzed with a semi-parametric proportional hazards statistical model using day of death as the dependent variable, and cross (reciprocals above), mtDNA, and their interactions as predictors. Mortality analyses were done on pooled data across the three replicate demography cages for each genotype. Survival curves were compared with log-rank tests, and tested for significance using JMP statistical software.
Age-specific mortality (μx) was reported as μx = –ln(1 – qx) where qx is the proportion of individuals that die in age class x (number of individuals that die, divided by the number of individuals that enter, age class x). We estimated age-specific mortality models for various lines using maximum likelihood (PLETCHER and GEYER 1999; PLETCHER et al. 2000). The Gompertz (G) model describes an exponential increase in mortality rate with age and is given by μx = e–?x, where μ(x) is the mortality rate at age x, is the initial mortality rate or intercept, and ? is the slope or age-dependent increase in mortality. The Gompertz–Makeham (GM) model is the Gompertz plus a parameter, C, that describes age-independent mortality. The Logistic (L) model is the Gompertz plus a frailty parameter, s, to describe late-life mortality rate deceleration. The Makeham and Logistic models can be combined with the Gompertz to produce a four-parameter Logistic–Makeham (LM) model. These models are nested, so likelihood ratio tests can be used to compare model parameters between different lines by allowing parameters to vary, or constraining them to be the same. Significance is determined using twice the difference in likelihood scores, distributed as a chi-square with degrees of freedom equal to the difference in the number of parameters. All such analyses were done with the Winmodest program (PLETCHER et al. 2000).
RESULTS
Longevities of intrapopulation crosses:
The longevity patterns for strains from within the Zimbabwe population (Zim 2, Zim 53) are shown in Figure 1. There are very significant differences between the inbred parental strains (Zim 2, Zim 53), but the longevities of the reciprocal F1 genotypes are very similar. The survivorship curves reveal a clear effect of heterosis, as the two F1 genotypes are significantly longer lived than the two parental strains. Reciprocal F1 females are not significantly different from one another (P > 0.1, log-rank tests), while males are (P < 0.001; reciprocal cross males also differ in any X-linked or Y-linked polymorphisms). The small mtDNA difference between these two strains (<20 bp substitutions; see MATERIALS AND METHODS) suggests that mtDNA haplotype effects cannot override the clear heterosis effects that result from reciprocal crosses between these two strains, which may have accumulated deleterious recessive mutations during laboratory culture (PROMISLOW and TATAR 1998; MACK et al. 2000; YAMPOLSKY et al. 2000).
Longevities of interpopulation crosses:
Survivorship and mortality plots for the interpopulation comparison of the two D. melanogaster strains from Crete and Four Town Farm, Rhode Island (Crete 10 and FTF 100) are shown in the top panels of Figures 2 (females) and Figures 3 (males). Similar data for the reciprocal crosses of these two strains to each of the two sim-mel interspecies introgression strains (sm21 and sm38) are shown in the lower panels of Figures 2 and 3, and will be discussed below. These data are presented in the same figure to facilitate comparison among the interpopulation (Crete 10 and FTF 100) and interspecies crosses (sm21 or sm38 crossed to either Crete 10 or FTF 100). The descriptive statistics of survival and mortality for all strains are presented in Table 1.
Significance tests of survivorship curves were done using log-rank tests, while significance tests of different mortality parameters were done using likelihood ratio tests (see MATERIALS AND METHODS). These tests can have different outcomes that reflect basic differences in survival vs. age-specific mortality. Survivorship curves display cumulative data so differences in survivorship between genotypes in one age interval are carried forward to other ages, which can inflate the effects of log-rank tests. Mortality parameters are a more accurate description of true aging effects since they focus on differences in the slope, intercept, or linearity of the mortality rate between genotypes. The data for all genotypes were contrasted using four mortality models [G, GM, L, and LM (see MATERIALS AND METHODS)]. Comparisons of alternative mortality models using the Winmodest package (PLETCHER et al. 2000) established that the four-parameter LM model was preferred over others for all genotypes presented in Table 1. We then compared each genotype to its reciprocal genotype to determine if any of the four parameters of the LM model differed between flies carrying alternative mtDNAs. This was done by determining the likelihood of a model that allowed each genotype to have a unique set of parameters (eight independent parameters; see Table 1, "unique" column) vs. a model that constrained one of the parameters to be the same for the two genotypes and allowed the remaining six parameters to vary (Table 1, "fixed ," "fixed ?," "fixed s," and "fixed C" columns).
Reciprocal F1 genotypes from crosses between two wild strains of Dmel, Crete 10, and FTF 100 show subtle but significant differences (Figures 2, A and B and 3, A and B). Log-rank tests show a significant difference between these two genotypes (P < 0.001), but likelihood ratio tests of mortality parameters do not show any significant differences. For Crete 10 and FTF 100 females, these likelihoods could not be distinguished (Table 1). In males there is a marginally significant difference in survivorship using a log-rank test (P < 0.02), and additional differences in mortality parameters (P < 0.01), neither of which is significant after correcting for multiple tests (see Table 1). In addition, it is not certain that this reciprocal cross effect in males can be attributed to mtDNA vs. X, or Y-chromosomal factors.
Comparisons between Dmel and Dsim mtDNAs:
Genotypes carrying either a Dmel or a Dsim mtDNA are shown in Figure 2, C–J (females), and Figure 3, C–J (males), representing reciprocal crosses between either Crete 10 or FTF 100, and the two "sim-mel" mtDNA introgression lines, sm21 and sm38. In general, the differences in longevity among flies carrying Dmel vs. Dsim mtDNA are much more pronounced than those carrying alternative Dmel mtDNAs (compare survivorship curves in Figure 2A to those in Figure 2, C, E, G, and I). However, the data clearly show that genotypes carrying the Dsim mtDNA are not consistently shorter lived than those carrying the Dmel mtDNA, as might be expected if Dsim mtDNA disrupts coadapted nuclear–mtDNA interactions in a Dmel nuclear background. Reciprocal crosses involving the "disrupted" mitonuclear sim-mel strains sm21 and sm38 are either shorter or longer lived than the reciprocal crosses between the two "native" mitonuclear Dmel strains FTF 100 and Crete 10 (see Figure 4). When the "sim-mel" introgression strains are crossed to the Dmel strain Crete 10, the sm38 (Dsim) mtDNA shows extended longevity, and sm21 mtDNA shows reduced longevity, relative to the Crete 10 (Dmel) mtDNA. The reverse is true when Dmel FTF 100 is used: sm21 mtDNA lives longer, and the sm38 mtDNA shows reduced longevity, relative to the FTF 100 mtDNA. The direction of these effects was the same in both sexes, although the magnitude of the effects was generally reduced in males (compare Figures 2 and 3; summarized in Figures 4 and 5).
Among the four pairs of reciprocal crosses involving Crete 10, FTF 100, sm38, and sm21, the effect of mtDNA haplotype on longevity is entirely epistatic, i.e., it depends on the allelic constitution of the F1 heterozygous nuclear background (Figure 5). This was quantified in a two-way ANOVA with nuclear background (reciprocal crosses) and mtDNA (Dmel vs. Dsim) as main effects, plus the interaction term. The nuclear x mtDNA interaction terms of these ANOVAs are all highly significant (see Table 2). Table 2 presents three different two-way ANOVAs: one involving the two pairs of reciprocal crosses of Crete to sm21 or to sm38, a second ANOVA involving the two pairs of reciprocal crosses of FTF to sm21 or sm38, and a third involving all four pairs of reciprocal crosses involving Crete, FTF, sm21, and sm38. Data from the three replicate demography cages were pooled for each genotype and a Cox proportional hazard model was used with day of death as the dependent variable and nuclear background and mtDNA plus interaction as the predictors. This treats the two reciprocal crosses with Dsim mtDNA as replicates (e.g., sm21 female x Crete male and sm38 female x Crete male). Thus, there is one degree of freedom for each factor in Crete or FTF ANOVAs, and three degrees of freedom for the nuclear reciprocal factors with all four reciprocals. The three replicate demography cages were generally in close agreement; ANOVA keeping them separate produced the same highly significant nuclear x mtDNA interaction terms (data not shown). To confirm the results of the ANOVAs, the analyses were done ignoring (1) the first 10 days, (2) the last 10 days, and (3) both the first and last 10 days of death events, and the results were qualitatively identical to the data reported in Table 2.
In females, the ANOVA shows strong variation among nuclear genotypes and in the nuclear x mtDNA interaction term, but no main effect of mtDNA. In males, the design violates a basic assumption of ANOVA since reciprocal crosses cannot really be considered replicated effects, so the results are presented for comparison only. Note that in males there are significant main effects of mtDNA, but this incorporates X- and Y-chromosome variation as well. As is evident in the figures, there is more nuclear and nuclear x mtDNA variation among genotypes in females than in males (female r2 = 13.1%, male r2 = 6.6%), suggesting that X- or Y-linked factors are reducing apparent effects of cytoplasmic term even if they are confounding it.
Demographic patterns:
Some additional comments are warranted about differences in mortality patterns among genotypes. Table 1 shows descriptive statistics for each of the reciprocal crosses involving the sim-mel introgression strains, with estimates of the LM terms that characterize mortality. The right side of the table presents the likelihood ratio tests of mortality parameters between reciprocal genotypes that quantify the differences shown in the right-hand panels of Figures 2 and 3 (mortality plots). In clear contrast to the results from the Crete x FTF cross (Dmel mtDNA only), the crosses with Dsim mtDNA strains show many significant differences in mortality parameters. The intercept (), slope (?), and late-life mortality deceleration terms (s) are all different for crosses to Crete 10. The effect of the latter term (s) can be seen in the Crete 10 crosses where a reduction of mortality occurs in mid to late life (Figure 2, C–F). It is also clear that crosses to FTF show fewer significant effects. This may be due to the fact that the sim-mel strains have largely Ore-R nuclear chromosomes, and these should be less differentiated from other North American strains such as FTF. Females show more significant differences than males.
Likelihood ratio tests between other pairs of genotypes further support the notion of complex nuclear–mtDNA interactions. For example, contrasts between the reciprocal crosses with sm38 females show no significant differences in , ?, or s (sm38 x Crete vs. sm38 x FTF), but contrasts between the reciprocal crosses with sm21 females show highly significant differences in , ?, and s (sm21 x Crete vs. sm21 XFTF; data available on request). Similarly, reciprocal crosses where FTF provided the mtDNA are significantly different for and C, but reciprocal crosses where Crete provided mtDNA are highly significantly different for all four parameters , ?, s, and C. Thus, the same Dsim mtDNA haplotype can have very different mortality patterns when crossed to alternative Dmel wild lines, and the same wild Dmel mtDNA has different mortality patterns when crossed to different sim-mel mtDNA introgression strains. These analyses show that nuclear–mitochondrial epistatic interactions can modulate age-specific survival in a variety of ways.
DISCUSSION
More than 90% of the functional mitochondrial genome is encoded in the nucleus. These nuclear-encoded mitochondrial genes (mitonuclear genes) arise either by transfer events from the mitochondrial to the nuclear genome or by recruitment of nuclear genes to a novel mitochondrial function through the acquisition of mitochondrial targeting sequences (RAND et al. 2004). This genetic architecture suggests that the mitochondrial phenotypes should be governed by (1) mitochondrial genes (mtDNA), (2) mitonuclear genes, or (3) genetic interactions between mtDNA and mitonuclear genes. Moreover, coadaptation among mitochondrial and mitonuclear genes predicts that the phenotypic effects of alternative mtDNAs should increase with increasing levels of DNA sequence divergence between native and foreign mtDNAs (e.g., mtDNA from different populations or species; RAND et al. 2004). The current study seeks to test these alternatives in the context of Drosophila aging by studying longevity among reciprocal crosses of strains carrying alternative mtDNAs.
Our results are qualitatively consistent with the coadaptation hypothesis: the inferred effects of alternative mtDNAs on Drosophila longevity increase in parallel with the degree of DNA sequence divergence. Differences in longevity were weak or absent between reciprocal F1 flies carrying alternative mtDNAs from within a Zimbabwe population, a subtle but significant difference was observed between an Old World and New World strain, and highly significant differences were observed among genotypes carrying mtDNAs from either D. melanogaster (Dmel) or D. simulans (Dsim). However, the results are not consistent with the hypothesis that flies carrying foreign mtDNA from a different species are generally disrupted and show reduced longevity. Our results clearly show that the effects of the foreign Dsim mtDNA on longevity depend strongly on the nuclear genetic background, i.e., there is nuclear–mtDNA epistasis for longevity.
Nuclear–mitochondrial epistasis and longevity:
Why are nuclear–mitochondrial epistatic effects stronger than mtDNA main effects on Drosophila longevity? Presumably main effects of mtDNA would be seen if a highly divergent mtDNA (say, from D. pseudoobscura) were carried on a Dmel nuclear background. One possibility is that the roughly 500 silent and 80 amino acid changes that exist between the Dsim and Dmel mtDNAs used in this study represent neutral or nearly neutral substitutions of nucleotide positions that are free to vary, rendering the two haplotypes nearly neutral variants with respect to longevity. Several lines of evidence argue against this. First, deleterious evolution of mtDNA is well established (NACHMAN 1998; RAND and KANN 1998), and non-neutral patterns of evolution have been documented for divergence of Dmel and Dsim mtDNAs (BALLARD and KREITMAN 1994; RAND et al. 1994; BALLARD 2000). Second, population cage studies with these mtDNAs indicate that Dsim mtDNA is indeed selected against relative to Dmel mtDNA (D. RAND, unpublished results). Moreover, less-diverged mtDNA haplotypes within D. simulans show repeatable differences in fitness effects of mtDNA (JAMES and BALLARD 2003; BALLARD and JAMES 2004). While these arguments do not establish that Dmel and Dsim mtDNA are non-neutral for longevity, it seems plausible that if mtDNA effects are detectable at an early age, there should be some additive mtDNA effects for these haplotypes at a later age when selection is less likely to have influenced their evolution.
A second possible explanation for the inconsistent main effects of mtDNA on longevity could stem from genomic imprinting of nuclear chromosomes. To infer an mtDNA effect on longevity, we compared reciprocal F1 genotypes carrying alternative mtDNAs. Reciprocal F1 females will be identically heterozygous and carry alternative mtDNAs, but could also differ in parent-of-origin effects on the nuclear alleles transmitted into the F1 flies studied. While imprinting has been reported for the Drosophila Y chromosome (MAGGERT and GOLIC 2002), this should not affect females. Notably, our results show that the differences in longevity among reciprocal F1 males was less than that for sibling females, so if imprinting was active in our lines it appears to have reduced, not increased, reciprocal cross effects (i.e., inferred mtDNA effects).
A third possible source of the strong epistatic effects is the retention of Dsim nuclear alleles in the mtDNA introgression lines carrying Dsim mtDNA. Introgression studies for a region of the second chromosome reveal that fertility and viability factors accounting for the isolation between Dmel and Dsim are densely distributed (SAWAMURA et al. 2000). During the initial generation of backcrossing between the Dsim C167.4 and Dmel In(1)AB, there may have been Dsim segments of the nuclear chromosomes that escaped the purging effects of hybrid unfitness in the increasingly Dmel nuclear background. Perhaps the presence of a Dsim mtDNA preferentially selected for individual Dsim alleles that are retained in some, but not all, of our sim-mel introgression strains (e.g., in sm21 but not sm38). While spurious selection effects during the backcrossing may also have contributed to the results, the strong reciprocal cross effects in females would require that the selection altered dominance patterns of certain nuclear alleles for a strictly nuclear effect to explain our results. The alternative mtDNAs carried in reciprocal cross females seems like a more parsimonious explanation.
Sequence analyses of 70 nuclear encoded subunits of mitochondrial OXPHOS subunits in all six of our sim-mel introgressions strains show them to have the Dmel Oregon-R allele (as expected from Oregon-R male backcrossing). However, there are hundreds of known mitonuclear genes that remain to be sequenced for these strains, plus many genes of unknown mitochondrial function that could retain putative Dsim alleles that alter longevity. Such loci could have epistatic interactions with the FTF 100 or Crete 10 alleles used in our hybrid crosses. Alternatively, the epistatic effects of these putative nuclear x nuclear interactions could be sensitized by the presence of the alternative mtDNAs in their respective F1 genotypes (ZEYL et al. 2005; WADE and GOODNIGHT 2006).
A further source of the nuclear–mtDNA epistatic effects we observed may stem from alterations of gene expression that result from the crossing of wild strains. Transcript profiling of reciprocal F1 genotypes from crosses between strains of D. melanogaster revealed that up to 33% of genes had significantly different expression patterns from the parental strains, and most of these effects were non-additive (GIBSON et al. 2004). This misregulation effect is also pronounced in interspecific hybrids (RANZ et al. 2004), with some genes showing expression outside the range of either parent.
If the two introgression strains (sm21, sm38) have retained distinct Dsim alleles during the backcrossing process, this could add further variation to the patterns of mis-regulated genes in reciprocal crosses of these strains to the two wild Dmel strains (Crete 10 and FTF 100). For this to account for our longevity data, these misregulation effects would have to be altered by the mtDNA haplotype present in the reciprocal F1 flies. Indeed, for this to occur in reciprocal F1 females, it suggests that mtDNA haplotype might alter the dominance of alternative alleles present in these hybrids. Mitochondrial-to-nuclear (retrograde) signaling modulates gene expression (BUTOW and AVADHANI 2004) and affects longevity in yeast (KIRCHMAN et al. 1999). In this context mitochondrial genotypes should be considered as novel "environments" for alleles and genotypes of nuclear loci. Since both nuclear x nuclear epistatic interactions and genotype x environment interactions affect Drosophila longevity (LEIPS and MACKAY 2000; VIEIRA et al. 2000), the effects of alternative mtDNA haplotype on the misregulation of genes in hybrid crosses is a potentially important component of variation in longevity among individuals in outbred populations, and warrants further study.
Nuclear–mitochondrial coadaptation:
The coadaptation hypothesis predicts that disrupted mitonuclear genotypes (e.g., Dmel nuclear chromosomes with Dsim mtDNA) should have reduced performance, and there is compelling evidence for this in a variety of systems (EDMANDS and BURTON 1999; RAWSON and BURTON 2002; MCKENZIE et al. 2003; SACKTON et al. 2003). It might follow that such genotypes would have reduced longevity due to disrupted OXPHOS functions, possibly resulting in elevated ROS production. However, the relationship between metabolic rates, electron transport and ROS production are not at all clear (SPEAKMAN et al. 2004).
In most species of Drosophila, metabolic rates do not change with age (PROMISLOW and HASELKORN 2002). There is no correlation between age and ROS production, and over-expression of the adenine nucleotide translocase (ANT) decreases ROS levels but shortens longevity (MIWA et al. 2004). Moreover, caloric restriction and reduced insulin signaling have no effect on metabolic rates or ROS production. It remains possible that the disruption of mitonuclear coadaptation could generate cellular states or gene expression patterns that extend longevity. An RNAi screen in C. elegans showed that extended longevity was associated with down-regulation of a number of mitochondrial genes, both nuclear and mtDNA encoded (DILLIN et al. 2002). In mice, extended longevity was associated with higher metabolism due to higher levels of uncoupling (SPEAKMAN et al. 2004).
Thus, it may be simplistic to assume that different sets of heterozygous flies carrying the same divergent mtDNA should show reduced longevity simply because an interspecific coadapted gene complex is disrupted. These genotypes may vary in the levels of expression of OXPHOS or uncoupling proteins that result in reduced or extended longevity in an mtDNA-dependent manner. For example, one pair of reciprocal crosses (sm38 x Crete 10) showed the greatest longevity extension, while a different pair showed the most reduced longevity (FTF 100 x sm21). These kinds of reciprocal crosses among mtDNA introgression lines provide excellent material for dissecting the mechanistic bases of altered longevity as they generate strong genetic effects that should have biochemical and cellular explanations.
Our results demonstrate that even with relatively divergent mtDNA alleles (mtDNA differing at over 700 point mutations including >80 amino acid altering sites), epistatic interactions with nuclear genetic background are a significant component of the mitochondrial genetics of aging. This kind of epistasis could explain why some mtDNA mutations can have very different phenotypic effects in different individuals, possibly obscuring the mtDNA effects in human aging and disease (JACOBS and HOLT 2000; ROSE et al. 2001). Studies that seek to identify mitochondrial genotype effects in aging should not seek to remove nuclear genetic variation, but should embrace it by replicating mitochondrial genotypes across several different nuclear genetic backgrounds. While the human species has relatively low nuclear genetic variation relative to Drosophila, two average people differ by 3 million nuclear base pairs, and 50 mtDNA point mutations. The significance of this vast array of possible mitonuclear genotypes should be considered in the context of the unpredictable nature of mitonuclear epistasis.
ACKNOWLEDGEMENTS
Larry Harshman, Jeff Hofmann, Chester McFly, Colin Meiklejohn, Kristi Montooth, Marc Tatar and Rebecca Wagaman and an anonymous reviewer provided helpful comments. The authors were supported by grants from the National Institutes of Health (GM-67862, AG-16632) and the National Science Foundation (DEB 0343464, DEB 9981497).
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Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Island 02912(David M. Rand, Adam Fry a)