Mitochondrial DNA variants in Drosophila melanogaster are expressed at the level of the organismal phenotype

Mitochondrion 11 (2011) 756–763

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Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o

Mitochondrial DNA variants in Drosophila melanogaster are expressed at the level of the organismal phenotype W.C. Aw a, C.C. Correa a, D.J. Clancy c, J.W.O. Ballard a, b,⁎ a b c

School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney 2052, Australia Evolution & Ecology Center, School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney 2052, Australia Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ , United Kingdom

a r t i c l e

i n f o

Article history: Received 20 January 2011 Received in revised form 30 May 2011 Accepted 28 June 2011 Available online 5 July 2011 Keywords Fecundity Starvation resistance Lipid proportion Physical activity

a b s t r a c t A plethora of experimental studies use mtDNA as a marker of demographic processes without questioning the possibility that selection may bias their interpretations. We studied four lines of Drosophila melanogaster that have a standardized nuclear DNA but variable mtDNA. We completed the sequencing of the mitochondrial genomes (excluding the A + T rich region) and compiled the differences. We then assayed male influence on oviposition, starvation resistance, lipid proportion and physical activity. We discuss these results in terms of the known differences between the lines and conclude that naturally occurring mtDNA variants in D. melanogaster are expressed at the level of the organismal phenotype. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction Batteries of experimental studies report that mitochondrial (mt) DNA does not always evolve in a manner consistent with a strictly neutral equilibrium model of evolution (Ballard and Melvin, 2010; Ballard and Rand, 2005; Ballard and Whitlock, 2004; Bazin et al. 2006; Dowling et al., 2008; Galtier et al., 2009). Despite these reports many phylogeographic studies assume mtDNA is an unbiased marker of demographic processes without testing the null hypothesis. If mtDNA is largely influenced by purifying selection, non-neutral mtDNA may alter some demographic or phylogenetic analyses. However, these alterations are expected to be relatively minor. If, on the other hand, mtDNA evolution is adaptive, interpretations of population histories may be both quantitatively and qualitatively biased. An important first step to test for the potential for mtDNA to be adaptive is to show that specific mutations can influence the physiology of the organism, and therefore can be selected upon. A second step is to quantify the evolutionary significance of the mtDNA mutations. The goal of this study is to test whether mtDNA changes influence four key physiological traits in male Drosophila melanogaster. Males are included because two lines of evidence suggest that functionally significant changes are easier to detect in males than females. First,

⁎ Corresponding author at: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia. Tel.: +61 2 9383 2029; fax: +61 2 9385 1483. E-mail address: [email protected] (J.W.O. Ballard).

mitochondrial diseases can be more severe, or more prevalent, in males than in females (Chinnery and Turnbull, 2001; Frank and Hurst, 1996; Innocenti et al., 2011). Secondly, when the mtDNA from Drosophila simulans is introgressed onto the nuclear background of Drosophila mauritiana, the disruption of COX (cytochrome c oxidase, complex IV) activity is more severe in males than in females (Sackton et al., 2003). This study does not quantify the evolutionary significance of the mtDNA mutations because females are not included. The direct impact of mtDNA variation on fitness has been measured in a variety of organisms including humans, mice, hares, finches, copepods and Drosophila (Ballard and Melvin, 2010; Ballard and Rand, 2005; Ballard and Whitlock, 2004; Dowling et al., 2008; Galtier et al., 2009; Meiklejohn et al. 2007). In humans, there is increasing evidence showing that human sperm motility is strongly dependent on the ATP supplied by oxidative phosphorylation (Ruiz-Pesini et al., 1998). Ruiz-Pesini et al. (2000) analyzed the distribution of mtDNA in Caucasian men having fertility problems and found that the sperm-motility phenotype was indeed conditioned by the mtDNA type. It has also been proposed that natural selection has shaped mtDNA variation in humans. Wallace and colleagues (Mishmar et al., 2003; Ruiz-Pesini et al., 2004) conducted complete human mtDNA genome analyses and hypothesized that certain haplogroups were positively selected as human's populated colder climates due to a decreased mitochondrial coupling efficiency. Decreased mitochondrial coupling is expected to lead to increased generation of heat. Amo and Brand (2007) present experimental biochemical data to test this intriguing hypothesis using cybrid cells that were constructed by fusing mitochondria obtained from healthy

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W.C. Aw et al. / Mitochondrion 11 (2011) 756–763

human volunteers with cells derived from human lung carcinoma A549 taken from a 58-year-old Caucasian male (Lieber et al., 1976). Contrary to the predictions of Wallace and colleagues (Mishmar et al., 2003; Ruiz-Pesini et al., 2004) mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups. This experimental result is intriguing, however, the functional significance of mtDNA haplotypes in a female genetic background is necessary to fully assess the evolutionary significance of specific mutations. Over the past decade a series of studies have considered the possibility that mtDNA substitutions influences the organismal phenotype of D. simulans and of D. simulans/D. mauritiana hybrids (Ballard and James, 2004; James and Ballard, 2003; Sackton et al., 2003). D. simulans is known to harbor three distinct mtDNA haplotype groups (siI, -II, and -III) with nearly 3.0% interhaplotypic divergence but b0.06% intrahaplotypic diversity (Ballard, 2000a). James and Ballard (2003) examined three physiological traits on fly lines with known sequence differences in the mtDNA genome after controlling the nuclear genome by reciprocal backcrossing. The authors found that flies with the siI haplotype are fastest developing and have the lowest probability of surviving to three experimental periods (2–6, 12–17, and 34–39 days of age). Wild-type males with siIII mtDNA were more active while disruption of specific coadapted mitochondrial–nuclear (mitonuclear) complexes caused a significant decrease in activity. Subsequently, Ballard and James (2004) examined the fitness of these three mitochondrial types in perturbation cages. Comparison of the pre-perturbation and post-perturbation data shows that the frequency of flies in population cages correlated with the observed worldwide distribution of the haplogroups (siII N − III N − I). In addition to mtDNA mutations, the efficiency of mitochondrial metabolism may be influenced by changes in nuclear encoded genes that produce proteins that are imported into the mitochondrion. These nuclear encoded proteins are essential for a multitude of functions including protein import as well as assembly and structure of complexes of the electron transport chain. Rand et al. (2006) assessed the relative contributions of (1) mtDNA, (2) nuclear genes, or (3) nuclear–mtDNA interactions in causing variation in Drosophila longevity. They compared strains of D. melanogaster carrying mtDNAs with varying levels of divergence within strains from one locality, between strains from different localities and introgression strains of D. melanogaster carrying mtDNA from D. simulans in a D. melanogaster Oregon-R chromosomal background. Strains from a single locality showed no difference in longevity while strains from different localities showed subtle but significant differences in longevity. The interspecific introgression lines showed very significant range of longevity differences that resulted in significant nuclear × mtDNA epistatic interaction effects. Dowling et al. (2007) similarly found sizable cytonuclear fitness interactions within D. melanogaster and present evidence suggesting that these effects were sex specific. One of the best-studied systems of coadaptation between the mitochondrial and nuclear genomes occurs in the marine copepod Tigriopus californicus. Populations of T. californicus exhibit high levels of mtDNA sequence divergence but retain the ability to produce viable offspring (Burton, 1986; Edmands, 1999). Laboratory crosses between T. californicus populations show a consistent pattern of F1 hybrid vigor and F2 hybrid breakdown for many metrics of physiological performance and fitness (Burton, 1986; Burton, 1990; Edmands, 1999). To test the hypothesis that disruption of nuclear–mitochondrial gene interactions can account for the reduced fitness of interpopulation hybrids Ellison and Burton (2008) backcrossed hybrids with low fitness to both maternal and paternal parental lines. The authors report that the low fitness of F3 hybrids is completely restored in the offspring of maternal backcrosses, where parental mitochondrial and nuclear genomic combinations are reassembled. Paternal backcrosses, which result in mismatched mitochondrial and nuclear genomes, fail to restore hybrid fitness. These results suggest that fitness loss in

757

T. californicus interpopulation hybrids is completely attributable to nuclear–mitochondrial genomic interactions. A limitation of all studies employing the backcrossing strategy to study mtDNA mutations is that the backcrossing may not have fully transferred and standardized the nuclear genomes of all organisms (Dermitzakis et al., 2000) because it may not have disrupted strongly coadapted mitonuclear gene combinations. Here, we include a set of D. melanogaster lines that were constructed by replacing wild-type line chromosomes with isogenic chromosomes of the w 1118 line (Clancy, 2008). The D. melanogaster lines vary with respect to the mtDNA but have a standardized nuclear genomic background (2008). One line has the w 1118 mtDNA. We include three additional lines that have the mtDNA from flies originally collected from Dahomey (former Benin), Japan or Alstonville (Australia). The sequence variation in the mtDNA protein coding region of these flies was determined by Clancy (2008). He then studied the rate of ageing of those four lines and showed that Dahomey has a reduction in mean lifespan of 4% while Japan has 15% shorter mean lifespan than Alstonville and w 1118, which do not differ. The rate of ageing is an important life history trait but it is questionable whether differences in this trait have the potential to directly influence the number of offspring in a species that typically only lives for a few weeks in nature. In this study, we first determine whether any mutations occur in the tRNAs or the rRNA regions. We then assay four physiological traits that may be important in nature. The traits we include are male influence on fecundity, starvation resistance, lipid content, and physical activity. Fecundity is an indirect estimator of sperm fitness because the persistence of increased egg laying in mated females depends on the viability of the stored sperm (Wolfner, 1996). Starvation resistance was included because individuals within many species must survive periods of starvation or exposure to suboptimal diets. Further, lines selected for longevity have a higher starvation resistance than unselected lines (Bubliy and Loeschcke, 2005), and differences in starvation resistance have been shown to differ between D. simulans mitochondrial haplotypes (Ballard et al., 2007). We assay lipid proportion because different mtDNA mutations have been shown to confer differences in bioenergetic efficiency (Gemmell et al., 2004) and this is predicted to influence patterns of food consumption and lipid storage (Simpson et al., 2004; Warbrick-Smith et al., 2006). Physical activity was included because it requires ATP from the electron transport chain and likely influences migration patterns. In Drosophila, Berrigan and Partridge(1997) showed that mean walking speed is correlated to metabolic activity. We show that when the nuclear genome is standardized but the mtDNA is allowed to vary male fertility, starvation sensitivity, lipid proportion and physical activity differ. 2. Materials and methods 2.1. Flies and husbandry Fly lines were created by chromosome replacement (CR) using balancers. Chromosomes X, 2 and 3 from wild type strains were replaced with homozygous chromosomes from the isogenic donor strain w 1118iso obtained from Bloomington (see Clancy, 2008 for crossing scheme). Homozygosity was tested using microsatellites and ‘phenotypic isogenicity’ for lifespan was tested by comparing unbackcrossed CR lines vs. F1 offspring of CR lines backcrossed to w 1118iso males. Crossing was done at large population sizes (~75 pairs/cross) although numbers in some strains became reduced at the double-balancer stage but underwent five generations of backcrossing to w 1118iso immediately prior to the commencement of the study. All strains had been tetracycline-treated prior to crossing to ensure they were free of Wolbachia. The strains were w 1118iso (Bloomington stock no. 5905, isogenic for chromosomes 1, 2 and 3, constructed by John Roote, Cambridge, UK, Canton-S mitochondria (Ryder et al., 2004),

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Alstonville (New South Wales, Australia, collected 2002), Dahomey (now Benin, Africa, derived from mass bred collected in 1970), and Japan (Jume, Japan from David Rand (Rand et al., 1994)). Cultures were maintained in overlapping generations at 23 °C in a 12 h day:12 h night cycle. For two generations before the study, and during all experiments, the density of flies in cages was strictly controlled. Eggs were collected by placing oviposition resources (solidified agar-based medium containing 4% agar and 10% molasses) in cages containing flies. The eggs were washed from the oviposition resources with water and then collected (Clancy and Kennington, 2001). About 170 eggs were transferred by pipette onto instant food (Carolina Biological Supply Company, 2700 York Road, Burlington, North Carolina 27215–3398, USA) in glass bottles. Flies were kept on a diet of adult food consisting of sucrose (114.5 g/l), yeast (93.57 g/l), agar (12 g/l), nipagen (0.4 g/l) and distilled water. To test for specific generational effects all studies were repeated with flies raised in distinct generations. Here we define these repeated studies as blocks. Unless otherwise stated all studies were conducted at 23 °C.

2.2. DNA sequencing Clancy (2008) reported the 13 mtDNA protein coding regions. In addition, several tRNAs were sequenced but not reported. Here we completed the mtDNA genome (excluding the A + T rich region) and report on the variation in all tRNAs and the rRNAs. Four regions of DNA were amplified and sequenced (Table S1). Region I was amplified using primers 19390+ and 350− and includes the tRNA Ile, tRNAGln, and tRNAMet. Region II was amplified using primers 1222+ and 1662− and includes the tRNA Trp, tRNACys and tRNATyr . Region III was amplified using primers 5932+ and 6546− and includes tRNAAla, tRNAArg, tRNAAsn, tRNA SerAGY, tRNAGlu and tRNAPhe. Region IV was amplified using 4 pairs of primers (12538+/13431−, 13346+/13947−, 13810+/14546− and 14328+/14912−) and includes tRNALeuCUN, 16S rRNA and the 12S rRNA. PCR products were sequenced in both directions and sequences were edited and aligned using Sequencher 4.5 (Gene Codes, Ann Arbor USA).

2.4. Starvation resistance This trait was measured because most Drosophila need to survive periods without food in nature. The starvation resistance protocol followed Kenny et al. (2008) but flies were assayed 11 days and 32 days after eclosion. We selected age 11 days because D. melanogaster are still in the first third of their lifespan (Minois et al., 2001). Age 32 days was chosen because it is before the point of the exponential decline in lifespan of w 1118 (Mourikis et al., 2006). Briefly, flies were cooled on ice and then transferred to new cages. After 2 h, wet cotton replaced food in the fly cages. To ensure the cotton pledget was fully saturated, 800 μL of distilled water was added every 24 h. Dead flies were removed and counted every 6 h until all flies died. The 50% and 10% starvation resistance rates determined for each line. ANOVA was completed using line, day and experimental block as random effects.

2.5. Lipid proportion We assayed lipid content because it has been shown to increase with starvation resistance in Drosophila (Ballard et al., 2008). However, increasing lipid reserves does not guarantee an increase in starvation resistance (Hoffmann et al., 2001). Lipid proportion was assayed 11 days and 32 days after eclosion. Our lipid extraction assay followed Hoffmann et al. (2005). Briefly, males were cooled on ice and collected in 1.5 ml eppendorf tubes. Flies were then frozen in liquid nitrogen and then transferred into a −80 °C freezer. After 4 days the flies were removed from the freezer and thawed for 20 min. Nine replicates of 10 flies/line were put in clean 10 ml glass tubes. They were dried for 48 h at 100 °C and the dry weight determined on a CP2P micro balance (Sartorius AG, Weender Landstraße 94-108, D-37075 Göttingen, Germany). Diethyl ether (7.5 ml) was added and the tubes were placed on a shaker at room temperature. After 24 h the remaining ether was removed and the flies were dried for a second 48 h at 100 °C. Flies were then reweighed, the difference between dry weight and final weight was considered the lipid. Lipid proportion is 1-final weight/dry weight. For ANOVA the lipid proportion was arcsine square root transformed to make the variance independent of the mean.

2.3. Male influence on female fecundity We assayed the influence on fecundity of male D. melanogaster flies harboring different mtDNA types when crossed with a common female genotype because mutations in mtDNA impact male fertility in many (Gemmell et al., 2004) but not all cases (Mossman et al., 2009; Pereira et al., 2007; Pereira et al., 2008). Virgin males and females were sorted within 4 h of hatching and each sex placed in a separate cage. Virgin females were kept in vials of 10 for 4 days to ensure they were unmated. Virgin males were kept in cages in groups of 50–100 for 4 days until they were placed individually with a female. This protocol is expected to maximize a male's ejaculate investment and the number of offspring (Bretman et al., 2009). After 5 days virgin w 1118 females were crossed with a single 5-day-old virgin male so that 12 pairs/line were included. After 24 h the males were removed and the females were transferred to new vials with adult food. Extreme care was exercised when changing adult food to minimize the risk of stressing and harming the fly by avoiding tapping or knocking the vials. The vials for females were replaced every 24 h. The total number of eggs in each vial was counted for 10 days, since a mated female fly harboring stored sperm is stimulated to lay eggs for up to 11 days after mating (Wolfner, 1996). Females that died during the study or did not produce any viable eggs were excluded. For statistical analyses these count data were normalized by square root (X + 1) transformation and analyzed with JMP Software (© 2007 SAS Institute, Cary, NC, USA) with day after mating treated as a continuous variable.

2.6. Physical activity In nature, the food needed for Drosophila survival and reproduction is an ephemeral resource. As a consequence, flies require ATP to move between these resources. We hypothesized that if the mtDNA mutations conferred a difference in ATP production then physical activity would also differ. The physical activity assay is based on the protocol of Melvin et al. (2007) but flies were assayed 11 days and 32 days after eclosion at 18 °C. Briefly, 24 flies of each line were anesthetized with humidified CO2 and transferred to 6.5 cm long glass tubes with 5 mm adult food and then 5 mm of wax seal. The other end was sealed with a piece of cotton so that the distance between cotton and food was 4.5 cm. Flies were given 10 h to acclimate. A TriKinetics Activity Monitor IV (TriKinetic Inc., 56 Emerson Road, Waltham, MA 02451, USA) was placed in a sealed incubator. This minimized disturbance from the environment. The number of light beam crossings was then counted in 5 min steps for 24 h, but only the day cycle data was taken for our assay. Flies that died during the experimental period or up to 24 h thereafter were excluded from the statistic analyses. The data were checked for outliers since flies may rest in the light beam. Statistical outliers were replaced by the mean of the remaining flies in that line for that step. The mean activity was calculated in Microsoft Excel and JMP was used for statistical analyses.

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3. Results

Alstonville

40

Variable positions were detected in three tRNAs (Table 1) and one 5 bp insertion was observed between tRNA Ala and tRNA Arg in Dahomey. We then used tRNAscan-SE v1.11 (Lowe and Eddy, 1997) to examine each variable tRNA. The mutation in the tRNA SerAGY of Japan occurred at the wobble position and has the potential to influence transcriptional accuracy and efficiency. The mutations in the tRNA Glu and tRNA Thr both occurred in the T loop (Sprinzl et al., 1998). Mutations in the T-loop are unlikely to influence the physiology of the organism. In humans, few of the mt-tRNA genes have pathogenic mutations that occur in the loop structures. The exception is the tRNA LeuUUR (McFarland et al., 2004). In the 12S rRNA, three substitutions were recorded (Table 1). In Japan, position 121 changes from a T to a C. This position occurs in stem 48 of the refined model of Drosophila yakuba 12s rRNA (Page, 2000) and may destabilize the structure. Position 546 changes from a T to a C in Alstonville. This region is not evolutionarily conserved and is in a region that includes a deletion in relation to D. yakuba (Ballard, 2000b). Position 708 changes from a T to an A in Alstonville. This position is conserved through D. yakuba and the D. melanogaster subgroup (Ballard, 2000b) and may represent a deleterious change. In the 16S rRNA, four changes were detected. These changes were aligned against the proposed D. melanogaster secondary structure and conserved motifs identified on the Escherichia coli model (Cannone et al., 2002). At position 829, there is a C to T transition in Dahomey. This change restores a Watson–Crick base paring in helix 1087 and may be expected to increase stability (Table 1). Two unique changes occur in w 1118 and one in Japan but they are not expected to be functionally significant (Table 1). The T to C change at position 72 in Drosophila occurs between helices 2043 and 2646. At, or around, position 317 there is a deletion of an A between helices 2259 and 2347. The consensus has 6 As while w 1118 has 5. In Japan, a deletion occurs at, or around, position 404. The consensus has 8 As while Japan has 7 As. This deletion occurs in the highly variable helix 2077 (Buckley et al., 2000). 3.2. Male influence on female fecundity Fecundity of w 1118 females when mated with the four experimental fly lines declined with day after mating in all cases. This decline was lower in Japan than in Alstonville, Dahomey, and w 1118 (Fig. 1). ANOVA showed the male influence on female fecundity was not significantly different between the lines (F3, 856 = 1.09, P = 0.35) or the experimental blocks (F1, 856 b 0.01, P = 0.98). There is, however, a significant effect of day after mating (F1, 856 = 101.55, P b 0.001) and a day by line interaction (F3, 856 = 3.39, P = 0.02). If the Japan line is excluded, ANOVA shows no significant day by line interaction

Numbers of eggs

3.1. DNA sequencing

Japan Dahomey w1118

30

20

10 0

2

4

6

8

10

Day Fig. 1. Male influence on female fertility decreased after day of mating in all fly lines but the rate of decline was lowest in Japan. Alstonville (Oviposition rate = − 1.41x + 31.77, R2 = 0.54), Dahomey (Oviposition rate = − 1.97x + 35.58, R2 = 0.80), w1118 (Oviposition rate = −1.59x + 31.29, R2 = 0.90) and Japan (Oviposition rate = −0.76x + 28.35, R2 = 0.44). Fertility rate of male Drosophila melanogaster lines Alstonville, Japan, Dahomey and w1118, age 5 days old after eclosion. Error bars indicated standard error of the mean.

(F2, 649 = 1.16, P = 0.32) suggesting that the decline in male influence on female fecundity in the Japan line differs from all others. 3.3. Starvation resistance For 10% survival, 32 day old flies have a 16.7% lower starvation resistance than 11 day old flies. Japan has the lowest starvation resistance which is 8.9% lower than the average of all other lines at 11 days and is 12.7% lower at 32 days of age (Fig. 2a). ANOVA showed that there is a significant effect of line (F3, 55 = 11.18, P b 0.001) and age (F1, 55 = 95.91, P b 0.001). There is no significant age by line interaction (F3, 55 = 0.51, P = 0.68) or experimental block effects (F1, 55 = 0.96, P = 0.33). For 50% survival, 32 day old flies have a 24.9% lower starvation resistance than 11 day old flies. Japan has the lowest starvation resistance, which is 10.28% lower than the average of all other lines at 11 days and 14.53% lower at 32 days of age (Fig. 2b). ANOVA showed that there is a significant effect of line (F3, 55 = 19.98, P b 0.001), age (F1, 55 = 227.25, P b 0.001), and an experimental block effect (F1, 55 = 14.30, P b 0.001). There is no significant age by line interaction (F3, 55 = 1.95, P = 0.13), line by experimental block interaction (F3, 51 = 0.13, P = 0.94) or age by experimental block interaction (F3, 51 = 0.06, P = 0.81). This block effect is due to an overall increase of survival of 6 ± 0.53% in all the treatments of the second block. Plausibly, the observed difference in survival rate may be caused by a slight variation in humidity that caused the cotton wool to dry at different rates. This would be expected to influence 50% starvation resistance to a greater degree than 10% starvation resistance.

Table 1 MtDNA encoded amino acid, tRNA and rRNA differences in the four Drosophila melanogaster strains included in this study. Complexa Gene Position Consensus w1118 Dahomey Japan Alstonville

I ND2 182 His ● ● Tyr ●

277 Leu Ile ● ● ●

RNAb ND4 248 Val ● Leu ● ●

III Cyt b 99 Val Met ● ● ●

IV COIII 40 Asn Asp ● ● ●

V ATP6 177 Asn Lys ● ● ●

180 Pro Ser ● ● ●

185 Met ● Ile ● ●

tRNA SerAGY 26 G ● ● A ●

Glu 50 C A ● A ●

Thr 48 T — ● ● ●

12S rRNA 121 546 T T ● ● ● ● C ● ● C

708 T ● ● ● A

16S rRNA 72 317 T A C — ● ● ● ● ● ●

404 A ● ● — ●

829 C ● T ● ●

Abbreviations for the genes are ND for NADH dehydrogenase subunits, CO for cytochrome oxidase and Cyt b for cytochrome b. Position is the position of the amino acid in the protein or nucleotide in the RNA. ● denotes the same as the consensus. Positions reported for the RNAs are in accordance to the published D. melanogaster mitochondrial genome (GenBank Accs. U37541.1). a Previously reported in Clancy, 2008. b Reported here.

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W.C. Aw et al. / Mitochondrion 11 (2011) 756–763

A

Alstonville Japan

10% Survival

80

A B

A B

B C

Dahomey w1118

A C D

60

B C

C D

D

3.5. Physical activity In all lines, 32 day old flies have a lower physical activity than 11 day old flies. This decline in mean walking speed with age was about 33% in w 1118 and Dahomey and 15% for Japan and Alstonville (Fig. 4). ANOVA showed a significant effect of age (F1, 372 = 50.17, P b 0.001) and an age by line interaction (F3, 372 = 7.68, P = 0.03). There was no main effect of line (F3, 372 = 4.52, P = 0.14) or experimental block (F1, 372 = 1.75, P = 0.15).

40 20 0

11d

32d

Age (d)

B

Alstonville

4. Discussion

Japan Dahomey

80

50% Survival

10% starvation resistance = 9.15 + 406.85 × lipid (r² = 0.60) and for 50% starvation resistance = 10.04 + 472.23*lipid (r² = 0.61).

A B

A B

B C

60

A

w1118

C D

D

D

40 20 0 11d

32d

Age (d) Fig. 2. The starvation resistance of male Drosophila melanogaster differ between fly line and decline with increasing age. Overall, the Japan line has the lowest survival rate. The fly lines included were Alstonville, Japan, Dahomey and w1118, aged 11 and 32 days (d) after eclosion. (A) 10% survival. (B). 50% survival. Bars represent survival and error bars indicated standard error of the mean. Letters above each bar indicate post-hoc significant differences as determined by Tukey's test with a = 0.05 and Q = 3.15.

3.4. Lipid proportion The 32 day old flies have a lower lipid proportion than 11 day old flies. Japan has the lowest lipid proportion which is 5.4% lower than the average of all other lines at 11 day old and 17.7% lower at 32 d of age (Fig. 3). ANOVA showed that there is a significant effect of line (F3, 135 = 35.96, P b 0.001), age (F1, 135 = 228.98, P b 0.001), and an age by line interaction (F3, 135 = 16.77, P b 0.001). ANOVA shows no significant experimental block effects (F1, 135 = 0.002, P = 0.96). As expected, lipid proportion is positively correlated to both 50% starvation resistance and 10% starvation resistance times (Fig. S1). For

Data presented here show that mtDNA variants are expressed at the level of the phenotype in males. Specifically, we show that when the nuclear genome is standardized but the mtDNA is allowed to vary male starvation sensitivity, male influence on female fecundity, lipid proportion and physical activity differ. Ballard et al.(2007) have previously shown that different D. simulans mitochondrial haplogroups with no subdivision in 13 nuclear genes have significant differences in weight, cytochrome c oxidase activity, egg size, cold resistance and starvation resistance. However in that study it was not possible to hypothesize which changes were functionally important. Here, we have three or fewer amino acid differences in a single complex and 18 differences in the protein coding and RNA genes. Of the four fly mtDNA genomes studied Japan is the most phenotypically distinct. Flies harboring this mtDNA type are unique in terms of male influence on female fecundity (Fig. 1), starvation resistance (Fig. 2ab) and lipid proportion (Fig. 3). They do not, however, show the greatest decline in physical activity suggesting that a higher rate of aging is unlikely to be a global explanation. Three mutations in flies harboring mtDNA from Japan may influence mitochondrial bioenergetics (Clancy, 2008). In complex I there is a Histidine to Tyrosine change at position 182 in ND2. This a change from a basic to neutral and polar amino acid (Lewin, 1997). Complex I, or NADH dehydrogenase, is the main entrance point of electrons and is the largest complex of the respiratory chain. It catalyzes the transfer of two electrons from NADH to ubiquinone and translocates four protons across the inner mitochondrial membrane. In humans, deficiencies of mtDNA-encoded complex I subunits are associated Alstonville Japan

4 Alstonville Dahomey

0.20

Lipid Proportion

A B

0.15

A B C

1118

A B

w

C

C D E

0.10

Physical Activity

Japan

3

A B C

A B

A

A B

Dahomey

B C C C D D D D

w1118

2

1 0.05 0 11d

0.00 11d

32d

32d

Age (d)

Age (d) Fig. 3. The lipid proportions of male Drosophila melanogaster differ between fly line and decline with age. The fly lines used were Alstonville, Japan, Dahomey and w1118, aged 11 and 32 days (d) after eclosion. Bars represent lipid proportion and error bars indicated standard error of the mean. Letter above the bar indicates significant differences between lines as determine by Tukey's test with α = 0.05 and Q = 3.08.

Fig. 4. Mean walking speed of male Drosophila melanogaster decline with age. The fly lines used were Alstonville, Japan, Dahomey and w1118, aged 11 and 32 days (d) after eclosion. w1118 line has a lowest mean walking speed compare with other line. Bars represent mean walking speed and error bars indicated standard error of the mean. Letter above the bar indicates significant differences between lines as determine by Tukey's test with α = 0.05 and Q = 3.05.

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with a range of clinical phenotypes including Leber hereditary optic neuropathy (LOHN) and reduced fertility (Selvi Rani et al., 2006). We observed an A to a C transversion in the anticodon (wobble position) tRNA SerAGY. This change has the potential to influence transcriptional accuracy and efficiency (Akashi, 1994). In Drosophila, tRNA SerAGY is four fold degenerate with the frequency of each mtDNA codon in D. melanogaster Oregon R being AGAN N AGUN N AGG = AGC (Ballard, 2000b). We also observed a T to C transition in position 121 of the 12s rRNA. This change occurs in stem 48 of the refined model of D. yakuba 12s rRNA (Page, 2000) and may destabilize the structure. Unlike DNA, which is almost exclusively encountered as a double helix with canonical Watson–Crick base pairs, RNA contains many non-canonical base pairs. Formation of non-canonical base pairs in RNA is essential, because—in contrast to Watson-Crick base pairs in RNA—they often play an important role, either functionally or structurally (Heus and Hilbers, 2003). The highest decline in physical activity occurred in w 1118 and Dahomey. No unique mtDNA mutations occur in both these lines. Both lines do, however, have changes in the ATP6 subunit of Complex V. Complex V, also called ATP synthase, is the final enzyme in the electron transport chain. The enzyme uses the energy stored in a proton gradient across the inner mitochondrial membrane to drive the synthesis of ATP from ADP and phosphate. A decrease in the function of complex V is considered plausible for two reasons. First, Holyoake et al. (1999) showed that a single amino acid transition from serine to proline in human ATPase6 was associated with a reduced male fertility. Second, Mathews et al. (1995) showed that BHE/cdb rats, which mimic the noninsulin-dependent (type II) diabetes mellitus in humans, have a single amino acid exchange in the ATPase6 subunit (Aspartic acid to Asparagine). This strain of rats has been bred specifically to develop impaired glucose tolerance at midlife. Numerous studies of their metabolic pathways as well as characteristic features of the liver, pancreas, and kidney have been conducted. Notable is a fatty liver with a greatly increased lipogenic and gluconeogenic activity (Berdanier, 1982; Lakshmanan et al., 1977). Flies with the w 1118 derived mtDNA have five additional differences from the consensus sequence. Of these, the substitutions in complex IV are more likely to have functional consequences. In complex IV, the substitution is from the neutral and polar Asparagine to the acidic Aspartic acid. Quaternary structure modeling of this complex shows that residue 40 is in a 5-amino acid loop between two alpha helices (Melvin et al., 2008). At 4 Å distance only residues within COIII are in close contact. At 6 Å distance residue 40 interacts with one residue of Cox6A and two residues of Cox7A. In the ATP6 gene of complex V, the change at position 177 is from Asparagine to the basic Lysine and at position 180 is from the neutral and hydrophobic Proline to the neutral and polar Serine. In addition to the complex V mutation, flies harboring Dahomey mtDNA have two additional unique mutations that may have physiological consequences. One of these occurs in the 16S rRNA and the other in ND4. It is possible that the C to a T transversion at position 829 of the 16S rRNA influenced mitochondrial bioenergetics. However, this mutation is more likely to be beneficial as it likely restored a Watson-Crick base pairing in helix 1087. The level of 16S rRNA reduces as Drosophila ages and in old flies it is only 20% of that present in young flies (Calleja et al., 1993). Moreover, the decrease in 16S rRNA levels is tissue specific with an important reduction of the amount present in highly demanding tissues such as brain and muscle (Calleja et al., 1993). The second mutation occurred at position 248 of ND4 and is predicted to cause a Valine to Leucine (hydrophobic) change. Both these amino acids are aliphatic, hydrophobic and neutral so the change seems unlikely to cause large bioenergetic consequences. There are at least three limitations of this study. First, the physiology of the mtDNA mutations was examined in a single genetic background and mitochondrial-nuclear interactions were considered

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negligible. Mitochondrial-nuclear coadaptation has been shown to be important in a number of studies (Ellison and Burton, 2008; James and Ballard, 2003; Rand et al., 2006) and standardizing the nuclear genome may have disrupted coadapted mitochondrial and nuclear gene complexes. To test the possibility that mtDNA interactions, and not the mtDNA alone, were causally responsible for the observed effects each haplotype could be placed in a second genetic background. Alternatively, or in addition, nuclear genes that are predicted to be interacting with specific mtDNA mutations could be sequenced to determine whether compensatory changes occur in the founding wild-type fly strains (Melvin et al., 2008). A second limitation of this study is that mutation(s) in the A + T rich region and/or in chromosome 4 may influence the phenotypic traits measured. The origin of replication occurs within the A + T rich region and the region can be highly variable (Saito et al., 2005). Balancer chromosomes could not replace chromosome 4, however, there were effectively thirteen backcrosses from w 1118 males during and following the crossing scheme. In D. melanogaster, chromosome 4 is not known to recombine under ordinary laboratory conditions (Sturtevant, 1951) and is achiasmate at female meiosis (Hawley et al., 1993). It is thought to contain 74 genes of which 12 have been characterized (Adams et al., 2000). A third limitation is that we conducted the study under one set of laboratory conditions and it is possible that small changes in these conditions may lead to different results. In Drosophila, temperature and diet are examples of environmental factors that are likely to influence mitochondrial bioenergetics and the concomitant physiological response(s). Pichaud et al. (2010), studied differences in mitochondrial performance and thermal sensitivity associated with mitochondrial haplotypes of D. simulans at four temperatures. Their results show that the globally distributed haplotype is better adapted to a large temperature range and specifically to extreme temperatures whereas the haplotype restricted to east Africa has a smaller thermal range. Diet has also been shown to be important in determining fertility and longevity in a variety of organisms including flies (Lee et al., 2008; Simpson and Raubenheimer, 2007) and it is easy to imagine that flies harboring different mtDNAs will respond differently to dietary changes. We suggest it is unlikely that the w 1118 line accumulated substantial variation in the nine generations between the culmination of the w 1118 inbreeding of this line and the completion of the back-crossing with other lines. Drosophila has an estimated amino acid mutation rate of 0.030 per haploid genome per generation (Keightley and Eyre-Walker, 1999). In this study, we have shown that mtDNA variants can be expressed at the level of the phenotype and conclude that these naturally occurring mtDNA mutations are not selectively neutral. The majority of changes are, however, predicted to be deleterious and not advantageous. The noted exception to this is the C to T transversion in helix 1087 of the 16S rRNA in Dahomey. A next step is to conduct biochemical tests to quantify the bioenergetic consequences of specific mutations in females through either a “top-down” or “bottom-up” approach. Essentially, the top-down approach involves the determination of the ADP:O ratio in normal and mutant flies and, if significant differences are found, the examination of specific OXPHOS complexes (Hafner et al., 1990). The ADP:O ratio, determined from extracted mitochondria, is the moles of ADP phosphorylated to ATP divided by the moles of oxygen consumed by mitochondria. The alternative approach to examining mitochondrial bioenergetics is to follow a more hypothesis driven strategy examining the activity of specific amino acid changes when the specific mutations of interest are known (Ballard et al., 2010; Kirby et al., 2007 ; Ruiz-Pesini et al., 2000; Smeitink et al., 2001; Wallace, 2002). Through such integrated genetic, biochemical and physiological studies it may be possible to determine whether any of these naturally occurring mutations are adaptive. Supplementary materials related to this article can be found online at doi:10.1016/j.mito.2011.06.012.

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Acknowledgments The work was supported by University of New South Wales internal grants and ARC Discovery Project – DP110104542. We thank David Rand and an anonymous reviewer for comments on a draft. Ines Ricafuente, Olaf Bressell and Richard G. Melvin helped in the laboratory and Robin Gutell advised us on RNA modeling.

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