Sympatric Drosophila simulans flies with distinct mtDNA show age related differences in mitochondrial metabolism

The primary causes of age-related changes in mitochondrial metabolism are not known. The goal of this study is to document the influence of naturally occurring mtDNA variation on age dependent changes in mitochondrial respiration, hydrogen peroxide (H(2)O(2)) generation and antioxidant defenses in the fly Drosophila simulans. Possible changes include an increase in rates of reactive oxygen species production with age and/or an age dependent decrease in antioxidant response. For this study we have used flies harboring distinct siII and siIII mtDNA types. Previously we have shown that males harboring siII mtDNA had higher rates of mitochondrial H(2)O(2) production from complex III at 11d compared to males with the siIII mtDNA type. Here, we corroborate those results and show that Drosophila harboring the siII and siIII mtDNA types exhibit significantly different patterns of pro-oxidant and antioxidant activities as they age. Flies harboring siII mtDNA had higher rates of mitochondrial H(2)O(2) production and manganese superoxide dismutase activity at 11 and 18d of age than siIII mtDNA harboring flies. Copper-zinc superoxide dismutase activity increased from 11 to 25d in siII flies while the accumulation of oxidized glutathione did not change between 11 and 25d. In contrast, siIII harboring flies showed an age dependent increase in H(2)O(2) production, reaching higher production rates on day 25 than that observed in siII flies. Copper-zinc superoxide dismutase activities did not change between 11 and 25d while the oxidized glutathione accumulation increased with age. The results show antioxidant levels correlate with pro-oxidant levels in siII but not siIII flies. These results demonstrate our ability to correlate mtDNA variation with differences in whole mitochondrial physiology and individual complex biochemistry.     

Functional conservatism among Drosophila simulans flies experiencing different thermal regimes and mitochondrial DNA introgression

Drosophila simulans possesses three different mitochondrial haplotypes (siI, II and III) that are nonrandomly geographically subdivided with a 3% interhaplogroup variation. The aim of this study was to determine whether perturbation of mitochondrial metabolism and ROS management by temperature variation and mtDNA introgression would influence the development of aerobic capacity and the intensity of oxidative stress in D. simulans at different ages. Environmental temperature divergences during development had few impacts on metabolic capacities. Our data suggested strong functional conservatism of mitochondrial haplotypes between the D. simulans lines studied. This conservatism was expressed by the low divergences in either mitochondrial or ROS buffering enzyme activities, or even markers of ROS damage even after disruption of coevolved genomes. Disruption of coevolved mitochondrial and nuclear genomes through mtDNA introgression induced no clear divergence on metabolic phenotype at any state of development. Reduction of cytochrome c oxidase activity that was observed after introgression of one mitochondrial haplotype will require further investigation to delineate whether it is associated with any modification of mito-nuclear interactions.     

Natural populations of Drosophila simulans show great uniformity of the mitochondrial DNA restriction map

The mitochondrial DNA of 21 Drosophila simulans isofemale lines of different geographic origins was digested with seven restriction endonucleases. All the lines, with one exception for one line and one enzyme, showed the same restriction patterns. The results confirm previous investigations showing great uniformity of the mtDNA genome in D. simulans.     

Multiple hybridization events between Drosophila simulans and Drosophila mauritiana are supported by mtDNA introgression

The study of speciation has advanced considerably in the last decades because of the increased application of molecular tools. In particular, the quantification of gene flow between recently diverged species could be addressed. Drosophila simulans and Drosophila mauritiana diverged, probably allopatrically, from a common ancestor approximately 250,000 years ago. However, these species share one mitochondrial DNA (mtDNA) haplotype indicative of a recent episode of introgression. To study the extent of gene flow between these species, we took advantage of a large sample of D. mauritiana and employed a range of different markers, i.e. nuclear and mitochondrial sequences, and microsatellites. This allowed us to detect two new mtDNA haplotypes (MAU3 and MAU4). These haplotypes diverged quite recently from haplotypes of the siII group present in cosmopolitan populations of D. simulans. The mean divergence time of the most diverged haplotype (MAU4) is approximately 127,000 years, which is more than 100,000 years before the assumed speciation time. Interestingly, we also found some evidence for gene flow at the nuclear level because an excess of putatively neutral loci shows significantly reduced differentiation between D. simulans and D. mauritiana. Our results suggest that these species are exchanging genes more frequently than previously thought.     

Intraspecific variation in survival and mitochondrial oxidative phosphorylation in wild-caught Drosophila simulans

Lifespans of organisms vary greatly even among individuals of the same species. Under the framework of the free oxygen radical theory of aging, it is predicted that variation in individual lifespan within a species will correlate with variation in the accumulation of oxidative damage to cell components from reactive oxygen species. In this study we test the hypothesis that variation in survival of three wild-caught Drosophila simulans fly lines (HW09, NC48 and MD106) correlates with three key aspects of mitochondrial bioenergetics. The rank order of median survival was HW09 > MD106 > NC48. Young HW09 flies (11-18 days) had (i) highest ADP:O (quantity of oxygen consumed by mitochondria when provided with a quantity of ADP) when metabolizing both electron transport chain complex I and complex III substrates; (ii) lowest rate of mitochondrial hydrogen peroxide production from complex III; and (iii) highest cytochrome c oxidase activity from complex IV. Rate of hydrogen peroxide production increased and cytochrome c oxidase activity decreased in all lines in the age range 11-25 days. This is the first study to correlate natural variation in organism survival with natural variation in mitochondrial bioenergetics.     

Mitochondrial DNA variation is associated with measurable differences in life-history traits and mitochondrial metabolism in Drosophila simulans

Recent studies have used a variety of theoretical arguments to show that mitochondrial (mt) DNA rarely evolves as a strictly neutral marker and that selection operates on the mtDNA of many species. However, the vast majority of researchers are not convinced by these arguments because data linking mtDNA variation with phenotypic differences are limited. We investigated sequence variation in the three mtDNA and nine nuclear genes (including all isoforms) that encode the 12 subunits of cytochrome c oxidase of the electron transport chain in Drosophila. We then studied cytochrome c oxidase activity as a key aspect of mitochondrial bioenergetics and four life-history traits. In Drosophila simulans, sequence data from the three mtDNA encoded cytochrome c oxidase genes show that there are 76 synonymous and two nonsynonymous fixed differences among flies harboring siII compared with siIII mtDNA. In contrast, 13 nuclear encoded genes show no evidence of genetic subdivision associated with the mtDNA. Flies with siIII mtDNA had higher cytochrome c oxidase activity and were more starvation resistant. Flies harboring siII mtDNA had greater egg size and fecundity, and recovered faster from cold coma. These data are consistent with a causative role for mtDNA variation in these phenotypic differences, but we cannot completely rule out the involvement of nuclear genes. The results of this study have significant implications for the use of mtDNA as an assumed neutral marker and show that evolutionary shifts can involve changes in mtDNA despite the small number of genes encoded in the organelle genome.     

Nucleotide sequence divergence in the A+T-rich region of mitochondrial DNA in Drosophila simulans and Drosophila mauritiana

We have determined the nucleotide sequences of two regions within the A+T-rich region of mitochondrial DNA (mtDNA) in the siIII type of Drosophila simulans and the maI type of D. mauritiana. The sequences of the two regions in siIII and maI are almost identical. The sequences include elements corresponding to the type I and type II repeats elements and the T-stretches as reported in D. melanogaster; an approximately 340-bp region (A region) adjacent to the tRNA(Ile) gene includes a part of the type II repeat element, and an approximately 440- bp region (B region) includes a central portion of the A+T-rich region between the type I and type II repeat arrays. Each sequence of the two species was compared with those of D. melanogaster and D. yakuba. The sequences of the A region are relatively well conserved among the four species. The alignment of the two sequences of the B region with those of D. melanogaster and D. yakuba requires numerous insertions/deletions. For both regions, nucleotide differences between D. simulans or D. mauritiana and D. melanogaster are similar to those between the two and D. yakuba. The tendency is obvious in a subregion within the type II repeat element in the A region. These findings suggest that the rate of nucleotide substitution in the subregion is accelerated in the lineage leading to D. melanogaster. Loss of functional constraint in the stem-loop-forming sequence is proposed for this acceleration.      

Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans

Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS–PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.     

Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

Evolutionary implications of mitochondrial genetic variation: mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies

The ancient acquisition of the mitochondrion into the ancestor of modern‐day eukaryotes is thought to have been pivotal in facilitating the evolution of complex life. Mitochondria retain their own diminutive genome, with mitochondrial genes encoding core subunits involved in oxidative phosphorylation. Traditionally, it was assumed that there was little scope for genetic variation to accumulate and be maintained within the mitochondrial genome. However, in the past decade, mitochondrial genetic variation has been routinely tied to the expression of life‐history traits such as fertility, development and longevity. To examine whether these broad‐scale effects on life‐history trait expression might ultimately find their root in mitochondrially mediated effects on core bioenergetic function, we measured the effects of genetic variation across twelve different mitochondrial haplotypes on respiratory capacity and mitochondrial quantity in the fruit fly, Drosophila melanogaster. We used strains of flies that differed only in their mitochondrial haplotype, and tested each sex separately at two different adult ages. Mitochondrial haplotypes affected both respiratory capacity and mitochondrial quantity. However, these effects were highly context‐dependent, with the genetic effects contingent on both the sex and the age of the flies. These sex‐ and age‐specific genetic effects are likely to resonate across the entire organismal life‐history, providing insights into how mitochondrial genetic variation may contribute to sex‐specific trajectories of life‐history evolution.     

On the nature of electron and energy transport in mitochondria. I. Multiple inhibition of mitochondrial respiration

1. A series of eight classical respiratory-chain inhibitors was studied. The slopes of State-3 respiratory rate versus dose plots are convex for antimycin, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), rotenone and sulfide, and concave for malonate, Amytal, cyanide and azide.

2. Plots of ADP: O ratio versus dose indicate uncoupling effects at higher concentrations of antimycin, HOQNO, cyanide and azide. On the other hand, sulfide and rotenone have no effect on the phosphorylating efficiency. Malonate increases the ADP: O ratio.

3. Two inhibitors can be combined in such a way that the total inhibition should be equal to the inhibition caused by the single inhibitors if each inhibitor affects respiration independently (additivity of inhibition). In practice, however, antagonism and synergism are also found.

4. Additivity of combined inhibition occurs where both inhibitors act on the same enzyme.

5. Antagonism is observed where the two inhibitors act on different enzymes of the same chain.

6. Synergism is found where the two inhibitors act on enzymes in different branches of a forked chain. This turns into normal additivity when the electron flow through both branches is made equal.

7. The results are compatible with the hypothesis that respiratory enzymes are arranged in chains. The possibility that the chains may be cross-linked or branched is discussed.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Differential fitness of mitochondrial DNA in perturbation cage studies correlates with global abundance and population history in Drosophila simulans

Mitochondria are often referred to as the powerhouse of the cell. However, research linking intraspecific differences in organismal fitness with genotypic mitochondrial DNA (mtDNA) variation has been hampered by the lack of variation in experimentally tractable species. This study examines whether fly lines harbouring three distinct Drosophila simulans mtDNA types (siI, -II and -III) exhibit differential fitness in laboratory perturbation cages. Comparison of the pre-perturbation and post-perturbation data shows that both the mtDNA and mitonuclear interactions have a significant and repeatable effect on the frequency of flies with specific genotypes in population cages (siII > -III > -I) and that coadapted mitonuclear interactions are greatest in the siI type. The rank order of mtDNA frequency correlates with the observed worldwide distribution of the haplogroups while mitonuclear interactions are most significant in the siI haplogroup that is likely to have been subject to repeated population bottlenecks. One possible explanation for the maintenance of the least fit siI haplogroup on Pacific islands is that it is protected from extinction by Wolbachia infection.     

Nuclear-mitochondrial epistasis and drosophila aging: introgression of Drosophila simulans mtDNA modifies longevity in D. melanogaster nuclear backgrounds

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 (approximately 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.     

Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans

We have analysed the viability of cellular clones induced by mitotic recombination in Drosophila melanogaster/D. simulans hybrid females during larval growth. These clones contain a portion of either melanogaster or simulans genomes in homozygosity. Analysis has been carried out for the X and the second chromosomes, as well as for the 3L chromosome arm. Clones were not found in certain structures, and in others they appeared in a very low frequency. Only in abdominal tergites was a significant number of clones observed, although their frequency was lower than in melanogaster abdomens. The bigger the portion of the genome that is homozygous, the less viable is the recombinant melano-gaster/simulans hybrid clone. The few clones that appeared may represent cases in which mitotic recombination took place in distal chromosome intervals, so that the clones contained a small portion of either melanogaster or simulans chromosomes in homozygosity. Moreover, Lhr, a gene of D. simulans that suppresses the lethality of male and female melanogaster/simulans hybrids, does not suppress the lethality of the recombinant melanogaster/simulans clones. Thus, it appears that there is not just a single gene, but at least one per tested chromosome arm (and maybe more) that cause hybrid lethality. Therefore, the two species, D. melanogaster and D. simulans, have diverged to such a degree that the absence of part of the genome of one species cannot be substituted by the corresponding part of the genome of the other, probably due to the formation of co-adapted gene complexes in both species following their divergent evolution after speciation. The disruption of those coadapted gene complexes would cause the lethality of the recombinant hybrid clones.     

Recombination and the frequency spectrum in Drosophila melanogaster and Drosophila simulans

Most "tests of neutrality" assess whether particular data sets depart from the predictions of a standard neutral model with no recombination. For Drosophila, where nuclear polymorphism data routinely show evidence of genetic exchange, the assumption of no recombination is often unrealistic. In addition, while conservative, this assumption is made at the cost of a great loss in power. Perhaps as a result, tests of the frequency spectrum based on zero recombination suggest an adequate fit of Drosophila polymorphism data to the predictions of the standard neutral model. Here, we analyze the frequency spectrum of a large number of loci in Drosophila melanogaster and D. simulans using two summary statistics. We use an estimate of the population recombination rate based on a laboratory estimate of the rate of crossing over per physical length and an estimate of the species' effective population size. In contrast to previous studies, we find that roughly half of the loci depart from the predictions of the standard neutral model. The extent of the departure depends on the exact recombination rate, but the global pattern that emerges is robust. Interestingly, these departures from neutral expectations are not unidirectional. The large variance in outcomes may be due to a complex demographic history and inconsistent sampling, or to the pervasive action of natural selection.     

High divergence among Drosophila simulans mitochondrial haplogroups arose in midst of long term purifying selection

We characterize the type of selection acting within and among mitochondrial lineages in five closely related Drosophila species. We focus on D. simulans, where three genetically distinct mitochondrial haplogroups show high interhaplogroup divergence and low intrahaplogroup polymorphism. Using maximum likelihood models we find that the branches leading to these three distinct mitochondrial groups show a significantly reduced rate of nonsynonymous relative to synonymous substitution. This interhaplogroup rate is significantly reduced compared to the intrahaplogroup rate, and closely resembles the rate observed between distinct species. The data suggest that slightly deleterious mutations segregating within D. simulans haplogroups are removed by selection prior to their fixation among haplogroups. We explore several hypotheses to explain how lineages within a single species can be compatible with this model of slightly deleterious mutation. The most likely hypothesis is that D. simulans haplogroups have persisted in isolation, perhaps due to association with the bacterial symbiont Wolbachia and/or demographic history, introducing a bias against the fixation of slightly deleterious mutations.