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Historically, mtDNA was considered a selectively neutral marker that was useful for estimating the population genetic history of the maternal lineage. Over time there has been an increasing appreciation of mtDNA and mitochondria in maintaining cellular and organismal health. Beyond energy production, mtDNA and mitochondria have critical cellular roles in signalling. Here we briefly review the structure of mtDNA and the role of the mitochondrion in energy production. We then discuss the predictions that can be obtained from quaternary structure modelling and focus on mitochondrial complex I. Complex I is the primary entry point for electrons into the electron transport system is the largest respiratory complex of the chain and produces about 40% of the proton flux used to synthesize ATP. A focus of the review is Drosophila's utility as a model organism to study the selective advantage of specific mutations. However, we note that the incorporation of insights from a multitude of systems is necessary to fully understand the range of roles that mtDNA has in organismal fitness. We speculate that dietary changes can illicit stress responses that influence the selective advantage of specific mtDNA mutations and cause spatial and temporal fluctuations in the frequencies of mutations. We conclude that developing our understanding of the roles mtDNA has in determining organismal fitness will enable increased evolutionary insight and propose we can no longer assume it is evolving as a strictly neutral marker without testing this hypothesis.

Background: Basenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits make understanding their genome structure relative to more modern dog breeds of great interest. Here, we report the de novo assemblies of two Basenji: a female, China, and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). We then align representative whole genome sequences from 58 dog breeds and show the importance of genome reference when assessing variation among dog breeds. Results: Here we present two high quality Basenji genome assemblies, CanFam_Bas (China) and Wags. CanFam_Bas is superior to CanFam v3,1 is terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection. Further, we generate a conservative list of structural variant calls using a consensus of both Pacific Bioscience and Oxford Nanopore long reads to identify large structural breed differences. Collectively this work highlights the importance the choice of reference genome makes in canid variation studies. Conclusions: The growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, as is increasingly being employed in other model organisms, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach.

Increasing evidence in animal species ranging from mammals to insects has revealed phenotypes that are caused by ancestral life experiences including stress and diet. The descendent phenotypes themselves are wide ranging, and include changes to behaviour, disease risk, metabolism, and growth. Ancestral dietary macronutrient composition, and quantity (over- and under-nutrition) have been shown to alter descendent growth, metabolism and behaviour. Several studies have identified inherited molecules in gametes which are altered by ancestral diet and are required for the transgenerational effect. However, there is less understanding of the developmental pathways in the period between fertilisation and adulthood that are altered by the inherited molecules. Here we identify a key role of the MAPK signalling pathway in mediating changes to Drosophila larval developmental timing due to variation in ancestral diet. We exposed grand-parental and great grand-parental generations to defined protein to carbohydrate (P:C) dietary ratios and measured developmental timing. Descendent developmental timing was consistently faster in the period between the embryonic and pupal stages when the ancestor had a higher P:C ratio diet. Transcriptional analysis of embryos, larvae and adults revealed extensive and long-lasting changes to the MAPK signalling pathway which controlled growth rate through regulation of ribosomal RNA transcription. The importance of these processes was supported by pharmacological inhibition of MAPK and rRNA proteins which reproduced the ancestral diet-induced developmental changes. This work provides insight into the role of developmental growth signalling networks in mediating non-genetic inheritance in the period between fertilisation and adult.Summary statementAncestral, diet-induced descendent developmental timing changes are caused by alteration of MAPK signalling pathways in the period between the embryo and pupal stages in Drosophila.

In this review, we provide evidence to suggest that the cost of specific mtDNA mutations can be influenced by exogenous factors. We focus on macronutrient-mitochondrial DNA interactions as factors that may differentially influence the consequences of a change as mitochondria must be flexible in its utilization of dietary proteins, carbohydrates, and fats. To understand this fundamental dynamic, we briefly discuss the energy processing pathways in mitochondria. Next, we explore the mitochondrial functions that are initiated during energy deficiency or when cells encounter cellular stress. We consider the anterograde response (nuclear control of mitochondrial function) and the retrograde response (nuclear changes in response to mitochondrial signaling) and how this mito-nuclear crosstalk may be influenced by exogenous factors such as temperature and diet. Finally, we employ Complex I of the mitochondrial electron transport system as a case study and discuss the potential role of the dietary macronutrient ratio as a strong selective force that may shape the frequencies of mitotypes in populations and species. We conclude that this underexplored field likely has implications in the fundamental disciplines of evolutionary biology and quantitative genetics and the more biomedical fields of nutrigenomics and pharmacogenomics.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased β-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson’s Disease., Author summary The detection and quantitation of mtDNA polymorphisms in populations and across whole habitats continues to be used as a central investigatory tool in evolutionary genetics. But, the approach is laden with assumptions about selection that are rarely examined. We present a series of studies that traverse the genotype to the phenotype. The studies were designed to experimentally test the interaction between diet and mitotype in Drosophila flies and provide a mechanism by which selection occurs. We start with population cage studies that include four laboratory diets and four mitotypes. We then directly compete two mitotypes (Alstonville and Dahomey) on a high protein and a high carbohydrate diet and show a flip in their relative fitness that is driven by differences in larval development. Next, we identify a single naturally-occurring point mutation, which drives the cage results. We track the ripple effects up to the level of the organelle (mitochondria), through the labyrinth of metabolic pathways and on to the phenotype. Notably, when flies were fed the high carbohydrate diet, energy metabolism was extensively remodelled in both mitotypes causing increased physical activity in Alstonville flies. These data invite an extensive experimental re-evaluation of the assumption that mtDNA inescapably evolves in a manner consistent with a strictly neutral equilibrium model. It also motivates investigation of genotype-specific dietary manipulation as an integrative treatment of human disorders involving mitochondrial metabolism and offers the potential for future therapeutic strategies.

Mitochondrial dysfunction and resulting energy impairment have been identified as features of many neurodegenerative diseases. Whether this energy impairment is the cause of the disease or the consequence of preceding impairment(s) is still under discussion, however a recovery of cellular bioenergetics would plausibly prevent or improve the pathology. In this study, we screened different natural molecules for their ability to increase intracellular adenine triphosphate purine (ATP). Among them, epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, presented the most striking results. We found that it increases ATP production in both human cultured astrocytes and neurons with different kinetic parameters and without toxicity. Specifically, we showed that oxidative phosphorylation in human cultured astrocytes and neurons increased at the level of the routine respiration on the cells pre-treated with the natural molecule. Furthermore, EGCG-induced ATP production was only blocked by sodium azide (NaN3) and oligomycin, inhibitors of cytochrome c oxidase (CcO; complex IV) and ATP synthase (complex V) respectively. These findings suggest that the EGCG modulates CcO activity, as confirmed by its enzymatic activity. CcO is known to be regulated differently in neurons and astrocytes. Accordingly, EGCG treatment is acting differently on the kinetic parameters of the two cell types. To our knowledge, this is the first study showing that EGCG promotes CcO activity in human cultured neurons and astrocytes. Considering that CcO dysfunction has been reported in patients having neurodegenerative diseases such as Alzheimer's disease (AD), we therefore suggest that EGCG could restore mitochondrial function and prevent subsequent loss of synaptic function.

Summary 1. The vast majority of studies employing mtDNA in evolutionary biology and ecology have used it as a means to infer demographic and historical patterns without pondering the underlying functional implications. In contrast, the biochemical and medical communities often aim to understand the influence of specific mtDNA mutations on mitochondrial functions, but rarely consider the evolutionary and ecological implications. 2. Ongoing research has shown that mtDNA mutations can profoundly affect mitochondrial function in humans and other animals. If the mutation (or set of mutations) is pathogenic, mitochondrial malfunction may be detected from early age. In nature, however, most mutations are not highly deleterious and may exist at intermediate frequency in populations. 3. In this review, we suggest that knowledge of the underlying biochemistry and functions of mitochondria can facilitate a more complete determination of the evolutionary dynamics of mtDNA and its influence on the life-history traits of organisms. With this approach, it is possible to use biochemistry to link the genotype with the phenotype. 4. After reviewing the literature, we conclude that there can be physiological and evolutionary trade-offs in the way that mitochondrial mutations can affect age classes and/or fitness components and that these effects may depend on the environment. Through these trade-offs, it may be possible for specific mtDNA mutations to have unequal fitness in different nuclear genetic backgrounds and also in different environments.

Cytochrome c oxidase (COX) of the electron transport system is thought to be the rate-limiting step in cellular respiration and is found mutated in numerous human pathologies. Here, we employ quaternary three-dimensional (3-D) modeling to construct a model for human COX. The model was used to predict the functional consequences of amino-acid mutations based on phylogenetic conservation of amino acids together with volume and/or steric perturbations, participation in subunit-subunit interfaces and non-covalent energy loss or incompatibilities. These metrics were combined and interpreted for potential functional impact. A notable strength of the 3-D model is that it can interpret and predict the structural consequences of amino-acid variation in all 13 protein subunits. Importantly, the influence of compensatory changes can also be modeled. We examine mutations listed in the human mutation database Mitomap, and in 100 older men, and compare the results from the 3-D model against the automated MutPred web application tool. In combination, these comparisons suggest that the 3-D model predicts more functionally significant mutations than does MutPred. We conclude that the model has useful functional prediction capability but may need modification as functional data on specific mutations becomes known.

The aim of the study was to determine the accuracy of near-infrared spectroscopy (NIRS) in determining species, gender, age, and the presence of the common endosymbiont Wolbachia in laboratory-reared Drosophila. NIRS measures the absorption of light by organic molecules. Initially, a calibration model was developed for each study. An independent set with flies not involved in initial cross-validation was then used to validate the accuracy of each calibration model. Flies from the independent sets were correctly classified into Drosophila melanogaster and Drosophila simulans with 94% and 82% accuracy, respectively, whereas flies were successfully classified by gender with accuracy greater than 90%. In the age grading test, correlation plots of the actual and predicted age for males and females of D. melanogaster and D. simulans were shown to be overlapping between the adjacent age groups. It is, however, possible to predict the age of flies as less than 9 days of age with 62–88% accuracy and flies that are equal to or older than 9 days of age with 91–98% accuracy. Finally, we used NIRS to detect the presence of Wolbachia in flies. Flies from the independent sets were successfully identified as infected or not infected with Wolbachia with approximately 90% accuracy. These results suggest that NIRS has the potential to quantify the species, gender, and presence of Wolbachia in fly populations. However, additional optimization of the protocol may be necessary before the technique can reliably estimate fly age.

Linking the mitochondrial genotype and the organismal phenotype is of paramount importance in evolution of mitochondria. In this study, we determined the differences in catalytic properties of mitochondria dictated by divergences in the siII and siIII haplogroups of Drosophila simulans using introgressions of siII mtDNA type into the siIII nuclear background. We used a novel in situ method (permeabilized fibers) that allowed us to accurately measure the consumption of oxygen by mitochondria in constructed siII-introgressed flies and in siIII-control flies. Our results showed that the catalytic capacity of the electron transport system is not impaired by introgressions, suggesting that the functional properties of mitochondria are tightly related to the mtDNA haplogroup and not to the nuclear DNA or to the mito-nuclear interactions. This is the first study, to our knowledge, that demonstrates a naturally occurring haplogroup can confer specific functional differences in aspects of mitochondrial metabolism. This study illustrates the importance of mtDNA changes on organelle evolution and highlights the potential bioenergetic and metabolic impacts that divergent mitochondrial haplogroups may have upon a wide variety of species including humans.

Enzymatic amplification of homologous regions of DNA using ‘universal’ polymerase chain reaction primers has provided insight into insect systematics, phylogeography, molecular evolution and species identification. One of the more commonly amplified and sequenced regions is a short region of the cytochrome c oxidase subunit I gene (COI), commonly called the barcoding region. COI is one of three mitochondrial-encoded subunits of complex IV (Cox) of the electron transport chain. In addition to the mitochondrial subunits there are nine nuclear-encoded subunits of the complex in Drosophila. Whereas a number of phylogenetic biases associated with this region have been examined and the quaternary structure of Cox has been modelled, the influence of protein–protein interactions on the observed patterns of evolution in this barcoding region of insects has never been examined critically. Using a well-resolved independently derived phylogeny of 38 Diptera species, we examined the homogeneity of the substitution processes within the barcoding region. We show that, within Diptera, amino acid residues interacting with nuclear-encoded subunits of Cox are evolving at elevated rates across the phylogeny. Furthermore, we show that codon position two is biased by protein–protein interactions. In contrast, third codon positions provide a less biased estimate of genetic variation in the region. This study highlights the need to examine the potential for systematic bias in DNA barcoding regions as part of the critical assessment of evidence in systematics and in biodiversity assessments.

Males and females age at different rates in a variety of species, but the mechanisms underlying the difference is not understood. In this study, we investigated sex-specific costs of a naturally occurring mildly deleterious deletion (DTrp85, DVal86) in cytochrome c oxidase subunit 7A (cox7A) in Drosophila simulans. We observed that females and males homozygous for the mutation had 30% and 26% reduced Cox activity, respectively, compared with wild type. Furthermore, 4-day-old females had 34%-42% greater physical activity than males. Greater physical activity in mutant females was correlated with a 19% lower 50% survival compared with wild-type females. Mutant and wild-type males had equal survival. These data suggest that females paid a higher cost of the mutation than did males. The data demonstrate linking population genetics and structural modeling to experimental manipulations that lead to functional predictions of mitochondrial bioenergetics and organism aging.

Linking naturally occurring genotypic variation to the organismal phenotype is critical to our understanding of, and ability to, model biological processes such as adaptation to novel environments, disease, and aging. Rarely, however, does a simple mutation cause a single simple observable trait. Rather it is more common for a mutation to elicit an entangled web of responses. Here, we employ biochemistry as the thread to link a naturally occurring two amino acid deletion in a nuclear encoded mitochondrial protein with physiological benefits and costs in the fly Drosophila simulans. This nuclear encoded gene produces a protein that is imported into the mitochondrion and forms a subunit of complex IV (cytochrome c oxidase, or cox) of the electron transport chain. We observe that flies homozygous for the deletion have an advantage when young but pay a cost later in life. These data show that the organism responds to the deletion in a complex manner that gives insight into the mechanisms that influence mitochondrial bioenergetics and aspects of organismal physiology.

Stress resistance traits in Drosophila often show clinal variation, suggesting that selection affects resistance traits either directly or indirectly. One of the most common causes of stress for animals is the shortage or suboptimal quality of food, and individuals within many species must survive periods of starvation or exposure to nutritionally imbalanced diets. This study determines the relationship between starvation resistance, body lipid content, and lifespan in five recently collected Drosophila simulans populations from four distinct geographic localities. Despite rearing under standard nutritional conditions, we observed significant differences in starvation resistance between sexes and between localities. If body lipid proportion is included as a covariate in statistical analysis the difference between the sexes remains (slopes are parallel, with males more susceptible than females to starvation across all lipid proportions) but the effect of locality disappears. This result suggests that flies from different localities differ in their susceptibility to starvation because of differences in their propensity to store body lipid. We observed a negative relationship between lifespan and starvation resistance in both males and females, suggesting a fitness cost to increasing lipid reserves. These data raise issues about the role of diet in maintaining life history trait variation within and among populations. In conclusion, we show many similarities and surprising differences in life history traits between D. simulans and Drosophila melanogaster.

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach.

The hypothesis that metabolic rate is inversely correlated with life span has long been debated. Another area of controversy has been the relationship between metabolic rate and aging. In most molecular studies key aspects of cellular metabolism have been shown to decline with age. Less attention has been focused on metabolic rate as an organism ages. We studied the survival of three Drosophila simulans fly lines and measured whole organism metabolic rate, mitochondrial DNA copy number and walking speed. Metabolic rate as assayed by CO 2 production did not correlate with median lifespan but increased by 0.43–1.14%/d. In contrast, mitochondrial DNA copy number decreased by 0.56–1.06%/d. Physical activity, as assayed by mean walking speed, did not change with age but was positively correlated with mitochondrial DNA copy number. One explanation for these data is that metabolic rate was increased, in the face of a reduced mitochondrial DNA copy number and capacity for oxidative metabolism, to maintain a constant bioenergetic demand (physical activity). Alternatively, metabolic rate may increase to provide energy for the repair of cellular damage or due to a shift in metabolic substrate use over time.

A common feature across all animals, including humans, is that mitochondrial bioenergetics is linked to oxidative stress, but the nature of these relationships with survival is yet to be properly defined. In this study we included 12 Drosophila simulans isofemale lines: four of each distinct mtDNA haplogroup (siI, -II, and -III). First, we investigated sequence variation in six mtDNA and 13 nuclear encoded genes (nine nuclear-encoded subunits, and the four known isoforms, of complex IV of the electron transport chain). As expected we observed high divergence among the three distinct mitotypes and greatest mtDNA variability in siII-harboring flies. In the nuclear encoded genes, no fixed amino acid differences were observed and levels of polymorphism did not differ significantly among flies harboring distinct mtDNA types. Second, 15,456 flies were included in mortality studies. We observed that mtDNA type influenced survival (siII approximately siIII > siI), flies harboring siII mtDNA had the greatest variation in mortality rates, and in all cases males were longer lived than females. We also assayed maximal rates of hydrogen peroxide (H(2)O(2)) production from complex III of the electron transport chain in mitochondria isolated from 11-day-old flies. Contrary to our prediction, rates of H(2)O(2) production tended to increase with mean survival. This result suggests that higher rates of H(2)O(2) production in younger flies may lead to an upregulation of antioxidants, age-dependent increase in the rate of H(2)O(2) production differ, and/or flies vary in their mitochondrial uncoupling. Alternatively, the whole organism may not regularly, if ever, experience maximal H(2)O(2) production rates.

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 si II and si III mtDNA types. Previously we have shown that males harboring si II mtDNA had higher rates of mitochondrial H 2 O 2 production from complex III at 11 d compared to males with the si III mtDNA type. Here, we corroborate those results and show that Drosophila harboring the si II and si III mtDNA types exhibit significantly different patterns of pro-oxidant and antioxidant activities as they age. Flies harboring si II mtDNA had higher rates of mitochondrial H 2 O 2 production and manganese superoxide dismutase activity at 11 and 18 d of age than si III mtDNA harboring flies. Copper–zinc superoxide dismutase activity increased from 11 to 25 d in si II flies while the accumulation of oxidized glutathione did not change between 11 and 25 d. In contrast, si III harboring flies showed an age dependent increase in H 2 O 2 production, reaching higher production rates on day 25 than that observed in si II flies. Copper–zinc superoxide dismutase activities did not change between 11 and 25 d while the oxidized glutathione accumulation increased with age. The results show antioxidant levels correlate with pro-oxidant levels in si II but not si III flies. These results demonstrate our ability to correlate mtDNA variation with differences in whole mitochondrial physiology and individual complex biochemistry.

This review explores the potential for changes in dietary macronutrients to differentially influence mitochondrial bioenergetics and thereby the frequency of mtDNA haplotypes in natural populations. Such dietary modification may be seasonal or result from biogeographic or demographic shifts. Mechanistically, mtDNA haplotypes may influence the activity of the electron transport system (ETS), retrograde signalling to the nuclear genome and affect epigenetic modifications. Thus, differential provisioning by macronutrients may lead to selection through changes in the levels of ATP production, modulation of metabolites (including AMP, reactive oxygen species (ROS) and the NAD+/NADH ratio) and potentially complex epigenetic effects. The exquisite complexity of dietary influence on haplotype frequency is further illustrated by the fact that macronutrients may differentially influence the selective advantage of specific mutations in different life-history stages. In Drosophila, complex I mutations may affect larval growth because dietary nutrients are fed through this complex in immaturity. In contrast, the majority of electrons are provided to complex III in adult flies. We conclude the review with a case study that considers specific interactions between diet and complex I of the ETS. Complex I is the first enzyme of the mitochondrial ETS and co-ordinates in the oxidation of NADH and transfer of electrons to ubiquinone. Although the supposition that mtDNA variants may be selected upon by dietary macronutrients could be intuitively consistent to some and counter intuitive to others, it must face a multitude of scientific hurdles before it can be recognized.

Mitochondria generate much of the cellular energy through the process of oxidative phosphorylation (OXPHOS). OXPHOS function may be affected by a mutation in any of the mitochondrial (mt) DNA encoded genes and/or nuclear encoded genes that produce proteins imported into the mitochondrion that form (or assemble) the five complexes located on the inner mitochondrial membrane. Decreasing mitochondrial OXPHOS efficiency, defined as the amount of inorganic phosphate incorporated into ATP per mole of oxygen consumed (ADP:O ratio), results in an increase in the production of reactive oxygen species (ROS). ROS damages mtDNA, cell membranes, and lipids and, according to the free radical theory of aging, the rate of its accumulation is a major factor affecting lifespan. In humans, studies of age-specific cohorts suggest that extended longevity is associated with specific mtDNA types (mitotypes). However, the influence of each mitotype appears to be dependent on the nuclear genetic background in which it resides. These association studies are intuitively appealing, but since the age groups are necessarily from different cohorts there is no rigorous means of eliminating spurious associations due to accidents of sampling or environment. The critical need is to determine the extent to which (i) mitochondrial encoded genes, (ii) nuclear encoded genes whose products are imported into the mitochondrion, and (iii) interactions among mtDNA and nuclear (mitonuclear) encoded proteins, influence mitochondrial metabolism and life history traits including survival. This need can be efficiently accomplished in the fly Drosophila simulans owing to its (i) high mtDNA diversity, (ii) ease of genetic and experimental manipulations, (iii) sequence of multiple complete genomes, (iv) short generation time, (v) ease of collection and (vi) ability to raise large numbers so that sex and age-specific effects can be determined. The utility of the fly model is supported by the conservation of the OXPHOS pathway and the mitochondrial genome in humans, mice and Drosophila.

Reconstructing ancestral geographic origins is critical for understanding the long-term evolution of a species. Bayesian methods have been proposed to test biogeographic hypotheses while accommodating uncertainty in phylogenetic reconstruction. However, the problem that certain taxa may have a disproportionate influence on conclusions has not been addressed. Here, we infer the geographic origin of Drosophila simulans using 2014 bp of the period locus from 63 lines collected from 18 countries. We also analyze two previously published datasets, alcohol dehydrogenase related and NADH:ubiquinone reductase 75 kDa subunit precursor. Phylogenetic inferences of all three loci support Madagascar as the geographic origin of D. simulans. Our phylogenetic conclusions are robust to taxon resampling and to the potentially confounding effects of recombination. To test our phylogenetically derived hypothesis we develop a randomization test of the population genetics prediction that sequences from the geographic origin should contain more genetic polymorphism than those from derived populations. We find that the Madagascar population has elevated genetic polymorphism relative to non-Madagascar sequences. These data are corroborated by mitochondrial DNA sequence data.

This study aims to unravel the biogeography of a model symbiont/host system by exploiting the prediction that a symbiont will leave a signature of infection on the host. Specifically, a global sample of 1,442 Drosophila simulans from 33 countries and 64 sampling localities was employed to infer the phylogeography of the maternally inherited alpha-proteobacteria Wolbachia. Phylogenetic analyses, from three symbiont genes and 24 mtDNA genomes (excluding the A + T-rich region), showed that each of four Wolbachia strains infected D. simulans once. The global distribution and abundance of the Wolbachia strains and the three mtDNA haplogroups (D. simulans siI, siII and siIII) was then determined. Finally, network analyses of variable regions within siI (584 bp from seven additional lines) and siII (1,701 bp from 383 lines) facilitated a detailed biogeographic discussion. There is little variation in siIII and the haplogroup is restricted in its distribution. These data show how the history of an infection can be mapped by combining data from the symbiont and the host. They say little about the organismal history of the host because the mtDNA genome is a biased representation of the whole genome.

We ask whether the observed mitochondrial DNA (mtDNA) population subdivision of Drosophila simulans is indicative of organismal structure or of specific processes acting on the mitochondrial genome. Factors either intrinsic or extrinsic to the host genome may influence the evolutionary dynamics of mtDNA. Potential intrinsic factors include adaptation of the mitochondrial genome and of nucleomitochondrial gene complexes specific to the local environment. An extrinsic force that has been shown to influence mtDNA evolution in invertebrates is the bacterial endosymbiont Wolbachia. Evidence presented in this study suggests that mtDNA is not a good indicator of organismal subdivision in D. simulans. Furthermore, there is no evidence to suggest that Wolbachia causes any reduction in nuclear gene flow in this species. The observed differentiation in mtDNA is not corroborated by data from NADH: ubiquinone reductase 75kD subunit precursor or the Alcohol dehydrogenase-related loci, from the shape or size of the male genital arch, or from assortative premating behavior. We discuss these results in relation to a mitochondrial genetic species concept and the potential for Wolbachia-induced incompatibility to be a mechanism of speciation in insects. We conclude with an iterated appeal to include phylogenetic and statistical tests of neutrality as a supplement to phylogenetic and population genetic analyses when using mtDNA as an evolutionary marker.

Here we determine the sex-specific influence of mtDNA type (mitotype) and diet on mitochondrial functions and physiology in two Drosophila melanogaster lines. In many species, males and females differ in aspects of their energy production. These sex-specific influences may be caused by differences in evolutionary history and physiological functions. We predicted the influence of mtDNA mutations should be stronger in males than females as a result of the organelle's maternal mode of inheritance in the majority of metazoans. In contrast, we predicted the influence of diet would be greater in females due to higher metabolic flexibility. We included four diets that differed in their protein: carbohydrate (P:C) ratios as they are the two-major energy-yielding macronutrients in the fly diet. We assayed four mitochondrial function traits (Complex I oxidative phosphorylation, reactive oxygen species production, superoxide dismutase activity, and mtDNA copy number) and four physiological traits (fecundity, longevity, lipid content, and starvation resistance). Traits were assayed at 11 d and 25 d of age. Consistent with predictions we observe that the mitotype influenced males more than females supporting the hypothesis of a sex-specific selective sieve in the mitochondrial genome caused by the maternal inheritance of mitochondria. Also, consistent with predictions, we found that the diet influenced females more than males.

In this study, we examine the dietary protein to carbohydrate ratio (P:C) on the mitochondrial functions of two Drosophila melanogaster mtDNA haplotypes. We investigated multiple physiological parameters on flies fed with either 1:12 P:C or 1:3 P:C diets. Our results provide experimental evidence that a specific haplotype has a reduction of complex I activity when the flies are fed with the 1:12 P:C diet. This study is of particular importance to understand the influence of diet on mitochondrial evolution in invasive and broadly distributed species including humans.

Variation and change in mitochondrial DNA (mtDNA) is often assumed to conform to a constant mutation rate equilibrium neutral model of molecular evolution. Recent evidence, however, indicates that the assumptions underlying this model are frequently violated. The mitochondria) genome may be subject to the same suite of forces known to be acting in the nuclear genome, including hitchhiking and selection, as well as forces that do not affect nuclear variation. Wherever possible, evolutionary studies involving mtDNA should incorporate statistical tests to investigate the forces shaping sequence variation and evolution.

Chromosome conformation capture (3C) is a powerful technique that allows for the generation of 3-dimensional transcriptome organisational maps. 3C facilitates the identification and quantitative measurement of distant chromosomal regulatory elements with proximal gene promoter regions. This is achieved by fixing protein onto deoxyribonucleic acid (DNA) followed by restriction enzyme digestion or sonication to shear the DNA prior to re-ligation of the fixed DNA region. PCR amplification of a chromosomal region of interest can then be performed and quantified. Next generation sequencing platforms on prepared 3C chromatin can provide information on whole genome regulatory networks. The technology and its modified derivatives provide high levels of resolution and throughput for determining nuclear organisation and allows for the identification of novel noncoding DNA regions that are associated with controlling gene expression. 3C is providing important insights into underlying mechanisms that may be responsible for various human diseases including cancer, muscular dystrophies, neurological and metabolic disorders. Key Concepts: Chromosome conformation capture (3C) is a key strategy used to determine distant chromosomal regulatory regions involved with controlling gene expression. Single nucleotide polymorphic (SNP) DNA variations in noncoding region DNA can now be interrogated to determine a functional role in altering gene expression. The 3-dimensional chromosome looping architecture can be dissected to determine multiple chromosomal regions involved with regulating gene expression through transcription factory interaction. Mutations in distant chromosomal regulatory regions may lead to uncontrolled repression of oncogenes allowing for increases in tumour growth and development. Identifying noncoding chromosomal regions associated with nuclear adaptive mechanisms in response to DNA mutation may allow us to determine critical regions involved with maintaining cellular homoeostasis. High-throughput sequencing strategies are allowing for unparalleled identification of DNA variation in the human genome. Keywords: chromosome conformation capture; gene regulation; SNP; cancer; human disease; mutation; malfunction; enhancers; repressors

The aim of this study was to investigate the effect of temperature changes on the functional properties of mitochondria from two sets of D. simulans fly lines harboring the siII and siIII haplotypes in a common nuclear genetic background. We studied four introgressed isofemale lines possessing the mtDNA of siII and the nuclear background of siIII (siII-introgressed) and four lines possessing siIII mitochondria with its native nuclear genome (siIII-controls). We assessed the catalytic capacities of electron transport system (ETS) at four different temperatures (12, 18, 24 and 28 ºC). The impact of temperature on the pyruvate dehydrogenase (PDH) activity, the mitochondrial respiration (coupled and uncoupled respiration), cytochrome c oxidase activity, as well as the excess capacity of complex IV (COX) were evaluated in these two sets of flies. Our results showed that the temperature coefficient values (Q(10)) measured for mitochondrial respiration in the lower range of temperatures (12 to 18 ºC) showed a 2 to 3 fold increase in siII-introgressed when compared to siIII-controls. This result shows that the impact of temperature on mitochondrial function is different between the two mitotypes studied. The Q(10) results seem to be linked to the apparent COX excess capacity of 193% for siIII-controls that is inexistent for siII-introgressed at 12 ºC. One explanation for these results is that the mitochondria can compensate for the disruption of mito-nuclear interactions at 24 ºC but not at lower temperatures. An alternate explanation would be that siII haplotype confer divergent kinetic properties to the ETS that translate to different temperature sensitivities.

In symbiotic associations such as those between Wolbachia and insects, the within-host symbiont density plays an important role in the maintenance of the infection in natural populations, as it relates to transmission fidelity and pathogenicity of the symbiont. Within-host density is speculated to be the result of complex interactions between the bacterial genotype, the host genotype and the environment, which may account for the substantial variation in Wolbachia titres among wild collected individuals compared to laboratory lines. Using quantitative PCR, we screened the Wolbachia gonadal density of individuals from 50 isofemale Drosophila simulans lines raised in standard conditions for at least two generations after collection from the wild. Although these newly collected lines displayed significant variation of ovarian Wolbachia titres, such variation was lost by F(19). Assaying these flies at different ages and under different environmental conditions indicated that symbiont titres in female gonads were not affected by the conditions tested. However, Wolbachia density in male gonads was consistently affected by these treatments in a line-specific way. We propose that the differences in Wolbachia densities among ovaries of F(4) flies are the consequence of large differences in the field-collected females caused by the variable environment, and carried over for at least four generations. In addition, we provide evidence of sex-specific dynamics of Wolbachia in gonads of females and males. In combination, our results support the view of sex-specific Wolbachia evolutionary interactions for males and females, which has been predicted by theory and observed experimentally.

Systematic analysis of 12S ribosomal RNA sequences supports the proposal that adults of Austrosimulium pestilens Mackerras and Mackerras and A. bancrofti (Taylor) s.l. may be distinguished by antennal segment number. However, there were too few informative positions within the region sequenced to resolve the phylogenetic affinities of the distinct cytological forms within A. bancrofti s.l. These data demonstrate that 12S rRNA can aid phylogenetic resolution of blackfly morphospecies but not cytological forms.

In ectotherms, the external temperature is experienced by the mitochondria, and the mitochondrial respiration of different genotypes is likely to change as a result. Using high-resolution respirometry with permeabilized fibers (an in situ approach), we tried to identify differences in mitochondrial performance and thermal sensitivity of two Drosophila simulans populations with two different mitochondrial types ( siII and siIII) and geographical distributions. Maximal state 3 respiration rates obtained with electrons converging at the Q junction of the electron transport system (ETS) differed between the mitotypes at 24°C. Catalytic capacities were higher in flies harboring siII than in those harboring siIII mitochondrial DNA (2,129 vs. 1,390 pmol O2·s−1·mg protein−1). The cytochrome c oxidase activity was also higher in siII than siIII flies (3,712 vs. 2,688 pmol O2·s−1·mg protein−1). The higher catalytic capacity detected in the siII mitotype could provide an advantage in terms of intensity of aerobic activity, endurance, or both, if the intensity of exercise that can be aerobically performed is partly dictated by the aerobic capacity of the tissue. Moreover, thermal sensitivity results showed that even if temperature affects the catalytic capacity of the different enzymes of the ETS, both mitotypes revealed high tolerance to temperature variation. Previous in vitro study failed to detect any consistent functional mitochondrial differences between the same mitotypes. We conclude that the in situ approach is more sensitive and that the ETS is a robust system in terms of functional and regulatory properties across a wide range of temperatures.

Modest dietary restriction (DR) prolongs life in a wide range of organisms, spanning single-celled yeast to mammals. Here, we report the use of recent techniques in nutrition research to quantify the detailed relationship between diet, nutrient intake, lifespan, and reproduction in Drosophila melanogaster . Caloric restriction (CR) was not responsible for extending lifespan in our experimental flies. Response surfaces for lifespan and fecundity were maximized at different protein–carbohydrate intakes, with longevity highest at a protein-to-carbohydrate ratio of 1:16 and egg-laying rate maximized at 1:2. Lifetime egg production, the measure closest to fitness, was maximized at an intermediate P:C ratio of 1:4. Flies offered a choice of complementary foods regulated intake to maximize lifetime egg production. The results indicate a role for both direct costs of reproduction and other deleterious consequences of ingesting high levels of protein. We unite a body of apparently conflicting work within a common framework and provide a platform for studying aging in all organisms.

Summary The goal of this study is to test the role of mitochondria and of mitochondrial metabolism in determining the processes that influence aging of female and male Drosophila. We observe that Drosophila simulans females tended to have shorter lifespan, higher levels of hydrogen peroxide production and significantly lower levels of catalase but not superoxide dismutase compared to males. In contrast, mammalian females tend to be longer lived, have lower rates of reactive oxygen species production and higher antioxidant activity. In both Drosophila and mammals, mitochondria extracted from females consume a higher quantity of oxygen when provided with adenosine diphosphate and have a greater mtDNA copy number than males. Combined, these data illustrate important similarities between the parameters that influence aging and mitochondrial metabolism in Drosophila and in mammals but also show surprising differences.

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.

The role of mitochondrial DNA (mtDNA) in mitochondrial metabolism is understudied yet humans harboring specific mtDNA types age at dissimilar rates, are unequally susceptible to various diseases, and differentially adapt to various environmental conditions. This study compares mitochondrial respiration, proton leak and electron transport of Drosophila simulans males with distinct mtDNA haplogroups ( si II and - III) that were collected in sympatry in Kenya. Despite the large divergence among haplogroups there is very low intrahaplogroup variation and no correlated variation in the nuclear genome has been detected. We show that repeatable bioenergetic differences exist between 11 d old males harboring si II and si III mtDNA. Males with si III mtDNA showed higher (i) state 3 respiration rates from isolated mitochondria for both complex I and complex III based substrates, and (ii) complex IV (cytochrome c oxidase) activity. Males harboring si III mtDNA had lower (i) hydrogen peroxide formation by both complexes I and III, (ii) proton leak from isolated mitochondria, (iii) mitochondrial ATPase activity, and (iv) mitochondrial cytochrome content. In combination, the results suggest that mitochondria isolated from si III mtDNA harboring males have more efficient metabolism than si II mtDNA harboring males.

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.

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.

Mitochondrial DNA (mtDNA) has been used to study molecular ecology and phylogeography for 25 years. Much important information has been gained in this way, but it is time to reflect on the biology of the mitochondrion itself and consider opportunities for evolutionary studies of the organelle itself and its ecology, biochemistry and physiology. This review has four sections. First, we review aspects of the natural history of mitochondria and their DNA to show that it is a unique molecule with specific characteristics that differ from nuclear DNA. We do not attempt to cover the plethora of differences between mitochondrial and nuclear DNA; rather we spotlight differences that can cause significant bias when inferring demographic properties of populations and/or the evolutionary history of species. We focus on recombination, effective population size and mutation rate. Second, we explore some of the difficulties in interpreting phylogeographical data from mtDNA data alone and suggest a broader use of multiple nuclear markers. We argue that mtDNA is not a sufficient marker for phylogeographical studies if the focus of the investigation is the species and not the organelle. We focus on the potential bias caused by introgression. Third, we show that it is not safe to assume a priori that mtDNA evolves as a strictly neutral marker because both direct and indirect selection influence mitochondria. We outline some of the statistical tests of neutrality that can, and should, be applied to mtDNA sequence data prior to making any global statements concerning the history of the organism. We conclude with a critical examination of the neglected biology of mitochondria and point out several surprising gaps in the state of our knowledge about this important organelle. Here we limelight mitochondrial ecology, sexually antagonistic selection, life-history evolution including ageing and disease, and the evolution of mitochondrial inheritance.

Drosophila simulans is known to harbor three distinct mitochondrial DNA (mtDNA) haplotype groups (siI, -II, and -III) with nearly 3.0% interhaplotypic divergence but

Within an individual, mitochondria must function in a range of tissue specific environments that are largely governed by expression of a particular suite of nuclear genes. Furthermore, mitochondrial proteins form large complexes with nuclear-encoded proteins to form the electron-transport system. These dynamics between mitochondrial and nuclear genomes have important implications in studies of within and among species genetic variation, and interpretation of disease phenotypes. Experimentally disrupting naturally occurring combinations of nuclear and mitochondrial genomes should provide insights into the coevolutionary dynamics among genomes.

The aim of this study is to examine the expression of cytoplasmic incompatibility and investigate the distribution and population frequencies of Wolbachia pipientis strains in Drosophila simulans. Nucleotide sequence data from 16S rDNA and a Wolbachia surface protein coding sequence and cytoplasmic incompatibility assays identify four distinct Wolbachia strains: wHa, wRi, wMa, and wAu. The levels of cytoplasmic incompatibility between six lines carrying these strains of bacteria and three control lines without bacteria are characterized. Flies infected with wHa and wRi are bidirectionally incompatible, and males that carry either strain can only successfully produce normal numbers of offspring with females carrying the same bacterial strain. Males infected with wAu do not express incompatibility. Males infected with the wMa strain express intermediate incompatibility when mated to females with no bacteria and no incompatibility with females with any other Wolbachia strain. We conduct polymerase chain reaction/restriction fragment length polymorphism assays to distinguish the strain of Wolbachia and the mitochondrial haplotype to survey populations for each type and associations between them. Drosophila simulans is known to have three major mitochondrial haplotypes (siI, sill, and siIII) and two subtypes (siIIA and siIIB). All infected lines of the sil haplotype carry wHa, wNo, or both; wMa and wNo are closely related and it is not clear whether they are distinct strains or variants of the same strain. Infected lines with the silIA haplotype harbor wRi and the siIIB haplotype carries wAu. The wMa infection is found in siIII haplotype lines. The phenotypic expression of cytoplasmic incompatibility and its relation to between-population differences in frequencies of Wolbachia infection are discussed.