1. Cell Biology
  2. Genomics and Evolutionary Biology
Download icon

A mitochondrial DNA hypomorph of cytochrome oxidase specifically impairs male fertility in Drosophila melanogaster

  1. Maulik R Patel Is a corresponding author
  2. Ganesh K Miriyala
  3. Aimee J Littleton
  4. Heiko Yang
  5. Kien Trinh
  6. Janet M Young
  7. Scott R Kennedy
  8. Yukiko M Yamashita
  9. Leo J Pallanck
  10. Harmit S Malik Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
  2. Howard Hughes Medical Institute, United States
  3. Vanderbilt University, United States
  4. University of Michigan, United States
  5. University of Washington, United States
  6. University of Washington Medical Center, United States
Research Article
Cited
6
Views
2,111
Comments
0
Cite as: eLife 2016;5:e16923 doi: 10.7554/eLife.16923

Abstract

Due to their strict maternal inheritance in most animals and plants, mitochondrial genomes are predicted to accumulate mutations that are beneficial or neutral in females but harmful in males. Although a few male-harming mtDNA mutations have been identified, consistent with this ‘Mother’s Curse’, their effect on females has been largely unexplored. Here, we identify COIIG177S, a mtDNA hypomorph of cytochrome oxidase II, which specifically impairs male fertility due to defects in sperm development and function without impairing other male or female functions. COIIG177S represents one of the clearest examples of a ‘male-harming’ mtDNA mutation in animals and suggest that the hypomorphic mtDNA mutations like COIIG177S might specifically impair male gametogenesis. Intriguingly, some D. melanogaster nuclear genetic backgrounds can fully rescue COIIG177S -associated sterility, consistent with previously proposed models that nuclear genomes can regulate the phenotypic manifestation of mtDNA mutations.

https://doi.org/10.7554/eLife.16923.001

eLife digest

Cell compartments called mitochondria are responsible for producing much of the energy that animal and plant cells need. Most of the proteins in mitochondria are produced from genes found in another compartment called the nucleus. However, some mitochondrial proteins are made from genes found in the mitochondria themselves.

Unlike the genes in the nucleus, which animals and plants inherit from both their mother and father, the mitochondrial “genome” is only passed on along the female line. Therefore, males represent an evolutionary dead-end for mitochondrial genes. Evolutionary theory predicts that this should result in the evolution and spread of mutations that can be harmful to males, providing they do not reduce the ability of females to survive and reproduce. Although such ‘male-harming’ mutations have been well studied in plants, it is less clear how common they are in animals.

Patel, Miriyala, Littleton et al. used fruit flies as a model system to identify and characterize male-harming mutations in the mitochondrial genome. The experiments isolated a mitochondrial genome with a single mutation in a gene that encodes an enzyme called cytochrome oxidase II. The mutation is said to be “hypomorphic” because it lowers the activity of the gene. The fertility of male flies with this mutation rapidly declined as they aged. However, the mutation did not appear to lower the fertility of female flies. In fact, apart from the lower male fertility, the mitochondrial mutation did not seem to affect any other traits in males or females.

Further experiments revealed that this hypomorphic mutation specifically impairs the development of sperm. Patel, Miriyala, Littleton et al. also found that the effect of the mutation on the fertility of the males depended on the genes in the nucleus of their cells, as some nuclear genomes were able to partially or completely suppress the mutation. This supports previous findings that the effect of mitochondrial mutations in animals and plants may be complex and can be strongly influenced by the genes in their nucleus.

Patel, Miriyala, Littleton et al.’s findings suggest that sperm development is particularly susceptible to defects in mitochondria, and that hypomorphic mutations may represent a broader category of ‘male-harming’ mutations in animals. A future challenge will be to find out whether such mutations occur in humans and whether they are associated with infertility in men.

https://doi.org/10.7554/eLife.16923.002

Introduction

The acquisition of the mitochondria by the ancestral eukaryote is one of the most remarkable instances of symbiosis in biology (Sagan, 1967; Schwartz and Dayhoff, 1978). This symbiosis gave the eukaryotic cell the ability to perform oxidative phosphorylation (Williams et al., 2013). In modern eukaryotes, oxidative phosphorylation is carried out by the electron transport chain, which comprises subunits encoded by both nuclear and mitochondrial genomes (mtDNA). Oxidative phosphorylation plays a fundamental role in many eukaryotes and is responsible for meeting most cellular energy demands. In humans, dysfunction of oxidative phosphorylation is associated with many diseases including cancer, diabetes, infertility, and neurodegenerative disorders (Wallace, 2001).

Despite the appearance of a symbiotic relationship, the evolutionary interests of mitochondria can be in conflict with those of nuclear genomes (Partridge and Hurst, 1998). This conflict arises from the differences in transmission between the two genomes. Whereas the nuclear genome is transmitted in a Mendelian fashion through both sexes, mtDNA is exclusively maternally inherited in most metazoans. Indeed, there are elaborate mechanisms to prevent the inheritance of sperm mitochondria (Birky, 1995; Al Rawi et al., 2011; Sato and Sato, 2011; Zhou et al., 2011; DeLuca and O'Farrell, 2012). Due to this uniparental inheritance, natural selection for mtDNA fitness can operate in females but not in males. As a result of this relaxed selection, 'male-harming' mtDNA mutations are expected to accumulate as long as they are beneficial, neutral or nearly neutral in females, a scenario referred to as the ‘Mother’s Curse’ (Frank and Hurst, 1996; Gemmell et al., 2004). However, since decreased male fitness is detrimental for the evolutionary success of the nuclear genome, outbred populations subject to accumulation of male-harming mtDNA mutations can rapidly evolve nuclear genome-encoded suppressors to restore male fitness (Rand et al., 2004; Dowling et al., 2008). Thus, evolutionary theory predicts a conflict-driven molecular arms race between mtDNA and the nuclear genome.

These evolutionary predictions have spurred interest into the molecular basis of how mtDNA mutations can manifest as specifically ‘male-harming’ particularly since mitochondrial function would be expected to be similar in most organs of both sexes. One of the best-understood examples of such ‘male-harming’ mtDNA mutations are cytoplasmic male sterility (cms) mutants described in many plant species (Budar et al., 2003; Chase, 2007). Such cms mtDNA can induce male sterility in hermaphroditic plants that can be cross-pollinated. By reallocating resources for pollen production instead for production of seeds, cms mtDNA increase female fitness – and consequently increase their own fitness (Budar et al., 2003). Molecular characterization of cms mutants shows that their ‘male-harming’ (and female-beneficial) properties are caused by novel chimeric genes that are created via recombination between mtDNA molecules. Nuclear-encoded suppressors of cms belong to the pentatricopeptide repeat (PPR) protein family and use RNA editing to effectively neutralize these male-harming chimeric genes in mtDNA (Budar et al., 2003; Chase, 2007; Castandet and Araya, 2012).

Animal mtDNA genomes are relatively small compared to those in plants, and the presence of novel chimeric genes is extremely rare in animal mtDNA. Despite these limitations, there is evidence that mutations in animal mtDNA genes might be male-harming. For example, mtDNA haplogroup T in humans is associated with reduced sperm motility (Ruiz-Pesini et al., 2000). Polymorphisms in mtDNA that cause this phenotype are hypothesized to be significant contributors to untreatable male subfertility, known to affect 7–10% of men (Baker, 1994). Recent findings implicate mtDNA variation as a significant contributor to shorter male lifespans in many taxa (Clancy, 2008; Camus et al., 2012). Although the phenotypic consequences of these mtDNA mutations are severe in males, it remains unclear whether they are benign in females, i.e., whether they are specifically ‘male-harming’ mtDNA mutations as predicted by the Mother’s Curse hypothesis.

Indeed, in spite of the theoretical predictions, male-harming mtDNA mutations have proven difficult to detect in animals. Several factors account for this difficulty. First, in natural populations, there can be significant indirect selection on mtDNA to maintain male fitness when the fitness of females is dependent on related males (those with the same mtDNA) (Wade and Brandvain, 2009; Hedrick, 2012). Such indirect selection against male-harming mtDNA mutations may be especially stringent in populations with high rates of inbreeding (Wade and Brandvain, 2009; Hedrick, 2012). Such indirect selection for male fitness might keep male-harming mtDNA mutations at low frequencies in natural populations, making them difficult to detect without deep surveys of natural polymorphism.

A second factor that may impede the discovery of male-harming mtDNA mutations in natural populations is their suppression by nuclear genome-encoded loci, which are under stringent selective pressure to restore male fitness (Rand et al., 2004; Dowling et al., 2008). Effects of detrimental mtDNA variants could thus only be evident if they were separated from their nuclear-encoded suppressors. With this goal, many researchers have created combinations of nuclear and mtDNA genomes in D. melanogaster, either from different strains or species (Rand et al., 2001; Maklakov et al., 2006; Rand et al., 2006, Dowling et al., 2007, 2008; Montooth et al., 2010; Clancy et al., 2011; Correa et al., 2012). Such combinations have revealed instances of dramatic incompatibility, even within species, which results in male but not female lifespan defects (Camus et al., 2012). Variation in mtDNA also affects sperm competitiveness in D. melanogaster (Yee et al., 2013). While these elegant experiments have revealed mtDNA-dependent affects on male lifespan and fertility, they did not comprehensively assess affects on female life-history traits; it is thus unclear whether these mutations are specifically male-harming. One further limitation of such mtDNA ‘swaps’ is that introduced mtDNA often have several co-inherited mutations. This linkage of different mutations makes it nearly impossible to implicate single mutations as being causal for the incompatibility or to understand their biological mechanism. An exception is a single point mutation in D. melanogaster mtDNA that has been causally linked to male sterility (Clancy et al., 2011). However, recent demonstration of this mtDNA haplotype’s negative pleiotropic effects on fertility and aging, both within and between sexes suggests it is not a specifically male-harming mutation (Camus et al., 2015). Thus, we currently lack clear examples of specifically male-harming mtDNA mutations in animals. This has also left unanswered the larger question of molecular mechanisms by which mtDNA mutations can manifest their ‘male-harming’ phenotypes.

Here, we isolated a mtDNA variant that harbors a single non-synonymous change in subunit II of cytochrome c oxidase (COIIG177S). We find that COIIG177S males have an age– and temperature–dependent decrease in fertility. This decrease in fertility is correlated with a drop in COII enzymatic activity, which remarkably does not result in defects in any other male or female phenotypic traits we measured. Cellular characterization reveals decreased sperm production and function in the mutant males. By combining evolutionary principles with detailed functional characterization, our study thus provides one of the clearest examples of a male-harming mtDNA mutation in animals, and provides insights into the stringent requirements for optimal mtDNA function in sperm development. We further show that the fertility defect in COIIG177S males can be completely suppressed by diverse nuclear backgrounds derived from various D. melanogaster strains, as predicted by the theory of cyto-nuclear genetic conflict.

Results

Recovery of a male-harming mtDNA mutation

To isolate male-harming mtDNA mutations, we devised an experimental evolution strategy that was partly inspired by previous strategies to decouple male versus female evolution in D. melanogaster (Rice, 1996; Rice, 1998). In our strategy, D. melanogaster females are prevented from mating with their male siblings. Instead, virgin females are mated to naïve males from an external stock every generation (Figure 1A). This strategy is predicted to eliminate indirect selection against male-harming mtDNA mutations because the fitness of the population is not dependent on the males carrying the mtDNA mutations; mtDNA are effectively absolved from supporting male fitness (Wade and Brandvain, 2009). Moreover, continuous replacement of a large fraction of the nuclear genome every generation via crosses to external males practically eliminates the likelihood of evolution of nuclear suppressors during the course of the experiment. In contrast, selection of mtDNA function for female fitness is stringently maintained over the entire course of the experiment. Thus, we anticipated this experimental evolution strategy might provide a permissive environment for accumulation and phenotypic manifestation of mtDNA mutations that are beneficial, neutral, or nearly neutral (only slightly deleterious) in females but cause defects in males.

Figure 1 with 1 supplement see all
An experimental evolution strategy to recover male-harming mtDNA mutations.

(A) In 12 D. melanogaster lines undergoing experimental evolution, 300 virgin female progeny in every generation were prevented from mating with sibling males (shown as being ‘crossed out’) and instead were mated with 100 males from the original stock. (B) In six lines undergoing coevolution, we allowed 300 virgin female and 100 male siblings to mate in every generation. Crosses for the 12 experimental and six coevolving lines were carried out for 35 generations.

https://doi.org/10.7554/eLife.16923.003

From an ‘original stock’ of w1118 flies, we established 18 independent lines, each consisting of 300 females and 100 males. In 12 of these lines (experimental lines), virgin females were collected every generation and mated with males from the original stock (Figure 1A). Female flies were subjected to this mating scheme continuously for 35 generations over approximately 70 weeks. For the remaining 6 lines (coevolving lines), we allowed sibling males to mate with the females over the course of 35 generations (Figure 1B). Based on published mtDNA mutation rate of 6.2 × 10−8 per site per fly generation (Haag-Liautard et al., 2008), we anticipated that multiple mutations per mtDNA would be sampled in 35 generations across the 18 lines. Any mtDNA mutations fixed at the end of the experiment must have either been pre-existing heteroplasmic mtDNA mutations that went to fixation (although the rate of fixation of such heteroplasmic mutations would be slow [Solignac et al., 1987]) or arose de novo in the experimental lines.

After 35 generations, we subjected flies to phenotypic analyses to assess whether any male-harming mutations had been sampled in the course of our experimental evolution strategy. To assess male viability, we first measured sex ratio in crosses between females with mtDNA subjected to experimental evolution (either ‘experimental’ or ‘coevolving’ lines) and control males (original w1118 stock maintained as a separate population). We did not observe a change in male to female ratio in any of the 18 lines, suggesting that any mutations in mtDNA acquired over the 35 generations did not alter male viability (Figure 2A). Next, we measured male fertility by mating males derived from the starting, experimental, and coevolving lines with females from the original stock. For 17 of 18 lines, we did not detect any significant differences in male fertility. However, we found that males from experimental line 7 (hereafter referred to as ‘EL7’) sired significantly fewer progeny compared to males from all the other experimental, coevolved and original lines (Figure 2B). In contrast, EL7 females do not have any significant decline in fertility (Figure 2C). This suggested the possibility of a male-harming mtDNA mutation in the EL7 line that was not deleterious to female fertility. We investigated whether the decline in male fertility we observed manifested over the entire adulthood of EL7 male flies. We found no obvious reduction in male fertility over the first few days of adulthood (note that F1 progeny do not emerge until day 12) in EL7 compared to the other experimental or control lines. Instead, we observed that the reduction in male fertility was age-dependent (Figure 2D). Our findings are consistent with previous observations in which mitochondrial dysfunction is more severe in aged individuals (Camus et al., 2012; Tower, 2015).

A single experimental line with male-specific fertility defects.

(A) We measured average sex ratio of progeny by mating five females from each of the original, experimental, or coevolving lines, with three males from the original stock. Average sex ratio was found to be close to 50:50 in each of the lines, suggesting that there were no gross viability differences between male and female progeny in any of the lines. All error bars represent standard error of the mean. All experiments were done in replicates of 10 per group. (B) We measured male fertility by mating three males from each of the original stock, experimental, or coevolving lines with five females from the original stock, and calculating average number of resulting progeny. Male fertility is represented as a normalized percentage of progeny relative to the original stock. Only one line, experimental line 7 (EL7) showed significant reduction relative to the others. All error bars represent standard error of the mean. All experiments were done in replicates of 10 per group. (C) We measured female fertility by mating five females from each of the original, experimental, or coevolving lines with three males from the original stock, and calculating average number of resulting progeny. Like in (B), female fertility is represented as a normalized percentage of progeny relative to the original stock. EL7 female fertility is not significantly different from the original stock. All error bars represent standard error of the mean. All experiments were done in replicates of 10 per group. (D) To gain further insight into altered male fertility in EL7 (B), we plotted the cumulative number of progeny sired by (three) males as a function of time since initial mating (10 replicates per line). We find that the cumulative number of progeny sired by EL7 males is normal until day 18 but is subsequently significantly lower than for all other lines. This finding suggests an age-dependent decline in male fertility in the EL7 line.

https://doi.org/10.7554/eLife.16923.005

A single missense mtDNA mutation results in male fertility decline

To identify the putative mutation responsible for decreased male fertility, we performed whole-genome sequencing of the original and EL7 D. melanogaster lines using DNA isolated from a pool of 100 flies for each line. We were able to achieve average coverage of over 1000-fold for the entire mtDNA, except for the highly repetitive, AT-rich ‘control’ region (Figure 3—figure supplement 1). Analysis of these mtDNA sequences revealed a single missense mutation in EL7 mtDNA, resulting in a glycine to serine substitution at position 177 in subunit II of cytochrome c oxidase (COIIG177S), the fourth complex in the mitochondrial electron transport chain (Figure 3A–B). Interestingly, the COIIG177S mutation did not arise de novo during the course of our experiment. Although 98% of the sequencing reads from EL7 harbored the COIIG177S mutation, 59% of the reads from the original stock also corresponded to the mutant allele. Sanger sequencing confirmed this difference in proportion of wildtype and mutant mtDNA (Figure 3—figure supplement 2).

Figure 3 with 3 supplements see all
A single missense mutation (G177S) in subunit II of cytochrome c oxidase underlies lower male fertility in EL7.

(A) The five complexes of the electron transport chain are schematized. These complexes are comprised of subunits encoded by both nuclear and mitochondrial genomes; the latter are indicated in pink. Subunit II of cytochrome c oxidase (COII) with the glycine to serine mutation at position 177 (G177S) in Experimental Line 7 is highlighted in bright pink. (B) Crystal structure of Bos taurus COII (in gold) in complex with COI (in blue) (PDB number: 2OCC). Glycine with its side chains at position 177 (G177) in COII is indicated. (C) Partial amino acid sequence alignment of COII from representative animal species highlights the conserved glycine residue at position 177 (boxed in yellow).

https://doi.org/10.7554/eLife.16923.006

A mixture of wildtype and mutant mtDNA can exist in the same population in two ways. First, a population can consist of homoplasmic (carrying genetically identical mtDNA) wildtype and mutant individuals. Alternatively, the two mtDNA can be present in the same individual in a state of heteroplasmy. Sanger sequencing of single individuals from our original line revealed the presence of heteroplasmic individuals as well as flies that were homoplasmic wildtype and mutant (Figure 3—figure supplement 3). Therefore, 59% reflects the average combined frequency of the COIIG177S mutation from both heteroplasmic and homoplasmic mutant flies in the population.

The mutant allele’s 59% frequency among reads from the original stock suggested that the COIIG177S mutation was present before initiation of the experimental lines. If this model is correct, we would expect that the COIIG177S mutation should also be found in some of the other control and experimental lines in addition to EL7. Indeed, we found this to be the case (Figure 3—figure supplement 2). However, EL7 is unique in being the only population in which the COIIG177S mutation is nearly fixed, whereas it varies from 0% to more than 50% in other experimental populations, which do not suffer any overt signs of male sterility at least at a population-level (Figure 2B, Figure 3—figure supplement 2). These data are consistent with the hypothesis that COIIG177S mutation causes defects in male fertility but only when present at very high levels. Our findings are consistent with similar observations in pathogenic mtDNA variants in human disease (Sobenin et al., 2014).

Our finding that EL7 nearly fixed a mtDNA mutation that was already pre-existing provided another opportunity to test the association of the COIIG177S mutation with decrease in male fertility. We reasoned that homoplasmic mutant males re-isolated from the original stock should also have reduced fertility despite the fact that these males have not undergone 35 generations of experimental evolution like EL7. Consistent with this expectation, we were able to re-isolate homoplasmic COIIG177S males, which sire fewer progeny compared to homoplasmic wildtype males derived from the same original stock (Figure 4A) (mutant and wildtype lines re-established from single females). These data demonstrate that the homoplasmic COIIG177S mutation is necessary and sufficient to explain the observed decreased male fertility in EL7 males. These re-isolated COIIG177S homoplasmic mutant mtDNA males also recapitulated the age-dependent decline in male fertility (Figures 2D,4B).

Males with COIIG177S mutation have decreased fertility at elevated temperatures.

(A) To measure male fertility, we compared the fertility of males homoplasmic for either wildtype or COIIG177S mutant mtDNA reestablished from the original stock. For each experiment, three males from homoplasmic stocks were mated with five females homoplasmic for wildtype mtDNA. Male fertility is presented as a normalized percentage of progeny produced, relative to the number produced by wildtype mtDNA males. COIIG177S mutant mtDNA males produce fewer progeny at 25°C, but this is further reduced at 29°C. To measure female fertility, five females from homoplasmic wildtype or COIIG177S mutant mtDNA stocks were mated with three males with wildtype mtDNA. Female fertility is presented as a normalized percentage of progeny produced, relative to wildtype mtDNA females. All error bars represent standard error of the mean. All experiments were done in replicates of 10 per group. (B) Number of progeny sired by wildtype or COIIG177S mutant males at 25°C is cumulatively plotted as a function of time since initial mating (10 replicates per line). Actual number of cumulative progeny from each replicate is indicated by dots at given time points. (C) Number of progeny sired by wildtype or COIIG177S mutant males at 29°C is cumulatively plotted as a function of time since initial mating (10 replicates per line). The reduction in male fertility is more significant at the higher temperature.

https://doi.org/10.7554/eLife.16923.010

We characterized the nature of the heteroplasmy in the re- isolated COIIG177S mutant mtDNA lines by sequencing a pool of COIIG177S mutant mtDNA and a separate pool of re-isolated wildtype mtDNA adult fly heads using a Duplex Sequencing strategy (Kennedy et al., 2014) followed by hybrid capture to significantly enrich for mtDNA (Figure 4—source data 1). Under this strategy, mtDNA is sequenced at very high depth of coverage, labeling individual DNA molecules and sequencing each one multiple times in order to distinguish true mutations from sequencing errors. This strategy allows for sensitive evaluation of heteroplasmic mtDNA mutations. We found no evidence for wildtype mtDNA sequence in a pool of flies that we had re-isolated to enrich for the COIIG177S mutant mtDNA (0 duplex consensus reads out of >7000). In contrast, we found no evidence for the COIIG177S mutant mtDNA in flies (0 reads out of >7000) that we had re-isolated from the same original w1118 stock to enrich for wildtype mtDNA. We were cognizant that although the pooled COIIG177S mutant flies showed no evidence of wild-type sequence, some heteroplasmy may nevertheless persist in individual flies. We therefore also sequenced 7 individual flies from the COIIG177S mutant mtDNA pool to high depth of coverage. For six flies, we found no reads (out of >8,000) corresponding to wildtype mtDNA, whereas in one fly we uncovered seven out of >11,000 reads corresponding to wildtype mtDNA (<0.1%). We therefore conclude that the re-isolated strains are almost completely homoplasmic for either COIIG177S mutant mtDNA or wildtype mtDNA. We therefore used these re-isolated strains for all subsequent phenotypic analyses.

Although we had set out to recover de novo mtDNA mutations that are male-harming using our experimental evolution strategy, we instead recovered what appears to be a pre-existing heteroplasmic mtDNA mutant, which fixed in only one of the 12 experimental lines. Therefore, we cannot attribute the isolation of this mutant to our scheme. Future work will be needed to determine the effectiveness of our experiment evolution scheme in recovering male-harming mutations, perhaps aided by experimentally increasing mtDNA mutation rates. For the rest of the manuscript, we focus on detailed characterization of the COIIG177S mutation to determine its cellular consequences and to determine whether it is specifically male-harming.

Decreased male fertility in COIIG177S mutants is temperature dependent

All our analyses so far suggest that COIIG177S is a specifically male-harming mtDNA mutation. Hence, we decided to further characterize the cellular and molecular basis underlying its detrimental effects. Previous experiments have shown that the phenotypic effects of mtDNA mutation can be exacerbated with increased stress, including higher temperatures (Hoekstra et al., 2013; Chen et al., 2015). We therefore investigated the effect of higher temperature on the fertility defect in COIIG177S males. We found that COIIG177S males are almost completely sterile when raised at 29°C instead of at 25°C (Figure 4A). Furthermore, we found that this defect manifests in both old and young males (Figure 4C). Thus, higher temperature provides a more sensitized condition to evaluate the various consequences of the COIIG177S mtDNA mutation as previously observed (Hoekstra et al., 2013).

COIIG177S mutants have impaired cytochrome c oxidase activity

Next, we investigated the molecular consequences of the COIIG177S mutation. COII is a subunit of cytochrome C oxidase (COX), which oxidizes the reduced form of cytochrome c. The glycine residue at position 177 is found in a loop of COII’s structure where it comes in very close proximity to subunit I of the enzymatic complex (Figure 3B). Based on the fact that G177 is highly conserved across metazoans (Figure 3C), we hypothesized that the G177S mutation affects either an intrinsic function of COII or its interaction with other proteins in complex IV of the electron transport chain. To understand the biochemical consequences of the COIIG177S mutation, we measured COX activity from whole fly lysates of flies grown at 25°C. Although the COX activity in COIIG177S mutant flies was slightly lower than flies with wild-type mtDNA, this defect was not statistically significant, even in old flies (Figure 5A). We therefore measured COX activity from flies raised at 29°C where male fertility is most significantly impaired. Our analyses revealed an approximately 20% decrease in COX enzymatic activity in COIIG177S mutants grown at 29°C in both male and female flies (Figure 5B). Given that the activity of the electron transport chain is coupled to ATP synthesis, we also measured ATP levels at 29°C. In contrast to COX activity, we did not observe a decrease in ATP levels in the COIIG177S mutants (Figure 5C). However, we did observe a mild but significant decrease in reactive oxygen species (ROS) levels (Figure 5D). Thus, impaired COX activity and reduced ROS levels, but not ATP production, appear to correlate with loss of male fertility.

Biochemical consequences of COIIG177S mtDNA mutation.

We measured COX activity from young (3–4 days) or old (21 days) flies raised at 25°C (A), or young flies raised at 29°C (B). (C) We measured ATP levels from 3–4 day old males raised at 29°C. (D) We measured ROS levels from 3–4 day old males raised at 29°C. 4–5 replicates per group for all experiments. Data is normalized to wildtype flies for each group in all experiments. Error bars represent standard error of the mean.

https://doi.org/10.7554/eLife.16923.012

The COIIG177S mtDNA mutant specifically impairs male fertility

Our finding that COIIG177S mutants result in lower COX activity of both males and females prompted us to evaluate several other phenotypes commonly seen in flies with mitochondrial impairment. Mitochondrial dysfunction is often associated with aging defects in many species (Cho et al., 2011; Tower, 2015). Naturally occurring variation in mtDNA is also known to affect aging in D. melanogaster (Rand et al., 2006; Clancy, 2008; Camus et al., 2012). We therefore assayed the lifespan of wildtype and COIIG177S mutant males and females at both 25°C and 29°C. We did not observe any differences in lifespan, even at the sensitized higher temperature between wildtype and mutant flies in either males or females (Figure 6).

Figure 6 with 2 supplements see all
COIIG177S mtDNA mutation does not significantly affect male or female lifespan.

We measured average lifespan of males and females at 25°C (A,B) or 29°C (C,D). N = 100 flies per group.

https://doi.org/10.7554/eLife.16923.013

Sensitivity to physiological stress and neuronal function are also associated with mitochondrial dysfunction (Fergestad et al., 2006; Pieczenik and Neustadt, 2007; Ugalde et al., 2007; Distelmaier et al., 2009; Celotto et al., 2011). We therefore assayed heat intolerance, and ‘bang sensitivity’, a measure of neuronal dysfunction (Burman et al., 2014). There were no statistically significant differences in these traits in either males or females, even in aged flies grown at 29°C, the highly sensitized and susceptible condition (Figure 6—figure supplements 1, 2). Thus, despite the 20% reduction in COX activity in both males and females, the phenotypic consequences of COIIG177S in D. melanogaster appear to be largely benign, with deleterious effects confined to male fertility alone.

COIIG177S impairs sperm development and motility

We next investigated the biological basis of the decreased male fertility in COIIG177S mtDNA-bearing males. Male sterility could either result from defects in the ability of males to mate or can be a result of defective sperm development/function or a combination of both. In order to distinguish between these two possibilities, we took advantage of the fact that transfer of male accessory gland peptides during mating induces egg-laying in females (Wolfner, 1997). We find that mating wildtype females with COIIG177S males induces egg-laying just as robustly as in females mated with males carrying wildtype mtDNA (Figure 7A). However, almost all of the eggs laid by females mated with COIIG177S mutant males were unfertilized and failed to hatch (Figure 7A). Taken together, these data suggest that the decreased fertility in COIIG177S males occurs due to defects in sperm development or function, and not due to mating ability. We therefore sought to determine the nature of cellular defects in COIIG177S male testes.

Figure 7 with 1 supplement see all
Normal mating but defective sperm development in COIIG177S mutant mtDNA males.

(A) We measured whether the COIIG177S mtDNA mutation affected the mating success of males. We measured eggs laid by virgin wildtype females that were either unmated, or mated with 2–5 day old wildtype, or COIIG177S mutant males at 29°C. We determined the fraction of eggs hatched by counting unhatched eggs and larva 24 hrs after eggs were laid. All experiments were done in replicates of 6 per group. Error bars represent standard error of the mean. Our results show that the number of eggs laid after mating to wildtype mtDNA males is not significantly different from those mated to COIIG177S mutant mtDNA males; in the latter case, most of the eggs are unfertilized and do not hatch. (B,C) We present maximum projection representative images of DAPI stained testis from 2–5 day old wildtype mtDNA male flies grown at 29°C at early (B), middle (B’), and late (B’’) needle stage of sperm development. Note that the sperm are organized during early needle stage (arrow) and then break up into individual sperm by late needle stage (arrowheads). We also present maximum projection images of DAPI stained testis from COIIG177S mutant mtDNA male flies grown at 29°C at early (C), middle (C’), and late (C’’) needle stage of sperm development. Note also that the sperm in COIIG177S mutant are ‘clumped’ and disorganized early in the needle stage and remain so through remainder of spermiogenesis (arrow). Scale bar, 20 μm. (D,E) Representative DAPI stained images of whole testis (outlined in dotted line) from 2–5 day old virgin wildtype mtDNA (D) and COIIG177S mutant mtDNA males (E) raised at 29°C. For orientation, in both images, we identify the tip of the testis (where germ stem cells reside) as well as the seminal vesicle (the storage organ for mature sperm). Note the much smaller seminal vesicle size in the mutant males. Scale bar, 50 μm. (F) Quantification of the seminal vesicle size, as measured by cross-sectional area, normalized to wildtype. Average calculated from 5–7 testes. Error bars represent standard error of the mean.

https://doi.org/10.7554/eLife.16923.016

Sperm development occurs within a cyst in which 64 sperm nuclei share a common cytoplasm. These sperm undergo individualization during the needle stage near the terminal epithelium of the testis. Examination of testes stained with the DNA marker DAPI revealed clear late needle-stage defects in COIIG177S males. These mutant sperm failed to individualize properly and instead formed tangled clumps (Figure 7B–C). Consistent with this developmental defect, there is a significant reduction in the number of mature sperm that are stored in the seminal vesicles of COIIG177S males raised at 29°C, as indicated by the decrease in the size of the seminal vesicles (Figure 7D–F). In addition, sperm that could be isolated from COIIG177S males have reduced motility (Videos 1,2).

Video 1
Sperm motility assays in w1118 males carrying wildtype mtDNA.

From a representative male grown at 29°C. Sperm stained with mitotracker Green (green), which stains immotile sperm and mitotracker CMS Rox (red), which stains motile sperm preferentially (related to Figure 8).

https://doi.org/10.7554/eLife.16923.018
Video 2
Sperm motility assays in w1118 males carrying COIIG177S mutant mtDNA.

From a representative male grown at 29°C. Sperm stained with mitotracker Green (green), which stains immotile sperm and mitotracker CMS Rox (red), which stains motile sperm preferentially (related to Figure 8).

https://doi.org/10.7554/eLife.16923.019

Although we used the higher temperature to provide a more sensitized assay to measure male fertility, we were aware of the possibility that the decreased fertility and the reduced seminal vesicle size could be solely the result of the elevated temperature (29°C). We therefore carried out similar experiments in aged males at 25°C, which also had displayed a reduction in fertility. Just like young COIIG177S males at 29°C, we found that aged COIIG177S males at 25°C also had reduced seminal vesicle size compared to aged males with wildtype mtDNA (Figure 7—figure supplement 1). In contrast, the seminal vesicle sizes of young males with wildtype mtDNA were the same, whether they were raised at 25°C or 29°C.Together, our results suggest that the decreased male fertility and reduced seminal vesicle size is not due to a confounding effect of higher temperature, but rather the result of the COIIG177S mtDNA mutation.

Examining the male infertility phenotype further, we found that mitotracker green, a mitochondrial membrane potential-independent dye, preferentially stains immotile sperm whereas mitotracker CMXRos, a mitochondrial membrane potential-dependent dye, preferentially stains motile sperm in wild type testes. Sperm from COIIG177S mutant males showed similar mitotracker green staining but significantly reduced CMXRos staining overall compared with wildtype sperm (Figure 8), suggesting that the lack of motility likely results from dysfunctional mitochondrial activity. Taken together, these data demonstrate that decreased COX activity in COIIG177S males correlates with the cellular defects in sperm development and motility in COIIG177S mutants.

Lower motility of sperm from COIIG177S mutant mtDNA males.

Sperm from wildtype mtDNA (A) and COIIG177S mutant mtDNA (B) males raised at 29°C stained with mitotracker Green (green), which stains immotile sperm and mitotracker CMS Rox (magenta), which stains motile sperm preferentially. Scale bar, 20 μm.

https://doi.org/10.7554/eLife.16923.020

Nuclear genome variation can suppress the male-harming effects of COIIG177S mtDNA

Male-harming mtDNA mutations like COIIG177S are predicted to be detrimental for the evolutionary success of the nuclear genome. In the face of such mtDNA mutations, it is expected that nuclear genomes might have evolved suppressors to restore male fitness (Rand et al., 2004; Dowling et al., 2008; Wolff et al., 2014). To test this evolutionary hypothesis, we generated males heterozygous for varied nuclear genomes by crossing COIIG177S mtDNA-bearing females with males from a number of D. melanogaster strains, collected from different global populations. We then assayed the fertility of the resulting male progeny that carried the COIIG177S mtDNA but were heterozygous for the nuclear genome. To maximize the sensitivity of our assays, we performed these fertility assays in young males at 29°C. Surprisingly, we found that the nuclear genomes from many of the strains we tested were able to completely restore male fertility in COIIG177S males (Figure 9A, Figure 9—figure supplement 1).

Figure 9 with 1 supplement see all
Dominant suppression of COIIG177S associated male fertility defects by diverse nuclear genome backgrounds.

(A) We measured male fertility of heterozygous males derived from crosses of w1118 females (carrying wildtype or COIIG177S mutant mtDNA) to males from a variety of different nuclear backgrounds (i.e., Oregon R, Arizona, Congo isoline 18, Congo isoline 26). In each case, three heterozygous males were mated with 5 w1118 females at 29°C (10 replicates per group). Male fertility was measured as average number of resulting progeny, normalized as a percentage of progeny of corresponding males with wildtype mtDNA. Raw progeny numbers are given in Figure 9—figure supplement 1. Most nuclear backgrounds show mild to complete suppression of the male fertility defects observed in w1118/w118 males carrying COIIG177S mutant mtDNA (**p<0.05). Error bars represent standard error of the mean. (B) We examined lifetime male fertility in the w1118/Oregon R background at 25°C (note that male fertility is generally higher at lower temperatures) to quantify the suppression of male sterility. (C) We measured male fertility of heterozygous males derived from crosses of w1118 females (carrying wildtype or COIIG177S mutant mtDNA) to males from six DGRP strains, at 29°C (10 replicates per group) as in (A). At least five of the tested DGRP strains show partial to complete suppression of male sterility.

https://doi.org/10.7554/eLife.16923.021

Male fertility is highly sensitive to temperature, with different strains exhibiting different threshold of tolerance (Rohmer et al., 2004; David et al., 2005; Hoekstra et al., 2013). Hence, the restoration of fertility that we observe could simply reflect greater tolerance to high temperature independent of the COIIG177S mtDNA. To address this possibility, we measured total fertility of males until 21 days of age at 25°C. We found that the Oregon R nuclear background suppresses not only the young male sterility at 29°C but also male fertility at 25°C (Figure 9B, Figure 9—figure supplement 1). Thus, we conclude that the Oregon R nuclear background encodes a bona fide suppressor of COIIG177S mtDNA-mediated male infertility. We currently don’t know the identity of the suppressor loci or whether these are specific only to the COIIG177S mtDNA mutation. For instance, these suppressors might employ a general mechanism that allows for rescue of male fitness defects caused by other mtDNA mutations. Under either scenario, we hypothesized that COIIG177S suppressor loci may be more abundant across D. melanogaster strains.

We used lines from the DGRP collection (Mackay et al., 2012), a set of fully sequenced inbred lines derived from a single natural population, to ask whether there is a lot of standing variation in the ability of nuclear genomes to suppress effects of the COIIG177S mutation. We found that DGRP line 861 was able to completely restore male fertility and most other lines were able to at least partially restore male fertility in males carrying COIIG177S mutation (Figure 9c, Figure 9—figure supplement 1). These data suggest that genetically dominant nuclear suppressors of the COIIG177S male fertility defects are widespread in natural populations. The different penetrance of the rescue also suggests that the suppression of COIIG177S associated male infertility is likely to involve multiple loci.

We examined in more detail the cellular basis of ‘rescued’ male fertility in OregonR-w1118 heterozygous males bearing the COIIG177S mtDNA. We found that sperm development and motility were partially rescued in young males raised at 29°C (Figure 10A–E, Figure 10—figure supplement 1, Video 3) and aged males at 25°C (Figure 7 —figure supplement 1), consistent with the restoration of fertility that we previously observed (Figure 9A,B). We also evaluated the biochemical consequence of such nuclear suppression of COIIG177S mtDNA’s male sterility to distinguish between two possible mechanisms of suppression. Under the first alternative, restoration of fertility in COIIG177S males might be due to higher baseline COX activity in the suppressor background, which can compensate for the hypomorphic COIIG177S and therefore be sufficient for sperm development and motility. Under this scenario, there would still be a difference in COX activity between wildtype and COIIG177S flies. A second alternative is that there is no difference in COX activity in flies carrying wildtype or COIIG177S mtDNA. When we measured COX activity in the suppressed nuclear background, we found the second scenario to be true i.e., COX activity is the same in wildtype and COIIG177S mtDNA-bearing males (Figure 10F). This result was also observed for three additional suppressor lines we tested (Figure 10—figure supplement 2). These data suggest that in the suppressed background, COIIG177S mtDNA is as functionally effective as wildtype mtDNA and is indicative of a compensatory mechanism that does not rely simply on higher baseline levels of COX activity.

Figure 10 with 2 supplements see all
w1118/OreR heterozygous males carrying COIIG177S mutant mtDNA show rescued sperm development and COX activity.

Representative DAPI stained images of the late needle stage of sperm development from w1118/w1118 males carrying either wildtype mtDNA (A), or COIIG177S mtDNA (B) compared to heterozygous w1118/OreR males carrying COIIG177S mtDNA (C). Numbers of examined males with defects in sperm development are indicated. Arrow points to ‘clumped’ sperm. Note the presence of many individualized sperm (arrowheads) in A and C but few in B. Scale bar, 20 μm. We stained sperm from homozygous w1118/w1118 males (D) and w1118/OreR heterozygous males (E), both carrying COIIG177S mutant mtDNA with mitotracker Green (green) and mitotracker CMS Rox (magenta), which stain immotile and motile sperm respectively. All flies were raised at 29°C. Scale bar, 20 μm. (F) COX activity measured from young (3–4 day old) w1118/w1118 males and w1118/OreR heterozygous males both carrying COIIG177S mutant mtDNA. All flies were raised at 29°C. Data is normalized to flies with wildtype mtDNA in the corresponding nuclear background. Error bars represent standard error of the mean.

https://doi.org/10.7554/eLife.16923.023
Video 3
Sperm motility assays in w1118/Oregon R males ('nuclear suppressor' background) carrying COIIG177S mutant mtDNA.

From a representative male grown at 29°C. Sperm stained with mitotracker Green (green), which stains immotile sperm and mitotracker CMS Rox (red), which stains motile sperm preferentially (related to Figure 10).

https://doi.org/10.7554/eLife.16923.026

Discussion

Evolution by natural selection rests on a simple but fundamental premise that the phenotypic manifestation of a genotype occurs in the same individual that transmits it to the next generation. Remarkably, the mitochondrial genome (mtDNA) does not abide by this rule since it is inherited and phenotypically manifests in both sexes, but is exclusively transmitted through females in most animals and plants. Thus, the phenotypic manifestation of mtDNA is decoupled from its transmission in males. Consequently, natural selection is ineffective at directly removing mtDNA mutations that are specifically harmful to males but not females. While the mtDNA phenotype/transmission decoupling clearly predicts existence of male-harming mutations (Mother’s Curse) (Frank and Hurst, 1996; Gemmell et al., 2004), a systematic study of such mutations and their underlying mechanisms is hampered by the relative paucity of such mutations in animals.

We devised an experimental evolution scheme intended to absolve mtDNA of supporting male function while both maintaining selection on female mtDNA function as well as reducing the possibility of nuclear suppression of male-harming mtDNA. While we did recover a bona fide male-harming COIIG177S mtDNA mutation via this scheme, we were subsequently surprised to discover that this was a pre-existing mtDNA mutation that was already present at moderate frequencies in our starting laboratory strain of w1118. It is possible that the experimental evolution scheme might have created the permissive conditions that allowed this mtDNA mutation to rise to fixation in one strain. However, the fact that it did not rise to fixation in the other experimental populations means that we cannot conclude that the experimental strategy itself was causally important for the fixation of this male-harming mtDNA mutant. Future work on fine-tuning the strategy can address its effectiveness. For example, the scheme can be improved by increasing mtDNA mutation rates to improve sampling of de novo mutations, perhaps by employing a mutator mtDNA polymerase before initiating the experimental evolution scheme (Trifunovic et al., 2004). Alternatively, random chemical mutagenesis might also be readily applied here in our scheme. Although such chemical mutagenesis will affect both mtDNA and nuclear genomes initially, backcrossing to ‘original stock’ males in our scheme will rapidly replace most mutated nuclear genes with wildtype versions, while enabling the transmission of mtDNA mutations that do not affect female fitness. Finally, combining the crossing scheme we propose with the targeted restriction endonuclease strategy to generate mtDNA mutations (Xu et al., 2008) might be a facile means to discriminate specifically ‘male-harming’ mtDNA mutations from those that grossly impair mtDNA function.

Our phenotypic assays did not provide any evidence for the COIIG177S mutation being beneficial in females. Furthermore, the mutation rose to high frequency in only one out of the twelve experimental lines, suggesting that the mutation is likely neutral or nearly neutral in females and therefore became nearly fixed through drift rather than selection. These data are consistent with the ‘selective sieve’ model, which predicts that even mutations that are neutral or nearly neutral in females but deleterious in males might be common because they cannot be effectively removed by natural selection (Innocenti et al., 2011). Our survey of the sequenced genomes from a large panel of 290 D. melanogaster strains and isofemale lines did not reveal any other lines besides our w1118 stock that harbored COIIG177S (Richardson et al., 2012). Thus, COIIG177S might be rare in natural populations.

The homoplasmic COIIG177S mutation results in a 20% decline in COX activity at 29°C. Remarkably, we observed no phenotypic effects of this decline in COX activity on other phenotypic traits in both males and females, even at high temperatures. The COX activity decline specifically impaired sperm production and function. This establishes COIIG177S as one of the first bona fide male-harming mtDNA mutations in animals. We considered the possibility that the phenotypic effects we have observed on male fertility are attributable to linked but unassayed changes in the 4-kb long AT-rich D loop control region of mtDNA. Indeed, the D-loop is one of the most rapidly evolving segments of mtDNA in D. melanogaster, but its highly repetitive nature challenges sequence characterization. However, our analyses allow us to directly implicate the COIIG177S mtDNA mutation as being causally linked to the phenotypes we have observed. Not only do we observe a perfect correlation of the male fertility phenotype with the reduction of COX activity, but we also observe a near complete restoration of male fertility upon replenishment of COX activity in the suppressor strains.

Surprisingly, the decline in COX activity in COIIG177S flies was not associated with a corresponding decline in ATP production. Since we were unable to reliably measure either COX activity or ATP production in testes, we cannot rule out the possibility that ATP production is specifically impaired in the male germline. However, our findings suggest a different molecular consequence of lowered COX activity may be responsible for defects in sperm development; we did observe decreased reactive oxygen species (ROS) production in COIIG177S mutants. Our findings suggest that perhaps alterations in ROS levels might underlie the defects we see in sperm production and function. ROS have been shown previously to act as a signaling molecule to control the cell cycle checkpoint as well as the differentiation of hematopoietic progenitors during development in D. melanogaster (Owusu-Ansah et al., 2008; Owusu-Ansah and Banerjee, 2009). These data leave open the possibility of ROS similarly acting as a signaling factor during sperm development.

We hypothesize that male fertility may be a common target of male-harming mtDNA mutations because the reproductive tissues are highly sexually dimorphic. This hypothesis is consistent with previous data, which showed that naturally occurring variation in D. melanogaster mtDNA largely affects expression of male-expressed genes in the testis and the accessory gland (Innocenti et al., 2011). Two separate mutations in mtDNA are known to cause male sterility in D. melanogaster (Xu et al., 2008; Clancy et al., 2011). A single amino acid mtDNA mutation (A278T) in Cytochrome B of complex III renders males sterile; the primary defect appears to be at the level of spermatid individualization (Clancy et al., 2011). In contrast, a single amino acid mutation in Cytochome Oxidase I (R301L) causes male sterility primarily due to a sperm storage defect. However, the effects of these mutations on female fitness have not yet been comprehensively addressed so we cannot attribute them to be specifically ‘male-harming’.

How might mtDNA function be different in testis compared to other tissues? It is possible that the testis has a differential requirement for COX activity. According to this hypothesis, although all tissues suffer from the relative reduction in COX activity, testis function is specifically impacted because it has a lower threshold of tolerance for a relative reduction in COX activity. In particular, COX activity might be required to facilitate the dramatic morphological changes that mitochondria undergo during sperm development in D. melanogaster (Fuller, 1998). If this hypothesis is correct, mildly hypomorphic mutations like COIIG177S might generally impair male fertility and thus represent a common mechanism of male-harming mtDNA mutations. Alternatively, the existence of a number of nuclear-encoded testis-specific components of the electron transport chain, including subunits of cytochrome C oxidase, provide a number of interacting partners that might exhibit testis-specific genetic incompatibility with the COIIG177S mutation (Tripoli et al., 2005; Gallach et al., 2010). Since we were unable to reliably measure COX activity in dissected testes of wildtype and mutant flies, we are unable to address whether the decrease in COX activity is more severe in testes than other tissues.

Our study has identified one mtDNA mutation, COIIG177S, whose phenotypic effects appear to be restricted to the male germline. However, other forms of sexual dimorphism might lead to sex-specific phenotypic manifestations of other mtDNA mutations beyond reproductive tissues. For instance, there is already evidence that mtDNA might harbor mutations that affect aging and lifespan, which is highly dimorphic between the sexes (Camus et al., 2012). At the molecular level, COX activity was shown to be differentially affected in male versus female flies based on their mtDNA haplotype (Sackton et al., 2003). Furthermore, almost 90% of the transcriptome in D. melanogaster is differentially expressed between the sexes (Ayroles et al., 2009). Amongst those differentially expressed genes, male-biased transcripts are enriched for mitochondrial energy metabolism, thus providing ample opportunities for sex-specific effects of mtDNA (Ayroles et al., 2009). Consistent with the nuclear background exerting an effect on the phenotypic manifestation of mtDNA mutants, we found that the COIIG177S -mediated sterility was suppressed in many of the D. melanogaster nuclear backgrounds tested. Thus, our study not only shows the existence and biological basis of a male-harming mutation, but also demonstrates importance of the intimate relationship between nuclear and mtDNA genomes for the phenotypic manifestation of mtDNA mutations.

Our finding that the phenotypic consequences of a specific male-harming mtDNA mutation are dependent on the nuclear genome also re-emphasizes the exciting possibility of uncovering the genetic basis of nuclear-mitochondrial incompatibilities and the molecular basis of buffering by nuclear genomes. Such buffering provides an important insight into the genetic basis of human mtDNA disease in which male-harming mtDNA mutations might initially increase in frequency in a 'suppressor' background while avoiding any negative fitness consequences. The phenotypic consequences of male-harming mtDNA mutations would only be exposed in a naïve nuclear genetic background upon introduction into new populations or even (in the laboratory setting) new species. Such nuclear-mitochondrial incompatibilities have been proposed to potentially serve as the basis of speciation (Burton and Barreto, 2012). Although only a few such nuclear-mitochondrial incompatibilities have been mapped in molecular detail (Ellison and Burton, 2006; Lee et al., 2008; Chou et al., 2010; Clancy et al., 2011; Meiklejohn et al., 2013), studies in which mtDNA swaps were made within and between species have revealed that many such incompatibilities exist (James and Ballard, 2003; Sackton et al., 2003; Ellison and Burton, 2006; Ballard et al., 2007; Lee et al., 2008; Montooth et al., 2010). Future genetic mapping experiments will help identify the nature and mechanism of nuclear suppression of COIIG177S and reveal whether they are specific or general suppressors of COIIG177S and other male-harming mtDNA mutations. Strategies such as the one we have outlined here, together with new advances in manipulating mtDNA genomes (Xu et al., 2008), provide an exciting means to uncover the dark side of one of the most ancient symbioses on the planet.

Materials and methods

D. melanogaster maintenance and strains

All D. melanogaster strains were maintained on standard molasses-cornmeal medium at 25°C. We obtained the w1118 strain of D. melanogaster free of Wolbachia contamination (kind gift of Susan Parkhurst, FHCRC). We obtained the D. melanogaster DGRP lines from the Bloomington Drosophila Stock Center (Bloomington, IN). The other D. melanogaster lines were either obtained from the Drosophila Species Stock Center (San Diego, CA) or from our colleagues (Daven Presgraves, John Pool, Chip Aquadro).

We confirmed absence of Wolbachia using PCR in the original w1118 stock. Briefly, six flies from each of the assayed strains were homogenized in 49 μl squish buffer (10 mM Tris-CL pH 8, 1 mM EDTA, 25 mM NaCl) and 1 μl Proteinase K (200 μg/ml), following which the samples were incubated at 37°C for 30 min, heated to 95°C for 10 min and cooled to room temperature for 10 min. We then used WSP (Wolbachia surface protein) primers (5’- GCATTTGGTTAYAAAATGGACGA-3’ and 5’- GGAGTGATAGGCATATCTTCAAT-3’) and performed PCR using Taq DNA polymerase (New England Biolabs, Ipswich, MA). We also assessed the status of Wolbachia infection after 35 generations of experimental evolution,in the re-isolated wt and COIIG177S mtDNA lines (Figure 1—figure supplement 1). All samples tested were found to be negative for Wolbachia.

Experimental evolution

An original stock was established by expanding the w1118 strain from one bottle to 36 bottles. We established 12 experimental lines and 6 coevolving lines in bottles by adding 300 virgin females and 100 males from the original stock. The bottles were flipped three times a day for two days after which the flies were discarded. To set up every subsequent generation, virgin females were collected over a course of seven days and then crossed with males from the original stock in the case of the experimental lines. For coevolving lines, males from the corresponding coevolving line were used. After 35 generations, all lines were maintained by flipping bottles allowing sibling mating.

Fertility and sex ratio assays

All fertility and sex ratio assays were done in vials with five virgin females and three virgin males that were all 2–5 days old. All assays were done in 10 replicates per group. Flies were transferred to new vials every 2–3 days and flies were discarded after the fiveth’ ‘flip’. In order to assess overall fertility, we counted all adult progeny to emerge from all of the vials.

Whole genome sequencing

100 females from the original stock and experimental line 7 were lysed in 100 μl squish buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 25 mM NaCl, 200 μg/ml Proteinase K) with RNase A at final concentration of 30ng/ml. Lysate was incubated at 55°C for 1 hr followed by 95°C for 10 min. Total DNA was then phenol-chloroform extracted from the lysates and the DNA pellet was dissolved in 25 μl H2O. Library preparation of samples and paired-end sequencing was performed by the Genomics Shared Resource core at Fred Hutchinson Cancer Research Center on an Illumina HiSeq 2500. Sequence data are available through NCBI's SRA database (project SRP057279).

Whole genome sequencing data was analyzed by aligning reads to a modified version of the D. melanogaster reference genome assembly (BDGP Release 5), where we masked a region of the unassembled chromosome (chrU:5288528–5305749) that harbors an alternative version of the mitochondrial genome sequence (in order to prevent ambiguous mapping of mitochondrial reads to the chrU region). Reads were filtered, quality- and adapter-trimmed, and aligned to our custom reference genome using GSNAP (Wu and Nacu, 2010). SNPs were called in the non-repetitive region of the mitochondrial genome (chrM:1–14196) using GATK's HaplotypeCaller algorithm (DePristo et al., 2011) with a 'sample_ploidy' setting of 20. A G-to-A change at position chrM:3611, encoding COIIG177S (Figure 3), was present in 98% of Experimental Line 7 reads and 59% of reads from the original stock. No other variants had greater than 5% higher read frequency in Experimental Line 7 than in the original w1118 sample.

Duplex sequencing

Duplex Sequencing of fly mtDNA was performed as previously described (Kennedy et al., 2014) with several modifications. Total DNA was purified from individual fly heads using a QIAamp DNA micro kit (Qiagen Inc., Germantown MD). The DNA was sonicated using a Covaris AFA S2 ultrasonicator (Covaris Inc., Woburn MA) with the following settings: Duty cycle: 10%; Intensity: 5; Cycles/burst: 100; Time: 15 s × 3. The sheared DNA was then end-repaired and ligated using the NEBNext Ultra 2 end-repair and dA-tailing kit (New England Biolabs, Ipswich MA) according to the manufacturer’s instructions. Duplex Sequencing adapters, described previously (Kennedy et al., 2014), were ligated to the DNA library using the NEBNext Ultra 2 ligation kit (New England Biolabs, Ipswich MA) according to the manufacturer’s instructions. 1.5 ng of total DNA was then PCR amplified using KAPA HiFi DNA polymerase (Roche Inc., Basel Switzerland) according to the manufacturer’s recommendations. After amplification, the mtDNA was enriched by targeted capture using xGen target capture probes (Integrated DNA Technologies Inc., Coralville IA) specific to the fly mitochondrial genome. The samples were then sequenced on a NextSeq500 machine (Illumina Inc., San Diego, CA) to generate 150 bp paired-end reads. The data were processed as previously described (Kennedy et al., 2014).

Establishing wildtype and COIIG177S lines from the original stock

15 virgin females were collected from the original w1118 stock. Each female was individually mated with males from the same stock. Status of the G177S allele was assessed by Sanger sequencing of the females after they were allowed to have progeny. One line that appeared to be homoplasmic wildtype and one line that appeared to be homoplasmic mutant were kept and constituted the re-established stocks.

Cox activity

six females or eight males from each group were gently homogenized on ice in 50 μl of sodium phosphate buffer with 0.05% Tween-20. Lysates were centrifuged at 4°C at 4000 × g for 1 min. Supernatant was collected and 20 μl were used to measure COX activity using a kit (ScienCell, Carlsbad, CA). COX activity was normalized to total protein concentration as determined by Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Data shown represent averages of 4–5 replicates per group.

ROS levels

10 flies 3–4 days old and raised at 29°C were homogenized on ice in 200 μl PBS with 0.1% Tween-20. Lysates were centrifuged at 4°C at 13,000 × g for 10 min. The supernatant was collected and 100 μl was incubated with 50 μM H2DCF (Molecular Probes). Fluorescence intensity was measured in an Infinite M1000Pro (Tecan, Switzerland) microplate reader using 490 nm wavelength excitation and 520 nm wavelength emission. ROS levels were normalized to total protein concentration as determined by Pierce BCA Protein Assay Kit. Data shown represent averages of eight replicates per group.

ATP levels

Five males 3–4 days old and raised at 29°C were homogenized on ice in 100 μl guanidine extraction buffer (6 M guanidine HCl, 100 mM Tris-HCl, pH 7.3). Samples were frozen in liquid nitrogen for 5 min and then incubated at 95°C for 5 min. Lysates were centrifuged at 4°C for 10 min at 12,000 × g. Supernatant was collected and 5 μl was diluted in 95 μl H2O. 10 μl of the diluted lysate was used to measure ATP levels using the ATP determination kit (Cat #: A22066, Molecular Probes). ATP levels were normalized to total protein concentration as determined by Pierce BCA Protein Assay Kit. Data shown represent averages of four replicates per group.

Mating-induced egg laying

Five virgin wildtype females were either kept alone, or mated with three wildtype or COIIG177S mutant males at 29°C in vials with grape plates. Flies were flipped into new vials every day for eight days. 24 hr after the flies were removed from a vial, number of unhatched eggs and larvae on the grape plate were counted. Data shown represents averages of six replicates per group.

Testis imaging

Immunofluorescence staining of testes was performed as described previously (Cheng et al., 2008 ). Briefly, testes were dissected in PBS, transferred to 4% formaldehyde in PBS and fixed for 30–60 min. The testes were then washed in PBS-T (PBS containing 0.1% Triton-X) for at least 30 min and mounted in VECTASHIELD with DAPI (Vector Labs). Imaging of whole testis was performed on a Leica SP8 confocal microscope.

Sperm imaging

Unfixed seminal vesicles were dissected in PBS mounted onto slides with PBS containing 1 µM Mitotracker Green and Mitotracker CMXRos (Lifetechnologies). Sperm were extruded from seminal vesicles using a tungsten needle. Imaging was performed immediately upon addition of a cover slip to minimize the effects of hypoxia. Still images were taken on a Leica SP8 confocal microscope and movies were captured using a resonant scanner on a Leica SP5 confocal microscope.

Suppressor assays

Males from wild strains including the DGRP collection were crossed with w1118 females homoplasmic for either wildtype or COIIG177S mtDNA. Resulting heterozygous male progeny were assayed for fertility, sperm development and motility, and COX activity.

Lifespan assay

An assay for lifespan was performed as previously described (Burman et al., 2014). Briefly, 100 flies from each group were divided into five vials with 20 flies each. Vials were flipped every other day and the number of dead flies was counted until all the flies had died.

Bang sensitivity

An assay for bang sensitivity was performed as previously described (Burman et al., 2014). Briefly, vials with two flies each were mechanically stimulated by placement in a bench-top vortex for 10 s at the maximum setting. The time for each fly to right itself after vortexing was recorded. Data shown represents average from 18–20 flies per group.

Heat tolerance

Assay for resistance to heat-induced paralysis was performed as previously described (Burman et al., 2014). Briefly, flies were assayed for heat-induced paralysis by placing them into pre-warmed vials maintained at 39°C. The time for flies to become paralyzed was recorded. After exposure to 39°C for 6 min the animals were then placed in new room-temperature vials (~20°C), and the recovery time from paralysis was recorded. Data shown represents average from 16 flies per group.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
    Mitochondrial genotype affects fitness in Drosophila simulans
    1. AC James
    2. JW Ballard
    (2003)
    Genetics 164:187–194.
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
    Sexually antagonistic cytonuclear fitness interactions in Drosophila melanogaster
    1. DM Rand
    2. AG Clark
    3. LM Kann
    (2001)
    Genetics 159:173–187.
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
    Drosophila Mitochondrial Genetics: Evolution of Heteroplasmy through Germ Line Cell Divisions
    1. M Solignac
    2. J Génermont
    3. M Monnerot
    4. JC Mounolou
    (1987)
    Genetics 117:687–696.
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73
  74. 74
  75. 75

Decision letter

  1. K VijayRaghavan
    Reviewing Editor; Tata Institute of Fundamental Research, India

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for choosing to send your work entitled "Experimental evolution uncovers a male-harming mitochondrial mutation in Drosophila melanogaster" for consideration at eLife. Your full submission has been evaluated by K VijayRaghavan (Senior editor), a Reviewing Editor and two peer reviewers and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

As you will see from the reviewers' comments below, one of the primary concerns was that your claim of establishing a novel experimental evolution scheme to isolate male-harming mitochondrial DNA mutations is not supported by data. Without more compelling evidence for this scheme, the current manuscript is not suitable for eLife. In addition, the link between the genotype of the missense mitochondrial mutation (COIIG177S) and phenotype described is complicated by the fact that the phenotype requires high temperature. Finally, in light of these concerns, the effect of nuclear modifiers was of great interest, but it would require further development, such as identification and characterization, to elevate the manuscript.

Reviewer #1:

One major concern is the claim that the experimental model was "successful" in isolating the male-specific phenotype. As the allele already existed in the parental stock, it may simple be that the authors were able to – by chance alone – fix the allele in 1-in-18 lines, and not due to the breeding scheme. Some added detail as to the state of the allele in the other lines would help us to determine if the breeding scheme can actually be said to be successful in driving the emergence of this phenotype, or if the experiment was simply very lucky to catch this one allele. Some additional arguments in support of their method would be very helpful.

The allele in question appears to have been present in the parental stock, and was isolated to homoplasmy in 1 of the 18 experimental lines. Is it possible for the authors to test the dynamics of the heteroplasmic transmission in the other 17 lines? While the authors are unable to detect any defects aside from the male fertility, if the transmission of the allele is consistent with neutral drift, the authors would clearly demonstrate that the allele had no fitness effects until fixed in the population and would very strongly support the claim of a specific phenotype. However, evidence of negative selection would show the some difficult-to-detect deficiency in the flies with the allele, and a positive selection signal would set up an interesting scenario of the allele being beneficial to the population, but detrimental to male fertility when fixed.

Reviewer #2:

This manuscript addresses an important issue in biology and describes a potentially interesting male semi-sterile mutation. The reported results can be divided into three areas. In the first, the manuscript describes a strategy described as "a novel experimental evolution scheme in Drosophila melanogaster to recover male-harming mutations". In the second, the phenotype of the temperature-enhanced sterility of the one mutation recovered is characterized. In the third, modification of the sterility affects by nuclear background is described. As discussed below, I found that the first part to harbor significant potential interest, but felt that the paper did not support the efficacy of the practice that it advocated. Nonetheless, they retrieved a male harming mutant, and second part of the manuscript describes its characterization. While this is an interesting mutant that fits the bill of a male harming mutant, I was somewhat uncertain about the value of the details of the phenotypic characterization. It seemed to me the details of the phenotype are likely to depend on the use of a high temperature to increase the expressivity of the defect. The analysis of enhancers in the third part of the paper is of great interest, but it was not advanced to level that gives us any insight into the nature of the interactions. In this regard, I was again concerned about the use of high temperature to enhance the phenotype. No attempt was made to disambiguate the influence of genetic background effects on the temperature range of fertility from a specific effect on the mutation, leaving a nagging suspicion that the effect of the nuclear background might not be very specific.

The strategy that was presented as a method to allow accumulation of male-harming mutations without hypothetical secondary counter selection due to male sterility was to simply out cross the females to an independent population of males. There are several issues with the claimed importance of this strategy and questions regarding its effectiveness that the manuscript fails to address.

One tangential, but in my mind important, consideration is that outcrossing may be an experimental condition in the laboratory but it is standard in the wild. If the rationale were correct, the inbreeding of confined laboratory stocks should be protecting them from the emergence of male harming mutants. In this regard, it would be interesting to compare the frequency of male harming mtDNA mutations in well-mixed wild populations, to the frequency in the confined breeding populations of laboratory flies. According to the rationale, one might expect a substantial frequency of male harming mutants in wild populations that then fail when established as more inbred laboratory stocks.

But the most important question is whether the paper shows that the strategy was effective. I was not convinced that it did. The only recovered mutation was in the starting population at a very high level. The paper suggests in its wording that this was a fleeting thing caught by the breeding strategy: e.g. It is suggested that "the mutant allele likely arose de novo in the original stock and transiently existed in a state of heteroplasmy before approaching homoplasmy in EL7", and "we devised an experimental evolution scheme to absolve mtDNA of supporting male function while both maintaining selection on female mtDNA function as well as reducing the possibility of nuclear suppression or male-harming mtDNA. Our scheme uncovered a COIIG177S mtDNA mutation[…]". The idea of fleeting mutation present in the original stock that was captured by the "experimental evolution scheme" might support the idea that it was effective. However, it seems very unlikely that the mutation was fleeting and there is no evidence that the scheme was needed to capture it. Even rare changes can go to fixation in small populations and one out six assayed flies in the starting population was homoplasmic for the mutation. Over 35 generations (plus an unspecified number of generations of additional crosses in which the flies were allowed to self) drift will lead to dramatic fluctuations in the abundance of the homoplasmic line and to random fixation. Fixation of an already abundant mutation in one of 18 lines is no surprise and there is no foundation for a conclusion that this possibility was enhanced by the "experimental evolution scheme".

The only data mentioned to support the idea that the mutation was present transiently in the original stock were not shown and the quoted finding does not seem directly relevant: "heteroplasmic flies were eventually lost over the course of a year from the original stock after their initial detection; later analyses revealed the population now only consists of individuals carrying homoplasmic wildtype or mutant mtDNA." The loss of the heteroplasmic flies is only indirectly relevant. Since the mutant persisted in the population as homoplasmic flies there is no indication that its presence was transient. Additionally, based on Solignac's (1984) detailed description of the gradual and statistical segregation of mtDNA genotypes one can make inferences about the behavior of a mutation in a stock – whether it is drifting randomly or is affected by selection. The only data shown regarding the distribution of heteroplasmy was an analysis of six flies of the original population. This is woefully inadequate for any detailed consideration of levels of heteroplasmy. A more thorough analysis might have revealed whether there was any constraint limiting the propagation of the mutant allele in the original stock, since such constraints, by selecting for different progeny classes, will alter the unbiased statistical changes in the distribution of heteroplasmy levels. Demonstration that a constraint existed would provide some foundation for the proposal that the "experimental evolutionary scheme" would have consequence-relief of this constraint. The crosses presented in this manuscript could have simply provided opportunity for random drift to fix an abundant pre-existing mutation. To make the claims regarding the "experimental evolutionary scheme", the manuscript would have to dismiss this null hypothesis and demonstrate the efficacy of the approach. At present, the appearance of one line out 12 with a mutant in the experimental sample and none in the 6 control lines is far from enough to argue that the outcrossing was essential to the recovery of the mutation.

The “experimental evolutionary scheme" was designed to bypass the influence of indirect counter selection due to reduced fertility of the males: but to what extent does such a selection influence outcome? It appears that the heteroplasmic male flies are fertile (not tested, but failure to see fertility depression in lines with high levels of mutant genomes suggest that mutant genomes can be carried without a phenotype) and hence that no such counter selection would operate early after the appearance of the mutant allele (when it is rare and heteroplasmic). The persistence of mutant flies (perhaps heteroplasmic, but untested) in three of the six coevolving/control lines suggests the absence of a significant counter selection preventing their maintenance. Consequently, male sterility could only come into play, if at all, very late after the mutation rose to be a dominant allele with frequent production of homoplasmic derivatives, and since the male sterility phenotype is weak at the temperatures used for propagation, it would not be surprising that secondary selection due to male fertility could prevent fixation of the mutant. Furthermore, since the scheme did not use unrelated males for outcrosses but used males from the starting population, which apparently harbored the mutant for many generations (many generations are essential to produce homoplasmic flies), these males might already have carried suppressors of the phenotype. Thus, I do not think that manuscript offers support for claim that an effective scheme for recovery of male-harming mutations is described. I would like to see direct evidence that it is effective and efficient.

The second component of the manuscript, the description of the phenotype of the male sterile mutation is detailed but I was not impressed that it gave us much insight into the defects and their connection to the mutation. One obfuscating issue is the use of high temperature to enhance the phenotype of an otherwise weak expressivity of the mitochondrial mutation. The highest temperature at which flies can be cultivated is generally limited by male sterility (e.g. David JR et al., 2005). For laboratory stocks, this highest temperature is usually taken as 29°C but populations tend to show differences in the temperature range across which they will grow-perhaps not surprisingly as environmental variables and adaptation would select for different ranges. At the temperature limit, defects in sperm development are usually seen as responsible for sterility (e.g. Rohmer C, et al., 2004). Interactions between nucleus, mitochondria and temperature have been described as affecting male sterility (Hoekstra et al., 2013). The manuscript shows an interaction of temperature and the mitochondrial mutation, but the study of the phenotype leaves an uncertainty whether the phenotype described is primarily that of the mutant or that produced at high temperature. That is, one might imagine that the mutation might sensitize the flies so that the high temperature defect shows up at 29°C, or one can imagine that 29°C increases the expressivity of the mutant phenotype, or something in between. In this regard, it would be nice to know if the sterile phenotype of w1118 wild type flies when raised at temperatures beyond 29°C, resembles or is distinct from the phenotype as seen in the mutant at 29°C.

The analysis of nuclear interactions is potentially exciting, but the reported analysis represents a first step. The loci responsible are not mapped or otherwise identified. Sequenced strains are analyzed but no mention is made of any finding emanating from this. I worry that modification might be the result of modification of temperature sensitivity. The things underlying this concern are that the maximal temperature of male fertility commonly vary between strains, genetic backgrounds that modified the reported phenotype are remarkably common, and the data shown for 29°C in the supplement of Figure 9 shows that w1118 is unusually weak even at the temperature selected as widely permissive. If the temperature is raised to say 31°C, the wild type w1118 stock is likely to be male sterile: I would like to know whether temperature tolerance is increased (i.e. this male sterility expected for the wild-type w1118 strain is suppressed) by out crossing of the wild type to the same strains that suppressed the mutant. This control might reveal that the recorded affects are due to expected widespread differences in thermo-tolerance. This concern that the modification may be due to a more generic affect is partly alleviated by the reported increase in Cox activity in suppressed mutant backgrounds.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "A mitochondrial DNA hypomorph of cytochrome oxidase specifically impairs male fertility in Drosophila melanogaster" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In their final submission the authors should address better previous criticism influenced by the fact the supposed approach to isolate male harming mutants was the most innovative part of the original submission. The new version is hence weaker, but still a nice report and should be published at eLife. Of two previous criticisms, the bigger one is that the data are not sufficient to document homoplasmy. While this could be serious flaw, it is very likely that the line is close to homogeneous and if it were not and the authors sampled flies that were heteroplastic in their tests of fertility they would have seen phenotypic variation. If the authors answer in this way, and put more guarded statements in their discussion, we would be satisfied on this account. The are other issues, on the phenotypic analysis which are more about making things more clear, particularly aspects that are buried in the supplementary data. Eliminating some unfounded discussion will bring more sharpness, but this is something that the authors are best placed to address. These are detailed below.

Essential revisions:

The presentation of the data has been changed substantially, while the data remain much the same. The change in the presentation has resolved one of the central concerns about the previous version of the manuscript that presented a crossing scheme as an effective selection for male harming mutations. The critiques, certainly mine, argued that this was not supported by the data and that the mitochondrial mutation preexisted and was uncovered by chance. The present draft still argues that this scheme should be effective, which I severely doubt based on pragmatics of mutation frequency and infrequent segregation of homoplasmic lines. I think that the unsubstantiated sentence in the current version saying that "we reasoned that multiple mutations per mtDNA would be sampled in 35 generations across 18 lines" should be eliminated or justified.This is completely untenable as shown by the mathematical analysis of the number of generations required for lines to progress to homoplasmy (See Solignac). Nonetheless, the major change in presentation has corrected the biggest problem with the previous submission.

The question now is, what is the merit of the present manuscript. The authors do report a maternally inherited partial male sterility trait that is linked with an SNP mt:CoII. The recognized SNP may well be the cause of the phenotype, but the possibility of linked changes in the unsequenced 4kb regulatory region was not eliminated (note that this is the region that changes the most rapidly and spontaneous variants of this region have been shown to arise in laboratory stocks – see Rand). Furthermore, identification of a mitochondrial DNA mutation altering an electron transport component that causes male sterility is not entirely novel (see Xu). The manuscript does report a fairly detailed effort to characterize the defect and shows suppression of this defect in crosses that introduce different nuclear backgrounds. In this regard, it goes beyond other reports that argued for nuclear modification of phenotypes caused by mitochondrial mutations. In our opinion these findings are of interest and could constitute a significant advance. We are however, left with some hesitancy because the merit ought to lie in the rigor in of the documentation of the principles, and we see some shortcomings.

One concern is that the data are not sufficient to rigorously demonstrate the genetic homogeneity of the lines examined. The details are below, but this view can be summarized as follows: the original plan treated the crosses as if they were a selection that would yield a pure male harming mutation, and suitable measures where not take to ensure the genetic homogeneity of the stock that was retrieved. Given the number of mt genomes carried by flies, a high sensitivity of detection of a rare allele (in a range approaching 0.1%) and analysis of single flies is needed to rigorously demonstrate homoplasmy.

A second issue concerns data on fertility demonstrating that in an early first mating the males exhibited no (at 25°C) or weak (29°C) deficits in fertility. At least in reading the text, the subsequent analyses did not seem to take this fully into account. Many of my concerns here could be dealt with by better reference to supplementary material and more careful wording. Nonetheless, it still seemed to me that this issue was not fully investigated and the impact of this finding on other analyses was not always considered.

Overall, this an interesting report that suffers from weakness in its details. These can be addressed in the revised final submission

https://doi.org/10.7554/eLife.16923.029

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

As you will see from the reviewers' comments below, one of the primary concerns was that your claim of establishing a novel experimental evolution scheme to isolate male-harming mitochondrial DNA mutations is not supported by data.

We completely agree with this criticism. While we obtained the COIIG177S mtDNA mutation via our experimental evolution scheme, our subsequent experiments were able to determine that this was a pre-existing heteroplasmic mtDNA mutation that manifested its male infertility only upon fixation in one of the experimental lines (el7). For the purposes of the current manuscript, we now describe the experimental evolution scheme simply to detail how we found a specifically male-harming mtDNA mutation. We also acknowledge that this mutation might have been obtainable without the scheme with a priori knowledge of the heteroplasmy (which we did not have), and that significantly more work is needed to determine whether the scheme will be effective. We have accordingly significantly edited our manuscript, including changing the title.

In addition, the link between the genotype of the missense mitochondrial mutation (COIIG177S) and phenotype described is complicated by the fact that the phenotype requires high temperature.

One of the reviewers raised the concern that the male fertility defects caused by the COIIG177S mutation might be confounded by the use of high temperature. We performed mutant characterization in the original submission at high temperature because the fertility phenotype is more pronounced in young males at high temperature. However, we agree with the reviewer concerns and have now performed additional experiments to address these points. We also showed in the original submission that the fertility phenotype is also present at 25°C but it manifests in older males. We now show that the cellular phenotype of the COIIG177S mtDNA mutation is similar in older males at 25°C to the phenotype in young males at 29°C. We further show that sterility of young males at 29°C is suppressed by a nuclear background (OregonR) in a manner similar to the suppression of old males at 25°C. Thus, we feel that we have completely addressed with our new experiments the reviewer concerns that our sterility phenotype and its suppression are confounded by a requirement of high temperature. It bears reiterating that we performed many life-history trait measurements at both 25°C and 29°C; male fertility was the only phenotype affected suggesting that the sterility defect associated with COIIG177S mtDNA is highly specific.

Finally, in light of these concerns, the effect of nuclear modifiers was of great interest, but it would require further development, such as identification and characterization, to elevate the manuscript.

There are two points to be made here. First, the abundance of nuclear suppressor(s) in D. melanogaster directly bears out the predictions of the theory of mito-nuclear genetic conflict and reinforces our finding that COIIG177S is a male-harming mtDNA mutation.. Second, we would love to know the genetic identity of the suppressor. But the reviewer is doubtless aware that this is a significant endeavor involving at least a few years of work. The best demonstration of this suppressor mapping was a recent paper (Meikeljohn et al. PLOS Genetics 2013), which found a nuclear-encoded tRNA synthetase that epistatically interacts with and suppresses a mitochondrial tRNA mutation. This work appeared three years after the original observation of the incompatibility (Montooth et al. Evolution 2010). Furthermore, the identity of this suppressor mutation was suggested by the very nature of the mtDNA mutation in a tRNA gene. Our preliminary experiments and the fact that we see both partial and completely restoration of fertility suggest that the suppression involves multiple genetic loci, whose identification will require 2-3 years of additional work. We intend to aggressively pursue this, but we feel that lack of this identification does not affect the novelty or importance of our findings in the current study.

Isolation of a bona fide male-harming mtDNA mutation

Although this is not a point explicitly mentioned by the editor, we would like to reinforce our claim that the identification of a specifically male-harming mtDNA mutation is novel and to our knowledge unprecedented in animals. The closest that any studies have come to characterization of ‘male-harming’ mtDNA variants are the elegant series of experiments performed by the group of Damian Dowling, who showed that mtDNA haplotypes from different D. melanogaster strains affect aging in males but not females (Camus et al. 2012 Current Biology), and competitive male fertility (Yee et al. 2013 Current Biology). These studies did not comprehensively test the role of all mtDNA haplotypes on female fitness. Just to highlight the novelty of our finding, while our paper was under review at eLife, one mtDNA haplotype that was previously shown to deleteriously affect competitive male fertility was also shown to decrease female lifespan (Camus et al., 2015 Current Biology); such a mutation could not be considered specifically ‘male-harming’. In contrast, our work examined the impact of COIIG177S mutation on both males and females in the context of fertility, lifespan, neurological function, and stress resistance, each at normal and elevated temperatures, and in young and old individuals. Hence, we can conclude with significantly greater confidence than any other study published so far that a single mtDNA variant in metazoans can be specifically detrimental to males but neutral in females. We believe that this comprehensive characterization of the COIIG177S mutation is the most novel and impactful finding of our manuscript. We have now revised our manuscript to emphasize this point.

We hope the reviewers will agree that the revised manuscript addresses all of their concerns, most of which arise from our initial claim regarding the ‘success’ of the experimental evolution strategy. We further hope the reviewers will agree that our demonstration of COIIG177S as a specifically male-harming mtDNA mutation in animals is highly significant and impactful.

Reviewer #1:

One major concern is the claim that the experimental model was "successful" in isolating the male-specific phenotype. As the allele already existed in the parental stock, it may simple be that the authors were able to – by chance alone – fix the allele in 1-in-18 lines, and not due to the breeding scheme. Some added detail as to the state of the allele in the other lines would help us to determine if the breeding scheme can actually be said to be successful in driving the emergence of this phenotype, or if the experiment was simply very lucky to catch this one allele. Some additional arguments in support of their method would be very helpful.

As discussed earlier, we agree with the reviewer. We now acknowledge that although we obtained the COIIG177S mutation via the experimental evolution scheme, it may have been possible to fix this allele to homoplasmy without our breeding scheme. We have significantly revised the beginning of the manuscript to reflect this acknowledgement, and to emphasize our recovery of the COIIG177S mutation, which appears to be specifically detrimental to males and is the most significant finding of our work.

The allele in question appears to have been present in the parental stock, and was isolated to homoplasmy in 1 of the 18 experimental lines. Is it possible for the authors to test the dynamics of the heteroplasmic transmission in the other 17 lines? While the authors are unable to detect any defects aside from the male fertility, if the transmission of the allele is consistent with neutral drift, the authors would clearly demonstrate that the allele had no fitness effects until fixed in the population and would very strongly support the claim of a specific phenotype. However, evidence of negative selection would show the some difficult-to-detect deficiency in the flies with the allele, and a positive selection signal would set up an interesting scenario of the allele being beneficial to the population, but detrimental to male fertility when fixed.

We do not have longitudinal data from all our experimental and coevolved lines, but the data we do have suggests that the fixation of the COIIG177S mutation did not occur until at least 25 generations through our experiment. Since our experimental lines would be expected to be homogeneous at the nuclear level given our backcrossing scheme, our findings that the viability and fertility of the EL lines (except EL7) are like wildtype despite being highly variable for the COIIG177S heteroplasmic mutation lead us to favor the conclusion that the mutation is neutral, not beneficial, to the population, but is detrimental to male fertility on homoplasmy.

Reviewer #2:

This manuscript addresses an important issue in biology and describes a potentially interesting male semi-sterile mutation. The reported results can be divided into three areas. In the first, the manuscript describes a strategy described as "a novel experimental evolution scheme in Drosophila melanogaster to recover male-harming mutations". In the second, the phenotype of the temperature-enhanced sterility of the one mutation recovered is characterized. In the third, modification of the sterility affects by nuclear background is described. As discussed below, I found that the first part to harbor significant potential interest, but felt that the paper did not support the efficacy of the practice that it advocated. Nonetheless, they retrieved a male harming mutant, and second part of the manuscript describes its characterization. While this is an interesting mutant that fits the bill of a male harming mutant, I was somewhat uncertain about the value of the details of the phenotypic characterization. It seemed to me the details of the phenotype are likely to depend on the use of a high temperature to increase the expressivity of the defect. The analysis of enhancers in the third part of the paper is of great interest, but it was not advanced to level that gives us any insight into the nature of the interactions. In this regard, I was again concerned about the use of high temperature to enhance the phenotype. No attempt was made to disambiguate the influence of genetic background effects on the temperature range of fertility from a specific effect on the mutation, leaving a nagging suspicion that the effect of the nuclear background might not be very specific.

The strategy that was presented as a method to allow accumulation of male-harming mutations without hypothetical secondary counter selection due to male sterility was to simply out cross the females to an independent population of males. There are several issues with the claimed importance of this strategy and questions regarding its effectiveness that the manuscript fails to address.

One tangential, but in my mind important, consideration is that outcrossing may be an experimental condition in the laboratory but it is standard in the wild. If the rationale were correct, the inbreeding of confined laboratory stocks should be protecting them from the emergence of male harming mutants. In this regard, it would be interesting to compare the frequency of male harming mtDNA mutations in well-mixed wild populations, to the frequency in the confined breeding populations of laboratory flies. According to the rationale, one might expect a substantial frequency of male harming mutants in wild populations that then fail when established as more inbred laboratory stocks.

This is an interesting suggestion and indeed, the main topic of a paper (Wade & Brandvain 2008 Evolution) that predicts exactly what the reviewer suggests – i.e. inbreeding selects against male-harming mtDNA mutations due to indirect selection. Indeed, this theoretical claim served as the rationale for our experimental evolution strategy. We took the predictions from this theoretical study and used it to motivate our scheme to isolate male-harming mtDNA mutations. The reviewer makes an interesting suggestion that we plan to follow up on by making use of the DGRP collection in the future. However, that work would be a significant undertaking that is beyond the scope of this manuscript.

But the most important question is whether the paper shows that the strategy was effective. I was not convinced that it did. The only recovered mutation was in the starting population at a very high level. The paper suggests in its wording that this was a fleeting thing caught by the breeding strategy: e.g. It is suggested that "the mutant allele likely arose de novo in the original stock and transiently existed in a state of heteroplasmy before approaching homoplasmy in EL7", and "we devised an experimental evolution scheme to absolve mtDNA of supporting male function while both maintaining selection on female mtDNA function as well as reducing the possibility of nuclear suppression or male-harming mtDNA. Our scheme uncovered a COIIG177S mtDNA mutation[…]". The idea of fleeting mutation present in the original stock that was captured by the "experimental evolution scheme" might support the idea that it was effective. However, it seems very unlikely that the mutation was fleeting and there is no evidence that the scheme was needed to capture it. Even rare changes can go to fixation in small populations and one out six assayed flies in the starting population was homoplasmic for the mutation. Over 35 generations (plus an unspecified number of generations of additional crosses in which the flies were allowed to self) drift will lead to dramatic fluctuations in the abundance of the homoplasmic line and to random fixation. Fixation of an already abundant mutation in one of 18 lines is no surprise and there is no foundation for a conclusion that this possibility was enhanced by the "experimental evolution scheme".

As we have responded to (points 1 and 5 above), we agree with the reviewer’s concern. We have significantly revised the manuscript to remove any reference to the ‘success’ of our experimental evolution strategy but simply to state that this is how we obtained the COIIG177S mutation. We remain confident that the mutation we did obtain is specifically male-harming and therefore its characterization important and, to our knowledge, unprecedented. As the reviewers point out, the effectiveness of the experimental evolution approach has yet to be rigorously tested. A full proof-of-principal test of the strategy will be the focus of future work.

The only data mentioned to support the idea that the mutation was present transiently in the original stock were not shown and the quoted finding does not seem directly relevant: "heteroplasmic flies were eventually lost over the course of a year from the original stock after their initial detection; later analyses revealed the population now only consists of individuals carrying homoplasmic wildtype or mutant mtDNA." The loss of the heteroplasmic flies is only indirectly relevant. Since the mutant persisted in the population as homoplasmic flies there is no indication that its presence was transient. Additionally, based on Solignac's (1984) detailed description of the gradual and statistical segregation of mtDNA genotypes one can make inferences about the behavior of a mutation in a stock – whether it is drifting randomly or is affected by selection. The only data shown regarding the distribution of heteroplasmy was an analysis of six flies of the original population. This is woefully inadequate for any detailed consideration of levels of heteroplasmy. A more thorough analysis might have revealed whether there was any constraint limiting the propagation of the mutant allele in the original stock, since such constraints, by selecting for different progeny classes, will alter the unbiased statistical changes in the distribution of heteroplasmy levels. Demonstration that a constraint existed would provide some foundation for the proposal that the "experimental evolutionary scheme" would have consequence-relief of this constraint. The crosses presented in this manuscript could have simply provided opportunity for random drift to fix an abundant pre-existing mutation. To make the claims regarding the "experimental evolutionary scheme", the manuscript would have to dismiss this null hypothesis and demonstrate the efficacy of the approach. At present, the appearance of one line out 12 with a mutant in the experimental sample and none in the 6 control lines is far from enough to argue that the outcrossing was essential to the recovery of the mutation.

The reviewer is correct in their argument that knowing the status of the heteroplasmy in the stock would have been important at the onset of the experiment. The best we can do is infer the heteroplasmy status in the stock at a time when we were evaluating and discovering the focal mtDNA mutation. We would be quite happy to characterize additional flies from the ancestral stock except that recognizing that this characterization is occurring >50 generations after the beginning of our experiment, the main point we wished to emphasize is that there was heteroplasmy in the original stock. We hope elimination of sentences regarding the transient nature of the heteroplasmy will minimize any confusion and avoid us overstating that we understand the dynamics of this particular mutation. While we agree that following the dynamics of this heteroplasmy would be very interesting, and would help bolster our claim of a “constraint-release” afforded by the experimental evolution scheme, such detailed characterization is beyond the scope of our paper. Even such a conclusion would be limited by the fact that the mutation became fixed in only 1 out of 12 experimental lines. Instead, we focus the bulk of the manuscript on what makes the mutation specifically male-harming.

The “experimental evolutionary scheme" was designed to bypass the influence of indirect counter selection due to reduced fertility of the males: but to what extent does such a selection influence outcome? It appears that the heteroplasmic male flies are fertile (not tested, but failure to see fertility depression in lines with high levels of mutant genomes suggest that mutant genomes can be carried without a phenotype) and hence that no such counter selection would operate early after the appearance of the mutant allele (when it is rare and heteroplasmic). The persistence of mutant flies (perhaps heteroplasmic, but untested) in three of the six coevolving/control lines suggests the absence of a significant counter selection preventing their maintenance. Consequently, male sterility could only come into play, if at all, very late after the mutation rose to be a dominant allele with frequent production of homoplasmic derivatives, and since the male sterility phenotype is weak at the temperatures used for propagation, it would not be surprising that secondary selection due to male fertility could prevent fixation of the mutant. Furthermore, since the scheme did not use unrelated males for outcrosses but used males from the starting population, which apparently harbored the mutant for many generations (many generations are essential to produce homoplasmic flies), these males might already have carried suppressors of the phenotype. Thus, I do not think that manuscript offers support for claim that an effective scheme for recovery of male-harming mutations is described. I would like to see direct evidence that it is effective and efficient.

The reviewer makes several good points here, which we address on-by-one. First, we can conclude with significant confidence that there are no suppressors of the sterility phenotype in the ancestral stock used to backcross. This is the same stock we did all our experiments to characterize the infertility and any suppressors would have been immediately evident. This is in spite of the fact that the mutation was present in this stock.

Second, the reviewer is doubtless aware that the presence of a cytoplasmically inherited male-harming mtDNA mutation would still be tolerated in the presence of the wildtype allele since only indirect selection acts on the mutant mtDNA. Nevertheless, we completely agree with the reviewer that the scheme we have proposed cannot be deemed essential for obtaining the COIIG177S mutation. We also agree with the reviewer that our findings show that the phenotype of COIIG177S only manifests when present at high levels of heteroplasmy, and that such penetrance issues will affect the detailed design of any experimental evolution strategy. To address the reviewer concerns, we have greatly de-emphasized the significance of the experimental evolution strategy and instead focus on the significance of finding and characterizing a male-harming mtDNA mutation.

The second component of the manuscript, the description of the phenotype of the male sterile mutation is detailed but I was not impressed that it gave us much insight into the defects and their connection to the mutation. One obfuscating issue is the use of high temperature to enhance the phenotype of an otherwise weak expressivity of the mitochondrial mutation. The highest temperature at which flies can be cultivated is generally limited by male sterility (e.g. David JR et al., 2005). For laboratory stocks, this highest temperature is usually taken as 29°C but populations tend to show differences in the temperature range across which they will grow-perhaps not surprisingly as environmental variables and adaptation would select for different ranges. At the temperature limit, defects in sperm development are usually seen as responsible for sterility (e.g. Rohmer C, et al., 2004). Interactions between nucleus, mitochondria and temperature have been described as affecting male sterility (Hoekstra et al., 2013). The manuscript shows an interaction of temperature and the mitochondrial mutation, but the study of the phenotype leaves an uncertainty whether the phenotype described is primarily that of the mutant or that produced at high temperature. That is, one might imagine that the mutation might sensitize the flies so that the high temperature defect shows up at 29°C, or one can imagine that 29°C increases the expressivity of the mutant phenotype, or something in between. In this regard, it would be nice to know if the sterile phenotype of w1118 wild type flies when raised at temperatures beyond 29°C, resembles or is distinct from the phenotype as seen in the mutant at 29°C.

We take advantage of the fact that the COIIG177S mutation affects male fertility in both age- and temperature- dependent fashion. We now show that the age-dependent infertility (performed at 25°C) is exactly what we would predict using additional cytological analysis of seminal vesicles, which we have shown is an accurate surrogate for male fertility. We find that the seminal vesicle size of COIIG177S mutant males is reduced compared to those with wildtype mtDNA, similar to what we found with young males raised at 29°C. Moreover, while w1118 males with wildtype mtDNA also show impairment at 29°C, their seminal vesicle sizes are not significantly reduced. Our additional experiments show that the infertility defect cannot be solely explained as a confounding factor with temperature, and that the temperature-dependent reduction of fertility in wildtype w1118 males is distinct from COIIG177S-dependent defect at 29°C. We hope that our additional experiments should satisfy the reviewer’s concerns regarding this criticism. Moreover, we must emphasize again that this is, to the best of our knowledge, the most detailed characterization of the phenotypic consequences of multiple life-history traits (aging, fertility, neural function) in young and old males and females, at two different temperatures.

The analysis of nuclear interactions is potentially exciting, but the reported analysis represents a first step. The loci responsible are not mapped or otherwise identified. Sequenced strains are analyzed but no mention is made of any finding emanating from this. I worry that modification might be the result of modification of temperature sensitivity. The things underlying this concern are that the maximal temperature of male fertility commonly vary between strains, genetic backgrounds that modified the reported phenotype are remarkably common, and the data shown for 29°C in the supplement of Figure 9 shows that w1118 is unusually weak even at the temperature selected as widely permissive. If the temperature is raised to say 31°C, the wild type w1118 stock is likely to be male sterile: I would like to know whether temperature tolerance is increased (i.e. this male sterility expected for the wild-type w1118 strain is suppressed) by out crossing of the wild type to the same strains that suppressed the mutant. This control might reveal that the recorded affects are due to expected widespread differences in thermo-tolerance. This concern that the modification may be due to a more generic affect is partly alleviated by the reported increase in Cox activity in suppressed mutant backgrounds.

The main conclusion we wish to draw from the nuclear suppressor findings is that there is a significant nuclear genetic variation to suppress the male-harming effects of mtDNA mutations like COIIG177S. We seriously considered the reviewer’s criticism that this could also be a pleiotropic effect due to the high temperatures we used to fully manifest the male infertility. Once again, using aged males, we show that the heterozygous OregonR nuclear background completely rescues (old) male fertility defects at 25°C that are caused by the COIIG177S mutation and that this fertility rescue is associated with suppression of the decrease in seminal vesicle size. Together with our earlier presented data, our findings strongly suggest that the OregonR nuclear genome harbors a ‘true’ suppressor of the COIIG177s-dependent male fertility defect and its mechanism of suppression is likely the same at 25°C and 29°C. We hope that our revised manuscript emphasizing our detailed characterization of how a hypomorphic mtDNA mutation can cause male-specific impairment will be viewed favorably by the reviewer. While we share the reviewer’s excitement about the potential mapping of these nuclear suppressors, we point out that this is not a trivial undertaking. The first author hopes to identify and characterize the nuclear suppressor mutation(s) in a future study which will take at least a few more years.

[Editors' note: the author responses to the re-review follow.]

Summary:

In their final submission the authors should address better previous criticism influenced by the fact the supposed approach to isolate male harming mutants was the most innovative part of the original submission. The new version is hence weaker, but still a nice report and should be published at eLife. Of two previous criticisms, the bigger one is that the data are not sufficient to document homoplasmy. While this could be serious flaw, it is very likely that the line is close to homogeneous and if it were not and the authors sampled flies that were heteroplastic in their tests of fertility they would have seen phenotypic variation. If the authors answer in this way, and put more guarded statements in their discussion, we would be satisfied on this account. The are other issues, on the phenotypic analysis which are more about making things more clear, particularly aspects that are buried in the supplementary data. Eliminating some unfounded discussion will bring more sharpness, but this is something that the authors are best placed to address. These are detailed below.

With new experiments (detailed below), we now show that the strains we analyzed are near homoplasmy (100% or 99.9%). We also address the other concerns raised by the reviewers.

Essential revisions:

The presentation of the data has been changed substantially, while the data remain much the same. The change in the presentation has resolved one of the central concerns about the previous version of the manuscript that presented a crossing scheme as an effective selection for male harming mutations. The critiques, certainly mine, argued that this was not supported by the data and that the mitochondrial mutation preexisted and was uncovered by chance. The present draft still argues that this scheme should be effective, which I severely doubt based on pragmatics of mutation frequency and infrequent segregation of homoplasmic lines. I think that the unsubstantiated sentence in the current version saying that "we reasoned that multiple mutations per mtDNA would be sampled in 35 generations across 18 lines" should be eliminated or justified.This is completely untenable as shown by the mathematical analysis of the number of generations required for lines to progress to homoplasmy (See Solignac). Nonetheless, the major change in presentation has corrected the biggest problem with the previous submission.

The reviewers express concern over our statement that multiple mtDNA mutations might be sampled over 35 generations across 18 lines. Note that we intentionally used the word ‘sampled’ to distinguish mutations that arise versus those that arise and drive to homoplasmy. In addition, while mtDNA segregation can take many generations to reach homoplasmy, dramatic instances of rapid shifts in heteroplasmy have also been observed in humans, cows, and flies (Brown et al., 2001; Hauswirth & Laipis, 1982; Olivo et al., 1983; Matsuura et al., 1990; Matsuura et al., 1991; Koehler et al., 1991; Jenuth et al., 1996; Matsuura et al., 1997; Sato et al., 2007; Fan et al., 2008; Lee et al., 2012; Freyer et al., 2012; Sharpley et al., 2012; Ma, Xu & O’Farrell, 2014; Ma & O’Farrell, 2015; Ma & O’Farrell, 2016). Nevertheless, to avoid any ambiguity, we now state that this period may not be necessary to drive mtDNA mutations to near homoplasmy and now cite the Solignac reference (Solignac et al. Genetics 1987).

The question now is, what is the merit of the present manuscript. The authors do report a maternally inherited partial male sterility trait that is linked with an SNP mt:CoII. The recognized SNP may well be the cause of the phenotype, but the possibility of linked changes in the unsequenced 4kb regulatory region was not eliminated (note that this is the region that changes the most rapidly and spontaneous variants of this region have been shown to arise in laboratory stocks – see Rand). Furthermore, identification of a mitochondrial DNA mutation altering an electron transport component that causes male sterility is not entirely novel (see Xu). The manuscript does report a fairly detailed effort to characterize the defect and shows suppression of this defect in crosses that introduce different nuclear backgrounds. In this regard, it goes beyond other reports that argued for nuclear modification of phenotypes caused by mitochondrial mutations. In our opinion these findings are of interest and could constitute a significant advance. We are however, left with some hesitancy because the merit ought to lie in the rigor in of the documentation of the principles, and we see some shortcomings.

The reviewer raises two points. First, there is a possibility of linked changes in the unsequenced 4 kb regulatory region- the so-called D loop. We now explicitly suggest this possibility in the Discussion. However, we would like to point out that the causal connection with the COIIG177S mtDNA mutation is quite strong. We have observed a biochemical defect in cytochrome C oxidase activity that correlates perfectly with the male infertility phenotype. This COX activity is rescued in the suppressor background – these data strongly suggest that the G177S mutation causes the COX defect, which in turn causes the fertility defect. This is as strong a statement that can be made with a causal connection with a single mtDNA mutation, the exception being the Xu et al. paper that used a restriction enzyme strategy to introduce mtDNA mutations. In all other cases that have mapped a phenotype to a single mutation in mtDNA they did not assay or sequence the A+T region (Camus et al. 2015, Meiklejohn et al. 2013, Clancy et al., 2011, Xu et al. 2008, and Clancy 2008).

Second, the reviewer is correct that the mt:CoIR301Lmutant in a previous report (Xu, DeLuca & O’Farrell Science 2008) was also associated with male sterility. However, this report did not quantitatively assess other aspects of male or female fitness. We now more fully discuss the mt:CoIR301Lmutant in our revisions, pointing out the biochemical and biological differences between this mutant and the one we characterize. Even though both mutants had a male sterility defect (mt:CoIR301Lwas completely male sterile), the authors observed no defects in cytochrome oxidase activity and spermatogenesis. Instead, the infertility was reported due to a defect in sperm storage. Thus, we have a unique manifestation of a male-harming mtDNA mutant. Additionally, we believe that our claim regarding the specificity of the G177S mutation in causing male sterility is based on one of the most comprehensive and quantitatively rigorous phenotypic characterizations.

One concern is that the data are not sufficient to rigorously demonstrate the genetic homogeneity of the lines examined. The details are below, but this view can be summarized as follows: the original plan treated the crosses as if they were a selection that would yield a pure male harming mutation, and suitable measures where not take to ensure the genetic homogeneity of the stock that was retrieved. Given the number of mt genomes carried by flies, a high sensitivity of detection of a rare allele (in a range approaching 0.1%) and analysis of single flies is needed to rigorously demonstrate homoplasmy.

We believe that the reviewers’ concerns can be remedied by clarified wording and one critical additional experiment that we have performed with the help of new co-author Scott R. Kennedy (now added). First, we now clarify in the text that subsequent to identification of the G177S mutation in experimental line 7, all experiments were done from a mutant and wildtype mtDNA stocks reestablished from the original w118 strain. Additionally, we have now sequenced pools and individuals of these stocks to show that the pools of flies are nearly homoplasmic:

“We characterized the nature of the heteroplasmy in the re- isolated COIIG177S mutant mtDNA lines by sequencing a pool of COIIG177S mutant mtDNA and a separate pool of re-isolated wildtype mtDNA adult fly heads using a Duplex Sequencing strategy (Kennedy, Schmitt et al. 2014) (Figure 4—figure supplement 1). […] We therefore used these re-isolated strains for all subsequent phenotypic analyses.”

A second issue concerns data on fertility demonstrating that in an early first mating the males exhibited no (at 25°C) or weak (29°C) deficits in fertility. At least in reading the text, the subsequent analyses did not seem to take this fully into account. Many of my concerns here could be dealt with by better reference to supplementary material and more careful wording. Nonetheless, it still seemed to me that this issue was not fully investigated and the impact of this finding on other analyses was not always considered.

All subsequent analyses to document infertility or its suppression were done in either aged males at 25°C or young males at 29°C; in both instances the deficit in fertility is robustly observed. Nevertheless, we have made changes to make sure that all references and caveats are clearly cited in our Results and Discussion.

Overall, this an interesting report that suffers from weakness in its details. These can be addressed in the revised final submission

We are grateful for the opportunity to suitably address these concerns and those listed below. We believe that the clarifications and the additional experiments regarding the status of heteroplasmy should satisfy many of the reviewer concerns.

https://doi.org/10.7554/eLife.16923.030

Article and author information

Author details

  1. Maulik R Patel

    1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    2. Howard Hughes Medical Institute, Seattle, United States
    3. Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    MRP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    1. maulik.r.patel@vanderbilt.edu
    Competing interests
    No competing interests declared.
  2. Ganesh K Miriyala

    1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Contribution
    GKM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Aimee J Littleton
    Competing interests
    No competing interests declared.
  3. Aimee J Littleton

    1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Contribution
    AJL, Acquisition of data, Analysis and interpretation of data
    Contributed equally with
    Ganesh K Miriyala
    Competing interests
    No competing interests declared.
  4. Heiko Yang

    1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
    2. Howard Hughes Medical Institute, University of Michigan, Ann Arbor, United States
    Contribution
    HY, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents
    Competing interests
    No competing interests declared.
  5. Kien Trinh

    1. Genome Sciences, University of Washington, Seattle, United States
    Present address
    1. Biogen Idec, Cambridge, United States
    Contribution
    KT, Acquisition of data, Analysis and interpretation of data
    Competing interests
    No competing interests declared.
  6. Janet M Young

    1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Contribution
    JMY, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0001-8220-8427
  7. Scott R Kennedy

    1. Pathology, University of Washington Medical Center, Seattle, United States
    Contribution
    SRK, Acquisition of data, Analysis and interpretation of data
    Competing interests
    No competing interests declared.
  8. Yukiko M Yamashita

    1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
    2. Howard Hughes Medical Institute, University of Michigan, Ann Arbor, United States
    Contribution
    YMY, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    YMY: Reviewing editor, eLife
  9. Leo J Pallanck

    1. Genome Sciences, University of Washington, Seattle, United States
    Contribution
    LJP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  10. Harmit S Malik

    1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    2. Howard Hughes Medical Institute, Seattle, United States
    Contribution
    HSM, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    1. hsmalik@fhcrc.org
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0001-6005-0016

Funding

Helen Hay Whitney Foundation

  • Maulik R Patel

National Institutes of Health (F30AG045021)

  • Heiko Yang

Howard Hughes Medical Institute

  • Yukiko M Yamashita
  • Harmit S Malik

National Institute of General Medical Sciences (GM104990)

  • Leo J Pallanck

G Harold and Leila Y. Mathers Foundation

  • Harmit S Malik

National Institute of General Medical Sciences (GM074108)

  • Harmit S Malik

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Lisa Kursel, Tera Levin, Mia Levine, Nitin Phadnis, and Benjamin Ross for comments on the manuscript. The authors were supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation (MRP), the Mathers Foundation (HSM), and US National Institute of Health grants F30 AG045021 (HY), GM104990 (LP) and GM074108 (HSM). YMY and HSM are Investigators of the Howard Hughes Medical Institute.

Reviewing Editor

  1. K VijayRaghavan, Reviewing Editor, Tata Institute of Fundamental Research, India

Publication history

  1. Received: April 14, 2016
  2. Accepted: June 30, 2016
  3. Version of Record published: August 2, 2016 (version 1)

Copyright

© 2016, Patel et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,111
    Page views
  • 332
    Downloads
  • 6
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Comments

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Microbiology and Infectious Disease
    Kenneth J Ertel et al.
    Research Article