Mitochondria provide most of the energy (ATP) and a significant proportion of the biosynthetic precursors and reduction potential (NADPH) needed for growth and development (Vander Heiden et al., 2009; Balsa et al., 2020). Given this centrality to development, it is hardly surprising that mitochondrial stress exercises downstream effects on signaling (Wallace and Fan, 2010; Holmstrom and Finkel, 2014), cellular differentiation (Kasahara and Scorrano, 2014), and cell death (Sweetlove et al., 2010; Vyas et al., 2016), which together impact on all aspects of fitness, health, and survival.

Mitochondrial energy transduction proceeds through the electron transfer system (ETS), generating ATP via oxidative phosphorylation (OXPHOS) with oxygen as the final electron acceptor (O₂ flux). Oxygen can also react directly with FeS clusters at several sites in the ETS complexes, giving rise to reactive oxygen species (ROS flux; Quinlan et al., 2013; Pamplona et al., 2021). Once perceived as harmful by-products of mitochondrial respiration, ROS are now appreciated for their role in regulating redox tone and gene expression (Holmstrom and Finkel, 2014). Far from simply correlating with O₂ flux, slow electron transfer through the ETS to oxygen tends to decrease ATP synthesis and increase ROS flux, as critical FeS centers become more highly reduced (Barja, 2013; Mota-Martorell et al., 2020). Slow electron transfer also impacts metabolic flux through the tricarboxylic acid (TCA) cycle, as NADH oxidation is impeded (Martínez-Reyes and Chandel, 2020). This in turn necessarily affects both growth and signaling, as TCA-cycle intermediates are key precursors for amino acid, fatty acid, nucleotide and sugar biosynthesis, as well as NADPH synthesis (Mullen et al., 2014; Bradshaw, 2019). Accumulation of TCA cycle intermediates such as succinate can induce epigenetic changes impacting on growth and development, which have been implicated in the metabolic rewiring characteristic of cancer (Deberardinis and Chandel, 2020). Clearly, anything that impairs electron transfer through the ETS could have profound effects on physiology, gene expression and fitness.

The importance of fast electron transfer for growth and development makes it all the more surprising that the respiratory complexes are mosaics of subunits encoded by two obligate cellular genomes, nuclear and mitochondrial (Blier et al., 2001; Rand et al., 2004). While the assembly and function of the respiratory complexes require precise coordination of the two genomes, there is potential for mitonuclear variation to be generated in every generation. In particular, nuclear alleles are recombined through sexual reproduction, whereas mitochondrial DNA (mtDNA) is normally inherited clonally down the maternal line, which can also produce sex-specific effects (Frank and Hurst, 1996; Gemmell et al., 2004). Equally confounding, mtDNA evolves at 10–50-fold the rate of nuclear genes (Neiman and Taylor, 2009), forcing the nuclear genome to adapt rapidly to new mitochondrial haplotypes in the population (Barreto and Burton, 2013b; Healy and Burton, 2020).

Severe incompatibilities between the mitochondrial and nuclear genomes are known to cause deleterious phenotypic effects by disturbing O₂ and ROS fluxes. Hybrid breakdown in crosses between highly divergent populations might even contribute to speciation (Lane, 2009; Barreto and Burton, 2013a; Gershoni et al., 2014; Pichaud et al., 2019). More subtle mismatches originating from single nucleotide polymorphisms (SNPs) may not produce a phenotypic response and circulate widely in natural populations. However, even a small number of SNPs in mtDNA can perturb the expression of hundreds of nuclear genes (Innocenti et al., 2011), and these effects could be exacerbated by environmental stress (GxGxE interactions). Given that mitochondria are increasingly recognized as important drug targets for various diseases, and that adaptation to changing environments stresses mitochondrial function, there is a pressing need to better understand the molecular basis of mitonuclear interactions and their influence on life-history traits in stressful changing environments. A number of investigations have focused on the effect of mitonuclear interactions on treatments ranging from nutrition (Camus et al., 2020a) to temperature (Towarnicki and Ballard, 2017; Montooth et al., 2019) and drug responses (Villa-Cuesta et al., 2014; Santiago et al., 2021) and indeed found significant effects, but the basis of these changes at a molecular level has been little explored.

In principle, subtle mitonuclear mismatches should slow electron transfer through the ETS complexes, lowering O₂ flux, increasing ROS flux from reduced FeS clusters, and slowing TCA cycle flux by impeding NADH oxidation (Lane, 2011; Barja, 2013; Martínez-Reyes and Chandel, 2020). Stressing mildly mismatched mitonuclear systems should amplify latent deficits and unmask new phenotypes. In this study, we probed how mitonuclear interactions modulate the response to distinct cellular stressors in a well-established Drosophila melanogaster model. Because larvae are constrained in their resource allocation for growth, the metabolic underpinnings of this stage in different mitonuclear genetic contexts are of particular interest. Developing larvae compete for limited food and must meet time-dependent developmental checkpoints or die (Meiklejohn et al., 2013; Rodrigues et al., 2015). Faster developing individuals have a competitive advantage, which has been linked to lower mtDNA copy numbers and higher O₂ flux (Salminen et al., 2017). We therefore examined the responses of Drosophila larvae to treatments that place a strain on mitochondrial function in relation to electron transfer: the glutathione–precursor N-acetylcysteine (NAC), which interferes with ROS signaling and redox balance; and a high-protein diet, which increases TCA-cycle flux and dependence on mitochondrial respiration.

Specifically, we compared larvae from three fly lines harboring distinct mtDNAs on an isogenic nuclear background, generated through backcrosses using balancer chromosomes (Clancy, 2008). These were: wild-type (WT; w^1118-5,095) with coevolved mtDNA and nuclear background; COX, possessing one SNP difference in the gene coding for the COXII subunit (Patel et al., 2016); and BAR, which has nine SNPs difference in protein-coding genes in its mtDNA, mainly in complexes I and IV (Clancy, 2008; Wolff et al., 2016). Adult BAR and COX flies have been shown to exhibit mild male subfertility at 25°C, which in the case of COX is exacerbated by higher temperatures (29°C), and accompanied by reduced complex IV activity and ROS levels (Patel et al., 2016; Camus and Dowling, 2018). Here, we show that stress does indeed amplify phenotypic differences between mitonuclear lines, and use fluorespirometry to demonstrate that these differences are largely attributable to variations in electron flux through complex I.

Serious incompatibilities between mitochondrial and nuclear genes, perhaps generated through introgression between divergent populations in changing environments, can cause hybrid breakdown and even speciation, with severe effects on mitochondrial physiology undermining fitness and survival (Wolff et al., 2014). However, the effects of more subtle mitonuclear mismatches, generated through sex within populations, on responses to mild stress are harder to predict. For example, subtle mitonuclear mismatches may generate a hormetic response that protects against the stress, while selection for mitochondrial function in specific environments (for example in relation to diet or temperature) could potentially offset the effects of subtle mismatches. Less work has been done on these ‘covert’ GxGxE interactions, yet their very unpredictability makes them potentially important in relation to both adaptation to changing environments and to health and personalized medicine. Because growth arguably places the greatest stress on all-round mitochondrial function (requiring balanced ATP synthesis, reducing equivalents in the form of NADPH and biosynthetic precursors), we considered the effects of mild stress on the development of Drosophila larvae. Specifically, we considered the effect of metabolic stress (a high-protein diet known to shorten the lifespan of adult Drosophila, Camus et al., 2020b) and redox stress (the glutathione precursor NAC) on developmental time, survival, and underlying mitochondrial function in one coevolved and two slightly mismatched Drosophila larvae lines. We show that mitonuclear interactions do indeed substantively influence responses to stress, with one mismatched line (COX) faring especially badly in response to redox stress, and another, BAR, consistently outperforming the coevolved WT line, despite the nuclear background being isogenic in all three lines.

When developing on control diet (standard molasses preparation), BAR flies developed significantly faster, had higher survival and tended to have a greater O₂ flux than the other two lines (Figures 1, 2A). The underlying mitochondrial phenotype showed no difference in their respiratory control ratio under control conditions (Figure 3A), but there was less reliance on glycerol phosphate as a substrate for respiration (through GpDH feeding electrons into the ubiquinone pool, Figure 2B) than the coevolved line. This hints at a higher contribution of complex I (nonsignificant, Figure 3B), and a lower (albeit nonsignificant) mtDNA copy number (Figure 5), a suggestion that was unmasked in response to stress. When fed the redox stressor NAC, BAR larvae maintained their high complex I contribution, whereas the complex I contribution of COX larvae fell to little more than half that of BAR larvae (Figure 3B). The robust complex I-driven respiration in BAR larvae could be linked with SNPs in complex I subunits ND4L and ND5, which have previously been associated with adaptive responses to climate in birds and humans (Balloux et al., 2009; Van Der Heijden et al., 2019). We note that the mean summer temperatures in Barcelona are 5–8°C greater than Oregon, imposing greater metabolic demands on BAR mitochondria (as the Q₁₀ suggests a doubling in metabolic rate for every 10°C rise in temperature). While the Oregon strain was collected in 1925, hence has had nearly a century to adapt to lab conditions, differences in heat tolerance at the species level can persist for decades in the lab, and correspond to differences in complex I and substrate use (Jørgensen et al., 2019, 2021). It is possible that BAR flies could have a respiratory architecture adapted to higher temperatures. Our results suggest that higher metabolic rates could be sustained by selection for the fastest and most efficient coupling of electron transfer to proton pumping via complex I-linked respiration, notably the rapid removal and oxidation of reduced ubiquinone from complex I. If so, then BAR flies may be better adapted to metabolic and redox stress than COX or WT flies due to climatic differences between their sites of origin, which outweigh the effects of mismatching mtDNA against the isogenic nuclear background; but whatever the reason, it is unequivocal that BAR flies have more robust complex I-linked respiration.

In contrast, COX larvae were especially vulnerable to redox stress. We used a NAC concentration in the mid-range of an earlier study (Brack et al., 1997) showing some lifespan benefits (at 1 and 10mg.ml⁻¹), and found a strong haplotype-specific response: WT and BAR were little affected by NAC in any parameters studied, whereas COX larvae had significantly slower development, lower survival, metabolic rewiring away from complex I-linked substrates, elevated ROS flux, and raised copy number of mtDNA. NAC appeared to cause oxidative stress associated with increased reliance on alternative substrates, notably succinate (which primarily feeds electrons into complex II) and proline (where ProDH transfers electrons to the ubiquinone pool and complex III (McDonald et al., 2018). ProDH is a recognized source of ROS production, mediating mitochondrial apoptosis and tumor growth (Kononczuk et al., 2015; Soares et al., 2015). ROS signaling in COX larvae was also perturbed, as illustrated by the high H₂O₂ flux in the maximal OXPHOS state and during inhibition of complexes I, II, and III (Figure 4). mtDNA copy number was also significantly raised in COX larvae on NAC compared with the other two haplotypes. This suggests a hormetic response, in which mitochondrial biogenesis partially offset the respiratory deficiency (Taylor and Turnbull, 2005; Lane, 2011) but was unable to fully protect against redox stress.

The seemingly paradoxical increase in ROS production produced by replenishing the matrix antioxidant glutathione using NAC is commonly referred to as reductive stress (Samuni et al., 2013; Korge et al., 2015). Slow electron transfer through complex I can drive reductive stress, as NADH oxidation is hindered, impacting on TCA-cycle flux and the regeneration of mitochondrial NADPH. Shifts in the NADH/NAD⁺ and NADPH/NADP⁺ ratios, as well as flavin reduction in the ETS, become the key factors determining the rate of ROS production (Korge et al., 2015). In COX larvae, the single SNP difference in the COXII subunit of complex IV has been shown to slow electron transfer at higher temperatures, when metabolic demands are greater (Patel et al., 2016). CIV passes on its electrons directly to oxygen, so it is not surprising that it exerts significant control over the overall rates of electron flow (Rodriguez et al., 2021). While the maximal rates of both coupled and uncoupled respiration were similar in COX larvae and the other fly lines (Figure 2B)—as well as complex IV activity itself (Figure 3C)—the shift in substrate usage and respiratory architecture plainly drove reductive stress when subjected to mild redox stress from NAC. This finding has critical ramifications for adaptation to stressful environments or pharmacological treatments, as the nuclear background of COX, WT, and BAR larvae are all isogenic. The only differences that we could measure—which had pervasive effects on development—were when COX larvae were mildly stressed with an antioxidant.

Feeding larvae with a high-protein diet consistently promoted faster development in the larvae of all three haplotypes, with BAR larvae once again outperforming the other lines in terms of increased survival and faster development (Figure 1). Compared with control and NAC-treated larvae, all protein-fed larvae had higher coupled O₂ flux on N-pathway substrates (Figure 2A), lower reliance on succinate and Gp pathways (Figure 2B), greater RCR (Figure 3A), and higher flux through complex I (Figure 3B). Curiously, the range values for virtually all respiratory parameters (Figure 2) were consistently smaller in protein-fed larvae, which might reflect tighter constraints on permissible respiratory architecture. Drosophila larvae have previously been reported to be more complex I-dependent than adults, which tend to rely more on complex III-linked substrates (Ballard and Youngson, 2015), potentially explaining the accelerated growth of BAR larvae. While ROS flux was not significantly impacted by protein treatment in WT and BAR flies (Figure 4), mtDNA copy number decreased in protein-treated COX compared with NAC and control larvae (Figure 5). This suggests a compensatory decrease in mitogenesis in COX larvae on this treatment, linked with lower ROS production. It is striking that ‘forcing’ COX larvae to increase flux through complex I was beneficial in terms of developmental time, survival, all respiratory parameters, ROS flux, and mtDNA copy number. From these results in larvae, it is interesting to contemplate why a high-protein diet should substantially decrease adult lifespan (Camus et al., 2020b), given that larval growth would seem to maximize demands on resource allocation.

Proteins are broken down into their amino acid constituents, notably glutamine. This is deaminated into glutamate and enters the mitochondria via the glutamate–aspartate carrier (Gnaiger, 2020). Glutamate is an anaplerotic substrate, which feeds into the TCA cycle at α-ketoglutarate to regenerate NADH, and hence support complex I respiration (Gnaiger and Group, 2020). Deficiencies in electron transfer through complex I increase the likelihood of reverse TCA-cycle flux, as increases in the NADH/NAD⁺ and α-ketoglutarate to citrate ratios stimulate reductive glutamine metabolism and ultimately lipid and lactate accumulation (Ballard and Youngson, 2015). Glutamine also regulates the mammalian target of rapamycin (mTOR) pathway promoting cellular growth (Altman et al., 2016). In diseases caused by complex I mutations, high-protein diets can exacerbate ROS production (Ballard, 2016) and could upregulate the mTOR axis, potentially driving quasi-programs linked with hyperfunction and diminished stress resistance (Blagosklonny, 2013; Wang et al., 2018). While we did not observe complex I defects in larvae exposed to high-protein treatment, it may be that damage to complex I later in adult life tends to drive reverse TCA flux, promoting an age-related growth phenotype that shortens lifespan in adult flies. Be that as it may, our results suggest that robust complex I function is indeed central to larval development and survival.

In conclusion, we report that the response of Drosophila larvae to mild metabolic or redox stress is strongly contingent on mitonuclear interactions. Ostensibly benign differences that do not manifest phenotypically in a standard rearing environment produce unpredictable outcomes depending on the type of stress and the mitonuclear background in question. Both NAC and high-protein treatment primarily affected flux at complex I, but the metabolic and phenotypic consequences were very different depending on the haplotype. Our study highlights the need to understand how subtle differences in mitonuclear interactions, amplified by stress, manifest through the rewiring of metabolic flux, signaling, gene expression and ultimately phenotype. These pervasive and fundamental effects are likely to hold important implications for health (personalized medicine) and biodiversity (adaptation and speciation) in a world where climate change will surely amplify mitonuclear stress.

All data are available on the Figshare Digital Repository; doi: 10.5522/04/16539723

ER, MC, and NL conceived the experiment. FG, ER, and MC collected and analyzed the data. All authors contributed to writing the manuscript.

This research was funded by the BBSRC (BB/S003681/1) and Leverhulme Trust (RPG-2019-109) grants to NL and MC.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.