Diet’s impact dictated by synonymous mitochondrial SNP interacting with nucleotype
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Abstract
Epistasis between mitochondrial and nuclear genomes can modulate fitness effects of nutrition. Are these “mitonuclear” effects deterministic with regard to optimal nutrition? Which nutrients and genetic loci participate? Here, in fruitflies, we show that mitonuclear epistasis repeatably dictates fitness effects of dietary lipids and amino acids, with a tripartite interaction emerging as an unprecedented source of quantitative variation. We also observed diet-dependent developmental lethality and parental effects, but only in specific mitonucleotypes. Associating phenotype to mtDNA variation implicated a non-coding RNA ( mt:lrRNA ), suggesting a novel mechanism, with epistasis between mitochondria-derived regulatory factors and the nuclear genome producing qualitative differences in how diet impacts fitness.
SUMMARY
Combined action of nuclear genotype and regulatory loci on mtDNA produces major differences in fitness impacts of nutrition.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study, Dobson and colleagues use the well-established Drosophila mitonuclear model to ask how diversity in mitochondrial and nuclear background impacts reproductive response to various diets (DMN, diet-by-mito-by-nuclear interactions). For this, they generated populations from three different geographical locations by introgressions to produce different mitonuclear combinations, which they produced in triplicates, and after preliminary analyses they excluded one geographical population (Canada), which brought down the total number of populations from 27 to 12. Then, they studied at the effects of 3 diets: a …
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study, Dobson and colleagues use the well-established Drosophila mitonuclear model to ask how diversity in mitochondrial and nuclear background impacts reproductive response to various diets (DMN, diet-by-mito-by-nuclear interactions). For this, they generated populations from three different geographical locations by introgressions to produce different mitonuclear combinations, which they produced in triplicates, and after preliminary analyses they excluded one geographical population (Canada), which brought down the total number of populations from 27 to 12. Then, they studied at the effects of 3 diets: a standardized control diet, a diet enriched in essential amino acids, and one enriched in lipids on various reproductive traits in two feeding regimes: one chronic (parents and offspring fed the diets), or parental (parents fed the experimental diets, eggs developed on a standard medium). They found that the magnitude of the dietary effects and the effect of parental nutrition on offspring fitness were dependent on mitonuclear genotype, with some diets unexpectedly deleterious in specific cases. They also show that this DMN variation is repeatable among independent replicates, and that mitonuclear epistasis plays a major role in the response to nutrition. Finally, they find an association between DMN interactions and a polymorphism in the mitochondrial gene long ribosomal RNA (mt:lrRNA), showing that a mitochondrial regulatory factor plays a role in DMN effects.
Major comments:
The authors have collected an impressive and very useful amount of data, with rigorous breeding regimes and statistical analysis. Their findings, interpretations and conclusions are in my opinion strongly supported by their experimental data. I have no major concerns with the study, and I believe it deserves to be in a wide-audience and high-impact journal. I only have some minor corrections and suggestions listed as follows.
Minor comments:
Have the authors thought about looking at rates of food consumption, and could some populations be consuming less (or none) of the experimental diets, possibly explaining lethality?
Line 136: this study's data does indeed show diet-dependent effects of mitonuclear interactions, but it not the first one, maybe rephrase? See refs (a couple are cited in the supplementary text, but it would be useful to add to the main text): Aw, W. C. et al (2018). PLoS Genetics doi:10.1371/journal.pgen.1007735 Camus, M. F., Moore, J., & Reuter, M. (2020). Biology Letters, 16(2), 20190891. doi:10.1098/rsbl.2019.0891 Camus, M. F., Kotiadis, V., Carter, H., Rodriguez, E., & Lane, N. (2022). bioRxiv, doi:10.1101/2022.02.10.479862 Cormier, R. P. J. et al. (2019). Scientific Reports. doi:10.1038/s41598-018-36060-5 Rodríguez, E. et al. (2021). Frontiers in Genetics. doi:10.3389/fgene.2021.734255 Towarnicki, S. G., & Ballard, J. W. O. (2018). Frontiers in Genetics. doi:10.3389/fgene.2018.00593
Figure 1D and results section lines 156-178: why is the term "eggs" included in development? I assume it refers to the measure of the amount of eggs, could the authors explain and mention it in the main text and/or figure legend? And why is it "eggs+1" in fig S2D, I could not find an explanation in the methods for this difference in the label.
Line 221, last sentence of the paragraph: I would make it clearer that this was in the "chronic" paradigm, I found it difficult to follow here.
Line 446 typo in the figure legend, should read "24h on..."
Figure 2A: "Genome origin" section of the legend could be placed on the top of the legend, I would find it easier to see as we read the figure from left to right.
Figure 2B: some kind of separation between T and C (dashed lines for eg.) would be appreciated to be able to compare better at a glance.
Figure S2D: boxplot lines seem very thin, could the outline be made clearer? It was especially difficult to see on a printed version.
Line 506, S2B legend: should "Benin" be used instead of "Dahomey"? Keeping the Dahomey mentioned only in Supporting text line 153.
Figure S3B and its legend text: the diets now appear completely different, except for EAA. Why is it "sya", should it be the same as "rearing diet" in S2B, or is it the "control" diet? "Mar", margarine, is to my understanding the lipid diet mentioned throughout the paper? I assume this is a matter of fixing the legend labels, unless I misunderstood this part. In the legend text lines 532-536, I could not find any explanation for this difference in labels.
Supplementary text line 43: "wild-type" meaning Benin flies?
Supp. text line 46: "development" medium is sya? See comment above about confusing diet names.
Supp. text line 51: I think the figures referenced are not the correct ones (S2B and S2C)? Should they be S2D and S2E?
Supp. text line 64: "AA1" is repeated twice in the parentheses, should it read "AA3" instead?
CROSS-CONSULTATION COMMENTS
I have read reviewer #1's comments and I see that we both agree on the quality and relevance of the paper, and the very minor corrections needed. Looking forward to seeing this published.
Significance
This study appears to me as very relevant in the context of predicting outcomes of dietary regimes (and more) given a certain mitonuclear background, which is very much needed in the field and in the context of personalized medicine. Repeatability of the effects gives confidence to the type of breeding regime and analyses done in the field of mitonuclear interactions (for example, in the research done by the authors cited previously). Pinpointing a synonymous SNP in a non-coding region of mtDNA is important in the context of deciphering the mechanism(s) behind DMN. As I come from the mitochondrial physiology and aging field, and currently work on mitonuclear interactions, these findings appear to me of great importance, but they will also appeal to a wider audience in the evolutionary biology and biomedicine fields; these results will contribute to advancing our knowledge of mitochondria-nucleus interactions in different environments, up to the human personalized medicine perspective.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Here, the authors investigate mitochondrial x nuclear x diet interactions in Drosophila melanogaster. They start by constructing D. melanogaster populations comprising fully-factorial combinations of mitochondrial and nuclear genomes from Australia, Benin, and Canada. This creates discrete populations with distinct combinations of mitochondrial and nuclear genomes. These populations are then exposed to various diets, notably a control diet, a diet high in essential amino acids (which promotes fecundity), and a diet high in plant-based lipids (which represses fecundity). They first screened for evidence of repeatable …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
Here, the authors investigate mitochondrial x nuclear x diet interactions in Drosophila melanogaster. They start by constructing D. melanogaster populations comprising fully-factorial combinations of mitochondrial and nuclear genomes from Australia, Benin, and Canada. This creates discrete populations with distinct combinations of mitochondrial and nuclear genomes. These populations are then exposed to various diets, notably a control diet, a diet high in essential amino acids (which promotes fecundity), and a diet high in plant-based lipids (which represses fecundity). They first screened for evidence of repeatable mitonuclear effects on fecundity, which they found. They then screened for additional fitness traits looking for influence of mito-nucleotype on response to chronic vs parental dietary changes. Once again, they were able to find evidence of phenotypic variation dependent on mito-nucleotype; however, the effect of mito-nucleotype on traits was variable. Certain mito-nucleotypes exhibited lower to near lethality progeny counts after amino acid feeding (which is thought to promote fecundity), while others exhibited normal counts. When quantifying the size of various effects, they found that mitonucleotype interactions often had comparable effect size to that of diet:mitotype interactions, diet:nucleotype interactions, and diet on its own. Finally, they were able to associate this mito-nucleotype interaction with a mt:lrRNA C/T polymorphism that had nucleotype-dependent effects on fertility.
Major Comments:
I find the key conclusions convincing, and I feel as if though the authors sufficiently show that there are mitonucleotype x diet interactions on fecundity/fertility traits. Furthermore, their ability to associate the phenotypic variation with a mt:lrRNA polymorphism is also of interest. I do not feel as if though the authors make claims that lack support in the paper. I do not feel as if though additional experiments would be essential to support their claims; however, I would be remiss not to acknowledge that the bulk of the experimental results came down to a 2 x 2 population exploration (Australia-Australia, Australia-Benin, Benin-Australia, Benin-Benin). This study would have been aided by more mitonucleotypes; however, I understand that to generate additional mitonucleotypes would have taken additional time and resources. As most of my comments are minor and should be easily addressed by the authors, I place them in the following section.
Minor Comments:
Below are minor comments and questions I had while reading the manuscript. I apologize in advance if any of my questions are answered in the main text, and I missed them while reviewing.
Page 3, line 35 - you state that variation in mitochondrial function can contribute to variation in dietary optima. I would add a citation here.
Page 4, line 74 - If possible, I would add a little more clarifying detail regarding the crossing scheme to the main text. I would just be clear that the generation of these mitonucleotypes takes advantage of the maternal transmission of the mitochondrial genome by crossing virgin females from population 1 to males from population 2, followed by continual backcrossing of virgin females each generation to males from population 2. You go into specific details in the materials & methods section, but I was surprised not to see it earlier. I know this is probably more detail, but I think it's better to be very clear about the crossing scheme used.
Page 5, line 90 - I would move the citations for the fact that enriching essential amino acids promotes fecundity from the supplement to the main text. You say that this is an established manipulation, and it would be best to point to examples of this manipulation, especially as some of your results find that fertility is negatively affected.
Page 8, line 129 - In this paragraph, you speak about the impacts of chronic EAA feeding in the AA, BA, and AB mitonucleotypes, but you do not coment on BB? Is there a reason for this? (This connects to one of my comments later)
General Questions - This may be outside the scope of your study, but I was surprised that there was no commentary on co-adaptation (or the potential lack of) between the mitochondrial and nuclear genome in the discussion. It's not immediately clear to me how isolated and for how long the Australian and Benin populations are, but I would expect there to be co-evolution/co-adaptation between the mitochondrial and nuclear genomes. Consequently, I would have expected AA and BB populations to show elevated fitness values; however, it actually seems as if though the AA mitonucleotype performs the worst when given the chronic EAA diet. I wonder if you could comment on the co-evolutionary potential between these two genomes.
Significance
It is becoming increasingly clear when studying mitochondrial x nuclear interactions that the environmental context of the study can significantly influence the results (see Camus et al. 2019, Rodriguez et al. 2021, and Towarnicki and Ballard 2018). This study furthers our understanding of mitochondrial x nuclear x environment interactions by exploring fully-factorial combinations of mitonucleotypes on two distinct diets (enriched for essential amino acids or plant-based lipids) and evaluating the fertility/fecundity of the different mitonucleotypes. They were able to identify a signle mt:lrRNA SNP that was associated with the measured phenotypes, raising the question of whether and how mitochondria-derived regulatory factors influence phenotypic variation. I also find the comparison to the omnigenic model compelling, particularly their commentary that mitochodnrial genes could contribute to the "core gene" set for several notable phenotypes.
I believe that this work would be of interest to those obviously studying the evolution and importance of mitochondrial x nuclear x environmental interactions, and I also believe that this work would be of interest to those interested in the effects of various nutrients/diets in both Drosophila and humans.
My expertise is as a theoretical population and evolutionary geneticist. I am primarily interested in genetic conflict, including between the mitochondrial and nuclear genome. I am interested in how these two genomes evolve to both cause and resolve genetic and sexual conflict.
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