Hypomagnetic Field Exposure Alters Iron–Sulfur Homeostasis and Oxidative Balance in a Frataxin-Deficient Insect System

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I find the work very interesting and comprehensive. However, I have a few comments that could improve the article.
Drosophila is the genus of the fruit fly (Drosophila melanogaster). Throughout the text, the authors refer to it as "Drosophila," which could indicate that they are referring to any species within the genus. Only in the figures do they specify the species. I suggest that at the beginning of the text they introduce the fruit fly as Drosophila melanogaster (line 21 and line 97) and that, for brevity, they use D. melanogaster instead of Drosophila.

In line 142, the authors report that the flies were housed in groups of 20 per tube, with three replicate groups per condition, but they don't indicate whether two or more independent experiments were conducted. I'm left wondering because in line 143 they mention that "Each experimental cycle consisted of a 72-hour exposure to either..." Could you clarify this?
On the other hand, regarding section 2.5, I'm still unsure how many flies were used for this analysis.
Finally, I would like clarification on what they mean by "...and only high-quality RNA was used for library preparation..." (line 189), and whether they obtained enough RNA for their analysis with only 5 heads per treatment.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript examines the interplay between hypomagnetic field (HMF) exposure and frataxin deficiency in Drosophila melanogaster, focusing on iron–sulfur homeostasis, oxidative stress, and transcriptional responses. The authors employ a technically advanced, multidisciplinary approach, integrating SR-XRF elemental imaging, ROS quantification, and RNA-Seq. The topic is both novel and timely, particularly in addressing how environmental stressors may exacerbate genetic metabolic vulnerabilities—a major concern for future biomedical research and space exploration. The use of live-tissue SR-XRF is a notable methodological strength, and the data presented are extensive. The manuscript is generally well organized.

Although promising and technically sound, this study's impact is limited by conceptual overreach, interpretational ambiguities, and unclear statistics. Addressing these issues—particularly in result interpretation, environmental framing, and methodological transparency—will enable the manuscript to contribute meaningfully to the field.

Major Comments

1. While the study highlights the importance of hypomagnetic fields in deep-space environments, it does not sufficiently justify their biomedical relevance to Friedreich’s ataxia (FRDA). The authors should more clearly explain why HMF exposure constitutes a meaningful biological stressor for terrestrial organisms and for FRDA pathophysiology. Currently, the discussion, especially the conclusions, overstates the translational implications without providing a strong mechanistic connection. The manuscript would be strengthened by explicitly framing HMF as an environmental perturbation that impacts redox balance and mitochondrial function, rather than implying direct relevance to patient exposure.
2. The SR-XRF measurements are a significant strength of this study, but the interpretation of "distribution" versus "content" is not sufficiently clear. The manuscript equates pixel count above a threshold with "distribution," yet this is only a semi-quantitative proxy and should be described more cautiously. At times, the authors interpret changes in iron content and spatial distribution as mechanistically distinct, but the absence of chemical speciation data (such as Fe²⁺ vs Fe³⁺) or subcellular localization makes these distinctions less definitive. The authors should clarify whether the observed changes reflect true redistribution or accumulation of iron and sulfur, or whether they may be influenced by altered detectability associated with tissue morphology. Including a schematic summarizing tissue-specific alterations in iron and sulfur under GMF versus HMF conditions would greatly improve overall clarity and interpretation for the reader.
3. In the manuscript, sulfur measurements are used as a proxy for Fe–S cluster biology; however, this link remains largely inferential. Total sulfur levels alone do not directly indicate the integrity of Fe–S clusters, and the authors should explicitly acknowledge this limitation to avoid overinterpreting sulfur changes as direct evidence of Fe–S disruption. To provide stronger context for their findings, the discussion should cite enzyme activity data from prior studies that depend on Fe–S clusters, clarifying the extent to which sulfur measurements can reflect Fe–S biology.
4. The use of DHE staining in this study raises concerns about specificity, as DHE oxidation is not exclusive to superoxide and can be influenced by other oxidants. The manuscript should acknowledge this limitation and temper claims about which ROS species are being detected. Additionally, the quantification methods—such as region of interest (ROI) selection and background subtraction—should be described in greater detail to enhance transparency and reproducibility. Despite these methodological concerns, the observed tissue-specific increase in ROS under HMF exposure in frataxin-deficient brains remains an interesting and credible finding.
5. The RNA-Seq dataset in this study is extensive, yet its interpretation is somewhat limited. The manuscript predominantly highlights selected differentially expressed genes (DEGs) without clearly distinguishing between primary effects of HMF exposure and secondary stress responses. Pathway enrichment results would benefit from a more systematic summary, such as listing the top Gene Ontology (GO) and KEGG pathway categories affected. The assertion that HMF “enhances transcriptional remodeling” should be supported by clearer quantitative data describing the extent and nature of this remodeling. Most importantly, transcriptional changes should not be interpreted as evidence of functional compensation unless substantiated by corresponding physiological or biochemical data.
6. The statistical section of the manuscript is somewhat convoluted and lacks clarity. Multiple statistical approaches are mentioned, but it is not always clear which tests were applied to specific figures or datasets. The manuscript should justify the use of Fisher’s LSD following ANOVA, as this method increases the risk of type I error. Additionally, the authors should clearly state the number of biological replicates for each assay, with particular emphasis on those conducted with SR-XRF, to ensure transparency and reproducibility of the results. Graphs should include number of biological replicates as number of dots.

 

Minor Comments

1. The title slightly overstates causality; consider softening “Disrupts” to “Alters” or “Perturbs.”
2. Several grammatical and typographical errors are present (e.g., spacing, hyphenation, inconsistent capitalization).
3. Figures 3–6 are information-dense; increasing font size and improving contrast would enhance readability.
4. The Abstract should explicitly mention tissue-specific effects earlier.
5. The Abbreviations section is incomplete (HMF definition formatting issue).

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors investigate the role of frataxin in iron metabolism and oxidative stress under an environmental stressor, specifically a hypomagnetic field. Their use of SR-XRF spectroscopy on live Drosophila tissues is particularly novel and informative. However, several aspects of the manuscript could be strengthened, including more rigorous quantification of the SR-XRF imaging data and clearer explanation and interpretation of the experimental results.

 

1. In the method section, please indicate the sex of flies used.
2. Please provide quantification for Figure 3, using fluorescence area or total fluorescence intensity and please include data points.
3. For figure 3, it’s hard to tell which region of the brain is shown, and whether the same region of the brain is used for quantification, an illustrative picture could be used to demonstrate this.
4. The decrease in iron content in the eye tissue is not obvious between control and fh-RNAi (Figure 4A), please use better picture.
5. The iron content in the eye tissues after frataxin silencing seemed to be increased (Figure 4B), is there a typo?
6. It was not clear to me what is the rationale of performing experiments in Figure 3 and Figure 4, I suggest the authors add a brief statement stating why experiments were performed, and what conclusions are reached.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf