Deciphering Ultra-High Dose Rate Irradiation with Drosophila melanogaster
, Riccardo Dal Bello 1, Stephanie Tanadini-Lang 1, Erich Brunner 2, Darlina von Salis 1, Damian Manetsch 1, Sandipan Tewary 2, Matthias Guckenberger 1, Jamie Little 2 and Martin Pruschy 1,*
Round 1
Reviewer 1 Report
In this study, the authors present how they use fruit flies to investigate the FLASH effect. The study is interesting and worth publishing. I am missing the description of dosimetry, which is not straightforward at these doses and dose rates. I recommend that a section describing their dosimetry approach is included. There is a large variability in the results between the different repeats. This needs to be discussed/addressed further.
Introduction, lines 40-42: Include results from FAST-01.
Introduction, 54-55: Rephrase. You even discuss one such study in the discussion. Include ref to this study: https://doi.org/10.1093/jrr/rrae079
Introduction, line 59: Remove "extremely"
MoM: Add section on dosimetry.
MoM: It would be good to move Supplementary figure 1 and 5 to the main paper rather than just showing in supplmentary.
MoM, lines 73-74: Parenthesis missing?
MoM, lines 74-75: Can you clarify what you mean here. Also, why limited to 99 pulses? The figure in supplementary would likely add to this clarification if moved to the main text.
MoM, line 79: "The phantom was placed at the interface mount..." What/where is this? Can you clarify?
MoM, line 82: "Error! Reference source" ?
MoM, lines 84-85: Why were different energies used? How well were the UHDR and CONV energies matched? Measured PDDs and profiles for the different beams would be valuable additions?
MoM, line 102: How long after IR was this?
Results, line 121: "Error! Reference source" ?
Results, line 126: "Error! Reference source" ?
Results, line 126-127: Something is strange/missing here...
Results, line 133: "Error! Reference source" ?
Results, lines 134-135: Also looking at Tables 1 and 2, Supplementary figures 3-5, why is the variation between repeats so large. Needs to be discussed/addressed. Especially as you claim that the model/assay is excellent...
Results lines 145-146: "Error! Reference source" ?
Results line 147: "Error! Reference source" ?
Figure 1: Should have the legend say "Split Dose" or similar rather than "Fractionated"
Tables 1 and 2: Why have them appearing twice?
Results, lines 171-173: Something is weird here...
Results, line 174: "Error! Reference source" ?
Results, line 176: "Error! Reference source" ?
Figure 2: Why caption above the figure rather than below? Again, should have the legend say "Split Dose" or similar rather than "Fractionated"
Figure 3: Would have the caption say "lipid peroxidation" rather than " ferroptosis"
Discussion, lines 213-215. Remove as repetition from lines 191-193
Discussion, line 223: Remove "Insofar"
Discussion, line 229: The total dose required to induce measurable damage is a problem when the FLASH effect is dose sensitive. How is what we see for these doses relevant for the much lower doses relevant for clinical treatments?
Discussion, lines 238-240: How does the model compare to other models, e.g., zebrafish embryos which have been used previously in FLASH research?
Discussion, lines 241-244: No reference to the discussed/relevant paper...
Discussion, line 253-257: You say "fraction" (and variations of this word) in several places here. I would avoid using this and in stead use Split Dose delivery or similar.
Discussion, line 264: I would not call this model "ideal". "suitable" would be a more accurate description.
Discussion, line 265: Here and in other places in the paper, you use "integral dose". It is not really easy to understand what you mean by this. I highly recommend that you change these everywhere to "total dose".
Discussion, last sentence, lines 270-272: This should be rephrased.
Conclusions, line 276: Again, I would change "integral" to "total".
Conclusions, line 278: "which is not approachable in in vitro cellular systems" It is not really clear what you mean or how you justify this statement. Either explain better or remove. Removing would probably be preferable.
Conclusions, lines 278-280: Rephrase the sentence.
Conclusions, line 280: Again, I would not call this model "ideal". "suitable" would be a more accurate description.
Author Response
Reviewer 1:
Major Comment:
In this study, the authors present how they use fruit flies to investigate the FLASH effect. The study is interesting and worth publishing. I am missing the description of dosimetry, which is not straightforward at these doses and dose rates. I recommend that a section describing their dosimetry approach is included. There is a large variability in the results between the different repeats. This needs to be discussed/addressed further
Answer related to dosimetry: We would like to thank this reviewer for these constructive comments. The dosimetry section has now been addressed in the revised manuscript, including two additional Supplementary Tables.
Answer related to variability: Indeed, we observed substantial variability in the lifespan of both irradiated and control flies across different experiments. These differences may have been influenced by variations in environmental conditions, food, and larval crowding, all of which are known to affect fitness-related traits, including lifespan. Additionally, stress during transport from the fly incubator to the University Hospital Zurich, where irradiation was conducted, may have impacted the flies, particularly in colder weather. Despite variability between control groups and experiments, UHDR consistently demonstrated improved survival compared to CONV irradiation across several independent experiments. Pooling the lifespan data helped to mitigate the effects of potential confounding factors, such as food contamination or environmental stressors, that may have influenced individual experiments.
We have addressed this issue in the Discussion section of the revised manuscript.
Minor Comment:
Point: Introduction, lines 40-42: Include results from FAST-01.
Answer: At this stage only an abstract on the results of the FAST-01 study is circulating. Thus, we prefer not to outline the results of this study.
Point: Introduction, 54-55: Rephrase. You even discuss one such study in the discussion. Include ref to this study: https://doi.org/10.1093/jrr/rrae079
Answer: We apologize for this confusion and corrected the text accordingly. We actually meant experiments perfomed with adult fruit flies.
Point: Introduction, line 59: Remove "extremely"
Answer: corrected accordingly
Point: MoM: Add section on dosimetry.
Answer:
We added additional text on dosimetry in the section describing the Irradiation with a clinical linear accelerator and dosimetry including a reference to a recent publication by ours. Furthermore, we added two additional Supplementary Tables 1,2 outlining additional irradiation parameters.
Point: MoM: It would be good to move Supplementary figure 1 and 5 to the main paper rather than just showing in supplmentary.
Answer: We agree with the reviewer that Supplementary Figures 1 and 5 are supportive of understanding the experimental set-ups. Accordingly, we have moved these Supplementary Figures as Appendices 1,2 into the main manuscript with reference in the Material and Method section.
Point: MoM, lines 73-74: Parenthesis missing?
Answer: corrected with integration of additional information
Point: MoM, lines 74-75: Can you clarify what you mean here. Also, why limited to 99 pulses?
Answer: The linac UHDR conversion was designed with a safety feature limiting the maximum number of pulses to 99 in a single beam-on session. This is now specified in the materials and methods.
Point: The figure in supplementary would likely add to this clarification if moved to the main text.
Answer: We agree with the reviewer that Supplementary Figures 1 and 5 are supportive of understanding the experimental set-ups. Accordingly, we have moved these Supplementary Figures as Appendices 1,2 into the main manuscript.
Point: MoM, line 79: "The phantom was placed at the interface mount..." What/where is this? Can you clarify?
Answer: We have clarified this issue in the revised version and specified that the interface mount is SSD = 60 cm.
Point: MoM, line 82: "Error! Reference source" ?
Answer: The correct reference is now inserted
Point: MoM, lines 84-85: Why were different energies used? How well were the UHDR and CONV energies matched? Measured PDDs and profiles for the different beams would be valuable additions?
Answer: The UHDR linac was designed to deliver both 16 MeV and 9 MeV electron beams. These two modalities were available during 2024. In 2025 linac was utilized for a clinical trial (NCT06549439) with 9 MeV, which required disabling the 16 MeV modality. Therefore, irradiation experiments with both 16 MeV and 9 MeV were performed in the first phase of the project, while only 9 MeV irradiations were performed in the second phase. This explanation has not been added to the manuscript to keep it concise and focused on the radiobiological experiments.
Measured PDDs and Profiles in air inside the vials are provided in the supplementary materials.
Point: MoM, line 102: How long after IR was this?
Answer: Dissected fly brains were fixed in 4% paraformaldehyde for 20 min 1 h following irradiation. These informations were added to the respective section in the manuscript.
Point: Results, line 121: "Error! Reference source" ?
Answer: This error is now corrected in the revised manuscript
Point: Results, line 126: "Error! Reference source" ?
Answer: This error is now corrected in the revised manuscript
Point: Results, line 126-127: Something is strange/missing here...
Answer: The missing part has now been completed.
Point: Results, line 133: "Error! Reference source" ?
Answer: The text has now been corrected accordingly
Point: Results, lines 134-135: Also looking at Tables 1 and 2, Supplementary figures 3-5, why is the variation between repeats so large. Needs to be discussed/addressed. Especially as you claim that the model/assay is excellent.
Answer: Indeed, we observed substantial variability in the lifespan of both irradiated and control flies across different experiments. These differences may have been influenced by variations in environmental conditions, food, and larval crowding, all of which are known to affect fitness-related traits, including lifespan. Additionally, stress during transport from the fly incubator to the University Hospital Zurich, where irradiation was conducted, may have impacted the flies, particularly in colder weather. Despite variability between control groups and experiments, UHDR consistently demonstrated improved survival compared to CONV irradiation across several independent experiments. Pooling the lifespan data helped to mitigate the effects of potential confounding factors, such as food contamination or environmental stressors, that may have influenced individual experiments.
We have addressed this issue in the Discussion section of the revised manuscript.
Point: Results lines 145-146: "Error! Reference source" ?
Answer: This error is now corrected in the revised manuscript
Point: Results line 147: "Error! Reference source" ?
Answer: The text has now been corrected accordingly.
Point: Figure 1: Should have the legend say "Split Dose" or similar rather than "Fractionated" Answer: This is now corrected in the revised manuscript
Point: Tables 1 and 2: Why have them appearing twice?
Answer: While Table 1 gives the Statistics for Individual Single High-Dose UHDR and CONV Irradiation Lifespan Assays, Table 2 gives the Statistics for Individual Single High-Dose and Split Dose UHDR Irradiation Lifespan Assays.
Point: Results, lines 171-173: Something is weird here...
Answer: We apologize for this mistake and have corrected the text accordingly.
Point: Results, line 174: "Error! Reference source" ?
Answer: The text has now been corrected accordingly
Point: Results, line 176: "Error! Reference source" ?
Answer: The text has now been corrected accordingly
Point: Figure 2: Why caption above the figure rather than below? Again, should have the legend say "Split Dose" or similar rather than "Fractionated"
Answer: We adjusted the figure and text accordingly
Point: Figure 3: Would have the caption say "lipid peroxidation" rather than " ferroptosis"
Answer: We have adjusted the text accordingly
Point: Discussion, lines 213-215. Remove as repetition from lines 191-193
Answer: We have adjusted the text accordingly
Point: Discussion, line 223: Remove "Insofar"
Answer: We have adjusted the revised manuscript accordingly
Point: Discussion, line 229: The total dose required to induce measurable damage is a problem when the FLASH effect is dose sensitive. How is what we see for these doses relevant for the much lower doses relevant for clinical treatments?
Answer: D. melanogaster exhibits higher radioresistance compared to mammals, a trait common among insects. Proposed mechanisms for the radioresistance of insects include efficient antioxidant systems, DNA damage protection, robust DNA repair pathways, altered stress responses, and resistance to apoptosis. The presence of only eight chromosomes may also contribute to the radioresistance observed in D. melanogaster. Furthermore, the radiosensitivity of D. melanogaster varies significantly across life stages. A study comparing γ-ray tolerance of different life stages revealed that the early pupal stage is particularly sensitive to IR, likely due to high rates of cell division and reorganization, despite a modest increase in antioxidant capacity. Notably, adult D. melanogaster displays the highest mortality threshold, as also shown in our manuscript - the postmitotic state of most tissues likely contributes to the high radioresistance of adult D. melanogaster flies. In this study, high doses of 1000 Gy and above were required for effectively inducing the UHDR effect in lifespan assays.
The strongly reduced radioresistance observed e.g. for third instar larvae could be attributed to the fact that sublethal doses for larvae can still impact developmental processes, ultimately affecting eclosion rates and the adult flies’ lifespan. However, results experimentally obtained with e.g. third instar larvae could be strongly affected by first and second instar larvae contaminations and will thereby give less clear effects. We therefore decided to perform experiments with adult fruit flies, which give more consistent results – eventhough only in response to high doses of ionizing irradiation. We agree with the reviewer that the results obtained in response to such high doses cannot be directly translated to the human situation. At the same time, it is of interest to determine these most probably highly conserved MoAs responsible for the differential response to UHDR and CONV irradiation at the endpoints investigated.
Paithankar JG, Deeksha K, Patil RK. Gamma radiation tolerance in different life stages of the fruit fly Drosophila melanogaster. International Journal of Radiation Biology. 2016;93(4):440-8.
Point: Discussion, lines 238-240: How does the model compare to other models, e.g., zebrafish embryos which have been used previously in FLASH research?
Answer: Thank you for this relevant question: All preclinical models so far used for UHDR irradiation have their advantages but also disadvantages. At the same time we have to keep in mind that preclinical experiments at this stage are primarily model studies. Eventually, UHDR irradiation will have to be probed on the clinical level - but based and optimized on the findings and indications gained from these model studies. Currently, a major topic of discussion among UHDR research is the question of the relevance of UHDR compared to CONV radiation therapy in terms of acute versus chronic side effects. These studies are primarily perfomed in mice. Zebrafish embryos are also advantageous for radiotherapy research due to their rapid ex vivo development, optical transparency, and genetic similarities to humans. Similar to D. melanogaster whole body experiments have to be performed with them with the advantage of increased radiosensitivity in comparison to adult D. melanogaster. On the other hand we could demonstrate that several endpoints can be addressed in the model organism of D. melanogaster (incl. survival), which could be even more extended on the intracellular level, e.g. DNA damage. Furthermore, genetic studies with different D. melanogaster strains can now be used to elucidate underlying MoAs. Of note, the fruit fly system is also considered more primitive. than the zebrafish. This indicates that the biological processes contributing to the UHDR versus CONV irradiation (on all the endpoints investigated in this manuscript) must be very conserved.
Point: Discussion, lines 241-244: No reference to the discussed/relevant paper...
Answer: We apologize for this mistake. We have now added this specific reference:
Hart A, Dudzic JP, Clarke JW, Eby J, Perlman SJ, Bazalova-Carter M. High-throughput, low-cost FLASH: irradiation of Drosophila melanogaster with low-energy X-rays using time structures spanning conventional and ultrahigh dose rates. J Radiat Res. 2024;65(6):836-44.
Point: Discussion, line 253-257: You say "fraction" (and variations of this word) in several places here. I would avoid using this and instead use Split Dose delivery or similar.
Answer: We have adjusted the text in the revised manuscript accordingly.
Point: Discussion, line 264: I would not call this model "ideal". "suitable" would be a more accurate description.
Answer: We have adjusted the text accordingly.
Point: Discussion, line 265: Here and in other places in the paper, you use "integral dose". It is not really easy to understand what you mean by this. I highly recommend that you change these everywhere to "total dose".
Answer: We have adjusted the text accordingly
Point: Discussion, last sentence, lines 270-272: This should be rephrased.
Answer: We have rephrased the text accordingly
Point: Conclusions, line 276: Again, I would change "integral" to "total".
Answer: We have adjusted the text accordingly
Point: Conclusions, line 278: "which is not approachable in in vitro cellular systems" It is not really clear what you mean or how you justify this statement. Either explain better or remove. Removing would probably be preferable.
Answer: We agree and have adjusted the text accordingly
Point: Conclusions, lines 278-280: Rephrase the sentence.
Answer: We have rephrased this sentence accordingly.
Point: Conclusions, line 280: Again, I would not call this model "ideal". "suitable" would be a more accurate description.
Answer: We have adjusted the text accordingly.
Reviewer 2 Report
This is a solid and complete study, with well written individual sections of the paper. I have reviewed many papers during the past two years, and rarely have come across so well designed manuscript.
I would definitely include supplemental materials in the Appendix or Appendices, if applicable. The authors would need to write some additional text, and then refer to the Appendix/Appendices in the main text.
Discussion section could be organized into subsections to better structure the section.
Author Response
Reviewer 2:
Major comments
This is a solid and complete study, with well written individual sections of the paper. I have reviewed many papers during the past two years, and rarely have come across so well designed manuscript.
Answer: We would like to thank this reviewer for these constructive comments.
Detailed comments
Point: I would definitely include supplemental materials in the Appendix or Appendices, if applicable. The authors would need to write some additional text, and then refer to the Appendix/Appendices in the main text.
Answer: We agree with the reviewer that Supplementary Figures 1 and 5 are supportive of understanding the experimental set-ups. Accordingly, we have moved these Supplementary Figures as Appendices 1,2 into the main manuscript with reference in the Material and Method section. Supplementary Figures 2,3,4 (now 1,2,3) are still kept as supplementaries as they do not provide additional information and results for the understanding of the main manuscript.
Point: Discussion section could be organized into subsections to better structure the section.
Answer: We have now addressed this issue in the revised manuscript and added two subsections.
We sincerely thank the reviewers and the editor for their thoughtful and constructive feedback, which has substantially strengthened our manuscript. We believe that the revised version now meets all requirements for publication in Antioxidants.
Round 2
Reviewer 1 Report
The authors have done a good job with the revision. The paper is now suitable for publication.
There is a "(Error! Reference source not found.B)" on line 179.
