Adaptation mechanism of the adult zebrafish respiratory organ to endurance training

Matthias Messerli; Dea Aaldijk; David Haberthür; Helena Röss; Carolina García-Poyatos; Marcos Sande-Melón; Oleksiy-Zakhar Khoma; Fluri A. M. Wieland; Sarya Fark; Valentin Djonov

doi:10.1371/journal.pone.0228333

Peer Review History

Original SubmissionSeptember 5, 2019
23 Oct 2019 Decision Letter - Eric A Shelden, Editor PONE-D-19-24766 Adaptation mechanism of the adult zebrafish respiratory organ to endurance training p, li { white-space: pre-wrap; } PLOS ONE Dear Prof. Dr. med. Djonov, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers are supportive of publication but have raised a number of points for your consideration. I think it is especially important to clarify the results of the BrDU study as both reviewers had questions about this aspect of the work. I also think some additional analysis of your data (as suggested by reviewer 2) is warranted, as is response to the request for clarification and additional citation from reviewer 1. The additional experiments suggested by reviewer 2 are an interesting suggestion that I hope you will consider. However, I do not consider them to be necessary for acceptance of a revised manuscript. We would appreciate receiving your revised manuscript by Dec 07 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Eric A Shelden, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, Messerli et al. report on the effects of exercise by swimming on the respiratory performance of adult zebrafish. The authors show that swimming training increases swimming performance as determined by maximum swimming speed and that this response is accompanied by physiological responses consequent with increases respiratory performance. Namely, the authors show that swimming training increases oxygen consumption and the growth of the gills in terms of increased gill volume, length of primary filaments and number of secondary filaments per primary filament. Finally, the authors show that swimming training increases the number of proliferating cells in the gills, suggesting a possible mechanism for the observed increased gill growth. The experiments are well conducted, following previous established swimming training conditions for adult zebrafish, use appropriate sample sizes and choose relevant methodological approaches to investigate physiological responses to swimming training. Therefore, the results are meaningful and make this study a valuable contribution to the field of exercise physiology. This reviewer recommends that the authors take the following several aspects into consideration: 1. The authors need to clarify aspects of the swimming training and respirometry experiments. First, details of the swim tunnels used in this study need to be provided (volume, model number). Second, a clear indication of whether the swimming training was conducted on individual fish or on groups of fish (if this was the case, please indicate exactly the number of fish in the swimming chamber) is needed. Third, please clearly state where the non-swimming fish were housed during the experiments. Fourth, please indicate the value of the training speed in body lengths per second. Fifth, please clearly indicate whether oxygen consumption was determined on individual fish in the Respirometry section of M&M. 2. A justification for the use of transgenic zebrafish is needed and related additional analyses are strongly recommended (point 3). First, the authors should confirm whether the fli transgenic line use corresponds to fli11a:eGFP, as indicated in the manuscript (line 119), or instead to the fli1a:eGFP line that is commonly used as a vascular endothelial cell marker. If the latter, please correct. Second, does the reference provided indicate the initial description of this line or the research group that provided the line? If the former, please indicate the origin of the line used in these experiments. 3. With regards to the use of this transgenic line, it is not clear why the authors did not exploit the GFP labeling of endothelial cells to determine if proliferating (BrdU+) cells are indeed endothelial cells or not. This reviewer strongly recommends the authors to reanalyze the immunofluorescence data and provide quantification of BrdU+/GFP+ (i.e. proliferating endothelial cells) and BrdU+/GFP- cells (i.e. proliferating non-endothelial cells) so that the possible nature of the proliferating cells can be discussed. 4. The authors conclude that their data represent the first evidence of the morphological adaptation of a respiratory organ to a physiological stimulus. This statement is incorrect (Fu et al. show increased lamellar surface area in response to swimming training) and should be removed or reworded to indicate that the authors provide further evidence to support it. 5. Line 376. The authors should state more clearly what they imply by “this phenomenon”. Are the authors referring to the stress-reducing effects of swimming? 6. Lines 415-416. The description of the behavioral effects of endurance training is poorly worded. This reviewer suggests to better describe the behavioral effects in terms of schooling. 7. Line 468. Common names are not italized. Reviewer #2: This is an interesting study and well written manuscript. OVerall, the authors appear to have conducted their studies carefully. I have only minor concerns. First, the authors state that BrDU staining is primarily located at the tip of gill filaments in trained fish, but it is unclear from Figure 7 what the authors mean by this. The stained cells appear to be distributed throughout the region shown in the figure. If the authors are arguing that the whole region shown in Figure 7 represents the “tip” of the gill fiber, then other images showing more distal regions of the gill should also be included for comparison. Alternatively, the images shown do not clearly look like the mitotic figures are concentrated toward the tips of the structures shown, and clarification of their conclusion and/or more images to support their conclusion, might be warranted. A second question concerns the data for mitotic index (Figure 7) and filament growth (Figure 3). While I accept that the average values for each group show significant increases in the trained fish, it certainly looks like many fish showed no changes versus controls. This should be commented on. In particular, I wonder if the authors can provide some data on which fish showed the best growth and mitotic index. For example, are these data correlated with overall growth of the animal? Could the authors not conduct regression analysis to show which variables do/do not correlate with gill growth? Finally, assuming that the authors are willing to conduct additional experimentation, it seems like the issue raised above could be addressed better if images of the gills of individual fish could be obtained at intervals during the course of the training period. If such images could be obtained at a resolution sufficient to visualize primary or even secondary gill filaments, this might provide still more compelling data with regard to the manner and location of filament growth. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0228333.r001
Revision 1
12 Dec 2019 Author Response Reviewer #1 1. The authors need to clarify aspects of the swimming training and respirometry experiments. First, details of the swim tunnels used in this study need to be provided (volume, model number). Details on the two used swim tunnels (Model #SW10100 for training and model # SW10000 for respirometry) have been added to the manuscript, including direct links to the relevant pages on the Loligo website. Second, a clear indication of whether the swimming training was conducted on individual fish or on groups of fish (if this was the case, please indicate exactly the number of fish in the swimming chamber) is needed. Swimming training was performed in 10/group. Maximum swimming capacity was assessed in individual fish. We updated the Materials and methods section in the manuscript accordingly. Third, please clearly state where the non-swimming fish were housed during the experiments. We specified details on general housing of the fish and where the control fish were kept (in their regular tanks) in the Materials and methods section. Fourth, please indicate the value of the training speed in body lengths per second. Except for the speed increase of the swim tunnel on line 138 all speeds were given in cm/s and body length per second. On line 138 we would like to leave the value since this is a machine setting and we think "increase of 0.5 cm/s every two minutes" is more illustrative than "increase of 1.71 bl/s every two minutes for body lengths measured before training". It is unclear if we should remove the cm/s altogether, please let us know if this is the case. The y-axis value of the left part of figure 1 was changed to show the speed in bl/s instead of cm/s. 2. A justification for the use of transgenic zebrafish is needed and related additional analyses are strongly recommended (point 3). The presented study is just one branch of a broader project on exercise-induced angiogenesis in skeletal muscle. The fli1a:eGFP line was thus chosen to label endothelium. With respect to the 3R-initiative (replacement, reduction, and refinement) for the ethical use and the reduction of the number of animals sacrificed for science, we've looked at other exercise-induced adaptations and noticed interesting changes in gills, which we present in the manuscript. A short sentence on the choice of transgenic line was added under the Animals section in the manuscript. First, the authors should confirm whether the fli transgenic line use corresponds to fli11a:eGFP, as indicated in the manuscript (line 119), or instead to the fli1a:eGFP line that is commonly used as a vascular endothelial cell marker. If the latter, please correct This was indeed an oversight from our side and has been corrected in the manuscript. Thanks for pointing this out to us. Second, does the reference provided indicate the initial description of this line or the research group that provided the line? If the former, please indicate the origin of the line used in these experiments. The reference indeed provides only an initial description of the zebrafish line. The fishes were originally procured from the Zebrafish International Resource Center. The text has been updated accordingly. 3. With regards to the use of this transgenic line, it is not clear why the authors did not exploit the GFP labeling of endothelial cells to determine if proliferating (BrdU+) cells are indeed endothelial cells or not. This reviewer strongly recommends the authors to reanalyze the immunofluorescence data and provide quantification of BrdU+/GFP+ (i.e. proliferating endothelial cells) and BrdU+/GFP- cells (i.e. proliferating non-endothelial cells) so that the possible nature of the proliferating cells can be discussed. As mentioned above, we were not intending to quantify the proliferating endothelial cells in the gills. However, it is possible to construct an eGFP+ surface in Imaris software and look whether the BRDU-positive spots lie within (see Fig 1, panels A and B). This figure has been added as supplementary material to the manuscript. Exact calculation of the endothelial surface is not trivial because of the unexpectedly high fluctuations in eGFP expression between fish. Uniform thresholding parameters (relative changes in eGFP intensities) inevitably cause over or underestimation of the endothelial surface in some samples. Nonetheless, our calculations provide evidence that the endothelial surface in filament tips of trained fish is significantly larger than in controls (+21.8 %, p-value 0.00053, n=12+12), while no changes in endothelial surface at the filament base was observed (p=0.16). The relative difference between swimmers-tips and controls-tips was observed independently on the thresholding parameters and we are thus confident that there is a real difference. However, the generated surface is just an estimation, e.g. not identical with the actual surface of the endothelium, and thus given in 'arbitrary units'. 4. The authors conclude that their data represent the first evidence of the morphological adaptation of a respiratory organ to a physiological stimulus. This statement is incorrect (Fu et al. show increased lamellar surface area in response to swimming training) and should be removed or reworded to indicate that the authors provide further evidence to support it. Our formulation was improper; we apologize. We cite the work of Fu et al. for the increase of lamellar surface but while they focused on the acute (and possibly transient, as the authors explain) adaptation to exercise, we've investigated more permanent effects that last at least for several days after the cessation of exercise. We aren't aware of any other publication on chronic (permanent) changes of gills after endurance training, but we've taken the formulation 'first evidence' out of the text to avoid misunderstandings, and clarified the differences between these two exercise models (48h without a break versus our 3- or 5-week regime) in the text. 5. Line 376. The authors should state more clearly what they imply by “this phenomenon”. Are the authors referring to the stress-reducing effects of swimming? We clarified the references to the objective stress-reduction signs. 6. Lines 415-416. The description of the behavioral effects of endurance training is poorly worded. This reviewer suggests to better describe the behavioral effects in terms of schooling. We updated and expanded the explanation of the observation of reduced objective signs of stress and linked to the movie about the behavioral effects already mentioned in the results section. 7. Line 468. Common names are not italicized. We have removed the italicization of Crucian carp on this line and from several other common names in the manuscript. Reviewer #2 First, the authors state that BrDU staining is primarily located at the tip of gill filaments in trained fish, but it is unclear from Figure 7 what the authors mean by this. The stained cells appear to be distributed throughout the region shown in the figure. If the authors are arguing that the whole region shown in Figure 7 represents the “tip” of the gill fiber, then other images showing more distal regions of the gill should also be included for comparison. Alternatively, the images shown do not clearly look like the mitotic figures are concentrated toward the tips of the structures shown, and clarification of their conclusion and/or more images to support their conclusion, might be warranted. We have added representative images from both the tip and base region of the gills to Fig07. 'Representative' means that the images correspond to the median value of the fishes in this row. Fig07 has been updated accordingly. Since this image is now more dense, we decreased the font size of the image label. To be consistent, the font size of all the labels on all the figures has been decreased to the same size. A second question concerns the data for mitotic index (Figure 7) and filament growth (Figure 3). While I accept that the average values for each group show significant increases in the trained fish, it certainly looks like many fish showed no changes versus controls. This should be commented on. In particular, I wonder if the authors can provide some data on which fish showed the best growth and mitotic index. For example, are these data correlated with the overall growth of the animal? Could the authors not conduct regression analysis to show which variables do/do not correlate with gill growth? The criteria for the quantification of mitoses was based on threshold values for intensities in BrdU and DAPI channels as well as spot size and geometry. Analysis was done in a batch process for all scans. Despite cautious and strictly uniform staining and imaging of the samples, our results show a large variability within the groups. Repeating the batch analysis with different selection criteria did not affect the ratio of the values between the groups; we're thus convinced that we see a true effect. As we didn't label nor train the fish individually, we are not able to calculate the growth or weight gain of the individual fish, we could only give an average of the whole group. This measurement was never a part of our study setting and animal experiment permit. We thus only have pooled data for the whole cohort. We plotted the regression of the different assessed parameters of the fishes. But apart from the obvious trends (fish weight and filament count or fish weight and gill volume), no significant or meaningful correlation was observed (as seen in Figures 3 to 6 below). Additionally, comparing the measured gill parameters prior and after training is not possible due to ex-vivo (destructive) character of the investigation. Please also see the answer to the next suggestion of yours for reasons to not perform more experiments. A combined figure of the regression plots below has been added to the manuscript as a supplementary figure. Finally, assuming that the authors are willing to conduct additional experimentation, it seems like the issue raised above could be addressed better if images of the gills of individual fish could be obtained at intervals during the course of the training period. If such images could be obtained at a resolution sufficient to visualize primary or even secondary gill filaments, this might provide still more compelling data with regard to the manner and location of filament growth. Due to the ex vivo imaging approach in this study, we cannot assess the gill parameters of individual fishes in a longitudinal study. The post-mortem method makes this impossible, we can only study the gill parameters at the study endpoint. In vivo tomographic imaging of fishes is possible but lacks the resolution needed to distinguish the secondary filaments. One possibility would be to could greatly increase the cohort size and remove a sufficient amount of fishes per timepoint to be studied (N>5) from the experiment for performing tomographic (and EM) imaging. This would then allow for assessing the data (pooled over the whole cohort) during the training period. Due to constraints in time, manpower and animal permit, we did not perform this in the present manuscript, albeit we fully agree that this would most probably provide still more compelling d https://doi.org/10.1371/journal.pone.0228333.r002
14 Jan 2020 Decision Letter - Eric A Shelden, Editor Adaptation mechanism of the adult zebrafish respiratory organ to endurance training PONE-D-19-24766R1 Dear Dr. Djonov, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Eric A Shelden, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: I believe the authors have adequately addressed the concerns that I raised in the previous round of review, and recommend acceptance. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0228333.r003
Formally Accepted
24 Jan 2020 Acceptance Letter - Eric A Shelden, Editor PONE-D-19-24766R1 Adaptation mechanism of the adult zebrafish respiratory organ to endurance training Dear Dr. Djonov: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Eric A Shelden Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228333.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .