PONE-D-19-15096

Candida utilis yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Local intestinal tissue and plasma proteome responses

PLOS ONE

Dear Dr. Øverland,

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.

Most importantly, you should pay attention to the statistical analysis of the proteomic data, as carefully explained by Reviewer 3. In addition, both Reviewers 1 and 2 express concerns about the discussion of the manuscript, which should be carefully revised. 

We would appreciate receiving your revised manuscript by Aug 23 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,

Annie Angers, 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. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

3. Please amend the manuscript submission data (via Edit Submission) to include author Leidy Lagos.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: The present work studies how the addition of C. utilis in soybean meal based diets impacts or reverts the changes induced when compared to a control fishmeal based diet. The results show that low levels of yeast reduced the severity of the enteritis caused by soybean meal based diets and C. utilis is proposed as an alternative protein source in functional diets for Atlantic salmon. In my opinion, this is a very attractive and complete study that combines different techniques in order to evaluate Atlantic salmon intestinal health. However, I have some comments and concerns that I believe should be addressed before considering this manuscript for publication.

Major concerns:

The experimental design seems carefully planned and complex, with many groups and variables. However, not all groups were used for all analyses and there is no explanation why. For example, it is stated that low doses partially prevent SBMIE, but these groups were not included in the gene expression or proteomic analyses. In addition, an explanation of the different effect that low and high levels of C. utilis have on inflammation should be included.

The discussion seems to focus on randomly selected proteins. There is the description of the function of some of these proteins but I fail to see the point and relevance in this particular study. In my opinion, the whole discussion should be revised to follow a particular relevance point regarding this research, animal model and application.

Other comments:

L71: This sentence looks weird. Maybe the authors meant: “…an inactive dry Candida utilis strain was used…” or “… an inactive dry yeast strain of Candida utilis… “.

L108: Vaccinated against?

When mentioning average body weight values the error should be included. For instance, L116.

L123: The feeding strategy was changed to what? In general, the experimental design is not clearly explained and even though there is a nice explanatory figure the text should be more clear.

L200: The amount of tissue used for RNA isolation should be stated.

L215: The concentration of the primers should be stated. Also, in the supplementary figure of the primer sequences the symbol for GDPH is GADPH, please unify the nomenclature.

L253: I think a reference to the salmon proteome should appear here.

L269: Why a series of PCA? Were several analyses performed? If so, they should be described. Also, verify the use of analyses instead of analysis for the plural.

L289: The average initial and final weight are mentioned (again I’m missing the errors), but there is no comparison among groups. Were there differences? Maybe a table should be included with the group values?

L303: “described in detail below” (remove the “ed”)

L293: section Histology: A panel of pictures showing the most significant changes (like the ones described in L308-312) would be nice. Also, how do the authors know exactly which cell types are present (L311-312) without specific markers? Some of these cells are difficult to identify in intestinal sections.

L323-324: Why where those groups excluded from the figure?

L366-367: The difference mentioned here is not represented in the figure, in fact, regarding the figure statistics these groups were not different in this parameter.

L387: The sudden change in nomenclature does not simplify the reading of the data, it actually complicates it. The nomenclature should be maintained along the manuscript (including the figures). In addition, why were the groups with low yeast dose excluded from this analysis? This should be explained in detail, particularly because the work concludes that the low doses are the ones having more positive effects but there are no results about them in this part of the study.

L403 and Fig6C: This heatmap seems to show differential expression values. If this is correlation I don’t understand against what each molecule is correlated. Please revise these results and explanations.

L409: “Analyses were carried out first with….” Why first? Was there a second analysis?

L445: These inconsistencies should be further explained.

L473: Delete DI.

L473-475: The statements about the role of aqp8 in homoeostasis should be further supported.

L489-492: I do not see the point of mentioning these particular roles of MHCI here. Maybe the authors are trying to make a point I do not understand.

L494: One study or several? If one, change for “A previous study”. Otherwise use plural.

L509: Downregulation in which tissue?

Figure 3: Scale bars should be added to the pictures.

Figure 5: The fold change is relative to what?

Reviewer #2: Review of PONE-D-19-15096 ” Candida utilis yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Local intestinal tissue and plasma proteome responses”.

This manuscript describes the effects of using a yeast strain as a functional protein source to boost gut health in fish at risk of intestinal enteritis when fed soy bean-based meal. The study tackles a highly relevant problem and is generally very well written and clear. Overall, this study will provide an important contribution to the field and fit well into the PLoS one journal. Below I provide some general and more specific comments to help the authors improve the manuscript for publication.

General comments

I want to applaud the authors for performing such a holistic analysis opposed to using just one single method. This is very much needed and sets a great standard for future studies.

However, what I miss a little is an attempt to better link all the data sets together in a biological way. Could simply be through discussing results, but in a way to e.g. link the genes tested for expression responses with the proteins analyses all the way to the tissue histological data. Maybe add a conceptual figure with the entire path from DNA -> RNA -> proteins -> tissues and the highlight where you have generated data and results. As is, the different data types are treated quite independently and in a somewhat random order.

For example, the discussion on lines 467-469 represents a good example of how gene expression data is integrated with insights from histology and immune response data.

Contrary, on lines 484-488 is an example where the protein plasma data is mainly discussed in isolation, whereas it would benefit from a stronger focus on the underling biological interactions with e.g. gene expression, histology and immune response data.

Specific comments

L. 108: What was the fish vaccinated against? Was it a standard vaccination program or inly for a specific disease? Pease specify.

L. 158: Which and how many fish were used for this immunohistochemistry analysis? Was it the same 8 fish as above or new ones? Please specify.

L. 224: Do you mean “… as index” instead of “… an index”?

L. 229 + 231 + throughout: Be consistent whether or not you add a space between the degree symbol and “C” when listing temperature values.

L. 253: Please provide a reference for the salmon proteome reference database.

L. 262-263: Can you clarify this description as it sounds like all individuals are already part of "a cluster" when you begin. So, when does the model know to end the analysis? Is it e.g. trying to minimise the number of clusters based on some preset value? This remains unclear as currently written.

L. 275: Add space before “[31]”.

L. 388-389: It is not clear to me how this "simplifies" except using fewer letters. The figures have plenty of space to write out the FM and SBM names respectively, and these are much more informative than having to cross read between the text here and the figure to know what D1-D4 represents. I would prefer to just keep the FM and SBM based names in the figures as well. Same for Fig. 7.

L. 438-444: Could this maybe also be due to individual responses among fish, and that these responses are partly controlled by the different genotypes among individuals? For example, if host genotype selects for different gut-microbiota communities, one could speculate that such different gut environments will differ in their ability to respond to the provided C. utilis strains? I agree this is very speculative, but the explanation do warrant mention as opposed to only focusing on how external parameters could explain the observed variation in gut responses; which seems to be the standard way of interpreting these types of gut data.

L. 503: re-write to "proteins with significant over expression in ... ". The proteins themselves are not significant, it is their expression level that is.

Figure 2: Please add labels for all Y axes.

Figure 3: Please clarify the different colors in the figure images. Are CD8 positive cells the purple colored ones? Or are both CD3 and CD8 colored brown? If so, what are the purple stains?

Figure 4: Discuss whether the low sample size (n=7) may cause type-2 errors for the non-significant comparisons?

Figure 5: Add both a and b letters between the two diet categories in the plots where there are significant differences.

Also, same concern as for figure 4 about regarding lack of statistical power with the relatively low sample sizes. Was any prior power analyses performed to inform the used sample sizes? There are some differences in e.g. plot C that could potentially be significant with more statistical power, which could for example be obtained through an increased sample size.

Reviewer #3: General Impressions:

In this manuscript, the authors performed a feeding trial on Atlantic salmon in which fish were fed fish meal or soybean meal-based diets that were supplemented with varying amounts of Canadis utilis yeast. Changes in intestinal morphology and the plasma proteome of the fish were monitored at several points in the study in order to monitor both intestinal and systemic effects of the different diets.

Overall, the experimental design appears valid and the data collection and analysis of the histological data is appropriate. However, in the eyes of this reviewer, the analysis of the plasma proteomics data could be improved to allow for more meaningful interpretation of the data. The current methodology does not provide significant evidence that the observed changes in the plasma proteome are related to the differences in diet. However, after the authors address the following comments, the manuscript would likely be suitable for publication.

Major Comments:

• The authors relied primarily on Spearman’s correlation coefficients to identify proteins of interest from the plasma proteomics data. Therefore, the authors are essentially looking for cases where protein abundances do or do not correlate across the different diets. However, since there is a clear control in the experiment (fish meal-based diet), it seems that comparing all protein abundance to that control would be more informative. Then, emphasis would be placed on proteins that have ‘abnormal’ expression relative to the fishmeal fed fish. This would also allow for volcano plots to be generated and included in the manuscript, which would aid reader understanding.

• Further description of how the quantitative values used in the proteomics analysis is needed. It appears that precursor abundance (from the MS1 scans) were used, but that is not explicitly stated. If that is the case, was peak intensity or peak area used? Also, was the ‘match between runs’ feature enabled in MaxQuant?

• A table showing all of the proteins identified in the plasma analyses and their quantitative values should be provided in the supplement. Ideally, fold change values should also be included. It’s possible that such a table was uploaded to PRIDE, but the PXD number provided does not bring up any results when searched. This is likely because the dataset has not been publicly released but the login info should be provided to reviewers.

• It is concerning that the fishmeal-based and soybean-based diets don’t cluster in either the heatmap or the PCA plots. It seems likely that such large differences in the feed would causes systemic changes in the blood which would lead to high correlation between the D1 and D7 samples, but that was not observed. This suggests that either the analysis method was flawed, or that individual variation was too large in order to adequately detect differences from the feed.

• Although this reviewer is not well versed in the VIP method used here, it seems that this technique is likely to put too much emphasis on proteins that are correlated purely by chance. Further justification of this technique should be included, or the analyses should be removed.

• It would be helpful if the reviewers included a few representative images of the changes in morphology observed between the groups that are summarized in Fig. 2. This could be included as an additional supplemental figure.

Minor Comments:

• L252: The fragment ion tolerance used here is much too large for orbitrap data. A tolerance of 0.02 Da would be more appropriate.

• L253: Where was the salmon proteome database derived from? Was it a public repository such as uniprot? If so, when was it downloaded?

• L316: No data is shown in this figure where SBM + C. utilis was fed first, followed by FM. This sentence says otherwise.

• Fig. 4 caption – L371: What is meant hear by ‘data are expressed as mean � SD for each individual’? Isn’t each point just representative of the mean for each fish? This should be clarified.

• L407-408: It doesn’t make sense to say that proteins ‘belong’ to a specific domain. Please revise.

• Fig. 7 caption – L424: What do the colors in panel C represent? Also, why are proteins with VIP scores < 2 included here?

• L473: Remove ‘DI.’

• L476: Change considering to considered.

**********

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

Reviewer #3: 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.

PONE-D-19-15096R1

Candida utilis yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Local intestinal tissue and plasma proteome responses

PLOS ONE

Dear Dr. Øverland,

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.

While all three reviewers consider the general response to comments satisfactory and that the manuscript is significantly improved, Reviewer 3 has underlined new concerns regarding the proteomic data analysis. Based on the possibility that the conclusion reached in the manuscript may be based on invalid interpretation of the results. The reviewer kindly suggests many viable options to overcome this conundrum, and I am therefore confident that the matter can be resolved.

Specific comments by both reviewers 2 and 3 should also be addressed prior to publication.

We would appreciate receiving your revised manuscript by Oct 28 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,

Annie Angers, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #3: (No Response)

**********

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

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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 have now been through this revised version of the manuscript. I am generally happy with all the authors responses to my original review. So I am happy to hereby recommend the manuscript for publication.

But I do have a few minor comments to help with some final improvements:

1)

Regarding the Y-axes labels on figure 2, it may be more appropriate to label them "Change" rather than "Scale" that seems not very informative.

2)

I suggest that the authors either delete or re-write the very kast sentence of the discussion:

"Further research is needed to evaluate the impact of yeast strain and the fermentation

and/or down-stream processing conditions of the yeast on functional properties in relation to gastro

intestinal health and systemic responses."

As is, this is not very informative. Consider adding some more concrete suggestions as to how such "Further research" can be designed to better help move this entire research field and add more values to readers of the paper.

Reviewer #3: The authors addressed a number of concerns raised by the reviewers and the manuscript has been significantly improved. Unfortunately, this reviewer still has significant concerns about the analysis of the proteomics data and the conclusions that are drawn from this data.

In the original manuscript, it was not made clear that the authors were utilizing spectral counting to obtain quantitative information from the proteomics data. This should have been stated in the manuscript, and needs to be added. The spectral counting method has been phased out in the field due to its low reproducibility and accuracy. When using a high resolution, accurate mass instrument such as the Q Exactive that was used here, MS1 intensity-based quantitation provides far superior quantitative values than does spectral counting. The reasons for this are well described in the literature, but are primarily the result of the somewhat stochastic nature of the data collection when data-dependent acquisition strategies are employed (as was done in this study). The fact that an MS2 mass tolerance was used for searching that was more than an order of magnitude higher than it should have been (0.8 Da rather than 0.02 Da) further reduces the quantitative accuracy of the spectral counting strategy used here.

For the reasons described above, the quantitative values presented should be treated with caution. The proteins shown to be differentially expressed in the volcano plots (Fig. 7) are likely valid, as this analysis incorporates both the calculated fold change, as well as a p-value, which incorporates other variables such as inter-sample variability. Unfortunately, many of the proteins that the authors focus on in the discussion (such as HMP, LYME, and HIIN) were not shown to be differentially expressed by this analysis, they simply didn't correlate well to the control. This alone is not adequate evidence of a change in protein abundance.

With these concerns in mind, this reviewer suggests the authors choose one of 3 options:

1. Reanalyze the data using MS1 intensity-based quantitation (as can be completed in MaxQuant or other software packages such as Scaffold Q+, Proteome Discoverer, etc.).

2. Focus the discussion only on proteins that showed differential abundance in the volcano plot analyses (Fig. 7)

3. Remove the proteomics data entirely and focusing on the gene expression, morphological, and other data presented in the manuscript.

Any of the above approaches would be valid and would make the publication suitable for publication in the opinion of this reviewer.

Specific Comments:

L262: The date the database was downloaded should also be included.

L403: Don't say 'expressed' here. The proteins were likely expressed in the samples, they just couldn't be detected by the mass spectrometer.

Figure 6: The Spearman's correlations done here do not provide sufficient evidence to say a protein is changing abundance. Panel C should be removed from the manuscript as well as all discussion of the results presented in this panel.

**********

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

Reviewer #3: 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.

PONE-D-19-15096R2

Candida utilis yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Local intestinal tissue and plasma proteome responses

PLOS ONE

Dear Dr. Øverland,

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.

The manuscript is much improved and almost ready for publication. However, the final concerns raised by Reviewer 3 still need to be addressed, and minor adjustments to the text are also needed.

We would appreciate receiving your revised manuscript by Dec 26 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,

Annie Angers, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

- Please address the possible data discrepancies between table S3 and table 2 referred to by Reviewer 3;

- Make sure that proteins that were not found in a replicate are not given a value of zero but treated as missing in your analysis (or clearly state that you did not use 0 in your analysis)

- Make the minor adjustments to text suggested by Reviewer 3.

[Note: HTML markup is below. Please do not edit.]

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 #3: (No Response)

**********

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 #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: 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 #3: 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 #3: 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 #3: General Impressions:

The authors have clearly addressed the concerns raised in the previous review, and the reanalysis of the proteomics data has dramatically improved the quality of the manuscript. I greatly appreciate the authors hard work and dedication to providing the highest quality data possible. I have noticed one discrepancy in the data analysis that should be addressed in the final version of the manuscript (described below), but I feel that the manuscript is otherwise ready for publication. I’ve also included a few very minor comments that I feel will improve the readability of the publication.

Major Comments:

The authors state that only protein that were identified in at least two of the four replicates were used for quantitative analysis. However, when looking at Table S3 and comparing that to the results presented in Table 2, there seems to be some discrepancies. For example, C0HAL2 (elongation factor 1-alpha) is reported as having a very significant fold-change between the FM (D1) and SBM (D2) in Table 2, yet no quantitative values for this protein are reported in the D2 group in Table S3. Why is this? Please ensure that your filtering is working as intended.

Additionally, proteins that were not identified in a replicate should not be assigned a quantitative value of 0. Instead, these values should be treated as missing (null), or missing value imputation should be used to provide baseline quantitative values. It’s difficult to determine how such values were treated in the analysis, and that should be stated outright.

As an aside for future experiments, use of the ‘match between runs’ feature in MaxQuant should dramatically decrease the number of missing values present in the dataset.

Minor Comments:

Line 241: It seems as if part of this sentence was accidentally removed as there is no mention of the addition of trypsin.

Line 243: Another sentence or two very briefly describing the column and MS parameters that were used would be useful here.

Line 261: Was a fragment mass tolerance of 0.8 used in the reprocessed data as well or was this a carryover from the previous version of the manuscript? As mentioned previously, a much tighter tolerance (~0.02 Da) would be more appropriate.

Lines 276-277: Saying that the average weights increased does not mean that every fish gained weight. Consider rephrasing.

Table 2: It would be nice if you included the p-value for each protein here as well.

Figure 6: As a note, the ‘presence’ of a protein in one sample and not in another is much more likely a result of the stochastic nature of the MS data collection rather than a protein actually being present in the sample or not. Therefore, comparisons such as the one shown here are often not very informative.

**********

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 #3: 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.

Candida utilis yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Local intestinal tissue and plasma proteome responses

PONE-D-19-15096R3

Dear Dr. Øverland,

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,

Annie Angers, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for the extensive revisions to the manuscript. I am sorry that the processed took so long, but I feel that the improved version was worth the efforts.

Reviewers' comments: