Mid and hindgut transcriptome profiling analysis of Atlantic salmon (Salmon salar) under unpredictable chronic stress

The intestinal epithelium is a selectively permeable barrier for nutrients, electrolytes and water, while maintaining effective protection against pathogens. Combinations of stressors throughout an animal's life, especially in agriculture and aquaculture settings, may affect the regular operativity of this organ with negative consequences for animal welfare. In the current study, we report the effects of a three-week unpredictable chronic stress (UCS) period on the intestinal morphology and transcriptome response of Atlantic salmon (Salmon salar) parr midgut and hindgut. Midgut and hindgut from both control and UCS fish were collected for histology and RNA-sequencing analysis to identify respective changes in the membrane structures and putative genes and pathways responding to UCS. Histological analysis did not show any significant effect on morphometric parameters. In the midgut, 1030 genes were differentially expressed following UCS, resulting in 279 genes which were involved in 13 metabolic pathways, including tissue repair pathways. In the hindgut, following UCS, 591 differentially expressed genes were detected with 426 downregulated and 165 upregulated. A total of 53 genes were related to three pathways. Downregulated genes include cellular senescence pathways, p53 signalling and cytokine–cytokine receptor pathways. The overall results corroborate that salmon parr were at least partly habituating to the UCS treatment. In midgut, the main upregulation was related to cell growth and repair, while in the hindgut there were indications of the activated apoptotic pathway, reduced cell repair and inhibited immune/anti-inflammatory capacity. This may be the trade-off between habituating to UCS and health resilience. This study suggests possible integrated genetic regulatory mechanisms that are tuned when farmed Atlantic salmon parr attempt to cope with UCS.


Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
This is a nicely presented manuscript with some interesting findings, relevant to better understand stress responses in Atlantic salmon. I would suggest some minor revision to clarify some statements made throughout the text, as outlined below.
Overall comments 1. I found that for several statements made in the introduction and discussion, no reference is mentioned. I would recommend reviewing these sections again and adding references in the appropriate places (examples: ll 38-39, ll 375-356). 2. Although no specific stress factor was tested, but rather random, changing events, it would be helpful if the rationale of these stressors was explained in context of the physiology of the fish, e.g. "natural" and "unnatural" stressors and how the fish's system may be inherently well equipped to deal with this. This could be put into the discussion. 3. The results from the histology suggest no difference between stressed and unstressed fish for both gut sections for the features analysed. I would suggest looking at more parameters that cover the whole gut section as sometimes damage has been found to be localised in certain areas. Furthermore, features not analysed here such as supranuclear vacuole appearance have been found to be good markers for tissue damage. I would recommend considering the scoring system proposed by Uran et al (2009) as additional parameters. 4. The manuscript would benefit from careful proofreading -in several places punctuations are missing. Furthermore, there are inconsistencies in the formating of "padj" and referencing authors in the text.
Methods section 1.It is not clear if you have assigned all identified genes a gene symbol (HGNC) prior to gene ontology and pathway analysis. Only the minority of loci of the salmon genome is annotated with a gene symbol, but the majority is only annotated with a LOC number, therefore you would lose a lot of differentially expressed genes in this analysis if you do not assign them a symbol. 2.You mention numbers for up and downregulated genes for each gut section, but it would be good if you would also mention how many of these are unique or duplicated genes and how many of these genes were fed into gene ontology and pathway analysis and if you analysed up and downregulated genes seperately or together as differentially expressed genes. 3. Furthermore it would be useful to know what cut-off you used, you mention a padj<0.05 but did you also filter by fold-change? Results 1. As mentioned above, when you provide the numbers for up and downregulated genes, it is useful to know what cut-offs you used. 2. Is the cortisol graph meant to go into the main text or supplementary material? Should be mentioned in the appropriate paragraph. 3. Table 3 and 4: it would be useful to translate the LOC numbers into gene names or symbols so the reader can easily understand what genes are associated with the kegg term. Also some indication if these are up or downregulated would be useful.

Discussion
1. The cortisol graph shows a dip in cortisol in the stressed group on day 5 relative to the other time points. It would be interesting if you could discuss why this may be in a few sentences. 2. Do you have any suggestions why tissue repair was evident from transcriptomic data and not histological data? May that be due to the only experimental timepoint being when there was already some signs to acclimatisation? 3. Could you please expand on the agenda of feeding the fish so shortly after having experienced stress? Would the fish have consumed food like the control fish if the feeding had taken place, let's say 2 hours after stress, therefore not had altered growth parameters? I understand it is impossible to include everything in an experiment, but I think it makes an interesting point for the discussion. 4. Lastly, I would suggest to check that you included all up-to-date references on this topic, a lot of references seem fairly old, and even when relevant, should be supplemented by the inclusion of the newest findings.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? No

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? Yes

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
Manuscript ID: RSOS-191480 This manuscript reports on a study conducted in 2013 that examined the mid and hindgut transcriptome profile of Atlantic salmon parr subjected to a 3-week unpredictable chronic stress. The study reports on results from samples collected at the last day of the study and compares control and stressed group. Ideally this study should have included in the transcriptome analysis more fish and sampling times. A comparison with samples collected at the start of the experiment would be beneficial to understand overtime changes.
Major comments, Please provide clearly the objectives of the study. The introduction is focused in explaining stress, chronic stress, and does not really cover transcriptome studies in fish during stress, and is somehow disconnected from the discussion.
The sampling protocol needs more clarification. A small sample size was used in this study (3 fish from the control group and 4 fish from the stressed group), and it is not clear where the samples are coming from. Are they coming from different tanks? Mid and hindgut samples were collected from the same fish. For example, control MID 1 and control HIND 1 are samples from the same fish. Ideally this study should have included in the transcriptomic analysis more fish and sampling times. A comparison with samples collected at the start of the experiment would be beneficial to understand overtime changes. Provide the following information (clarify in the material and methods): were the gene counts filtered to only keep genes that were present in all replicate samples of the same treatment? Did you keep only the genes that had a normalized expression over for example 1 count per million? Normalization of genes by counts per million controls for differences in library size, reducing bias.
Summarize the gene expression results. The authors should include a table demonstrating the top genes up and down regulated, and possibly present the genes that were similarly up and down regulated in both mid and hind gut. The manuscript is well written, but some elements of the discussion can be moved to intro or removed, as it is rather lengthy. The discussion would also benefit of exploring further the lack of significant results on histo when compared to gene expression. Is it possible that longer periods of stress would have caused changes on the morphometric parameters? Or is it possible that you did not collect samples at the right time to show morphological changes? (discussion L321-338). What is the significance of all these transcriptomic/pathways changes if no morphological changes occurred? Are these transcriptional changes consistent in all samples?

04-Dec-2019
Dear Ms Løvmo, The editors assigned to your paper ("Mid and hindgut transcriptome profiling analysis of Atlantic salmon (Salmon salar) under unpredictable chronic stress") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that have been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-191480 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. We have received the feedback from two reviewers, both of which agreed about the merits of this study. Although there were some concerns about low sample size, I think this manuscripts will fit within the scope of RSOS upon revision. Both reviewers provided constructive feedback that should help the authors to improve their manuscript. I agree with the reviewers that the manuscript would benefit from a broader conceptual context that is rooted on previously published studies. In addition, clarifying some gaps in the methods and analytical approaches is necessary.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) This is a nicely presented manuscript with some interesting findings, relevant to better understand stress responses in Atlantic salmon. I would suggest some minor revision to clarify some statements made throughout the text, as outlined below.
Overall comments 1. I found that for several statements made in the introduction and discussion, no reference is mentioned. I would recommend reviewing these sections again and adding references in the appropriate places (examples: ll 38-39, ll 375-356).
2. Although no specific stress factor was tested, but rather random, changing events, it would be helpful if the rationale of these stressors was explained in context of the physiology of the fish, e.g. "natural" and "unnatural" stressors and how the fish's system may be inherently well equipped to deal with this. This could be put into the discussion.
3. The results from the histology suggest no difference between stressed and unstressed fish for both gut sections for the features analysed. I would suggest looking at more parameters that cover the whole gut section as sometimes damage has been found to be localised in certain areas. Furthermore, features not analysed here such as supranuclear vacuole appearance have been found to be good markers for tissue damage. I would recommend considering the scoring system proposed by Uran et al (2009) as additional parameters.
4. The manuscript would benefit from careful proofreading -in several places punctuations are missing. Furthermore, there are inconsistencies in the formating of "padj" and referencing authors in the text.

Methods section
1.It is not clear if you have assigned all identified genes a gene symbol (HGNC) prior to gene ontology and pathway analysis. Only the minority of loci of the salmon genome is annotated with a gene symbol, but the majority is only annotated with a LOC number, therefore you would lose a lot of differentially expressed genes in this analysis if you do not assign them a symbol.
2.You mention numbers for up and downregulated genes for each gut section, but it would be good if you would also mention how many of these are unique or duplicated genes and how many of these genes were fed into gene ontology and pathway analysis and if you analysed up and downregulated genes seperately or together as differentially expressed genes.
3. Furthermore it would be useful to know what cut-off you used, you mention a padj<0.05 but did you also filter by fold-change? Results 1. As mentioned above, when you provide the numbers for up and downregulated genes, it is useful to know what cut-offs you used.
2. Is the cortisol graph meant to go into the main text or supplementary material? Should be mentioned in the appropriate paragraph.
3. Table 3 and 4: it would be useful to translate the LOC numbers into gene names or symbols so the reader can easily understand what genes are associated with the kegg term. Also some indication if these are up or downregulated would be useful.

Discussion
1. The cortisol graph shows a dip in cortisol in the stressed group on day 5 relative to the other time points. It would be interesting if you could discuss why this may be in a few sentences.
2. Do you have any suggestions why tissue repair was evident from transcriptomic data and not histological data? May that be due to the only experimental timepoint being when there was already some signs to acclimatisation?
3. Could you please expand on the agenda of feeding the fish so shortly after having experienced stress? Would the fish have consumed food like the control fish if the feeding had taken place, let's say 2 hours after stress, therefore not had altered growth parameters? I understand it is impossible to include everything in an experiment, but I think it makes an interesting point for the discussion.
4. Lastly, I would suggest to check that you included all up-to-date references on this topic, a lot of references seem fairly old, and even when relevant, should be supplemented by the inclusion of the newest findings.
Reviewer: 2 Comments to the Author(s) Manuscript ID: RSOS-191480 This manuscript reports on a study conducted in 2013 that examined the mid and hindgut transcriptome profile of Atlantic salmon parr subjected to a 3-week unpredictable chronic stress. The study reports on results from samples collected at the last day of the study and compares control and stressed group. Ideally this study should have included in the transcriptome analysis more fish and sampling times. A comparison with samples collected at the start of the experiment would be beneficial to understand overtime changes.
Major comments, Please provide clearly the objectives of the study. The introduction is focused in explaining stress, chronic stress, and does not really cover transcriptome studies in fish during stress, and is somehow disconnected from the discussion.
The sampling protocol needs more clarification. A small sample size was used in this study (3 fish from the control group and 4 fish from the stressed group), and it is not clear where the samples are coming from. Are they coming from different tanks? Mid and hindgut samples were collected from the same fish. For example, control MID 1 and control HIND 1 are samples from the same fish. Ideally this study should have included in the transcriptomic analysis more fish and sampling times. A comparison with samples collected at the start of the experiment would be beneficial to understand overtime changes.
Provide the following information (clarify in the material and methods): were the gene counts filtered to only keep genes that were present in all replicate samples of the same treatment? Did you keep only the genes that had a normalized expression over for example 1 count per million? Normalization of genes by counts per million controls for differences in library size, reducing bias.
Summarize the gene expression results. The authors should include a table demonstrating the top genes up and down regulated, and possibly present the genes that were similarly up and down regulated in both mid and hind gut. The manuscript is well written, but some elements of the discussion can be moved to intro or removed, as it is rather lengthy. The discussion would also benefit of exploring further the lack of significant results on histo when compared to gene expression. Is it possible that longer periods of stress would have caused changes on the morphometric parameters? Or is it possible that you did not collect samples at the right time to show morphological changes? (discussion L321-338). What is the significance of all these transcriptomic/pathways changes if no morphological changes occurred? Are these transcriptional changes consistent in all samples?

Author's Response to Decision Letter for (RSOS-191480.R0)
See Appendix A.

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The manuscript should be once more very carefully proofread for proper wording, grammar and punctuation. Examples: Line 147. Missing a period at the end of the sentence. Also should the number of that reference be between brackets? Line 152: Sections we stained with.... Should be Sections were stained.... Line 179: change respectfully, with respectively.
Decision letter (RSOS-191480.R1) 24-Jan-2020 Dear Ms Løvmo: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-191480.R1 entitled "Mid and hindgut transcriptome profiling analysis of Atlantic salmon (Salmon salar) under unpredictable chronic stress" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-191480.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. The authors have satisfactorily addressed previous reviewer concerns, and this manuscript is acceptable for publication after some minor revisions. I have a few additional comments that might help ti improve the clarity of the figures:  It is a pleasure to accept your manuscript entitled "Mid and hindgut transcriptome profiling analysis of Atlantic salmon (Salmon salar) under unpredictable chronic stress" in its current form for publication in Royal Society Open Science.
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Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. Reviewer: 1 Comments to the Author(s) This is a nicely presented manuscript with some interesting findings, relevant to better understand stress responses in Atlantic salmon. I would suggest some minor revision to clarify some statements made throughout the text, as outlined below.
Overall comments 1. I found that for several statements made in the introduction and discussion, no reference is mentioned. I would recommend reviewing these sections again and adding references in the appropriate places (examples: ll 38-39, ll 375-356). 2. Although no specific stress factor was tested, but rather random, changing events, it would be helpful if the rationale of these stressors was explained in context of the physiology of the fish, e.g. "natural" and "unnatural" stressors and how the fish's system may be inherently well equipped to deal with this. This could be put into the discussion.
R: The rationale behind the stressors in the UCS regime is mostly to induce several different stressful events that prevented fish from adapting over time, however without exposing the fish to severe physical treatment (introduction, line 69-75). The stressor in this trail are not intended to mimic "natural" or "unnratural" events, but only to develop a model of UCS in order to asses a prolonged stress response.
3. The results from the histology suggest no difference between stressed and unstressed fish for both gut sections for the features analysed. I would suggest looking at more parameters that cover the whole gut section as sometimes damage has been found to be localised in certain areas. Furthermore, features not analysed here such as supranuclear vacuole appearance have been found to be good markers for tissue damage. I would recommend considering the scoring system proposed by Uran et al (2009) as additional parameters.
R: As suggested by the reviewer, we have gone back and reviewed all histological pictures of the intestinal segments for vacuolization, and we can see no difference between the UCS group and the