Transcriptome Analysis of Juvenile Black Rockfish Sebastes schlegelii under Air Exposure Stress
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress
Liu et al,
This manuscript investigated DEGS after air exposure stress in the black rockfish. The experimental design was one control and two treatments (in triplicate).
Comments:
The manuscript is well written, but it requires organization and editing in several parts. Additionally, it requires “expanding” and explaining the results obtained.
Abstract:
The abstract is not quite informative of results. It requires re-writing and editing.
It says:
“This RNA was then used for transcriptome sequencing with the Illumina NovaSeq 6000 platform. Differential gene screening was performed, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.”
“A total of 9 transcriptome sequencing samples were processed, resulting in 58.5 Gb of clean data. The quality of the sequencing data was high, with percentages of 20 bases with Q20 and Q30 scores exceeding 97.61% and 93.51%, respectively. The sequencing yielded 92,469 transcripts and 58,397 unigenes, with N50 values of 2,772 bp and 2,478 bp, and N90 values of 631 bp and 534 bp, respectively.”
In my opinion, the above paragraphs can be omitted. They do not give any information to the reader.
Then it says:
“The GO functional enrichment analysis indicated that these DEGs were predominantly enriched in categories related to peptidase activity and the extracellular region. Meanwhile, the KEGG pathway enrichment analysis highlighted pathways such as metabolic pathways, protein digestion and absorption, and antibiotic biosynthesis.”
In my opinion, the above information has to be expanded related to anti-stress. For example, I do not catch why protein digestion is related to anti-stress. Once these issues are expanded, the reader could understand the last paragraph of the abstract.
Lines 79 -96:
All this information is not useful to the reader. It should be placed in discussion (and perhaps no even there). The above is because it does not reveal anything regarding air exposure in fish. For example: low temperature stress or hypoxia-stress are quite different from air exposure stress.
Suggestion: write what is known regarding air exposure stress in fish. And then continue with the general objective and its justification. (from line 103).
Lines 97-103:
This section belongs to M and M, not the introduction. It should be deleted because M and M explains the experimental design.
Lines 158-161:
A normal electrophoresis does not show RNA integrity (28S and 18S). These band will be degraded. If electrophoresis is used, the gel has to be pre-treated with 1% bleach and then solidified. Google bleach electrophoresis.
As the Agilent 2100 Bioanalyzer system was used, it gives you a pick of RNA integrity. No electrophoresis is needed.
Figure 2 is fuzzy. It has to be edited for clear reading.
Discussion.
Avoid repeated methodology and results in the discussion.
Line 436: Expand why peptidase activity is one of the first responses to air exposure stress.
Line 481 it says:
“By elucidating the molecular signatures of the stress response, this work paves the way for the development of targeted strategies to mitigate the adverse effects of water stress and improve the resilience of juvenile Sebastes schlegelii in aquaculture settings.”
I really do not know how will you mitigate air exposure stress by just having your main results. Please elaborate more. Additionally, I do not know how will you improve the resilience in aquaculture settings.
Stress produces cortisol, which is produced by the Hypothalamus, pituitary gland and adrenal glands axis.
What concerns me is that what was found (peptidase activity, protein digestion, etc) could be a secondary consequence originated from a main and different stress response, for example some endocrine pathway involving cortisol (perhaps) and then, cortisol affecting other metabolic pathways.
Author Response
Dear Reviewers:
Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress” (ID: fishes-3033711). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
Reviewer1:
Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress
Liu et al,
This manuscript investigated DEGS after air exposure stress in the black rockfish. The experimental design was one control and two treatments (in triplicate).
Comments:
The manuscript is well written, but it requires organization and editing in several parts. Additionally, it requires “expanding” and explaining the results obtained.
Reply: Thank you for your constructive feedback on our manuscript. We greatly appreciate the time and effort you have invested in reviewing our work.
In response to your comments regarding the organization and editing of the manuscript, we have undertaken a thorough revision. We have carefully restructured several sections to ensure a more coherent flow and have polished the language for clarity and precision.
Abstract:
The abstract is not quite informative of results. It requires re-writing and editing.
It says:
“This RNA was then used for transcriptome sequencing with the Illumina NovaSeq 6000 platform. Differential gene screening was performed, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.”
“A total of 9 transcriptome sequencing samples were processed, resulting in 58.5 Gb of clean data. The quality of the sequencing data was high, with percentages of 20 bases with Q20 and Q30 scores exceeding 97.61% and 93.51%, respectively. The sequencing yielded 92,469 transcripts and 58,397 unigenes, with N50 values of 2,772 bp and 2,478 bp, and N90 values of 631 bp and 534 bp, respectively.”
In my opinion, the above paragraphs can be omitted. They do not give any information to the reader.
Then it says:
“The GO functional enrichment analysis indicated that these DEGs were predominantly enriched in categories related to peptidase activity and the extracellular region. Meanwhile, the KEGG pathway enrichment analysis highlighted pathways such as metabolic pathways, protein digestion and absorption, and antibiotic biosynthesis.”
In my opinion, the above information has to be expanded related to anti-stress. For example, I do not catch why protein digestion is related to anti-stress. Once these issues are expanded, the reader could understand the last paragraph of the abstract.
Reply: Thank you for your constructive feedback on the abstract of our manuscript. We have taken your suggestions to heart and have made the necessary revisions to provide a clearer and more informative summary of our study's results.
In response to your comments, we have restructured the abstract to focus more directly on the key findings and their implications. We have omitted the detailed technical descriptions that you felt did not contribute to the reader's understanding and have instead expanded upon the significance of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in the context of the anti-stress response in juvenile Sebastes schlegelii.
We have elaborated on how the identified DEGs and enriched pathways relate to the fish's ability to cope with air exposure stress, providing a more coherent narrative that links our molecular findings to the biological mechanisms of stress response.
We believe that these revisions have significantly improved the abstract, making it a more concise and informative summary of our research. We are grateful for your guidance and appreciate the opportunity to enhance the clarity and impact of our work.
Lines 79 -96:
All this information is not useful to the reader. It should be placed in discussion (and perhaps no even there). The above is because it does not reveal anything regarding air exposure in fish. For example: low temperature stress or hypoxia-stress are quite different from air exposure stress.
Suggestion: write what is known regarding air exposure stress in fish. And then continue with the general objective and its justification. (from line 103).
Reply: Thank you for your insightful comments and suggestions. We appreciate the guidance on focusing the content more precisely to align with the study's objectives. In response to your feedback, we have revised the manuscript accordingly. We believe these revisions enhance the clarity and relevance of our manuscript, ensuring that the focus remains on air exposure stress in fish and the significance of our research in this area.
Lines 97-103:
This section belongs to M and M, not the introduction. It should be deleted because M and M explains the experimental design.
Reply: We appreciate your keen observation and are grateful for the opportunity to refine our work. Your feedback has been invaluable in enhancing the overall organization and presentation of our research.
We have taken the necessary action and removed the section from the Introduction. This adjustment ensures that the flow of information is logical and that each section of the paper focuses on its respective purpose, maintaining the clarity and integrity of the manuscript.
Lines 158-161:
A normal electrophoresis does not show RNA integrity (28S and 18S). These band will be degraded. If electrophoresis is used, the gel has to be pre-treated with 1% bleach and then solidified. Google bleach electrophoresis.
As the Agilent 2100 Bioanalyzer system was used, it gives you a pick of RNA integrity. No electrophoresis is needed.
Reply: Thank you for your insightful comment regarding the assessment of RNA integrity. We acknowledge the importance of accurately representing the methods used to confirm RNA quality, especially in light of the Agilent 2100 Bioanalyzer system's capabilities.
In response to your guidance, we have revised the paragraph to accurately reflect the use of the Agilent 2100 Bioanalyzer system for assessing RNA integrity. The updated description is as follows:
"The integrity of the total RNA was ascertained using the Agilent 2100 Bioanalyzer system, which provides a precise measurement of RNA quality. This system rendered the traditional agarose gel electrophoresis method unnecessary, as it offers a comprehensive analysis that includes the evaluation of the 28S and 18S ribosomal RNA bands, ensuring the absence of RNA degradation or contamination."
We have removed the reference to the 1% agarose gel electrophoresis and the treatment with bleach, as these were not part of our methodology. We trust that this amendment aligns with the best practices in RNA quality assessment and accurately represents the methods employed in our study.
We appreciate your expertise and are grateful for the opportunity to enhance the accuracy of our manuscript.
Figure 2 is fuzzy. It has to be edited for clear reading.
Reply: Thank you for bringing this to our attention. We have reviewed Figure 2 and agree that its resolution is not up to the standard required for clear reading. We apologize for any inconvenience this may have caused.
To address this issue, we have taken the following steps: We have re-imported the original high-resolution images into the manuscript to ensure that all details are visible and legible.
Discussion.
Avoid repeated methodology and results in the discussion.
Line 436: Expand why peptidase activity is one of the first responses to air exposure stress.
Reply: Thank you for your question. We appreciate the opportunity to delve deeper into this subject. Our understanding is as follows:
When fish are exposed to air, they experience a sudden and severe stress due to the lack of water, which is essential for their respiratory process. This immediate stress triggers a rapid response from the body to meet the energy demands for survival. Peptidases, as enzymes that break down peptides into amino acids, play a critical role in this process by accelerating protein catabolism to quickly release energy.
We believe that this rapid mobilization of energy through the action of peptidases is a crucial survival mechanism for fish during periods of air exposure. The swift breakdown of proteins into amino acids not only provides the necessary energy but also supplies the building blocks for the synthesis of stress-related proteins and other essential biomolecules that aid in the fish's recovery and adaptation to stress.
Line 481 it says:
“By elucidating the molecular signatures of the stress response, this work paves the way for the development of targeted strategies to mitigate the adverse effects of water stress and improve the resilience of juvenile Sebastes schlegelii in aquaculture settings.”
I really do not know how will you mitigate air exposure stress by just having your main results. Please elaborate more. Additionally, I do not know how will you improve the resilience in aquaculture settings.
Stress produces cortisol, which is produced by the Hypothalamus, pituitary gland and adrenal glands axis.
What concerns me is that what was found (peptidase activity, protein digestion, etc) could be a secondary consequence originated from a main and different stress response, for example some endocrine pathway involving cortisol (perhaps) and then, cortisol affecting other metabolic pathways.
Reply: Thank you for your insightful questions. Firstly, by elucidating the molecular signatures of the stress response, we have identified key genes and pathways that are affected by water stress. These include changes in peptidase activity and protein digestion, which, as you rightly pointed out, could be secondary consequences of a primary stress response. Our hypothesis is that these changes are indeed linked to the hypothalamic-pituitary-adrenal (HPA) axis and cortisol production, which are central to the body's stress response.
We acknowledge that our findings are a starting point, and further research is needed to fully understand the complex interplay between the stress response and the various metabolic pathways. We are committed to continuing this research to develop effective strategies that can improve the welfare and productivity of Sebastes schlegelii in aquaculture.
We appreciate for Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.
Sincerely,
Li-Guo Yang
2/6/2024
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript titled " Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress.”
The study investigates differentially expressed genes (DEGs) in juvenile Sebastes schlegelii under air exposure stress. The experiment included a control group and two air exposure groups , with three biological replicates each. DEG analysis revealed significant gene expression changes, with GO and KEGG analyses showing enrichment in peptidase activity, extracellular region, metabolic pathways, and protein digestion. The findings suggest air exposure induces stress responses in Sebastes schlegelii, elucidating underlying biological mechanisms.
The science in this manuscript is solid, considered and adds valuable knowledge to managing environments.
Introduction:
Lines 80-81: Could other transcripts regulating osmoregulation, such as the "NCC" cell transcripts identified in Zebrafish, also be involved in these mechanisms?
Results:
Line 230: If you had 100% mortality, does that mean you collected samples from the deceased for transcriptomic analysis? If so, please specify and clarify whether the RNA was not degraded.
Table 1: Note that no mortality was observed in the control group.
Table 2: Indicate the standard errors (SE).
Figure 5: Specify the conditions compared in the Venn diagram.
- Discussion
The first paragraph is too general; it could be moved to the introduction. It is recommended to start a Discussion with a brief general summary of your main results linked to your objectives.
Author Response
Dear Reviewers:
Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress” (ID: fishes-3033711). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
Reviewer2:
The manuscript titled “Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress.”
The study investigates differentially expressed genes (DEGs) in juvenile Sebastes schlegelii under air exposure stress. The experiment included a control group and two air exposure groups, with three biological replicates each. DEG analysis revealed significant gene expression changes, with GO and KEGG analyses showing enrichment in peptidase activity, extracellular region, metabolic pathways, and protein digestion. The findings suggest air exposure induces stress responses in Sebastes schlegelii, elucidating underlying biological mechanisms.
The science in this manuscript is solid, considered and adds valuable knowledge to managing environments.
Reply: Thank you very much for your positive and constructive comments on our manuscript titled “Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress.” We are pleased to hear that you found the science in our manuscript to be solid and that it adds valuable knowledge to the field.
Your recognition of the study's contribution to understanding the stress responses in juvenile Sebastes schlegelii and the elucidation of the underlying biological mechanisms is highly appreciated. It is encouraging to know that our research is considered valuable for managing environments and could potentially benefit the aquaculture industry.
Introduction:
Lines 80-81: Could other transcripts regulating osmoregulation, such as the "NCC" cell transcripts identified in Zebrafish, also be involved in these mechanisms?
Reply: Thank you for your question. In order to provide a more comprehensive and easily understandable explanation, we have rephrased this part of the content.
Results:
Line 230: If you had 100% mortality, does that mean you collected samples from the deceased for transcriptomic analysis? If so, please specify and clarify whether the RNA was not degraded.
Reply: Thank you for your inquiry regarding the collection of samples at 100% mortality in our study. To clarify, at no point in our experiment did we observe 100% mortality among the juvenile Sebastes schlegelii specimens used for transcriptomic analysis. The conditions applied were carefully designed to induce stress without causing immediate lethality, allowing us to collect samples for analysis while the fish were still alive.
The RNA samples used for transcriptomic analysis were collected from live fish that had been subjected to the air exposure stress conditions as outlined in our experimental design. We ensured that the RNA was of high quality and not degraded by using the Agilent 2100 Bioanalyzer system, which provides an accurate assessment of RNA integrity, as previously mentioned in the manuscript.
We apologize for any confusion that may have arisen and appreciate the opportunity to provide further clarification. We will make sure to specify these details in the manuscript to avoid any misinterpretation.
Table 1: Note that no mortality was observed in the control group.
Reply: Thank you for your observation regarding Table 1 in our manuscript. We appreciate your attention to this detail.
We confirm that the statement in Table 1 correctly indicates that no mortality was observed in the control group throughout the duration of the experiment. This is an important detail as it establishes the baseline for comparison with the air exposure groups, where differential gene expression was analyzed in response to stress.
We assure you that the control group was closely monitored and maintained under optimal conditions to ensure their health and survival, providing a clear reference point for our study's findings.
We will ensure that this point is highlighted in the manuscript to emphasize the contrast with the experimental groups and the significance of the control group in our research.
Table 2: Indicate the standard errors (SE).
Reply: Thank you for your request to indicate the standard errors (SE) in Table 2 of our manuscript. However, we regret to inform you that we are unable to provide the standard errors for the transcriptome sequencing statistics as initially expected.
Upon consultation with the sequencing facility, we have been advised that the standard errors are not available from the sequencing data provided. We understand that this information is valuable for a comprehensive analysis, and we apologize for any inconvenience this may cause.
We would like to assure you that the data presented in Table 2 is accurate and has been rigorously validated through the sequencing process. While the standard errors are not available, we are confident in the quality and reliability of the sequencing results.
Figure 5: Specify the conditions compared in the Venn diagram.
Reply: Thank you for your request for clarification regarding Figure 5. We have now provided a detailed explanation within the figure caption itself, specifying the conditions compared in the Venn diagram. This additional information should offer the necessary clarity for readers to understand the comparisons being made.
Discussion
The first paragraph is too general; it could be moved to the introduction. It is recommended to start a Discussion with a brief general summary of your main results linked to your objectives.
Reply: Thank you for your question. To provide a more comprehensive and easily understandable explanation, we have removed most of the ineffective descriptions from this part of the content.
We appreciate for Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.
Sincerely,
Li-Guo Yang
2/6/2024
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress (Revision 1)
Liu et al,
This manuscript investigated DEGS after air exposure stress in the black rockfish. The experimental design was one control and two treatments (in triplicate).
Comments:
The manuscript has improved its organization and editing. However, explaining the results is still limited.
The relationship between peptidase activity and stress (by air exposure) is still not clear. I have read literature, and peptidase activity is related to multiple processes. Particularly, peptidases are related with oxidative stress response, activating oxidative enzymes.
For example, in Procambarus clarkia, oxidative damage occurs (line 86)
However, neither GO nor KEGG showed oxidative stress.
Please consult:
Malovan et al.2023. The emerging role of dipeptidyl peptidase 3 in pathophysiology. FEBS.
GO showed peptidase activity and KEGG metabolic energy management, but also endocrine and immune systems (Fig.4).
There must be many other genes (differentially expressed) that will have to be search “manually” on data in order to give a more detailed information. These genes of course, will not be on the top ten of enrichment (GO and KEGG).
In summary. I consider that more detailed information of specific genes related to the endocrine and immune systems, and oxidative stress could be “search and extracted”. I guess that it will give more results and a better explanation of them.
In the other hand, the manuscript can be accepted for publication as its now.
Author Response
Dear Reviewers:
We are grateful for your correspondence and the insightful remarks provided by the reviewers on our manuscript, "Transcriptome Analysis of Juvenile Black Rockfish Sebastes schlegelii Under Air Exposure Stress" (ID: fishes-3033711). The feedback is of considerable value and has been instrumental in refining and enhancing our work, as well as providing crucial guidance for our research endeavors.
We have thoroughly examined the reviewers' comments and have diligently undertaken the necessary revisions, which we believe will be satisfactory. The revised sections have been highlighted in red within the manuscript. Below is a summary of the principal amendments made to the paper, along with our responses to the reviewers' observations:
Reviewer1:
Transcriptome analysis of juvenile black rockfish Sebastes schlegelii under air exposure stress (Revision 1)
Liu et al,
This manuscript investigated DEGS after air exposure stress in the black rockfish. The experimental design was one control and two treatments (in triplicate).
Comments:
The manuscript has improved its organization and editing. However, explaining the results is still limited.
The relationship between peptidase activity and stress (by air exposure) is still not clear. I have read literature, and peptidase activity is related to multiple processes. Particularly, peptidases are related with oxidative stress response, activating oxidative enzymes.
For example, in Procambarus clarkia, oxidative damage occurs (line 86)
However, neither GO nor KEGG showed oxidative stress.
Please consult:
Malovan et al.2023. The emerging role of dipeptidyl peptidase 3 in pathophysiology. FEBS.
GO showed peptidase activity and KEGG metabolic energy management, but also endocrine and immune systems (Fig.4).
There must be many other genes (differentially expressed) that will have to be search “manually” on data in order to give a more detailed information. These genes of course, will not be on the top ten of enrichment (GO and KEGG).
Reply: Thank you for your continued guidance and constructive comments on our manuscript. We have taken your feedback to heart and have made the following revisions to address the concerns raised:
We acknowledge that our previous explanation of the results was limited. To enhance clarity, we have expanded our discussion to provide a more detailed interpretation of the results, focusing on the implications and potential mechanisms.
We understand that the connection between peptidase activity, specifically DPP3, and stress induced by air exposure was not sufficiently clear. In response, we have included additional literature to support the role of peptidases in the oxidative stress response and have incorporated a more thorough analysis of how DPP3 activity might be modulated under stress conditions.
We have now included a discussion on the involvement of peptidases in oxidative stress pathways, particularly focusing on the Keap1-Nrf2 pathway, which is a key mediator of the cellular response to oxidative stress. This addition aims to clarify the role of DPP3 in this context.
If our previous submission did not adequately address this, we have now included additional data (Table S1-3) and discussion to highlight the role of DPP3 in oxidative stress response pathways as indicated by GO and KEGG annotations.
We believe these revisions have significantly improved the manuscript and provided a clearer understanding of the role of DPP3 in the context of oxidative stress and its relevance to peptidase activity under stress conditions.
We appreciate the opportunity to refine our work and hope that our revisions meet with your approval.
In summary. I consider that more detailed information of specific genes related to the endocrine and immune systems, and oxidative stress could be “search and extracted”. I guess that it will give more results and a better explanation of them.
Reply: Thank you very much for your suggestions. We have uploaded all the differentially expressed genes for the comparisons between BS vs BC (40-second air exposure vs control), BD vs BC (2 minutes and 30 seconds air exposure vs control), and BD vs BS (2 minutes and 30 seconds air exposure vs 40-second air exposure) as supplementary tables. We believe this additional information will provide readers with more comprehensive results and a clearer interpretation.
In the other hand, the manuscript can be accepted for publication as its now.
Reply: Thank you for your time and consideration.
We appreciate for Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.
Sincerely,
Li-Guo Yang
6/6/2024
Author Response File: Author Response.pdf
