Comparative Analysis of Blood Transcriptome in the Yangtze Finless Porpoise (Neophocaena asiaeorientalis)
Round 1
Reviewer 1 Report
One major concern now that the method sections contains more information is that the read with an adapter sequence were discarded completely rather than removing the adapter sequences? If the authors could shed some light on why they did this, that would be great. I agree that in some cases adapter trimming is not needed but I don’t quite understand why you would discard an entire read. Based on the 150bp however I would recommend removing these prior to alignment as adaptor contamination will lead to errors and increase the number of unaligned reads (as they are synthetic).
I only have very minor edits to recommend for the rest of the manuscript. See below.
Ln 34: Still not clear what the 3 recommendations are here
Ln 42: Recent surveys indicated that there is an existing unbalance in sex
Ln 78: transferred to the library at -80 °C for – do you mean laboratory?
Ln 95: short reads were filtered based on the phred score
Ln 102: needs a reference for DESEQ2 and R. Also version of R is needed.
Ln 115: note that this references is in a webpage instead of a referenced manuscript.
Ln 124: that the thickening of blubber is an
Ln 125: Literature showed that TAG synthesis or
Ln 124-132: this section needs references
Ln 136: in the response you said these were from the same porpoise but here it reads that you did total RNA from additional males and female. I would suggest clarifying it here.
Ln 214-..: this looks really great! Thank you for adding them. It is great to see that age here is more of a driving factor. Just out of curiosity, any idea why male 1 is such an outlier?
Ln 246-277: I think here it is great you are placing this in the cetacean blubber thickening context but you need to elaborate a bit more on why males and females would have different expressions.
Ln 280-307: This has not addressed my previous comment related to adaptive ability and you rather picking up differences in genes that are known to change with age. This needs to be addressed.
Response RE GO: if you for example do the overrepresentation analysis as you did here, in R you should be able to pull out an csv file or an excel sheet with each GO term, pvalue and the genes that are of your dataset overrepresented in that specific term. (Sorry I can’t see table S1 on my reviewers interface so my apologies if this is what you are showing in that table – I get an error message). You can then do a simple venn diagram that shows you the overlap. This can be done in R but I tend to use this website as it is click only. http://bioinformatics.psb.ugent.be/webtools/Venn/ . For the scope of the paper, I don’t think it is needed to include this but might be worth for future studies.
Author Response
Dear reviewer,
Thank you very much for your considerate and rigorous comments to the Ms.
We carefully read the comments and have now completed a revision of the manuscript that addresses your concerns. Thorough and careful modifications have been made throughout the manuscript following the suggestions point to point.
In the revision manuscript, we proofread our manuscript (highlight in yellow for some revised sentences) that described the suggested revisions. Some spelling mistakes and grammatical errors were also corrected for this time. We hope and believe that the revised paper is now acceptable for publication in Fishes. We are looking forward to good news from you!
If there are some inadequacies we will make our efforts to revise our manuscript.
Thank you for your kindly and considerate comments again!
Best Regards,
Yours sincerely,
Di-an Fang
Response to Reviewer 1#’s comments
One major concern now that the method sections contain more information is that the read with an adapter sequence were discarded completely rather than removing the adapter sequences? If the authors could shed some light on why they did this, that would be great. I agree that in some cases adapter trimming is not needed but I don’t quite understand why you would discard an entire read. Based on the 150bp however I would recommend removing these prior to alignment as adaptor contamination will lead to errors and increase the number of unaligned reads (as they are synthetic).
Answer: Now sequences we get from sequencing companies are often 5’-adapter trimmed. To filter “sequences with adapters”, we mean 3’-adapter. Only when the insert sequence (target) is really short (or fragmented) can the adapter be found in a read. So, after trimming the 3’ adapter, the remaining sequences are often too fragmented to provide enough information for contig assembling or analysis. In addition, sequences with 3’-adapter usually account for only a small fraction of all generated reads. If we discard them, we still have enough clean data as shown in Table1.
I only have very minor edits to recommend for the rest of the manuscript. See below.
Ln 34: Still not clear what the 3 recommendations are here
Ln 42: Recent surveys indicated that there is an existing unbalance in sex
Ln 78: transferred to the library at -80 °C for – do you mean laboratory?
Ln 95: short reads were filtered based on the phred score
Ln 102: needs a reference for DESEQ2 and R. Also version of R is needed.
Ln 115: note that this reference is in a webpage instead of a referenced manuscript.
Ln 124: that the thickening of blubber is an
Ln 125: Literature showed that TAG synthesis or
Ln 124-132: this section needs references
Ln 136: in the response you said these were from the same porpoise but here it reads that you did total RNA from additional males and female. I would suggest clarifying it here.
Ln 214-..: this looks really great! Thank you for adding them. It is great to see that age here is more of a driving factor. Just out of curiosity, any idea why male 1 is such an outlier?
Ln 246-277: I think here it is great you are placing this in the cetacean blubber thickening context but you need to elaborate a bit more on why males and females would have different expressions.
Ln 280-307: This has not addressed my previous comment related to adaptive ability and you rather picking up differences in genes that are known to change with age. This needs to be addressed.
Answer: We have rewritten some sentences according to your suggestive comments. Also we have revised the manuscript following your kindly recommendations (seen in yellow highlights).
Response RE GO: if you for example do the overrepresentation analysis as you did here, in R you should be able to pull out an csv file or an excel sheet with each GO term, pvalue and the genes that are of your dataset overrepresented in that specific term. (Sorry I can’t see table S1 on my reviewers interface so my apologies if this is what you are showing in that table – I get an error message). You can then do a simple venn diagram that shows you the overlap. This can be done in R but I tend to use this website as it is click only. http://bioinformatics.psb.ugent.be/webtools/Venn/ For the scope of the paper, I don’t think it is needed to include this but might be worth for future studies.
Answer: Thank you for the detailed instructions. We tried to represent the overlaps of all 64 terms, but it seems to be an impossible mission. A venn diagram containing more than six groups is nearly unreadable. Here, we drew a venn showing 5 GO terms, including positive regulation, negative regulation, and regulation of biological processes, as well as multicellular organismal and multi-organism processes for your reference. This venn diagram can be provided as Supplementary Figure.
Reviewer 2 Report
Authors analyze new transcriptome data from Yangtze finless porpoises. General and specific comments on each section are below.
Abstract
General comment. Needs to be written in a better English. Briefly, some background, methods used, results, conclusion. Abstract is meant to draw attention on the work. I don’t understand what the ms is about from this abstract. It is not clear what samples have been used in what analysis, nor the question and hypothesis behind the work.
Specific comments:
Line 11: ‘RNA-Seq technology has been widely applied to genomics, transcriptomics and functional gene study’.
I think that should be: genomics, transcriptomics and functional genomics.
Line 17-18: ‘Combined with the microsatellite DNA technology for maleage identification, DEGs for two groups of males and female were analyzed’.
What is combined with the microsatellite DNA technology? Samples are two groups of males and two groups of females? OR two groups, one male and ine female.
Line 15-16: ‘Diverse and extensive expressed gene catalogs have been sampled for the Yangtze finless porpoise. DESeq2 was used to analyze differential expression genes (DEGs) obtained from the assembled transcriptome.’
I am not sure what this sentence means. Are different samples being compared?
Keywords
Line 22: ‘Comparative transcriptome’? What does it mean?
Introduction
General comment: The introduction can use some rewriting to improve grammar. I have included some suggestions below.
Specific comments:
Line 25-26: ‘is also listed as an endangered 25 species in the IUCN…’
Please note that Neophocaena asiaeorientalis is not endangered on the IUCN list, it is CRITICALLY ENDANGERED (https://www.iucnredlist.org/species/43205774/45893487).
Line 42-43: ‘Recent surveys indicated that is an existing unbalance in sex proportion between the 42 male and female Yangtze finless porpoise.’
Something is missing in the sentence: ‘Recent surveys indicated that THERE is …
Line 59: Add a specific reference for marine mammals/dolphins transcriptomic technique:
Annalaura Mancia, Chapter 11 - New Technologies for Monitoring Marine Mammal Health,
Editor(s): Maria Cristina Fossi, Cristina Panti, Marine Mammal Ecotoxicology, Academic Press,
2018, Pages 291-320, ISBN 9780128121443,https://doi.org/10.1016/B978-0-12-812144-3.00011-5.
Line 60-61: ‘As such in this study, we performed de novo comparative transcriptomic analysis of the blood in the Yangtze finless porpoise, N. asiaeorientalis.’
Comparing what exactly? The adjective ‘comparative’ expresses a difference; comparative analysis is a process of comparing items to one another and distinguishing their similarities and differences. Rewrite the sentence.
Line 62-63: ‘Our results provide comparative and comprehensive data on male-child relationship and we identified differential gene expression patterns and signaling pathways’.
Child? Maybe you meant calf? Or males and calves? Also, the differential gene expression patterns and signaling pathways are relative to…? Please explain.
Line 64-65: ‘This could increase our knowledge to which regulatory pathways drive the biological adaptation processes for the Yangtze finless porpoise.’
This sentence needs rewriting and some explaining: how did we go form a male and a calf to adaptation processes?
Material and Methods
Line 72-75: ‘Using the microsatellite DNA technology for parentage identification, three male and three female individuals (Male 1, 5 years old; Male 2, 2 years old; Male 3, 2 years old; Female1, 4 years old; Female 2, 2 years old; Female 3, 5 years old) were verified, the female was not pregnant’.
Please rewrite, for example: Three male and three female individuals (N = 6) were identified using the microsatellite DNA technology for parentage identification. Specifically: Male 1, 5 years old; Male 2, 2 years old; Male 3, 2 years old; Female1, 4 years old; Female 2, 2 years old; Female 3, 5 years old. The females were not pregnant.’
How do you know females were not pregnant? Did you do an ultrasound? Hormone testing? How did you the age of dolphins? Aldo, please, add a reference for the microsatellite DNA technology for parentage (?) identification techniques used.
Line 78: ‘After sampling, animals were released to the Anqing west river section for further conservation.’
Remove ‘for future conservation’: After sampling, animals were released in the west river section of the Anqing river.
Line 124-132: Why is this part in the methods? First, it needs references. Second, it needs to be moved in the results or discussion (for example in Line 241 where male and female DEGs are discussed or Line 246 where you finally explain what TAG means…).
Line 133: Which genes were tested by real time PCR? Where are the primers? Isn’t the RNA used for RT the same RNA used for RNA-sequencing?
Results
Line 191-192: ‘Signal transduction, cancers: overview and immune system were the top three enriched pathways with 9975, 7341 and 6700 genes annotated (Figure 2, Table S2).
Where? I do not see those pathways in Figure 2, maybe you meant Figure 3?
Line 203: ‘When the two assembled transcriptome combined, DGEs enrichment pathway analysis?
Now there are two assembled transcriptomes…one from three males and on from the three females?’
Please, do explain.
This part is very confusing, and Figures are wrongly cited.
Line 220: ‘The second group includes the mother of the offspring’
The second group includes Female1 and Female3, which one is the mother? Which animals are the offspring? And how do you know?
Line 221: ‘..while the third group is Male 1, which is the same father of the younger’.
Who is the younger and once again, how do you know he is the father?
Line 236: ‘It has been proven that the Yangtze finless porpoise adaptive process involved series of temporal, intricate and developmental stages, in which, like other biological processes, blood is also accurately regulated in a temporal and spatial manner, so that specific genes would be turned on and off accordingly.’
Who has proven what? I don’t understand…Rewrite the sentence.
Line 239: ‘Interestingly, KEGG Genes between male and female blood transcriptome were analyzed and listed in Table S6.’
Remove ‘interestingly’ or add the interesting part of the result.
Line 241-243: ‘Lipid metabolism was focused and identified in the assembled transcriptome, in which triacylglycerol (TAG) synthesis related genes, are expressed in the indentified transcriptome differentially. It would indicate that their potential regulation function in their blubber thicken process.’
I imagine that amongst male vs female DEGs they have found genes involved in lipid metabolism?? Please explain better.
Line 248. Explain what DGAT1 and DGAT2 stand for.
Line 250-251: ‘Remarkably, some regulatory genes related to TAG synthesis were also determined to be under positive selection (Fig. 5)’
There is no evidence of genes going under positive selection in Figure 5 or anywhere else in the analysis and result of this MS. Positive selection is the process by which new advantageous genetic variants sweep a population. I don’t see any analysis related to the nature and frequency of allele diversity, nor a codon analysis based on comparing patterns of synonymous and nonsynonymous changes in protein coding sequences.
Line 256-258. Please explain your results …what findings?
Line 258-260. Add references.
Line 275-276: ‘…which suggested the convergent evolution of TAG metabolism-related genes for the Yangtze finless porpoise during their adaptation to the aquatic environment.’
Absolutely not, please mind the terms you are using. The differential expression of few genes within a single species does not suggest convergent evolution. Convergent evolution is the independent evolution of similar features in species of different periods or epochs in time. Convergent evolution creates analogous structures that have similar form or function but were not present in the last common ancestor of those groups.
Line 277-278. ‘These data were consistent with the positive selection view of the other cetaceans.’
Once again, what are talking about, what positive selection? What other cetacean? Maybe you want to add references and discuss them?
Line 287: ‘These results confirmed that the expression differences of most studied transcripts among groupers revealed by both methods were generally consistent.’
What??? Rewrite please.
Line 306-307. ‘In the sampled Yangtze finless porpoises, the older individual have better adaptive ability to the environment than their younger’s.’
Why do you say that? Based on what?
Line 313: Table 4 and primer sequences and real time PCR methodological details go in the methods section.
Conclusion
Line 327-328: ‘ The present study is the first comprehensive and systematic analysis of the comparative transcriptome of the Yangtze finless porpoise…’
It is not really a comprehensive analysis the way it is.
Line 331-333: ‘Some DEGs are highly expressed could potentially be functioned in its biological behavior, and functional studies on these DEGs will be clarified as regarding their molecular function in the Yangtze finless porpoise living.’
Please write in proper English.
Author Response
Dear reviewer,
Thank you very much for your considerate and rigorous comments to the Ms.
We carefully read the comments and have now completed a revision of the manuscript that addresses your concerns. Thorough and careful modifications have been made throughout the manuscript following the suggestions point to point.
In the revision manuscript, we proofread our manuscript (highlight in yellow for some revised sentences) that described the suggested revisions. Some spelling mistakes and grammatical errors were also corrected for this time. We hope and believe that the revised paper is now acceptable for publication in Fishes. We are looking forward to good news from you!
If there are some inadequacies we will make our efforts to revise our manuscript.
Thank you for your kindly and considerate comments again!
Response to Reviewer 2#’s comments
Abstract
General comment. Needs to be written in a better English. Briefly, some background, methods used, results, conclusion. Abstract is meant to draw attention on the work. I don’t understand what the ms is about from this abstract. It is not clear what samples have been used in what analysis, nor the question and hypothesis behind the work.
Specific comments:
Line 11: ‘RNA-Seq technology has been widely applied to genomics, transcriptomics and functional gene study’.
I think that should be: genomics, transcriptomics and functional genomics.
Line 17-18: ‘Combined with the microsatellite DNA technology for maleage identification, DEGs for two groups of males and female were analyzed’.
What is combined with the microsatellite DNA technology? Samples are two groups of males and two groups of females? OR two groups, one male and one female.
Line 15-16: ‘Diverse and extensive expressed gene catalogs have been sampled for the Yangtze finless porpoise. DESeq2 was used to analyze differential expression genes (DEGs) obtained from the assembled transcriptome.’
I am not sure what this sentence means. Are different samples being compared?
Keywords
Line 22: ‘Comparative transcriptome’? What does it mean?
Answer: We have rewritten the abstract according to your suggestive comments. Also we revised the abstract following your specific comments (seen in yellow highlights).
Introduction
General comment: The introduction can use some rewriting to improve grammar. I have included some suggestions below.
Specific comments:
Line 25-26: ‘is also listed as an endangered 25 species in the IUCN…’
Please note that Neophocaena asiaeorientalis is not endangered on the IUCN list, it is CRITICALLY ENDANGERED (https://www.iucnredlist.org/species/43205774/45893487).
Answer: Yes, we have revised this sentence.
Line 42-43: ‘Recent surveys indicated that is an existing unbalance in sex proportion between the 42 male and female Yangtze finless porpoise.’
Something is missing in the sentence: ‘Recent surveys indicated that THERE is …
Answer: Yes, we have added it.
Line 59: Add a specific reference for marine mammals/dolphins transcriptomic technique:
Annalaura Mancia, Chapter 11 - New Technologies for Monitoring Marine Mammal Health,
Editor(s): Maria Cristina Fossi, Cristina Panti, Marine Mammal Ecotoxicology, Academic Press,2018, Pages 291-320, ISBN 9780128121443,https://doi.org/10.1016/B978-0-12-812144-3.00011-5.
Answer: Yes, we have added it.
Line 60-61: ‘As such in this study, we performed de novo comparative transcriptomic analysis of the blood in the Yangtze finless porpoise, N. asiaeorientalis.’
Comparing what exactly? The adjective ‘comparative’ expresses a difference; comparative analysis is a process of comparing items to one another and distinguishing their similarities and differences. Rewrite the sentence.
Line 62-63: ‘Our results provide comparative and comprehensive data on male-child relationship and we identified differential gene expression patterns and signaling pathways’.
Child? Maybe you meant calf? Or males and calves? Also, the differential gene expression patterns and signaling pathways are relative to…? Please explain.
Line 64-65: ‘This could increase our knowledge to which regulatory pathways drive the biological adaptation processes for the Yangtze finless porpoise.’
This sentence needs rewriting and some explaining: how did we go form a male and a calf to adaptation processes?
Answer: Yes, we have rewritten this sentence and added some explains according to your suggestive comments (seen in yellow highlights).
Material and Methods
Line 72-75: ‘Using the microsatellite DNA technology for parentage identification, three male and three female individuals (Male 1, 5 years old; Male 2, 2 years old; Male 3, 2 years old; Female1, 4 years old; Female 2, 2 years old; Female 3, 5 years old) were verified, the female was not pregnant’.
Please rewrite, for example: Three male and three female individuals (N = 6) were identified using the microsatellite DNA technology for parentage identification. Specifically: Male 1, 5 years old; Male 2, 2 years old; Male 3, 2 years old; Female1, 4 years old; Female 2, 2 years old; Female 3, 5 years old. The females were not pregnant.’
Answer: Yes, we have rewritten these sentences.
How do you know females were not pregnant? Did you do an ultrasound? Hormone testing? How did you the age of dolphins? Aldo, please, add a reference for the microsatellite DNA technology for parentage (?) identification techniques used.
Answer: We have done the routine ultrasonic test. Furthermore, the microsatellite DNA technology for parentage is the unpublished data by our research team.
Line 78: ‘After sampling, animals were released to the Anqing west river section for further conservation.’
Remove ‘for future conservation’: After sampling, animals were released in the west river section of the Anqing river.
Answer: We have revised it.
Line 124-132: Why is this part in the methods? First, it needs references. Second, it needs to be moved in the results or discussion (for example in Line 241 where male and female DEGs are discussed or Line 246 where you finally explain what TAG means…).
Answer: We have added reference and moved it to the result part.
Line 133: Which genes were tested by real time PCR? Where are the primers? Isn’t the RNA used for RT the same RNA used for RNA-sequencing?
Answer: This information is in the result part. We have revised it.
Results
Line 191-192: ‘Signal transduction, cancers: overview and immune system were the top three enriched pathways with 9975, 7341 and 6700 genes annotated (Figure 2, Table S2).
Where? I do not see those pathways in Figure 2, maybe you meant Figure 3?
Answer: Yes, we have rewritten these sentences.
Line 203: ‘When the two assembled transcriptome combined, DGEs enrichment pathway analysis?
Now there are two assembled transcriptomes…one from three males and one from the three females?’
Please, do explain.
This part is very confusing, and Figures are wrongly cited.
Answer: Yes, we have rewritten these sentences.
Line 220: ‘The second group includes the mother of the offspring’
The second group includes Female1 and Female3, which one is the mother? Which animals are the offspring? And how do you know?
Answer: We have rewritten these sentences.
Line 221: ‘..while the third group is Male 1, which is the same father of the younger’.
Who is the younger and once again, how do you know he is the father?
Answer: Yes, we have rewritten these sentences.
Line 236: ‘It has been proven that the Yangtze finless porpoise adaptive process involved series of temporal, intricate and developmental stages, in which, like other biological processes, blood is also accurately regulated in a temporal and spatial manner, so that specific genes would be turned on and off accordingly.’
Who has proven what? I don’t understand…Rewrite the sentence.
Answer: Yes, we have rewritten these sentences.
Line 239: ‘Interestingly, KEGG Genes between male and female blood transcriptome were analyzed and listed in Table S6.’
Remove ‘interestingly’ or add the interesting part of the result.
Answer: Yes, we have removed it.
Line 241-243: ‘Lipid metabolism was focused and identified in the assembled transcriptome, in which triacylglycerol (TAG) synthesis related genes, are expressed in the indentified transcriptome differentially. It would indicate that their potential regulation function in their blubber thicken process.’
I imagine that amongst male vs female DEGs they have found genes involved in lipid metabolism?? Please explain better.
Line 248. Explain what DGAT1 and DGAT2 stand for.
Answer: We have explained them.
Line 250-251: ‘Remarkably, some regulatory genes related to TAG synthesis were also determined to be under positive selection (Fig. 5)’
There is no evidence of genes going under positive selection in Figure 5 or anywhere else in the analysis and result of this MS. Positive selection is the process by which new advantageous genetic variants sweep a population. I don’t see any analysis related to the nature and frequency of allele diversity, nor a codon analysis based on comparing patterns of synonymous and nonsynonymous changes in protein coding sequences.
Line 256-258. Please explain your results …what findings?
Answer: Yes, we have rewritten these sentences.
Line 258-260. Add references.
Answer: Yes, we have added it
Line 275-276: ‘…which suggested the convergent evolution of TAG metabolism-related genes for the Yangtze finless porpoise during their adaptation to the aquatic environment.’
Absolutely not, please mind the terms you are using. The differential expression of few genes within a single species does not suggest convergent evolution. Convergent evolution is the independent evolution of similar features in species of different periods or epochs in time. Convergent evolution creates analogous structures that have similar form or function but were not present in the last common ancestor of those groups.
Answer: Yes, we have rewritten these sentences.
Line 277-278. ‘These data were consistent with the positive selection view of the other cetaceans.’
Once again, what are talking about, what positive selection? What other cetacean? Maybe you want to add references and discuss them?
Line 287: ‘These results confirmed that the expression differences of most studied transcripts among groupers revealed by both methods were generally consistent.’
What??? Rewrite please.
Line 306-307. ‘In the sampled Yangtze finless porpoises, the older individual have better adaptive ability to the environment than their younger’s.’
Why do you say that? Based on what?
Answer: Yes, we have rewritten these sentences.
Line 313: Table 4 and primer sequences and real time PCR methodological details go in the methods section.
Answer: Yes, we have removed it to the methods section.
Conclusion
Line 327-328: ‘ The present study is the first comprehensive and systematic analysis of the comparative transcriptome of the Yangtze finless porpoise…’
It is not really a comprehensive analysis the way it is.
Line 331-333: ‘Some DEGs are highly expressed could potentially be functioned in its biological behavior, and functional studies on these DEGs will be clarified as regarding their molecular function in the Yangtze finless porpoise living.’
Please write in proper English.
Answer: Thank you very much, we have rewritten these sentences. We think it will be better understood for this time. If there are some inadequacies we will make our efforts to revise our manuscript.
Thank you for your kindly and considerate comments again!
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Introduction - The introduction can use some rewriting to improve grammar. I have included some suggestions below.
Ln 35-37: …ecosystem functions that are related to the Yangtze finless porpoise habitat…
Ln 37: what are the 3 recommendations?
Ln 40-42: captive breeding programs served as a valuable and effective protection method and provide vital help to the conservation work in this species.
Ln 42-43: For captive animals to breed successfully in ex-situ conservation area, their ecological adaptive mechanisms must be fully understood.
Ln 44-45: Not sure what you are trying to say here. Is this what you meant? Recent surveys indicated that is an existing unbalance in sex proportion between the male and female Yangtze finless porpoise.
Ln 45-48: From 1993 to 2004, the survey result showed that the mature female-male sex ratio was 0.86:1, while the puberty 46 female-male sex ratio was 0.33:1. This indicated that there was a decrease in female birth rate, which may be one of vital reasons for the Yangtze finless porpoise remarkably decline.
Ln 49: Furthermore, the Yangtze finless porpoises populations also showed a low genetic diversity…
Ln 50-53: Blood contains and can produce informative biological messages that is reflective of the environment that the animal lives in.
Ln 53-58: Animal adaptation processes are highly well-organized and complex [12]. Functional genes are expressed in orderly patterns during different time of adaptation processes. These functional genes expression patterns are determined by both extrinsic cues and intrinsic genetic programmed blood cells [13]. Stringent temporal genes expression in blood are thus radically important to ensure the highly ordered biological adaptation activities both in the transcriptional and translational levels [11].
Ln 61-63 Despite recent advances in genomic data resources, the Yangtze finless porpoise lacks any existing genomic data. As such in this study, we performed de novo comparative transcriptomic analysis of the blood of the Yangtze finless porpoise.
Ln 63-67: Our results provide comparative and comprehensive data on parent-child relationship and we identified differential gene expression patterns and signalling pathways. This could increase our knowledge to which regulatory pathways drive the biological adaptation processes for the Yangtze finless porpoise.
Ln 67: I would add a few sentences how identifying those pathways can help the conservation efforts.
Materials and Methods - The method section could use more details, see comments below. It is currently unclear which groups were compared with each other and how certain analysis were done.
Ln 72: how did you identify if they were healthy. Did you base this on certain parameters or assessment? How many were sampled and what age stage? Reproductive stage?
Ln 88. What are the details of the sequencing, how base pairs? Pair ended? Double ended? Sample was pooled but how many runs where done on the HiSeq? Any technical replicates.
Ln 90: how short are the short reads? Quality based on prhed score? Were adapters trimmed.
Ln 108: the title is very confusing as this is talking about off spring vs parent but the title says male vs female. Also higher up in the method you say that these samples were pooled together. Were they pooled per sex then? how did you determine the mother vs offspring identity? How old was the offspring? These things need to be clarified better in the method sections as currently written it is confusing to follow the results. Did you compare 3 male vs 3 female, or pooled data vs offispring?
Ln 119. I don’t like the word adaptation used in this context. If you were doing comparative genomics between different species, then you can use the word adaption to describe evolutionary changes and thus adaptations. Or if these animals were exposed to a certain stressors, you can then discuss their adaptation to that encounter. As the data is collected the way it is, it is a snap shot in time and therefore it is hard to disentangle what normal biology is and what is considered local adaption. For example, same species between 2 different locations comparison would be more suited to use the word adaptation. As you used transcriptomics and only comparative between males vs females, or offspring (?age). what you might be picking up is a difference in biology and difference in age and thus not evolution and adaption.
Ln 121: how did you select the genes, and which housekeeping genes did you use to optimise the PCR given that this a novel transcriptome.
Ln 123: it would be good to have more information here how this was verified and how many animals were sampled.
Ln 133 – where these from the same samples as the RNA-seq data? If no, how can you verify the genes if they are from different samples?
Ln 138 – the details of the data deposition is currently in the results section ln 150-153 and need to be moved here.
Results and Discussion – I have some issues with how the data is interpreted and from the DESeq analysis, some basic analysis is missing to make an informed conclusion. One of the issues is that the results are hard to follow as a lot of details are missing in the method section. It is currently unclear exactly which groups were compared to what. Clarification in the method section would already improve the result section. Lastly the section focussing on TAG, I have some major concerns about.
Ln 146 - first time you are mentioning 150 bp. This needs to be included in the method section.
Ln 157-159: unclear here what you mean with pooled. Did you pool the samples and then sequenced them? Or you sequenced the data and then pooled this? Very unclear what type of analysis was done.
Ln 161: what type of enrichment analysis did you use? Did you use any parameters or cut-off (eg. size of the GO terms), did you base significance on p-value, enrichment score, adjusted enrichment score? In general, it would be good to see how many enrichment terms show overlap. For example, negative regulation of biological process, positive regulation of biological process and regulation of biological processes may show a significant overlap in the genes they contain.
Ln 188-189: here you talk about the 2 transcriptomes combined. Which 2? Did you pool all females and males separately and then did sequencing of either or sex? Also with DESeq2 which did you compare? More information is needed here. Also what were the parameters for DGE? Did you adjust for library size and normalise the data, did you filter out any low expressed genes to avoid over estimation? These things need to be added to method section. Clarity on the comparison is needed.
Ln 247: oxidative stress needs to be placed in the concept of diving as this is transcriptomics in the blood. The red blood cells will have gone through adaptations to deal with the diving cycles. Many of the comparative genomics papers show key adaptions to deal with reactive oxygen species. IN general, the results need to be placed in the tissue you are looking at. This is gene expression in blood and the discuss need to be written to reflect that.
Ln 252-267: As you are dealing with 3 vs 3 in this section, more explorative plots are needed to assess the variation between the groups and thus how valid the results are. I would strongly recommend that the authors include a PCA plot, a heatmap and a clustering diagram based on the transcriptome response. These plots will be very informative to readers. These are included in the DESeq 2 manual and should not take too much extra time to construct but will greatly benefit the paper. It also not clear from the method section that you compared male vs female as it sounds like you pooled the blood samples together to sequence them.
Ln 290 -313: I really have issue with this section and the use of adaptive behaviour. The data is not set up to reflect this interpretation. There are couple of things why you could be picking up differences in the TAG metabolism. You compared adults vs offspring here? This again is not clear from the start and needs to be better explained. What was the age of offspring and what is the age of the adults. The conclusion that one group is better adapted to their environment is in my opinion an over generalisation that is not supported by the data. The data showed an upregulation of a select few genes in the older population. Both AGPAT1 and PPARA are two key genes that are involved in the ageing process. AGPAT1 is linked to fatty liver syndrome and markers of that disease is shown to increase with age in dolphin populations. In addition, many stranding records of cetaceans also show that older individual often have a fatty liver. PPARA is also a key gene involved in ageing. For example mice knockouts of PPARA are linked to longevity. Altered expression of PPARA is linked to increased with oxidative stress induced ageing and also increased in inflammatory responses. Thus, the fact that you are picking up differences between offspring and parents may not be due to what you call a better adaptive ability to the environment, but might in fact be just a hallmark of ageing and being older. Currently with the data it is very hard to disentangle what you are seeing is due to age or other factors contributing to this.
In general, I think that this manuscript has tremendous potential to improve our knowledge of the study species and aid in any further conservation biology studies.
Reviewer 2 Report
In this study, the Yangtze finless porpoise transciptome has been analyzed through RNA-sequencing of blood samples from 6 individuals (3 males and 3 females). Authors deeply analyzed gene functional categories and use qPCR to validate the data obtained.
The study has potential. Data can be valuable to better understand the biology of the Yangtze finless porpoise, which could be beneficial to the species conservation.
But the way the authors present the data and the English grammar makes the comprehension of the work very hard. It is not clear what is the hypothesis behind the work, what is the meaning of parental and offspring in a male versus females analysis of DEGs, what is the goal of the work, specific objectives? I suggest re-writing in a more simple way.
Reviewer 3 Report
Fang et al. analyze new transcriptome data from Yangtze finless porpoises. Some comments are below which might help improve the manuscript if addressed properly.
1) English grammar, style, word choice, and typos should be touched up throughout the manuscript. e.g., just in the abstract:
- line 13, change “got” to “generated”
- line 14 change “Totally” to “In total”
- line 16, change “A diverse” to “Diverse”
- line 17, change “analysis” to “analyze”
- line 18, change “The result indicated that DEGs were functional annotated, which have close relationship with…” to “DEGs that were functionally annotated have close relationships with…”
- line 19, change “This transcriptome data” to “These transcriptome data”
2) line 200, I did not really understand the basis for the statement, “Metabolic pathways by KEGG analysis indicated that the most important pathways 200 that may be relevant to the Yangtze finless porpoise adaptive biology process including 201 Mitogen-Activated Protein Kinases (MAPK) signaling pathway, Calcium signaling 202 pathway, Neuroactive ligand-receptor interaction, Wnt signaling pathway, RNA 203 transport, GnRH signaling pathway, TGF-beta signaling pathway, PPAR signaling 204 pathway, and so on.” Why is this so? What is an “adaptive biology process”? The authors should define this term, in particular why the adjective “adaptive” is used. This has a very particular meaning in evolutionary biology. Do the authors instead mean that lots of the gene expression in the blood is related to these pathways listed above, or is there more to it than that? The authors say that these issues will be dealt with in future papers, but then what is the purpose of this paper? The following text from the authors speculates on a bunch of things but there is not really much, if any, scientific hypothesis testing. The manuscript just seems to describe some stuff. Is that enough for this journal?
3) line 281, the authors should describe in more detail why the SSRs are being quantified for this study in their transcriptome datasets.
4) line 290, the authors discuss how some genes are possibly characterized as having evolved adaptively in comparative analyses across marine mammal species, but I did not get how this relates to within species adaptation at the molecular level in finless porpoise. The transference from among species patterns to within species adaptation does not compute really.
