Transcriptome Analysis Reveals Differences in Gene Expression in the Muscle of the Brown-Marbled Grouper (Epinephelus fuscoguttatus) with Different Growth Rates

Round 1

Reviewer 1 Report

The authors describe a characterization study of differentially transcribed genes associated with muscle growth in an economically important fish species. The rationale for the study was presented and the experimental design for the analyses of the production traits are reasonable. The authors selected fish from the fastest growing and fish from the slowest fish for their differential transcriptomic study. The experiments were well described with the minor exceptions presented below. The data led the authors to conclude that fish that ate more grew faster. This is not an unexpected observation. While it is important to continue to investigate the transcriptional and translational mechanisms of fish growth, the use of these data was not discussed by the authors. For instance, were they looking for biomarkers for broodstock selection or did the study help inform nutritional strategies? Because the mechanisms of muscle growth, especially in indeterminant growers is of great importance to the elucidation of the potential for resolving issues like sarcopenia, cachexia, space flight, muscular dystrophy, and spinal cord injury these types of studies are still important. Therefore, a couple of issues should be addressed by the authors if they choose to revise the manuscript.

1. The use of a single reference gene is always problematic since not all references genes are stable under different conditions, like differential nutritional planes. The authors should discuss the use of a single reference gene based on the study of Jacob, et al. 2012 (attached).

2. There are English grammar issues that need to be addressed. These are not serious, as the manuscript is well written except for these minor issues.

3. Were the melting curves and efficiency of the primers evaluated. Since it appears that all primer/template reactions were conducted under the same conditions it is important that all of the primers behaved the same under those same conditions. This is important for providing a basis for confidence in the interpretation of the data from the experiments.  For a discussion of this issue a publication by Bustin, et al. 2009 is provided. It is suggested that the authors response reflects the suggestions of MIQE presented in the Bustin publication.

4. line 151. The authors state that they used 10ul of DNA. The amount of sample which would be conveyed by volume (concentration) of the sample is more informative and allows replication of the experiments in independent laboratories.

Comments for author File: Comments.pdf

 

There are English grammar issues that need to be addressed. These are not serious, as the manuscript is well written except for these minor issues.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors present an RNA-seq analysis of muscle tissue from brown-marbled grouper.  The experimental design compares half-sibling fast and slow-growing groups.  The authors found relatively few genes that were differentially expressed between the groups.  They used various analyses to identify if there was an enrichment of different types of genes that were differentially expressed.  While I thought the methods utilized were fairly standard and good, I thought the experimental design and the discussion were not appropriately matched.  I think that some of the claims were not supported by the study.  I think the manuscript could still be publishable, but changes to these claims should be made.  Please see my notes below.

Issues needed to be addressed before I support publication:

1) Grammar issues should be addressed throughout the manuscript.
2) Over extrapolation throughout the manuscript should be removed.
3) An introduction that includes reviews on growth physiology and gene expression (with a focus on how growth is regulated in muscle tissue).
4) A discussion should be included to describe the strengths and weaknesses of the experimental design, and how this influences the interpretation of the data (see notes).



Specific notes:

Abstract: Grammar issues throughout.  The authors conclude that the up-regulation of ghr and tnni2 and down-regulation of stc2 led to a growth advantage.  This oversimplifies the biology of growth.  For example, what if you found genes that were differentially expressed in another tissue in these same groupers?  Would they also be the genes responsible for the growth advantage?  This is also contradicted later in the abstract as other pathways are suggested.  I suggest that the authors more carefully phrase what the findings are and how this impacts our understanding of the differences in growth between the two groups of grouper without over extrapolation.  Also, it would be good to better state what the goal of this project was.  Is it to find what mechanisms are responsible for muscle growth differences between genetically identical grouper or genetic differences that led to growth differences?

Introduction: Grammar issues throughout.  I suggest using "ray-finned fish" instead of "fish" as that term can ambiguous (there is an argument that humans could be called fish if coelacanths are called fish).  I think the authors may oversimplify growth and suggest that they include a more thorough description of the physiology.

Lines 54-61 does not seem to be focused on the topic under investigation.  
Lines 62-75 a few articles are cited, but they do not give the reader a good understanding of organs, tissues, or genes involved in growth.  There are reviews the authors could cite that would help with summarizing what is known about the physiology and gene expression involved in growth.

Methods: Please describe where muscle tissue was collected from (part of the body) and measures to prevent variables that might influence gene expression (such as only including muscle vs. muscle and nerve/blood vessel tissue).  If you collected muscle from one area of the body on one fish, but from another area on a different grouper that could cause differences in gene expression that have nothing to do with growth.  The same could happen if you included nerve tissues in one, but not the other.  Describing this information will also make it easier for any researcher to try and replicate this study.

Please also describe why a half-sibling design was used.  I'm not sure what the goal of this project was as I stated above.  The half-sibling design would introduce more genetic differences between samples.  Without knowing which family the grouper came from, it makes it more difficult to disentangle environmental (e.g., feed availability), genetic (e.g., family differences), and environmental x genetic influences (maternal influence).  Also, it would be good to mention the sex of the fish and how this might impact the study (are you looking at differences between males and females or the other factors above).  Please note for the reader if there has been an observed growth difference between male and female grouper.  Indeed, in the results we see genes related to the regulation of estrogen.

Please include parameters used in the analyses so that it is repeatable.

Lines 125-126: Does this mean they were aligned to proteins?  Please describe this in greater detail.
Lines 139-142: What software was used to align the reads?

Please describe the goal of looking for SNPs in the transcriptome.  Why would we expect these differences be related to growth differences?  How are environmental and gene x environment factors controlled for in this analysis?

Results:
Line 197: This is not the same grouper mentioned earlier.
Figure 2: Please describe this better.  What do the different elements mean?
Table 1: When it states the top 20 pathways, does that mean the ones with the lowest p-values?  Please clarify in the text.

Line 206-207: This seems like it should be in the methods section.  Please describe why a q-val < 0.25 was used.

Discussion:
Line 257-258: Ray-finned fish are also animals so it might make more sense to say mammals or something along those lines instead.
Lines 268-270:  This may not be true, what if the SNP is a silent mutation?  We do not know that this SNP influences growth or expression at all so it is over extrapolating to say that it is important for growth regulation.  There are probably hundreds of thousands or millions of SNPs between the groupers, why would SNPs in the transcript make them more likely to influence growth?
Lines 267-287:  Why were these specific specific genes discussed out of the others identified?  Please describe why in the text.

There were many grammar errors throughout the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript has significantly improved, but there are still some issues that would be good to address.

Introduction:

Line 49-51:  Growth depends on all tissues, not just muscle.  This oversimplifies a complex trait.  It would be better to rephrase.

Methods:

It might be good to mention that these groupers are monandric protogynous hermaphroditism to let the reader know that all these individuals should be the same sex (immature females), but that might change as they grow past 70 cm (https://onlinelibrary.wiley.com/doi/full/10.1111/j.1095-8649.2007.01545.x).

If a program used default settings, it would be good to note that for reproducibility.

If a q-value of 0.25 is used, that means that as many of 25% of the significant findings are false-positives.  That seems pretty high.  I think a more common threshold is 0.05 (which was used for the study's gene expression analysis).  If the authors would like to keep this value, please add some citations to show that there is a precedent for using this high of a value.

I'm worried that the analysis of SNPs does not identify anything meaningful.  Most studies that attempt to link genotype and phenotype use hundreds of individuals and a statistical framework to find associations.  In the current study, we are presented with a set of SNPs that are different between a few individuals.  Since there are so many SNPs between individuals, there will almost always be a certain number that differ between randomly selected groups of individuals.  That is why many individuals are required to make these associations robust.  I recommend removing this analysis from the current study.

Results:

Figure 2: For circle 2, I don't understand how there can be 193 DEGs if there were only 125 DEGs from the analysis (line 210).  Also, the figure is labelled as p-value instead of the q-value

Figure 4: A, B, and C are not labelled on the figure.

Figure 4 came before Figure 3, but it would be good to make it go in numerical order.

Discussion:

Line 296-297:  While tnni2 may be involved in regulating growth, there isn't enough evidence from the current study to say this.  Just because there is a correlation, it doesn't imply there is causation.  Another way this statement could be phrased that would be more accurate would be: "However, our results still indicate that the tnni2 gene is associated with growth differences in brown-marbled grouper."

There is still a noticeable amount of grammar issues throughout the manuscript.  This does not impact interpretation.

Author Response

Please see the attachment

Author Response File: Author Response.pdf