Molecular Characterization and Expression Analysis of TAK1, TAB1 and TAB2 of Golden Pompano (Trachinotus ovatus)

Round 1

Reviewer 1 Report

The authors reported the molecular cloning of TAK1, TAB1, and TAB2 of golden pompano (Trachinotus ovatus) Furthermore, they presented basic sequence analysis and tissue-specific expression data, phylogenetic analysis. The manuscript should be edited by a native speaker for English grammar.

Please take into account the specific comments below:

Keywords: Change gene clone with gene cloning

Line 25: Kinase is duplicated

Line 28: Body environmental stress is not a protein or receptor. Stress could regulate literally everything in the organism so I think its obvious. Better to be removed.

Lines 35-37: Crustaceans lack an adaptive immune system so I think that the example is not comparable with fish.

Line 65: Change to bacteria [32], viruses [33] and parasites [34, 35],

Line 67: To the prevention.

Line 89: Agarose electrophoresis is not an adequate method for RNA quality (integrity) assessment.

Lines 99 and line 125: Primers’ concentrations should be mentioned.

Line 129: How beta actin was chosen as the reference gene. It is better to use the geometric mean of two reference genes for normalization.

Lines 135-137: How did the authors handle these data with ANOVA. Did the data follow the ANOVA assumptions (normality and equality of variance)? This analysis can lead to statistical errors and results misinterpretation.

Line 135: data were

Line 137: were regarded

Line 152: These domains which were conserved

Lines 196-197: The methodology of phylogenetic reconstruction should be dully written in the methodology part (line 119). In the legend, the numbers on each node should be mentioned. Possibly by molecular type the authors mean the gene name.

Figures 2 and 3: I don’t find a reason to add the first category of measurements that are actually 1 (muscle tissue and 0h). The authors should exclude these from the figures and write that the measurements are relative to these tissues (or time points) that represent 1 in relative expression. Furthermore, data reflect small variations within groups. Did the 3 samples in each group handled separately or were the RNA samples pooled?

The discussion is missing arguments and should be backed up by more text on results interpretation and literature in the field.

Supplementary files: Domains annotated with the arrows are a little bit confusing, especially in Supplementary Fig. S4. The domains should be defined carefully and remove errors.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In the present manuscript authors worked on the molecular characterization and expression identification in vivo of TAK1, TAB1 and TAB2 proteins in the golden pompano, a marine fish with great economic value. The present study is well organized and presented by the authors. However, extensive language editing should be conducted. 

Minor issues regarding Materials and Methods:

Lines 77-80: Are the 4 normal golden pompano used for total RNA extraction from spleens are different from the other 4 used for basal gene expression analysis in different tissues. Please clarify.

Lines 118: The authors should give details regarding the parameters applied for phylogenetic analysis.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper of Xie et al. set off to characterize the TGF-β-activated kinase 1 (TAK1) and two related binding proteins in T. ovatus.

The subject is of clear interest to physiologist focused on teleost immune response progression and this Journal is a good fit.

I identified some points that can be addressed by the authors in order to increase the quality of the study and/or presentation of scientific results. In general, the latter is quite poor, and most of the tables or figures were included as supplementary information, even those that should be the main output of the study.

The use of English must be revised.

At last, the introduction and Discussion lack a deeper perspective. I think that linking these results to improved farming practices is a long stretch.

Please find my comments as follow, with corresponding line numbers:

L9: edit "important molecular".

L18: remove "important". You have just demonstrated that gene expression is modulated, not showed any actual evidence of pathway involvement

L28: what is "body environmental stress"?

L30: use the passive form for the verb "involve", here and throughout the text

L32: Drosophila

L35: Why such a large focus on arthropods? As per text in L57-61, information on the molecules of interest are available for fish species. Move the focus to other phyla to the Discussion section.

L41: Authors should at least introduce molecules that may not be of common knowledge

L50: This sentence can be expanded

L63: Remove "delicious"

L63-67: Linking the content of the present paper to a boost in farming practices is a long strecth. I suggest revising this sentence and reformulate these lines

L70: Vibrio alginolyticus. Correct throughout the text

L80: Were these the same specimens from which spleen was isolated?

L82: Authors should justify why the control group received a PBS injection

L97: GenBanck accession numbers should be provided, if not here at least in a figure or table caption to be included in the main text

L118-119: Which sequences were used as outgroups for rooting purposes?

L125: Primers at what concentration?

L126-128: Useless sentence

L129: MIQE guidelines recommend the use of multiple housekeeping genes. Also, on which grounds was b-actin chosed as housekeeping? Which software was employed for assessing consistency of expression?

L133: Move list and sequences of primers to the main text

section 2.6: Authors did non mention the verification of the fullfilment of ANOVA assumptions. Also, I suggest to visualize data with boxplots, in order for readers to get a picture on data distribution 

 L144: This figure and similar ones (e.g. protein domain analysis) are the main output of the paper, why not including it in the main text in the form of a panel?

L149: "higher" than what?

L152-153: reformulate sentence

L189: greater amberjack, not great

section 3.5: Why was statistics not applied to such baseline transcritptional study?

L202: With wPCR you obtain (relative) quantities of transcripts, not distribution. That is achieved in a presence/absence manner with conventional RT-PCR

Figure 3: I find it remarkable that at 0 h the standard error of expression data is so small. Please verify and upload raw data to response to reviewers. Also, is significance referred to the expression data of the same experimental group at 0 h? Nowhere is this indicated.

L244: grass carp and black carp, not carb

L245: I'm afraid you have not expanded any knowledge with regards to immune functions of these transcripts, but just provided evidence of their modulated gene expression following certain challenges.

L257: when discussing, use present tense. PP2C is still a protein phosphatase

L265: might be involved

L277: were constitutively

L281-283: What is the term of comparison here? Also, with a relative gene expression data, one can only comment on the pattern, not the actual expression quantities themselves. Therefore, I don't understand what "higher" refers to.

L286-287: Since they touched upon it, authors should try to discuss or justify why TAB1 expression was highest also in heart and what is the difference in tissue expression between TAB1 and TAB2 caused by.

L303: Why only antiviral, specifically ? Authors have challenged pompanos with LPS, Poly I:C and V. alginolyticus, and in all cases an upregulated expression of genes, although with different kinetics, was observed

Regards 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

I have noticed an increase in quality of the manuscript, especially in the discussions, which now benefits from expanded literature data.

However, I must say that the Authors did not address all comments as adequately.

For instance, I don't understand the response to my original comment on statistics applied to baseline expression. "Secondly, it is really true that the expression of the same gene in different tissues was different. In our study the application of statistics to this baseline transcriptional study is not significant.". Have you run a statistical test and did not obtain any statistically-significant results? This should be stated and take into account when reporting and discussing the results. No significance? Then, according to the data, all tissues have similar gene expression.

I would also not indicate the extraction quality of total RNA as a possible explanations of differences among fish species expression profiles (line 330).

Please enrich data visualization (figures, tables) in the main text

Kind regards

Author Response

Dear reviewer,

Thank you very much for your patience and careful review of our manuscript. We apologize for not adequately addressing all comments of the first round.

For instance, I don't understand the response to my original comment on statistics applied to baseline expression. "Secondly, it is really true that the expression of the same gene in different tissues was different. In our study the application of statistics to this baseline transcriptional study is not significant.". Have you run a statistical test and did not obtain any statistically-significant results? This should be stated and take into account when reporting and discussing the results. No significance? Then, according to the data, all tissues have similar gene expression.

Response: We have made correction according to the Reviewer’s comments. Statistical analysis was applied to basal tissue expression.

I would also not indicate the extraction quality of total RNA as a possible explanations of differences among fish species expression profiles (line 330).

Response: We have made correction according to the Reviewer’s comments. We have removed “the extraction quality of total RNA”.

Please enrich data visualization (figures, tables) in the main text

Response: We have made correction according to the Reviewer’s comments. We have moved the sequences and primers to the main text.

Other changes:

Line 146-148: Remove the useless sentence “In order to obtain the three genes Cycle threshold (Ct), the Roche LightCycler®480 PCR system was used to analyze the qPCR results”.

Line 280-281: Add “Bars that do not share a letter mean significant difference (P<0.05) of the same gene among different tissues”.

Special thanks to you for your good comments.

Yours sincerely,