Round 1

Reviewer 1 Report

This MS studies the cDNA sequences of metallothioneins (MTs) in various Antarctic teleost families. This protein has roles in heavy metal detoxification and ROS concentration mediation. The aim is to study the evolution of MT genes in these fish.

 

It’s an interesting study of relevance to Antarctic fish clades facing climate change, and by extension polar biomes in general. The paper is very well written... I particularly liked the background on the Antarctic current and how it served to isolate marine animals and drive their adaptation in part 4.1 of the discussion. I’m not an evolutionary molecular biologist, and so I can’t offer an opinion on whether the programs chosen to compare nucleotide and amino acid sequences are appropriate. From a biochemistry perspective, the way the authors described the tests indicated that their use was appropriate, e.g., comparing nucleotide changes within codons to changes in amino acid content in the MT protein, and giving less weight to AA mutations that are similar chemically, like Gly→Val.

 

I would have liked to see a phylogenetic tree with the current consensus view (e.g., that considers a range of housekeeping/constitutive and inducible gene sequences) of the relatedness of the teleost species examined in this study. Figs. 1 to 3 show us how related they are for the MT gene(s) in their coding region, 3’-UTR and 5’-UTR, but comparing this with an overall cladogram (as referenced in lines 402-4) would be useful. As the authors point out, sequences of UTRs are almost always more divergent than that of the coding sequence due to their importance in the regulation of gene expression. 

 

The only other concern I have about the methodology is that the authors’ hypotheses are entirely based on the mRNA sequences they obtained. To more definitively demonstrate their ideas, they should measure whether MT protein parallels that of mRNA in the species examined. This requires looking at protein expression and MT activity in vitro, e.g., using immunoblotting and enzyme assays, respectively. It would also be good to determine the relative activity of MT-1 vs. MT-2, and whether each has assumed a specialized biochemical role since the gene duplication event that gave rise to them. The authors suggest MT might be especially important in ROS scavenging in oxygen-rich Antarctic waters… it would be good to measure SOD activity and ROS levels to corroborate such hypotheses. Such work would be a useful followup study. The authors’ conclusions are a bit too definitive given the limited amount of mRNA-only data they used  in their study.

 

I notice the calculated evolution rates don’t include errors, e.g., the SOD amino acid substitution rates in notothenioids (lines 408-9). Calculated rates are also stated to be higher or lower than others based on these error-less numbers. Can an error be calculated, and if so, it be used to calculate a P-value to back up implications that one rate is (presumably “significantly”) higher or lower than another?

 

Re RNA integrity (line 105): what was the minimum quality gate that allowed a sample to “pass” and be used for cDNA synthesis? E.g., was it an A260/A280 ratio of >=2, or some other measure?

 

For table 1, tree length should be expressed as either small or capital L in both the legend and the table itself.

Author Response

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Reviewer 2 Report

This manuscript described that analysis of the coding region and untranslated (UTR) sequences of MT genes in Antarctic notothenioid fish indicated the presence of two MT clades, corresponding to MT-1 and MT-2, respectively. The authors indicated that MT gene evolution was characterized by strong purifying selection, suggesting the possibility of negative selection. The explanations for the presented results were logical and sound. However, there are some points which the authors should address as follows;

 

1.     The abstract did not clearly state what was new. Please state the novelty plainly.

2.     In “Introduction”, please explain the isoforms, roles, binding heavy metals, and relationship with stress of MT that have been experimentally clarified so far in Antarctic notothenioid fish.

3.     It is not clear why the authors focused on metallothionein (MT). In particular, in the last paragraph of "Introduction", please describe more specifically what you were trying to learn by clarifying the molecular evolution of MT.

4.     It was difficult to understand the concept of molecular clocks. Please explain in more detail.

5.     Why were there two isoforms of MT in Antarctic bony fish? Was it physiologically necessary?

6.     Although the relationship between substituted amino acids and environmental adaptation was well-explained, it seemed unclear why the amino acid substitution rate was higher than the nucleotide substitution rate.

Author Response

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Reviewer 3 Report

Dear Authors,
The paper submitted for review titled:''Molecular evolution of metallothioneins of Antarctic fish: a physiological adaptation to peculiar seawater chemical characteristics'' has a correct structure and presents correctly performed and interpreted results. The discussion and conclusions are also structured correctly.
The only shortcoming of the paper seems to be the lack of a bioethics committee approval number (the authors only mention that they conducted the study in accordance with legal requirements without giving specific approval).
Therefore, after correcting the deficiencies, the work meets the requirements and can, in my opinion, be published.
Yours sincerely

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Reviewer 4 Report

The topic of this manuscript is within the scope of Journal of Marine Science and Engineering. It is a well-structured paper with some novelty. However, there are too many grammatical errors throughout the manuscript, the authors should seek help from language editing service to improve grammar and readability.

Author Response

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Reviewer 5 Report

Journal: Journal of Marine Science and Engineering

Manuscript Title: Molecular evolution of metallothioneins of Antarctic fish: a physiological adaptation to peculiar seawater chemical charac-teristics

Manuscript Number: jmse-1971753-supplementary

Section Abstract

1. L 17: Revise “low–molecular weight sulphur–rich” to “low-molecular weight sulphur-rich”. Please pay attention to similar problems.

2. L 19-20: Revise “study investigates” to “study aimed to investigated”.

3. L 20: Revise “this protein” to “the proteins”.

4. L 25-26: Revise “the coding region and untranslated (UTR) sequences indicate” to “coding region and untranslated (UTR) sequences indicated”.

5. L 27: Revise “two MTs clades” to “two MT clades”. Please pay attention to similar errors.

6. L 27: Revise “MT–1 and MT–2” to “MT-1 and MT-2”. Please pay attention to similar problems.

7. L 27: Revise “results indicate” to “results indicated”. Please pay attention to similar problems.

8. L 29: Regarding “bioinformatics analyses”, Please supplement specific methods.

Section Introduction

9. L 48-55: Please compress this part and delete the content that has little to do with this study.

10. L 56-58: Please compress this part  and delete the content that has little to do with this study.

11. L 59: Revise “increases the dissolved” to “increases dissolved”.

12. L 60-61: Please provide references to support the following information: “this condition can increase the rate of reactive oxygen species (ROS) formation,”.

13.  L 82: Revise “suggest that” to “suggested that”.

Section Materials and methods

14. L 102: Revise “contaminants [12,13” to “contaminants [12,13]”.

15. There is no part 2.2.

16. L 108: Revise “70°C” to “70 °C”.

17. L 110: Revise “30 min at 42°C” to “at 42 °C 30 min”.

18. L 188: Revise “To analyze for positive” to “To analyze positive”.

Section Results

19. L 257: Revise “(MYA) is shown” to “(MYA) was shown”.3

Author Response

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Round 2

Reviewer 4 Report

In the revised version, the authors have sufficiently answered all the questions raised by the reviewers. The revisions are well reflected in the revised manuscript.