Review Reports

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

The authors have reported a comparative transcriptomic analysis of the gills, mantles and digestive glands of the Antarctic scallops (A. colbecki) stabling at different water temperatures for different period of times. No significant change in transcriptome was observed upon the thermal stress. The transcriptomics changes observed in the digestive glands were thought to be associated with the stabling stresses.

Major comments:

1. The authors have tried to assemble the transcriptomes of mantle, gill and digestive gland into 1 reference transcriptome and then map raw reads from each sample to the reference to generate the read counts for each transcript in the samples for subsequent differential gene expression analysis. Unfortunately, no significant change was observed upon the thermal stress. Have the authors tried to build the reference transcriptomes for each organ and tried to do the differential gene expression analysis for each organ upon the thermal stresses? May I know the reason the build a reference library for all 3 organs instead of building one by one?

 

Low coverage is a well-known issue for de novo transcriptome assembly, which may significantly impact the quality of assembled transcripts, leading to excessive fragmentation. We decided to produce a single reference by combining all samples in order to improve the coverage of individual transcripts, thereby maximizing the completeness of the assembly. This approach was expected to lead to an assembly with significantly better metrics, allowing the retrieval of a higher proportion of full-length transcripts, even in the case of mRNAs expressed at uneven levels across tissues. Moreover, in absence of a reference genome, this strategy improves the functional annotation of transcripts and enables a direct comparison of the results between tissues, since it does not require to align sequences from different assemblies to one another to build an index of matches. We have used the very same strategy on a high number of occasions and we can confirm that this strategy invariably results to be far superior -in terms of assembly quality- and far less complex -in terms of biological interpretation- to the alternative strategy proposed by the reviewer.

In terms of quantification of gene expression levels and statistical analysis, having only one reference does not pose any issue. In fact, the pre-processing step removes from the analysis any non-expressed gene, which is functionally equivalent to using tissue-specific reference transcriptomes, with the great advantage of not requiring any post-processing for the comparison among tissues.

Nevertheless, we recognize that this point may have been somewhat obscure in the original submission, and we have consequently addressed this issue in the revised text by clarifying the role of the preprocessing step (rows 167-170).

 

2. Have the authors filtered the transcripts with low counts in the samples before the differential gene expression analysis? Have you normalized the read counts before the analysis? The authors have not provided these details in their methods.

 

Yes, this was done as part of the standard edgeR analysis pipeline. We changed the text to explicitly state that such technical passages were performed (rows 167-170, 175-176).

 

3. In line 46 to 49, the authors mentioned that the scallops were unable to response to stimuli when water temperature was 2 C and the lethal temperature should be around 3 C. The temperature was 0.6 C in the thermal stressed group in the study. May I know the reason for choosing 0.6 C instead of 2 C? Even I assume the assembly and differential gene expression analysis were done correctly and the results presented in the manuscript were correct, I still cannot claim the transcriptome of the scallop was unresponsive to thermal stresses. It is because we have not stressed the scallops with higher temperature.

 

We choose a moderate heat stress, well below the known thermal limit of the species, in order to simulate the effects of the ocean warming in the upcoming years.

We deliberately decided to apply a thermal stress of this magnitude to investigate the transcriptomic alterations in a realistic near future scenario. Most likely, Adamussium colbecki will never ever face temperatures in the range of 2°C in its natural environment, unless we somehow manage to alter planet Earth’s climate beyond repair.

In summary, the aim of the experiment was not to investigate the molecular processes triggered near the mortal thermal limit of the Antarctic scallop, but rather to assess whether any type of response could be observed in a realistic warming scenario. In addition, please note that as mentioned in the introduction, previous experiments clearly indicated that Antarctic scallops display significant behavioral alterations when water reaches temperatures close to 0°C, i.e. a temperature lower than the one used in the present experiment.

We changed the introduction to clarify the aims of this study, explicitly explaining that the goal was to investigate the response of A. colbecki to non-lethal thermal stress, using an experimental temperature in line with current forecast about a possible future Antarctic scenario (rows 81-84).

 

4. May I know which group of the samples were plotted in heat map (figure 2)? Only the control group? OR a mix of both control and thermal stressed groups? If the samples were come from both groups, then both temperature and day were the variables in the differential gene expression analysis. If the authors really want to test the stabling stress on the scallops, then the authors should only use the transcriptomes from the control group, but not the thermal stressed group.

 

As stated in the methods (line 187-189), only the control and naive samples were used in the stabling stress analysis, so the treated samples were not considered, as the reviewer correctly pointed out. We changed the caption of the heatmap to clarify that only control (+naive) samples were plotted.

Line to line comment:

1. Please label the temperature and day for each sample plotted on MDS in Figure 1.

The metadata layers requested were added to the figure and the caption has been changed accordingly.

 

2. Please label the temperature for each sample plotted on the heat map in Figure 2.

This would not be needed as the temperature is the same in all samples represented in the figure. Nevertheless, we changed the caption to clarify this point.

Reviewer 2 Report

Title: Add author name and year of discovery. 

Abstract: Please add one implication sentence at the last of the abstract section. 

Introduction: Most of the sentence is long and need to be split some sentences. Make sure one sentence is expressing not more than two senses. When it's too much complex by adding "and", "but", and "ing", it's becoming hard to track for the reader. The reader will lose their concentration and the information in the manuscript will be misunderstood.

-The objective of the study is written but in a hidden manner. The objective should be clear and must be aligned with the result. 

Methods:

-A map of the sampling site will be helpful for understanding the location of the sample collection. 

- Subheading "2.2", please cite previously published research to reduce texts. NO citations were observed. 

-Instead of describing the statistical analysis  under 2.3 and 2.4, please add a separate sub-heading as "statistical analysis"

Results: 

"ONLY" describe your results in this section. For discussion, another brand new section (discussion section) is there after this section. 

OR

Write your results and discussion section together. 

References: 

Check the species names. make sure they are italicized. 

Comments for author File: Comments.pdf

Author Response

Title:

Add author name and year of discovery.

 

We changed the title according to your suggestion

 

Introduction:

Most of the sentence is long and need to be split some sentences. Make sure one sentence is expressing not more than two senses. When it's too much complex by adding "and", "but", and "ing", it's becoming hard to track for the reader. The reader will lose their concentration and the information in the manuscript will be misunderstood.

-The objective of the study is written but in a hidden manner. The objective should be clear and must be aligned with the result.

 

We have carefully revised the introduction and modified some sentences to improve the readability of the text. In response to this comment and another similar remark provided by reviewer #1, we have modified the final part of the introduction to clarify the objective of the study (lines 81-84).

Methods:

-A map of the sampling site will be helpful for understanding the location of the sample collection.

We added a figure with a map showing the location of the sampling (figure 1)

- Subheading "2.2", please cite previously published research to reduce texts. NO citations were observed.

We have updated this section, mentioning that the methodologies used for library preparation and sequencing were the same as previously reported in another recently published study. Moreover, part of the protocol was based on the information provided by kit manufacturers (e.g. no specific literature reference was available): in such cases, we added a sentence reading “following the manufacturer’s instructions” (rows 112,113).

 

-Instead of describing the statistical analysis  under 2.3 and 2.4, please add a separate sub-heading as "statistical analysis"

Thank you for the suggestion. We splitted the contents of those two subsections and created a proper separate subsection for statistical analysis.

 

Results:

"ONLY" describe your results in this section. For discussion, another brand new section (discussion section) is there after this section.

OR

Write your results and discussion section together.

Thank you for this suggestion. We recognize that the first part of the results section, dealing with the detection of exogenous contamination, contained several sentences that would fit much better in the discussion section. We have considerably shortened this results section and moved several sentences to the first part of the discussion (rows 254-272).

References:

Check the species names. make sure they are italicized.

species names were italicized in the references

Round 2

Reviewer 1 Report

Thanks for the responses from the authors. 

I am okay with most of them except for the title of the manuscript. The title, to me, basically claims that the transcriptomes of the scallops are unresponsive to any thermal and captivity stresses at all, not matter how high the sea water temperature is. As the authors wanted to investigate the effects of increased sea water temperature on the scallops which closely resemble to real world scenario, so please amend the title and make it more precise. 

Author Response

Thank you for this suggestion. We agree with the view of the reviewer and we have consequently changed the title to recognize the fact that the current experimental design does not allow to establish whether A. colbecki will display a significant transcriptomic response to higher temperature increases. The updated title now reads as follows: "The Antarctic Scallop Adamussium colbecki (Smith 1902) is Unable to Transcriptomically Respond to Captivity and
Moderate Thermal Stress"