Seasonal Stability Assessment of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalization in Bombus terrestris

Round 1

Reviewer 1 Report

The manuscript entitled "Seasonal Stability Assessment of Reference Genes for qPCR 2 Normalization in Bombus terrestris" provides a valuable study on reference gene stability for gene expression and genomics data analysis. However, there are critical issues that need to be addressed. 

While the Materials and Methods and Results sections are adequately written, the Introduction and Discussion sections require intensive improvement. Currently, there is a lack of relevance between the first paragraph and the title. The discussion on the availability of genomics information, eusociality, and the physiological background of the transition from solitary to social behavior appears entirely unrelated to the title and the intended focus of the research.

Moreover, the purpose of the research is unclear. It is essential to explicitly articulate the research question, objectives, and hypotheses in the final paragraph of the introduction to provide a clear roadmap for the study. This will ensure that readers can grasp the significance and direction of the research, fostering a better understanding of the study's aims and expected outcomes.

The most critical aspect of this research involves studying the stability of reference genes under different seasons while all colonies were kept under constant environmental conditions inside greenhouses. Given that the colonies are maintained inside the greenhouse, it is essential to elaborate on how collecting samples in different seasons could influence gene expression. Understanding the impact of seasonal variations within the controlled environment is crucial for interpreting and contextualizing the gene expression data.

Best regards.

Comments for author File: Comments.pdf

The Introduction and discussion sections require extensive English revision. 

Author Response

We express our gratitude to all the reviewers for dedicating their time, effort, and keen interest to critically appraise our submitted research work. We believe that the manuscript has undergone significant improvement based on the valuable comments provided by the reviewers. We trust that the revised manuscript now meets the standards for publication in the "Current Issues in Molecular Biology" Journal and hope for your favorable consideration

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments

1.      The MS seems to be an advertisement of the capabilities of the web-based tool RefFinder and the others.

2.      An abbreviation table is needed at the start of the MS and the explanations should not be repeated in Figure legends and Tables.

3.      The entire idea of MS is challenged by the fact that it is stated in a reference -cited paper 12- that the gene Kr-h1 fluctuates at the life stages of the insect. It has a high abundance in juvenile instars and lower levels in the final instar and pupal stage while it reappears in adults. So, it is expected that it will fluctuate at seasonal times and of course in the various organs and tissues. The authors indicated the mode of this fluctuation which lowers the novelty and importance of this work.

4.      Plagiarism is very difficult to detect in this type of work since the tools of the web and the trade are restricted and followed by all scientists. I suspect that some plagiarism appears from a neighboring citation already cited. However, it is difficult to say further because it is a serious comment for the authors.

I suggest the authors should re-examine and rewrite the MS. They should consider all the comments, some of them are written as sticky notes in the MS and supplementary material file. They should pay attention to the figures and the MV, SV, SD abbreviations which must be explained in the M&M session.

I encourage the resubmission of the MS in the CIMB journal.

Author Response

We express our gratitude to all the reviewers for dedicating their time, effort, and keen interest to critically appraise our submitted research work. We believe that the manuscript has undergone significant improvement based on the valuable comments provided by the reviewers. We trust that the revised manuscript now meets the standards for publication in the "Current Issues in Molecular Biology" Journal and hope for your favorable consideration

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear authors, 

I am delighted to announce that the revised manuscript has undergone significant improvements and is now deemed acceptable for publication.

Kind regards

Author Response

We would like to extend our sincere gratitude to the reviewers for their dedicated time, effort, and keen interest in critically appraising our submitted research work. Their valuable comments and insights have been instrumental in enhancing the quality of our manuscript. We truly appreciate the constructive feedback provided, which we believe has significantly contributed to the improvement of our research.

We have carefully considered and incorporated the suggestions made by the reviewers into the revised manuscript. We are confident that the changes made align with the high standards set by the "Current Issues in Molecular Biology" Journal. We believe the manuscript is now better positioned for publication, and we hope for your favorable consideration.

Thank you once again for the opportunity to contribute to your esteemed journal, and we look forward to your decision.

Reviewer 2 Report

Comments

1.       Lines 76-79: The MS states “The assessment encompasses the diverse seasons of the Republic of Korea (Spring, Summer, Autumn, and Winter) and spans four distinct tissues of the bumblebee. Additionally, we confirmed target gene expression profiles in the ovary, fat body, thorax, and head”: What tissues does the assessment span? Are these tissues located in the ovary fat body, thorax, and head? These four items are not tissues, they are organs and have many tissue types. I resolved this point in the legend of Fig. 1 This point must be clear here because it forms the basis of the assessment.

2.       Fig. 3: I think that this figure contains all the stability values of genes in all seasons and organs. All other figures confuse the reader. Of course, because the MS cannot be reformed at this point of revision the authors should reform this point in the discussion session of the MS. Better still! The authors should take in mind that Tables 1 and 2 contain all the information contained in Fig. 1-3. I suggest retaining only these tables and removing Fig. 1-3.

3.       Line 328: Pl. replace ‘are’ with ‘is’.

4.       Last paragraph: The passage ‘Through a thorough analysis of gene stability, we found that AK and S28 were the most reliable reference genes in the spring, while S28 and GAPDH exhibited high reliability in the summer. In the autumn, S18 and EF1 emerged as the most stable reference genes, whereas AK displayed the least stability across all samples. For the winter season, ACT and S28, along with ribosome genes S28 and S18, supported the notion that utilizing a suitable reference gene across diverse seasons is preferable over relying on one unstable reference gene.’ Is the most important part of MS and it is expected to aid the readers.

I suggest publishing the MS provided that the authors took into mind the comments.

Author Response

We would like to extend our sincere gratitude to the reviewers for their dedicated time, effort, and keen interest in critically appraising our submitted research work. Their valuable comments and insights have been instrumental in enhancing the quality of our manuscript. We truly appreciate the constructive feedback provided, which we believe has significantly contributed to the improvement of our research.

We have carefully considered and incorporated the suggestions made by the reviewers into the revised manuscript. We are confident that the changes made align with the high standards set by the "Current Issues in Molecular Biology" Journal. We believe the manuscript is now better positioned for publication, and we hope for your favorable consideration.

Thank you once again for the opportunity to contribute to your esteemed journal, and we look forward to your decision.

Author Response File: Author Response.pdf