Transcriptomic Analysis Following Artificial Selection for Grasshopper Size

Round 1

Reviewer 1 Report

The authors present in this paper the results of an artificial selection experiment carried on during 11 in a colony of the grasshopper Romalea microptera. The paper focus on the study of the divergent phenotypes of two R. microptera populations selected by size, as well as the associated gene expression profiles induced by these changes. To support the hypotheses presented in the study, the authors performed meassures of the thorax ob populations that were selected for large or short thorax size, and after 11 generations, they sequenced the RNA from five female adults of each colony and performed differential gene expression and funcional genomics analyses.

This study proves how quick artificial selection can shape divergent populations of a same species, as well as its consequences in gene expression. In addition, it presents the first transcriptome for R. microptera, which increases the knowledge on orthopteran genomics. 

Overall, the text is acceptable for publication. However, there are a few points that should be addressed for the article to be accepted. The issues pointed by line number are listed below. Marked in bold are points that I find crucial to include or modify. In italics I include typos that the authors might want to change. The rest of the issues are left to the authors to decide if are worth modifying or not:

Line 68: "...at least 16 additional traits..." is a result. You could remove this sentence from introduction and mention in the results section. I cannot see anywhere in the paper which traits are these or if there is a publication where these traits are listed. This datum is mentioned in several lines (220, 287) and it is never addressed to a publication or to a table with the affected traits.

Line 74: "In total, we detected 693 differently expressed genes in our two treatment groups" is a result. You could remove this sentence from here and keep it mentioned in the results section. 

Line 91: Add full stop at the end of the sentence.

Line 97: Change "American" by "America" or an appropriate region name.

Line 120: You mention "pronotum length" instead of "thorax length". Is the pronotum length the proxy meassure for thorax length? Please clarify or switch pronotum by thorax to keep the words as similar as possible. In addition, Table 1 is addresed to this datum when Table 1 never mentions pronotum length.

Line 129: Is there a way you could show the ratio between thorax and other size-related variable (i. e. hind femur length)? If the ratio is similar between both colonies, you could say you where selecting by size instead of thorax length.

Line 135: Having RNA extracted from adults seems to show the maintenance of difference sizes in the same species. However, differences in developmental genes would have been more clearly detected in nymphs. Is there a specific reason for having used adults instead of juveniles?

Line 150: This section lacks a lot of crucial information. You never mention if there is already a sequenced genome/transcriptome of Romalea microptera or if you assembled your own transcriptome. Besides, you do not explain how did you map the reads to the transcriptome or how did you test for differential gene expression. You must include the software and parameters you used to run these analyses in order to make the study replicable for other researchers. Or if you followed a protocol described in the reference #55 (cited in line 164), please mention it in this section.

Line 151: You should explain how did you obtained your unigenes. By raw sequence similarity clustering? Or by removing redundant contigs with the same BLAST result? Please explain further. I am guessing you used BLAST to compare the sequences to the specified databases, so cite BLAST and specify the parameters you used to run it.

Line 159: "Romalea microptera" should be in italics.

Line 164: I miss assembly statistics like N50, unigene length distribution or sequencing depth. If these statistics are not in other paper, please include them in this section. These statistics will show the quality of the assembly.

Line 192: I suggest to change the title of the subsection to "functional characterization (or analysis) of differentially expressed genes", since the "differentially expressed genes" are already mentioned in the subsection 3.1.

Line 220: Again you mention the 16 traits, so I refer to the comment from line 68. In addition, It would be interesting if you mention how DEGs are putatively linked to or affecting some of these traits.

Line 275: "Carboxylesterase has multifunctional functions" is redundant. How about just saying "Carboxylesterase has several functions"?

Once that at least the changes in bold are dealt with, I think the paper will be ready to be accepted.

Author Response

Response to Reviewer(s) comments is hereby attached. We appreciate the comments of the anonymous reviewers. With Regards Corresponding Author(s)

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript titled “Transcriptomic analysis following artificial selection for grasshopper size” reports the differences between the transcriptomes of two selected lines of the grasshopper Romalea microptera—one selected for large size and the other for small size.

The idea of the work is worthy, the experimental design is well thought, the analyses are standard and I can’t see any flaws in them.

Based on that, I think that the manuscript is worth publishing although I happen to disagree with the authors as to splitting their data so that they can be published in more than one manuscript—but that is the authors’ decision and, as I don’t see the present work as incomplete, I respect it.

The text of the manuscript is well written and I will only highlight a typo at line 182 (the symbol should be (R)).

I encourage the authors to specify whether it was whole bodies (as the text implies) or specific tissue(s) what was used for RNA extraction. In fact, the discussion of the results and the inferences that can be made from them would be different depending on the tissue used for comparison.

For reproducibility, the authors should write in the main text (methods section) about the assembly software, procedure and parameters used. The same for the gene expression level determination and for the statistical analyses used for detecting differential expression (these are vital info for an article of the kind).

Explaining why they chose females for RNA-Seq and not males would also be a good thing.

Table 1 could perfectly be part of the results section—I see it as the result of the 8-years long selection experiment. However, I leave this aspect to the authors’ criterion (I is my general reviewing philosophy that it is the authors’ work, their names are the ones that will appear and stay in it forever so, as long as it is sound and acceptable, the work should be published as its authors want it to be).

The authors specifically discuss a few particular genes (collagens, F-spondin...). I suggest that they explain better why these genes and not others (is it a question of magnitude of differential expression? Something else—expert decision?).

I think that the manuscript is of interest and worth publishing.

Author Response

We appreciate the comments, suggestions of the honourable reviewers. Attached is the point-wise response to each reviewer. Indeed we appreciate the efforts of the anonymous reviewers. 

With Regards

Corresponding Authors

Author Response File: Author Response.docx

Reviewer 3 Report

Overall, I found this manuscript to be quite engaging and its content very interesting. I have no doubt that writing up the results of an 8-year study are quite satisfying and the fact that this one yielded some intriguing, and surprising, results is even better. The results are relevant to multiple scientific fields and leave the door open for several future investigations. Plus, I must add that it's nice to see such evolutionary experiments carried out with organisms beyond fruit flies.

I endorse this manuscript for publication with very minor revisions as indicated in the edited version of the manuscript that I have attached. I have included 74 edits/comments that I recommend addressing before final acceptance. The majority are simply grammatical issues with some occasional comments; in my opinion, all will make this paper even stronger. I look forward to seeing this work published!

One final thought: assuming there was one, did you consider performing transcriptomic analyses on the colony that had no selection pressure? The results of this and of a wild population (or more, from different regions) would be interesting comparisons to these results, I think. Maybe for a future study!

Comments for author File: Comments.pdf

Author Response

We appreciate the comments, suggestions of the honourable reviewers. Attached is the point-wise response to each reviewer. Indeed we appreciate the efforts of the anonymous reviewers. 

With Regards

Corresponding Authors

Author Response File: Author Response.docx