All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
Thank you very much for having considered the comments from both reviewers carefully. I hope this editorial experience has been constructive for you and your co-authors.
While they have not provided a formal review, the reviewer agrees that the authors have done their best, short of completely redoing the work. We have agreed that it can be accepted in accordance with PeerJ's publication criteria. The manuscript is now fine and has been much improved, even though it could have been better if the authors had a stronger background in genomics and related fields.
[# PeerJ Staff Note - this decision was reviewed and approved by Nigel Andrew, a PeerJ Section Editor covering this Section #]
Please consider the queries posed by Reviewer#1 when you prepare your rebuttal, as they seem very relevant to grant the replicability of the study. Specifically, authors should clearly show how they improved the manuscript considering the points raised regarding transcriptome assembly and GST identities.
I really appreciate the effort made by the authors in the improvement of the manuscript; I think that present version is much robust than previous ones in the presentation of results. However, published scientific literature must contain enough information to be replicated by readers, and this is still lacking in the manuscript. I feel that authors are not familiarized with the bioinformatics field, and for this motive this part of the work is weak and must be corrected for publication:
Lines 103-106: Raw reads must be assembled in a transcriptome for database searches: please provide basic information on how this transcriptome was assembled, at least which program was used. Also, I suppose that you performed Blast searches with all the transcripts as queries, to obtain the information provided in Supplementary file 3. Otherwise you could not make searches using key words. The information on how searches were performed to assign a hit for each contign must also be provided. It is not the appropriate way to make database searches by key words; a scientific work should make blast searches that are the correct approach. In Blast searches you can evaluate false positives and false negatives; not all the sequences of interest will be named according to a key word. However, if you identified the sequences by key words (which is not correct but is already done in this work), you must check that they are not false positives, by performing blast searches in other databases (NCBI for example). I have tried to check it for your sequences but they are not still available in Genebank. In the case of GST gene family, you cannot affirm that is a delta GST without a phylogenetic analysis. At best, you can provide the best hit in the Blast search, and inform the level of sequence identity.
Authors inform that the contig 16150 has a hit with glutathione S-transferase D1 [Apis mellifera]. This is not informative without further information on e-values.
Authors write that they used “GST” as key word for database searches. Using this key word a single hit is obtained (Supp file 3) in contig3573, which seems to be a glutathione-s-transferase theta from Aedes aegypti. However, using the key word “glutathione-s-transferase”, you can have several hits. One of this hits is the mentioned glutathione-s-transferase delta from Apis mellifera, but this hit is not obtained using the key word mentioned by authors. Even though I know that this is not the focus of the work, the information provided is not enough, confuse and/or wrong.
In my previous review I posed the following questions: a) provide complete information about how the database was generated and sequenced (how the transcriptome was assembled, how many million reads where used, paired or single reads, etc); b) make public the raw reads in a public repository such as SRA in NCBI; c) specify how the transcript in the transcriptome where identified (i.e the method used to assign a name for each transcript in the database); d) provide the complete sequences of the transcripts (“genes”) that they are using for primer design.
Point a is partially addressed (information lacks on how samples were prepared and how the transcriptome was assembled). Point c has not been addressed; without this information the description of the methods is incomplete and the manuscript is not suitable for publication.
Throughout the text, please take in account that insect species have tens of GST encoding genes; avoid generalizations about “GST”. You are working in the better case with a particular GST gene.
see my comments
see my comments
see my comments
Ok.
Ok.
Ok.
Thank you for explaining and taking into consideration the suggestions. I would suggest for the future that the authors avoid transforming data and explore the use of GLM and GLMMs for dealing with troublesome data. Also, to try and use other software like R or python for analyzing data.
Please consider the comments from both reviewers carefully into consideration, as I understand they are correct and these questions seem fundamental for proper analysis and future reproducibility.
no comments
no comments
no comments
Unfortunately, the gene-identification procedure is not clear in the revised manuscript. Given that this is a central part for the interpretation and reproducibility of the results, this should be clarified in the work in order to be suitable for publication. Authors should: a) provide complete information about how the database was generated and sequenced (how the transcriptome was assembled, how many million reads where used, paired or single reads, etc); b) make public the raw reads in a public repository such as SRA in NCBI; c) specify how the transcript in the transcriptome where identified (i.e the method used to assign a name for each transcript in the database); d) provide the complete sequences of the transcripts (“genes”) that they are using for primer design. Furthermore, is not clear why the authors speculate that a particular GST is involved in the response to temperature stress; they cannot affirm that this is a delta GST without a phylogenetic analysis.
Lines 109-110: “Expression levels were normalized using reference genes GAPDH and EF-1 for high and low temperature stress, respectively”. As I stated in my previous revision, both house-keeping genes must be used as reference genes for all the determinations (at least two reference genes must be used in qRT-PCRs studies for comparisons on gene expression), not alternatively for different experiments.
I cannot find supplementary file 3.
In conclusion, unfortunately the authors did not address any of the previous requirements that I posed in order to consider the manuscript suitable for publication.
no comment
no comment
no comment
Although some more detail on the statistical analysis was included, I would like authors to specify if assumptions of the methods were checked. And, if those were not met, how did they managed to analyse them.
Also, they declare in the rebuttal that: " When analyzing the data, if there is an obvious large deviation between one data and the other three data, we will choose to delete this data to ensure the accuracy of the final result."
What would be obvious large deviation? and, more importantly, why would you even discard data. I am uncomfortable with this procedure and would never recommend it.
Please take all comments from two independent reviewers into consideration when preparing your resubmission. I highlight that many refer to methodological issues and statistics, which are central for proper manuscript comprehension and adequacy.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
The authors studied the effect or thermal stress on the activity and expression of three enzyme/superfamily: SOD, POD and GST in the invasive species Frankliniella occidentalis. The experimental work performed to meassure enzymatic activity is technically correct. However, the meassurements of gene exprssion present important issues. Please see my comments below.
The more important observation is that GST is not a single enzyme, but an enzymatic superfamily with tens of representatives in each insect genome (see for example Shi et al. Genomics 100 (2012) 327–335. Furthermore, GST superfamily is classified in different clades (Delta, Epsilon, Omega, Theta, Sigma and microsomal clades). Each one of this clades has been associated with different physiological role, particularly in insects. Hence, when authors describe results on “GST gene”; one particular GST gene can be responsible of a small part of GST activity, depending on the process under study. They should explain wich GST gene they are studing, to wich clade it belongs, and why they decided the study of this particular GST from the complete GST repertoire of the species. Otherwise all the results on GST expression cannot be interpreted and are not robust enough to be published.
Lines 98-99: How the sequences where searched in the transcriptome? Wich sequences were used as queries? Details on this experimental part of the work are relevant in order to understand the complete work. Moreover, positive hits on the database search must be reported as complete sequences. Also, information about the database should also be maked public: completeness of the transcriptome, how it was generated (from which tissues? wich life stage? wich sequencing platform was used? provide the basic parameters of the sequencing strategy (long of reads, million of reads generated, assembly strategy, etc.). If the authors prefer to not publish their transcriptomic data, they shoul work with the published genome of the species, but not refer as “unpublished data”, given that all the data used must be published in order to admit reproducibility of the results.
qPCR: a minimum of two house keeping primers is the standard for the technique. Information on efficiency for every qPCR primer must be provided.
as stated above, the transcriptomic and genetic analysis fail to meet the standards
werease the enzymatic activity assays could be vaild, the genetic analysis must be revised, as I stated above.
no comments
no comment.
Methods should be described with sufficient information to be reproducible by another investigator. This manuscript fails at this point. Procedures are referenced to other papers but should be explained here, at least its basics.
The statisitical analyses is where the main failures are evinced. No information regarding how the 4 replicates were taken into account. Clear and detailed information on the ANOVA should be stated. In fact, it seems that a GLMM would be better suited for this analyses to take into account possible effects of the replicates.
Also, the F statistics of the ANOVA have different number of degrees of freedom between the 3 enzymes and it is unclear why.
Survival or mortality results are not reported but mentioned in the experimental design.
no comment.
This paper adds relevant information of effects of thermal stress on an agricultural pest. The rationale behind the experiments are clear and the objective fulfilled. However, some decisions on which treatments were used as controls and details on experimental procedures have not been mentioned.
Additionally, mortality results have not been reported. The main flaw of this paper is the poorly detailed statistical analyses. Authors should invest much more effort in detailing the models used, albeit GLMM seem to be the correct approach to take into account the effect of replicates.
In general, this paper is a good report on the dynamics of expression of the enzymes and genes involved in thermal stress. Perhaps more contextual framework would help understanding the importance of the results.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.