Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS AS FEATURE EXTRACTORS TOWARD IMPROVED MALARIA PARASITE DETECTION IN THIN BLOOD SMEAR IMAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I am very happy to see that the work done by the authors are tested with sufficient dataset collected from real patients unlike many manuscripts (which often report results on cultured cell dataset / an insufficient dataset). The manuscript is written in good English.
Experimental design: Yes. The the work done by authors meet the aim and scope of the journal. Though the novelty is minimal (other than they have worked on real patient dataset), I think the manuscript can be substantially improved by addressing the comments that I have raised in 'validity of the findings' section reported below.
Validity of the findings: 1. As you have pointed out (Lines 104 - 106), Liang et al. has reported that they have found (at least for the dataset that they were using), the custom designed CNN worked much better than the pretrained model (here, your choice). It would be interesting to see the behavior for your dataset. i.e., you design a CNN that works on the same set of images used to test the pretrained model and report the accuracy (sensitivity, specificity).

2. As you have pointed out in 141-142, RBC segmentation is an important and challenging task.  You are claiming that the segmentation algorithm is novel. But this  reviewer could not assess the novelty (from your part). For me, it appears that you have just combined two popular models. This thought, may be due to little details that you have provided on cell segmentation. Please elaborate.

 For example, in Fig.1 you can see neutrophils. How good your algorithm is in segmenting WBCs? What strategy you have used to distinguish a WBC (say a lymphocyte) from a heavily infected RBC?

You must give details on cell segmentation and on the technique that you have used to assess the accuracy of segmentation? For example,  manual annotation with the Dice coefficient as performance measure will do an independent evaluation.

3. Have you come across the following recent papers.?

"Image analysis and machine learning for detecting malaria, 2017 Elsevier Translational Research"

"Convolutional neural network-based malaria diagnosis from focus stack of blood smear images acquired using custom-built slide scanner. 2017 Wiley journal of Biophotonics"

4. Line 160 - 163, the authors are claiming that they have ensured sufficient data for training (generalized training) and sufficient data for testing? How did you conclude that the particular split is sufficient?

5. As you have pointed out in Line 107, you are also doing spatial scaling. Did you study its effect? It would be interesting to see the result of this study.

6. Can you justify/at least speculate on the behavior that you have reported in Lines 213 & 214?

7. When it comes to practical testing scenario, the number of infected cells is going to be very less compared to the uninfected cells (say in 1% parasitemia). Since the number of RBCs is very high in 1 microlitre of blood (~5 million), we need a system offering ~1 specificity and a very high sensitivity (say 0.995). In such case (especially for a medical system) rather than simply reporting sensitivity and specificity it is preferred to report a combined measure such as MCC as well.

8.In the practical scenario (in Point # 7), it would be interesting and beneficial if you add the following analysis

Given a system (with p sensitivity, r specificity), how many RBCs need to be analyzed to confidently decide that a person is infected by malaria (given he has q% parasitemia).

With the above reasoning/study, report how many RBCs you need to analyze to confidently decide (statistically significant) a person (with q% parasitemea) is infected or not by the best system that you have developed (say with DenseNet 121).

Also report by the same system, what is the lower level of parasitemea you can diagnose.
Additional comments: I appreciate your work. As I have pointed out in above sections, the novelty is limited. However if you address all of my comments listed above, it is going to be a good paper and I believe that you can contribute great to the field (in building a usable device for malaria screening).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS AS FEATURE EXTRACTORS TOWARD IMPROVED MALARIA PARASITE DETECTION IN THIN BLOOD SMEAR IMAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article evaluated the performance of five well known pre-trained CNN models as feature extractors for automated malaria screening. This paper is well written and the performance of the proposed method seems to be good.
Experimental design: In general, the experimental design was good and clearly written. However, this article needs some major revisions.
Validity of the findings: No Comment
Additional comments: Major points:
1) In the article, you have mentioned that:
"In this study, we instantiated the convolutional part of the pre-trained CNNs, everything up to the fully-connected layers and trained a fully-connected model on top of the stored features." (lines 180-182)

However, after reviewing your code (pre_trained_CNN_feature_extraction.py), I don't see the parts by which you store features, or freezing (fixing) the pre-trained layers in order to train just your defined fully-connected model. What I see in your code is that you have retrained (not fine-tune) the pre-trained model alongside your randomly initialized fully-connected model.

When you adding a randomly initialized fully-connected model on top of a pre-trained model and training them all together (without freezing pre-trained layers), the large gradient updates triggered by the randomly initialized weights would wreck the learned weights in the pre-trained model. And as a result, you cannot claim that you have used a pre-trained model.

2) It worries me that you might have used your test data as your validation data too. In line 161, you have mentioned that:
 "In total, we had 24,760 cell images for training and 2730 for testing."
and in your code (load_data.py - lines 9, 10 and 11) the numbers are:
nb_train_samples = 24760 #  training samples
nb_valid_samples = 2730 # validation samples
nb_test_samples = 2730 # test samples
and there is no validation folder in your shared dataset file. Could you explain how you chose validation images?

Minor points:
3) Can you provide examples of images on which the algorithm has failed?
4) In the first row of Figure 1, the input image (A) does not match with other images (B, C and D)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS AS FEATURE EXTRACTORS TOWARD IMPROVED MALARIA PARASITE DETECTION IN THIN BLOOD SMEAR IMAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is improved considerably.
Experimental design: Experimental design looks fine now, except for the validation of the segmentation/cell detection accuracy.
Validity of the findings: The findings are valid but need to be more specific in reporting especially for the qn 8 in their rebuttal letter.
Additional comments: For qn. 1 in rebuttal letter: Did you consider any other model? Why did you choose the specific architecture?)

For qn. 2 : From the points, I believe that you did not evaluate the accuracy of segmentation. May be because you don't have manually annotated images for segmentation. I wonder how you measured the precision and recall for cell detection just by identifying the centroids. How did you compare the true centroids with the detected one? i.e., did you compare the exact pixel location or you had a tolerance? Make these things explicit.

For qn. 3: Since there are many experiments done in the paper, when you report sensitivity and specificity, you are bound to specify the number of samples in the test set and for what cross validation/experiment the reported result is.

For qn 8: I am not completely satisfied with the response for Qn 8 in the rebuttal. Suppose you are analyzing 1000 cells from 1% parasitaemic individual. Out of these cells, it is reasonable to believe that 990 cells are healthy and 10 cells are infected. Your best system with 0.947 sensitivity and 0.972 specificity will then behave something like this : It will detect 1 out of 10 infected cells as healthy and detect 27 healthy cells as infected.  This means that for all samples, the system will identify infected cells. This is true even for the samples from a perfectly healthy individual. So the question is, how will it be useful? Or how many cells has to be analysed to ensure that 99.99% chance is there that we have identified really an infected cell among the so many identified infected cells.

Every run in cross validation experiment (provided in Table 1) ideally should take same number of samples? Why there is a difference in number of samples used across the runs? How did you select the samples for each run?

Any reported result in this paper to substantiate the claim made in line 291?

How does the diagnostic odds ratio (line 298) differ from sensitivity?

Minor comments:
Fig. 1 : 2nd row Images placed one over the other and need to be corrected.
Fig.2 looks a little odd. It could have drawn more professionally.

Please proof read the document carefully. There are missing spaces (such as line 41), repeated words (such as improving improved in line 184)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS AS FEATURE EXTRACTORS TOWARD IMPROVED MALARIA PARASITE DETECTION IN THIN BLOOD SMEAR IMAGES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: I think the paper has now been substantially improved and deserves publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRE-TRAINED CONVOLUTIONAL NEURAL NETWORKS AS FEATURE EXTRACTORS TOWARD IMPROVED MALARIA PARASITE DETECTION IN THIN BLOOD SMEAR IMAGES
Review round: 3
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLYING FOLIAR STOICHIOMETRIC TRAITS OF PLANTS TO DETERMINE FERTILIZATION FOR A MIXED PINE-OAK STAND IN THE QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is devoted to N and P contents in forest stands with several tree species. N and P contents were determined in foliar, soil litter layer and in mineral soil samples. The authors used stoichiometry to asses N and P demand by forests and recommended a rate of fertilizer application. The most intriguing aspect for me was that despite of the fact the research sites are in the subtropics the stands appear to be N limited. Also interesting is the correlation between soil N and foliar N:P ratios, suggesting that soil N status affects tree P nutrition.  I think that the paper contains enough interesting information and worth publishing after minor revision.
Experimental design: The experimental design is nice.
Validity of the findings: Interesting data
Additional comments: 1)	I would not recommend using the term “tree layer”, it sounds awkward. Better use “foliage” or vegetation, or plants instead.
2)	The recommended N application rate seems too high. It should be compared to the rates applied in the region. Also important how you suggest to apply this amount of N: at once or distribute it across several years?
3)	Line 34: not ”was added” but “should be added”
4)	Line 185: Not relationship but correlations between
5)	Line 195: Correlation, not relationship
6)	Line 196: Replace the depth to the end of the sentence
7)	Line 293: The recommendation of N fertilization must be softened. For example, the stoichiometric approach we used in the present study reveals that approximately 1.49 t/ha of N should be added if we want to achieve big timber cultivation. However, these rates should be further confirmed by a field fertilization experiments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLYING FOLIAR STOICHIOMETRIC TRAITS OF PLANTS TO DETERMINE FERTILIZATION FOR A MIXED PINE-OAK STAND IN THE QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Several redundant figures such as figure 2 and 3. Author should consider another  presentation methods.

Author should add the statistic analysis in Table 3 and also description in M&M section.

L224-229 How about N of P. tabuliformis and N and P of P. armandii ? Author should indicate the leaf life span of three species.

L261-267 There are arbitrary inferences in this paragraph. Need more evidences and supporting by other researches.
Experimental design: Need more detail information about species in M&M section.

L97-98 Author claim that the average stand age is 42 years. But the stand height only 9.2 m in average?
Validity of the findings: no comment
Additional comments: Several mistakes in unit should be corrected such as L25, L27,and L151.

The format of unit should be consistent such as mg.g-1 in Table 3 or mg g-1 in L187.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLYING FOLIAR STOICHIOMETRIC TRAITS OF PLANTS TO DETERMINE FERTILIZATION FOR A MIXED PINE-OAK STAND IN THE QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: My review with comments are attached
Experimental design: The experimental design was of high quality
Validity of the findings: See my review comments
Additional comments: The review contains my comments for the author. Additionally, Figures 2 and 3 need major revision. Although the research results contribute to current knowledge of N, P and N:P of forested ecosystems, the findings did not statistically support previously known behaviors of these elements in forested ecosystems of China. The authors should have concluded as such. All figure statistical p-values did not support the positive correlations the authors concurred.Trend lines or regression lines can point up or down but if they are not supported by statistical values, they do not mean much. The authors should not simply concur with previous conclusions elsewhere when they did an excellent work in a forested area whose climate and edaphic factors may have affected forest physiological ecology, and so the different results they found.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLYING FOLIAR STOICHIOMETRIC TRAITS OF PLANTS TO DETERMINE FERTILIZATION FOR A MIXED PINE-OAK STAND IN THE QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English is now sufficient. The text is well written and structured.
Experimental design: Excellent
Validity of the findings: Important and interesting findings
Additional comments: The authors have answered all my and other reviewers comments and properly addressed them in the text. Well done! I suggest to accept the manuscript for publishing.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLYING FOLIAR STOICHIOMETRIC TRAITS OF PLANTS TO DETERMINE FERTILIZATION FOR A MIXED PINE-OAK STAND IN THE QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Author has revised throughout the manuscript.
The line number is confused in WORD file. It's difficult to find the revised sentences.
Experimental design: I suggest that author should consider other statistical methods such as ANOVA and post hoc tests in Table 3.
Validity of the findings: no comment
Additional comments: How different is the upper/lowercase alphabet  in Table 3?
According to the note description, it seems lowercase maybe enough. However, the footnote is too redundancy. The description in the last paragraph of M&M is enough.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TRANSCRIPTOME PROFILING OF ANTHOCYANIN-RELATED GENES REVEALS EFFECTS OF LIGHT INTENSITY ON ANTHOCYANIN BIOSYNTHESIS IN RED LEAF LETTUCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript’s structure conforms to PeerJ’s standards and the figures are relevant and of sufficient quality. However, there are a number of issues regarding the reporting that need to be addressed:

1. The NCBI SRA datasets SRR5868088 and SRR5943715 are not accessible. Trying to access them results in an error message stating that “This record has not yet been released. If you found reference to this accession in a publication, please let us know”. This is not in accordance with journal policy and the raw data should be made accessible for review.

2. The sentence structure and phrasing requires considerable editing. Some instances where the language/phrasing is imprecise or ambiguous are listed below:
2a) When referring to the identified lettuce gene homologues, it should be made clear that these are putative anthocyanin structural or regulatory genes as their involvement in the pathway is only inferred from phylogeny and correlation of gene expression. This is done in most cases, but not everywhere (e.g. lines 174, 183, 214, 281)
2b) It is not always clear whether a statement refers to anthocyanin biosynthesis, accumulation or the expression of anthocyanin-related genes (e.g. lines 64, 76)
2c) On several occasions the text reads “anthocyanin-regulated” genes (i.e. regulated by anthocyanins) when it should refer to genes regulating anthocyanin levels (lines 38, 188, 195, 201, 268).
2d) The sentence referring to Unigene12000_All and Unigene10814_All in lines 180-182 is not comprehensible.

3. The introduction could benefit from adding a few additional pieces of information which will make the article more accessible to non-experts
3a) A sentence or two stating what anthocyanins are and from what they are synthesized might be helpful. The abbreviations EBGs and LBGs should be introduced here.
3b) Two putative homologues of anthocyanin transporters are mentioned in the results and in the abstract. Thus, anthocyanin transport should be mentioned in the introduction and references for this should be given.
3c) References should be given for the statement in lines 59-61. Given their importance in the manuscript, it could also be established here that the MYB transcription factors and HY5 control light-dependent expression of the structural genes in other species.

4. There appears to be some confusion regarding the term “-fold” or “fold change”. While fold change (FC) in transcript levels, i.e. a ratio of gene expression values, is often stated for qPCR results, log2FC is commonly used to refer to gene expression changes in RNA-seq analysis. It should be clearly stated whether the numbers mentioned in the text represent FC or log2FC so that readers do not underestimate the observed differences.

5. Gene names should be stated in full the first time they occur.
Experimental design: The presented research is within the scope of the journal. The experimental design of this study is sound, the applied methodology and bioinformatics analyses are routinely used. Below are some suggestions that may further improve the quality of the manuscript.

1. Has the sequencing data been examined for contamination? As the lettuce is grown in non-sterile conditions, bacterial, fungal etc. transcripts can represent a significant amount of the detected sequences. Checking the species distribution among the BLASTX hits could reveal the extent of potential contamination. If there is a large amount of reads that represent hits for non-plant sequences, filtering the data against the respective genomes should be considered.

2. The authors might consider to annotate the differentially expressed genes not only for KEGG pathways, but also for GO terms or COG categories, which are commonly used alongside KEGG in the literature. Alternatively, they should state why KEGG is the database of choice for their analysis.

3. The software packages used for differential gene expression analysis need to be stated. Version numbers should be stated for all mentioned software packages.

4. Quantification of anthocyanin levels and basic statistics on this could be included in Figure 1.

5. A reference for the CTAB method (line 92) should be given.
Validity of the findings: The results obtained in this study are clearly related to the previously defined research question and significantly advance the understanding of the regulation of anthocyanin accumulation in red leaf lettuce. Some conclusions however need clarification:

1. A putative lettuce homologue of Arabidopsis HY5 has been identified – is there phylogenetic information supporting this? Is HY5 the best hit when BLASTed against Arabidopsis proteins and is Unigene19629_All the best hit when the AtHY5 sequence is BLASTed against the assembled unigenes?

2. Why do the authors contrast the lack of change in lettuce COP1 transcript levels in lines 256-259 with the regulation of COP1 in Arabidopsis? Light does not strongly affect Arabidopsis COP1 transcript, but affects the protein’s interactions, activity and subcellular localisation. Hence, a major effect of light on lettuce COP1 transcript would not be expected.

3. The paragraph in lines 260-266 is not clearly laid out and the hypotheses stated in it are vague – what exactly are the PSI and PSII pathways? It could be left out of the discussion or – if the authors feel it represents an important conclusion drawn from their data – should be more carefully explained with precise hypotheses and arguments.
Additional comments: In their manuscript, Zhang et al. compare transcriptomes of low and high light-grown red leaf lettuce to detect transcripts correlated with increased anthocyanin content under high light; using gene annotation and phylogenetic analysis they identify genes encoding putative anthocyanin biosynthesis enzymes, anthocyanin transporters and transcription factors regulating anthocyanin accumulation.

Overall, the manuscript presents valuable information on light-regulated anthocyanin biosynthesis in red leaf lettuce that may also serve as basis for further investigations. There is a number of issues with regard to the reporting of the findings that require editing; however, if these issues are appropriately addressed and if the data sets are made accessible, the manuscript should be considered for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TRANSCRIPTOME PROFILING OF ANTHOCYANIN-RELATED GENES REVEALS EFFECTS OF LIGHT INTENSITY ON ANTHOCYANIN BIOSYNTHESIS IN RED LEAF LETTUCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript, "Transcriptome profiling of anthocyanin-related genes reveals effects of light intensity on anthocyanin biosynthesis in red leaf lettuce" attempts to investigate the genetic basis of light-mediated anthocyanin biosynthesis in red leaf lettuce using transcriptomic approach.

The authors first establish the effect of increased light intensity on the coloration of red leaf lettuce, followed by transcriptomic analysis to identify genes associated with increased anthocyanin accumulation under higher light intensity.

The manuscript needs improvement at a few places for general writing and meaning.
- Line 71-75: Rephrase the sentence for better readability.
- Line 280-282: Sentence may be broken down to two sentences.
- Very long sentences with many commas should be avoided.
- Line 52: "The enzymes encoded by these gene..."
- Line 38 and 60: "anthocyanin-regulatory"
Experimental design: -  Line 88 mentions that, "leaves from five individuals were pooled in each group...". But there is no mention of number of biological replicates used for the study. There must be at least three biological replicates for the statistical analysis, and FDR cutoff used in the study. Authors must include a scatter-plot showing the distribution if replicates for each sample.
- What is the reasoning for doing two independent assemblies for low light and high light conditions? Since the organism and tissue type are same, it would have been better to do a single assembly combining all the reads to increase the chances of getting full length ORFs.
- Did authors do something to check the quality of assembled transcriptome? It is currently not mentioned in the manuscript. Perhaps estimating the percentage of full length ORFs as explained in Trinity manual might be performed.
- Line 107-109: Blastx can not be performed against nucleotide (Nt) database. Authors should make sure about the correctness of the statement. I feel Blastx might be performed against Nr, SWISS-PROT and InterPro databases as also mentioned in line 156-158.
- Figure 3: Legend mentions white and blue colors, but figure has green and red.
Validity of the findings: From the transcriptomic analyses, authors find some relevant anthocyanin biosynthetic and transport genes upregulated in response to increased light intensity. In addition, authors also find the differential expression of some key transcription factors regulating the anthocyanin biosynthesis. The authors validate some of those results using qRT-PCR. However there are many sections in the discussion that seem to be over-statement.
- There is no results presented about the photosystem (PS) I and PS II. Hence, the discussion paragraph (line 260-266) seems to be out of place.
- The discussion about COP1/SPA complex also doesn't well, as the paper talks about the transcript levels. But COP1/SPA mediated regulation happens at the level of protein. Therefore, the section should be rephrased to make it more contextual.
- There is not enough evidence for the discussion regarding the light-mediated increase in sugar levels, which in turn increased anthocyanin levels. Either quantification of sugar levels under different light conditions or expression analysis of critical sugar-related genes need to be performed to justify the statements.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TRANSCRIPTOME PROFILING OF ANTHOCYANIN-RELATED GENES REVEALS EFFECTS OF LIGHT INTENSITY ON ANTHOCYANIN BIOSYNTHESIS IN RED LEAF LETTUCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made the changes requested and the language of the manuscript has improved considerably. There are two minor issues that need to be addressed:

1. The authors have clarified when referring to fold change (FC) or log2FC. However, I think the wording for log2FC needs to be slightly altered; levels can be “x-fold higher/upregulated”, but not “x-log2 fold change higher/upregulated” (lines 40, 209, 216, 229, 233, 244). As a log2FC > 0 already implies increase, the text could simply read “genes showed a log2FC of 2.7-9.0” (example for line 40).

2. It appears that two different unigenes are referred to as Unigene10814_All (Table 1 and lines 210 and 216), one a 3GT and the other a GST homologue. This needs to be clarified.
Experimental design: No additional comments
Validity of the findings: No additional comments
Additional comments: The authors have satisfactorily addressed my previously raised concerns and made the recommended changes.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TRANSCRIPTOME PROFILING OF ANTHOCYANIN-RELATED GENES REVEALS EFFECTS OF LIGHT INTENSITY ON ANTHOCYANIN BIOSYNTHESIS IN RED LEAF LETTUCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors have addressed the comments properly, and the revised manuscript is significantly improved over the previous version.
Experimental design: Authors have sufficiently addressed my comments for previous version of the manuscript, except for the usage of the replicates in the experimental design. If the authors have taken one pool of each sample for RNAseq library preparation and analysis, I am not sure about the usage of FDR cutoff for selecting the genes. Without having three replicates for the analysis, the analysis is just qualitative and FDR cutoff doesn't have any meaning. Since the authors have checked the candidate genes by qPCR, the interpretation in the paper OK. However, authors should be careful using FDR cutoff in text.
Validity of the findings: Authors have done a few new experiments ( e.g. sugar content measurement) and analyses (e.g. prediction of full lenght ORFs) to justify the validity of findings in the revised manuscript. Further, authors have removed some of vague and speculative statements from the discussion regarding the findings.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EVOLUTION OF DOPAMINE RECEPTORS: PHYLOGENETIC EVIDENCE SUGGESTS A LATER ORIGIN OF THE DRD2L AND DRD4RS DOPAMINE RECEPTOR GENE LINEAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: Please see attached PDF.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EVOLUTION OF DOPAMINE RECEPTORS: PHYLOGENETIC EVIDENCE SUGGESTS A LATER ORIGIN OF THE DRD2L AND DRD4RS DOPAMINE RECEPTOR GENE LINEAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: I believe that the data support the conclusions of the study
Experimental design: Not being a evolutionary biologist I cannot judge of the experimental design
Validity of the findings: As far as I understand the data are acceptable. I only find that from an outside point of view little discovery is revealed in this manuscript
Additional comments: The manuscript is well written. I cannot judge of all the data but they seem to be expected. I only would like that the authors define what is D2l and D4rs. This is difficult for pharmacologist as D2l relates to the long form of the D2 receptor and D4rs is reminiscent of the different D4 subtypes. Also the comments on the role of the D4 in psychiatric disorders should be toned down as thus far the D4 receptor has not been proven to be associated with any disorder


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EVOLUTION OF DOPAMINE RECEPTORS: PHYLOGENETIC EVIDENCE SUGGESTS A LATER ORIGIN OF THE DRD2L AND DRD4RS DOPAMINE RECEPTOR GENE LINEAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The authors have addressed my comments, but I think a bit more detail still needed, specifically:

1. For the BLAST search performed, which sequence was used as the seed sequence? Which BLAST algorithm was run? How was "conserved synteny" identified? How were sequences manually identified if a software was also used for the same step? The details here are improved but are still too thin. In addition, any code written and employed for these tasks MUST be released with the paper either as supplementary information or on a linked github repository, or similar.

2. Running tree inference "multiple times" is not a specific number. Is this 5 times? 10 times? 100 times?You need to give a real value and as needed infer the tree again so that you can give a specific value.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EVOLUTION OF DOPAMINE RECEPTORS: PHYLOGENETIC EVIDENCE SUGGESTS A LATER ORIGIN OF THE DRD2L AND DRD4RS DOPAMINE RECEPTOR GENE LINEAGES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is acceptable
Experimental design: No issue
Validity of the findings: Acceptable
Additional comments: You mention the variability in the DRD receptor nomenclature as stemming from the fact that these receptors were originally not looked from a phylogenetic standpoint. DRDs are most important in neuropharmacology and as such are discussed as pharmacological entities by most.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EVOLUTION OF DOPAMINE RECEPTORS: PHYLOGENETIC EVIDENCE SUGGESTS A LATER ORIGIN OF THE DRD2L AND DRD4RS DOPAMINE RECEPTOR GENE LINEAGES
Review round: 3
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: I feel some clarification is still needed, in particular:

1. My comment regarding this sentence was not answered: "Dopamine receptor genes were manually annotated by comparing known exon sequences to genomic pieces using the program Blast2seq (Tatusova and Madden 1999)."

Please explain how you can manually annotate while using a program - what is the role of this program vs your manual annotation?

2. The updated sentence "Phylogenetic analyses were 20 times in order to better explore the tree space." needs a verb, i.e. "were PERFORMED 20 times".

3. The level of explanation regarding "synteny analysis" is still far too thin. It does not sound as though any synteny analysis was performed, just that the authors read a paper which claimed to identify synteny. The authors MUST be PRECISE about their analysis here.

4. The authors may not have written code, but they have certainly used software for which specific parameters must be specified. The authors must therefore include the following in the manuscript:
- specific *parameters* used in the tblastn search (i.e. evalue, database searched, etc.)
- specific arguments, and all input/output files generated, for the molecular dynamics simulations
- details with this sentence: "Sequences were aligned using Vector NTI 169 Express (Thermo Fisher)". What alignment algorithm is used in this program? What arguments were specified?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TWEET SUCCESS? SCIENTIFIC COMMUNICATION CORRELATES WITH INCREASED CITATIONS IN ECOLOGY AND CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Paper is generally well-written, references provided seems adequate.
Experimental design: It is still not very clear on why the authors have chosen to focus on ecology and conservation (noting that other papers have attempted similar studies linking altmetrics and traditional citation means). Are they doing this just because this hasn’t been explored in the literature? (I hope not!) I’d like to see a stronger framing of the paper’s research focus – what are the questions and why is it necessary for us to find answers for them, and particular so in the context of ecology of conservation?

Additionally, it would be useful if the authors provided more details of their search methodology, given that a search with such generic terms like "ecolog" and "conservat" will end up with a huge number of irrelevant papers (and quite a lot of systematic work would be needed to eliminate these), notwithstanding that effort have been made to cull review papers and letters from the core analysis.
Validity of the findings: Statistical framework seems sound, and is clearly explained.
Additional comments: Social media is playing an increasingly important role in the communication of research findings, especially in the disciplines of ecology and evolution, and biodiversity conservation. As a researcher working in these fields, I fully appreciate the need to regularly use social media platforms to convey interesting findings in digestible snippets, in the hope that some of these information would eventually be utilised by decision makers and policymakers. This paper explore and assesses the relationships between more traditional citation metrics in relation to altmetrics (and counts of media sources) in the context of ecology and environmental research. The authors found that altmetrics were able to predict citation rates until a particular threshold in two time periods. Broadly, the paper is well written, and offers some new insights on how the real-world influence of ecology/conservation research papers could be measured, long before it accumulates citations. The authors have also evaluated a large amount of material before proceeding with their analysis, and their statistical approach seems very sound. I would like to offer a few minor suggestions to frame the paper more clearly (to distinguish it from other papers doing similar things), and strengthen its discussion.

Minor comments
L63 – The (applied) role of ecology/conservation research could be made clearer here, perhaps an example on links between conservation research and policymaking, and a citation on what kinds of environmental problems
L75 – Are there actual instances where policy making processes were shaped from increased lobbying resulting from social media exposure of an environmental/conservation issue
L87 – “showed that tweets” – good to maintain consistence tense
L88 – “ornithology” – please correct this and other spelling mistakes throughout the paper
L90 – Not clearly explained here why is there is need to identify high-impact papers rapidly
L92 – “covered under the umbrella of…”
L95 – I think a more recent citation is needed, since you say it “continues to determine professional success”
L108-111 – Authors say what the paper is going to do, but did not make clear why this is of interest. This section of the paper needs more work to show it is not just another data exploratory paper.
L114 – What kind of descriptive data on these articles?
L123 – Suggest highlighting here that the focus of the paper is on media discourse in the English language – Note that large amounts of similar scientific discourses pass through social media in other language medium (e.g. weibo, weixin)
L124 – More information needed on how the papers were selected after the initial searches – how did you ensured that paper covered content specifically relevant to ecology and conservation? Checking of the abstracts? Checking with the key words of papers? Both disciplines (and search terms) are quite broad and you would likely have had to throw away a lot of non-relevant papers
L133 – you mean “research article”
L136 – “the generalised linear model”
L140 – “GLMs” – please remove the apostrophes throughout – this and for “BRT’s”
L146 – “fitted”
L151 – Rephrase sentence, awkward phrasing
L152 – “percentage”
L188 – please correct spelling of “altmetric” throughout the article
L204 – Not wrong to say this, but conservation is certainly more “applied” compared to ecology, where a lot of research output remains as theoretical or empirical studies
L250 – Perhaps provide an example to show how media outreach by institutions may be revised in response to these patterns
L256 – Could there be other factors involved – that drives up citation rates (e.g. search results algorithms)
L256 – “can transform”
L264 – L269 – This section could be improved by revisiting the findings more in relation the study’s focus on ecology and conservation, the current discussion is a bit generic.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TWEET SUCCESS? SCIENTIFIC COMMUNICATION CORRELATES WITH INCREASED CITATIONS IN ECOLOGY AND CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well thought out paper with a specific goal achieved through a correlational analysis of data extracted from two databases – one traditional citation based and the other altimetric based. The authors found relationships between altimetrics and citations.

Which suggest that publicity received by papers has an impact on future citations.
However, there are a few issues that need to be addressed.

-	There is a need to clearly define terms as and when they first appear. This would greatly help the reader later in the text. A clear example is the need to clearly define altimetry at the start.

-	The study is actually of a specific subset of scientific articles that have both the keywords Ecology AND Conservation. The author should consider changing EC throughout the text to E&C. As the data extracted from the databases were using this operator. Which also means that the study is of a specific smaller subset of a larger field of study. This specific subset of data provides great power for the analysis that the authors have carried out. But it does at the same time only allow inferences for papers written in other parts of the larger field of study. This needs to be acknowledged at the start and in the discussion.

-	In Figure 1C, the authors illustrate an important finding of the changing contributing proportion of different publicity channels over time. This is quite an interesting finding worth elaborating on.

-	While not a specific issue, could the plateauing of citations with impact factors above 10 be an artefact of the E&C journals themselves? Consider that E&C journals have impact factors below 10 and only a few top tier journals that periodically publish E&C papers (e.g. Nature, Science, PNAS) have impact factors above 10.
The study warrants publication subject to addressing the relatively minor issues highlighted in this review.
Experimental design: The study is well designed to address the relevant question.

More details in the attached PDF.
Validity of the findings: Line 173:
The number of articles found appears to be very low. It could also be because the authors are using AND instead of OR. This means the study is really looking at only a very specific subset of research that include both the keywords Ecolog* and Conservat*.

Line 187-190:
How were discipline-specific (i.e. conservation vs ecology) differences found if the original data search was using the AND operator?

Line 224-240:
One of the key issues not dealt with in the discussion is that the scope of this study did not include the content of the specific E&C articles. Previous work by Papworth et al  and Wijedasa et al. (Papworth et al., 2015; Wijedasa, Aziz, Campos-Arceiz, & Clements, 2013) found that the specific content of the articles were more likely to get featured. While not part of this study, it is a limitation.

Line 263-265:
The levelling off that you noted could also be because there are very few journals in E&C with impact factors greater than 10. So this result may be an artefact of the fact that many journals in E&C have impact factors lower than 10. This is also in contrast to what Papworth et al (2015) found, where the likelihood of being featured in news or twitter was higher for high impact factor journals (e.g. Nature, Science, PNAS).
Additional comments: Well written paper. Most comments made are minor.
Do consider tightening up your results as conclusions drawn on the results as the study is of a specific subset of papers in the field (i.e. those that have both keywords Ecolog* AND Conservat*). This gives considerable power to the study as it may reduce noise.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: JOINT-LEVEL ENERGETICS DIFFERENTIATE ISOINERTIAL FROM SPEED-POWER RESISTANCE TRAINING—A BAYESIAN ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No Comment.
Experimental design: No Comment
Validity of the findings: See General Comments.
Additional comments: The primary aim of this study is in my opinion two-fold and can be summarised as
1)	Comparison of 2 different intervention: Isoinertial training vs. speed power training
2)	Validity of Bayesian inference to compare between group difference.
The study design is fairly appropriately designed to test for the 2 different interventions. The findings are interesting, the use of Bayesian statistics is novel (and much needed) and the discussions logical.
No objections to publications and in general support its publication. There are a few suggestions below, which I recommend the authors address to strengthen the article.

1) Normalisation of Training (line 107 to 123)
Training programs are logical and sound. It is suggested that the authors include an explanation on how they normalise/equalise the training (e.g. volume etc) between two programs. While I understand this might be hard (especially between the 2 different modalities), doing so will help to convince the readers that the results are more due to the modality and not simply due to the difference in training variables.

2) Leg Stiffness (line 99 to 103)
While, it is understood, that this study is part of a larger study, It is suggested that the authors still include the actual leg stiffness values for 2 main reasons. The first is that the as the authors have mentioned, the study was based (statistically powered) to detect a significant change in leg stiffness (line 99 to 103). Second, while indirect indicators were included (e.g. power absorption, line 205), including the actual leg stiffness values would be useful as it might further reinforce the strength of the results that you shared with the readers.

3) Increase in CMJ depth
It is suggested that the authors try address the increase in CMJ depth a bit further, especially since the difference is one way (greater) and jump power is one of the dependent variables in this study. This is because a greater CMJ depth tends is likely to lead to a greater jump power, though not necessarily jump height (Jidovtseff, Quievre, Harris, & Cronin, 2014). Greater depth can possibly lead to a greater jump power through 1) Greater torque and 2) Increased time for activation and a higher starting force. Since jump power is one of the main dependent variables in this study, it is recommended, that the authors elaborate this slightly further.

On a side note, I concur with the discussion point that the increased preload might explain the greater CMJ height is logical.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: JOINT-LEVEL ENERGETICS DIFFERENTIATE ISOINERTIAL FROM SPEED-POWER RESISTANCE TRAINING—A BAYESIAN ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	L 32: Please provide a reference for the statement: “squat jumps and countermovement jumps are the most commonly assessed vertical jump types”
2.	L 46: Paragraphs should not start with the word “yet”
3.	L 49-51: The authors cite several previous studies that have compared interventions. They decided that another study utilising Bayesian methods was the best way to identify the small effect sizes previously observed. Would a meta-analysis of these studies have been a better way to pick them up?
4.	L 52: “have” should be “has”
5.	L 56: Would “qualitative” be better described as “descriptive”?
6.	L 59 (and elsewhere): “clinical” – should this word be deleted? The decisions aren’t necessarily in a clinical setting
7.	L 75: Consider adding “if any” after “which”
8.	L 81: Clarify that you hypothesise increases in both CMJ and SJ jump height.
9.	L 81: Please define an improvement in energetics
10.	L 200: The data in brackets reads as SJ height change not the diff between isoinertial change vs speed-power change. Please clarify
11.	L 254: Consider deleting the word “presently”
12.	L 255: “was” should be “has been”
13.	L 259-60: Consider changing “which does not mean it was the only method of Bayesian inference” to “however it was not the only method of Bayesian inference used”
14.	L 267: Insert “the” after “that”
15.	L 288: Consider changing “This was” to “Although this theory is”
16.	L 291: Add a full stop after “2010b)”
17.	L 311: Consider changing “active ingredients” to “specific parameters” (no quotes needed)
18.	L 335: Change “ultimate” to “ultimately”
19.	Figure 1: Correct spelling of “enrollment” to “enrolment”
20.	Figure 1: Expand or otherwise clarify “mocap”
21.	Figure 2 & 3: Provide more detail on the calculation of the location of the dotted lines – e.g. is this from both groups combined?
22.	Figure 2 & 3: 18 graphs on one page was too many for easy evaluation. Consider superimposing to allow easier comparisons. Some graphs could maybe go into the supplementary material. Shading was also too light when printed out (black/white). The mean line was not visible.
Experimental design: 1.	The sample size calculation is for an outcome (leg stiffness) not reported in the current study (as this analysis was part of a larger study) and therefore it’s utility is not clear. While I am unsure if there are validated power calculations for Bayesian analyses; it would inform the reader if the authors could state the sample size that would have been needed if jump height was considered the primary outcome (using the minimum meaningful difference that they hoped to be able to detect / expected from previous studies).

2.	L 120: How does the volume, intensity, repetitions and time to complete the CMJ and SL exercises compare to the resistance training? Could your results be due to differences in these rather than the modality?

3.	This study utilises a similar intervention methodology to other papers; but differs by the type of analysis (Bayesian vs frequentist). The introduction implies that a Bayesian analysis was chosen a priori however it is not clear why a larger sample size was not considered given the small effect sizes found in previous studies.

4.	Would a comparison to the frequentist methodology be more appropriate to demonstrate superiority of the Bayesian method?

5.	L 17 & 167 & Results: While I am not overly familiar with the use of Bayesian methods (as I expect many readers will be as well), it surprised me that a level of significance was defined in a similar way to frequentist methods. From my understanding Bayesian methods aim to get away from the binary classification of results so that small effects are not dismissed but given a level/range of confidence/credibility.

6.	A copy of the visual 3D model file and analysis pipelines would improve reproducibility

7.	L 153: Please explain / reference the unified-deformable foot method

8.	L154-5: Please define/reword your use of the terms eccentric and concentric as muscle action was not measured directly

9.	L 183-4 & 190: How are the number of burn-in and inference samples decided? Why do they differ between scalar and waveform variables?

10.	L 192: The correct analysis of left/right sides has been debated (Menz 2004, Stewart 2018 - http://www.gaitposture.com/article/S0966-6362(17)30974-8/fulltext ). How does your analysis compare to the Stewart approach (other than being Bayesian)?

11.	Table 1: p-values are described (frequentist analysis) but the tests used are not in the methods. Why use frequentist for some analyses and not others? Given the use of males and females, were body mass and height data binomially distributed? Were these analysed differently?

12.	Table 1: Resistance training experience is reported but no mention of speed-power training experience (e.g. from circuit training).

13.	Table 1: Does the lower resistance training experience and recent training in the isoinertial group explain the greater increases observed?

14.	Table 2: Is the total time to carry out each session the same for isoinertial vs speed-power?
Validity of the findings: 1.	L 22: no variability is reported for the increase in power absorption. Is the value reported a mean?
2.	L 205-208: Please include CrIs. Are these mean values?
3.	L 225: “meaningful” – please define in the introduction and/or in relation to the sample size calculation
4.	L 238: “average” – is this a mean?
5.	L 242: “may be large” – consider adding “or have low variability”
6.	L 247: Did the Wilson study have a similar sample size?
7.	L 253: “may be plausible” - Can you be more definitive than this?
8.	L 286: “qualitatively similar” – this is measured in quantities – consider stating instead that the means were within 5 degrees.
9.	L 319: I presume these reports used frequentist methodology – were the effect sizes similar directions and magnitudes but just not detected?
10.	Table 3: Please include info on CMJ and SJ height
Additional comments: No further comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF METFORMIN MONOTHERAPY ON SERUM LIPID PROFILE IN STATIN-NAÏVE INDIVIDUALS WITH NEWLY DIAGNOSED TYPE 2 DIABETES MELLITUS: A COHORT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: see below
Validity of the findings: See below
Additional comments: In this paper, the authors demonstrated that metformin monotherapy improves serum lipid profile in statin-naïve individuals with newly diagnosed T2DM.
I have a major comment that needs to be addressed:

Major concern
As underlined by authors, the strong limitation of the present study is the lack of a control group treated with lifestyle changes only. In fact, not only lipid profile changes as a consequence of anti-hyperglycemic drugs (Buse JB, et al. Diabetes Obes Metab 2004), but also the beneficial effect of exercise (Balducci S, et al. Diabetes Metab Res Rev. 2009 Sep;25 Suppl 1:S29-33. doi: 10.1002/dmrr.985) and diet (Greco M, et al. Mediators Inflamm. 2014;2014:750860. doi: 10.1155/2014/750860) on lipid profile is well known. These observations should be added in the discussion (line 204). Also, any information about clinical and biochemical features at the end of the study is lacking. The authors must indicate at least body weight and HbA1c in the three treated cohorts at the end of the study and discuss this point.

Minor concerns
Bibliography should be revised. Authors could have provided something more specific than the whole text by Melmed (line 256), while references at lines 234 and 260 could be substituted, or accompanied by more prominent studies.

Grammar and typo errors deserve further re-check. For example, in line 160, “improved”  should be “improve”.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF METFORMIN MONOTHERAPY ON SERUM LIPID PROFILE IN STATIN-NAÏVE INDIVIDUALS WITH NEWLY DIAGNOSED TYPE 2 DIABETES MELLITUS: A COHORT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: EXCEPT FOR SCATTERED STYLISTIC EDIFICATION _FOR ABIDING BY PEERJ GUIDELINES_ THE STUDY SIGNIFICANCE AND IMPACT OF CLINICAL RELEVANCE OF METFORMIN MONOTHERAPY IN DRUG NAIVE DM2 TO DYSLIPIDEMIA IS NOVEL AND UNPRECEDENTED!
Experimental design: SCIENTIFICALLY SOUND AND COMPREHENSIVE
Validity of the findings: FINDINGS ARE VALIDATING THE HYPOLIPIDEMIC EFFICACY OF METFORMIN MONOTHERAPY ON A TITRATION SCALE_THIS VASTLY REFLECTS ON ITS RE-PURPOSED PHARMACOLOGY AS A HYPOLIPIDEMIC PHARMACOTHERAPEUTIC AGENT
Additional comments: ACCEPT AFTER MINOR REVISIONS


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF METFORMIN MONOTHERAPY ON SERUM LIPID PROFILE IN STATIN-NAÏVE INDIVIDUALS WITH NEWLY DIAGNOSED TYPE 2 DIABETES MELLITUS: A COHORT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: Accordingly with my requests, the paper has been improved and it is now susceptible for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRESENT AND FUTURE ECOLOGICAL NICHE MODELING OF GARTER SNAKE SPECIES FROM THE TRANS-MEXICAN VOLCANIC BELT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript of Fernández and colleagues (#22530) uses clear and unambiguous, professional English. Overall, the literature provided is appropriate for setting the field background/context. The structure of the manuscript is professional and figures and tables are relevant. The manuscript is clearly self-contained with relevant results to hypotheses. Still, in section General Comments to Authors below, it is listed a series of improvements that should be made to the manuscript.
Experimental design: The manuscript of Fernández and colleagues (#22530) is original primary research that falls within Aims and Scope of the journal. Please see section General Comments to Authors for details on how Research questions and methods should be improved.
Validity of the findings: The conclusions of the manuscript of Fernández and colleagues (#22530) are well stated, linked to original research question, and limited to supporting results. There are no obvious speculations.
Additional comments: The manuscript of Fernández and colleagues addresses the ecological niche modelling of a snake community in a global biodiversity hotspot under climate and habitat change scenarios. The subject is timely and important for the conservation of important biodiversity. Overall, the manuscript is well structured, well written, and main conclusions are well expressed. However, there are multiple aspects that affect the general patterns found. These methodological aspects may have deep effects in the distribution models produced and, thus, serious effects in the predicted patterns of species distribution. Below these aspects are detailed, together with minor details, which need to be addressed in a revision of the manuscript.

L63-L64: "it is the second biogeographic zone with the highest herpetological richness and the most important region in endemic amphibian and reptile species". Which is the scale here used? Is it at global level? Specify in the text the scale of the observation.

L81-L84: The explanation on the why using Thamnophis snakes here should be developed. Given that the TMVB is a biodiversity hotspot, as authors evidenced, then why focusing the study on these 5 snake species? Couldn't the work be developed with a larger faunal assemblage? If not, then authors could explain better the ecological differences among the five species, so that the patterns observed for each one of them may provide indications about expected range shifts in other fauna inhabiting identical ecological conditions. The Introduction could include another paragraph expressing ecological differences and similarities in these five snakes and how representative they may be of other faunal assemblages.

L84-L86: The objectives of the study should be detailed and presented as a list of research questions. These questions should be based on hypotheses that are well explained in the preceding Introduction.

L92: why were selected only observations from the last 20 years?

L93-L94: the information in this sentence is not clear because we need to understand first which is the pixel size to be used in the ecological models. This is information is not given in the manuscript (I could not find it) and it is crucial. Concerning this sentence, if the pixel size of the ecological models is 1x1 km (as I suspect), then this filtering process does not reduce spatial autocorrelation of the presence data, but in fact it eliminates duplicated observation from the same 1x1km pixel. Please revise this sentence for clarity.

L113: which is the pixel size of these variables?

L118-L120: there are important information for the fine-tune of Maxent models, which involves testing distinct parameters (DOI: 10.1111/j.1600-0587.2013.07872.x). Authors should check this publications and apply the guidelines provided.

L122-L124: the models developed in this study are based in partial distributions. In other words, the modelled present range is based in a portion of the ecological conditions where the species range. I assume here that none of them is endemic to the study area; this should be well explain in the Introduction (see above comment for L93). There are abundant literature about the potential effects of modelling partial ranges of species under simulations of future range according to potential climate change impacts (DOI: 10.1111/j.0906-7590.2004.03673.x; DOI: 10.1111/j.1600-0587.2010.06181.x). However, there are conditions when the use of partial range models may be the best option (DOI: 10.1111/ddi.12115). Authors should explain the criteria used to develop ecological models based in partial ranges under climate change scenarios.

L152: replace "see" by "evaluate"

L161-L190: this is a very long section that describes the patterns observed in the figures or tables, species by species. Authors need to make this text more dynamic and interesting. For instance, base this text in the variables and not on the species. Express which are the most important variables, i.e. which variables are more frequently related to the distribution of the five snakes. The same rationality should be applied to figure 1. Authors should combine in the same plot the response curves of multiple species along the same variable. Or alternatively, combine present and future curves for the same species. This way it will be easier to observe differences/similarities among species (in the first option) or across time (second option). Remove from the main text the percentages given in brackets; these values are expressed in the table.

L198-L201: this information should be given in the methods section.

L199: where are the most important variables defined in the manuscript? The methodology for their selection should be given in methods and they should be listed here in Results section

L252: replace "T. Scalaris" by "T. scalaris"

L257-L267: this section is too focused on a exception to the general predictions and not on the "Present and future potential distribution". Authors need to develop this section accordingly.

L306: are the patterns observed in this study anyway representative of any general pattern that could be expected for other fauna inhabiting the region under identical ecological conditions?

FIGURE 3: Include a description of the meaning of the grey colour scale. What is the meaning of the darker and lighter pixels?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRESENT AND FUTURE ECOLOGICAL NICHE MODELING OF GARTER SNAKE SPECIES FROM THE TRANS-MEXICAN VOLCANIC BELT
Review round: 1
Reviewer: 2

Output:
Basic reporting: I believe the study can be a valuable contribution to the field of niche modeling and to understand how climate changes and land use influence the distribution of suitable habitats for snakes. However, the manuscript needs some crucial improvements. My greatest criticism is related to the way the authors express themselves, mainly when interpreting niche modeling results. I highlighted several examples of this error in the abstract, but they can be found throughout the text. The authors must review these issues carefully before resubmitting the manuscript.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRESENT AND FUTURE ECOLOGICAL NICHE MODELING OF GARTER SNAKE SPECIES FROM THE TRANS-MEXICAN VOLCANIC BELT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript of Fernández and colleagues (#22530) uses clear and unambiguous, professional English. Overall, the literature provided is appropriate for setting the field background/context. The structure of the manuscript is professional and figures and tables are relevant. The manuscript is clearly self-contained with relevant results to hypotheses. Still, below in section General Comments to Authors, it is listed a series of improvements that should be made to the manuscript.
Experimental design: The manuscript of Fernández and colleagues (#22530) is original primary research that falls within Aims and Scope of the journal.
Validity of the findings: The conclusions of the manuscript of Fernández and colleagues (#22530) are well stated, linked to original research question, and limited to supporting results. There are no obvious speculations.
Additional comments: The manuscript of Fernández and colleagues addresses the ecological niche modelling of a snake community in a global biodiversity hotspot under climate and habitat change scenarios. I had the opportunity to revise the initial submission and to provide a series of suggestions to improve the quality of the manuscript. Authors provide a new improved version. However, there are still a few aspects that were not addressed in the review. These are listed below.

L103-L129: It was suggested that authors explain better the ecological similarities/differences among the five Thamnophis species under study and how representative they may be of other faunal assemblages occurring in the region. Authors have included a long text about the ecological traits of the genus, but no reference to the two subjects mentioned above. This section (L103-L129) is now very long and does not reply to the two issues raised before. It should be revised

L130-L134: The objectives of the study should be detailed and presented as a list of research questions. These questions should be based on hypotheses that are well explained in the preceding Introduction.

L214-L239: this text is still very long and mostly describing the patterns observed in the figures or tables. As example, in L217-L218 it is repeated the information (%) given in Table 1. In L223-L226, the text provides a simple description of the plots, which readers can observe by themselves. In L250-L252, the AUC values are repeated from Figure S1. Delete either from main text or from figure S1.
Please do not repeat information given in Tables/Figures in the main text, and vice-versa.

L354-LL364: I acknowledge the effort to turn the predictions for genus Tamnophis comparable to other fauna, but this text (L354-L364) is very long, providing many species names, but without explaining why are the “land-use and climate change in the TMVB” expected to cause changes in these other species. The examples listed in L356-L364 could be reduced to 2 or 3 maximum.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF MIXING EGGS OF DIFFERENT INITIAL INCUBATION TIME ON THE HATCHING PATTERN, CHICK EMBRYONIC DEVELOPMENT AND POST-HATCH PERFORMANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The quality of written English is basic, often with missing words and unconjugated verbs. This is visible throughout the whole text but particularly noticeable in lines 9, 16-18 (abstract), lines 26-36 (introduction) and lines 158 and 163 (discussion).
The methods section in the abstract chapter requires more detail.
The introduction section is not focused and does not fully explain the background supporting the work.
Information that should be in the Materials and Methods section (such as the fact that the eggs were laid on the same day and stored for no longer than 2 days) is instead featured in the Discussion, in lines 155-156.
Experimental design: The research is original and the research question is well defined.
However, the description of the methods does not allow for sufficient detail to replicate the study as the exact number of eggs in each study group is not divulged (only the number of chicks used for the post-hatching trials), although it can be estimated from raw data. Also, there is no mention of mortality rates or even how (or if) the EMI and LMI eggs were marked so that they could be later separated for hatching. There is also no explanation offered on why these two groups were separated for the hatching stage.
Validity of the findings: The data is robust and statistically sound.
However, it is not mentioned that different numbers of eggs were incubated/set to hatch for each group (as calculated from the number of eggs hatched vs hatchability per group - EI=151, EMI=126, LI=139, LMI=118) and it is not discussed how a larger number of eggs could influence the hatch window.
Discussion of the results is mostly supported by speculation around sound stimulation and CO2 stimulation of embryos. However, it does not address the fact that multi-stage incubators are still often used, mixing eggs which have started incubation 24, 48, 72 hours apart. Is this not one of the main reasons why a study like this would be meaningful?
The conclusions taken are limited and not quite clear.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF MIXING EGGS OF DIFFERENT INITIAL INCUBATION TIME ON THE HATCHING PATTERN, CHICK EMBRYONIC DEVELOPMENT AND POST-HATCH PERFORMANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study that provides additional information about ways of decreasing the hatch window and increasing the uniformity of chicks in order to ensure good post-hatch performance.
The manuscript is generally well-written although I have made many minor suggestions for changes to increase the clarity and readability of the paper.  Sufficient background and context are provided and the results are relevant to the stated aims.
Experimental design: The experimental design is appropriate to the stated aims of the study.  The study appears to have been performed to a high technical and ethical standard.
Validity of the findings: The reported data appear to be robust and the discussion and conclusion are consistent with the findings.
Additional comments: The manuscript is generally well-written.  However, for authors for whom English is not the first language, it is always helpful to have the manuscript read by a native English speaker, if at all possible.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF MIXING EGGS OF DIFFERENT INITIAL INCUBATION TIME ON THE HATCHING PATTERN, CHICK EMBRYONIC DEVELOPMENT AND POST-HATCH PERFORMANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: English still needs revising as there are still various points where words are missing or in excess. For instance, the sentence in lines 39 to 42 is not a sentence, as it lacks a verb.
Likewise, in the Materials and methods section, line 66, the EI group is wrongly labeled as the LI group.
Experimental design: The total mortality should be divided between: 1) dead-in-shell embryos and 2) unhatched eggs.
Validity of the findings: No comments
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF MIXING EGGS OF DIFFERENT INITIAL INCUBATION TIME ON THE HATCHING PATTERN, CHICK EMBRYONIC DEVELOPMENT AND POST-HATCH PERFORMANCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is improved by the authors' adopting the changes suggested by the two reviewers.
Experimental design: The experimental design is clearly stated and ambiguities identified by the reviewers have been addressed.
Validity of the findings: The reporting of the findings has been modified to clarify identified ambiguities.
Additional comments: The manuscript is now suitable for publication in PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING INTRASPECIFIC GENETIC DIVERSITY FROM COMMUNITY DNA METABARCODING DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear English yes.
Relevant literature references provided.
Code shared, good figures.
Objective needs to be clarified, see comments in the attachment.
Experimental design: Original primary research within scope, yes.
Research question should be re-framed, see attached comments.
Good preliminary investigation, see attached comments.
Wet lab methods not described in sufficient detail.
More clarity needed for bioinformatics methods, see attached comments.
Validity of the findings: Title and abstract should be edited to reflect the discussion which provides a more thoughtful and balanced perspective for using partial CO1 sequences for assessing intraspecific genetic diversity.  See attached comments.
Additional comments: This paper is nice but would benefit from the addition of method details.  This paper could be a particularly timely contribution to the literature if the objective is re-framed as looking at the increased resolution provided by ESVs compared with OTUs.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING INTRASPECIFIC GENETIC DIVERSITY FROM COMMUNITY DNA METABARCODING DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is timely and useful research. I'm intrigued by it's potential and I look forward to trying this haplotype analysis in my own work.

I have one main question/concern: For the multi-species samples, were they IDed morphologically prior to metabarcoding? and if so, were the OTUs found in line with what had been found based on morphology?

My only other comments are minor grammatical changes:
Line 16: Change 'loosing' to 'losing'
Line 18: Hyphenate 'abundance based'
Line 33-34: Change 'to capture' to 'of capturing'
Line 49-50: There is a strange break before the period. Perhaps an improper line break/enter?
Line 53: Change 'milestone' to 'leap'
Line 57: Place a comma after 'management'
Line 74: Move 'collected' to between 'we' & 'multi-species'
Line 83: I am not sure why there's a '(1)'
Line 100: Change 'into' to 'in'
Line 114: Place commas before and after 'in fact'
Line 180: Hyphenate 'metabarcoding based'


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING INTRASPECIFIC GENETIC DIVERSITY FROM COMMUNITY DNA METABARCODING DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language quality is good, except in the M&M section where some typos and unprecisions were present.
L93 First mention of expected error (ee) should be written out.
L70 hyphen between metabarcoding and based
L74 “Subseqeuntly, we multi-species metabarcoding data…” might be a word missing in this sentence.
L88 brackets are in odd number.
L95, L98, L102. Different names for the same thing, respectively “monitoring bulk samples”, “bulk samples” and “monitoring samples”. Please pick one and use it throughout the MS.
L97 is it dereplication or deduplication?
L102 multi instead of Mulit
L100-102. What was the reference chosen for the Finnish multi species samples mapping?. Also with Vsearch or another mapper (Usearch?)

The background information provided in the introduction and the discussion of the results are appropriate and helpful for placing the present study into context.
Experimental design: This regards self containment and design both: to be able to reach a broader audience, this paper lacks in clarity and it is difficult for somebody new to the analysis to fully understand the steps taken. It helps to read the documentation available on Github for the JAMP package, but I am critical of papers where you actually have to read two other papers and a few Github commits to be able to follow. I am also puzzled by the Github readme of JAMP, which says: “At this point it is intended for internal @leeselab use ONLY”. Isn’t it a bit contradictory?

I have concerns about the material and methods section, which is essential in this paper since it is mostly a methodological one. One of the key criteria at PeerJ is a good description of the experimental design. While the choice of study sites/datasets and the various steps taken to test the several hypotheses are appropriate, the reporting isn’t.
I would recommend revisions, particularly in the very abundant supplementary material, to make it more understandable and eventually ensure a better dissemination. I did look at everything, and while I appreciate the authors’ openness, it is not usable as of now. In particular, it is not clear at first glance what is used as an input for what step. Also, sometimes I have the feeling several versions of the same script were uploaded. An example is denoise, I found denoise v6 and denoise v7. Which one is the appropriate one? Are they really different?
I am aware it is somewhat unusual to put so much emphasis on suppmats, but in this case, the work product is a software package, where documentation is essential.

I woudl like to make clear at this point that I am sure the authors did well and I do not doubt the results at all, only the amount/reporting of details provided.
Validity of the findings: The authors also honestly and clearly identify the limitations of their study or more generally of the approach itself. Furthermore, they point out how an approach of this kind could help identify interesting study sites/populations.
Additional comments: Elbrecht et al present a tool to mine the wealth of data „hidden“ in metabarcoding approaches. These are very widely used to describe community’s composition or subsets of these communities, and are based (most of times) on sequences of the cytochrome oxidase subunit I when analysing animal DNA. Elbrecht et al want to show that the intraspecific diversity can be described as well in a reliable manner, thus allowing going one step further in monitoring. I commend the initiative and think this study is very interesting from a personal point of view, and would be useful to a broader community.
The issues with the M&M section need to be addressed.

Suggestion for Figures:
I would increase the contrast in Fig1. A, the grey and blue symbols are very light. Furthermore, the extensive use of green and red make Panels A and C unsuitable for color-blind readers. Please consider using magenta and blue instead in Panel C. Same goes for the haplotype maps


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE GENES IN HAEMOPHILUS PARASUIS ISOLATED FROM PIGS IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study screened multiple antimicrobial resistance genes by PCRs in 143 Haemophilus parasuis isolates in piglets from China. The molecular epidemiology of isolates was also analyzed.
This study is a purely epidemiological survey, and the information provided is limited. The manuscript was not prepared well. Data presentation (in the text and especially in tables) and discussion should be more concise and effective. The English language needs a thorough check, preferably by a native speaker.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Many sentences are difficult to understand and many parts need rephrasing for example but not limited to
Abstract
line 14: "Haemophilus parasuis is a common porcine respiratory disease". Haemophilus parasuis is a pathogen but not a disease.
line 23: "blaTEM-1" not "TEM-1". Resistance genes "rmtD, aadA1, aac(3’)-ⅡC" should be writtern in italic. Similar errors exist in Supplemental S2.
line 27: "pulsed-field gel electrophoresis". The letter "p" should be capitalized.
line 30: "high-level antibiotic resistance" would be better.
line 32: The author stated that "GyrA gene mutation also was the most important role in quinolone resistance". Actually, whether these gyrA mutations in this study cause quinolone resistance has not been validated.

Materials and Methods
line 71: A total of 143 or One hundred and forty-three.
line 92: change including to mediating


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE GENES IN HAEMOPHILUS PARASUIS ISOLATED FROM PIGS IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Several grammatical shortcomings have been identified throughout the manuscript. I suggest the manuscript should be revised by a native English speaker.
Experimental design: Some additional tests should be added, for example S1-PFGE and plasmid conjugation.
Validity of the findings: no comment
Additional comments: The manuscript describes antimicrobial resistance genes in H. parasuis isolated from pigs by PCR and pulsed-field gel electrophoresis. Some interesting resistance genes such as rmtB, blaROB, and qnr were found, which probably lead to the high antibiotic resistance of H. parasuis in piglets, but all of them lacked in-depth analysis. The reported data may be interesting for the readership. However, major revision need to be made before it will be accepted for publication.

The amendments are as follows,
Several grammatical shortcomings have been identified throughout the manuscript. I suggest the manuscript should be revised by a native English speaker.

In sections of the methods, “Bacterial isolates”: please add a brief description about how to obtain the samples and the detailed methods for isolation the strain.

In sections of the methods, if possible, the authors should study the location and horizontal transfer of some of the important antimicrobial resistance genes.

In the results section, antibiotic MIC values for 143 H. parasuis isolates should list the detailed data.

The following specific comments also need to be considered in the revised version of the current manuscript.
•	The resistance-related protein and antimicrobial resistance genes need to be discriminated, such as ROB-1 to blaROB-1 and TEM-1 to blaTEM-1.

•	Line 36: Abbreviations should be defined when they first appear, such as “Haemophilus parasuis is the etiological agent of Glässer’s disease that….” to “Haemophilus parasuis (H. parasuis) is the etiological agent of Glässer’s disease that….”.
•	Line 71: Paragraphs should not begin with numbers. Please, rephrase the follow sentence: “143 H. parasuis strains were isolated from diseased swine suffering ….”.
•	Line 152-153: The name of bacterium should be in italic type.
Tables and Figures
•	Table 4 This table is too long, remove the Isolate column, re-design the table.
•	Table 5 The expression forms of MICs should be unified. Please, “——, —” by “-”; The unit of MICs should use μg/mL.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE GENES IN HAEMOPHILUS PARASUIS ISOLATED FROM PIGS IN CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is scientifically sound, professional and interesting. It reports new findings, that merit publication.
Figures and Tables should be clarified as stated in my report, e.g. Supplemental Material should be a Table in the manuscript.
Experimental design: No concerns at all. The study is well designed.  Information of the antimicrobial susceptibility of all isolates should be included, if available.
Validity of the findings: Data are robust and relevant. The presentation of the results is somewhat confusing. With suggestions about Figure 1 and Supplemental Table, linking the data, will very much help interpretation of the results.
Additional comments: The manuscript by Zhao et al -Characterization of antimicrobial resistance genes in Haemophilus parasuis isolated from pigs in China- is of great scientific relevance and very interesting. Some clarification in the data would largely facilitate the Reading and interpretation of the results.

Has an antibiogram been performed for all the 143 isolates? Only 73 had one of the genes tested? The identification of the gens is interesting, but one should know if the genes are expressed and confer resistance or not.
No integrons have ever been identified in Pasteurellaceae. Please do not mention or discuss this, as in L212.

Do the mutation in the QRDR correspond to presence/absence of qnr genes?

Table S2 is much mor important and informative than Tables 1 and Table 2. Please include Table 3 in the main text. Otherwise the Pulsotype in Figure 1 is not interpretable.

Figure 1. Include a column on the right of the strain name with the genes present in each isolate. This will help the reader to identify clonality of the isolates.

Minor comments: L217 (…) in Gram negative and Gram-positive bacteria from pigs such as Salmonella spp., E. coli or Streptococcus suis (Cao et al 2017, Escudero JA et al Antimicrob Agents Chemother. 2007 Feb;51(2):777-82.)

L182 (…) and resistance is increasing among Gram-negative pathogens (Du et al 2009), being sometimes clonal spread of a single pulsotype (Hopkins KL et al. Emerg Infect Dis. 2010 Apr;16(4):712-5. ). In our case, a clone bearing rmtB HP118 and HP037, was present in two different regions.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE GENES IN HAEMOPHILUS PARASUIS ISOLATED FROM PIGS IN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed most comments. But, it still needs attention in some places, for example, in Table 1, resistance genes should be written in italic. In addition, please add the information on how to identify H. parasuis. Other suggestions are presented as the following.
Experimental design: No concerns at all.  if possible,  the information on how to identify H. parasuis. should be added.
Validity of the findings: no comment.
Additional comments: (1)	For Abstract, no information was on description of fluoroquinolone antimicrobial susceptibility testing in Methods and Results.
(2)	Lines 70-71: “Verbal informed consent was obtained from the owners of the pigs before this study” should be deleted.
(3)	Lines 115-116: “Information of the fluoroquinolone antimicrobial susceptibility of all isolates are listed in supplemental S1. The results of fluoroquinolone antimicrobial susceptibility testing of 143 H. parasuis isolates towards six fluoroquinolone antibiotics showed that 82.52% were resistant to nalidixic acid and 55.94% were resistant to enrofloxacin.”
It could be changed into “the results of the fluoroquinolone antimicrobial susceptibility of 143 H. parasuis isolates are listed in supplemental S1. It showed that 82.52% and 55.94% of all isolates were resistant to nalidixic acid and enrofloxacin, respectively.”
(4)	Lines 196-197: “However, until now, few studies have described the presence of the armA and rmtB genes in food animals.”
It could be changed into “However, until now, few studies have described the presence of the armA and rmtB genes in H. parasuis isolates, although they have been frequently reported on Enterobacteriaceae from food animals.”
(5)	Table 1, 2 and 3 are not very useful and can be supplementary files.
(6)	Table 1,
1)	“TEM” represents the name of enzyme. “blaTEM ” represents the name of resistance gene. “TEM” should be changed into blaTEM”, please checked other genes.
2)	“fosA3” confers resistance to fosfomycin, not macrolids.