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: CYTOLOGICAL ANALYSIS OF TRACHEAL WASH AND BRONCHOALVEOLAR LAVAGE FLUID IN HEALTH AND RESPIRATORY DISEASE IN DROMEDARY CAMELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are many spelling and grammatical errors throughout the manuscript. The reviewer indicated some of them but the authors should read carefully the entire manuscript to correct all mistakes.
Experimental design: The study design is straightforward however, the diseased group should be better characterized in the results section. For example, the clinical score should be reported (mean +/- SD or median, IQR as appropriate) for both diseased and healthy camels. Also, what were the final or presumptive diagnoses based on the work-up performed at the hospital (e.g. viral, bacterial, fungal, or parasitic infections or non-infectious respiratory disease)? Pneumonia vs. upper airway infection? Bacterial vs. viral diseases? This information may be provided in table format.
Validity of the findings: Mast cell granules do not stain with Diff Quick (Leclere et al. J Vet Int Med 2006). How did you differentiate mast cells from other cells?
Additional comments: PeerJ #54751
Cytological analysis of tracheal wash and bronchoalveolar lavage fluid in dromedary camels
The authors report their findings on cytology of respiratory secretions collected in dromedary camels by tracheal wash (TW) and bronchoalveolar lavage (BAL). Animals sampled included 9 clinically healthy camels and 18 presenting signs of respiratory disease. Tracheal washes and BAL were performed trans-orally under endoscopy guidance in sedated camels. Samples were processed within 1 hour of collection, stained with Diff Quick and differential count obtained by counting 400 cells.
The main finding was that in healthy camels, neutrophil and epithelial cell proportions were higher in TW than BAL samples while lymphocyte proportions were lower in TW than in BAL.  Camels with respiratory disease exhibited higher proportions of neutrophils in both TW and BAL compared to healthy camels.
According to the authors, this is the first report of cytological analysis of respiratory secretions collected by TW and BAL in dromedary camels. As such, these findings provide valuable information concerning normal airway cytology as well as abnormalities commonly identified in camels with clinical respiratory disease.

General comments:
This reviewer was able to find 2 manuscripts reporting TW cytology in dromedary camels during a cursory Google Scholar literature search:
-	Habasha FG and Hussain MH. Cytological analysis of transtracheal washes from healthy camels in Al-diwaniya province. AL-Qadisiya Journal of Vet. Med. Sci.2014 (13) 1.
-	Habasha FG. Clinical & Diagnostic Study of E-coli from Camels with pneumonia using VITEK 2 Compact, histopathology & conventional PCR. - qu.edu.iq
It does appear that this study is the first to report BAL cytology in camels and the authors should be congratulated for such an accomplishment.
It would be helpful in the introduction to be more about specific about the type of respiratory diseases responsible for high mortality rate (bacterial vs. viral infections? PI-3, pasteurellosis, etc.?). What respiratory diseases are the most commonly diagnosed in camels in Saudi Arabia?
The discussion of sampling of respiratory secretions in the introduction needs to be more nuanced. In horses, TW is preferred for diagnosis of respiratory infections (both cytology and microbiologic culture) whereas BAL is preferred for diagnosis of non-infectious pulmonary diseases. However in cattle, BAL was shown to be equivalent to TW for the diagnosis of common bacterial and viral respiratory pathogens (Doyle et al. J Vet Int Med 2017). Also, the authors should talk about the fact that respiratory cytology may provide a presumptive diagnosis of infection rapidly (Jocelyn et al. Equine Vet J 2018) but ultimately, identification of the etiologic agent requires bacterial or viral culture or PCR.
The study design is straightforward however, the diseased group should be better characterized in the results section. For example, the clinical score should be reported (mean +/- SD or median, IQR as appropriate) for both diseased and healthy camels. Also, what were the final or presumptive diagnoses based on the work-up performed at the hospital (e.g. viral, bacterial, fungal, or parasitic infections or non-infectious respiratory disease)? Pneumonia vs. upper airway infection? Bacterial vs. viral diseases? This information may be provided in table format.
Mast cell granules do not stain with Diff Quick (Leclere et al. J Vet Int Med 2006). How did you differentiate mast cells from other cells?
Presumably, some diseased camels were affected by bacterial pneumonia. In such cases, a large portion of BAL neutrophils would be expected to show degenerative changes and potentially intra- and extra-cellular bacteria. Please provide those findings in the results section and comment in the discussion.
Data regarding potential complications or side-effects related to TW/BAL observed during this study (or lack thereof) should be presented in the results section.
The discussion related to neutrophils should appear first after discussion of total cell count (e.g. line 209) and before discussion of macrophages, lymphocytes, etc. The reason is that the neutrophilia is the main cytological finding of importance. The other changes are related to the fact that if neutrophil proportion increases then, other cells will be present in smaller proportions (max 100%).
There are several spelling and grammatical errors throughout the manuscript. The reviewer indicated some of them but the authors should read carefully the entire manuscript to correct other mistakes.

Specific comments:
-	Line 51: One article cited in the introduction in support of the statement that camels are susceptible to a variety of respiratory pathogens is not related to respiratory diseases and should be removed (Scaglione 2017).
-	Line 92: It is preferable to say “… lung sounds and rate of breathing were recorded”.
-	Line 96: consider writing “Camels with a clinical score less than 3 points were considered healthy while camels with a clinical of 3 points or higher were considered diseased.”
-	Line 99: consider replacing “fixed” by “positioned”.
-	Line 102: Replace “application of xylazine” by “injection of xylazine”. How was xylazine administered? Intramuscularly or intravenously?
-	Line 103: spelling mistake: “mouth gauge” should be replaced by “mouth gag”.
-	Line 104: consider “…to keep the mouth open to protect and allow easy passage of the endoscope”.
-	Line 109: the TW was done via endoscopy using a catheter to instill and recover saline solution. Why did you advance the endoscope and catheter into bronchi (e.g. first of second generation) when you explain subsequently that you sampled mid-cervical trachea? Also, if saline is infused mid-cervical region, the fluid would flow to the lowest point of the trachea (thoracic inlet) and the tip of the catheter would have to be advanced further in order to aspirate it back. Please explain.
-	Line 114: spelling mistake; replace “preformed” by “performed”.
-	Line 117: correct “mouth gag”
-	Line 123: spelling mistake; replace “installed” by “instilled”.
-	Line 120: consider “As soon as the tube was wedged into the bronchus, the cuff…”
-	Line 129: what is a Barker’s chamber? Total cell counts are usually determined using a hemocytomer.
-	-line 135: Reference to Fig. 3 should be moved to the results section.
-	Line 153: specify how many camels required use of the endoscope to pass the BAL tube.
-	Line 157: cells labelled macrophages in Fig. 3 appear to be lysed and possess more neutrophil than macrophage morphological attributes. Because of the degree of cell lysis, these cell should not be identified. Another photomicrograph would be preferable.
-	Line 167: “oral squamous epithelial cells”.
-	Line 171: “cells/µl”
-	Line 177: “epithelial cells…”
-	Line 194: Others have perform trans-tracheal wash to bypass the upper airway thereby, decreasing the likelihood of oral contamination (see Habasha 2014). This possibility should be discussed.
-	Line 239: Please discuss this finding of ciliocytophthoria as potential indicator of viral disease (Gelardi et al. 2016).

Figure 3: Indicate magnification or insert a scale for each photomicrograph (photo II is clearly at a lower magnification compared to the other photos.)
Figure 4: A scatter-plot with mean and SEM would be more appropriate to display the results and provide the range of values. Also, only indicate  p-values < 0.05 on the graph.


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: CYTOLOGICAL ANALYSIS OF TRACHEAL WASH AND BRONCHOALVEOLAR LAVAGE FLUID IN HEALTH AND RESPIRATORY DISEASE IN DROMEDARY CAMELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Much improved version. Some minor editorial comments, as below

Line 76-78 Sentence: “Although respiratory cytology may provide a presumptive diagnosis of infection rapidly but ultimately, identification of the etiologic agent requires bacterial or viral culture or PCR (Jocelyn et al. 2018)”
suggest rephrase to: “Although respiratory cytology may rapidly provide a presumptive diagnosis of infection, identification of the etiologic agent requires bacterial or viral culture or PCR (Jocelyn et al. 2018)’”

Line 107-110 Sentence “ Clinical score points were recorded by two authors, but not blindly, so it was not possible to ask the owner of the diseased camel about the case history and repeat the examination in the Veterinary Teaching Hospital. ”  confusing, please reformulate.

Line 116 , 120 Dose of xylazine, one place uses0,1 mg/kg, the other 0.1 mg/kg. Please use one or the other for consistency

Line 117 “ a special mouth gag… (not gage)

Line 192   symbol…     ± 39.2 cells/µlL)

Sentence  line 212-215 The percentages of neutrophils in TW (78.83 ± 3.89 %) and BAL (31.42 ± 4.33%) samples from the camels with high rectal temperature were higher than their percentages in the TW (51.22 ± 5.9%) and BAL (14.76 ± 1.99%) samples from the animals with normal rectal temperature.
Discussion: lines 219-234. Much improved.
Line 235 … with saliva might have resulted from….

Line 285…..” cilia, which could have originated from the inflamed airways”
Lines 290-294  suggest rephrasing as
Finally, one of the limitations of the present study is the cell identification method. Although it is widely used for cytological analysis in several species (Jackson et al. 2013), staining with Diffquick may fail to reliably identify all cell types (Leclere et al. 2006). Specifically,  the estimated frequency of mast cells in the BAL samples may have been affected by Diffquick staining


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: CYTOLOGICAL ANALYSIS OF TRACHEAL WASH AND BRONCHOALVEOLAR LAVAGE FLUID IN HEALTH AND RESPIRATORY DISEASE IN DROMEDARY CAMELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been revised to the satisfaction of this reviewer.
Experimental design: The manuscript has been revised to the satisfaction of this reviewer.
Validity of the findings: The manuscript has been revised to the satisfaction of this reviewer.
Additional comments: Thank you for correcting the manuscript in response to this reviewer's comments. The only minor correction to make is to insert units Line 93 & 96 (e.g. years, kg)


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: CYTOLOGICAL ANALYSIS OF TRACHEAL WASH AND BRONCHOALVEOLAR LAVAGE FLUID IN HEALTH AND RESPIRATORY DISEASE IN DROMEDARY CAMELS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Minior comments:
Line 121,  please provide the route of  the 0.05 mg/kg xylazine (I presume it was also IV as per induction?

Line 145, typo- "hemocytometer.
Experimental design: ok
Validity of the findings: ok
Additional comments: as above


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: CYTOLOGICAL ANALYSIS OF TRACHEAL WASH AND BRONCHOALVEOLAR LAVAGE FLUID IN HEALTH AND RESPIRATORY DISEASE IN DROMEDARY CAMELS
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: The authors have duly revised the manuscript.


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: ANALYSIS OF CERNA NETWORK OF DIFFERENTIALLY EXPRESSED GENES IN FADU CELL LINE AND A CISPLATIN-RESISTANT LINE DERIVED FROM IT
Review round: 1
Reviewer: 1

Output:
Basic reporting: -English style needs a bit of improvement. There are a few typos in the following lines: 30-csplatin, 47-cord, 119 sift.
-Line 94, use script for 103.
-Scale bar missing in Fig 1A and 1B. Please include those!
-Some names in Fig 3 and 4B are overlapping, hence rendering them unreadable.
 Please redo these figures.
Experimental design: At the moment, there is no information on how many biological replicates each experiment has. Please provide 'n' for each experiment.
Validity of the findings: -Fig 1D and 5 have the error bars. From how many experiments these data are represented? Are these the technical replicates or the biological replicates and how many experiments?
-For the statistics in Materials and Methods, the authors did not mention whether they used paired t-test or unpaired t-test. Please indicate that.
Additional comments: -For the qPCR results in line 186, the representation of the results in a table format is recommended.
-In the discussion, please mention the limitations and future directions for this study.
-For Fig 2B, the indicated p-value range is from 0.1 to 0.5. Why these p-values are so large? What are they representing?


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: ANALYSIS OF CERNA NETWORK OF DIFFERENTIALLY EXPRESSED GENES IN FADU CELL LINE AND A CISPLATIN-RESISTANT LINE DERIVED FROM IT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The title, abstract, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the experiments are well conducted, and analysis is well performed. The figures are relevant, high quality, well labelled and described.
Experimental design: The experimental design is original and the research is within the scope of the journal.. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
Validity of the findings: All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.
Additional comments: This paper investigates the function of expression of competitive endogenous RNAs (ceRNAs) in cisplatin-resistant hypopharyngeal cancer by comparing differentially expressed genes (DEGs) between cisplatin-resistant hypopharyngeal cancer cell lines and ordinary hypopharyngeal cancer cell lines. The authors identified  a bunch of miRNAs, lncRNAs, circRNAs and  mRNAs that have greater enrichment. To explore further about the related pathways, the authors demonstrate that these genes are functionally related to TNF IL-17 and JAK-STAT signaling pathway utilizing DAVID. To make the findings more convincing, the authors chose several critical miRNAs and mRNAs to perform the validation experiments.

Editorial Criteria
BASIC REPORTING
The title, abstract, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the experiments are well conducted, and analysis is well performed. The figures are relevant, high quality, well labelled and described.
EXPERIMENTAL DESIGN
The experimental design is original and the research is within the scope of the journal.. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
VALIDITY OF THE FINDINGS
All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.

Overall, I think this paper is novel and will be of interest to the community of hypopharyngeal cancer and cisplatin resistance in cancer in general. The statistical part is valid and makes sense. The authors make it comprehensive by integrating in vitro experiments and analysis of pathways enrichment. The main strengths of this paper is that it addresses an interesting and unexplored question, finds a novel discovery based on a carefully selected set of bioinformatic and experimental procedures. Some of the weaknesses are the not always easy readability of the text. Another limitation is that the sample size is relatively small. In general, the work is convincing except some major and minor comments below:

Major Comments:

I’m wondering if there are any ongoing clinical trials focusing on the miRNAs and mRNAs identified in this study in hypopharyngeal cancer or head and neck cancer? It will be very strong evidence for the significance of the current study if so.

I’m just wondering if there is any TCGA or GEO hypopharyngeal cancer dataset with drug resistance and transcriptome profiling data. It will be great if the results could be validated using an external dataset. Please also check if there is cell lines expression data with drug response information (eg., IC50) to be able to utilize as validation dataset, like the CCLE dataset (Cancer Cell Line Encyclopedia https://portals.broadinstitute.org/ccle ) and GDSC (Genomics of Drug Sensitivity in Cancer https://www.cancerrxgene.org/ ).

Please add the limitations of this study and future directions in the Discussion part.

Minor Comments:
Background of the Abstract: please add some background and prognosis of hypopharyngeal cancer. Then introduce cisplatin and cisplatin resistance.

It is great that a session of abbreviations was there to list all the abbreviations used in the study. I would recommend to also include abbreviations like FaDu, DEG, KEGG, ceRNA etc in that list.

All the gene names should be italic for all the gene names.

Line 30: typo for “csplatin”.


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: ANALYSIS OF CERNA NETWORK OF DIFFERENTIALLY EXPRESSED GENES IN FADU CELL LINE AND A CISPLATIN-RESISTANT LINE DERIVED FROM IT
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors addressed my points well. The manuscript can now 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: COMPARATIVE ANALYSIS OF FATTY ACID METABOLISM BASED ON TRANSCRIPTOME SEQUENCING OF WILD AND CULTIVATED OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. weak. the manuscript contains several spelling mistakes as well as odd sentences which obfuscate than help to explain findings and discussion.
2. I recommend deeper and more focused on fungi metabolism selection of references. All cited references should be checked again for proper and uniform formating. Correct in line 202, 327 reference “GERHARDT”; it should not be capitalicized . I don’t think the publication on fatty acid metabolism in higher plants is a good reference here. One should refer to either fungi or related eukaryota metabolic pathways.
3. structure ok
4. Line 124 -  the publications by Zhihui et al., 2017 is not indexed in NCBI, nor in other non-chinese speaking platforms. I would like to see much detailed description of the method as I could not verify approach applied in this study.
Experimental design: proper
Validity of the findings: Line 191-193 – this is not a cycle. Both proceses are spatially and temporarly separated and regulated not to form a futile cycle. Remove!
Line 210-211. Fatty acids are not sources of PL and GL. FA are used to synthesize these lipids, and in turn they can be degraded and release FA. The sentence is false.
Figure 4. Fatty acid oxidation (FAO) takes place in mitochondria or/and  in peroxisomes in Ascomycota. There is no consensus about the localization of the pathway in various fungi groups. According to Shen&Burger 2008 Sordariomycota, to which O. Sinensis belongs, utilise mitochondrial and hybrid peroxisomal FAO pathway. As stated before, fatty acids (palmitic acid) synthesis is located in cytosol and it is regulated differentially than FAO. In the bottom of the figure authors suggest that elongation of an acyl chain by two carbons consumes one acetyl-CoA. Wrong. In yeast (and other eukaryotes, thus probably in O. sinensis too), a malonyl-CoA unit is consumed to add 2C. Mitochondria of O. sinensis might possess a conserved pathway of FA synthesis – used to form eight-carbon-long lipoate, a cofactor of KGDH (e.g. Nowinski et al Curr Biol. 2018). The figure 4 suggest that de novo synthesis and FAO coexist and function as opposite processes. This is wrong and needs to corrected.
There are spelling mistakes in this wrong figure.
Line 206 the subclause: ...” and can also be used as a branched-chain amino acid (Ile, Leu, and Val)  metabolic enzymes” has little meaning here – remove it. There are dehydrogenases specific for branched  amino acids and they are not part of the FAO, so as ACAD does not necessarily takes part in BCAA catabolism.
Line 216-219 These two sentences little sense. Butyrate metabolism wasn’t mentioned before. What is the relevance of butyrate metabolism in O. sinensis? Is this one of key synthesized metabolites of it? How “alanine aspartate metabolism” is related with acetyl-CoA. Is FAO the main mitochondrial acetyl-CoA producing pathway in the fungus? What is the proportion of glycolytic pyruvate or degradation of host proteins (supply of TCA intermediates from amino acids degradation) ? How elevated expression of biosynthetic and catabolic genes would supply more energy to the wild fungus?
Additional comments: Line 99 – invalid URL, is “cdu" should be “edu”
Line 136 – invalid URL
Line 193 – Acetyl-CoA should be abbreviated Ac-CoA. Correct throughout the manuscript.
Line 207 “homeopathic”
Line 213 “electronic”, spelling mistake
Line 233 what are  these “previously reported conclusions”?
Line 258 what is meant by the term “strong fruiting bodies”? Clarify.
Line 283-286 rephrase the sentence as it makes little sense at the moment


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: COMPARATIVE ANALYSIS OF FATTY ACID METABOLISM BASED ON TRANSCRIPTOME SEQUENCING OF WILD AND CULTIVATED OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript entitled “Comparative analysis of fatty acid metabolism based on transcriptome sequencing of wild and cultivated Ophiocordyceps sinensis”, the authors aimed to investigate the differences of O. sinenesis by performed morphological and transcriptomic comparisons between wild and cultivated O. sinensis. The manuscript generally includes the appropriate references to describe the background of this study. However, it is needed to inform and describe the specimens collection for further DEGs analysis and statistical analysis. Besides, some figures poorly represented. They are needed to have large improvements.
Experimental design: The authors should describe detailed methods to provide reliability and reproducibility of the analysis. Some points did not relate to each other between methods and results, the suggestions are in the comments. In particular, when you collected specimens from wild samples and cultivate samples, the authors should inform the substrates for cultivation as well e.g., cultivated O. sinesis. Since DEGs will change upon nutrients. Moreover, for natural growing O. sinesis, the authors also need to describe more details.
Validity of the findings: The authors found that key DEGs related to fatty acid metabolism and were upregulated in wild O. sinensis. The expression level of these genes was validated with the qRT-PCR result. Also, the fatty acid content was determined using GC-MS. These results well support each other. The conclusion is stated and linked to the research question.
Additional comments: Major comments
1. Due to environmental factors affect to O. sinensis, the authors should add more details in the specimen collection part, which season that wild O. sinensis were harvested. Also, the condition which was used for cultured samples should be defined, such as medium or host, temperature, light intensity, and humidity.
2. Which part of O. sinesis (worm body or fruiting body) was chosen for total RNA extraction to perform RNA sequencing and qRT-PCR, and for fatty acids extraction to determine the content of fatty acids?
3. To ensure that an international audience can clearly follow experiment. The method of sample preparation and fatty acid extraction should be explained in your manuscript because, in the previous study (Zhihui et al., 2017), it was reported in the Chinese language.
4. Which criteria were used to select the DEGs? In this manuscript, there are both “FDR ≤ 0.05 and log2(FC) ≥ 1”, and “FDR ≤ 0.01 and fold change > 2”. The statistic method are needed to add in manuscript in details.
5. How many biological replicates were performed for RNA sequencing in each group? The authors stated that “three biological replicates were performed for each sample” in the materials and methods section, but the heatmap of DEGs in Figure 2D shows only two data in each group. Also, the raw data of cultivated O. sinensis which were deposited in SRA has only two accession numbers.
6. The authors stated that “DEGs were calculated based on FPKM” (line 164). However, the differential expression analysis using the DESeq2 R package use median of ratios as a count normalization method, not FPKM. These are needed to address in manuscript.
7. The authors didn’t describe about the results related to Figure 3B., while Figure 3C was mentioned but it’s not presented in your manuscript.
8. In Figure 4., the authors presented the metabolism and degradation of palmitic acid in O. sinensis. I also suggest that the expression value of DEGs involved in the palmitic acid biosynthesis pathway should be included in this Figure.
9. In Figure 6B., The total ion chromatograms show only the peaks of the OSW_S1 sample and the content of fatty acids between the wild and cultivated O. sinesis were presented without statistical value. So, I suggest that the data of the content of all detected fatty acids in wild and cultivated O. sinesis should be presented as mean ± S.D. (n = 3 biological replicates in each group). The authors need to add statistics in the results.


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: A SYNTHETIC DEFECTIVE INTERFERING SARS-COV-2
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript is very well written. The introduction was clear and covered the most important literature.

Specific comments:
• For line 49, the authors should cite this paper (https://doi.org/10.1371/journal.ppat.1009226) as it has been recently shown that defective viral genomes can be found in SARS-CoV-2. It would be interesting to compare the DI made by the authors to the DIs found naturally in the paper above.
• For line 77-78: Just to clarify…were any aberrations found?
• The resolution of figures was not great.
Experimental design: The concept of using DIs as an antiviral is not novel by itself and has been applied to many viruses already but not too much work has been done yet on SARS-CoV-2. So this manuscript is a good addition to the antiviral DI literature. Overall, the data looks very convincing but there are lots of missing gaps that would make it more convincing that DIs could be used as an antiviral against SARS-CoV-2.

Specific comments:
• Have the authors tested the DI on another cell line? VERO-E6 are not an ideal model as not human, not lung and not having “normal” type I IFN signaling. It would be interesting to see if this DI would be even more interfering in a cell line that has functional type I IFN signaling. DIs have been shown that they can also be immunostimulatory.
• The DI was tested on one SARS-CoV-2 variant. As they are more and more variants have the authors tested if the DI would also be inhibitory against other variants? Is the effect specific to this variant?
• Virus have been shown to evolve to become resistant to DIs (https://doi.org/10.1016/0092-8674(83)90022-3). Is this something authors have observed throughout passages? If sequencing was done, did any new mutations appear?
• The antiviral effect of DI works when DIs are added since the beginning of the infection based on the authors experiments. This is of course not a very realistic scenario in patients. It would be interesting to see if the authors would add the DI at different time points if it affects the antiviral ability of the DI.
• DI have been shown to be antiviral short term but also long term to cause persistence. The authors state that virus is still present in their last passage. Have the authors done more passage and observed if the virus disappears completely at some point? Important to address this as line 255-257 DIs are only portrayed as antiviral.
Validity of the findings: It is encouraging to see that the authors were able to demonstrate that the DI as antiviral approach might also work for SARS-COV-2. Overall, current experiments are well designed but some clarifications are needed.

Specific comments:
• For passage 1, the authors could have generated purified DI particles (DIPs) stocks instead of doing transfection (for example: https://doi.org/10.1093/infdis/jiz564). Having DIs as purified DIPs form would make it easier (as combination of transfection and infection can be very challenging sometimes) also to answer more questions such as the time point comment in the previous section.
• An MOI of 10 was used for original infection. DIs generally are generated at high MOI. Do the authors have any concern that in addition to synthetic DIs you might also detect naturally occurring DIs?
• What did the author do to make sure the different primers have about the same efficiency? This is important as this could bias results.
• Figure 1:Based on methods all these experiments were done in triplicates, but most conditions have more of different amounts of dots. What do these represent? Biological repeats? Technical repeats? What type of statistic was use?
• Line 224-226: This sentence “We were unable to measure the absolute WT/DI ratio because the amount of DI was below the level detectable by digital PCR.” is unclear. How were authors able to make graphs if DI not detectable? Please clarify.
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: A SYNTHETIC DEFECTIVE INTERFERING SARS-COV-2
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: 1. It is unclear from the results whether DI_0 genomes could be detected at any time after transfection. Is it possible that despite their relatively small size that they were unable to be transfected into cells?

2. Lines 145-148, 221-227. It was noted that the DI genome was detected across all four passages (Line 222), but the amount of DI was below the level detectable (Line 225); these statements appear to be contradictory.  Was there a monotonic increase in DI genomes or monotonic decrease of WT genomes across the four passages? It is unclear at what point (which passage) the DI and/or WT genomes fell below the level of detection or quantification.
Additional comments: 3. There are multiple references throughout the report to “DIs” or "synthetic DIs” without specifying whether these are intended to refer to DI genomes or DI particles. Please specify in each case the intent.

Language

1)	Lines 16-17 could be reworded for greater clarity - (in order to replicate and infect other cells, viruses must…)
2)	Also in lines 16-17, clarify precisely what is being referred to as “they” (i.e. viruses, virions, etc.)
3)	In lines 18-20, sentence could be split into two, creating a more succinct flow that makes it easier for the audience to follow
4)	In line 23, specifically in the part that states “enable it to be replicated and packaged”- what is being referred to as “it”? Perhaps more specification would make this part clearer
5)	Verbose sentence in lines 38-41; difficult to follow, may consider restating so that sentence ends after the phrase “full length viruses” and then proceed to elaborate on what would be needed for replication and packaging
6)	The word “they” should be removed from line 42; end sentence after “packaging” in line 42 and begin next sentence with “given” for better flow and clarity
7)	Lines 45-50 should be broken up into multiple sentences → transition into SARS-CoV-2 should be marked by a new sentence, as the information provided in this sentence is quite dense
8)	Perhaps adding broader information regarding SARS-CoV-2 and its relation to the COVID pandemic would be beneficial, as it would provide more context
9)	Remove comma in line 51, specify that “they” refers to the DI genome in line 51
10)	After line 52, add a broader concluding sentence that specifies how replication and  packaging of the DI genome could “impair the growth of the wild type (WT) virus”
11)	In line 67, semicolon should be replaced with comma
12)	Reword line 85 for greater clarity → (i.e. which were later infected with SARS-CoV-2)
13)	Lines 190-194 are verbose; consider removing last part “as most of the RNA cannot replicate and is quickly degraded” because this is redundant (already stated in beginning of the sentence → “Because of the fast degradation of the synthetic RNA inside cells”)
14)	In line 195, restate what “its” is referring to
15)	In lines 195-196, “reduced the amount of SARS-CoV-2 by approximately half” could be stated in a way that better relays the decrease; moving the part that states “within 24 hours of transinfection” so that it is placed immediately after the colon may allow for greater clarity
16)	In line 214, replace “must be” with “is likely” unless results prove this
17)	Remove “it” from line 242, as it is not needed
18)	In line 242, last part could be reworded for greater clarity, so that is effectively relays that the amount of packaging does not meet the threshold for detection
19)	Sentence in lines 257-258, “By enabling replication…” can be omitted, as it is previously stated and thereby redundant
20)	For better clarity, reword lines 261-263; brief discussion of why this “spill over” to humans could be significant might provide helpful context
21)	Consider rewording lines 267-268 (i.e. DI therapy has been attempted to be used on viruses such as HIV and influenza; however, these viruses are not ideal…)
22)	End sentence at the end of line 272
23)	Figure 2 adds helpful context and allows for better understanding of what the primary goal of the study is
24)	Methods are described thoroughly, yet concisely, and provides information that  sufficiently relays primary aspects of the experimental design


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: A SYNTHETIC DEFECTIVE INTERFERING SARS-COV-2
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The author have not performed any new experiments, which would have helped address some of the questions of the reviewers. However, they have made significant changes to the manuscript to focus on 3 main points:
- SARS-CoV-2 DIs can replicate in coinfections (faster than the WT genome)
- SARS-CoV-2 DIs can be packaged in coinfection (as efficiently as the WT genome)
- SARS-CoV-2 DIs interfere with the WT genome (but still not known whether this would be enough for therapy)
The data in this manuscript convincingly address these 3 points. Furthermore, the authors have added many clarifications throughout the manuscript.


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: TAXONOMIC REVISION OF THE TEMNOTHORAX SALVINI CLADE (HYMENOPTERA: FORMICIDAE), WITH A KEY TO THE CLADES OF NEW WORLD TEMNOTHORAX
Review round: 1
Reviewer: 1

Output:
Basic reporting: Matthew Prebus has produced a spectacular taxonomic revision of the Temnothorax salvini clade. It is a remarkable body of work, being informed by molecular phylogenetics, but making full use of morphological data to best characterize species and to provide keys. This is one of the best alpha taxonomy works I have ever seen.

I waive anonymity. I contacted the author and obtained a Word version of the manuscript, so that I could make detailed edits using Track changes. There were scattered minor edits of spelling and formatting, and some missing references. I returned the edited manuscript directly to the author.

A couple of general comments:

The penultimate paragraph of the Introduction needs expansion. It treats the three morphological groups in the salvini clade: macromischa, pergandei, Myrfant-like. Later in the manuscript it is made clear that a molecular phylogeny of the clade is in the works. The molecular work has informed the definition of the clade, but diagnosing it morphologically requires a combination of traits. The manuscript provides a key to clades and a morphological diagnosis of the salvini clade. But this is not clear in the Introduction. You need to tell readers here that these resources are part of the paper.

Also, Prebus 2020 appears in the Methods, but needs to be integrated in the Introduction.
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: TAXONOMIC REVISION OF THE TEMNOTHORAX SALVINI CLADE (HYMENOPTERA: FORMICIDAE), WITH A KEY TO THE CLADES OF NEW WORLD TEMNOTHORAX
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Dear Dr. Matthew M. Prebus,

I would like to congratulate the author for the initiative and hard work in conducting the Taxonomic Revision of the Temnothorax salvini Clade.

The manuscript provides a taxonomic revision of the salvini clade of ant genus Temnothorax, in which 35 new species are described and other nomenclatural acts are taken. The manuscript is complete and well-founded. In addition to taxonomic contributions, the manuscript provides insights into new issues to be investigated, such as the relationship between the size of the sting and the stratum that species occupy.

My comments are mainly focused on the structure of the species descriptions, on the standardization of small inconsistencies that I detected. My revision is available in the attached file.

My thanks to the author for his effort to make this taxonomic revision available to the scientific community.


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: TAXONOMIC REVISION OF THE TEMNOTHORAX SALVINI CLADE (HYMENOPTERA: FORMICIDAE), WITH A KEY TO THE CLADES OF NEW WORLD TEMNOTHORAX
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is a species-level taxonomic revision of a large group of ants (Temnothorax salvini clade) occurring in the Nearctic and northern Neotropical regions. It is a notable accomplishment, treating the group carefully and comprehensively. This is essentially a taxonomic monograph—if PeerJ is open to publishing such large papers, then that is welcome. I have to say that, despite two attempts, I was unable to download the full complement of files. The zip file was too large, it seems. So, my review is based on examination of the main text of the paper and selected images (about 80 of them) that I downloaded individually.

I do not have any major criticisms of this study. It is a stellar contribution to Temnothorax systematics, and it stands as a model for modern, intelligently-executed, species-level taxonomy. Much of the recent taxonomic literature on ants consists of short papers describing one or two new species, without a broader synthetic overview, so it is a pleasure to see a really comprehensive treatment of a species-rich group, over a large geographical area. The work is grounded in detailed examination of morphological variation in a large sample of specimens, and backed up by an impressive molecular (phylogenomic) dataset. The paper is also copiously illustrated, with high quality specimen images and and distribution maps. The use of unique specimen identifiers is exemplary, as is database management of the specimens examined.

My main concern is a practical one: will this paper, with its full set of illustrations, be capable of being easily downloaded, and will it be kept to a reasonable size? Unlike other papers, for which online access is generally sufficient, there is a specific utility to having taxonomic papers available as offline copies. One might be in the field, for example, without internet access, and want to use the keys. Indeed, while I don’t want to devalue the detailed and careful species descriptions, let’s be honest: the parts of this paper that will be most frequently consulted by future users are the illustrated keys and the differentiating comments in the species accounts. So, I hope that in the production of this paper the issue of portability is kept in mind. This might require some discussion between PeerJ and the author—perhaps reducing the resolution of some images and having them available as high-resolution images in supplementary material.

I have quite a few other comments and corrections, but they are mostly minor and should be readily addressable.

In the Abstract (and References) you transcribe “Neotropical” in two different ways (capitalized and not). I would capitalize throughout.

Lines 44-46. The placement of the last phrase (“by evolutionary convergence”) leads to some ambiguity. Rewrite for clarity:
“…have arrived by evolutionary convergence at a morphological syndrome with characteristics that were used to diagnose the former genus Macromischa.”

Line 62. Change “longcaulis” to “longicaulis”.

Lines 100-101. Reposition “in”.

Line 112. Change “has” to “have”.

Line 163. Delete “in”.

Lines 164 et seq. The name “Palearctic clade” emphasizes that most members are found in that region, but it is a bit misleading in that the clade is actually Holarctic. I guess that the name is now entrenched, but “Holarctic clade” would have been a better moniker.

Line 175. Insert “of” after “majority”.

Line 178. For clarity change “certain species of the Mediterranean” to “certain species of the Mediterranean region of Europe” (if that is what you mean). See also line 6720.

Line 208. “ocarinae” is misspelled.

Line 209. Change “only known” to “known only”.

Lines 311-312. “Diagonal petiolar length in lateral view; measured from the junction between the apex of the subpetiolar process to the posterodorsal corner of caudal cylinder”.
This is confusing. What do you mean by “from the junction between   to”? This would make more sense if the phrase “the junction between” was removed:
“Diagonal petiolar length in lateral view; measured from the apex of the subpetiolar process to the posterodorsal corner of caudal cylinder”.

Lines 314-316. The definition of NOL (petiole node length) is a bit odd. I think it would be better defined by the reference line that you describe in lines 318-320.

Lines 321-323. It is probably worth adding that this measurement of the length of the postpetiole excludes the helcium.

Lines 350-351 repeat lines 348-349.

Lines 388-389. “In case where coordinates were not given on the specimen labels, they were estimated from label data with Google Earth; these coordinates are provided in brackets, with a standard error of one minute.”
You cite decimal coordinates so it is curious that you here cite a standard error of “one minute”.

Lines 404 et seq. Please provide locations for collections BEBC and MMPC (even if they change in the future).

Line 429. BMNH is the more common (and better recognized) acronym for the Natural History Museum, London.

Line 533. You cite Fig. 12f as an example of a long node, yet the dashed lines in that figure signify a different aspect of node length than NOL (cf. Fig. 6f).

Lines 536, 539. Change “short a” to “a short”.

Lines 681-620. “The species groups used in this study correspond to morphologically diagnosable groups of species which are supported by molecular evidence from the studies mentioned above.”
It is a bit vague to just say “supported by molecular evidence”. Presumably you mean “supported as monophyletic groups”? It is worth being explicit about this.

Lines 623-763. The illustrated key to clades of New World Temnothorax is a great feature of this paper. I tried the key and it generally worked well. I can appreciate the challenges faced in making this key, especially distinguishing between the salvini clade and the hypervariable sallei clade.
I think that couplet 8 (lines 674-679) may cause some difficulties, however. Measurement of several obturator-like workers from Mexico, with broader postpetioles than Texas obturator, yielded PWI values of 150-154. Some additional characters may be needed at this couplet to direct the user to the obturator clade.

Line 676. Change “PW” to “PEW” (PW is pronotum width).

Line 698. “posterior of head with ten or more erect setae”.
Do you mean the posterior margin, as seen in full-face view, or some more vaguely defined posterior region?

Lines 783-1115. Key to species of the Temnothorax salvini clade.
I had little material available to test this key, but what I had went through the key smoothly.

In couplet 9 of the key, T. aztecoides is said to have the “anterior half of first gastral tergite lightly sculptured”. In contrast, the species diagnosis (p. 278) states that the “entire first gastral tergite” is sculptured (and this is said to be a differentiating feature from T. aztecus). The species description (p. 280) is different again. In any event, this character needs to be checked. The gaster appears to be smooth and shiny in at least some specimens referable to T. aztecoides (Fig. 152).

Line 1727. Change “acutispinosus” to “altinodus”.

Line 1856. Change “five species (four of which are described here)” to “five species (four of which are newly described here)” because you do describe the fifth species.

Lines 1873-1874. In this and other lectotype designations, it is worth bolding the word “lectotype”, to make the action more obvious. I.e.,
“Syntype workers. Cuernavaca, Mexico. One worker here designated <b>lectotype</b>.”

Line 2860. Change “dead stalk of an aster” to “dead stalk of Asteraceae” (cf. line 3219).

Lines 2874-2875. This cannot be a syntype gyne (and therefore a paralectotype) because Forel’s (1901d) original description refers to workers only.

Line 2876. Macromischa is misspelled.

Lines 2878-2880. Leptothorax augusti was a replacement name for Leptothorax foreli (Aguayo) (nec Leptothorax foreli Santschi), not a replacement name for Macromischa petiolata Forel.

Line 4254. Correct the misspelling of “distribution”.

Line 4255. Change “may be revealed” to “it may be revealed”.

Line 4614. Specify the caste of the syntype selected as lectotype.

Line 5107. Change “examined” to “examine”.

Line 6018. Elsewhere in the manuscript this section, which provides information on distribution and biology, is titled “Comments”. For consistency do the same here.

Line 6271 Insert “of” after “inspection”.

Lines 6713-6720. In introducing the pergandei group, it seems worth mentioning that for a long time the nominate species was placed in its own subgenus (Dichothorax).

Lines 7985-7986. It seems questionable to interpret a Winkler sample as a “nest collection”.

Line 8694. Change “which are known” to “which is known”.

Lines 8694-8696. You seem to be saying that T. flavidulus is known only from the types collected off the northern coast of the Dominican Republic, which is not true.

Line 8704. Out-of-place paragraph return.

Line 8921. Change “which are known” to “which is known”.

Lines 10492-10493. “…recovered all of the known specimens used in this study.”
For clarification insert “of T. wilsoni” after “known specimens”, if that is what you mean.

Line 19063. Change “discovered in” to “discovered”.

Lines 11276, 11278. Specify the castes of the syntypes selected as lectotypes (the information is provided later but it should be here too).

Line 11586. Correct misspelling of Isthmus.

Line 12403. What is the meaning of “Paratype worker gyne”? Should there be a comma after “worker”?

Lines 12607-12608. “The types of this species were found in dead twigs, live hollow stems, and by beating vegetation.”
It is a bit odd to speak of the “types” of this species, since there is only one primary type specimen. In any event, the holotype and two paratypes come from a single nest series, so the statement is inaccurate.

Line 12619. Change “Paratype workers” to “Paratype worker”, since there is only one.

Line 12948. Remove “made”.

Line 13317. Capitalize “mountains”.

Line 13980. Correct the misspelling of “generalized”.

Line 13892. Change “membership to” to “membership in”.

Line 14203. Correct the misspelling of “tricarinatus”.

Lines 14268-14269. “Zoology” is misspelled. Moreover, this should be cited as a serial (as you do for Wilson, 1955):
Bulletin of the Museum of Comparative Zoology 104:1–585.

Line 14291. Volume number is missing.

Line 14320. Capitalize “sociaux”.

Lines 14332-14334. Citation appears to be incomplete.

Lines 14357-14358. A doi is needed for this reference since “250” is the article number, not a page number. Also, “evolutionary” and “biology” should be capitalized.

Lines 14359-14361. Now that the final version of this paper has appeared, with volume and page numbers, it should be cited that way. I.e., Prebus (2021) Systematic Entomology 46:307-326. Be sure to replace Prebus (2020) with Prebus (2021) throughout the text.

Line 14386. Insert “of the” before “Entomological”.

Lines 14395-14440. There is some mix-up here. A paper with the same title, journal, volume, and page numbers is listed twice and with different years: Vásquez-Bolaños (2011) and Vásquez-Bolaños (2015b). Remove the duplicate (which is apparently the second one) and then check to see that the paper is properly cited in the text.

Lines 14397-14398. As cited, this paper has 523 pages. I think it should be:
Métodos en Ecología y Sistemática 10(1): 1-53.
If this is the only 2015 paper by Vásquez-Bolaños that you cite then it needs to be changed from 2015a to 2015.

Line 14406. Here you cite the journal as “Psyche (Cambridge)”, for other papers just “Psyche”. The former is preferable.

Lines 14413-14414 and 14417-14418. Wheeler & Mann (1914) is cited twice, slightly differently. The second one is the correct citation.


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: WHOLE BLOOD FATTY ACID CONCENTRATIONS IN THE SAN CRISTÓBAL GALÁPAGOS TORTOISE (CHELONOIDIS CHATHAMENSIS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) I can say nothing about the linguistic shortcomings because several of co-authors are from universities in USA or Australia.

2)  I could not find the hypothesis behind the work in the section "Introduction". Prior literature has not appropriately been used to prepare the section Introduction and to discuss findings.

3) I think the paper structure follow the journal guidance. Table should be improved, in the General comments for the author I explained about that. Figures are redundant, they are presenting nothing more than the table, just a data repetition. Raw data are not bad.
Experimental design: 1) I could not find a question in the paper. The paper tried to uncover a gap, but was not successful. When there is not a question without any hesitation there is no an answer.

2) The work cannot address the minimum standards of a research program. In fact, this is not a research program, it is just a GC.

3) There are serious missing in the section Materials and Methods I explained some of them in the General comments for the author. The main technique of used in the work is GC although there is no description about that!
Validity of the findings: The simple GC of carried out in the work cannot support the matters claimed in the discussion and conclusion. Generally, the sections are prepared based on the section "result"  and the early studies, but I didnt find any result in the work, I saw  just a GC output .
Additional comments: This is a work in which authors collected blood from several tortoises (without any specimen selection criteria) and then the blood samples subjected to a GC to determine their fatty acid composition.

General idea

	The surprising point is that the work have seven co-authors. What is their roles in the GC?

	I provided the work with a series of comments although the comments cannot improve the scientific level of the work.

Comments

Title
	This is not a title of a research project. This is just a title for a test (GC), nothing more.

Abstract
1)	The abstract consist of 144 words. Seventy two words (50%, lines 27-33) of the 144 are background.

2)	The abstract says nothing about the hypothesis behind the work.

3)	Lines 33-34: “very little information about baseline values for fatty acids in this species exists”. → What does mean the phrase “very little”? → This means there is few studies, if yes, why didn’t cite in the section discussion and didn’t consider them to discuss your findings.

4)	Lines 34-36: “It was noted that the ω-6/ω-3 ratio was higher for the breeding center than for the ecological farm and that overall polyunsaturated fatty acids (PUFAs) did not have any significant differences”. → This doesn’t show the result of the work, this is just a GC output.

5)	Lines 36 – 38: “This study presents these fatty acid profiles to establish this information and allow future studies to continue expanding the understanding of their influence on husbandry in managed-care facilities”. → This is an irrelevant and redundant sentence for abstract. This can be used in the end of the section introduction. In the part of the abstract, authors should add a sentence that in which the general concept of authors about their results is presented.

Introduction
The end of the section is important for me where authors should clearly let readers know the hypothesis behind the work, but I could not find their hypothesis for the work. They just say that “this study presents the fatty acid profile of C. chathamensis from both a captive breeding center and ecological farm”. In fact, they say that they just determined the blood FA composition of several specimens.

Materials and Methods
1)	Lines 70 – 95: Please provide the section with a map and indicate the sampling sites on it.

2)	Lines 70 – 95: When the samples were collected? (sampling time - season)

3)	Lines 70 – 95: Please add the environmental parameters in the sampling sites.

4)	Line 70 – 95: Please provide the section with the criteria that have been considered to select the specimens.

5)	Line 70 – 95: Please let us know the age, size, and the sex (I think all were male) of the specimens of selected.

6)	Lines 104 – 107: This is not enough, authors should provide the section with details about lipid extraction, methylated esters preparation, and GC, and the methods should be provided with suitable references.

Results
1)	It is better to delete the columns related to median of the FA values in the table 1.

2)	What is the unit of the FA values in the table 1?

3)	Please add the FA values as mean±SD

4)	In the footnote of the table 1 you need to add that results sharing the same superscript letter are not statistically significantly different.

5)	The FA values as biochemical data should be correlated to various endpoints at molecular and organismic levels to uncover their effects on the biology of the animals, otherwise the values are just accounted for a summarize of the GC out put.

Discussion
1)	Lines 127-149: the lines are just a story, irrelevant and redundant.

2)	Lines 149-150: a repetition from the result section.

3)	Line 151: the data presented in the current paper cannot support the claim. To support the claim the diet FA profile should be determine.

4)	Lines 151-153: Since most models that have studied adverse effects of ω-6/ω-3 are mammalian, extrapolations to reptiles must be drawn cautiously. → What does mean the sentence?

5)	Lines 153-154: “it is likely that this altered ω-6/ω-3 would have some effect on immune system regulation and the inflammation cascade in the SCGT from the present study”. → You cannot approve the claim just based on a rough GC output.

6)	Lines 155-156: “the animals in our study were all healthy and were not likely in an inflammatory state”. → What were the health criteria? Please introduce them in the “materials and methods” and then present related data in the “result” section.

7)	Lines 156-157: “if these animals were from a rehabilitation facility, there could be implications for alterations to this ω-6/ω-3 ratio”. → What does mean the sentence?

8)	Lines 159-162: “highly unsaturated fatty acids (HUFA) are those that contain multiple double bonds in their carbon chains and therefore are a specific type of PUFA. It has been established that plants and phytoplankton are among the only organisms that can synthesis these PUFAs”. → This is an old story, irrelevant and redundant.

9)	Lines 169-172: “it is interesting to hypothesis why the ω -3 HUFAs of the SCGTs at the breeding center were at such high levels and whether this impacted any of their membrane functionality in terms of fluid balance, immune system, or reproductive capability. → Your data cannot support the claim.

10)	Lines 174-191: “since several tortoises in the present study came from a breeding center, it is essential to discuss the role of FA in reproduction. There have been several reproductive studies conducted to examine the role of fatty acid concentrations on fertility in vertebrates (Cartland-Shaw et al., 1998; Lin et al., 2016; Len Van Tran et al., 2017; Samee et al., 2018). In one study using zebrafish, it was concluded that the addition of extra virgin olive oil extracted from Koroneiki that was rich in oleic acid (18:1 ω-9) and linoleic acid (18:2 ω-6) improved the reproductive capability of both males and females in terms of sperm quality (motility, fertility, and hatching), frequency of follicular development, and accelerated hatching (Samee et al. 2018). Another study looked at the importance of ω-3 and ω-6 PUFA for male reproductive health in ruminants, including parameters of sperm membrane integrity, maintaining viable sperm during the chilling and freezing process, and testicular development (Len Van Tran et al., 2017). The ω-3/ω-6 ratio in boar’s was examined and determined to improve testicular development and possibly chances for breeding selection overall libido in animals fed a 1:1 ω-3/ω-6 ratio (Yan Lin et al., 2016). While these references are promising developments to be correlated to reproductive success, the SCGT values in the present study were not examined for fecundity. This may be a future avenue to explore once normal FA have been established for this species”. → Irrelevant and redundant, this is just a story.

Conclusion
1)	Lines 194-195: “the objective of the study was to present FA values for the Galapagos Tortoise endemic to San Cristóbal Island”. → This is not an objective.

2)	Lines 195-197: “previous studies conducted in other reptilian species showed patterns of higher overall PUFAs and ω-6/ω-3in-managed animals compared to wild animals. → In the conclusion section you should summarize your findings no discuss them”. Please delete the sentence.

3)	Lines 199-200: “future studies could examine a larger population to validate baseline FA values further. Additionally, parameters influencing the differences of these fatty acids such as diet, sex, and physiologic status, could be examined”. → In a weak work you cannot talk about outlooks


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: WHOLE BLOOD FATTY ACID CONCENTRATIONS IN THE SAN CRISTÓBAL GALÁPAGOS TORTOISE (CHELONOIDIS CHATHAMENSIS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: The research question (use of fatty acid profile to check the nutritional status, immune function and reproductive capabilities of turtles) is well formulated
The experimental design and methods used for taking the samples and for lipid and fatty acids analyses are correct, as well as the statistical analysis. Ethical standards are defined. Method for fatty acid analysis is just cited  (AOCS, 2005) and carried out in an external laboratory
Validity of the findings: The findings are too basic to get any new information about the nutritional and immune status of the turtles, neither to assess the reproductive capability. There is no much information published about the fatty acid profile in turtles but the resutls obtained are not easy to discuss, it is just a list of fatty acids found in the blood without knowing which diet the turtles have taken (ingested) or what was the reproductive status (immature, mature) or sex (females, males). The authors centered most of the discussion in the ration n-6/n-3 but in some animals (i.e. fish) a high ratio means low n-3 and high n-6 and that is not good at all for the animals. For mammals is different but we are talking about aquatic animals, reptles.
In marine animals N-3 (omega 3) fatty acids are very important for neural development, growth and health status. In marine turtles should be more or less the same, depending on their food ingested (micro and macro algae have a lot of n-3 fatty acids). In this case the higher proportion of fatty acids came from n-6, and mostly arachidocnic acid (involved in reproduction in marine fish) and linolenic acid (18:2n-6) typical from plants.
As a summary the results show the first fatty acid profile of these turtles as a first step but it should have included a comparison not only between stocks (farmed and kept in a breeding center) but also between different diets used for feeding and between different reproductive status or size of the animals. It is too basic....
Additional comments: Continue with the work, this is just a preliminary analysis of what can be done, but the information provided is too basic and without any conclusive results to be used neither for growth of the animals , for reproduction or to assess the health status. It is just showing the fatty acid profile in the blood.


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 MANNAN OLIGOSACCHARIDES ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, RUMINAL FERMENTATION AND HEMATOLOGICAL PARAMETERS IN SHEEP
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the manuscript highlighted the importance of feed supplementation with prebiotics like Mannan Oligosaccharide (MOS) in sheep diet which improves nutrient utilization and decreases ammonia-nitrogen production in the rumen. The following observations were made:
1.The article should include sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge because the majority of the previous work cited about the role of MOS are related to monogastric and poultry species which have different anatomy and physiology with ruminants (Sheep). It should also state clearly the statement of the problem, hypothesis, and justification for conducting such research. Therefore, relevant prior literature should be appropriately referenced.
Experimental design: 1. The methodology adopted needs to be reference from previous studies for each procedure either with modification or not because it wasn’t mentioned in the manuscript.
2. In line161-162 of the manuscript, the side of blood collection used (Cervical vein) is not appropriate for blood collection in sheep rather (jugular vein). Additionally, blood collected in non-heparinized tubes will be clotted leaving only serum and not suitable for hematological examination.
Validity of the findings: 1. The results presented were Satisfactory.

2. There is an incorrect heading for Table 3. Where serum was harvested from blood but hematocrit parameters were presented. Therefore, no result for serum biochemistry presented.
Additional comments: I suggested to the author to improve on defining his research question and proving his hypothesis and all references of previous work given should be consistent with his research objectives.


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 MANNAN OLIGOSACCHARIDES ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, RUMINAL FERMENTATION AND HEMATOLOGICAL PARAMETERS IN SHEEP
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: i) I recommend the authors to further clarify how MOS increased nutrient digestibility.

ii) In line 226, citation or reference is needed for the statement "MOS are a type of carbohydrate that can be degraded by ruminal microbes."

iii) Generally, elaborate discussion of the results is recommended.


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 MANNAN OLIGOSACCHARIDES ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, RUMINAL FERMENTATION AND HEMATOLOGICAL PARAMETERS IN SHEEP
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well prepared and the tables are nicely configured. Authors just need to work on the decimal places in the tables to make it neater. Literature are well cited and adequate background information provided
Experimental design: The design is ok
Just a little concern with the statistical analysis
Validity of the findings: no comment
Additional comments: Review of manuscript 'effects of manna oligosaccharides on growth performance, nutrient digestibility, ruminal fermentation and hematological parameters in sheep.
Generally, the study is well laid out, properly designed with adequate number of animals. There are just a few issues I will like to point out, for example in the statistical analysis, the authors can use the mixed model approach to analyse the data because the ingredients in the formulated diet are varied. Also there are improvement in the DM, OM, Ash etc apparent digestibility of animals fed the MOS treatments but no effect on the average daily gain. Mixed Procedure of SAS will be able to account for some of the errors.
Table 1
Looking at the ingredient table, normally animals on 1.6 and 2.4% MOS will have better performance taking account the feed intake which will be more palatable considering extra 50kg maize, 30 kg soybean, lesser maize straw and malt sprout. Although authors tried to balance in terms of chemical composition, but there is a little concern with the feed formulation. Authors could have included a treatment as positive control with a commercial supplement vs MOS, while presenting the ingredient composition
The chemical composition g/kg should be presented in terms of DM composition. The DM should be removed and other nutrient presented in terms of the DM
Why was acid detergent fibre not determined?
Table 2
Average intake or average daily intake?
The unit should of average daily intake should be in g/d
Initial weight is missing in this table

Table 3
The title is not properly written and carried no information about the treatments
Values in the table should be standardized, 1117 instead of 1117.94, and 367.9 instead of 367.8, in that way at least there will be 4 numbers and the table becomes neater

Line 120, How was the doses determined?
Line 154, how did you ensure rumen fluid collected were not contaminated with saliva? And How were you able to take rumen fluid samples from 3 regions of the rumen when using stomach tube?

Experimental diets
The authors claim in line 120 that the experiment was a single-factor design, meaning the only introduction was the 4 different doses of MOS, whereas in line 130, authors formulated 4 diets and varied the composition of ingredients in the diet. The different proportion of ingredient composition could have one way or the other influence response.

Line 135 to 139, Authors carried out digestion and metabolism trial during the short 5 weeks study. Authors could have carried out the digestion and metabolism trial after the growth performance trial as the break for the digestion trial could have influenced the experimental animals

Line 144 to 145, How did the authors determine the DM of the faecal samples

Blood sample analysis, authors left out serum biochemical parameters, parameters such as blood urea nitrogen and liver enzymes such as ALT, ASP could give an indication of the health status of the animals

Line 187, delete “The ash apparent digestibility of sheep fed the 0.8% MOS diet was higher than those in sheep fed the 2.4% MOS diet (P = 0.034)”
Line 196, “MOS did not affect the physiological or biochemical parameters of sheep serum”, authors did not test the serum biochemical indices of sheep
Line 201 to 209, unusually long sentence, too long, try and breakdown into shorter sentences
Line 219 to 222, “This may be because ruminal microbes degrade MOS in the rumen, preventing it from affecting the rest of the body (although it can still improve nutrient utilization and ruminal fermentation). This indicates that MOS are suitable for nutritional regulation purposes in adult ruminants, but not for growth performance enhancement”
This is self-contradictory, authors need to rephrase
Line 225 to 228, I don’t really understand what the authors meant by the statement. Of course most carbohydrates can be degraded in the rumen, this is useful for the microbes to multiply and make their cells, this also leads to production of volatile fatty acids which contributes mainly to the energy requirements of the animal, and for milk production. Authors need to bring clarity to this.


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 MANNAN OLIGOSACCHARIDES ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, RUMINAL FERMENTATION AND HEMATOLOGICAL PARAMETERS IN SHEEP
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript now looked scientifically good and interesting as the introductory part identified the research problem and clearly stated the hypothesis in question. I believe this manuscript merits publication but I have  only a few comments and suggestions for the authors
Experimental design: No any comments
Validity of the findings: Satisfactory
Additional comments: In the last review, I suggested these observations to the author but still, they appeared again in the second review, therefore, 1. The authors should give clarification of mentioning four doses instead of three (3) and control because for the first dose no MOS was added rather a MOS-free diet.

2. The author should have mentioned twenty-four (24) animals per group instead of 6 replicate with 4 replicate each.

3. The authors should give the scientific basis for using non-heparinized vacuum tubes in collecting blood and how he measured hematological indices with clotted blood.

4. The author should differentiate between the terms he used in the abstract hematological parameters, in methodology blood parameter, and in result physiological and biochemical parameters as to whether they mean the same or different meaning?


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 MANNAN OLIGOSACCHARIDES ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, RUMINAL FERMENTATION AND HEMATOLOGICAL PARAMETERS IN SHEEP
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: the validity is ok
Additional comments: I have read through the manuscript again. Although the manuscript has been improved from the original state, I still have some concerns I raised before, especially with the feed formulation. Formulation of feed for ruminants is not only about the chemical composition but also the acceptability. It became obvious that there will be a confounding effect. I can judge this without even conducting the experiment based on more maize, soybean, lesser barley, and wheat straw. Six ingredients have been varied out of 12 ingredients used.
Secondly, I think there is a need to send the response letter to English language editor as I struggle keeping up with some of the responses.
Thirdly, the authors need to motivate in the manuscript why carrying out digestibility study during a 5 week trial is good, preferably linking it with a reference


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 MANNAN OLIGOSACCHARIDES ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, RUMINAL FERMENTATION AND HEMATOLOGICAL PARAMETERS IN SHEEP
Review round: 3
Reviewer: 1

Output:
Basic reporting: Satisfactory
Experimental design: The study and its experimental design falls within the aims and scope of the journal, the hypothesis are well defined and the objective clearly indicated. Standard methods have been used to carry out the experiment
Validity of the findings: Satisfactory
Additional comments: The authors responses to review suggestions and criticism are satisfactory


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: HETEROGENEITY OF GUT MICROBIAL RESPONSES IN HEALTHY HOUSEHOLD DOGS TRANSITIONING FROM AN EXTRUDED TO A MILDLY COOKED DIET
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please find the full report below.
Experimental design: Please find the full report below.
Validity of the findings: Please find the full report below.
Additional comments: Jirayu Tanprasertsuk and colleagues investigated the effect of a mildly cooked diet for 4 weeks in healthy household dogs. I found the paper confusing and of limited relevance to current knowledge in the field. Dog owners’ perceptions of changes in health outcomes are ambiguous and almost impossible to interpret, as the authors noted it themselves. Moreover, the fact that some parameters were not available in all dogs (e.g. in Table 1) makes the results questionable. The paper is also often difficult to grasp, particularly the use of values from the PCo1 axis of Bray Curtis dissimilarity only, and the categorization of dogs based on the degree of PCo1 (or 4?, see line 775, label of Figure 2) axis values. I always encourage the use of novel alternatives to analyze the gut microbiota, but the authors give little information to back up their approach. Finally, there are sections in the manuscript that appear too careless (e.g. references, font types).

General comments
The authors use the word “species” a lot, and do not use (or even mention) OTU or amplicon sequence variants. Read this: https://pubmed.ncbi.nlm.nih.gov/12142474/ and consider rephrasing the entire manuscript.
The squares, for missing symbols, is very distracting. The authors should have reviewed the final PDF before submitting.
In the abstract generated by PeerJ, page 4 of PDF, is written the word “rho”, instead of the Greek letter. This is the only time they do so.
How can you discard the possibility, or the effect, of natural variation over time? See this paper in humans: https://doi.org/10.1186/gb-2011-12-5-r50.
The results need to be carefully re-organized, there are way to many headings (e.g. 3.4 Gut microbiome) and sub-headings (3.4.1 Diversity).

Title
In the title, “heterogeneity of responses” sounds ambiguous and confusing, please rephrase, focusing on a title that is both short and informative regarding the nature of this research work.

Abstract
Lines 56-57. This is confusing, is there any other way to write this? Please remember that the paper needs to be easy to understand even for people outside of the field of gut microbial ecology.
Line 59. Are these percentages before and after? If so, could you use, e.g., 61% vs 39%?
Line 62. What is the relevance of including these percentages here? Also, it is not clear for me whether these values come from day 28.
Line 74. This statement is too strong, even a strong correlation with dietary protein may be related to other parameters (e.g. liver metabolism etc) and not necessarily related to the possibility of personalized nutrition. This is concerning, because it goes along with one of the main purposes of nomnomnow.

Introduction
In the first paragraph of the introduction, where is reference 2?

Material and methods
Line 305. Could you please back up this approach?

Results
Line 316. What does “dog-weeks” mean exactly?
Lines 339-340. Could you add the numbers of dogs as well (e.g. 19 out of 31 dogs for the 61%?)? Also, and more importantly, these perceptions are ambiguous and almost impossible to interpret.
Line 350. This section is very confusing, the authors should make an effort to be clear and concise. From my point of view, the authors dug too deep.
Line 377. Why citing only these two studies? This can go into the discussion, with a more general overview of the many other studies that have studied the gut microbiota in dogs.
Line 380. Here it says abundances, and in 3.4.2 says relative abundances. I am not sure if the authors are aware of the issues related to the use of relative abundances: http://dx.doi.org/10.3402/mehd.v26.27663.
Line 391. This looks saturated, please be concise. Also, please note that the names are ultimately meaningless:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1857736/pdf/rstb20061914.pdf
Line 413. Do you really think is necessary to include all this information? Please back up your answer with appropriate references.
Line 422. I do not think this argument is strong enough to classify the subjects. Please provide useful references or arguments to back up this approach.
Line 457. Please see my comment above about heading and subheading. In this line, it looks strange that this subheading is not numbered. I would re-organize the entire manuscript, focusing on the most relevant results and observations only.
Line 512. All the subsections are confusing and out of place.
Line 524. This weakens the entire argument about the relationship with protein or any other nutrient in the diet.
Line 544. This is subjective and confusing, I would remove it.

Discussion
Eight and a half pages for the discussion is way too much. I admit that PeerJ do not have a page limit, but the authors should nonetheless be concise, clear, and explicit. I would re-organize the entire manuscript, and cut the discussion by half.
Line 568. In the title, you placed GM first. Please be consistent.
Line 577. I am not sure whether this is true, particularly the point on “taxonomic resolution and accuracy”. Besides the reference provided above, please note that even identical marker genes can be found in bacteria with highly divergent ecophysiologies (DOI: 10.1128/AEM.70.8.4831–4839.2004) and this reference is better for taxonomic resolution: https://doi.org/10.1038/s41587-020-0501-8 (please us this).
Line 589. Cite those other studies.
Line 681. Do not use flora.
Line 682. It is surprising that this wonderful reference is only used here, and in that context.

References
Be consistent, e.g. all first letters capitalized or not, same font type (ref. 62).

Figures
There is way too much emphasis on HR, MR, and LR, e.g. in figure 2C. I personally would not place too much emphasis on such ambiguous categorization.

Tables
The information presented in tables 3, 5, 6 and 8 is excessive. It is strange that the font of the title of Tables 3, 4, 5, 6 and 8 is different. Even more strange, the mixing of font types in the title of Table 3.


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: HETEROGENEITY OF GUT MICROBIAL RESPONSES IN HEALTHY HOUSEHOLD DOGS TRANSITIONING FROM AN EXTRUDED TO A MILDLY COOKED DIET
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper studies the impact (compared to a baseline kibble/extruded diet) of feeding a mildly cooked diet on the microbiome, and owner-assessed health outcomes in the dog.

The paper is well written and considerable thought appears to have gone into the statistical  analysis and interpretation of the results.

The authors have used the available literature widely.

In order to support the hypothesis, some background/introduction on the effects of mildly cooked/fresh diets on health aspects, even if anecdotally, could be included (I note it is discussed in the literature).
Experimental design: The experiment utilises dogs in home, and therefore relies on owner assessment and reporting. The diets used are commercial diets, and as such there is not treatment per say, instead a baseline sample (taken from dogs consuming a variety of commercial extruded/kibbled diets). This lends the study to make generalisations about what was observed; a fact acknowledged by the authors in L747-754.

Methodology: I would like to see data on the cooking process/nutrient composition of the MC diets provided as main Tables rather than supplemental data. This is because the diet is the main driver of the changes in the microbiome. Sequencing/analytical methodology is described well and would be repeatable for any study.

Regardless the authors have considered their data set in novel ways (ie the identification of low and high responders) which adds to our knowledge base on the impacts of diet on the microbiome of the dog.
Validity of the findings: Overall, the findings reported link back to the hypothesis/study undertaken.
Additional comments: Major comment:
Regarding the term 'mildly cooked' - this needs to be defined (especially in the materials and methods). Similarly, when referencing the literature that use the term 'mildly cooked' the authors should stipulate what this was defined as if the temperatures etc differ from the current study. My preference would be to remove the statement on L121 "also commonly known as fresh diets" as the words cooked and fresh contradict each other. This may seem pedantic, however one issue arising from assessing the impacts of diet on the microbiome is that we as a research community use a variety of terms which, when reading articles in depth, do not reflect the treatments investigated.

Questions arising were:
The age of the dogs is large (1-14 years); given age affects the microbiome, and 'ageing' is affected by body size (as a rough guide) were any considerations taken to assess either breed or 'dog size' in terms of the results?

The MC diets had a range of primary proteins used; studies in the dog/cat and rat have shown that protein source can affect the microbiome; was this taken into consideration?

Minor comments:
The term 'transition' is used (eg L568-569); this (to me at least) implies that the microbiome was assessed at multiple time points AND/OR the microbiome is not considered to be stable. Indeed, your study of 28 days would we would expect to see a stable population.

In your discussion on L625-640: I would expect most high-quality kibbled diets to include many of these ingredients.

I understand the process around looking at the baseline/kibble protein content of the diet on the results is and agree with it in principle. However given 'on pack'/reported protein levels can vary up to 10% from actual it would be good to see a statement along those lines.

In general, I look forward to seeing this work published, and seeing more research into the concept of 'high' and 'low responders' in the future.


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: HETEROGENEITY OF GUT MICROBIAL RESPONSES IN HEALTHY HOUSEHOLD DOGS TRANSITIONING FROM AN EXTRUDED TO A MILDLY COOKED DIET
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors aimed to determine the effect of variety of differences between diets on dog microbiome. The study also observed a shift in the diversity and abundance of GM upon diet change adn also the fecal outputamong the enrolled dogs. The resuts indicates a a significant shift in the GM profile of 73 healthy dogs. The magnitude of shift was associated with baseline GM and dietary protein, and 74 may provide a basis of personalized nutrition in companion dogs. These results support future 75 investigation of the impact of a MC diet on health maintenance given its increasing popularity. These findings maybe useful in the understanding for canine foods.
Experimental design: The experimental designs were clear and the statistical analysis was suitable. However, the sample size of the dogs needd to be calculated, in orer to obtain the significant sample sizze for analysis.
Validity of the findings: The diversity of the gut microbe will be a good indications for the health status. However, the quantitative analysis of the gut microbe maybe needed to be determined because these microbes are associated with the efficacy of microbe to utilize and synthesize the essential elements for dog.
Additional comments: The study sounds clear and useful in term of canine gut microbiome. However, the quantitative analysis of the gut microbe maybe needed to be determined because these microbes are associated with the efficacy of microbe to utilize and synthesize the essential elements for dog.


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: HETEROGENEITY OF GUT MICROBIAL RESPONSES IN HEALTHY HOUSEHOLD DOGS TRANSITIONING FROM AN EXTRUDED TO A MILDLY COOKED DIET
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all comments either by altering the body of the manuscript or via the rubuttal
Experimental design: Appropriate
Validity of the findings: Appropriate
Additional comments: N/A


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: HETEROGENEITY OF GUT MICROBIAL RESPONSES IN HEALTHY HOUSEHOLD DOGS TRANSITIONING FROM AN EXTRUDED TO A MILDLY COOKED DIET
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment.
Validity of the findings: No comment
Additional comments: The revised manuscript has already revised and the improvments are satisfied. My decision is the manuscript is suitable 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: NEW AUSTROLIMULID FROM RUSSIA SUPPORTS ROLE OF EARLY TRIASSIC HORSESHOE CRABS AS OPPORTUNISTIC TAXA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The basic paper is very good. I would have liked to see more phylogenetic context for Attenborolimulus (i.e. the discussion of Limulitella).
Experimental design: The morphometric analysis is a little bit of pattern searching without a hypothesis driving it. I think this could be fixed with a little reframing of the analysis.
Validity of the findings: If you are going to talk about austolimulids diversifying in the wake of the end Permian extinction, you might briefly consider their origins--do they originate in the late Paleozoic? Early Mesozoic? Do they just *pop* into existence in the early Mesozoic?
Additional comments: Pay close attention to the comments in the reviewed 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: NEW AUSTROLIMULID FROM RUSSIA SUPPORTS ROLE OF EARLY TRIASSIC HORSESHOE CRABS AS OPPORTUNISTIC TAXA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment, all OK.
Experimental design: All OK
Validity of the findings: All OK
Additional comments: This manuscript describes a new species of fossil horseshoe crab and places it in its wider context as part of a putative radiation of taxa after the Permian mass extinction even. Principal component analysis is also applied to help resolve its systematic placements. The manuscript is well written, the descriptions of the new species are of a high standard and the figures are very good. I think the manuscript is a useful contribution to the systematics and evolution of Mesozoic horseshoe crabs and is acceptable for publication subject to MINOR REVISION as outlined below.

TITLE

I’m not sure “uncovers” is the correct word here. A single species alone is not really evidence for all horseshoe crabs being disaster taxa. Maybe “…supports the role of Triassic horseshoe crabs…” would be better?

ABSTRACT

line 21: “…these bizarre forms illustrate that…”

INTRODUCTION

line 33: “…recovery of distinct the palaeoecological…” Do you mean “…of the distinct…”.

MATERIALS AND METHODS

line 92: “A backscattered electron detector…“

line 95: “…presented in Lerner et al., (2017), Bicknell (2019)…etc.”

line 99: “…material falls in morphospace…”

SYSTEMATIC PALAEONTOLOGY

lines 139/143: style question? Should these sentences end in a full stop?

line 197: What are “clam shrimps”, I’m not familiar with this term so maybe you need to add the scientific name of the group.

line 2017: “Furthermore, as Belinurina went extinct by the end-Permian…” maybe qualify this by adding “on current data” or something like that.?There is always the chance that a Triassic belanurid could be found later in which case age alone would not exclude this taxon.

line 230: “B Based on this…” [delete “B”?]

RESULTS

line 245: “The PCA plot…”

REFERENCES

line 377: should “Wein“ be “Wien“ (for Vienna?).

line 481: italicise “Tarracolimulus rieke“

line 481: Is the species name correct? In Table 1 (p. 33) it is listed as „rieki”?

FIGURES

line 555: there seems to be part of the legend missing in the text here. Later with the figures we have the information, i.e.

“(A, B): Photograph and interpretative drawing. Image credit: Sergey Bagirov. Abbreviations: Car: cardiac lobe; Eye: lateral compound eye; Fla: thoracetronic flange; Fs: fixed spine; Med: medial thoracetronic lobe; Oph: ophthalmic ridge; Pa: prosomal appendage; Pro: prosoma; Thr: thoracetron; Tel: telson.”

line 573: “Three examined xiphosurid families in…“


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: NEW AUSTROLIMULID FROM RUSSIA SUPPORTS ROLE OF EARLY TRIASSIC HORSESHOE CRABS AS OPPORTUNISTIC TAXA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the story is straight forward, expanding the diversity of Triassic representatives of Xiphosurida. My two main suggestions are rather simple: 1) Avoid Linnean ranks wherever possible. You can only do it wrong, or better, there is no right way as ranks are a matter of opinion and nothing else. 2) Throw out the 'disaster taxa'. I do not have the impression that this is a well developed concept, but just the attempt to push another buzz word.

Hence I suggest to accept the paper after minor revision.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General: Linnean ranks are subjective categories. There is no objective criterion when which rank should be applied. I can therefore only suggest to not use these categories.

Title: This is not a geologists' journal, most readers (including me) will have to look up what “Olenekian” is. “Disaster taxa” is not a well known concept, in fact I am not sure whether it is indeed a well formulated one. I suggest to avoid it altogether and use an easily comprehensible phrasing.
Throughout the manuscript the geological ages need to be accompanied by rough estimations of age for non-geologists.

Abstract: The first sentence includes many general statements of which I am not sure if they are correct. Why are they archetypical, the majority of extant forms of Chelicerata sensu stricto living in the marine realm (more than 1000 species of sea spiders) look quite different. In which way is the fossil record exceptional? Compared to which other lineages?

As pointed out I would prefer to see no use of Linnean ranks. Yet, using such ranks demands for following specific rules. For example, the expression “xiphosurid” as used here is ambiguous. In most lineages the ending '-id' is derived from the family name, hence '-idae' (as it is done in other instances in the text). Yet here it is, as far as I get it, derived from Xiphosurida. Hence it should correctly be derived as “xiphosuridan”.

Line 129: Especially here the presented ranks are highly subjective, and many authors would disagree on the here assigned ranks. Just leave them out.

Line 155: not entirely sure, but I guess that this is not the prosomal shield

Line 159: if the anterior tagma is called prosoma, why then use here a malacostracan term (cephalothorax)? This is in my view inconsistent.

Line 205: what is 'complete expression of tergites'? Does this refer to them not being articulated? Please express in simple words.

Line 278: I do not get the concept of disaster taxa. It seems not a widespread known term. Also as it is explained here I personally would think they are more something like “post-disaster taxa”. Also the term “taxon” makes the expression strange as it might well read as referring to a single species. I do not see why the story gets better with this term.

Line 302: Maybe it is because I am a German native speaker, but Bauplan is a very unfortunate term in these days. Since Intelligent Design is around, we need to avoid any reference to a planer of evolution.