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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Authors present a paper, in which they evaluate the association between appendectomy and increased risk to develop chronic Kidney disease (CDK) and end-stage renal disease (ESRD). The Authors conclude saying that appendectomy is a new risk factor for CKD because it contributes to intestinal dysbiosis in patients undergoing the surgery procedure. However, in my opinion, there are too many limitations that affect the paper results. First of all, considering that Taiwan has a high incidence and prevalence rates of CKD and ESRD, the missing information regard some important and predominant risk factors such as family history of renal disease, uric acid concentration, proteinuria, use of drugs and herbal medicine, and data on chronic glomerulonephritis and chronic interstitial nephritis are crucial to interpret the results correctly.
Experimental design: Data regarding well-established risk factors information for CKF and ESRD are missing.
Validity of the findings: In this study, the Authors hypothesize and conclude that appendectomy causes gut dysbiosis leading to CKD. However, no evidence is reported to demonstrate the correlation between the impairment of gut microbiota and appendectomy procedure. No uremic toxins are measured in the serum samples to demonstrate the link between appendectomy and gut dysbiosis.

The increased risk for CKD and ESRD in appendectomy patients are not supported by important information  on risk factors.

How do The Authors demonstrate that the inflammation in diabetic patients is altered by appendectomy and not by a chronic dysmetabolic profile?
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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Literature references, especially in the discussion section but also in the introduction, should be considerably expanded. Authors aim to demonstrate a potential link between appendectomy and the incidence of CKD, mediated by a putative appendectomy-induced gut microbiota dysbiosis. Although intriguing, the causative role of gut dysbiosis in the aetiology of CKD has not been demonstrated yet, and this research, although providing a novel and interesting result, does not demonstrate this mechanism. Authors should stress this aspect, dampening the speculations about this point. In CKD, a complex bi-directional relationship between gut microbiota and kidney is present, I suggest to expand the description of the scientific context, both in introduction and discussion sections (lines 76-84 and 275-282), providing more literature to support this hypothesis.

The manuscript is written in clear and correct English. Anyway, I would suggest to re-formulate long paragraphs (such that at lines 88-92) into shorter sentences in order to improve their readability.
Experimental design: Although the research topic is innovative and well conducted, the research gap should be better discussed and the research question must be completely revised. Indeed, this is an observational study whose aim cannot be to demonstrate that “appendectomy causes gut dysbiosis and that this dysbiosis results in CKD”. Authors should be aware that this is beyond the possibilities of their study and should better discuss this point in the study limitations. It is necessary to modify the sentence at lines 98-99. I would rather state that authors aim to evaluate the presence of an association between an increased CKD risk in those patients undergoing a past appendectomy.
I recommend authors to contextualise the putative link with gut microbiota dysbiosis as their own speculation, also because data on gut microbiota composition or at least on uremic toxins are not included in their research. For this reason, the presented results do not allow to draw conclusions on microbial dysbiosis as the causative link between the two pathological events. Any link with gut microbiota dysbiosis should be moved to the discussion and referred to only at speculative level.

Please provide a justification for the use of a 1:4 ratio instead of a one-to-one matching between the appendectomy and the propensity score-matched cohort. This is not clearly explained.
Validity of the findings: The present research demonstrates an association between a past appendectomy and an incident CKD, especially in diabetic patients.
In spite of the novelty of the research, there are some apparently inconsistent results which are worthy of authors’ comments in the discussion:

1. along with the increased CKD and ESRD occurrence, appendectomy patients seem to display a reduced mortality (Table 1)
2. the risk for CKD results higher only in male patients and, strangely, only in those without comorbidities and not with comorbid conditions (Table 3)
3. diabetes seems to be a risk factor only for ESRD and not for CKD (I would expect a similar risk also for CKD) (Table 4)
4. although not significant, the interaction analysis seem to suggest that comorbid factors such as stroke and use of statins abolish the association between appendectomy and increased CKD or ESRD risk (Fig 3), resulting as a kind of “protective” factors.

I suggest to provide, along with the “microbiota hypothesis”, other potential hypotheses explaining the association they found. For example, the authors could include in their discussion other possible links between appendectomy and incident CKD, such as the presence of a previous renal subclinical alteration (Tal 2017, doi: 10.1002/jum.14480) or the probability of AKI after appendectomy (Kim 2014, doi: 10.1213/ANE.0000000000000425) or the evidence that diabetic patients basally present a complicated clinical frame (Bach 2016, PMID: 27657594), even if a reference to the latter point is present in the manuscript (290-294). I recommend to further expand this point. Furthermore, dietary habits, in particular a scarce intake of dietary fiber, could represent a common risk factor for both conditions. Please add all these points to the discussion and to the limitations of the study (dietary habits).

Please correct CKD with ESRD at line 234.
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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All 3 previous comments have been addressed.


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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have no further comments, and all the issues raised in the first revision process have been successfully addressed. The manuscript is improved.
Experimental design: I have no further comments, and all the issues raised in the first revision process have been successfully addressed.
Validity of the findings: I am satisfied with the modifications made by the authors. They provide a novel and interesting resultand although remain research gaps and limitations. I have no further critical comments.
Additional comments: The manuscript is improved, I appreciate the authors' response to my 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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is written in clear and correct English, and the revisions made are ok. Please change “diabetics” in “diabetes” (line 304). Raw data are available.
Experimental design: The authors solved many of the raised questions.
I would rather rephrase the sentence in lines 96-98 into “we evaluated the correlation between a past appendectomy and the occurrence of CKD and ESRD”.
Validity of the findings: The authors solved many of the raised questions.
Just few remaining issues:

Table 1 - along with the increased CKD and ESRD occurrence, appendectomy patients seem to display a reduced mortality.
Thank you for the further analysis you performed, that I suggest to include in the manuscript.

Table 4 - Diabetes seems to be a risk factor only for ESRD and not for CKD (I would expect a similar risk also for CKD).
Ok, but I suggest to add a comment in the manuscript about the apparent inconsistence of the interaction between diabetes and ESRD and not with CKD.

The revisions made in the discussion section are ok. I suggest to re-add a reference already present in the first version of the manuscript (Sabatino 2017, doi: 10.1007/s11892-017-0841-z), thoroughly discussing gut dysbiosis and inflammation in CKD and diabetes, as underlying and linking factors between the two pathologies. In the context of this manuscript, it could be useful to the authors to support a putative hypothesis explaining the association they found between appendectomy, ESRD and diabetes. Indeed, appendectomy could have a cumulative effect on the pre-existing dysbiosis in diabetic patients, and increase the risk of ESRD occurrence. Clearly, this link needs to be validated in more specifically designed studies.
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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: Authors: We thank the reviewer for their kind reminders. We revised “diabetics” to “diabetes” in line 304.

The correction was made in a wrong place, please correct “diabetics” in “diabetes” at line 312 (I refer to the Word manuscript with tracked changes) and leave the adjective “diabetic” referred to "patients" at line 317 (the original version was correct).
I suggest to perform a last language revision by a native English speaker, especially for the added revisions (for example, the sentence “The interaction effect of DM with appendectomy was appeared inconsistence, the observable subgroup effect on ESRD, but not on CKD” is not understandable).
Experimental design: No comment
Validity of the findings: The revisions of Table 1 and conclusion are ok.
Please correct the syntax of the sentence "The interaction effect of DM with appendectomy was appeared inconsistence, the observable subgroup effect on ESRD, but not on CKD."

Thank you for adding the suggested reference. Anyway, in Sabatino et al, a causative relationship between appendectomy and dysbiosis is not reported.

For this reason, a modification of the sentence:

"The microbiota in the large bowel can change after appendectomy and lead to dysbiosis.9 Evidence suggests that appendectomy impairs gut microbiota and results in a pathogenic inflammatory effect.18”

into:

“The microbiota in the large bowel can change after appendectomy and lead to dysbiosis9. Gut dysbiosis and inflammation are underlying and linking factors between CKD and diabetes18. Appendectomy could have a cumulative effect on the pre-existing dysbiosis in diabetic patients, increasing the risk of ESRD occurrence.”

would be more appropriate.
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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 4
Reviewer: 1

Output:
Basic reporting: The authors solved all the raised questions
Experimental design: No Comment
Validity of the findings: The authors solved all the raised questions
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: EVALUATING THE UTILITY OF THE FEMALE-SPECIFIC MITOCHONDRIAL F-ORF GENE FOR POPULATION GENETIC, PHYLOGEOGRAPHIC AND SYSTEMATIC STUDIES IN FRESHWATER MUSSELS (BIVALVIA: UNIONIDA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: There are certain problems with the definition of research problem, as explained in detail in the General comments.
Validity of the findings: As a consequence of problems with Experimental Design the validity of findings is also questionable, as explained.
Additional comments: Dear Author,
Your manuscript describing the pair of M and F mitogenomes from two Lampsilis species is intriguing.
While reading, I encountered the following problems (referenced by line lumbers):
12: you mentioned population genetic studies yet you report only four new sequences from one locus. This is way below the required >100 from more than one locus.
14-15: the conclusion is not supported by the presented data; figure 2 is hardly promising, with very poor support throughout the tree.
31-33: do you mean freshwater mussels or DUI bivalves in general?
41: the sentence should be split at “because” (the word itself is not needed)
49: italicize “orf”
49-56: the relationship between declared aims and presented data is problematic. You did compare M and F mitogenomes from two (putative?) species – well, at least from four individual representatives of these two species. However, you have not provided any data for population genetics studies, at least nothing serious enough to consider the conclusions. The last objective (phylogenetic utility of f-orf) is not very convincing. You sequenced ca. 30000 bp from each species yet you tested if only 300 bp is usable for phylogenetic analysis? Why? Moreover, you have not used the obtained mitogenomic data in phylogenetic analysis at all… I admit I am confused as this would give you much better context for discussing the possible scenarios leading to the observed polymorphisms. I suggest you re-thing the objectives and focus on the presented new data.
53: check authority
73: how?
83..: I suggest including the whole contents of supplementary material S1 here (both tables, text and references), in the main body of text. Currently the choice of data for comparative analyses is unclear – it is better to have this information in the main body of the ms, particularly if the whole text is rather short and there are no limits on the length.
86: why don’t you use exact numbers?
88-89: the mentioned features are given in the context of which mitogenomes (used as reference)?
90: “are presented”?
97: “are in Genbank” is not acceptable. Also, the explanation in bracket is weak. Please re-think your arguments and rephrase. The real reason behind using this data set is that you previously generated it (this is obvious when looking at Table S2) for your MBE 2011 publication. Very few individual f-orf sequences have been deposited after that, and they are mostly derived from mitogenomic data. This raises the question: why not use the whole mitogenomic data instead? Is it really much cheaper and yet efficient to sequence only f-orf? If you argue it is then why do you present only old data in this context?
121: jargon…
131-139: this paragraph is rather speculative. All mitochondrial markers are linked so looking for positive selection signatures in any particular gene is risky at best. Consider also “draft feedback”...
149-153: the conclusion from this should then be that short fragments of mtDNA are poor phylogenetic markers… which is not very surprising… Why don’t you use the whole mitogenomes??
154-159: while potentially interesting this part is not acceptable. By looking at table S2 I see very few sequences derived from single species. These are more like pairs of sequences than like proper population level data sets. Moreover, it is not clear if cox1 and f-orf sequences are derived from the same individuals. Therefore, the reported difference between intraspecies and intragenus polymorphisms for these two genes may well be random effects.
Figure 1 is way too small, almost unreadable
Table S2: there is an error in accession number – HM84985 should really be HM849385

Good luck with your 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: EVALUATING THE UTILITY OF THE FEMALE-SPECIFIC MITOCHONDRIAL F-ORF GENE FOR POPULATION GENETIC, PHYLOGEOGRAPHIC AND SYSTEMATIC STUDIES IN FRESHWATER MUSSELS (BIVALVIA: UNIONIDA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is very well written, the bibliography is adequate and provide a good context to all sections.
The figures and tables are also approptiate for the manuscript.
All aims were met with success and properly discussed.
Experimental design: The present manuscript is well inside the scope and aims of PeerJ.
The main aims are well defined and of importance to the field. There is clearly a need for fast evolving markers for population genetics in invertebrates and particularly in freshwater mussels.
The science is sound and well executed with novel methodological approaches. All experimental setup is clear and of high scientific level.
Validity of the findings: The paper is of high scientific value and the data robust and well analysed. Conclusions are  well supported.
Additional comments: Dear Authors
This paper presents an interesting study about the potential usage of the recently discovered F- and M- mitochondrial ORFs of Freshwater mussels for population genetics and systematics, with a case study on the subfamily Ambleminae. The paper is very well written, the bibliography is adequate and provide a good context to all sections. The present manuscript is well inside the scope and aims of PeerJ.
The main aims are well defined and of importance to the field. There is clearly a need for fast evolving markers for population genetics in invertebrates and particularly in freshwater mussels.
The science is sound and well executed with novel methodological approaches.


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: EVALUATING THE UTILITY OF THE FEMALE-SPECIFIC MITOCHONDRIAL F-ORF GENE FOR POPULATION GENETIC, PHYLOGEOGRAPHIC AND SYSTEMATIC STUDIES IN FRESHWATER MUSSELS (BIVALVIA: UNIONIDA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: GENERAL COMMENTS

The title should be changed, because it indicates that both sex-specific mitochondrial orf genes were analyzed and this is not true.

The authors write about the use of the orf sequence in taxonomy (orf is a gene indicating high variability), but is it consistent with the morphology-based taxonomy?

The introduction provides information that taxonomy based on shell morphology can be problematic due to phenotypic plasticity. This thread is missing in the discussion.

The introduction lacks data about introgression via genome hybridizations.

In-text citations should be formatted according to the requirements of the journal.

Standardize and consistently use everywhere the same symbols.

Some adjustments need to be performed.
Introduction
36-38: And what is the point of this? Have there been such studies in other mussels?
44-46: specify which F- and M-type DNA sequences were useful (e.g. which genes)
46: add some information in the sentence: “Several studies have used both F- and M-type mtDNA sequences to answer systematic, phylogenetic (Krebs et al., 2004, 2013; Doucet-Beaupré, et al. 2012) and phylogeographical (Mioduchowska et al., 2016) questions about freshwater mussels.”
Mioduchowska M, Kaczmarczyk A, Zając K, Zając T, Sell J. (2016) Gender-associated
mitochondrial DNA heteroplasmy in somatic tissues of the endangered freshwater mussel Unio crassus (Bivalvia: Unionidae): Implications for sex identification and phylogeographical studies. J. Exp. Zool. A Ecol. Genet. Physiol. 325: 610-625.

49-50: „Our aim is to evaluate the potential of sex-specific mitochondrial orf genes for population genetic and systematic studies in freshwater mussels” – this aim is wrongly formulated, and thus not implemented (lack of data for all freshwater mussels and few analyses for Ambleminae, in addition based only on f-orf sequences).

Discussion
Lines 147-149: this information seems to be suitable for the results section, but not discussion
Line 149: add the number of figures where you present ML and BI trees

Conclusion
164: In the line 133 information is provided about positive selection – then what kind of selection is it?

In the supplementary information you wrote: ”Samples were identified as Lampsilis siliquoidea or L. powellii based on geographic location”. Since the introduction provides information about the difficulty of species identification based on morphology, what was your confidence that the specimens were properly determined at the species level? Are the barcoding and orf sequences consistent with the systematics based on morphological features?

I would recommend final proof-reading by an English native speaker before submission. Some examples where the language could be improved:

Introduction
18: except for Antarctica
20: In fact, approximately 70%
22: filtration capabilities, and the production
31: The key feature
32: gene in addition to the 13 typical genes
33: the fastest evolving
39: Molecular techniques are commonly used to study
39: remove “is not uncommon”
40: add comma before “since”
41: add comma after “subfamilies”
41: conditions can affect
42: taxonomic identification
46-47: Therefore, it is justified to hypothesize that mitochondrial genes
47: associated with DUI regulation may be

Materials & Methods
61: Complete mtDNAs were PCR amplified, according to the method of Breton et al. (2011)
61: “in two halves”? Rephrase these unclear sentence, please.
62: primers listed in Table S1

Results
86-87: delate “gene” before order and revise the sentence to “… order of genes and their composition in Lampsilis spp. …”
90: are given in Fig. 1.
90: Our results show, consistently with the degree
92: delate “our results show”
97: add comma after “gene”
97-98: because many f-orf sequences are available in GenBank (each tissue except for sperm will contain f-orfs, making them easier to obtain).
102: delate “The” before ML

Discussion
124-125: delate “protein-coding”
125: genes encoding proteins
126: the role of proteins
126: delate “roles”
129: replace: “As such” with “Therefore”
130: it evolves in a different manner than
131: replace: “Taken together” with “In summary”
132: delate comma after “Ambleminae”
132: and they are ideal
134: replace: “hypothesized” with “hypothetical”
135-139: revise the sentence: “The general research trend has shown the involvement of sex-linked genes in reproductive isolation (Qvarnström and Bailey, 2009), therefore, demonstrating the participation of F- and M-ORF proteins in sex determination, as predicted by Breton et al. (2011), would particularly corroborate the potential of mitochondrial genetic speciation mechanisms.”
141: variability, but at the same time it should be sufficiently conserved to reduce phylogenetic noise
141: analyses have suggested
144: the systematic usefulness
146: delate comma after “support”

In Table S1, please change “Primer” to “Primer name” and  “Sequence (5’ to 3’) ” to “Primer sequence (5′-3′)”.

In dendrograms (Figure 2 and Figure S2), species names should be written in italics. Apply in Figure 2 the gray scale to indicate Lampsilini, Pleurobeminijak and Quadrulini as in Figure S2.
Experimental design: Materials & Methods
62: How big was the sample? True, the details are given in Table S1 but here, however, at least concise information should be provided.

Results
98: How many m-orf and f-orf genes are in GenBank? Perhaps it is also worthwhile to conduct such analyses on limited m-orf data, because the title suggests that such analyses have been carried out.

In the supplementary information you wrote: “Total DNA was extracted from male gonads or female mantles” – why did you use different tissues? Please, explain this point.
Validity of the findings: 'no comment'
Additional comments: REVIEW OF MANUSCRIPT #24609

I read with interest the manuscript entitled “Using sex-specific mitochondrial orf genes for population genetic and systematic studies in freshwater mussels (Bivalvia: Unionida)”.
I found that the draft included some new findings. However, there should be made some adjustments in order to make the reading logic and more fluid. Please see my comments and suggestions. After modification I hope the draft will be beyond the standard of PeerJ.


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

Input:
Title: EVALUATING THE UTILITY OF THE FEMALE-SPECIFIC MITOCHONDRIAL F-ORF GENE FOR POPULATION GENETIC, PHYLOGEOGRAPHIC AND SYSTEMATIC STUDIES IN FRESHWATER MUSSELS (BIVALVIA: UNIONIDA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I accept the rebuttal
Experimental design: It is acceptable
Validity of the findings: I still doubt it but if you insist this is just a pilot suggesting the possibility then I will not object.
Additional comments: I am disappointed you decided not to discuss the M genomes at all and barely did any mitogenomic comparisons but focused your ms on f-orf only instead. Still, this is your work, not mine...


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: EVALUATING THE UTILITY OF THE FEMALE-SPECIFIC MITOCHONDRIAL F-ORF GENE FOR POPULATION GENETIC, PHYLOGEOGRAPHIC AND SYSTEMATIC STUDIES IN FRESHWATER MUSSELS (BIVALVIA: UNIONIDA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the corrections made by the Authors. The weak points which were included in the preceding version were removed. Now I find this draft impressive and suitable with the standard of PeerJ.


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

Input:
Title: SYNERGISTIC AND ANTAGONISTIC EFFECTS OF IMMUNOMODULATORY DRUGS ON THE ACTION OF ANTIFUNGALS AGAINST CANDIDA GLABRATA AND SACCHAROMYCES CEREVISIAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript investigates drug interactions between antifungal and immunomodulatory drugs in C. glabrata and S. cerevisiae, in the context that these drugs can be co-administrated in a clinical setting. Overall the structure and concept of this manuscript is easy to read and comprehend. However, parts of this manuscript are unclear and need clarification in writing and further experimental procedures. Please see the following:
INTRODUCTION
•	More background information is required for C. glabrata in the clinical setting. Because you focus on C. glabrata and not the major agent of candademia, more information is required to focus on why C. glabrata is important for this study. Are there differences between C. glabrata and C. albicans in terms of treatment, predominant population they affect?
•	More context is needed for the mechanisms of drug resistance in Candida, especially to azoles as you focus on drug resistance in your manuscript
•	It is ambiguous why S. cerevisiae was included in your study as you do not make further mention of S. cerevisiae past the checkerboard results. Will an antagonistic S. cerevisiae response to FLC+MPA be studied later and presented in another publication? Further justification of why S. cerevisiae was included is needed aside from that fact that it is a great model to understand the evolutionary basis it presents in understanding drug resistance development
•	Please expand on the scope of drug combinations investigated by the scientific community. There’s plenty of literature that investigate drug and chemical library screens that synergise with antifungal drugs
•	More background information is needed in terms of the types of immunomodulatory drugs that are encountered in the clinical setting
•	More background information is required for MPA which becomes your main focus – is it known to have antifungal properties, what pathways does it target etc
•	Most studies screen for synergistic drug interactions to understand the mechanism behind them. You focused specifically on antagonism for which your conclusions for doing so at the discussion and conclusion is sound. However, you need to justify why you are looking at antagonism in the introduction
•	The end of your introduction need results and conclusions
Experimental design: A random selection of clinical C. glabrata along with pathogenic S. cerevisiae were tested. Antagonism was found prevalently between MPA and FLC, therefore MPA was further screened with other azoles. Because azole resistance is predominantly associated with increased expressed of efflux pumps, mostly Cdr1, the gene expression of this efflux pump as well as Pdr1 which regulates Cdr1 was investigated in MPA and FLC individually and when combined.
Both the CLSI based checkerboard assay and BI are accepted methods of investigating drug interactions. However, there are experimental methods that need clarification, more information and need to be conducted to a rigorous standard.
Below are the amendments needed:
•	It would be beneficial to state the MICs of your selected C. glabrata and S. cerevisiae strains to drugs tested in Table 1.  There are trends where organisms with high MICs show interactions with synergy compared to organisms with low MICs. Strain Sc6 is from sorghum beer, please make amendments that this strain is not a clinical isolate or justify why this strain was tested.
•	Lines 116-118. Please state the stock concentrations of all drugs used.
•	Lines 121-123. Please state the modifications made to the CLSI checkerboard assay - the CSLI protocol prepares all final drug concentrations in RPMI/test media and are performed in microtitre plates.
•	Lines 125-126. Different cell concentrations were used for singular and combined drug susceptibility assays. While both 3x103 and 5x103 cells/mL both fall into the range needed for inoculating microtitre drug plates, is there a reason why these tests need different cell inocula? Justification is needed. Is the reference Zupan et al., 2013 used for the cell counting technique or a higher cell inoculum? If the latter, this reference is not suitable.
•	Line 129. Is there a specific reason why assays were incubated at 37 °C when strains were grown at 35 °C? Usually assays are incubated at either 35 or 37 °C.
•	Line 134. What is the genotype of the Cgpdr1Δ mutant?
•	Line 134. Why was C. glabrata ATCC2001 not used in the initial drug screening and only for antagonism with azoles and immunomodulatory drugs? If you were to do further screening, it makes no sense to use a strain that was not in the initial drug screening, especially if the further screening only involves one strain. I would recommend testing C. glabrata ATCC2001 with the other antifungal + immunomodulatory drugs used in the first screen and incorporating these results in Table 1.
•	Please state the concentration ranges tested for antifungal and immunomodulatory drugs
•	Line 135. MICs change with media type. MICs tend to increase in rich media (YPD) compared to minimal media (RPMI). What was the reason the switch in media was made? Auxotrophy? If so, please substitute the required amino acids into RPMI and redo the MPA+FLC checkerboard with Cgpdr1Δ mutant.
•	Line 144. Azoles have a trailing effect and their MICs can be read at either MIC50 or MIC80. Which one was used? Please state this for immunomodulatory drugs as well if a trailing effect is seen.
•	Line 165. The concentrations used for FLC and MPA in the gene expression experiments may have been predetermined but they do not correspond to Figure 2A. Please supply the preliminary growth tests and checkerboard assay results are supplementary data.
•	Line 168. Please state your reasoning behind choosing timepoints 2 and 4h? Are there literature showing the expression of drug efflux can be seen at 2 and 4 h in particular environments? If so, please reference them.
•	Lines 181-182. Statistical analysis needs to be performed for the comparison of qPCR data, please include this. Also state the number of replicates used.
Validity of the findings: Reporting of findings were skeletal and lacking in robustness. Interpretation of results need to be defined for consistency for drug interactions. There are inconsistencies with what is reported and represented in the figures/tables. Please see the amendments:
•	Line 187. Please state the hours of incubation as three incubation times were used.
•	Line 188. Please state the range of concentrations strains exhibited resistance to FLC and ITR
•	Line 191. State the strain and the MICs obtained
•	Please reformat Table 2 so values and headings are legible in one line. State if MIC values are range, geomean or mode. State in footnotes MIC ranges that are considered susceptible, resistant etc. Alternatively reference this
•	Lines 197-199. Please quantify drug interaction findings instead of stating a majority showed synergy etc. E.g., 1 out of 6 strains exhibited synergy with drug A and drug B.
•	Lines 200-201. Please clarify ‘mixed responses’. Do you mean the interactions are indifferent? Or that across all strains tested, drug interactions were strain specific? All drug combinations are covered except for MPA+ITC. Please comment on this combination
•	Lines 202-203. Please clarify ‘BI scored more hits’. Does this mean more synergy/antagonism was seen with BI vs FICI? Is there are good correlation between FICI and BI? Interpretation of drug interaction with these two models combined as you have done in Figure 1 needs to be defined. E.g., Cg3 AMX+FLC FICI show indifference, but BI shows synergy. Is this combination considered synergistic because at least one model is? This needs to be clarified for Figure 1 and in methods.
•	Figure 1 needs a better key. A gradient in colours may be misread as level of drug interaction and not the level of consensus between FICI and BI. Perhaps separate out the colours as a separate key (you’re using a colour gradient) and the placements of FIC/SYN/ANT.  Please state if FIC max or FIC min is used.
•	Tables 3 does not state MIC results at RPMI, YPD, different temperatures or different time points as stated in text. Please amend this. Footnote of ‘n’ is ambiguous. Do you mean number of replicates? What is meant by ‘among 49 drug combinations for each strain’ when you only tested MPA + azole combinations with ATCC2001?
•	Line 210. MPA MIC should be in a table format with RPMI YPD different temperatures tested etc
•	Line 211. How did you assess timepoints as early as 4h? Did you assay this through back plating? If so, this needs to be stated in methods as to what protocol was being used and the results need to be shown. Please justify/reference the reasoning behind the choice of timepoints examined
•	Line 212. Last sentence is too big a conclusion to draw, especially as these results are drawn from one strain alone. Restate this sentence.
•	Line 222. Is there a statistically significant difference between gene expressions in the different treatment types? Incorporation of statistical significance is required
•	Line 223. Rearrange sentence to make it clear that you are comparing between the two different timepoints
•	Line 225. Rewrite paragraph so comparison of results do not jump across different parameters being measured
•	Lines 229-238. As it was shown Pdr1 is not central to MPA+FLC antagonism, was Cdr1 confirmed to be central to this antagonism? qPCR results will be more robust with testing a Cgcdr1Δ mutant with MPA+FLC. Have you considered the expression of other drug efflux transporters?
•	Figure 2. Please rearrange A B C D data in consecutive order they were discussed in the results. Antagonism was stated to be emphasised at MPA 8ug/ml and FLC 4 ug/ml. Why were qPCR experiments not performed at these drug concentrations? Please justify this lack of consistency in your methods
•	Figure 2A. The interpretation of antagonism in the graphs given is not immediately clear. What are the cutoffs in growth for the MICs of both drugs tested? Is the interpretation of antagonism a 2 fold increase in MIC for FLC? If so, this does not follow FICI e.g., if The MIC of FLC alone is 64 ug/ml, having an MIC of 128 in combination with MPA at 16 ug/ml is not considered antagonistic. Please also state the media used for this test
•	Figure 2B.  Same with the comment above. The stated antagonistic combination of 4 ug/ml FLC + 8 ug/ml MPA is not considered antagonistic according to FICI. Please justify this. Better representative data is needed
•	Line 235. Better to show BI interaction surface for Cgpdr1Δ mutant with MPA+FLC. Please comment on the decrease in FLC MIC seen here and not just in the discussion
DISCUSSION
More information is required for a robust discussion, specifically why you focused on the drug antagonism and the antagonism between MPA+FLC in the clinical setting. Additionally, the possibility of other drug efflux mechanisms that may play a role in MPA+FLC antagonism and antagonism from the MPA side of the story. Please also see the following:
•	It is not clear why pathogenic S. cerevisiae strains were included in the initial screen between antifungal and immunomodulatory drugs as you make no mention of them in the discussion. Please clarify this.
•	Line 245. Drug interactions can also be dangerous because of adverse reactions in the host. It is a more rounded argument to discuss this as well as the unwanted side effects of inducing drug resistance in a pathogen
•	Line 248. Clarity is needed as to how FICI and BI correlated well – need to define this in the methods and interpretation of results. Are there literature that you can cite which talks about the correlation of FICI and BI to support your claims?
•	Lines 255-257. You did not explore mechanisms of drug synergy is your study and therefore cannot make the claim that your ‘evaluation and interpretation of azoles and immunomodulatory drugs are on track’. Please rephrase this sentence. There are quite a lot of literature that report synergism between antifungal drugs and calcineurin inhibitors so reference these.
•	Lines 263-264. Please clarify which drug the strains have a high resistance to
•	Lines 264-265. Please clarify what the mechanisms of drug resistance may be
•	Lines 262-268. This section does not sound like a discussion and you don’t talk about the validity of your findings here. Discuss about the potential consequences of MPA+FLC antagonism in the clinical setting here. What is the frequency of MPA+azole use in the clinics? Why would this combination be important?
•	Line 272-274. Statistical analyses is required to support this claim
•	Line 277 – 278. This can be further supported by qPCR-ing CDR1 in Cgpdr1Δ
•	Lines 280-281. Clarification or a better representation of data is further needed for this method and the interpretation of these results as antagonistic
•	Lines 286-287. You may also want to investigate antagonism from the MPA side of drug interactions, not just solely on the FLC side
•	Lines 296-297. ‘We can speculate that this antagonism could be connected to this increase in fungal infections’ is an invalid claim. MPA is an immunosuppressant and the incidences of fungal infections as secondary complications are reviewed in Ritter & Priofski 2009. There is no claim to an increase in fungal infections with the use of MPA, let alone with MPA and azole drugs in combination. Additionally, there is no account of patients receiving antifungal and immunomodulatory drugs at the same time in the review and associated literature within. Please rephrase this sentence
•	Lines 297-300. Please rephrase this sentence. Which drug combination is being referred to? What do you mean by ‘entire set of drugs for a prescribe treatment’?
CONCLUSION
I like the end of the conclusion and the statements presented mostly connect to the original questions investigated. Please find the following amendments and also make amendments according to those from the discussion section above:
•	Line 306. ‘discovered an antagonistic interaction between MPA+azoles which is probably due to increased expression of drug efflux pump’. This sentence needs to be rephrased as you confirmed antagonism between MPA and azoles and performed qPCR experiments in only ATCC2001
Additional comments: Overall English language is legible. There are some sentences that are ambiguous and need clarification. Please see the following:
•	The aims for the manuscript in the last paragraph need to be tidied up. Expanding on the mentioned points in the introduction will certainly help the aims/goals of this manuscript.
•	Be consistent when referring to checkerboard/combination assays – choose one way to refer tests
•	Line 24. Common to clinical environment meaning they are already in use or that they are common due to nature of diseases seen in clinics?
•	Line 63. Resistance development and evolution – some resistance to drugs are transient
•	Lines 60-62. Not just the side effects, the time and money taken to develop new drugs is problematic.
•	Line 62. Synergistic and additive drug treatments not drug interactions
•	Line 64. Be clear about functions of pathogen – mechanisms of pathogenesis?
•	Line 65. Drug combinations currently used in clinics? Potential drug combinations?
•	Line 68. Drug combinations in the clinical environment are overlooked?
•	Line 75. What are these issues – does this belong to previous paragraph or existing paragraph? State issues first then state goals of project
•	Line 77-78. Do you mean ‘To explore the modulatory effects of immunomodulatory and antifungal drugs in vitro’?
•	Line 84. Please clarify this sentence so it corresponds to your methods
•	Lines 94-95. Make it clear which strains are QC strains for drug testing and which one is used for antagonism studies
•	Line 124. Suggest to state percentage of saline water, not 8.5g/L NaCl
•	Line 144. Please reference Meletiadis et al., 2005 instead of Liu et al., 2014
•	Line 162. Please change ‘Freshly sub-cultured C. glabrata ATCC2001 grew in liquid YPD overnight at 37 C’ to: C. glabrata ATCC2001 was grown in YPD overnight at 37 C
•	Line190. Change ‘did no reach MIC’ to ‘did not obtain an MIC’
•	Line 208. State what azole antifungals were tested
•	Line 243. Drug interactions is not a practice. Please amend sentence
•	Lines 246-247. Ambiguous sentence, needs to be clarified
•	Line 259. Please clarify what ‘they’ is
•	Line 283. Change exited to existed
•	Line 287. Rephrase ‘Antagonism: Clinical application’ as antagonistic drug combinations will not be applied in clinical settings
•	Line 296. Please cite original paper for increased fungal infections with MPA use


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: SYNERGISTIC AND ANTAGONISTIC EFFECTS OF IMMUNOMODULATORY DRUGS ON THE ACTION OF ANTIFUNGALS AGAINST CANDIDA GLABRATA AND SACCHAROMYCES CEREVISIAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: English is sound, throughout the manuscript. However, the paragraph starting in line 185 should be completely re-written. It is an absurdly incomplete, superficial and poorly writen description of results. For example, the sentence starting in line 187 mentions "some strains" and "certain antifungals". Nothing could be more vague!

Minor points:
Table 2 - what is meant by cross-reference "a"? If nothing, remove it.
Table 3 - cross-reference "a" is not mentioned in this table?
I suggest that in both tables only the acronyms are described, with no need for "a" or "b".
Experimental design: The experiments were carefully planned and professionally executed. However, the following few issues should be addressed:

1 - The scope of the paper is on C. glabrata. The addition of S. cerevisiae can clearly be justified for its role as a model organism. But its mention as a pathogen should be very carefull. Please be less emphatic in sentences starting in lines 20 and 56.
2 - The introduction section should be more clear in terms of what is already known on the use of drug combinations. The authors mention "several drug combinations ..." (line 65). How do previous studies compare to yours? Have the same immunomodulatory drugs been tested before for combinatorial effect with antifungals?
3 - The selection of the 6 out of nearly 50 clinical isolates is not well justified in the paragraph starting in line 88. Authors should present the MICs for the whole collection to justify the selection of the 6. And given their selection, results should be analysed in light of the MIC exhibitted by each strain.
4 - Other Candida species mentioned in lines 95 and 96 were not used, were they? If not, discard them from the M&M section.
5 - line 101 - Strains were not preserved in just glycerol, were they? Please specify medium and % of glycerol.
Validity of the findings: There is a problem with the interpretation of the results in table 2. The major finding of this manuscript, that is the antagonism between MPA and FLC is not observed for all the tested strains. Indeed it is only seen for 3 out of 6 Cg strains, while for 2 of the remaining there seems to be a synergistic effect! A more clear discussion on these results must be included, and I suggest additional work in this point:

 - given the proposed model that Cdr1 up-regulation is the reason for the antagonistic effect of both drugs, Cdr1 expression studies should be extended to the 6 Cg strains. By doing so the authors could establish a more clear correlation between Cdr1 responsiveness and the antagonistic effect, or absence of it in some strains. The fact that the effect might be strain dependent decreases the impact of this finding!

- please add statistical analysis to the results displayed in figures 2C and 2D.

- one of the interesting findings of this study is the suggestion that TFs other than Pdr1 may control CDR1 expression in the presence of MPA. Which TFs (line 237-8)? A few could be suggested based on the info gathered in the PathoYeastract database.

- the conclusion stated in line 250 that BI is more sensitive that FICI seems innapropriate. It would also be possible to argue that it is not more sensitive, it is more misleading, wouldn't it? If the authors have strong arguments for their point, please provide them.
Additional comments: Nothing to add.


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: SYNERGISTIC AND ANTAGONISTIC EFFECTS OF IMMUNOMODULATORY DRUGS ON THE ACTION OF ANTIFUNGALS AGAINST CANDIDA GLABRATA AND SACCHAROMYCES CEREVISIAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for making the corrections, the manuscript has significantly improved. There are still a few things that need to be added and corrected. Please see the following suggestions:

Introduction

•	Line 57. Does ‘a higher rate of antifungal use and the resistance of C. glabrata’ refer to innate resistance or developed resistance? Please clarify.
•	Line 78. ‘activating mutations in the gene PDR1’. Do you mean mutations that result in the overactivation of this gene? Please clarify this sentence.
•	Lines 81-83. Are all these genes in the genus Candida or specific to C. glabrata? Please clarify.
•	Line 86. Please capitalise and italicise Erg11.
•	Lines 88-91. Please clarify this sentence, it does not follow on from the previous sentence and the ending doesn’t make sense.
•	Lines 94-98. Are you drawing similarities between S. cerevisiae and C. glabrata in the first sentence? Please clarify the first sentence or delete it as it is redundant.
•	Lines 118-121. Please clarify that both AMB and 5FC are antifungal drugs. Is this combination used for Candida? Or is this combination used in general for mycoses?
•	Line 124. What studies is being referred to here?
•	Line 125. What inhibitor is being referred to? Of a protein of interest?
•	Line 142.Does MTX have the same antifungal mode of action as calcineurin? If not, please amend sentence.
•	Line 145. Does mycophenolic acid have an abbreviation?
•	Line 160. Please change ‘usually’ to commonly.
Experimental design: Please see the following suggestions:

•	Line 230. ‘200 to 2 mg/l’. Please make this consistent with how the other ranges are written.
•	Line 236. Please clarify if the triplicates are biological or technical.
Validity of the findings: Results:

•	Line 296. Please clarify ‘expected range’. Do you mean of all the antifungals tested?
•	Line 300. Please reference CLSI.
•	Line 314. How many S. cerevisiae strains and C. glabrata strains?
•	Line 316. What is the measure of uniformity here? 8/12 strains? Was it the same S. cerevisiae and C. glabrata strains in the 8/12 strains?
•	Lines 318-322. Are there any strains that showed synergism/antagonism across MTX or AMX?
•	Line 324. What are Cg5 and Cg6 resistant to?
•	Line 353. Please replace ‘two genes’ with PDR1 and CDR1.
•	Line 353. Please state how you did this in the methods. How did you normalise your data?

Discussion and conclusions

•	Line 386. Drug combinations in clinical settings are often unintentional and just occur and not achieved. Please amend sentence.
•	Line 390-391. Are these 8/12 strains the same strains that were antagonistic in FLC+MPA and synergistic in AMB+MPA? If not, please clarify.
•	Line 411-413. Please clarify sentence, it is not clear what you want to convey here.
•	Line 420. Please clarify what is meant by ‘this effect’.
•	Lines 421-423. This sentence makes it sound as though efflux pumps are not studied in S. cerevisiae or C. glabrata. Please amend this sentence.
•	Line 430. Please state increased resistance to what.
•	Line 435. Please state what the combination is for clarity.
•	Line 463. Please change ‘tolerance’ to resistance.
Additional comments: Previous suggestions and responses. Please see my responses starting with '>':

•	It would be beneficial to state the MICs of your selected C. glabrata and S. cerevisiae strains to drugs tested in Table 1. There are trends where organisms with high MICs show interactions with synergy compared to organisms with low MICs. Strain Sc6 is from sorghum beer, please make amendments that this strain is not a clinical isolate or justify why this strain was tested.
We agree, the MICs would be a good addition to the Table 1. However, in our opinion the addition would make the Table 1 too crowded and unclear. This is the reason we made a separate Table 2, where we have strains with their MICs. Additionally, we added a list of all the tested strains for the drug susceptibility (the ones mentioned in lines 90–91) to Supplemental Files (“List_of_strain.xlsx”).

We have added an explanation, why we picked Sc6 (from sorghum beer; it displayed high tolerance to fluconazole for a non-clinical isolate) and corrected it throughout the text (mostly we replaced “clinical isolates”, since they are only 11 out of 12, with “selected strains”).

> It is great that you have provided the MICs of the strains you have used. Please refer this data to the supplementary file in your manuscript so readers can refer to it if they wish to.

•	Lines 116-118. Please state the stock concentrations of all drugs used.

We can add the stock concentrations in the Supplement Files. However, we think that they aren’t as relevant to the publication and would distract from the clarity. Each experiment had a different set of stocks, the stock solution was usually prepared for each experiment individually depending on the final concentration of drugs, keeping in mind, that the relative DMSO value at the end concentration was always below 1 %. Publications with similar methodology (for example (Homa et al., 2016; Moreno-Martinez et al., 2015; Reichert-Lima et al., 2016; Valentín et al., 2016)) usually only have stated test ranges for each drug (we added this), but the stock solution concentrations are left out, since the data doesn’t add anything really scientifically important.

> Please put these in the supplementary files. It is for the benefit of other who want to replicate your methods from start to finish.

•	Lines 125-126. Different cell concentrations were used for singular and combined drug susceptibility assays. While both 3x103 and 5x103 cells/mL both fall into the range needed for inoculating microtitre drug plates, is there a reason why these tests need different cell inocula? Justification is needed. Is the reference Zupan et al., 2013 used for the cell counting technique or a higher cell inoculum? If the latter, this reference is not suitable.

There wasn’t a particular reason for the different concentrations, both were inside the range defined by the CLSI checkerboard assay. The susceptibility to a single drug was performed first and in later for the checkerboard assays, we used the slightly higher concentration. We haven’t notice this to have any effect and to be completely frank, we aren’t really sure why this change happened.  The focus was just to stay inside the prescribed range. We also argue, that this difference doesn’t have an impact.
We could maybe write it in a less confusing way, similar as in (Reichert-Lima et al., 2016): “The inoculums were 0.5 x 103 to 2.5 * 103 CFU ml-1”. Or in our case something along the line: “Inoculum was prepared by diluting yeast cells in RPMI medium to 3 x 103 to 5 x 103 cells/ml using the automatic ImageJ-counting technique...”.
The Zupan et al., 2013 reference is used for the cell counting technique.

> It is true it will not make a difference, but to not distract from clarity, please write it according to Reichert-Lima et al.

•	Line 135. MICs change with media type. MICs tend to increase in rich media (YPD) compared to minimal media (RPMI). What was the reason the switch in media was made? Auxotrophy? If so, please substitute the required amino acids into RPMI and redo the MPA+FLC checkerboard with Cgpdr1Δ mutant.

We confirmed that the antagonistic effect is present in both media – results were added in the Supplement Files (“ATCC_2001_RPMI_YPD.xlsx”), and this was respected for further experiments (especially for the future studies of the mechanism via chemogenomics/transcriptomics). The MICs in YPD after 48 h were higher than in RPMI, but the induced tolerance was still present at the boarder concentrations of fluconazole.

> Please state that you have done this in RPMI as well as reference to it as supplementary material.

•	Line 144. Azoles have a trailing effect and their MICs can be read at either MIC50 or MIC80. Which one was used? Please state this for immunomodulatory drugs as well if a trailing effect is seen.

MIC50 for azoles and immunomodulatory drugs, and MIC90 for amphotericin B. Added in the text.

> Why MIC90 for AMB? CLSI uses MIC of 100 for AMB.

•	Line 165. The concentrations used for FLC and MPA in the gene expression experiments may have been predetermined but they do not correspond to Figure 2A. Please supply the preliminary growth tests and checkerboard assay results are supplementary data.

Added a Supplement file (“Growth_curve_time_points_for_gene_expression.docx”), which includes the growth curve and the references for the time points.

> Please refer to this in manuscript text.

•	Please reformat Table 2 so values and headings are legible in one line. State if MIC values are range, geomean or mode. State in footnotes MIC ranges that are considered susceptible, resistant etc. Alternatively reference this.

Reformatted that the headings are in one line. It already states that the MIC values are range (in the heading), if this isn’t what you meant, we will require further explanation. In the footnote, the MIC ranges have the interpretation with added reference for the breakpoints. We haven’t changed how we marked the resistant strains. If this approach isn’t appropriate, please let us know.

> All good, no further clarification is needed.

•	Line 222. Is there a statistically significant difference between gene expressions in the different treatment types? Incorporation of statistical significance is required

Added the analysis (in methods, changed figures 2C, 2D), corrected in the results.

> Please add p values in figures and manuscript text so readers know which comparisons show the significance of difference between gene expressions.
Another thing you must include for qPCR is the normalization of gene expression data with your house keeping genes. Please include in your method for this.

•	Line 124. Suggest to state percentage of saline water, not 8.5g/L NaCl

Corrected.

> This reads as 8.5 g/L salt. Please change NaCl to saline solution.


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: SYNERGISTIC AND ANTAGONISTIC EFFECTS OF IMMUNOMODULATORY DRUGS ON THE ACTION OF ANTIFUNGALS AGAINST CANDIDA GLABRATA AND SACCHAROMYCES CEREVISIAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: All the previously raised issues were adequately addressed.
Experimental design: All the previously raised issues were adequately addressed.
Validity of the findings: All the previously raised issues were adequately addressed.
Additional comments: All the previously raised issues were adequately addressed.


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: IDENTIFYING THE PREDISPOSING FACTORS, SIGNS AND SYMPTOMS OF OVERREACHING AND OVERTRAINING IN PHYSICAL EDUCATION PROFESSIONALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I commend the authors for their work. The manuscript is clearly written in professional, unambiguous language. The paper is easily-read and the findings have important implications for this population. The theoretical background provided is enough and article structure is professional and clear for readers.

However, there are some concerns. Mainly, related to the sample size (and this might affect to the potential and impact of this paper). Additionally, some minor corrections have been requested.
Experimental design: Some comments are described below:

*Abstract:
- The reviewer is not a native english speaker, but some mistakes have been detected. Please, check the entire manuscript according to this.
- These abreviations must be explained here (and not in the next sentence)

*Introduction:
- I really enjoyed reading this intro section. It is well organised and easy for readers to undersntand the importance of this study.
- Lines 69-76: Any previous study to support this?

*Material and methods:
-Sample: I am not convinced about the convenience of this sample size. The paper would be reinforced if the authors provide the power of this sample size. This population (sports and exercise professionals) is huge, and a N=132 is not close to an infinite sample size...
- Lines 91-92: Many papers have been previously published about OVT or OVR in athletes. All those papers report training loads much higher than 4 h/wk. Take a look, for example, to this paper in which the author describe the training plan of a professional triathlete:
https://journals.humankinetics.com/doi/abs/10.1123/ijspp.2013-0245
Based on that, the criterion used for the authors to talk about OVR or OVT seems not to be enough... Could you report any cases of OVT or OVR with similar training loads?
- experimental design: It is quite controversial to talk about overtraining from a cross-sectional design...even more, if physiological (HRV, HR...) nor biochemical parameteres (cortisol, testosterone...) were nor monitored or measured.
- Questionnaires: Please, as well as the validation paper, provide the psychometric properties of both questionnaires
Validity of the findings: *Conclusions: lines 293-294
Please, add a reference to support this sentence (scientific consensus)
Additional comments: I commend the authors for their work. The manuscript is clearly written in professional, unambiguous language. The paper is easily-read and the findings have important implications for this population. The theoretical background provided is enough and article structure is professional and clear for readers.

However, there are some concerns. Mainly, related to the sample size (and this might affect to the potential and impact of this paper). Additionally, some minor corrections have been requested.


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: IDENTIFYING THE PREDISPOSING FACTORS, SIGNS AND SYMPTOMS OF OVERREACHING AND OVERTRAINING IN PHYSICAL EDUCATION PROFESSIONALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Title.
- In regards to the title, it is not clear what is meant by "sports and exercise professionals". It can be understand in many who for example: athletes or any professional dedicated to working with athletes or those who exercise (sports psychologist, sports nutritionist, etc.). I suggest that the title be more specific as to those who this research is about: personal trainers, coaches or other. I am suggesting that all wording written as: "sports and exercise professionals / workers", be clarified so as to understand,without a doubt, who you are talking about.

English language.
-The English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include line 100 : practice (no practise).

Abstract.
- Line 24 does not describe the abbreviation of OVR and OVT. These appear on lines 26 but should be on line 24, which is where these two abbreviations first appear.

Introduction.
- Line 78: I suggest:  ... symptoms of OVR and/or OVT ...

Discussion.
- Line 237-238-239, talks about shock microcycle training (high intensity) that improved the performance of athletes (tennis players). I believe they do not have much relationship with sports professionals (trainers). I suggest that you can include  some study that is related to the coaches.

introduction, discussion.
On many occasions it is mentioned that personal trainers/coaches intentionally modified the athletes (volume / intensity) load training but then talk about sports and exercise professionals (coaches or personal trainers) are exposed to symptoms of overtraining due to their own personal trainings, in addition to possibly be experiencing personal problems.  This is not clear, because first it refers to athletes and then to coaches.
Experimental design: The Borg scale has been modified. I suggest including some reference regarding the validity of this modified scale. For example, the Borg CR100 scale has been used as a complement to CR10 in some studies.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: - It is important to know/ask how many years the personal trainer/coach has been performing the work routine? Have you considered this question in your survey?


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: IDENTIFYING THE PREDISPOSING FACTORS, SIGNS AND SYMPTOMS OF OVERREACHING AND OVERTRAINING IN PHYSICAL EDUCATION PROFESSIONALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thanks to the authors for taking my comments and suggestions into consideration. I really think the paper has substantially improved.
The writing is much clearer and definitively easier for readers to follow and understand the text; and the changes done in the methods section (sample size and new references for the questionnaires used) along with the information added to the discussion section, improved the quality of the manuscript.
Congratulations on your work!
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thanks to the authors for taking my comments and suggestions into consideration. I really think the paper has substantially improved.
The writing is much clearer and definitively easier for readers to follow and understand the text; and the changes done in the methods section (sample size and new references for the questionnaires used) along with the information added to the discussion section, improved the quality of the manuscript.
Congratulations on your work!


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: IDENTIFYING THE PREDISPOSING FACTORS, SIGNS AND SYMPTOMS OF OVERREACHING AND OVERTRAINING IN PHYSICAL EDUCATION PROFESSIONALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you. All suggested changes have been modified.
Experimental design: thanks for including the reference.
Validity of the findings: Thank you for including these questions in the table 1.
Additional comments: Good job with this new version.


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: SEASONAL DYNAMICS ALTER TAXONOMICAL AND FUNCTIONAL MICROBIAL PROFILES IN PAMPA BIOME SOILS UNDER NATURAL GRASSLANDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please, see General Comment
Experimental design: Please, see General Comment
Validity of the findings: Please, see General Comment
Additional comments: This is a noteworthy, sound and welcome contribution to the field of soil microbial ecology. It is well conceived and designed, uses modern metagenomic methodologies, results are very ably analysed, and pertinent conclusions are drawn.
In my view, three general conclusions stand out:
1) the soil microbiota functional and taxonomic composition responds to seasonal and location variations;
2) Abundance of specific taxons is more sensitive to environmental change than functional capabilities of the microbiota;
3) a core fraction of the microbiota, that probably defines the specific biome being studied, persists over time and space.

The fact that the ms. does not need to go in-depth into specificities of the taxonomic and functional characterisation attests to the relevance of this study.

Among the strong points in the manuscript are the use of metatranscriptomics to analyse the functional composition of soil microbiota, an adequate level of replication, and a thorough statistical treatment of the significance of the data obtained.

Despite the overall, highly positive impression, I must note some specific points that, in my opinion, would lead to an improvement of the ms. if taken into consideration:

1. The abstract is written in a rather obscure and convoluted manner, and it is difficult to understand. I believe it would strongly benefit from a rewriting along the lines of what is more clearly stated in the Conclusions section.

2. In the introductory section, when authors introduce available scholarship on the effect of environmental factors on soil microbiota, they omit pH, a major driver of microbial diversity in the soil. I believe some of the current scholarship on that factor should be mentioned.

3. Throughout the ms.  authors run into an apparent contradiction. They claim to expect a higher abundance and diversity in warm seasons (l. 87), and when they get quite the opposite (l. 244-248), they rationalize it by stating that this is due to an increased abundance of fast growing microorganisms in summer (l. 330-338) without disappearance of other taxons. If this were indeed the case, I believe the observed results would be a direct consequence of a not-deeep-enough sequence coverage, and authors should elaborate on that.

4. I was a little surprised that authors chose to use functional metatranscriptomics, a decision to be applauded, but chose to amplify 16S rDNA rather than 16S rRNA, which would have allowed them to infer the metataxonomics of functional microorganisms.

5. I may have missed it, but, did authors compare metataxonomic assignments derived from amplicon metagenomics with those derived from metatranscriptomics? This would enrich the ms.

6 . Discussion on growth on nucleic acids (L. 323-329) is probably irrelevant, based on a  reference (Tozzi et al) that is probably not appropriate, and could probably be left out.


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: SEASONAL DYNAMICS ALTER TAXONOMICAL AND FUNCTIONAL MICROBIAL PROFILES IN PAMPA BIOME SOILS UNDER NATURAL GRASSLANDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Barboza et al. have used Next Generation Sequencing to compare the taxonomic (metataxonomics) and functional (met transcriptomics) profiles among soil samples from two subtropical natural grasslands located in the Pampa biome (Brazil) considering location and marked seasonal environment variation. They reported effects of season on both microbial community structure (abundance and richness) and functions. Also, the authors state that the seasonal variation mainly come from oscillation in the relative abundances of specific taxa along seasons. Finally argued the existence of a stable taxonomical and functional core microbiota associated to the biome.

The article is well set and described in all its parts (with few exceptions pointed below). The results and discussion section are accurately written and takes into account the data and justifies them. This kind of research increase our perception about microbial community ecology and physiology dynamic interaction with environmental factors, being a step forward to build the complex operational concept of soil healthy.
Experimental design: a. The experimental setup cover two location (SM and SG), two seasons (cold and warm) with 3 replicates. For the same season, there are two samples (may and august -cold) and (nov and feb - warm). Why there is no data comparing possible changes in microbial phylotypes and/or microbial functions? It would represent a knowing variation for local effect. We see no data in the article for this. why?
Validity of the findings: I would like to raise some points related to the validity of the findings:

a. The authors properly claimed that environmental factors such as temperature and moisture are main drivers of microbial community assembly. And the former is decisive to explain the experimental finds. Environmental factors are assumed to shape structural and functional diversity of microbial communities living in soil. However, for soils under influence of plant root community (rhizosphere effect), a new drive force emerges. Do the results obtained in this study coming from soils free of root exudates of the plant community? If not, the data interpretation related to location and seasonal effect will be strongly biased.

b. PCA graphic representation had shown clear distinction for phylotypes in location and season. Should be interesting highlight the contrasting pattern for presence/absence and abundance related to microbial functions. (Pg. 10, lines 3-5)

c. For PERMANOVA analysis (table 1) my first point is that for the microbial phylotype parameters, location and season interaction was significant at 5% probability. It means that we cannot infer the data for location and season separated. The community structure shifts emerge from the interaction factors. I think it has an impact in the data interpretation. For microbial function, there is no interaction and even location effect, so the evaluated microbial functionalities were preserved despite of the changes in microbial community. In this case, seasonal effects have a role, being the quantitative effect more pronounced than the qualitative. For functionality, it would be possible combine two location to strength the interpretation of season. Please show your view about the comment raised.

d. Even considering the same biome, climate and soil taxonomy for soil sampling, soil microscale variation will be an important factor, so it is no possible to avoid location effect over microbial structure as stated in pg. 10 (lines 9-14).

e. Why the cut-off of relative frequency greater than 1%? How representative is the sum of the others below 1%? We could group them in highest taxonomy order and bring them to be analyzed?

f. Table 2 show the major groups. Why the results were expressed sometimes for species, genera, or families? Why the diversity of predominant groups is low? The establishment of a core in this situation could be critic. Why do not consider the group of less frequency to compose the core?

g. Pg. 11 - lines 8 - 11: The authors mentioned that" In agreement with the differential abundance analysis, alpha diversity measurements indicated similar microbial richness (number of observed OTUs) during the seasons, but greater microbial diversity during the cold season (Fig. 3)". My comment: It is not so clear for Shannon index applied to taxa diversity.

h. Pg. 11 (lines 10--12): "Cold temperatures allowed for greater evenness while warm temperatures decrease the diversity of taxa" Why we cannot see it for observed alpha diversity? (Fig 3)

i. Pg. 11 -lines 12-13: The statement "The diversity of microbial functions presented similar trends, displaying a core set of microbial functions along the year in both locations (Fig. 3 and Table 3)". My comment: What you mean similar trend in this context?
Additional comments: Some minor points:
a. Soil samples were collected under natural grassland, currently used for grazing of cattle. Question: Is the sampled soil free of plant influence?

b. Please check the statement (pg. 5 - lines 14 -17) and compare with the raw data fig 1 plus fig 1. for example, rainfall regime for February 15th (check averages)

c. Pg. 5 - Lines 18 to 22: What is the relevance of this information for the study?

d. Note that PDF article submitted version fig 2 and fig 3 were misplaced (referred at pg. 10, line 2-3)

e. Figure 3 Legend seems to be incomplete

f. Pg. 11 (line 22-23): Legend Table 3. Is it correct? “Number highlighted in bold represent greater abundance during either cold or warm season.”

g. Pg. 12-13. line 24 and following:  Explain why? "Our sampling strategy allowed us to obtain a highly tractable model, with true landscape level of biological replications, for verifying whether soil microbial functions follow the changes in the abundance of taxa during seasonal variations"


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: SEASONAL DYNAMICS ALTER TAXONOMICAL AND FUNCTIONAL MICROBIAL PROFILES IN PAMPA BIOME SOILS UNDER NATURAL GRASSLANDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My assessment of the previous version of this manuscript was, overall, highly positive. This version has taken into consideration the reviewers' suggestions and has clarified some of the unclear points mentioned in my previous review, making it a better, more balanced contribution.
Experimental design: Some of the doubts arising out of the previous version have been satisfactorily solved. No further comment.
Validity of the findings: Again, this is a well rounded manuscript reporting on relevant observations, well carried out, analysed and discussed. Overall, a worthwhile addition to soil microbiomics
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: SEASONAL DYNAMICS ALTER TAXONOMICAL AND FUNCTIONAL MICROBIAL PROFILES IN PAMPA BIOME SOILS UNDER NATURAL GRASSLANDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors covered properly criticisms and question raised. As anticipated in my first review the contribution is scientifically sound and deserve a place on Peer J.
Experimental design: After R1-version of the manuscript, I have no additional comment related to experimental design.
Validity of the findings: After R1-version of the manuscript, I have no additional comment related to validity of the findings.
Additional comments: No additional comments


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

Input:
Title: CAN NON-DESTRUCTIVE DNA EXTRACTION OF BULK INVERTEBRATE SAMPLES BE USED FOR METABARCODING?
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: I think one important point that could also be discussed is whether you observed any issues with cross-contamination when trying to amplify/sequence individual specimens post bulk non-destructive extraction. To my mind, this would be one of the major confounding factors for re-using specimens after bulk treatment – i.e., residual mixed DNA from the bulk extraction retained on each individual body that might result in co-amplification of barcodes in what should be an individual reaction. If this was not observed to be a problem, that is worth mentioning in the results and discussing at greater length. Especially given the comment early in the paper on the ability to use the ethanol from sample vials for metabarcoding - akin to an eDNA approach.
Additional comments: The paper is well written, clear, concise, all figures and tables are easily interpretable, and provides sufficient introduction to concepts and citation of relevant literature. I have no problem recommending this for publication pending some very minor changes.

Minor comments
Introduction – maybe worth including a citation for Krosch & Cranston (2012, Chironomus)?  Authors can decide relevance – this paper reported a modified protocol for non-destructive DNA extraction from chironomid pupal skins that expands the barcode-able collection of biomonitoring programs.
Line 108 – ‘for  intended for’, remove first ‘for’
Line 226 – change ‘primers’ to ‘primer’
Line 392 – change ‘such those’ to ‘such as those’
Line 394 – remove ‘as’
Line 402 – ‘trialled’
Line 460 – change ‘i.e.’ to ‘e.g.’
Table S1 – amend column widths to avoid split words (maybe landscape layout would be better). Some genus names are italicised, some not – please make consistent


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: CAN NON-DESTRUCTIVE DNA EXTRACTION OF BULK INVERTEBRATE SAMPLES BE USED FOR METABARCODING?
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Carew et al. aims to test if freshwater macroinvertebrates used for non-destructive DNA-extraction are damaged beyond subsequent species recognition, and if DNA can be extracted in a second round. The findings are interesting to a wide audience and of importance for future development of DNA metabarcoding for monitoring and assessment of freshwater ecosystems. Although I have heard people talk about trials at conferences, I have not seen any published studies presenting this proof-of-concept before.

The paper is written in a clear and concise language and I think the authors do a very good job in presenting the results from a complex experimental setup. The use of references is very good and the background information sufficient for the reader to get a relevant impression of the field.

The use of figures and tables is extensive, but adds value to the paper by presenting results in a clear and easy-to-understand manner.
Experimental design: The experimental design is quite complex, but makes sense in the light of the primary research questions asked. The most problematic issue is the lack of true replicates to evaluate the variance often observed in multiple extractions of the same bulk sample. I know this can be resource demanding to achieve, but the good thing is that the authors have one technical replicate in experiment 2 (with 120 individuals).

I am wondering if residue ethanol in the bulk sample before adding lysis buffer might have influenced the DNA extraction success of "hard" animals. In the methods the authors describe that ethanol was pipetted out, but not if the sample was dried completely before buffer was added. If residue ethanol was present inside beetles and other hard shelled animals, it would prevent lysis and extraction from these specimens.
Validity of the findings: The discussion is mostly adequate and good, but should include considerable more detail on the issues mentioned under experimental design. The lack of replicates reduces the explanatory power of the results and this should be mentioned. Also, there are some differences observed between the two replicates that the authors do present. Please discuss this with reference to papers that argue for multiple DNA extractions of the same bulk sample when doing metabarcoding.

Please also discuss the eventual influence of the ethanol removal step in the extraction of DNA from bulk samples.
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: CAN NON-DESTRUCTIVE DNA EXTRACTION OF BULK INVERTEBRATE SAMPLES BE USED FOR METABARCODING?
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some editing is required through the text. It is hard to follow, some sentences are weak and the way in which they are written makes the paper very dense. I understand that as a methodological paper, many details need to be provided. However, I recommend some editing to make some parts more concise. For example, line 235: …., and were amplified in triplicate using…. [skip ‘amplifications were performed as three replicates’]. The PCR replicates were pooled and cleaned…

Also, the flow of the procedure is very confusing. I found quite hard following the direction of the experiment (Component 1). I suggest the authors re-writing this section following a unique direction from Extraction 1 to Extraction 2, without going back again to Experiment 1 and then to Experiment 2 many times. Also, to be consistent between Fig 1 and Table 1, consider renaming Experiment 1a and Extraction 1; and Experiment 1b and Extraction 2 using the same term. It is quite difficult to follow. In addition, I recommend moving Tables 2 and 3 to the results section.

In contrast, in Component 3 more detail in the text is needed. Many steps depicted in the figure are not explained and makes it difficult to follow.

Figure 1. Add accordingly a, b, c because it is mentioned in the text but not in the figure.

Table 1. As mentioned above, try to be consistent with the nomenclature between Table 1 and Figure 1 to ease the flow. Also, the information provided by Aims and Question is redundant. To make the table more informative, I recommend the authors, skip one of them and add a third column with the answer to each question/aim.
Experimental design: I thank the authors such a detailed experimental design to address many questions crucial for the better understanding of metabarcoding methods for environmental monitoring. I recognize the big amount of work that this experiment requires and the detailed manipulation of animals needed to mimic the samples (especially in Component 2). Sufficient detail is provided (except for Component 3 - see comment above)

The questions of the paper are clear enough but the introduction lacks a sentence with the hypothesis/aims/questions of this research. Which then can be followed by the procedures in Line 86-96
Validity of the findings: The findings regarding the amplification success greater than 88% (Line 329) is not reflected in the material indicated for that (table 5). Table 5 only provides the number of reads obtained for each sample of known composition, but not for the amplification success. Please provide.

The findings regarding Component 3 must be supported with figures/tables. The figure supporting the statement that "Overall, most taxa were detected in samples (Line 361-362)" must be provided/mentioned.

The discussion is well stated and linked to the original question. I highlight the fact that the possible reasons for some failures in the method are discussed. Yet, I highly recommend extending the discussion focussing on the pros and cons of this method in the case it is aimed to be implemented in routine monitoring. If so, a high economic investment (cost of consumables and time) would be required.  An attempt is done in lines 321-323, but it should be better explained.
Additional comments: I congratulate the authors for the nice experimental design of this research. I provide some minor comments that should be addressed.

It is expected that the digestion in pk in Extraction 1 will degrade some tissues making it difficult for the subsequent taxonomic identification of specimens. That might be problematic if, in practice, this procedure is followed. Wouldn’t be possible swapping the order of the steps and performing the morphological identification first and then going for the DNA extraction? Clarify this, please.

Move lines 113-115 to the beginning of methods section

Line 132: specify the target gene (COI). Also, it is well known that the degenerated versions of this primer set perform better than the Folmer. Why did not the authors use those?

Explain what sort of technical replicates (i.e. metabarcoding samples twice).

Line 221: this sentence leads to misunderstanding. It appears that the DNA barcode region has only three amplicons. Please re-write to give sense to the sentence.

Line 228-230: move to line 240. It interrupts the flow as you are still describing the first round of PCR.

Line 266-268: specify the search in the databases. All barcodes belonging to freshwater invertebrates?

Line 279: Fig 1a shouldn’t take place here; it is only methodology.

Table S1: provide it in a different format so that the test in the column fits properly.

Table S2: From 80, 100 and 120 individuals in Sample A, B and C, only 2, 8 and 6, respectively, could be DNA barcoded? Explain the reason.


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: DYNAMIC SUCCESSION OF SUBSTRATE-ASSOCIATED BACTERIAL COMPOSITION AND FUNCTION DURING GANODERMA LUCIDUM GROWTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: With pleasure I have been reading the article ‘Dynamic succession of substrate-associated bacterial structure and function during Ganoderma lucidum growth’. The article is well written in a clear and scientific manner. This study is in my opinion quite novel since it looks into the bacterial community that is growing together with the mushroom Ganoderma lucidum. In general, there have not yet been many studies that look into the bacterial composition on mushrooms and on its substrate. In addition, the mushroom Ganoderma lucidum has for the past few decades attracted attention with scientists all over the world because of its medicinal properties, which makes a study on this mushroom species very interesting.

I do have a number of points of feedback. I believe these are necessary to address to make the article easier to understand for the reader. In addition, I have feedback on the scientific aspects of the study.

To start, the title should not include the term bacterial structure, but rather should include the term bacterial composition.

It would be nice to include a more extensive discussion/conclusion, in which for example available literature on bacterial cultures on mushrooms and substrates are studied. This can be nice to compare the current study with. Also, it would be nice to include speculations as to why particular genera are more or less abundant in later stages of the mushroom’s life. For example, it is possible that Ganoderma lucidum defends itself against bacteria, because it produces many compounds including antibacterial ones and as such might be able to shape its own bacterial community. This is just an example and does not have to be included in the article.
Experimental design: The bags were sterilized using autoclaving, how can it be that bacteria start to grow on the substrate/mushroom during the hyphal stage?  Were the polypropylene bags open on one side or did the bags have filters that allow bacteria to enter? This is currently not clear and needs to be explained, since these aspects have a tremendous impact on bacterial composition. It would help if picture(s) can be included that show the reader what the growing conditions look like. Pictures of taking samples would also really help. For example, it is currently not completely clear from what parts of the substrate/mushrooms the samples are taken and what the material looked like. Also, the inclusion of information on the humidity in the room might be important, as moisture/condensation on the substrate/mushroom is a perfect breeding area for bacteria and as such can have a huge impact.
Keep in mind that with the information provided in this paper other researchers should be able to replicate this experiment.

In line 112 it is mentioned that 25-27 cycles are used during the PCR. Since the impact of the number of PCR cycles is high for the outcome, it might be nice to indicate which samples received what number of cycles.

Were the PCR products purified before being pooled? If yes, please include this in the methods.

It would be nice to include a method of quantifying the amount of bacteria, instead of only having compositional data.

It is very nice that a chemical analysis was performed on the substrate material, but what I am missing is the measurement of acidity over time. It is commonly known that mushroom substrates become more acidic over time because of digestion of the substrate and as such particular bacterial species will thrive on this more acidic substrate. This is why I expect acidity to be a large confounder and it would be nice to include in the current study. Same applies to the moisture content of the substrate.
Validity of the findings: It is very nice that a KEGG pathway analysis was performed, but what I would like to see is not only the dry observations of the pathways changing over the growing phases, but rather also include what the biological reasons might be that drive the change in pathways.

In figure 2, a cladogram is shown including 12 samples in 4 groups. This results in 3 samples per group, which I believe is a very low number to make conclusions.

From the article I understand that one single fruiting body was used for sampling, which means that the power number is 1 (N = 1). With such a low power number, it is hard to come up with meaningful conclusions.

In the heat map it seems that for each group of samples, the average is being presented. I think it would be better to include all samples, rather than the average. This will give the reader more information in terms of similarity between samples within a group and will give an idea of the variability of particular bacterial groups.

At the y-axis of the barcharts it says Richness, I believe this should be Relative abundance.
Additional comments: Interesting research and good luck with the revisions!


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

Input:
Title: DYNAMIC SUCCESSION OF SUBSTRATE-ASSOCIATED BACTERIAL COMPOSITION AND FUNCTION DURING GANODERMA LUCIDUM GROWTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the purpose of the study was to investigate the interactions between fungal growth stages and soil bacterial community, how soil bacterial community shapes fungal growth and vice versa. The objective is to be commended and provides new insights into fungal-bacterial dynamics.
However, there are several issues with the approaches utilized and the presentation of the data.
Experimental design: Why was the substarte autoclaved?  Higher diversity at later stages  may be due to bacterial growth after colonization at cultivation sites..

Line 84: It is not clear what and where the cultivation sites are.

Lines 87-99: three replicates per growth stage makes 4 fungal samples. How many substrate samples were obtained? Throughout the results and discussion, references are made to differences between substrates and between substrates and growth stages, yet no results to indicate this.

Line123: why the 0.001% cut off?
Validity of the findings: Lines 139-144. State the statistical values for results that are stated to be significant and vice versa, in the sections.

Lines 147-148: What does 295 OTUs shared in substrate between four growth stages mean? Again, state the associated statistical values for reports that are significantly different or not.

The results are present for both phyla and genera. Pick one. And revise discussion and other results sections accordingly.

Lines 168-174: There were no significant differences in fungal bacterial communities in across stages, despite the clear separation in the nmds plot. How does this relate to Table 2? Again, state the associated statistics.

Lines 187-199: Not entirely sure the heatmap analysis is required. Also, how did the 50 top bacterial OTUs get determined? Are these the OTUs that differed among samples from the LEfSe analysis?

Difficult to track results, for a variety of reasons.
1. There are phyla and genera results, but discussion crried out at phyla level.
2. There are different things mentioned but not presented. Substrates, substrate and growth stages, and growth stages. However, only growth stages are shown. If these were needed, they should be in results and not in supplementary materials

In the discussions, limit the speculations regarding functions of bacteria ( which were done at the phyla level), and avoid using words like co-evolved ( line 283) in this instance. The results do not speak to this.
Additional comments: Overall, I found the paper of values. I however, don't like the combination or rather a partial combination of substrate and fungal associated data. Partial because we don't see any actual substrate data. I would recommend focussing on only the fungal stages, doing the analysis at the genera level and discussing the results at the genera level.

Sterilizing the substrate prior to inoculation ensures a common starting point, but this impacts how bacteria can be said to improve fungal growth. However, in the 35 days it takes for the spores to germinate, colonization of the substrate would have occurred. I believe, just tracking the differences ( alpha and beta diversity) among soil samples at each sampling point and doing the same for fungal samples is a better approach, than comparing whats in the soils with whats in the fungus. You do not have any direct evidence of how this might be the case. Explain the sampling process for fungi and substrate properly. Minimize speculation of function based solely on this descriptive study, unless u can add quantitative data (RT-qPCR or qPCR) on the abundances of some of these bacterial genes in fungal samples.