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: EXERCISE CAN IMPROVE SLEEP QUALITY: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: Dear Authors
Thanks for replying the previous comments
one major point is not well considered in your research. ac cited before, your title refers to considering the sleep quality, and neither specific age group nor population hasn't been categorized to be considered, so, its highly recommended to consider two main categories (elderly people and athletes) who have been enormously studied in different investigations. since exercise plays the key role in sleep cycle of athletes and elderly, more relevant references in this context must be applied, therefore the following reference are also recommenced to be referred.
1- Monleon C, Hemmati Afif A, Mahdavi S, Rezaei M. The Acute Effect of Low Intensity Aerobic Exercise on Psychomotor Performance of Athletes with Nocturnal Sleep Deprivation. Int J Sport Stud Hlth. 2018;1(1):e66783.
2- Irandoust K, Taheri M. The Effect of Strength Training on Quality of Sleep and Psychomotor Performance in Elderly Males. Sleep and Hypnosis. 2018;20(3):160-5.
3- Taheri M, Valayi F. Aerobic Exercise Improves Attention and Quality of Sleep Among Professional Volleyball Players.


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

Input:
Title: EFFECTS OF ENVIRONMENTAL HYPOXIA AND HYPERCARBIA ON VENTILATION AND GAS EXCHANGE IN TESTUDINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.) Overall, the study was well framed and the data/meta-analysis make a nice contribution.  However, I suggest the authors thoroughly proofread the paper for proper English grammar and syntax.  For example, lines 82-83 read, “It has been shown, that the normal response to either of the changes…” Lines 97-98 read, “C. carbonarius was chosen because it is a wide-spread South American tortoise that not had its respiratory physiology investigated previously…”  These kinds of errors were prevalent throughout the paper.
2.) Additionally, my view is that the paper would read more clearly if it included distinct “Results” and “Discussion” sections.  As written, the discussion/interpretation is intertwined with the results.  This makes assimilating trends observed in a large number of respiratory variables across multiple species rather difficult.
3.) It would be helpful if Figure 1 was, or included, raw data showing a “zoomed in” series of breaths (and also breath-to-breath O2 consumption) with all of the respiratory pattern parameters assessed in this study clearly labeled.  This will improve the reader’s ability to follow along during the presentation of the results.
4.) In text citations with more than two authors were inconsistent.  Some citations were reported as “Author A et al., date” while others were reported as “Author A, Author B, and Author C, date.” Please choose one style and adhere to that format.
Experimental design: 1.) Although references to methods are cited, please include more methodological detail specific to the experiments performed for ventilation and O2 consumption measurements. (1) What was the approximate dead space volume in the mask and funnel and what were the flow rates for each experimental set up? Since the mask and funnel were, presumably, small volumes, flow rates need to be sufficiently high to prevent rebreathing.  If flow rates were not high enough, rebreathing of the gases in the mask or funnel could alter interpretation of the present results.  (2) How was the calibration and analysis for O2 consumption performed? Based on the references, it seems that the O2 fraction measured for each breath was determined from a calibration curve with known gas concentrations injected into the mask.  Was this the case?  A bit more detail would improve the ability to understand the experiments performed.  (3) For calibration of the pneumotach, is the volume obtained from integrating the flow signal linearly across the physiologically relevant range of flow rates described in the results (~5-60 ml s-1; Fig. 5)?  Clarification of each of these points in the text of the manuscript would improve my confidence in the data presented.
2.) Please clarify the numbers of animals used in each analysis.  The authors indicate that 8 T. scripta and 6 C. carbonarius were used in this study.  However, lines 144-147 read, “Experimentation started around 8:00 am and ventilation and gas exchange were measured under normoxic conditions, followed by progressively decreasing hypoxic (9, 7, 5, 3% O2) or progressively increasing hypercarbic (1.5, 3.0, 4.5, 6.0% CO2) exposures.”  Was each animal exposed to both progressive hypercarbia and hypoxia, or were only some of the animals from each species exposed to either hypercarbia or hypoxia?  If, say, half of the animals from each species were exposed to either hypoxia or hypercarbia, the “n” of each experiment is likely to be very low.  Please clarify these points in the text of the manuscript and mark the “n” for each analysis in figure legend.
Validity of the findings: 1.) With respect to interpreting the results from hypercarbia/hypoxia from the experiments and the meta-analysis, it is worth mentioning that intracardiac shunt patterns in reptiles can have unpredictable effects on blood gases during hypoxia and hypercarbia,  Lines 345-346 state that variation in ventilatory sensitivity to gases observed among species may “suggest varying regulatory mechanisms of breathing pattern during hypoxia and hypercarbia and between species.”  Although this may be true, it is also possible that mechanisms of breathing control are similar across species, but differences in intracardiac shunt patterns produce arterial gases that lead to differences in stimulatory signals at chemoreceptor sites during hypoxia/hypercarbia and therefore contribute to the range of ventilatory responses across species.  For example, at least in toads, the right-left cardiac shunt decreases during hypoxia (Gamperl et al., 1999, J. Exp Biol. 202, 3647–3658).  Thus Po2 and the magnitude of the ventilatory response to hypoxia, and hypothetically hypercarbia, is likely to be influenced by ventilatory “chemosensitivity” and cardiac shunt patterns.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF ENVIRONMENTAL HYPOXIA AND HYPERCARBIA ON VENTILATION AND GAS EXCHANGE IN TESTUDINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very detailed descriptive study on the effects of hypercapnia and hypoxia on the ventilatory pattern in two species of chelonians. The study provides novel data on a hitherto un-described tortoise (Chelonoidis carbonarius) and make comparison to the much more well-characterized Trachemys scripta (the “white rat of reptile physiology”). The study shows that the differences in breathing pattern amongst terrestrial and aquatic testudines is not as marked as previously reported and this conclusion is of interest. Secondly, the manuscript provides an excellent review of the available data on the ventilatory responses to CO2 and hypoxia in turtles. This is a very useful resource for future studies.

I have made some minor suggestion in the annotated PDF that the authors may consider when revising the manuscript. Some of these are merely suggestions to improve wording or clarity; there are also other types of suggestions.
My only main concern is the very descriptive nature of the study and the manner in which the manuscript is written. Is would be desired if the authors could provide a more defined rationale for the study – what are the specific goals. Then, the discussion should follow up on these problems and make more firm statements based on the extensive literature review.

Tobias Wang
Experimental design: well performed
Validity of the findings: no concerns
Additional comments: This is a very detailed descriptive study on the effects of hypercapnia and hypoxia on the ventilatory pattern in two species of chelonians. The study provides novel data on a hitherto un-described tortoise (Chelonoidis carbonarius) and make comparison to the much more well-characterized Trachemys scripta (the “white rat of reptile physiology”). The study shows that the differences in breathing pattern amongst terrestrial and aquatic testudines is not as marked as previously reported and this conclusion is of interest. Secondly, the manuscript provides an excellent review of the available data on the ventilatory responses to CO2 and hypoxia in turtles. This is a very useful resource for future studies.

I have made some minor suggestion in the annotated PDF that the authors may consider when revising the manuscript. Some of these are merely suggestions to improve wording or clarity; there are also other types of suggestions.
My only main concern is the very descriptive nature of the study and the manner in which the manuscript is written. Is would be desired if the authors could provide a more defined rationale for the study – what are the specific goals. Then, the discussion should follow up on these problems and make more firm statements based on the extensive literature review.

Tobias Wang


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: 3D SKELETAL MUSCLE FASCICLE ENGINEERING IS IMPROVED WITH TGF-Β1 TREATMENT OF MYOGENIC CELLS AND THEIR CO-CULTURE WITH MYOFIBROBLASTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript was well written.  The organization was logical and statements supported by data or the literature.  The authors should have cited Am J Physiol Cell Physiol 2001;280:C288-95  since this work describes the importance of the addition of fibroblasts to for functional myotubes in 3D scaffold-free cultures.

Line 154.  The reference Baaijens et. al was not provided.  Note  the phrase is "et al." not "et. al".

Figure 1 should indicate when TGFbeta was added relative to the start of differentiation.  Also, clarify in the methods section whether the time of TGFbeta addition was the same in 2D and 3D culture.
Experimental design: The experimental design largely follows that of of other studies and the hypothesis is not novel.  The methods are generally well described and can be reproduced with one comment noted below. As noted above Dennis et al. (2001) showed the importance of adding fibroblasts to enhance myotube formation   and increase contractile force generation in 3D scaffold-free muscle cultures.  The studies with TGFbeta largely replicate and validate the work of Weist et al.  Consequently, the investigators do not provide a novel hypothesis or indicate what new information will be sought.

Figure 1 should indicate when TGFbeta was added relative to the start of differentiation.  Also, clarify in the methods section whether the time of TGFbeta addition was the same in 2D and 3D culture.
Validity of the findings: Overall, this study was technically well done but adds little to our understanding of the role of TGFbeta on the function of 3D muscle cultures. The effect of TGFbeta on myofibroblasts in 3D is rather modest given the large number of nuclei shown in the supplemental figure and does not appear to effect function  or  the ability to maintain 3D cultures for 24 hours or longer.  The effect of TGFbeta on myoblasts is significant and agrees with the earlier work of Weist et al.   Further,  Dennis et al. (Am J Physiol Cell Physiol 2001;280:C288-95) have previously shown the benefits of adding fibroblasts to scaffold-free 3D cultures, although the current study   provides some additional details of the impact of fibroblasts to myoblast 3D cultures.  The new information is the effect of the various treatments on the Young's modulus, the dimensions of the constructs and the organization of myotubes with added fibroblasts.

The authors argue that removal of the  3D cultures from the posts is analogous to the slack test applied to muscle fibers to measure the zero force velocity.  Removal of the  muscle rings from the posts does eliminate tension arising from forces required to maintain the diameter of the post.  However, since the  dimension of the ring depend on conditions, a tension remains  after removal of the post due to the passive forces arising from differences in constructs.  Cutting the ring would remove any internal force and replicate the slack test.

The enhancement of myofiber formation is consistent with the work of Weist et al.,  although they observed more myofibers present in the absence of TGFbeta, possibly a reflection of the use of primary cells.

The manuscript would be stronger if new mechanistic of functional insights were obtained.  As is, the study is largely duplicative and does not increase our understanding about the role of TGFbeta in 3D muscle cultures.
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: 3D SKELETAL MUSCLE FASCICLE ENGINEERING IS IMPROVED WITH TGF-Β1 TREATMENT OF MYOGENIC CELLS AND THEIR CO-CULTURE WITH MYOFIBROBLASTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: 1)	The title of the article states ‘Skeletal muscle Facile engineering is improved by co-culture with myofibroblasts and TGF-β1’. The term " co-culture" can be used only if two cell types are cultured together and do not apply to co-culture with growth factors or supplements. Hence the title should be restructured to include another cell type or changed to supplementation of TGF-β1 and not co-culture. Also, it should also include that it specifically in 3D culture and not just co-culture.

2)	The authors should explain in detail about their self-assembled agarose tissue culture since it is not mentioned in the methods section. They mentioned that the custom agarose rings with central mold, but it is not clear why they chose that design. I strongly suggest them to provide a clear picture of the mold and explain the structure in detail.

3)	Also, authors have mentioned that the cell seeding density was 350k cells/millimeter of post diameter, but did not explain why this seeding density was chosen or is there any data that supports this cell density. The self-assembled structure design and the cell density in the structure are highly critical because this is the basis of the mechanical testing results that the authors had obtained, so this has to be explained clearly and thoroughly.

4)	The authors treated the cells with 1ng/ml TGF-β1 in this study. Will the authors be able to provide data on what will be the dose-response of TGF-β1 in myotubule expression on the cells, to select the optimal amount of TGF-β1 required? Also, if this optimal amount varies between 2D and 3D cultures.

5)	In 2D cultures of the fibroblasts, the authors stated that there is no difference in collagen 1 transcription level during TGF-β1 treatment but reported that increased collagen expression in immunostaining. There was no explanation given as to why they observe this or why they think that post-translation collagen expression is higher.

6)	The authors did not provide any information on the size of the self-assembled agarose 3D cultures or collagen 3D hydrogels. It might be a fact that some of the downregulated genes that the authors are observing may be because the gels are diffusion limited. Live/dead staining images of the cells in the hydrogels to see the percentage of the cells survive in the hydrogel environment would be very informative. Authors should consider perfusion systems for culturing 3D tissues otherwise the cultures may be diffusion limited preventing the mass transfer of nutrients and gas exchange.

7)	The authors mentioned that the expression of collagen is downregulated by fibroblasts in 3D cultures compared to 2D cultures. In fig H they show that just by the addition of fibroblast to myoblasts in 3D cultures Young's modulus of the hydrogel increases. If this indeed is a factor why does the modulus is increasing although the collagen 1 expression is down-regulated in 3D cultures? The authors should provide a reasonable explanation for this.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: 3D SKELETAL MUSCLE FASCICLE ENGINEERING IS IMPROVED WITH TGF-Β1 TREATMENT OF MYOGENIC CELLS AND THEIR CO-CULTURE WITH MYOFIBROBLASTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: The issues I raised in my original review 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: TAXONOMIC ANNOTATION ERRORS INCORRECTLY ASSIGN THE FAMILY PSEUDOALTEROMONADACEAE TO THE ORDER VIBRIONALES IN GREENGENES: IMPLICATIONS FOR MICROBIAL COMMUNITY ASSESSMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The study is useful in its research area and suited for publication.


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

Input:
Title: TAXONOMIC ANNOTATION ERRORS INCORRECTLY ASSIGN THE FAMILY PSEUDOALTEROMONADACEAE TO THE ORDER VIBRIONALES IN GREENGENES: IMPLICATIONS FOR MICROBIAL COMMUNITY ASSESSMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: In this paper, Lyndon and Lipp call the field’s attention to errors in recent versions of the Greengenes 16S rRNA gene taxonomic database. Sequences corresponding to Vibrio and Pseudoalteromonas are often misclassified using Greengenes, as evidenced by cross-checking with SILVA, RDP, and NCBI’s 16S database. The purpose to the paper is to provide evidence for the errors, raise awareness of the issue, and to call the field to better curate these databases.

The paper has two main results - (1) evidence that the errors are present in the database and (2) evidence that these errors have impacted the field boradly.

Result 1

The authors mention in the discussion there could be two types of errors - Vibrio that are misclassified as Pseudoalteromonas and Pseudoalteromonas that are misclassified as Vibrio. However, their data appears to only explore the latter problem.

I feel the strongest way to communicate result 1 would be a phylogenetic tree of the Alteromonadales and Vibrionales 16S sequences from the Greengenes database with indication on the tree the taxonomic classification of each sequence based on the different methods the authors tried.

In the methods section, it says BLAST, SeqMatch, and SINA were used but does not indicate how they were called. Was this done using web interfaces with default settings?  A supplemental table explaining which parameters were chosen would aid in reproducibility.

Result 2

The authors used 86 papers to assess the impact. A table that lists each of these papers should be included.

Other comments

I think the introduction and discussion sections could spend more time explaining the ecological significance of Vibrio and Pseudoalteromonas and explain why correct classification could matter. Both genera are gamma proteobacteria, mostly aerobic, heterotrophic, conditionally rare taxa that often have the potential to eat a wide range of carbon sources. What are some broad ways they are different? In particular, I feel the first paragraph of the discussion doesn’t flow - the hypothesis of how the errors could have occured in Greengenes feels like a non-sequitur. However, the second paragraph is stronger and has a clear call to action.


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

Input:
Title: TAXONOMIC ANNOTATION ERRORS INCORRECTLY ASSIGN THE FAMILY PSEUDOALTEROMONADACEAE TO THE ORDER VIBRIONALES IN GREENGENES: IMPLICATIONS FOR MICROBIAL COMMUNITY ASSESSMENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors perform a comparison of annotation of Pseudoalteromonas by Greengenes Database and found an important problem in this Database. This study merits publication after a few corrections.

I enjoyed this short paper, but have a few remarks to improve the quality of the study.
1. The authors mention (Line 73) type strains, the most relevant material to compare with and to prove the possible annotation errors. However, the information is hidden in the supplemental material, a large excel table. In this table I could not find the the closest matches for the 164 Pseudoalteromonas sequences mentioned (Table 1). Which are the closest type strain for those 164 sequences? Which are the closest type strains for those 46 sequences of vibrionaceae (Table 1) ?
It is important to give more taxonomic information on those aspects and expand the taxonomic analyses using type strains. Table in supplemental material is not the way to present to the reader. It is not easy to read and interpret..
2. The taxonomic information will imply in ecological information that could be gained. Eg if Vibrio cholerae CT+ is present in the samples, one would argue for a possible risk for human health. If Vibrio coralliilyticus is present, one could argue for a possible risk for coral health. If many studies perform the wrong taxonomic affiliation they may came to wrong ecologic conclusions. Can the authors pinpoint a few example of such problems in the literature and expand the discussion?
3. Perhaps the authors could also include a phylogenetic analysis/tree with sequences from the Greengenes and from type strains. these will help to easier interpret the data and implications.
Experimental design: ok.
Validity of the findings: ok.
Additional comments: ok.


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

Input:
Title: TAXONOMIC ANNOTATION ERRORS INCORRECTLY ASSIGN THE FAMILY PSEUDOALTEROMONADACEAE TO THE ORDER VIBRIONALES IN GREENGENES: IMPLICATIONS FOR MICROBIAL COMMUNITY ASSESSMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revisions are fine.
Experimental design: The revisions are fine.
Validity of the findings: The revisions are fine.
Additional comments: The revisions are fine. Thank you.


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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is written in clear professional English. It is of a good standard with adequate referencing, and professional handling of data, tables and figures.
Experimental design: The experiment is pseudo-replicated, in that the plots for each treatment are located together spatially. This limits the applicabilitity of the findings. The problem is not fatal, but needs to be acknowledged in the interpretation of the results. The experimental layout leaves open the possibility that the differences (increases) in SOC and nutrients with age of plantation could have been inherited from the sites. This possibility needs to be discussed.
Validity of the findings: In Lines 260 - 276, the authors argue that there has been no N fixation, but as the plantations age, the N cycle becomes more open with enhance N volatilization. So how does N increase with time. This query also applies to the Conclusions in section 5. If N fixation is not occurring, how can you argue that the increasing N is coming from N cycling in leaf litter? Where is the N ultimately coming from? It was low to start with - so what was the source? If it was leaves going into leaf litter, then where ultimately was the source for the leaves? Could there be something wrong with the logic. Have a read of Cook and Dawes-Gromadzki 2005 Landscape Ecology 20: 649-660 who looked at some of these issues in an Australian acacia landscape. This paradox needs to be addressed. At the moment it is self-contradictory.
Additional comments: Line 118: Eragrostis (no "e")


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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have no major comments with regards to basic reporting (although I have included some minor concerns in the general comments below). The manuscript was fairly clear and generally well structured.
Experimental design: See comments under general with regard to methodology queries
Validity of the findings: See general comments below
Additional comments: The manuscript by Abaker et al. describes nutrient (N, P, K) and soil carbon stocks in the soil and vegetation under grassland and Acacia senegal plantations of different ages. This study reports data from a poorly studied region with some very pressing environmental and land management issues. The authors have provided a fairly clear and comprehensive discussion of their dataset and the implications of plantation establishment for recovering soil fertility.
I have a number of concerns with some of the methodology descriptions and analyses that I have presented below.
1.	Soil sampling and analyses:
The authors mention that soil samples were dried prior to analysis at 105 degrees. This is unfortunate, as it is likely that some organic matter was volatilised at this high temperature (see Reuter, D.J., Robinson, J.B., Peverill, K.I., Price, G.H. and Lambert, M.J. (1997). Guidelines for collecting, handling and analysing plant materials. In: ‘Plant Analysis: An Interpretation Manual, 2nd Edition’, pp. 53-70, (D.J. Reuter and J.B. Robinson Eds) (CSIRO Publishing: Collingwood)). It might be helpful to discuss the implications of this for your results.
Secondly, the authors mention on line 153-154 that the apparent bulk density of composited soil samples was determined using the ‘laboratory packing method’. I am unfamiliar with this method and I would like to see a more detailed explanation of what it entails. My main concern is that by compositing soil samples much of the sample structure has been lost, making accurate bulk density measurements quite difficult. This may then have a substantial impact on your nutrient stock calculations presented in Table 1. However, I may be mis-interpreting this paragraph and perhaps bulk density determinations were performed on individual intact soil samples?
My final comment is in regard to comparison of soil nutrient and carbon stocks between different land uses that may vary considerably in terms of bulk density. When SOC and STN stocks are being compared across sites where bulk density varies and soils are sampled to a fixed depth, SOC and N stocks will need to be re-calculated to an equivalent soil mass before comparisons are valid, otherwise differences between sites in nutrient content may be solely caused by differences in volume of soil sampled. Re-calculation of SOC and STN stocks to an equivalent soil mass (usually calculated from the site with the lowest bulk density) is performed by determining the relationship between cumulative soil mass and cumulative C or N stock at each site (see Gifford and Roderick 2003; Lee et al. 2009). It might be helpful to discuss this potential issue in your dataset, particularly if there was substantial soil impacts or compaction during plantation establishment.
Gifford RM, Roderick ML (2003) Soil carbon stocks and bulk density: spatial or cumulative mass coordinates as a basis of expression? Global Change Biology 9, 1507–1514
Lee J, Hopmans JW, Rolston DE, Baer SG, Six J (2009) Determining soil carbon stock changes; Simple bulk density corrections fail. Agriculture, Ecosystems and Environment 134, 251-256.
2.	Sample types
The authors mention that above and belowground samples of ground vegetation were taken and analysed for nutrient content. I was curious as to how root samples were removed – eg. was the main root ball dug up, was it rinsed prior to dying etc.? There was also an indication on line 146 that these samples were analysed separately, but only a single value was reported in Table 4- is this a combination of below and aboveground biomass samples or just aboveground samples? If below ground samples were combined with aboveground, it is unclear then why root nutrient contents were not sampled for the trees as well.

My other concern was the similarity between this manuscript and a previous manuscript by Abaker et al (2016) that also reports soil organic carbon (SOC) stocks and contents under the same plantations. Parts of Figure 3 and Table 2 are identical to SOC results reported in this publication, and this should perhaps be noted or these results excluded and referred to in the discussion only. This would also remove any emphasis in the current manuscript on the SOC results, as the implications of this dataset have already been discussed and published elsewhere.

A number of minor concerns are presented below:
Abstract line 43: the ‘nutrient uplift’ effect was not directly measured in this manuscript so it may be best to rephrase this sentence as “We speculate that the higher mineral nutrient concentrations….”
Introduction line 71: the wording of this sentence is a little unclear in terms of how SOM contents are related to increased nutrient contents and cycling under tree canopies (it would seem that these processes result in higher SOM contents rather than the other way around).
Introduction line 78: It might be helpful to start a new paragraph focused on N fixation here.
Introduction line 104: change hypothesized to hypothesize
Results line 186: The reference to Fig. 2 should be removed as this does not show nutrient concentrations with depth and age
Results line 190: Remove “as could be expected”
Discussion line 235 to 238: Reference and re-iteration of the results should be removed.
Discussion line 246-255 and 340-342: It might be helpful to indicate that you speculate that ‘nutrient uplift’ may be the mechanism enhancing nutrient contents in surface soils
Discussion line 346: I was not entirely clear how improving soil fertility under Acacia plantations would benefit the local community. Is it because they will then be cleared and used for food production? Or is it because it improves the diversity and palatability of ground vegetation that is used for grazing? It might be helpful to either remove this sentence or place the context of plantations and soil fertility and benefits to local communities into the introduction.
Figure 1: It would be helpful to include a map of the study sites that includes their location in reference to the whole of Sudan (similar to the Abaker et al. 2016 reference).
Figure 3: Would it be possible to include error bars here
Table 1 to 5: Could you include number of replicates (where applicable) for values where standard deviations have been calculated in the Table labels e.g. “Mean soil stocks (g m-2; 0-50cm ± (standard deviation), n = 3)…”
Table 2: the formatting of the superscript letters in the SOC column is a little confusing.
Best wishes in addressing these concerns,
Dr Anna Richards


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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study builds on Abaker et al. (2016) — which investigated biomass and soil carbon in various aged plantations of Acacia senegal — be exploring potential co-correlates with important plant nutrients.
This is a very well-written and well-structured article that clearly explains the importance of the research.  Figures are relevant, correctly labelled etc. There is one issue that the authors need address: contextualising the other studies on the same system (i.e. Abaker et al. 2016 and Abaker et al. In Press). There are hints throughout the manuscript about these studies, but the reader is expected to piece them together to figure out that these studies share the same study system, and I assume the same research program. Please tell the reader up front in the introduction that this study system has been explored along different avenues and summarise those avenues. That helps contextualise this contribution in relation to the other two. (I continuously had this nagging feeling that the other two studies may have looked at some other area).
There is an issue with the species name used: Acacia senegal. The new scientific name for this species is Senegalia senegal (L.) Britton. Although I strongly dislike the manner in which the name change for African Acacia occurred, we do need to maintain the correct taxonomic links. Officially, I should ask the authors to change the name throughout, but I think they could make a convincing argument that the old name has local importance and association (e.g. with government officials?) and so the old name is more accessible. If the authors can provide a convincing argument for using the old name, then the new name does need to be included in the abstract and text at some point, else the new name should be used throughout.
My last major comment is that keeping track of samples sizes from the methods to the results is tiresome and difficult. Please include the sample sizes in the tables (even if this is simply in the table heading).
A difficult comment to implement, but there seemed to be much repetition between the results text and tables, so this could be shortened. Please ensure only major main trends are reported in the results text.
In terms of maintaining a predictable order for the reader throughout, the introduction sets up the aims (e.g. L98-100), but the methods and results don’t follow this order (e.g. 148-164). It is minor, but keeping the order consistent helps the reader.
Minor comments:
Line 111: please explain what is “gum arabic”?
L118: “Eragrosties” to “Eragrostis”
Line 152: Please include model and manufacturer for the spectrometer.
Lines 148-159: These methods are not referenced. One I am not familiar with so I think providing a reference for all is necessary.
Line 164: I’ve used carbon and oxygen isotopes before, and these are referenced against international standards. Is there an international standard for nitrogen (atmosphere, maybe?)? Please include.
Please consider including an appendix figure with ground-level photos of the different sites to give international readers further insight into the study system.
Line 319: replace “simply” with “by process of elimination”. (Yes?).
Line 364: “of of”
Figure 3. Please include information about replicates.
Experimental design: The experimental design is sufficient to answer the questions raised, and is sufficiently described for experimental reproduction. Although I don’t believe it is important, the shape and size of plots varied across different treatments (e.g. grass vs plantation). Please could a brief explanation be included as to why this was done, and also any potential (or not) influence this might have had. Again, I don’t think this is important, but the change in design was odd.

Line 204: Although unlikely in such a dry environment, fire can influence SOM rates. Please briefly mention whether fires occur in these landscapes (at least to rule this out as influencing factor, as it does play a role in more mesic environments). If fires do occur, then this needs to be considered…
Validity of the findings: The interpretation is consistent with the results, i.e. the findings are valid.  No further comments required.
Additional comments: This was a well-thought out study and well put-together.  One way to make your results accessible to a general reader is to provide a summary table of soil carbon stocks and rates of carbon sequestration in relation to other studies. This does not need to be a comprehensive list, but a table with stocks, rates, vegetation type and mean annual precipitation will allow the reader to immediately gauge your values in comparison to other systems. One such study that is at the same MAP is Mills&Cowling (2010; J. Arid Environments, 74: 93-100), but there are many more that can be easily harvested from Google Scholar. This little additional effort will help the reader, and likely increase the citation rate for the article.


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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors need to address the issue of pseudo-replication in their design.
Experimental design: The experiment is pseudo-replicated. The authors disagree that this is an issue and chose not to address it. I believe it still is and have raised a number of ways it could affect the results. The fact that studies were done at two locations is irrelevant to the argument because they are not included in a formal analysis. The authors must address the issue and justify their analysis with evidence that starting conditions did not bias their analyses.
If the authors cannot address this issue in some way, the paper should be rejected.
Validity of the findings: With pseudo replication, one can only infer that a trend may be the result of a treatment. It is not conclusive. The way the results are presented is not valid. The analyses can legitimately say that site x is different to site y, but not that it is due to the age difference of the plantations. It could be due to landscape position, prior landuse or several other possibilities.
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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The authors have addressed a number of my concerns raised in my previous review of their manuscript titled "Linkages between soil carbon, soil fertility and nitrogen fixation in Acacia senegal plantations of varying age in Sudan". However, I still have one outstanding issue that I would like to draw attention to. In my previous review I mentioned an issue of comparing nutrient stocks measured to a fixed depth across sites with different soil bulk densities. The authors chose not to address this concern but to refer to this issue in the discussion.

I am afraid, however, that I would like to bring this point up again. I have looked at the underlying dataset for the manuscript and, while bulk density is not highly variable between sites, there are still differences that could impact overall stock calculations and the significance of differences in nutrient stocks between sites reported in Table 2. The authors mention that time restrictions and consistency of results with previous publications was their reason for not performing these calculations. However, the time to do these calculations is not onerous (1 day perhaps) and the authors are already reporting results from previous publications in this manuscript so there is little need to refer to the previous published work (with respect to nutrient stocks). If the authors could show that they had performed these calculations and that there were no differences in site nutrient stocks compared to the fixed depth calculation then I would be happy to accept that Table 2 remains unchanged. However, without this evidence it is difficult to support the statement made by the authors that the fixed depth values are sufficiently accurate. I am sorry to labour this point and hope that it can be resolved relatively easily.

Best wishes,
Dr Anna Richards
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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The limitations of the experimental design have been acknowledged appropriately and the interpretation of the results takes this into account.
Validity of the findings: The interpretation of the findings takes appropriate account of the limitations of the design.
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: LINKAGES BETWEEN SOIL CARBON, SOIL FERTILITY AND NITROGEN FIXATION IN ACACIA SENEGAL PLANTATIONS OF VARYING AGE IN SUDAN
Review round: 3
Reviewer: 2

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


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

Input:
Title: EVALUATION OF STANDARD IMAGING TECHNIQUES AND VOLUMETRIC PRESERVATION OF NERVOUS TISSUE IN GENETICALLY IDENTICAL OFFSPRING OF THE CRAYFISH PROCAMBARUS FALLAX CF. VIRGINALIS (MARMORKREBS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: See "general comments for the authors".
Experimental design: See "general comments for the authors".
Validity of the findings: See "general comments for the authors".
Additional comments: In their manuscript “Volumetric evaluation of standard fixatives and imaging techniques on nervous system preservation in genetically identical offspring of the crayfish Procambarus fallax cf. virginalis (Marmorkrebs)” Emanuel S Nischik and Jakob Krieger compare the impact of different fixation methods on the tissue preservation of a crustacean brain. The authors use the uprising model-organism Procambarus fallax cf. virginalis, which reproduces by parthenogenesis and thus produces genetically identical offspring. Three fixation methods and two imaging techniques are investigated, all cutting edge techniques, not only in the (neuro-)morphological community. The authors concentrate their investigation on the volumetric changes of three distinct brain areas, the DCL, the AcN, and the midbrain. Nischik and Krieger demonstrate that µCT procedure is the least disruptive method, whereas fixation in Karnovsky solution leads to a volume decrease of 24 %, and Zinc-formaldehyde fixation to a decrease of 19%. This is the first investigation generating basic data to obtain conversion factors in order to allow interspecific comparisons of volumetric data and thus represents an important contribution to the literature.
Overall, the study is well executed and the datasets provided are convincing and well supported by the 5 figure plates and one table. The raw data of all scanning series can easily be accessed via MorphDBase as described in the manuscript. The introduction shows the context of this research and interpretations in the discussion are well supported by the presented datasets. However, the text is often confusing and seems unstructured (see major issues point 1). Additionally, I have several other major issues. If these and the minor points below are addressed I would definitely recommend this paper for publication in PeerJ.
Major Issues:
1.)	The paper needs some structural modifications. In the following I will give some examples, but the authors should check the whole manuscript for possible modifications.
a)	The abstract could be more effective in clarifying the purpose of the paper. In my opinion some sentences concerning an introduction or motive for the study might be necessary at the beginning. It should be mentioned that volumetric data is an important tool in neuroanatomy. However, due to the application of different methods these data are hardly comparable and that this account starts the first approach for a posteriori comparison of already existing data in the literature (it is nicely described in the introduction).
b)	Results: The results start with a subheading “MicroCT” (line 238). If I understand it right, in the second part of this subheading the authors present volumetric data of all three methods applied (Line 248, starting with …”While the volume of the accessory lobes…”). However, this should be done in the section where all methods are compared, e.g., lines 276, section MicroCT versus cLSM. And actually, the same data are already compared here, namely the volume of left and right hemispheres of paired lobes. Compare:
Lines 249-251: “…no difference in the volume of the left and right hemispheres of paired lobes could be found in any given treatment (paired Student’s t-test: DCL: p = 0.2691; AcN: p = 0.1107; n = 20).
Lines 288-289: “No significant differences for left and right lobes (paired Student’s t-test: DCL: p = 0.2691; AcN: p = 0.1107; n = 20) have been detected.”
c)	Discussion: In Lines 361-364, the authors discuss that µCT-preparations feature the closest in vivo coherence, but are influenced by the fixation and dehydration leading to shrinkage. While reading the manuscript for the first time, at this point I was wondering why no in vivo data have been generated. The discussion of in vivo methods however follows on the next page, starting with line 391, too late in my opinion. It might help shifting the whole paragraph to line 364.

2.)	Lines 69-71: The authors mention several aspects of tissue processing, which influence its preservation. I think they missed the factor temperature at which the fixative is applied, as it influences the speed of penetration. In this context, I noticed that this information is sometimes missing in the M&M section (e.g., lines 168/169 or 185-188). The authors should mention all relevant settings in the M&M section, in order to allow other scientists to repeat their experiments. This is especially important regarding the scope of this paper (comparison/evaluation of volumetric data/methods). For example, in line 147 it is not clear if the 3 steps of 99.5% ethanol are incubated for 30 min as well, and the concentration of NaN3 is missing (line 186). Furthermore, supplier of reagents are not given consistently, e.g., supplier of the Synorf-Antibody is missing.

3.)	In the context of the previous issue, please indicate in the M&M section how many specimens were used for each experiment. This information is given for µCT, but not for the other cLSM studies. This information should also be included in the Figures 1 and 2.

4.)	The authors make great effort measuring volumetric data of three different brain regions. However, they do not show the raw data. As the “n” is not too large they should add a Table with their results for each brain. In the same context, the authors have to be aware that the data are based on a small “n”. That should be taken into account when discussing the results and statistics…

5.)	Lines 277/278 (see also 297-299): The authors state that all three tested methods differ to the in vivo congruent. But this is not documented by their volumetric data, as in vivo measurements have not been performed. The authors should rephrase this statement into a more careful and hypothetical sentence. It is just an assumption made due to the reasons explained in the next sentences. By the way, in my opinion, these explaining sentences (lines 278-281: Starting with: …. Since all samples at least temporarily….) are part of the discussion.

6.)	The intriguing idea of this paper is “to obtain conversion factors in order to deduce approximate in vivo volumina” (lines 414-415) in order to “allow for a posteriori standardization of determined nervous tissue volumes in malacostracans” (lines 416-417). The authors mention some works from the literature, where volumetric data concerning the DCLs are given (Table 1). My question is: can the authors apply their conversion factors (percentage of shrinkage) to the cited studies? I guess it is still not possible to apply these conversion factors, as there are too many variables (fixation time, temperature, etc) and the authors should make clear that this study has to be interpreted as a pioneering study.

7.)	In the same context, an often applied fixation method is 4% PFA for several hours or overnight as done in the cited literature (Beltz et al., 2003; Krieger et al., 2010). Would it not be logically consistent to obtain a conversion factor for this methodological setup (I assume that fixation in Karnovsky versus PFA might result in different shrinkage levels)? Could you explain why you have not conducted a fourth experiment with a fixation in solely 4% PFA?

8.)	Does the entire nervous tissue shows the same degree of shrinkage or do different areas/neuropils show different degrees of shrinkage? This should be made clear to the reader, as a putative conversion factor might not apply for all parts of the brain.

9.)	I am wondering if the fact that the brain for the µCT is incubated in fixative solution without further dissection prevents shrinkage. Maybe the surrounding tissue supports a more careful preservation of nervous tissue? Do the authors have data concerning Karnovsky- or ZnFA-fixed brains inside the head capsule without dissection?

Minor Issues:
1.)	In-text citations concerning three-author-papers are inconsistent: Sometimes they are abbreviated (e.g. line 60: Akkari et al., 2015), sometimes all authors are listed (e.g. line 341: Durst, Eichmüller & Menzel, 1994).

2.)	I am wondering why the mixture of glutaraldehyde and PFA is not termed Karnovsky’s solution throughout the manuscript, as done for Bouin’s solution.

3.)	Line 36: Please replace “Zinc-Formaldehyde” by “zinc-formaldehyde”

4.)	Line 60/61: Please rearrange order of references chronologically (the same issue applies for line 91)

5.)	M&M section: Please replace “second antibody” by “secondary antibody” throughout the manuscript.

6.)	Line 133: Please add a period at the end of the section.

7.)	Line 193: Please replace “3c11” by “3C11”.

8.)	Line 207: Please replace “Cy3-conjugats” by “Cy3-conjugates”.

9.)	Line 267: Please replace “statically” by “statistically”.

10.)	Line 273: The cross reference to Fig. 3D is wrong. It should by Fig. 4D.

11.)	References: Some references are cited with the DOI, several are not.

12.)	Legend of Table 1: Please add the abbreviation of “deutocerebral chemosensory lobe” (DCL).

13.)	Table 1: The correct citation for Birgus latro is “Krieger et al., 2010”.

14.)	Table 1: Please indicate if the mean of DCL volume for Procambarus is the mean of all three methods used. And would it not be more appropriate to separate between the three fixation methods considering that there is a significant difference at least between two methods (Figure 3)?

15.)	Legend of Figure 1: “accessory lobe” should be changed to the plural form.

16.)	Legend of Figure 2: The abbreviations DCLs and AcNs should be introduced when the corresponding term is used first.

17.)	Figure 4: Some lettering is hardly readable in this Figure (for example CA, TN, 9/11, 10 in A, and 9/11 in B). Maybe a contrasting contour might help here.

18.)	Legend of Figure 5: Explanations of the following abbreviations used in the Figure are missing in the legend: AIINv, LAN.


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: EVALUATION OF STANDARD IMAGING TECHNIQUES AND VOLUMETRIC PRESERVATION OF NERVOUS TISSUE IN GENETICALLY IDENTICAL OFFSPRING OF THE CRAYFISH PROCAMBARUS FALLAX CF. VIRGINALIS (MARMORKREBS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: In my opinion some of the conclusions cannot be drawn based on the current experimental design. This point will be elaborated in detail in the "general comments to the author" section. I make some suggestions on how to re-phrase the conclusions and how to sharpen the interpretation of the findings.
Additional comments: The manuscript “Volumetric evaluation of standard fixatives and imaging techniques on nervous system preservation
in genetically identical offspring of the crayfish Procambarus fallax cf. virginalis (Marmorkrebs)” by Nischik and Krieger is a valuable contribution to the field of microscopic volumetric analysis of biological soft tissue samples based on 3d images. It provides a comparison of image quality and sample shrinkage between standard preparation protocols for two of the most widely used microscopic 3d imaging techniques: confocal microscopy and x-ray microCT. Evaluation is based on measurements obtained from different parts of the central nervous system (brain) of the crayfish Procambarus fallax cf. virginalis. While the intention of the study is clear, the conclusions drawn by the authors suffer from some severe technical shortcomings based on the study design. First, the experimental design does not allow evaluating differences in tissue shrinkage based on different fixatives (see major concerns, point 1). Second, no measure of in vivo brain volume is provided, so there is no estimation of absolute shrinkage (see major concerns, point 2).

Despite these shortcomings, several aspects of this study are positive and should be highlighted:
- The quality of the image data is very good.
- I highly appreciate the fact that all data were deposited at the online repository MorphDBase! Making the original image data available is great for several reasons! It also helped me with interpretation of the data while doing this review.
- The presented data highlights some characteristics of the used tissue preparation protocols and imaging techniques. It shows that the tested protocols provide good image contrast.
- The data also shows that microCT imaging is much more suitable for doing volumetry than confocal microscopy, because several technical limitations yield an anisometric image quality in optical sectioning. This is very obvious when looking at the original image volumes (see major concerns, point 4).

Taken together the positive aspects and the technical shortcomings, I suggest to make clear that…
- the present experiment does not allow to make some statement about the contribution of the used fixative to total specimen shrinkage
- no attempt was made to use or develop a protocol that minimizes specimen shrinkage (see major concerns, point 3)
- the present data only allows for an evaluation of currently used standard protocols in terms of final specimen size and image quality
- one of the main findings of this study is the difference in image volume quality (isotropy) between confocal and microCT images

Further I have one more suggestion: The authors should consider scanning some (5-10) fresh brains after dissection with microCT (see major concerns, point 2a) and calculate the average volume of a fresh brain (central brain) at the investigated developmental stage. This would yield two major benefits. First it would allow calculating absolute shrinkage values for the tested protocols. Second, this data would provide a baseline for future studies for refining fixation and staining procedures in order to minimize shrinkage.
I know that asking for additional experiments during a revision is somehow problematic. The authors would have to have immediate access to (i) fresh specimens and (ii) the microCT scanner. In the present case I think that this requirements could be fulfilled. If not, the authors can state why they are not able to do it.  The scan resolution does not need to be very high (e.g. acquired with a detector binning of 4) so the time for a tomography could be roughly 15 minutes. This would allow the authors to collect data from 10 brains within one or two days (dissection and scanning). With maybe 2 days for image segmentation, volumetric data for whole brain volume could be generated within one week. Personally I think that this data would be absolutely valuable and would dramatically increase the scientific value of this manuscript.

Despite my criticism, I think that after a serious revision the paper would be a valuable contribution to your journal. Before publication I ask the authors to consider the following more detailed comments when revising the manuscript.

Major concerns:

-1- This is my main criticism. In the abstract the authors state that “Fixation in paraformaldehyde of dissected brains lead to a volume decrease of 36 24 %, whereas fixation in Zinc-Formaldehyde resulted in a shrinkage of 19 %.” (referee comment: this decrease is calculated relative to the volumes measured on Bouin’s material scanned with microCT, not as a decrease from in vivo tissue volumes). However, the measured differences in volumes reflect shrinkage occurring during the whole sample preparation protocol, and not only during fixation. Using the present experimental design, an evaluation of different fixatives concerning volume shrinkage cannot be done. For doing this, a much simpler experimental design is needed: testing a couple of fixatives and keeping the rest of the workflow (intact head specimen vs. dissected brain, washing steps, staining, clearing, mounting, etc.) constant. In the present experiment multiple variables are present. Many of the steps very likely introduce some tissue shrinkage, and just from the final volume measurements it is not possible to say which steps contributes most to the shrinkage finally observed in the image volume. Thus no statement about the contribution of fixative to shrinkage is possible.
Several more detailed comments should underline this statement:
-a- dehydration to absolute ethanol as used in the microCT protocol is known to introduce tissue shrinkage (traditional histological paraffin wax embedding protocols...)
-b- staining with iodine introduces shrinkage due to changes in osmolarity as proven before (see for example Buytert et al 2014, Microscopy and Microanalysis)
-c- clearing with methyl salicate introduces shrinkage as well (see for example Bucher et al 2000, J Neuroscience Methods)
-d- microCT images were acquired from intact heads, confocal images were acquired from dissected brain specimens. Besides possible artefacts coming from the dissection procedure, the dissected brains might respond differently to chemicals and finally the mounting of dissected brains for confocal microscopy might introduce some further mechanical distortion

Taken together these facts, the authors should consider to re-phrase the title and to re-structure the presentation of their findings. What the authors do show is that the applied standard protocols yield strong differences in tissue preservation, shrinkage, and volume image quality. What the authors don´t show is how the fixatives specifically contribute to these findings.

-2- The second main criticism is that there is no measurement of absolute tissue shrinkage, as no data on fresh tissue volumes before fixation is provided. This point is well aware to authors and they consider “direct volumetry using Archimede´s principle or high resolution MRI” (page 19, line 399) as promising approaches to measure in vivo brain volumes. In principle I agree, but in practice I think that direct volumetry using Archimede´s principle is very hard to do with such small volumes and might be prone to measurement errors, and MRI might fail to provide sufficient resolution on native tissues (without use of paramagnetic contrast agents). The authors should consider microCT as an alternative method to measure in vivo or at least fresh (unfixed) total brain volume. Two approaches seem feasible:
-a- Fresh dissected brains could be mounted in conical containers (e.g. pipette tips) in air. Some water/buffer is added at the bottom of the tube and the tube is tightly sealed. This will provide a moist atmosphere and prevent drying of the specimen. A quick tomography can be done just to see the boundary of the brain. Most scanners will do such scans in 10-15minutes by using detector binning (if you plan to further process the sample, the brain would be fixed after the scan; the overall time from dissection to scanning to fixation is still below 30min, which might be acceptable for many kinds of analysis).
-b- Alternatively the authors could consider scanning intact heads of freshly killed specimens with in-line phase contrast. This might give sufficient contrast to discriminate between boundaries on brain to surrounding tissues, although it might be hard to do this scans quick enough with laboratory microCT setups (they could however be done at synchrotron beamlines).

-3- I understand the point of using “standard” protocols for estimating tissue shrinkage, as the overall goal of this study is to make volume measurements comparable even between different preparation protocols and imaging modalities. The presented protocols (fixatives, contrast agents) have been utilized in the past in many studies and have proven to provide excellent tissue preservation and image contrast for different kinds of qualitative morphological evaluations. Despite this fact, I doubt that the presented protocols are suitable for avoiding volume shrinkage.  I will elaborate this point on one example: the microCT protocol including Bouin´s fixation and iodine staining. While iodine staining on Bouin´s fixed material has yielded excellent qualitative morphological data in the past (Sombke et al 2015, J Comparative Neurology), I don´t think that the protocol presented in the present paper is the best choice for reducing tissue shrinkage artefacts. This is for three main reasons:
- a- Previous quantitative studies did show that tissue shrinkage and tissue deformation in Bouin’s fixed tissue is quite severe (Schmid et al 2010, BMC Developmental Biology) and worse compared to other fixatives.
-b- Dehydration to 100% ethanol
-c- Using 2% Iodine is not a good idea in my opinion. Many studies in the past used 1% elemental iodine (Metscher 2009, Developmental Dynamics; Sombke et al 2015, J Comparative Neurology). The concentration of the contrast agent will affect osmolarity, and higher concentrations will cause more shrinkage.
Instead, other protocols could be considered. For example, Wong et al (2013, PLOSOne) showed that shrinkage in staining with Lugol´s iodine potassium iodide solution could be reduced to a minimum by embedding samples in a hydrogel mesh before staining. Given the data in the literature that already addressed these issues, I think the authors should re-phrase their main argument.

-4- Different imaging techniques have different kinds of bias and physical limitations. This is a general shortcoming that makes it very hard to compare microCT data with confocal data. For microCT data we can assume that image geometry is correct. For confocal data we cannot simply assume that, based on somewhat limited z-resolution depending on the PSF of the system, a possible refractive index mismatch, …

I looked into the original image stacks deposited at MorphDBase with some detail. The quality of the images is good compared to recent standards in microCT and confocal imaging! Still, if you reslice these stacks in in XZ or YZ plane, the geometric issues with confocal data become very well visible. While the brains have a very “nice 3d structure” in the microCT scans, they look much more “flat and squeezed” in the confocal images. Many factors may contribute to this phenomenon, including dissection, squeezing during mounting, optical artefacts, and others.
While this makes it even harder to evaluate contribution of different chemical procedures to tissue shrinkage it obviously points towards another fact: tomographic techniques (like microCT) are highly superior compared to optical sectioning techniques (like confocal microscopy) regarding geometric image isotropy. This means that tomographic techniques are highly superior for tissue volumetry, despite current limitations in image resolution and specific labelling techniques.

Minor concerns:

-5- I don’t understating the difference between general sample preparation (Line 135) and sample preparation for microCT / cLSM. What does general mean? Does It mean that every specimen was fixed in 4% PFA before being fixed in Bouins?

-6- Please check the consistency in wording. One example is the alternating use of microCT and µCT throughout the whole text. Please use one or the other, but not both.

-7- Page 12 Line 206-209 Which confocal microscope was used? Company/model? Inverted or upright? Pinhole size/section thickness? Slice interval? This is relevant for interpretation of image data and volume measurements.

Some more suggestions on wording:

Page 9 line 125 Change “Juveniles started feeding individually” to “juveniles started autonomous feeding”

Page 11 Line 188 Rephrase “abundant primary antibody” by “excess primary antibody”

Page 12 Line 217 The material statistics tool uses the voxel data and not the surface to calculate volumes

Page 12 Line 219  Remove information on threshold, as no explanation is provided for image intensities (8 bit/16bit, byte scaling during reconstruction, calibration, beam hardening correction,…)

Page 16 Line 320 Resolution and image noise do not necessarily correlate. Resolution is limited by the microCT scanner (minimal spot size, specifications of the scintillator and optical detector…) while using longer exposure times can increase the SNR.


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: EVALUATION OF STANDARD IMAGING TECHNIQUES AND VOLUMETRIC PRESERVATION OF NERVOUS TISSUE IN GENETICALLY IDENTICAL OFFSPRING OF THE CRAYFISH PROCAMBARUS FALLAX CF. VIRGINALIS (MARMORKREBS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this revised version, the authors have made a serious effort to modify their manuscript and responded to all issues of prior reviews. In particular, they have streamlined the text, added important information as well as supplementary data, and addressed the major criticism concerning missing in vivo data by a more detailed discussion and additional experiments.
Therefore, I strongly recommend this revised manuscript for publication in PeerJ.
I have some very minor suggestions, which could further improve the manuscript (line numbers refer to the clean version without tracked changes):
1.)	I would prefer including the supplementary tables into the main body of the manuscript as they contain important data and the journal is not restricted by page limitation. This will be far more comfortable to the readers. However, I leave this decision over for the editor.
2.)	Please check introduction of used abbreviations. For example, in line 175 “room temperature” is abbreviated by RT, but written out throughout the rest of the text. HEPES buffered saline and zinc-formaldehyde are abbreviated first in lines 201/202, but are already mentioned before (just two examples, there might be more issues).
3.)	Line 148: Change Boiun’s to Bouin’s.
4.)	Line 334: ‘thevc’ should be changed to whatever the authors meant to write here.
Experimental design: see above
Validity of the findings: see above
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: EVALUATION OF STANDARD IMAGING TECHNIQUES AND VOLUMETRIC PRESERVATION OF NERVOUS TISSUE IN GENETICALLY IDENTICAL OFFSPRING OF THE CRAYFISH PROCAMBARUS FALLAX CF. VIRGINALIS (MARMORKREBS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Some findings must be re-interpreted after considering an appropriate method for correcting z-axial scaling in confocal images.
Additional comments: In the revision of the manuscript “Evaluation of standard imaging techniques and volumetric preservation of nervous tissue in genetically identical offspring of the crayfish Procambarus fallax cf. virginalis (Marmorkrebs)” by Nischik and Krieger, the authors managed to resolve most of the concerns raised by the two reviewers. I highly appreciate the fact that the authors carried out an additional experiment for measuring fresh brain volumes and for estimating tissue shrinkage before and after Bouin´s fixation and microCT staining.

However, one main criticism was not yet resolved. This issue concerns the topic “optical artefacts in confocal microscopy” and has to be addressed before the paper is ready for publication. This topic is of central importance to this manuscript, thus in my opinion justifying a second major revision.

In my review of the initial submission I referred to this point as “Major concern 4”. I criticized the fact that optical sectioning techniques produce different artefacts compared to µCT, related to shrinkage, specimen squeezing, refractive index mismatch, etc. The authors tried to resolve this topic in the revised manuscript, but in opinion they do not treat the topic “refractive index mismatch” adequately. They recognize this problem and try to correct for z-axial scaling in the revised paper (conversion factors, supplementary tables), as seen in some passages of the revised text, such as:

Lines 412- 417 “However, using the correction factor for the refractive mismatch for the use of methyl salicylate provided by Bucher et al. (2010) of 1.581 (for a 10x dry objective with a numerical aperture of 0.40) resulted in a drawn-out appearance of the brains and led to an aberrant ratio between the dimensions in xy-axis and z-axis. By manually adjusting the voxel z-dimension of confocal image stacks by a factor of 1.188, led to spatial congruence of the brain dimensions of µCT-scans”

However, I argue that “manually estimating” a correction factor is not a scientifically correct approach. How was this congruence with µCT measured?  Based on physical principles the correction factor for the present study should be somewhat close to the number calculated by Bucher et al (identical  media = air/methyl salicylate, identical magnification = 10X, identical numerical aperture = 0.4). I also checked one of the morphdbase datasets (M64-001.1) and to me a correction factor of 1.58 looks quite plausible on this dataset.
 I have three suggestions how to resolve this issue:
-a- Calculate a theoretical correction factor using one of the established approaches (Hell et al 1993, Journal of Microscopy 169:3, 391-405)
-b- Practically measure the correction factor with some PMMA spheres under identical conditions as in brain measurements (Besseling et al 2015, Journal of Microscopy 257:2, 142-150)
-c- Use the values from Bucher et al as an approximation, which is in my opinion acceptable for reasons mentioned above (identical  media, identical magnification, identical numerical aperture).

However, if the z-axial correction factor is really higher then 1.5, the main conclusion resulting from this is the following: the two tested confocal preparation protocols shrink the sample less than the tested microCT preparation protocol.  This finding would need to be embedded into the manuscript. The whole paper is about volume measurements, so it needs a very serious treatment of that issue. The discussion could be somehow structure like this:
-i- These are the volume measurements I get from the uncorrected image volumes (µCt measurements are larger than confocal measurements)
(ii) This is for several reasons, including sample prep artefacts, imaging artefact etc.
(iii) Confocal data can be corrected for refractive index mismatch using different approaches
(iv) These are the volume measurements I get after correcting confocal volumes
(v) Finally discuss all the strengths and pitfalls of the different preparation and imaging protocols

Despite this major concern I have one more minor comment:  the authors should state clearly why they did not use same aged individuals for measuring fresh brain volumes. While the main analysis was done on juvenile brains, adult animals were used for measuring fresh volumes. Brain volumes of adults are roughly 100 times larger compared to the  juvenile animals. This could be a problem, because the chemical composition (e.g. protein or lipid content) of adult brains could be different, which would mean that they might also respond differently to fixation, dehydration, etc.


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: ‘WE LIKE IT WET’: A COMPARISON BETWEEN DISSECTION TECHNIQUES FOR THE ASSESSMENT OF PARITY IN ANOPHELES ARABIENSIS AND DETERMINATION OF SAC STAGE IN MOSQUITOES ALIVE OR DEAD ON COLLECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The manuscript of Charlwood and colleagues present a comparative evaluation of two microscopy techniques for the determination of parity status of Anopheline mosquitoes and other basic information about their population dynamics. The English is appropriate but needs to be revised, as I noticed several typos in the text. The manuscript, despite its main question is worth to be investigated, shows several limits and needs to be improved in many aspects. First of all, I don’t agree with the approach to use only field specimens to assess the quality of the two techniques: the field conditions, which are by definition unknown, are not ideal to evaluate the best method to assess the parity of the mosquitoes, as some unknown environmental/physiological factors could have affected the status of the ovaries. Moreover, the test is based on the assumption that the wet dissection is the gold standard but no justification are provided for this choice. The comparison of two techniques should be based on the postulation that one of the two is the best known approach in order to assess the outcome to be detected. The authors should provide bibliographic information to support their choice. Anyway, there are no sure information about the oviposition event of the field mosquitoes collected. The only added information could be the presence of sperm in the spermathaeca, which could give information about mating but no guarantees that an oviposition event happened or not. So, how the authors are sure that the mosquitoes were parous or nulliparous? An insectary study based on mosquitoes that have done single oviposition as positive control would have been a better approach. With the protocol adopted in this study it is impossible to determine the false positives and false negatives, which is the basis of the determination of sensitivity and specificity of a technique compared to another. For all these reasons, I strongly suggest to repeat the study also in insectary conditions in order to have a complete picture.
Other comments:
- in methods section it is not reported how concordance, specificity and sensitivity is calculated, please describe the formulas citing the appropriate references.
- lines 192-193 (and also 250-251): it is not clear to me what is the aim of the comparison between Culex quinquefasciatus and Anopheles arabiensis performance of the techniques. What is adding this test to the manuscript? Moreover, the better quality of the former species is not based on objective evaluation and is not described how a reader could evaluate this quality in order to use this result for other studies. I suggest to better integrate this experiment or to remove it from the manuscript.
- line 194: the numbers of A. arabiensis dissected here is 238, while in the abstract is reported 389 collected. So, why it is not included the number of mosquitoes collected in Results? Moreover, what about the remaining 151 mosquitoes?
- lines 200-201: the 286 mosquitoes collected are from which site? In table 1 is reported a total of 289 mosquitoes, while here 211 are reported. Please correct.
- lines 206-212: This is a comment that should be moved into Discussion. Also, it could be interesting to specify how many nights of collection have been done, as the total number of mosquitoes should be 389. I suppose that the number of nights would be not more than 3-4. So, it is possible that the difference in parous rate is just due to a sampling bias. The authors should take into account this possibility in the interpretation of this result.
- lines 215-217: please describe the other species of the A. gambiae complex found (I suppose just A. gambiae s.s.)
- lines 218-220: the numbers in the text and in table do not seem to match (3649 in text, 3239 in table 2). Please check.
- line 225, 242, 244, 246, 247: please add the percentage of mosquitoes used for the Chi-square test.
- Discussion: I don’t find any specific comment on the conclusion of the authors about the results of wet vs. dry technique. Which one is the best?
- lines 262-265: the proportion of out vs in collected virgin females could be just a consequence of the interception of newly emerged mosquitoes and not a behavoiural effect due to mating.
- figures 2-3-4: please add the captions
- table 1: please add the caption
- table 2: please describe better the table: add “collection method” to the first column and “status” to the second one. Please describe the acronyms in caption.
- tables 2 and 3: which method has been eventually chosen to define the parous rate?


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: ‘WE LIKE IT WET’: A COMPARISON BETWEEN DISSECTION TECHNIQUES FOR THE ASSESSMENT OF PARITY IN ANOPHELES ARABIENSIS AND DETERMINATION OF SAC STAGE IN MOSQUITOES ALIVE OR DEAD ON COLLECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written. The background was addressed appropriately, methods were described concisely and results presented in an easily understandable manner.
There is one section which is difficult to follow and can be improved (see other comments to the author).
Experimental design: The experiment was well designed and methods explained with sufficient detail to allow its replication. The comparison of the two methods presented is of importance to researchers and health authorities wanting to measure mosquito survival in the field and therewith evaluate the impact of vector control interventions. The rationale was well described in the introduction. Although dry dissections are considered "easier" the wet method has been thought to have higher accuracy. The results presented in this article indicate dry and wet dissections provide similar information in regard to parity status of a malaria mosquito. Ethics approval was sought.
Validity of the findings: Conclusions are well stated.
Additional comments: Line 101: “For example, a change from a two-day cycle to a three-day one produces a four-fold
increase in the potential numbers of vectors.” This is very interesting, could the authors please clarify how this conclusion was reached? Increasing from two-day cycle to three-day cycle would also reduce the number of potentially infected blood meals a mosquito would take along its life.

Line 167 The authors talk about different types of sacs (a,b,c) it is not clear what these are. Please define them earlier in the text.

Line 166 – 171 This section is difficult to follow. It seems like there is some important information missing. Please explain what the authors mean by the “sac” “no sac” mosquitoes, is this the egg sac? Isn’t the presence of a sac just indicative that eggs have been laid recently? How can the length of the gonotrophic cycle be inferred from this observation?

Line 204 One of the clear limitations of dissections is the level of dexterousness needed to perform the technique and the difficulty of standardizing the technique since it depends on each individual’s talent. Also, there is an arguable degree of subjectivity. This should be mentioned given that sensitivity and specificity of the technique will vary according to whoever is performing the dissections.

Line 216 “Among 183 A. gambiae s.l. from Kyamyorwa identified to species by PCR 152 (83.1%) were A. arabiensis” What were the remaining 17%?

Line 224 “Among the nulliparous insects, virgins survived better than those with mating plugs (Chi-Square 5.4373, p = 0.020).” Do the authors think there is a biological explanation to this finding?

Line 231 “Hence, newly emerged insects were more likely to attempt to feed outdoors rather than indoors.” I believe this finding has been seen in other studies however there is no consensus why younger mosquitoes prefer to feed outdoors, do the authors have a theory? Is it relatable to “experience” of the vector? Do the authors think anophelines can learn and remember? I think this is a very interesting topic that isn’t discussed enough. Perhaps the authors would like to share their hypothesis backed up by their data.

Line 234 “Among parous insects the proportion with ‘a’ sacs was significantly greater in insects that were dead” So is this indicative that females that have recently oviposited are more fragile and likely to die than others?

Line 298 I would say the gold standard rather than the method of choice.


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: ‘WE LIKE IT WET’: A COMPARISON BETWEEN DISSECTION TECHNIQUES FOR THE ASSESSMENT OF PARITY IN ANOPHELES ARABIENSIS AND DETERMINATION OF SAC STAGE IN MOSQUITOES ALIVE OR DEAD ON COLLECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Well prepared paper, clear and concise.

Literature cited left the author to conclude that they were the first group to use both dried and wet preparations to determine age in anophelines.   While this paper may be the first to compare each method as a stand alone assessment of parity, several previous papers have combined both methods to ascertain parity [dry methods] and number of ovipositions [wet method].
Experimental design: Well presented.

The only flaw in the work, is that they really don't know which method is the gold standard.  The senior author [a well respected expert] selected the wet method based on his experience.  It may have been useful to prepare a known set of standards similar to Hugo et al.
Validity of the findings: It might have been interesting to present the data as 2-way tables comparing nulliparous and parous by each method to show where the difference lie.  This was addressed statistically on L204-205.
Additional comments: As discussed by the authors, estimates of parity and therefore survivorship can be biased by the collection methods used, time specimens were held and source of the material dissected.  The authors did not discuss the impact of the pregravid blood meal on using the dry method.  Here, the small blood meal often taken by virgin females in lieu of sugar advances the ovarioles to Stage IIa and this yolk can make it difficult to see the tracheoles.  It also is important to understand the ecology of the species.  Some anopheles rest indoors but egress to feed outdoors; they also oviposit and refeed the same night, and rapidly transition from sacculate to dilation.  If they fail to find a blood meal the same night, they will appear parous but not sacculate the following evening.


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: ‘WE LIKE IT WET’: A COMPARISON BETWEEN DISSECTION TECHNIQUES FOR THE ASSESSMENT OF PARITY IN ANOPHELES ARABIENSIS AND DETERMINATION OF SAC STAGE IN MOSQUITOES ALIVE OR DEAD ON COLLECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: All comments have been addressed. The manuscript is acceptable as it is.


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: MOLECULAR CHARACTERIZATION OF CLONAL LINEAGE AND STAPHYLOCOCCAL TOXIN GENES FROM S. AUREUS IN SOUTHERN NIGERIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Tables are not well presented. Especially table 2. No title provided and some contents are written in short forms and no legends to define them. In the text, it has been indicated that 2 MRSA isolates were detected, and in the table only one is indicated.
I would  suggest to have one more table that provides social demographic information of the the clinical S.aureus isolates, perhaps poultry too. from the table it should be indicated dates of isolation, source and health status (in case of human s.aureus) and if the patient was admitted or not. this is important as it might tell the which spa types are related to certain conditions,,but also we can see if certain spa types are related with hospital environment or community
Experimental design: Since it has been indicated that t064 was more prevalent in HIV individuals from previous studies  ; it  was wealth in the current study to include the suggested . this information will provide information on  spa types in relation to disease condition. I understand it is not the main aim,but this will add  value to this article .
Validity of the findings: The conclusion is not well stated.This conclusion repeats the results. It would be better to give out what you think this report has contributed and importance of this report to the locality of the study and globally
Additional comments: The manuscript is about: Molecular characterization of clonal lineage and staphylococcal toxin genes from S. aureus in Southern Nigeria.
It is well written.
The data is very important but you must state this locally generated data will offer to the settings. considering that this kind of methodology is not part of routine diagnostics in resource limited settings. by having this information what do you think are the benefits?


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: MOLECULAR CHARACTERIZATION OF CLONAL LINEAGE AND STAPHYLOCOCCAL TOXIN GENES FROM S. AUREUS IN SOUTHERN NIGERIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors typed 47 Staphylococcus aureus strains for staphylococcal enterotoxin genes previously presented in other studies in Southern Nigeria. The aim of this study was to characterize the S. aureus isolated from human and poultry by determining spa types and the presence/absence of enterotoxin genes, mecA/C, and pvl.
I appreciate the authors focused their study on such a subject, but there are many questions and problems remaining to be clarified.

1. Abstract. An abstract must be a concise synopsis of the key parts of the research, at least contains a brief conclusion summary. There are no any study conclusions that satisfy the criteria of the journal;
2. On bacterial strains. Isolation history and detailed profiles of strains whose “lineages” was determined are important for better understanding or interpreting the information presented;
What is the relation between the poultry strains and the humans? Why have been chosen these strains? The authors should provide detailed information about the isolates used in this study, including details of the carriers, isolation history etc. (year, date, city, country, etiological foods if it was a food-borne outbreak, fatalities, etc);
3. Nowadays, there are many techniques for typing S. aureus. The methods presented are not sufficient for the “clonal lineage” study. In fact the author stressed about this term but there is no genetic study clearly presented. Would be interesting present a minimum spanning tree with hypotetical node computing the spa-types. A comparative analysis with the strains presented is necessary to support some conclusions.
Other phenotypic or genotypic typing methods must be used to discriminate the different S. aureus isolates, for host attribution or to determine the relatedness of different isolates.
In addition, the analysis of the polymorphic X region of the protein A gene (spa typing) was presented in detail. It is sufficient to cite the paper from Koreen et al., (2004) for reference. The same for the other methods cited, as soon as they are not novel or presented and used with variation on the protocol.
4. The authors carried out staphylococcal enterotoxin genes typing, this is a very important point of the study but many information of enterotoxins was lacked or wrong. This point is critical and cannot be overlooked. The authors’ data lacks reliability due to the fact that there were no positive and negative controls mentioned in the manuscript for all the analysis performed. Another problem is lack of information about the previous study cited as the master paper as source of the isolates.
5. Genetic analysis was not enough. It is not clear the reason why the virulence factors included were considered important for the conclusion of the study. In addition the term used are not correct and there are many errors in the classification of the toxins.
The authors focused their study on the toxin genes, as seen in their title. But many information of enterotoxins was lacked or wrong. This point is critical and cannot be overlooked.

Conclusion.
The conclusions are not clear and there is not relevance for the few data presented. All genes of enterotoxin except for SElX, SElY and SElZ are located on mobile genetic elements or genetic clusters, and are highly variable, which might be closely relevant with their dynamic evolution (Sato’o et al. 2014 JCM 52:2637-2649, Suzuki et al. 2014. MAI. 58:570-580, Suzuki et al. 2015. JAM 118:1507-1520. Suzuki et al. 2017. Int J Food Microbiol. 262:31-37). The authors must give more space in the discussion for this subject.

Evaluation scheme:
1. The language is not clear and several time is ambiguous, intense professional English editing is needed.
 2. The article must be written in English and must use clear, technically correct text. The article lacks in this standard. Strong conforming process is required.
3. The Literature references are not sufficient. The authors cannot avoid citing the most recent relevant papers published on MRSA and enterotoxins for support the area of research they speculated and give a context to support their conclusions.
The article should include detailed introduction and background to demonstrate how the work fits into the broader field of staphylococcal enterotoxins. Relevant prior literature should be appropriately referenced.
4. The manuscript is not well structured and there are several formatting errors, including on figures and tables. The raw data shared as a supplemental file is not clear on the information presented. What toxins the authors referred to? Are maybe referring to the genes instead?
5. The structure of the article is conform to an acceptable format of ‘standard sections’, but it clearly need a formatting editing.
Experimental design: The experiment is very easy but the methods are described in a useless way.
The investigation must have been conducted rigorously, in this case very low standards have been followed that do not meet the technical standard. The analysis must include negative and positive controls at all levels.
Validity of the findings: The data are very poor and there is not a clear representation of the results. The conclusions are not well stated and there are limited supports results.
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: MOLECULAR CHARACTERIZATION OF CLONAL LINEAGE AND STAPHYLOCOCCAL TOXIN GENES FROM S. AUREUS IN SOUTHERN NIGERIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Language not clear and ambiguous use of the English.
Literature reference must be improved, in particular in the discussion section (see notes for the authors).
Experimental design: No comment
Validity of the findings: Minor revision and more information related to the speculation of the impact and the novelty of the study.
Additional comments: Suggest a discussion that take in consideration the already published studies:
About the spread of the MRSA in poultry:
- Bhedi KR, Nayak JB, Brahmbhatt MN, Roy A, Mathakiya RA, Rajpura RM. Detection and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Obtained from Poultry and Poultry House Environment of Anand District, Gujarat, India. International Journal of Current Microbiology and Applied Sciences Vol. 7 No. 2018;2:867-72.

About MRSA in ovine:
- Macori G, Giacinti G, Bellio A, Gallina S, Bianchi DM, Sagrafoli D, Marri N, Giangolini G, Amatiste S, Decastelli L. Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in the ovine dairy chain and in farm-related humans. Toxins. 2017 May 16;9(5):161.

About the approach for a population structure study, correlation between spa-types and a food-chain sector.
- Johler S, Macori G, Bellio A, Acutis PL, Gallina S, Decastelli L. Characterization of Staphylococcus aureus isolated along the raw milk cheese production process in artisan dairies in Italy. Journal of dairy science. 2018 Apr 1;101(4):2915-20.