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: RESIDENCY, MOVEMENT PATTERNS, BEHAVIOR AND DEMOGRAPHICS OF REEF MANTA RAYS IN KOMODO NATIONAL PARK
Review round: 2
Reviewer: 2

Output:
Basic reporting: - The language use is this paper is overall clear and unambiguous.

- Figure 1 still need a minor improvement. As I suggested previously, the label of Raja Ampat is still misplaced. I suggest using a dot and line to show exactly where it is. Need to add unit for bathymetry (in the legend).

- Good to see that the authors have made significant improvement in the literature references. However, in some references, the authors made incorrect citations by cherry-picking findings/statements from the cited papers to support findings in the manuscript. For example, in line 574, the authors cited partially some findings on the number of immature individuals from Raja Ampat.

Setyawan et al 2020 (page 62) stated “In total, 153 juvenile M. alfredi (individuals ≤ 2.4 m DW) were documented in Raja Ampat between 2011 and 2019….” and this statement followed by another statement “Focusing on YoY individuals (≤ 2 m DW), we recorded a total of 65 YoY individuals between 2011 and 2019…”.

Here the authors made incorrect comparison as they compared immature individuals in Komodo and Nusa Penida with only YOY individuals, but not including other juveniles in Raja Ampat. Moreover, Setyawan et al 2020 focused on the number of juveniles and YoYs sized ≤ 2.4 m DW), and didn’t summarise the number of ALL immature individuals >2.4 m. Please note that males were estimated to reach maturity at 270-280 cm in DW (Stevens 2016).

Another example for incorrect statement can be found in line 43-45, where the authors stated "In 2013, manta ray fishing was banned in the waters within and adjacent to Komodo NP, paving the way for a nationwide manta ray fishing ban in 2014.".

Setyawan et al 2022 wrote in details about the manta conservation in Indonesia. While the authors wrote some statements about manta conservation milestones in line 115-120, the statement in the Abstract (lines 43-45 was incomplete and misleading).

I strongly recommend the authors to fix those incorrect and incomplete citation and misleading statements. They authors have a significant effort to fix many overstated claims in the the previous version of the manuscript, and therefore similar effort should be taken to fix those in the final manuscript.
Experimental design: The research questions are well defined and are useful to fill knowledge gap on the absence of population level studies investigating manta ray demographics and habitat use within Komodo National Park. I also commend the authors for strong analyses performed in this study. However, there is an important issue regarding the methods described, in particular size estimation that later in the discussion the authors made a conclusion about the potential nursery in CL.

While the authors described in details about manta identification and maturity status in lines 195-208), the authors didn’t add any more information about size estimation, although they mentioned in line 189 that size estimation was also collected by the approximately 20 local dive operator staff as stated in line 186.

In this study, I wonder if the local dive operator staff did collect the size estimation data while dealing with guests, which is their main responsibility I believe? Furthermore, how to maintain accuracy and minimize biases of the size estimation given many local dive operator staff and citizen scientists involved in the data collection. What is the resolution of size estimated?

While the authors clearly admitted the lack of size estimation accuracy in study (line 347), they have to stated in details on how size estimation was done. The authors didn’t report the numbers of individuals with size estimated as well.

I strongly recommend adding the details of size estimation as they did for manta identification and defining maturity.
Validity of the findings: The authors made an improvement in the validity of the findings in this manuscript. However, the authors (in lines 588-590) made invalid conclusion about the possibility of CL as a nursery area using criteria proposed by Heupel at al 2007 on nursery area for newborn and young-of-the-year/YoYs (in this manuscript, the authors use yearlings to represent those newborn and YoY individuals.

With insufficient survey effort at CL and biases and inaccuracy in size estimation (if there were size estimations done), the authors do not have any qualification to test the criterias proposed by Heupel et al 2007 to conclude that CL is a potential nursery area of manta rays.

The authors noted (in line 585-587) “….we did not have the data to be able to discern yearlings from generally immature individuals, an important distinction when considering nursery habitats”, this is a clear message that the authors are not able test the criteria. It is unclear weather the immature individuals encountered in CL were yearlings or other immature individuals which sized were up to 270 cm for males (Stevens 2016). IF the immature individuals were in fact yearlings, the authors didn’t have data to fulfil criterion (2), which is the yearlings remain in the area for extended periods, as they didn’t have continuous monitoring effort in CL, but only opportunistic effort.

Given these, there is no point of testing the criteria for nursery in this manuscript. While identying manta nursery area is critical for conservation (Stewart et al 2018), much more significant and dedicated effort is required. Relying heavily on citizen science and inconsistent survey effort (survey period and data collector) are not sufficient, even for generating too early conclusion on potential nursery area.

Finally, in this manuscript, I strongly recommend the removal of discussion about CL as potential nursery area as well as the assertive by too early effort to test the criteria for nursery area.
Additional comments: In conclusion, I appreciate the authors that have made significant effort in addressing issues raised in the previous version on the manuscript. However, there are some critical statements and conclusions requiring serious effort from the authors to fix/address for the acceptance of this paper.

Overall, I recommend this article should be accepted after the authors addressing and fixing the following main issues: 1) incorrect/inaccurate statements/citation as described in details above; 2) size estimation - accuracy and biases; and 3) assertive effort to test criteria for nursery area.


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: SEARCH FOR MORPHOLOGICAL INDICATORS THAT PREDICT IMPLANTATION BY PRINCIPAL COMPONENT ANALYSIS USING IMAGES OF BLASTOCYST
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Mashiko et al evaluated the utility of classifying blastocyst morphology into grainy and non-grainy for the purpose of successfully predicting implantation or pregnancy more accurately, as indicated by positive pregnancy tests. The results indicated that successful pregnancies had blastocysts with grainy morphology and unsuccessful pregnancies had non-grainy morphology. Additionally, there were no significant difference in Gardner scores between grainy and non-grainy blastocysts, indicating that this morphology criteria can be used independently and/or as a supplement to the Gardner score.
This manuscript is applying for publication in PeerJ’s “The Journal of Life and Environmental Sciences”. Since this journal is not specific to reproductive health and technology, there are some terms that should be defined, however briefly, to be meaningfully to an audience that may not be familiar with reproductive health research.
The introduction highlighted a deficiency of study into classifications outside the Gardner score, placing a spotlight on an important area of research in reproductive health and suggests that complacency with the Gardner score may be blinding individuals, such as clinicians and patients, to the possibility of a useful, if not better, selection criterion for embryos. This could have been stated explicitly to be more informative to individuals who have a vested interest in this research.
After the title, line 38 introduces the reader to principal component analysis (PCA). There was no description of what PCA is other than line 67 mentioning that it is an effective method for expressing image features. It would be informative if the author summarized PCA. The use of PCA in face recognition software would have served as a supportive example for the use of PCA for expressing image features of grainy versus non-grainy blastocysts. Also suggested is to represent the findings more definitively within the abstract, there are some major significant findings (e.g. lines 170-174; 185-192) in the results section not stated in the abstract.
Line 58: what other “notable parameters”?
Line 59: it was stated the outcome of single-embryo transfer will be improved by more accurate morphological evaluation of blastocysts. Perhaps a brief statement on the practical benefits of improving embryo selection and success rates of single-embryo transfer should be included, such as reducing instances of multiple gestation and associated perinatal complications.
Line 75: maternal factors were brought up, but besides subject age, mentioned on line 99, there were no other maternal factors listed. Clarification of what is meant by maternal influences should be included, such as genetic influence, socioeconomic status, employment status, and available social support.
Line 77: what “morphological parameters”? a quick literature search found others.
Line 104: in brief, please provide some information regarding parameters on embryo transfer since it is known that differences can exists in outcomes between techs/physicians
Lines 117-118: all embryos included in the study were ≥3B Gardner grade. A brief rationale for this criterion should be included, such as if this Gardner grade was the industry standard for embryo selection or if it was recommended by an embryologist.
Line 120 states that 64 x 64 pixels was the chosen resolution to normalize image size. Embryo-scopes are capable of higher resolution images, an explanation for the chosen 64 x 64 resolution should have been included.
Lines139-140: it was stated the blastocyst images were cropped to normalize the position of observation. It should be clarified what was cropped out and why, such as if the images were cropped to allow the blastocyst to take up as much of the 64x64 pixel space as possible. Or if there was another rationale for how each image was cropped.
Line 156: In addition to the classification of blastocysts into grainy and non-grainy, the authors should have mentioned if there were other notable parameters considered and rejected. Perhaps a notable criterion such as actual diameter of blastocyst.
Line 218: it was stated research to search for other parameters had not been conducted, but it was stated in the introduction at lines 61-62 that attempts have been made that focused on parameters not included in the Gardner score, that is degenerative tissue. Additionally, there has been research into the presence of cellular fragments, a criterion not used in the Gardner score, in the perivitelline space a predictor of expanded blastocyst quality: https://www.frontiersin.org/articles/10.3389/fcell.2020.616801/full
Lines 245-247 stated clinical implications of evaluating grainy and non-grainy in blastocyst. This criterion would be useful in selecting embryos for transplantation from multiple blastocysts and that this evaluation may be used to reflect fragmentation in clinics that do not perform time-lapse imaging. It would be informative to mention that time-lapse imaging (TLI) is an additional cost and some research does not enthusiastically support the use of TLI in standard care as mentioned in this article: https://trialsjournal.biomedcentral.com/articles/10.1186/s13063-020-04537-2.
As previously mentioned, including brief descriptions of the terms noted in the article would reduce the workload of the reader and enhance the reading of the article. The mention of clinical implications in the abstract would greatly improve the impact of the abstract.


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: SEARCH FOR MORPHOLOGICAL INDICATORS THAT PREDICT IMPLANTATION BY PRINCIPAL COMPONENT ANALYSIS USING IMAGES OF BLASTOCYST
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript titled “Search for morphological indicators that predict implantation by principal component analysis using images of blastocyst”, the authors came up with a new parameter based on the morphology of blastocyst to evaluate the blastocyst capacity. The results showed the blastocysts from the mothers who showed higher human chorionic gonadotropin (hCG) presented the grainy morphology, which seemed more accurate than the prediction of implantation by Gardner score. Furthermore, authors suggested that graininess also can be combined with Gardener score to improve implantation prediction accuracy.
Generally, the manuscript was written well and data are clearly presented. The finding is interesting for current reproductive technologies. I have several concerns regarding the manuscript in its current form which are listed below.
Major comments:
1.	Authors divided the blastocysts into grainy and non-grainy according to exceeding thresholds and non-exceeding thresholds. However, based on the morphology in Figure 3B, grainy blastocysts seemed to show late developmental stage of blastocysts with bigger expansion and cell number as already described in Gardner score. Therefore, the graininess criteria seems similar to Gardner score except more calculation based on images. Authors should explain more and display the typical difference between the graininess criteria and Gardner score.
2.	In line 173, authors revealed the correlations between graininess and embryo fragmentation, and hastily concluded that squashed cellular debris caused the failure of graininess. Apparent debris could be the feature or indicator for the failure of graininess, not the mechanisms. Authors should show more solid data, for example add some cellular apoptosis assay, to support this mechanism.
3.	In Figure 4, authors only present the summary of prediction accuracy and abortion rate after prediction, which appeared graininess was more accurate in prediction. It would be supportive if authors provide more details about maternal conditions, such as individual age, hormone levels, BMI and times of embryo transfer.
Minor comments:
4.	In Figure 2 and Figure 3, the number of patients or embryos should be added in legends.
5.	In line 75, some references should be cited when discussed maternal factors like hCG. Besides, this paper (10.1007/s10654-015-0039-0) probably will be helpful to explain why chose hCG.
6.	In discussion part, the paragraphs format is different from introduction.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: SEARCH FOR MORPHOLOGICAL INDICATORS THAT PREDICT IMPLANTATION BY PRINCIPAL COMPONENT ANALYSIS USING IMAGES OF BLASTOCYST
Review round: 1
Reviewer: 3

Output:
Basic reporting: Attached
Experimental design: Attached
Validity of the findings: Attached
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: SEARCH FOR MORPHOLOGICAL INDICATORS THAT PREDICT IMPLANTATION BY PRINCIPAL COMPONENT ANALYSIS USING IMAGES OF BLASTOCYST
Review round: 2
Reviewer: 1

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


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

Input:
Title: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is interesting and important paper on heavy-metal-induced malformations in the Late Devonian brachiopods from western Junggar, NW China

The presented material has not been published previously, either in part or in full. The text is properly organized and presented and all objectives and conclusions are clear.
 I did not find any errors of facts. I found that all interpretations are competent and sufficiently backed with available data. The list of references is OK.
I found that the length of the manuscript is appropriate. I don’t think that there are any sections that could be shortened or, to the contrary, need to be expanded for clarity.
The figures are necessary, and no additional ones are needed. All illustrative materials are of good quality and adequately explained in the text.
Experimental design: The research questions would be more clearly stated such as "The aims of current study are:   "
Methods are described with sufficient information and are reproducible by another investigator.
Validity of the findings: The findings are well presented and I don't have doubt in their validity.  The data on which the conclusions are based are provided.
Additional comments: Can be published after minor/moderate revision.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is nicely prepared and well-written (although I am not a native speaker). It is rally an interesting topic which fit to the journal very well. The rest of the comments are below.
Experimental design: The authors retrieved many geochemical data which potentially may be used for explanation of the problem they want to resolve. The supplemental data containing the elements and their quantity is also provided.
Validity of the findings: The paper submitted by Ruiwen Zong and Yiming Gong concerns an interesting aspects of the occurrence of malformed brachiopods. The authors link the origin of malformations with hostile palaeoenvironment caused by, as the authors stated, toxicity of heavy metals, especially by lead (Pb).
Although the toxicity of the palaeoenvironment is possible, its link to the occurrence of malformations in the brachiopods' shells should be well-supported. The authors indeed found that the high content of Pb correlates with the occurrence of the malformed brachiopods, and the highest Pb content falls within the interval when the brachiopods disappeared. Ok, but some questions arise.
1. Quite high Pb content also occur in the interval when the brachiopods recover, but there is no signs of malformation. Why? And why the whole population of the bachiopods do not bear malformations within this specific horizon? Please explain.
2. Very high content of another very toxic element, arsenic (As), occur below the horizon with malformed brachiopods where actually the brachiopods are very abundant and where the Pb is on much lower level. In the horizon with malformed specimens, As strongly drops. Thus, the authors should explain such discrepancy. Does arsenic cause such malformations, or not? It should be explained.
3. The authors said: "Some athyrids from the Upper Devonian of western Junggar bear parasitic organisms, such as trumpet corals and bryozoans". However, I think these epibionts were not parasites. If so, please provide the evidence.
4. The authors also stated that malformations were unlikely caused by parasites as no epibionts are preserved on the malformed specimens. However, many parasites which could caused shell malformations could be soft-bodied organisms which either lived on or inside the host brachiopods. In that way, such epi/endobionts would not fossilize.
5. Redox proxies. Ni/Co and V/V+Ni are bad redoks proxy. V/Cr is also discussed as not reliable. One of the best I think is U/Th. Vanadium and Nickel proxy often show anoxia in oxic paleoenvironments, and the Ni/Co shows the opposite. So, I would not rely the interpretations solely on these two proxies. If You can, please check it using U/Th, especially that You have the data. Please try and check how their relate to the other proxies used.
Additional comments: Summarizing, this is interesting paper and suitable for the journal. I think many readers would be interested in the problem. However, to strengthen the conclusions, the authors should look at some specific points presented by me above to avoid any oversimplifications and overinterpretations. I would use the phrases like "possible link with toxicity" etc. That's a shame that authors did not analyse the section in a more detail. Now the data are rather very scattered throughout the section. Anyway, having even these which are presented, the authors may retrieve conclusions in elegant, but careful, manner.
For me the paper needs a MODERATE/MAJOR revision before it is published.
Best wishes.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: see: 4. Additional comments
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have added some few (minor) suggestions and comments for improving the style of the manuscript text directly into the original word document. The authors should go through that and suggest whether these comments can be helpful for them.
The same accounts to my suggestions concerning additional references which for the better presentation of the research carried out at the Hongguleleng should be included. - However, finally it is upon the authors to decide! In case the authors do need some of the suggested literature, please feel free to contact me via: suttner.thomas@gmail.com.

The recognition of malformed brachiopods is a very important observation which has not been recognized for fossils from the Hongguleleng before! - I have experience in seeing similar malformation in Middle Devonian conodonts - but triggers and causes are further speculative. - The presented study by Zong and Gong could be one possible explanation.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript is generally written in clear professional English. In few places it would benefit from minor corrections, but nothing that impacts on the understanding of the science. The introduction and background is OK for some parts, but lacks information on the regional / local geology. Particularly info on the depositional environment is needed to help the interpretation of the findings. The figures are OK, but Figure 2 could be simplified (the pie charts are not needed and the histograms could be clearer). Figure 4 shows a lot of geochemical data of which some are not discussed in the text (e.g. Ba, Ag, Cd etc.) so these could be removed. Raw data are shared in the form of spreadsheets with geochemical values and brachiopod measurements and listings of malformations. However, see below for a critique of why this is insufficient.
Experimental design: As far as I understand from the manuscript, lead was measured from bulk rock samples, so it remains unresolved where the lead sits within the rock. If an argument is made about the concentrations of lead causing malformations, it is necessary to demonstrate that the lead was in the water – many volcanic minerals will contain some lead, but remain inert in the sediment. If there was significant lead in the water, it would accumulate in the shells of the brachiopods. So these need to be analysed. 5ppm lead from bulk rock probably means that a fraction of this was in the water. But even if 5ppm would have been in the water it would have been too little to account for the reported percentages of malformations.
Validity of the findings: Malformations of Devonian shells induced by lead poisoning is a very challenging hypothesis to assess and needs to convince the reader of: (1) malformations – how are they defined? (2) Existing malformations cannot be explained by expected processes such as space constraints or repair of predation damage; (3) that the lead was (3a) in the water (and not in sedimentary grains) and (3b) was in sufficiently high concentrations. Unfortunately, the manuscript falls short of many of these critical points.

Malformations: While the manuscript qualitatively describes what is considered as malformations, no precise definition is being provided. To what degree does the shell have to deviate from the ‘perfect’ shape to be considered a malformation? What is the natural variability of the shell shape? Shells were binned by size, and the measurement was given as ‘height’. Figure 2C indicates that the author’s ‘height’ is really the length (as defined in volume 1 of the Brachiopod-themed volumes of Treatise on Invertebrate Paleontology). In the absence of a precise definition of what constitutes a malformation and what is considered a healthy shell, it is not possible for the reader to fully evaluate the results. It is also rather difficult to explain away the apparent absence of ‘malformations’ from other groups of fossils in the same strata. Even if different taxa have different degrees of sensitivity – serious Pb concentrations in the environment should leave their mark in the total fauna.

Alternative explanations of the ‘malformations’. Some of the possible alternative causes of the malformations are brushed away too easily. The slight deviation of growth caused by space constraints are discounted because the specimens don’t occur closely together. However, no data is provided about the depositional environment. Where the specimens transported? How do the authors know that they did not grow in a constrained space? Furthermore, repair of predation-induced damage is not discussed at all, when at least one of the figured specimens looks like it suffered that type of damage (Fig. 1H). The ‘tectonic’ influence on the specimens is discussed in too much detail – why not list some papers on the tectonic structures of the region? As long as there is no cleavage / strain present all is fine. The image in Fig. 3D is not an example of tectonic damage – this is just a shell that cracked before lithification of the sediment.
Additional comments: The brachiopods are well documented and I do not doubt that there are malformations in these shells. But I think that you are trying to force the issue with Pb without having evidence. The Devonian biodiversity crisis is a very exciting time when significant paleoecological restructuring is happening. I suspect that a lot of your shell damage is down to predation, which is rarely reported (see for example: Alexander 1986, Journal of Paleontology, v. 60(2), 273-285) and can tell you something about paleoecological relationships. Identifying and quantifying predatory shell damage (and asking why some taxa are more strongly affected than others) might be a more interesting way to evaluate your fossils.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is well written and interesting paper on the malformations in Late Devonian brachiopods. The authors have improved the MS. Now it can be published in its present form.
Experimental design: Research question are well defined, relevant & meaningful. Rigorous investigation have been performed to a high technical standard. Methods are described with sufficient detail.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound. Conclusions are well stated, linked to original research question and are supporting results.
Additional comments: Can be published in its present form.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I see that the authors made substantial improvements to the previous text. Although they still prefer the toxic metal poisoning as a possible driver of brachiopod shell malformations, they do not entirely 'escape' from epi/endoparasitic factors. I think that the toxic metal, especially Pb, influence is possible and interesting hypothesis and may be presented as such, but still the data is not as supportive as one should wish them to be. The higher content of Pb, for example, is only significantly higher in one brachiopod species, while in the other the its content, as the contents of other metals, is rather similar or insignificantly higher. The same concerns the Cd content in the horizon with malformed shells and the horizon below: they are very similar and not significantly different.
Anyway, the conclusions are balanced and the hypothesys possible, at least concerning the influence of Pb, so I think it is worth to show the results. Thus, I recommend the publication, let it be read and discussed.
Experimental design: OK. I don't see any flaws here,
Validity of the findings: OK.
Additional comments: No.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 2
Reviewer: 3

Output:
Basic reporting: Thanks for sending me a revised version of the manuscript! It reads much better now!
I have added only a few remarks (style/typing errors) directly into the PDF file.

One important thing is to change also the headings in <Supplemental file 1.>, in order that these conform with the corrections made in the running text:

normal/abnormal specimens

Please consider the newly added corrections - as soon as that is finished the paper is ready for publication from my side!

All kind regards,
Thomas S.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: see 1. Basic reporting


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 2
Reviewer: 4

Output:
Basic reporting: The manuscript has been significantly improved by including the discussion of parasites, providing more information on the geological setting, nature of malformations, and toning down the discussion on the influence of metal poisoning.
Experimental design: The new analysis of four brachiopod shells is rather meaningless as grinding up the total shells for analysis would have included the sedimentary infill or internal cement. What is really needed is the careful analysis of shell material. This can be done by micro-drilling the shell, or by LA-ICP-MS of sectioned shells and should be accompanied by a careful evaluation of shell diagenesis before analysis – see Baeza-Carratala et al. 2021 (Estudios Geologicos, 77(2): e141) for an example.
Validity of the findings: I appreciate that the authors analysed some brachiopods to look for support for their interpretation that the malformations are linked to metal poisoning. However, I remain highly unconvinced for the following reasons:

1.	What constitutes excessively high metal values in limestone? What values would be considered average / normal for metals such as Pb, Cd, or As that are known to induce tetatomorphies? I had a quick look and found two studies on similar rocks providing relevant data: Franchi et al (2015, Chemical Geology, 409: 69-86) describe the elemental composition of Devonian limestone from seamounds and associated sediments, and Babek et al. (2021, Sedimentary Geology, 420: 105934) describe the elemental composition of Ordovician orthoceratide limestone (see tables below). Furthermore, Baeza-Carratala et al 2021 (Estudios Geologicos, 77(2): e141) analysed many individual brachiopods shells from the Lower Jurassic and their values also suggest that the metals of the current study are quite average. Clearly the current study has very similar values for the relevant elements as these studies (see attached table), which raises the question if this is not just the normal background amount of heavy metals to be expected in such settings.
2.	The increase of heavy metals described for the section is closely linked to samples BL-5, BL-6, and BL-7, all of which are from tuffites (all other samples are from bioclastic limestone). It is not a big surprise to get higher values of heavy metals in these sediments.
3.	See experimental design above
Additional comments: The way forward to provide a robust analysis of whether the malformations were induced by metal poisoning is to carry out is to focus on the brachiopod shells and analyse them with the methods and care outline above. And this should not just include the shells from the layer with the malformations, but older/younger layers with similar brachiopods as well. I would only be convinced if the shells from this particular horizon with the malformations contains contain significantly higher metal concentrations than other horizons that lack malformations. In the absence of such evidence, I would strongly recommend to remove any statements that suggest that the malformations are linked to metal poisoning. The topic of metal poisoning can still be discussed and the relevant data presented. But rather, the discussion should conclude that there is no evidence that metal poisoning was responsible for the observed malformations.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 3
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English is used throughout. Literature references, sufficient field background/context are provided. Authors interpretations are supported by the data.
Experimental design: Original primary research within Aims and Scope of the journal. Research question are well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Validity of the findings: All underlying data have been provided. The data are fine. Conclusions are well stated.
Additional comments: This paper is well written and deserves to be published in its present form.


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: MALFORMATIONS IN LATE DEVONIAN BRACHIOPODS FROM THE WESTERN JUNGGAR, NW CHINA AND THEIR POTENTIAL CAUSES
Review round: 3
Reviewer: 2

Output:
Basic reporting: Geberally ok. I think, that in the chapter Geological settings should be use and cited papers: Shen et al. (2021- Review of Palaeobotany and Palynology 295) and Stachacz et al. (2021-Acta Geologica Sinica).
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Editor,

I was reading with real satisfaction this manuscript by Ruiwen Zong and Yiming Gong entitled Malformations in Late Devonian brachiopods from the western Junggar, NW China and their potential causes. In the present manuscript, Ruiwen Zong and Yiming Gong did nice work to explore the Late Devonian brachiopods shell malformation caused by epi/endoparasites infestation. An interesting topic and this paper will be quite good for PEERJ. I recommend it can be published after minor revisions after adding the comments I proposed below. The main issues I am concerned are:

1.	In my opinion, these malformations are connected rather with biotic interactions with parasites. Oxygen depletion is not a reliable factor in the investigated section in the Hongguleleng Formation. We can exclude anoxic conditions. The U/Th ratio can be confusing especially in pure limestones, and higher values of these ratios are often connected with impoverished with detrital fractions represented by Th. But it did not reflect true redox conditions. This ratio was proposed by Jones and manning (1994) to investigate ancient mudstones rocks in fact. Generally, it is very important to look at directly U contents and enrichment factors. In fact, the better way to decipher redox changes is trace element enrichment factors (EFs). Therefore caution and controlling it by other proxies are needed when we use simple ratios such as U/Th, V/Cr for detail see Algeo & Liu, 2020(Chem. Geol. vol. 540) and Rakociński et al., 2021(3-Paleo vol. 566).

2.	In the investigated sections U contents are very low < 1 ppm in beds with malformed brachiopods (samples BL-3 and BL-4). UEF in BL-3 and BL-4 reached 0.67 and 0.18 (in comparison to average limestones according to Wedepohl, 1970). U is depleted which is indicative of oxic conditions in fact. It is confirmed by very low Mo contents < 1 ppm in the whole section. According to Suttner et al. (2014), the TOC contents in the whole Honggulelung Fm is very low with max values of 0.22 wt. %. Generally close to 0.1 wt. % TOC. These sediments are organic poor, and they consist of a lot of benthic biotas which is indicative of oxic conditions. Additionally, Stachacz et al. (2021-Acta Geologica Sinica) described rich ichnofossils in the lower part of Honggulelung Fm which reflected not oxygen restricted benthic environment.

3.	High levels of heavy metals or toxic elements can also lead to malformation of organisms. Yes, that is true, but it is rather not in the investigated case. Authors stated that “The levels of some heavy metals (e.g., cadmium, lead, barium, and zinc) in the layer with the malformed athyrid shells are relatively high compared to the levels in the lower part of the Lower Member, particularly cadmium and lead.”
However, values Cd and Pb are lower in samples BL-3 and BL-4 than in the upper part of sections (see Supplementary files). On the other hand, Pb can be enriched in sediments during diagenesis. Additionally, Pb values 5 ppm are not high in fact. Cd values also are not too high, I have higher values e.g. in pure fossils-rich limestones (crepida Zone at Kadzielnia in the Holy Cross Mountains) reached values between 0.1 to 4.1 ppm. Additionally, barium, lead, and zinc can be enriched during diagenesis by hydrothermal fluid migration, but in the investigated section they are not very enriched in fact. In summary, I think the best way to explain these brachiopod malformations by authors is biotic interactions.

4.	I know, that it is not a geochemical paper, but I think, that in results you could short describe more important geochemical data, which were used in the discussion?

5.	In the chapter Geological settings should be use and cited papers: Shen et al. (2021- Review of Palaeobotany and Palynology 295) and Stachacz et al. (2021-Acta Geologica Sinica).

Other minor remarks:
Line 32: cephalopods: could cited e.g. House, 1960-Palaeontology, Klug, 2007-Acta Palaeontologica Polonica, De Baets et al., 2011-Acta Palaeontologica Polonica; especially I recommend cite here De Baets et al. 2015-Ammonoid Paleobiology.

Lines 74-79: That is not true, we know many successions rich in fossils from Lower Famennian. Reef organisms are rare generally to Middle Famennian, but other groups are numerous and diverse in fact. What is important according to Shen et al. (2021-Review of Palaeobotany and Palynology vol. 295) Hongguleleng Formation starts from crepida conodont Zone and is lack triangularis Zone, this is a recovery interval after the Frasnian-Famennian crisis, therefore biodiversity increases.

Lines 199-203: It is worth mentioning that TOC contents in the studied formation are very low (for detail see Suttner et al. 2014).
Lines 252-253: “The lead contents are 1.3 and 2.2 ppm in the lower part, but 5.1 and 5.2 ppm in the upper part.” But in higher samples, the Pb contents increase > 7 ppb.
Lines 254-256: I think that such small variations in trace metals are irrelevant.
Lines 263-268: I’m not sure whether it is a statistically significant sample. However, it could be interesting.
Lines 272-273: Good point. I agree with this conclusion.
Lines 283-290: Very good point and future challenges!
I think, that in the future may be a good way will be cutting malformed shells to try to find parasites (see De Bates at al. 2011-Acta Palaeontologica Polonica).

Kind regards,
Michał Rakociński
University of Silesia in Katowice
Faculty of Natural Sciences
Będzińska 60
41-200 Sosnowiec, Poland
E-mail: michal.rakocinski@us.edu.pl


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: GENOME-WIDE IDENTIFICATION AND RESPONSE STRESS EXPRESSION ANALYSIS OF THE BES1 FAMILY IN RUBBER TREE (HEVEA BRASILIENSIS MUELL. ARG.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Based on the rubber tree database, the authors used bioinformatics methods to characterize the physicochemical properties, gene structure, cis-elements and expression patterns of rubber trees. The language of the main text should be substantially improved. There are numerous examples of sentences that are unclear, or even seem contradictory. In some cases, the overall meaning is understandable, but the sentences themselves require rephrasing. As such, a thorough proof-reading is recommended before acceptance. Furthermore, there are some major issues in the manuscript.
Experimental design: In the materials and methods section, the author used three paragraphs to describe RNA extraction, inversion, and final quantification. It is recommended that the author condense this part into a paragraph. Furthermore, for RNA-seq data and HbActin, please note the accession number.
line 123: The author downloaded the HMM model of the domain, not the domain. Please correct.
line 126: The author wrote "based on these two methods." This is very unnecessary. Just write one method for the identification of gene family members, and the other method is just for you to verify whether your results are correct.
In “Plant materials and treatments” section, How to get the concentration used in different disposals, whether to conduct a preliminary experiment or refer to previous studies, please add an explanation.
“Identification of BES1 genes in rubber tree” section lacks description of subcellular location.
Validity of the findings: 1. In “Results” section: What is the difference between the research results of “Heatmap of HbBES1 genes in different tissues and treatments” section, and “Tissue expression specificity HbBES1s in rubber tree” section?
The grouping of the gene LOC Os01g08180 in the phylogenetic tree is not accurate, and it is obviously closer to the I and II groups.
Figure 7: it was not cited in the main text. An abscissa title should be included.

2. The discussion section: line 318: the authors concluded that “the number of BES1 transcription factor in different species have no significant variation account for there are relationship in different species.” What does this mean? This sentence does not seem to reveal the relationship between the number of its members and the evolution of the species. The authors should make a collinearity analysis diagram within and between species to reveal the evolutionary relationship more accurately.
Line 291: The author came up with “This result indicated that HbBES1 genes response to stresses through different patterns.”, but used hormones such as ABA, ETH, SA, JA, and even H2O2 in this experiment. The author’s experimental results were too subjective and did not objectively consider the significance of the experimental design.
Lines 336-339: “The number of each group in rubber tree was different with Arabidopsis and rice on account of the retention of duplicates in different groups under different evolutionary constraints was different in Arabidopsis, rice and rubber tree.” This sentence is very confusing. Please reorganize the language expression.
The abbreviated form should be used for brassinolide in discussion.
Additional comments: 1. The abstract is very informative but, lacks on developments from a concern. It is best to have this as the opening sentence.
Line 48-50: Please check sentence grammar issues

2. The introduction is well written and has strengths on developing awareness. However, it is best to integrate the components of the proposed aim within the context of literature so that readers can piece the communication into a proposed idea. This helps maintain the reading pleasure as well.
Lines 78, 86:  Arabidopsis thaliana? Please check for similar errors in the full text. Furthermore, use the full name for the first occurrence of the Latin name, and replace it with A. thaliana for subsequent occurrences.
There are many grammatical errors in the introduction, such as lines 60, 79, 67-69, 96-99, 89-92 and 109-111. Please re-check this section and correct it. “neGA3tively”, “elonGA3tions”?


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: GENOME-WIDE IDENTIFICATION AND RESPONSE STRESS EXPRESSION ANALYSIS OF THE BES1 FAMILY IN RUBBER TREE (HEVEA BRASILIENSIS MUELL. ARG.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, the BES1 family genes in rubber tree were identified and characterized, including physicochemical properties, gene structure, cis-elements and expression patterns. These results lay the foundation for further studying the function of HbBES1 gene in rubber tree. The results presented here provide interesting data about HbBES1. This manuscript is suitable for publication in PeerJ. However, the manuscript requires some revisions based on the following points.
Some of the statements are not clear and complete. For example, ‘To realize the function of BES1 family in rubber tree, based on rubber tree database, we used bioinformatics to characterize physicochemical properties, gene structure, cis-elements and expression patterns’, etc. The manuscript needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure so that the goals and results of the study are clear to the reader.
There are many ‘GA3’ in word, for example ‘Brassinosteroids (BRs) is a natural plant hormone and is located in plant orGA3ns of pollen, seed, stem, leaf and so on to widely involved in various plant regulation processes, such as stem elonGA3tions’, Please check whether it is a spelling mistake or a conversion problem.
References: Some References are incomplete. For example ‘Li T, Kang X, Lei W, et al. SHY2 as a node in the regulation of root meristem development by auxin, brassinosteroids, and cytokinin [J]. Journal of Integrative Plant Biology. 2020, 62(10).’ The reference style should be formatted according to the journal style. Please check carefully, and arrange all of your references according to the standard reference style of the journal.
Experimental design: Plant materials and treatments: More information or details should be given. Tissue culture seedings were treated with SA, etc. How to treat？Spray leaves? ‘All samples harvested immediately frozen’, which tissue? Leaves?
Validity of the findings: ‘The molecular weight of members ranged from 30,524.99 to 79,071.07 kDa’, Please check this result.
Additional comments: Quantitative real-time PCR assay: ‘95℃ 15 seconds’, ℃ and seconds should be ℃ and s.
‘CATA S73397’ should be ‘CATAS73397’.
‘ETH (Ethephon)’ and’ Ethylene (ETH)’, I think all should be ETH (Ethephon).


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: GENOME-WIDE IDENTIFICATION AND RESPONSE STRESS EXPRESSION ANALYSIS OF THE BES1 FAMILY IN RUBBER TREE (HEVEA BRASILIENSIS MUELL. ARG.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: See additional comments
Experimental design: See additional comments
Validity of the findings: See additional comments
Additional comments: 1.	BES/BZR transcription factors are key components of BR signaling in plants, so that the biological functions of HbBES in BR response should be investigated.
2.	AtBES1 and AtBZR1 are homologous gene with high sequence similarity (88%). Their biological functions have many similarities. However, the manuscript is mainly focus on the studies of AtBES1. We think the writers should provide some related data to explain the question.
3.	The gene name of BES/BZR transcription factors in figure 2 should be labeled with the published gene name, such as AtBZR1. In addition, the evolutionary relationship of HbBES and OsBZRs also should be analyzed.
4.	The Specific expression of 9 HbBES1 genes in different tissues (Figure 6) should be analyzed by qRT-PCR assays, but not semi RT-PCR assay.
5.	There are many grammatical mistakes in the manuscript, please correct them


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: GENOME-WIDE IDENTIFICATION AND RESPONSE STRESS EXPRESSION ANALYSIS OF THE BES1 FAMILY IN RUBBER TREE (HEVEA BRASILIENSIS MUELL. ARG.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thanks for addressing all the revisions and corrections requested.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND RESPONSE STRESS EXPRESSION ANALYSIS OF THE BES1 FAMILY IN RUBBER TREE (HEVEA BRASILIENSIS MUELL. ARG.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have revised the manuscript carefully according to the comments and suggestions.
Experimental design: The design of the experiment is reasonable.
Validity of the findings: The results presented here provide interesting data about HbBES1.
Additional comments: The revised manuscript is suitable for publication in PeerJ.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND RESPONSE STRESS EXPRESSION ANALYSIS OF THE BES1 FAMILY IN RUBBER TREE (HEVEA BRASILIENSIS MUELL. ARG.)
Review round: 2
Reviewer: 3

Output:
Basic reporting: The content of the article meets the requirements of journal publication
Experimental design: The experimental design are reasonable
Validity of the findings: Research results are reliable
Additional comments: No


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: ANATOMICAL INDICATORS OF EUCALYPTUS SPP. RESISTANCE TO GLYCASPIS BRIMBLECOMBEI (HEMIPTERA: APHALARIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. I am not completely competent to evaluate because I am not a native speaker. I have found some errors and I believe that a revision of the text is needed
Experimental design: 1. yes
2. Authors hypothesize that it is possible to compare potted plants with adult plants. Without some preliminary data that relate young plants to mature forest ones, the results obtained are random and, at most, preliminary.
Validity of the findings: Without some preliminary data that relate young plants to mature forest ones, the results obtained are random and, at most, preliminary.
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: ANATOMICAL INDICATORS OF EUCALYPTUS SPP. RESISTANCE TO GLYCASPIS BRIMBLECOMBEI (HEMIPTERA: APHALARIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Major flaws in the introduction and discussion sections.
Experimental design: Experiments established to check the anatomical characters are fine but more experiments should have been established to validate the resistant characterstics.
Validity of the findings: This is a combination of qualitative and quantitive research. Findings are validated according to the 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: ANATOMICAL INDICATORS OF EUCALYPTUS SPP. RESISTANCE TO GLYCASPIS BRIMBLECOMBEI (HEMIPTERA: APHALARIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work deals with an innovative and extremely important subject, since G. brimblecombei is a limiting factor in the production of eucalyptus. The article has scientific merit in looking for species resistant to the insect. However, some points should be improved and are highlighted below:

General:
a) Abstract very well described, with clear objective, concise material and methods, and well-marked results.
b) The introduction is quite compact, it needs further elaboration, supporting the work's hypothesis.
c) The literature used is outdated, only 20% of the references (6 out of 30) are from the last 5 years. I suggest a data update.
Specific:
L. 61 - Improve the connection between the sentences, explore more the productivity of eucalyptus and its importance, then describe the insect pest.
L. 76 - I suggest further supporting the hypothesis of the work. Are there other works/papers in this area? Are there studies with other insects and eucalyptus, evaluating the resistance of this plant?
L. 76-79 - Explore this information further, it is very important to support the work.
L. 83-86 - This sentence is more consistent in the introduction, also because it approach part of the work's hypothesis.
Experimental design: General:
a) Material and methods propose a differentiated but important methodology to achieve the proposed objectives.
b) Was the Quantitative variables of leaf anatomical characteristics methodology was based on any author/manuscript? If yes, please cite. If it is self-authored, it needs to be more detailed, so that it can be repeated.
Specific:
L. 87-89 - In this paragraph more details are needed, I believe that some information is missing, as it is confusing.
L. 97 – Tall or height? Please check the most suitable term
L. 100-102 - It is not clear whether the insecticide was used on all seedlings. I suggest it be restructured, detailing this information
L. 116-117 - Please put the scientific name in italics
Validity of the findings: General:
a) The work has highlighted importance for the sector, however, this importance is not clear in the justification of the work. It is very important to improve the justification, bringing out the potential, scientific and technical merit of this work;
b) Figures must be numbered in the sequence in which they appear in the text. I suggest rearranging the number of figures.
c) The titles of the tables must be clear and self-explanatory, however, they must not contain excessive information. In this case, the descriptions of the 18 variables are already in the table, being unnecessary and redundant. But in the title of table 1 it is necessary to include the material in which all this was analyzed, it is not mentioned that it was made from eucalyptus leaves. On page 17 of the pdf document, the first sentence would connote a perfect title for table 2 and in this same line it would be for table 1
d) The beginning of the discussion is confusing because it is not clear that the information is referring to the data of the present work and, therefore, comparing to the literature. It is important to make it clearer, to make it explicit when the information was observed in the present work. I reread it several times to understand that it was referring to the data of the present work. Initially it just looked like a literature review. (L. 185-196)

Specific:
L. 138 - I suggest reviewing the subtitle, as what is described in the results does not refer to quantification itself, only observations were described.
L. 142 - Please explain this sentence better
L. 149-152 – Please explain this sentence better. I suggest rewriting.
L. 153-156 - How did you come to this conclusion, especially for group 2? I suggest explaining better
Fig. 4 - I suggest taking the legend out of the graphic. Position below or leave the text box highlighted, so as not to confuse it with the data.
L. 226-227 - Does this information refer to the present work or to Brennan's article?
L. 234-236 - What is the relationship of this information with the data obtained in the present work and with the histological analyses?
L. 249-250 - Was any analysis performed in this work to reach this conclusion? I believe this information would fit more as a discussion.
Additional comments: Please check the scientific nomenclature. The first time it is mentioned in the text, the genus must be in full, as well as at the beginning of sentences. In other quotes, it should be abbreviated. Check the lines: 91, 105, 185.

Suggestion of references that may help authors (it should be noted that the reviewer is not the author or co-author of these):

1. Bush et al. (2020) 10.2989/20702620.2020.1824556
2. Jere et al. (2020) 10.1111/aje.12686
3. Pereira et al. (2021) 10.18671/SCIFOR.V48N126.20
4. Silveira et al. (2021) 10.1007/s10973-021-11027-3


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

Input:
Title: ANATOMICAL INDICATORS OF EUCALYPTUS SPP. RESISTANCE TO GLYCASPIS BRIMBLECOMBEI (HEMIPTERA: APHALARIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout: I am not completely competent to evaluate because I am not a native speaker, but the Authors provided a revised text by Dr. Phillip John Villani (University of Melbourne, Australia);

Literature references are sufficient field background.

The structure of the article is professional with figures and tables. Raw data shared.

As the authors themselves assert, they are self-sufficient with pioneering results according to the hypotheses.
Experimental design: It is original research within the scope of the journal.
The submission clearly defines the research question.
The investigation has been conducted rigorously.
Methods are described with sufficient information to be reproducible by another investigator.
Validity of the findings: The data on which the conclusions are provided are in an acceptable discipline-specific repository. The data are statistically sound.
Conclusions are well stated and limited to supporting results.
Additional comments: The authors clarified well the perplexities highlighted with the first review.


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: ANATOMICAL INDICATORS OF EUCALYPTUS SPP. RESISTANCE TO GLYCASPIS BRIMBLECOMBEI (HEMIPTERA: APHALARIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made the changes. I am still not very comfortable with the text.
For example, although abaxial and adaxial terminologies are used in some sections but in other sections, awkward usage of 'lower side' 'lower plant parts' occurs (lines 238-239).

I believe still work on language is needed.

Under 'discussion'
"Glycaspis brimblecombei is a sucking insect and its damage produces a large amount of honeydew, causing the development of sooty mold (Reguia & Peris-Felipo, 2013), increasing  the humidity inside the lerps, which can reduce the nymph development and survival."
this does not make sense. Firstly damage does not produce honeydew. Honeydew is secreted by G. brimblecombei. Secondly, what is the connection between increased humidity, plant resistance, and this study?
References are not cited correctly, Brennan 2001 is not cited correctly in the text (a and b are missing).
  Antibiosis and antixenosis is still not discussed enough.
Experimental design: NA
Validity of the findings: NA
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: TRACING RNA VIRUSES ASSOCIATED WITH NUDIBRANCHIA GASTROPODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presents a study in which the author has mined available transcriptome data in NCBI that originates from Nudibranchs, to elucidate viral diversity. This approach has been used extensively in the past elsewhere to survey RNA viral diversity in invertebrates (e.g. Shi et al 2016 Nature), but to the best of my understanding has never been applied to this charismatic group of marine invertebrates. The authors recovered a small number (31) of ‘bona fide’ viral contigs, most of which were similar to unclassified viruses. Interestingly, most identified/classified viruses were similar to a viral genome recovered from an ‘echinoderm’ (no further info available in NCBI). The author also points out considerable contamination of viromes with dinoflagellate and cnidarian genetic material.

Basic reporting is OK. Materials and methods are sufficient for another scientist to follow and repeat. I found that Fig 1, while somewhat informative, was not key to understanding the conclusions of the manuscript, so perhaps could be included as a supplemental figure. I would also promote supplemental Table 1, which includes accessions to consulted data to the main manuscript.
Experimental design: The experimental design is straightforward; the author consulted 47 metatranscriptomic databases through NCBI, assembled them, then used a couple of algorithms to highlight potential viral contigs, then used comparisons against ncbi’s databases to determine if these were ‘bona fide’. This is an appropriate pipeline as a first-order survey of novel viruses in transcriptomes. One thing that caught my eye: the authors do not perform any phylogenetic analysis of detected viruses, which would greatly benefit the manuscript. Of the 11 contigs matching Beihei picornavirus 75, it is impossible to know how closely related these contigs are. The author should perform alignment and maximum likelihood or other analysis to establish how closely related these contigs are (is it 1 across all transcriptomes? Or multiple? If the former, I’d be a bit suspicious of a contaminant).
Validity of the findings: The findings of novel viral contigs are valid. However, the discussion relating to their relative abundance is not valid in my opinion (Line 173 onwards). Just because a contig recruits a certain number of reads does not necessarily equate to abundance, since there can be un-quantified and un-expected biases introduced into the sequencing pipeline. For example, were the transcriptomes amplified by any approach? Amplification biases can lead to skewed virome data based on A+T vs G:C content and length. Were extraction protocols the same between all transcriptomes? The author mentions about 2% of reads in a library recruiting to a contig as potential evidence of significance. Viral RNAs are encapsinated and thus decay more slowly than free RNAs from hosts. Hence, the absolute representation in the library may reflect variable length of time between collection, preservation, and sequencing, and I really can’t put much stock into its virological significance. Recommend deleting this, and focusing instead on the phylogenetic analysis of new viruses detected.
Additional comments: Interesting data mining paper which elucidates new viruses from an underexplored host. Note however that even if viruses are found in host transcritpomes they may not actually infect the host, but may also be either viruses of microbes in the host microbiome or contaminants in reagents or from the surrounding environment.

As a footnote, the author should avoid "Nudivirome" as my first instinct was to see this as a paper about nudiviruses. Perhaps "Nudibranchviriome" might be more appropriate.


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: TRACING RNA VIRUSES ASSOCIATED WITH NUDIBRANCHIA GASTROPODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English writing has some small issues that could be corrected.
Experimental design: Please see 4 below.
Validity of the findings: Please see 4 below.
Additional comments: The manuscript by Rosani presents an analysis of transcriptome data from nudibranchs in an attempt to uncover viral RNAs and provide insight into viruses infecting these organisms. The analyses have been carefully performed and the results are interesting and of value to the virology community, at least as a starting point to perform future targeted work on viruses in these hosts. Some comments for possible improvements to the manuscript are below.

1. It is somewhat surprising that so few transcripts could be assigned as potentially coming from DNA viruses. There seems to be only 1 such case? I believe there are quite a few known invertebrate-infecting DNA viruses, so it seems reasonable to expect some here. This could be discussed more (in the discussion section)?

2. On line 137, is “non-redundant” meant as opposed to “redundant”?

3. Table 1 - the taxonomic names should be checked for correctness - there seem to be 2 different typos in instances of Picornavirales and should “Negarviricota” be “Negarnaviricota”? Is the 1 putative DNA virus in this table or are these all RNA viruses?

4. Figure 2b would benefit from a scale to indicate the numbers of sequences the circle sizes represent - or just put the numbers into the circles.

5. Figure 3b - it is hard to make much of this with the way the virus sequences are named on the right - could they perhaps be in the same descending order as in table 1 with an indication that is so in the legend? I am assuming this is all of the sequences from the tope section of table 1? Similarly, maybe the bottom labels would be more informative as the animal species designations, or something other than the meaningless current version.


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

Input:
Title: TRACING RNA VIRUSES ASSOCIATED WITH NUDIBRANCHIA GASTROPODS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Clarity of language: It is appreciated that the narrative is mostly written in an uncomplicated style that facilitates understanding the content. There are several unusual or non-idiomatic words or phrases, and some have been highlighted in the text. For example, line 56 “assignation” has a meaning totally different from the one the author intends, I am sure.
Literature for the content described is appropriately cited and referenced. Although the paper is self-contained, it does need a wider scope in the introduction that sets up the significance of this taxon or its virome.
Data are presented clearly and the supplementary tables are appreciated and helpful as searchable references.

Fig. 1. It is not clear that the figure is needed.  If this fits into a significant question or next step, this should be justified in the text. Presenting numerical data as a figure is helpful, but is best done when that information needs to be understood by the reader to appreciate the significance of that data versus some other data set in the paper or in the field of study. Figure 1 seems like a graphical representation of numbers that are mostly record-keeping.
Fig. 2a. The tree is useful, but looks like a standard output of a software package, without a lot of guidance for the reader where the significance is. In the tree or legend, it should be made more clear which of the taxa have representation in the data analysis. This relates to the Additional comments regarding the need for more synthesis within the introduction and discussion. The names on the ends of the branches are very small.
Fig. 2b seems un-necessary. Do the circle overlaps signify shared sequences? If that is all, the list of numbers in the text conveys this well enough.  In the discussion, the merits of using blastx versus the software packages was helpfully described. If this figure is there to support that comparison, it needs to be made more evident.

Supplemental Figure 1 appears to lack citation in the text. The figure appears to be a standard output of a software package.  It is not clear whether this figure helps deliver the main point of the paper. The essential facts, of how many virus-like annotations were found, is easily extracted from the text.

There was a  focus on a behai picorna-like virus. Please make note in the discussion that this virus-like sequence was itself assembled from a large data-set, and has no other evidence of an actual virus. While it is most likely that these viruses do exist as infectious particles, one should be accurate in all descriptions of putative virus genomes. Therefore supplementary S2 figure is not needed, and appears to be a comparison of alignments without much context. How does it carry forward the ideas about viromes?
Experimental design: This is original work that applies available bioinformatic tools to capture more knowledge from existing data sets. However it could use more context and grounding in important questions related to the study, as explained in Additional Comments.

I did not see an analysis using BLASTp or tBLASTx to search for viruses.  BLASTx has a lower sensitivity to finding virus-like sequences. Virus genomes are mostly intron-free, so coding regions are rich and will give you more certainty that there is a virus there. Did you do a search or uninterrupted ORFs?  Did you investigate the possibility that there would be evidence of active transposons, and how might that affect the analysis?
If the analysis did not include a translated search for matching sequences in nt/nr, it should either have one, or explain why it is not informative to do so. If the bioinformatics packages of VirSorter and checkV use protein alignments, it would be good to have that described.
Validity of the findings: The findings are valid, and the analysis conducted is a step towards understanding the virome of a lightly-studied taxon. The author does not make unsupported claims. However the findings could be more complete.
Methods are described with sufficient detail to repeat and evaluate.
The significance of the study would be greater if there were a central question or two that this study helps answer, or a future direction for research that it opens up.
Additional comments: Applying current bioinformatic tools to extract new knowledge from existing datasets is applauded. The description of putative virus genomes from metagenomic sequence data of nudibranchs is a potentially valuable resource for understanding issues related to the health and ecology of nudibranchs. The paper, in general, does a good job of describing the data used, the methods employed, and the putative virus genomes detected. However it has a deficit of synthesis and analysis within the biological, ecological or evolutionary disciplines that are relevant.
For example, the word disease appears not at all in the paper, yet viruses and disease are a major emerging problem in marine ecology, especially in invertebrates.
The introduction should include more than a passing reference to the  paucity of data on nudibranch virome study. For this paper to bring the field forward, it should make the reader aware of at least one or two nudibranch- related issues. For example, is there a nudibranch mortality event that is unexplained? Are there concerns about them as invasive species, or are there concerns of diseases being moved into nudibranch habitats? Or, are the symbiotic relationships between the mollusc and other taxa important in interesting ways. How does this analysis bring us forward on an issue regarding this taxon.


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: THE MICROPHENOTRON: A NOVEL METHOD FOR SCREENING PLANT GROWTH-PROMOTING RHIZOBACTERIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1)The text requires grammatical polishing. In its current station it is difficult to understand.
2)The images presented  have extremely low resolution. I would encourage the submission of high quality figures. The narrative is quite simple so it would benefit from having decent resolution figures.
3) I think the data presented in the Tables should be transformed into figures. I find it quite weird that those experiments are summarized as tables and not as figures. This is particularly relevant for the tables in which the auxin and ethylene lines were tested.
4) I could not see any reference to raw data. Please provide it
Experimental design: 1)The introduction requires formatting. The research question is well defined but still it is difficult to grasp from the introduction. The question that the authors formulate is valid despite lacking novelty.
2) The clear relevance of the paper is that the authors implement a novel protocol to maximize the throughput of PGP traits.
3)The methodology is clearly stated
Validity of the findings: 1)Authors do not mention how many times each experiment was performed nor mention how reproducible their 96 well plate assay is. I am curious to see how reproducible are the measurements they perform. Due to the fact that the major novelty of this article is this trough-put screening I would encourage that they asses how reproducible and reliable their measurements are. This should be added to the article.
2) The authors mention a validation of the approach using a green house experiment. Nevertheless, despite mentioning this they never actually compare the trends observed in their high trough-put system in comparison to the green house experiment. I want to see a correlation performed between both approaches, if their high trough-put assay is proper enough we should expect to see high correlation between the results obtained by both approaches. Specifically, the author should correlate a specific trait (e.g, fresh weight)  between both approaches and show it quantitatively. Due to the fact that their paper is based on this methodological novelty it is quite relevant that they show its validity.
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: THE MICROPHENOTRON: A NOVEL METHOD FOR SCREENING PLANT GROWTH-PROMOTING RHIZOBACTERIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript is generally well written but does need some English editing. Figures and tables need to be improved significanly and the methods need more details, specifically about replication. The table say that the values are means of 3 reps which is severly insufficient.
Experimental design: There are problems here, as I point out in my general comments. Some controlls are missng in my opinion, replication is not sufficient, and some experiments need to be preformed side by side.
Validity of the findings: no comment
Additional comments: In this manuscript, the authors screen 10 bacterial isolates for plant growth promoting traits.
These isolates are screened in vitro for a number of traits associated with plant growth promotion (more on that later), then screen for their activity in a lab-based plant growth phenotyping system, and finally validate their findings in soil. The authors also include the use of phytohormone insensitive and hypersensitive Arabidopsis mutants, to test whether hormonal manipulation indeed plays a role as a pgpr mechanism.
 This is a widely used experimental pipeline, with the main point of novelty being the use of the “micro-phenotron” system. It seems like an intriguing system, but its main advantage of allowing high throughput screening is not demonstrated here. Only 10 bacterial strains are tested with 3 only 3 replicates, which is not sufficient power to deduce anyting about plant growth promotion. Furthermore, this paper does not address the two underlying assumptions: (1) that the in vitro plant growth promotion screens are predictive of activity in plants and (2) that the phenotron results correlate with results in soil. This is especially acute in the case of auxin production, which is many cases is actually harmful for the plant. Curiously, all the tested strains seem to possess the growth promotion traits screened for and all seem to produce larger plants than the uninoculated control (critically – it is not clear whether the phenotron remains axenic or not), so there is no true negative control of plants inoculated with non pgpr strains.
The use of hormone insensitive mutants is interesting, and it seems to support the notion that the growth promotion observed is not phytohormone related, but these experiments need to be performed with wt and the mutants side by side in order to be informative, and this does not seem to be the case.
Finally, the data presentation in this manuscript needs to be improved, as does the number of replicates. I recommend presenting the data using boxplots with scattered dots to show the true distribution of the data.

Specific comments.

The introduction focuses on bacterial IAA production as a reliable measure for plant growth promoter potential. I do not agree with this premise. IAA production can actually be harmful for the plant, and the evidence cited by the authors to support their claim is not sufficient.

Line 234 It’s not entirely clear how taxonomy was assigned to the isolates. What database were they compared to and in what method. How similar are they to the nearest hit?
In addition, the tree is not informative if it only includes your own isolates. You could make a combined tree with related database sequences to inform the reader about the relatedness of your isolates to known sequences. Furthermore, it is not clear how you have 2 isolates on the tree with seemingly identical sequences but different genus names assigned (Psychrobacter and Moraxella).

Line 245 There is too much text here to explain something trivial. Just say that reduction in auxin production with increasing salinity is explained by reduced growth. Figure 2 is barely readable.

Line 268 If I understand correctly, all isolates tested were positive for all pgpr traits tested? I find this 100% success rate puzzling. Also, the lack of negative strains does not help in assessing the predictability of these in vitro screens for actual plant growth promotion, which is an ongoing lacuna in the field.

Line 277 Make sure all figures are called in the text. Figure 5 needs to be improved. Present it as a box plot showing all datapoints. Again, it is puzzling that all strains were positive for growth promotion. This makes me wonder whether this is an artifact of the system. Would E. coli also produce growth promotion in this system?  A proper negative control is needed (e.g bacterial stains with no growth promotion activity.


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: THE MICROPHENOTRON: A NOVEL METHOD FOR SCREENING PLANT GROWTH-PROMOTING RHIZOBACTERIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Potted plant experiments are costly, difficult to reproduce and often don’t translate to the field. To address this Forde’s group at Lancaster University developed a “microphenotron” that uses a model plant grown in modified 96 well plates. As I understand it the microphenotron, was developed for screening metabolites. The application of this technology to screening rhizobacteria is interesting but I am not sure if the authors clearly evaluated, or at least presented, this application of the microphenotron. Discussion includes a statement that “system can be used to screen beneficial rhizobacteria” but that statement is not supported by data or a reference (line 336).

Introduction should be modified to state if and when the microphenotron has been applied to screening live bacteria, versus metabolites and small molecules. This should be done in a dedicated paragraph that justifies using this technology. The last paragraph of Introduction should state what was done and what was observed. Results should include a comparison of the results obtained with the microphenotron and potted plants and Discussion should state whether the system worked in this application.

Line 26. Start Abstract and Introduction with background on why the microphenotron is used.
Line 29. Replace “would allow” with “allows”
Line 30. Replace “were able to degrade” with “degraded”
Line 58. It is not clear here if you are talking about the model plant (Arabidopsis) or the microphenotron.
Line 72. Introduction needs reorganization. Replace “species” with “strains,” delete “have been reported to” and move that and line 72-75 to the start of the 1st paragraph, where you introduce the field of PGPR.
Line 76. Here and throughout, delete fillers like “Now it is established through numerous studies...” and “It was established from different studies (Line 347). Also, replace “have been considered as” with “is”, unless you have doubts about that science.
Line 89 – 99. This section needs revision, following the above general comments. Delete “that would” and “under specified..effect.”
Line 93. Here and throughout, the name is Forde not Ford.
Line 96-99. This discussion of root morphology doesn’t belong here.
Line 104. Provide location of sampling areas and mode of collection.
Line 135. Why cap Indole?
Line 148. Provide (City, State) for USA locations.
Line 169. Why in the plate reader identified here but not in line 133.
Line 177. Sterilized.
Line 210. Revise to 6:1:1
Line 235 and 240. Move “Sequences were... BLAST” and “The sequences have...KJ011879.” to Methods.
Line 246. Start each paragraph in Results with a topic sentence that states the key result. Lines 246 – 248 provide no results. Similarly, move Line 269 -271 to Methods.
Line 282. Tell the reader what the figure shows in terms of the treatment effect.
Line 322. Replace “showed stimulatory effects on rosette growth biomass” with “stimulated rosette growth”
Line 328. Change to “controls”
Line 363. Delete “parameters”
Figure 5. Box, whisker, violin or similar plots are preferred to bar charts.
Experimental design: What is an appropriate control for this application? The control is not identified in Table 2 or Methods, so I presume it is water. I am not sure if that is an appropriate control for tests of live bacteria. All of the isolates significantly increased root growth relative to water, so the system might reveal traits not associated specifically with PGPR. In other words, a more appropriate control might be a strain that does not promote plant growth. This would control for the fertilization effect of adding microbial biomass.
Validity of the findings: I am concerned that the each 8-well strips used in the system corresponded to one treatment or the control. Some might argue that each strip should be treated as one replicate, as the treatments are not randomly assigned. I would be interested in the strip to strip variation for duplicate treatments.
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: THE MICROPHENOTRON: A NOVEL METHOD FOR SCREENING PLANT GROWTH-PROMOTING RHIZOBACTERIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I appreciate the inclusion of the phylogenetic tree of isolates assayed in this study.
Some legends in the figures are not legible still.
Experimental design: Ok
Validity of the findings: The authors lacked the clear correlation against a proven system.
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: THE MICROPHENOTRON: A NOVEL METHOD FOR SCREENING PLANT GROWTH-PROMOTING RHIZOBACTERIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: -	My concerns regarding sample sizes and phylogenetic placement were addressed. On the other issues, I stand by my original assessment. I think the Microphenotron system can be a great system for screening PGPRs. I also think that using hormone-insensitive mutants is an elegant way to test whether PGPR in these strains is hormone-dependent. However, the experiments as they are presented don’t really address the questions that the authors set out to answer. The authors say that these 10 strains were selected since they were found to have  many PGPR traits a-priori. I would like to know what happens in their system with strains that were not found to have these traits. In other words, how does PGPR in their system correlate with the results of their pre screening on one hand, and to soil based assays on the other. Since all strains in this study seemed to have a positive effect, the only negative control is uninoculated plants and I do not think that is sufficient. Why weren’t strains that were NOT shortlisted, meaning that they didn’t perform as well in the in-vitro assays, used as a negative control in the in-planta assays?

The authors and I are in some disagreement as to the role of rhizobacterial auxin
production as a plant growth promoting mechanism. I think this is a legitimate debate, and certainly not ground for rejection. However, they do not really provide literary support for their claim. In line 90 they write: “For agricultural applications, screening of rhizobacteria based on auxin production has provided a reliable tool for the selection of PGPR” and provide Pavlovic, 2018 as a reference for this statement. The paper that is referred to did not study PGPRs at all.

Finally, I suggested that the authors use boxplots and indeed they Figure 7 was added with boxplots in it. But this figure presents what by all accounts is a meaningless comparison.

I am sorry I cannot be more supportive, but I do not think that this manuscript should be published as it is presented. It seems like a more methodical comparison of their system could be made with both pot-based assays and PGPR trait screenings.


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: THE MICROPHENOTRON: A NOVEL METHOD FOR SCREENING PLANT GROWTH-PROMOTING RHIZOBACTERIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised manuscript presents a very important data set. As far as I know, this is the first study to apply the microphenotron to the screening of rhizobacteria for the ability to promote plant growth. The parallel measurement of plant growth promotion in potted plant trials, which is current standard, adds particular power to this work. As in the first version, the authors need to improve the organization of the manuscript to present this key contribution clearly.
Experimental design: The research question needs to be clearly defined. The manuscript does not make it clear that this is a novel application of the microphenotron.
Validity of the findings: Results should have a paragraph that starts with a topic sentence that states something like what the rebuttal says “Strains that performed well in ‘Microphenotron’ bioassay also showed good results in pot trials for wild-type A. thaliana.” That paragraph should provide a figure that compares the microphenotron to potted plant trials. The x-axis should present plant growth response in the microphenotron (with errors bars that indicate the variation between three different experiments) and the y-axis presents the result of the potted plant trials (with error bars for within treatment variation). Each point should correspond to an individual isolate.
Additional comments: As many of my previous comments were ignored or glossed over, I am not going took a fresh look at the manuscript.
Reorganize Introduction into the following paragraphs.
1.	Provide background why we need more PGPR and the limitations of the current methods (in vitro traits and potted plant trials) of screening for them. Move lines 75 – 97 to this first paragraph.
2.	Start this paragraph with a topic sentence that describes the microphenotron. Provide a topic sentence that clearly sone way or another if this system has been evaluated for screening bacterial isolates for plant growth promotion. In other words, is this work novel?
3.	State what you did: Rhizobacteria were isolated from halotolerant plants because ___. Strains tested with in vitro assays for traits associated with PGPR, potted plant trials and a microphenotron. Salt concentrations was varied in in vitro bioassays because _____ and different KO mutants of Arabidopsis were selected because _____. Then summarize the results, the microphenotron agreed/disagreed with potted plant trials.

Specific comments.
Line 27. Replace “deliver…” with “test the response of seedlings of Arabidopsis to natural products”
Line 28. Delete “of shoot…where”
Line 29. Delete “can be assessed”
Line 31. You cannot assume they are PGPR. Replace with “rhizobacteria”
Line 32. Add comma before “which”
Line 38. Revise to “extracts, including..”
Line 39. Here and through write actively by starting the sentence with the subject (rhizobacteria) and the verb what the subject does (produced ___). I suggest “Psycrobacter ___ and ____ produced the highest amount of IAA….” Leave the amount they produced to Results.
Line 50. Which results show that microphenotrons are effective? The current standard (potted plant trials), which was used to validate the results, is not mentioned in Abstract. As I understand it the “experiments with mutant lines” were potted plant trials designed to elucidate the mechanism of plant growth promotion for these isolates but potted plant trials with the wild type was used to validate the microphenotron.
Line 75-76. Comma before “such”  and replace “or” with “and.”
Line 77-79. Delete “After screening…phytohormones but”
Line 81-85. Why go into so much about detail about one trait (auxin) and not the others (HCN, P..). The discussion of tryptophan and auxin is distracting. Delete it.
Line 176. Delete “the method suggested by”
Line 238. As stated in my earlier review, move “Sequences were... BLAST” and “The sequences have...KJ011879.” to Methods. Start each paragraph in Results with a topic sentence that states the key result.
Line 241. The term “similarly” and the phrase “On the other hand” add nothing.
Line 290. As stated in my earlier review, tell the reader what the figure shows then cite the figure.
Line 293-295. Move this to Methods and/or Introduction. Also, make it clear in the Tables how the data was generated (microphenotron or potted plant).
Line 304. Revise to write directly “Application of B. thuring… significantly enhanced rosette…in the auxin resistant mutant.”
Line 321. Delete “showed” here and in line 323.
Line 339. Delete “were found to have the ability to” and change to “promoted”
Line 351. Delete phrases like “It was established from different studies that,” which have no content. State the fact and cite the source.
Line 462. Carefully check references for content and consistency of format. For example, Martinez (2020) lacks a year and some references provide the issue (line 458: 86(1):1-..” and some do not (line 466: 2:49-…).
As stated in review 0, “Figure 5. Box, whisker, violin or similar plots are preferred to bar charts.”