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

Input:
Title: A SPATIOTEMPORAL ENSEMBLE MACHINE LEARNING FRAMEWORK FOR GENERATING LAND USE/LAND COVER TIME-SERIES MAPS FOR EUROPE (2000–2019) BASED ON LUCAS, CORINE AND GLAD LANDSAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presents an approach for continental-scale annual land-cover mapping by integrating several machine learning tools and existing independently collected training data. The manuscript is hard to follow. Consider adding hierarchical section numbering (1, 1.1, 1.1.1). It is very hard to distinguish a section from a sub-section, which makes the document structure unclear. I have concerns about project methodology (which is not always fully presented in the paper). I also have concerns about the findings and interpretation of the results, which contradicts previously published reports. Overall, I do not think that this manuscript is ready for publication in PeerJ.

1. Project methodology
L 151 – I do not see “m” in equation 1., Y and X are not explained (land cover and annual covariates?)

It is not clear why such a complex model was implemented rather than using a single approach (e.g., CART or CNN). The authors suggest that the proposed EML model is beneficial compared to a single machine learning approach, however, no valid comparison is presented to prove this point. It would be beneficial to compare the author’s results with a result from a single approach. Specifically, it is important to confirm if the class probability based on a single model (i.e. CART ensemble) will be substantially different from the obtained results. Is it also important to understand why such a complex model was needed: due to temporal inconsistency of source data, or errors in training data, or inconsistency between land cover and land use thematic classes?

The EML model design and application described very superficially, precluding detailed understanding and possibilities for replication (L185-198). Such description may be suitable for dataset metadata, but not for a scientific publication. It is not clear how the results of all different machine learning tools were integrated and what was the weight of each of the outputs to the final decision.

The purpose of “prevalent change mapping” is not clear. Why was it done and how was it validated and used?

Table 2: I think, the journal standard is to use comma, not period, as a thousands separator. Please correct.

Table 4 – Is it 30 m^2 (equivalent to 5.5 m spatial resolution), or 900 m^2?

L515 – I disagree. First, each algorithm represents an ensemble of models calibrated using random subsets of training data. Did you check that the covariate’s weight stays the same if a new random training set is used? Did you check the difference between the models in each ensemble?. Second, most of the Landsat-based metrics are correlated with each other. The selection of one metric over another may be a function of the model parameters or code specifics. Finally, even if these models have differences in the weights of covariances, that does not prove that integrating results from multiple models are more effective and accurate than implementing a single algorithm.

The conclusion from the test results for comparison of the spatial and spatiotemporal models is not clear. Can you provide an explanation on why the results of the model trained using multiple years are better? Is it because of Landsat data availability, or incorrect normalization of the ARD products, or training data errors?

2. Uncertainty Metrics and Validation
The authors suggest that the per-pixel class prediction uncertainty is an important feature of their method and the lack of per-pixel uncertainty reporting is a limitation for similar methods (L97). I disagree with this position. The model-based per-class uncertainty depends on multiple factors, including the annual cloud-free data availability and training site quality and distribution. By training data quality, I understand both temporal consistency (land cover changes correctly represented by training class) and thematic consistency (training data correctly represent the class that has a characteristic land cover and that can be identified using multispectral data). The per-pixel uncertainty reflects the ability of the model to replicate the training data, not to predict the actual class distribution. In case when training data consist of thematic or temporal uncertainties, the per-pixel uncertainty may be inappropriate for quantification of the map accuracy. That is why I see limited benefit from per-pixel uncertainty reporting in this or other similar projects.

The “accuracy” term is not always used correctly. For example, L98-99: “Mapping accuracy is provided as a general number (average performance) for the whole area, although in practice prediction accuracy often varies from class to class.” I see a contradiction here: if the mapping accuracy provided for the whole area, by each class, that should be sufficient to describe per-class accuracy variation, correct?
I suggest to authors provide conventional User’s and Producer’s accuracy metrics. This way, the accuracy of your product can be compared with earlier publications. Also, does the validation data were collected using a probability sampling design?
Table 5. The reclassification table seems very superficial. Why have you selected vineyards as a separate category, but omitted orchards? Why did you exclude peat bogs, which category are they on your map? Which category did you assign the mixed forests to?

The average accuracy of the method (Tab. 8) is below most of the published European products (Tab. 7). What is the benefit of the presented approach?

3. NDVI trend
The Landsat ARD product provides normalized reflectance, not surface reflectance calculated using the radiative transfer function. As such, the NDVI calculated from the ARD should not be directly used to estimate the vegetation vigor. Another issue related to changes in Landsat data availability during the 2000-2019 interval. The data in the early years (before Landsat 8 launch) were collected following a data acquisition plan which prioritized growing season observation. As such, the annual NDVI metrics may overestimate vegetation vigor at the beginning of the interval. Finally, the analysis of long-term trends in vegetation cover and/or NDVI requires statistical analysis to quantify trend significance (see Song et al., 2018, for example). The trend maps (Fig. 8) display both significant and subtle insignificant changes. All these factors may be responsible for the observed NDVI decrease in the northern latitudes, which contradicts earlier finding that shows the NDVI increase (Myers-Smith et al., 2020). Fig. 8 also demonstrates artifacts (e.g., a Landsat scene outline visible over Poland) in the NDVI trend. I suggest removing the NDVI trend analysis or re-doing this analysis using temporally consistent surface reflectance data.

4. “Deforestation” in Scandinavia
The “deforestation" term used in the manuscript is misleading. It suggests that the forest cover in part of Europe (specifically, in Scandinavia) is decreasing. This is not true and contradicts other findings.

“Deforestation” term this term is usually used for land-use conversion, while forest change in Scandinavia is (mostly) due to logging operations in managed forests. The net loss of forests in Scandinavia is not validated, and mist probably an artifact of the method. It seems that the forest regeneration was not quantified correctly by the authors. As such, it may demonstrate the weakness of the present approach and its unsuitability for continental-scale land cover change assessment.

5. Incorrectly used and omitted references
L 105-106. That is incorrect. Several publications specifically address the drivers of land cover change. See Tyukavina et al. 2017; 2018, for example.

The reference to Hansen et al., 2013 for this comparison (L 690-691) is wrong. The GFW product and the referenced paper specifically pointed out that the forest loss and gain products are not balanced, and the gain is underestimated. This comparison here is misleading.

When the authors refer to Ceccherini et al. (2020) to confirm their results, they should also refer to the recent replies to this publication (Picard et al., 2021; Palahi et al., 2021) that disproof results published by Ceccherini et al.
Experimental design: see above
Validity of the findings: see above
Additional comments: No additional comments


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

Input:
Title: A SPATIOTEMPORAL ENSEMBLE MACHINE LEARNING FRAMEWORK FOR GENERATING LAND USE/LAND COVER TIME-SERIES MAPS FOR EUROPE (2000–2019) BASED ON LUCAS, CORINE AND GLAD LANDSAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The provided manuscript agrees with PeerJ polices and conforms to the required format and structure. However, there is a lack of the Conclusion section that should clearly and concisely summarise the stated objectives and findings that support them. This could be made by selecting appropriate subsections of the Discussion part.
It is also written in good English with some typos that should be corrected. It is advised that the manus is checked by English native speaker to assure flawless reading.
The manus provides sufficient introduction to the topic and includes the most important and necessary references to literature. Most of the included figures and tables are very useful and allow better understanding of the content. However, some of them were not cited in the text. So they should be checked and removed as unnecessary material or referenced correctly in the text.
Experimental design: According to my knowledge the presented research is novel and similar approach have not been published elsewhere. The authors identified aims of the work and indicated direction of study clearly. It seems that the authors performed a lot of hard work to put all the pieces together. However, there should be clearly stated benefits coming from this work as compared to other studies addressing similar topic.
The description of the work is long but there is still may issues that seems to be explained not enough clearly. They are pointed out in  section ‘General comments for the author’.
Validity of the findings: The authors describes the real findings which are in most cases correctly identified and supported by results. However, I provide some comments and ideas that may require some additional changes in the manus and I think they should be considered.  There are also numerous questions in my comments which should be answered in order to make the manus easier to understand and follow. This would make the findings more consistent with the conclusions and comments from the authors.
Additional comments: In general speaking I appreciate the presented work as very interesting but some aspects should be corrected and explained more clearly. More detailed comments are provided below:
- line 76 – 7 years of what?
- lines 78-81 – the sentence is not grammatically correct
- Table 1 – why the table is not filled fully? There is some information missing.
- EO is not defined
- line 138 – 20TiB – I would suggest to use the most commonly used unites: TB
- equation 1 – first, adding such equation seems to be useless in such manuscript; second, the notation ‘m’ is not present in the equation
- lines 170-180 – I would put this and other codes as supplementary materials (appendix) to not make the manus too long.
- line 196 – it is not clear how the tuning was performed. Was it carried out on the entire Europe? Or only on some selected 30x30 km tiles? Where the 30x30 tiles come from?
- lines 205 – 208 – what is the different between point 1 and 3? How was p. 1 derived? How p. 3 is derived if there are different classes found in the same pixels by the three classifiers used? This is not clear
- general questions – how the Landsat images were analysed? Per tile? Or were they mosaicked?
- line 245 – ‘covariates’ – why to introduce a new name? usually the terms like input variables or classification features are used in the field of remote sensing. The term ‘variable’ is also used in Figure 1. I would suggest to not complicate it.
- line 247 – which years of Landsat data were analysed? 1999-2020 or 2000-2019??
- lines 252 – 255 – what is the reasoning behind this division? Any scientific clues? The season differ in different regions of Europe so where the one used here come from?
- lines 259-266 – this description is not clear, some figure would make it easier to understand
- lines 297-300 – this is also unclear, why LUCAS got 100% and CLC 85%? And how the weights were used? The lines 229-300 are totally unclear
- lines 312-317 – this section and the use of OSM is difficult to understand
- line 321 – where values 101-200 come from?
- Table 4 caption – 30 m2? Should be rather 30 m pixels size
- lines 362 – 366 – section quite unclear and there is repetition in lines 395-400;
- line 362 – ‘For each dataset’ – what dataset??
- Lines  - 365-366 – both ‘spatial’ and ‘spatiotemporal’ models use multi-temporal data!
- lines – 380-394 – all description of the validation methods and comparison strategy is quite confusing and should be supported with some figure/workflow to make it clear, there are too many combinations and years mixed
- lines 404-406 – the sentence here is too long and at least some commas should be used.
- line 413 – OLS??? Please provide full explanation of this abbreviation
- lines 420 – 446 – I suggest to add it as supplementary materials
- Figure 4 – is this illustration representing analysis for only one class??
- lines 459 – 463 – not much informative
- lines 467-470 – very confusing sentence
- Table 7 – why there are different number of classes provided in the table for the same land cover products but compared with training data from different years?
- line 509 – ‘This proves…..’ what exactly means ‘this’?
- - line 520 – please correct the structure, it should be ‘…accuracy for 33 classes at level 3, …’ by the way, this results are for which classification? spatial or spatiotemporal? On which training dataset? CLC, LUCAS or combination of them?
- line 529 – what is the positive rate??
- line 541 – reclassifying…
- Table 9 – it is nice summary of the results. But it is not clear of which results? Which year? Which set of training data used? What is the ‘Accuracy’ at the bottom of the table? Please also use the same unit of data. In the text you write in %, e.g. 62% but in the table you provide number with decimal places, why?? The table shows that the accuracy of most of the classes at the level 3 received very week accuracies and cannot be considered as reliable result. There are just six out of 33 classes exceeding 70% of F1-score, therefore a clear statement should be provided addressing this issue and informing that the map should not be used in other analyses as very erroneous.
- lines – 560 – which results exactly is described here?
- lines – 568-569 – correct the numbers of figure…
- Figure 7 – not referenced in the text.. remove if not necessary. The pictures and notations are too small.
- Table 12 – not referenced in the text, remove if not useful
- line 575 – Fig. 4??,
- Figure 8 – not referenced in the text!! Pictures too small
- Figure 10 – not reference in the text
- line 623-626 – how the findings mentioned here can be seen in table 8?
- line 630 – it must be mentioned here that the result of 87% was met only for the classification with level 1 – five classes!!!
- lines 632-634 – very confusing sentence. It is actually not so comparable. The similar results are met only for level 1, with less classes. And the threshold of 85% was met for level 1 so another confusion. If you mention about 10 m resolution then you cannot do this because there is no product resulting from your methods with such resolution.
- line 729 – 731 – this is obvious because land use classes with heterogonous cover are very difficult to map automatically with ML and in many cases such attempt is not logical. This is why you received so low accuracy for the level 3 classification. Many of this classes are too complex to be resolved with ML and Landsat data.
- figure 11 – not reference in the text!!! By the way, it is a bit unclear what is presented here. If the figure stays in the manus, please explain its content.


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

Input:
Title: A SPATIOTEMPORAL ENSEMBLE MACHINE LEARNING FRAMEWORK FOR GENERATING LAND USE/LAND COVER TIME-SERIES MAPS FOR EUROPE (2000–2019) BASED ON LUCAS, CORINE AND GLAD LANDSAT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper was much improved by the authors. It is easier to read and is much more consistent. Most of the issues that I highlighted have been addressed. I still have a concern about the NDVI trend analysis presented here, and I suggest the authors will consider removing it from the publications. I am leaving this decision at the authors’ discretion.
NDVI trend analysis issue.
Fig. 16. The map of NDVI trends clearly shows some artifacts in the data. For example, the visible NDVI gain over one Landsat path/row in Poland. The change from positive to negative NDVI trend by latitude at around 59 degrees N may be an artifact. The NDVI decline in the Alps is also a possible artifact. I still believe that this analysis may be compromised by the ARD data quality and the method of trend extraction. I think fig 12 and fig 17 are adequate to represent dominant land cover change trends. I suggest considering removing NDVI trend analysis and fig 16 altogether.
Fig 19 shows that the average NDVI after 2013 is lower than before 2013. This requires additional investigation. In particular, a comparison with other NDVI products from MODIS and VIIRS will be informative.
Experimental design: NA
Validity of the findings: NA
Additional comments: Other recommendations.
Check references format. Using the first name (J. Chen et al., 2015) is not recommended by the Journals formatting rules (https://peerj.com/about/author-instructions/).
L318. Edit for clarity. I suggest using parenthesis: (1) the same season; (2) ... , etc.
L396. I am confused... Here, you say “our model was not trained to predict peat bogs”. Yet, you have peat bogs as a class in Table 3, and you stated that Peat bogs have high accuracy (L516). Can you explain this inconsistency?
L515-525. You probably should explain that while you use the water frequency from Pekel et al. as the input, it is not correct to say that your model “predicts” water class. The class label is clearly identified by one of the model predictors, so you have a circularity here.
L627: replace “:” with a period.
L 642. Capitalize “Europe”
L710-711. Hansen et al. does not provide data on balanced forest loss and gain 2001-2018. Where does the data come from? I think you are interpreting the GFW data incorrectly.
L771 Should be spelled “stakeholder”.


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

Input:
Title: A SPATIOTEMPORAL ENSEMBLE MACHINE LEARNING FRAMEWORK FOR GENERATING LAND USE/LAND COVER TIME-SERIES MAPS FOR EUROPE (2000–2019) BASED ON LUCAS, CORINE AND GLAD LANDSAT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The already relatively good structure of the first version of the manuscript has been improved and contains now all important parts. English and different typos has also been corrected.
I regret, however, that the authors provided a document called ‘peerj-61104-PeerJ_land_cover_dataset_EU_article_diff.tif’, which is so messy that cannot be used during revision. I do not know if this results from the journal’s requirements or it was up to the authors.
Experimental design: No comments
Validity of the findings: This is a very lengthy manuscript, which even though written in proper English require numerous reading for understanding its contents. In general most findings are correctly identified and supported by results. However, it seems that one of the very important message coming from this research is not highlighted enough clearly. In my opinion this research confirms that AUTOMATIC mapping of land cover or/and land use at a large scale like continental, is still very difficult when considering large number of classes. This most often results in low mapping accuracy. The authors highlighted the issue of relatively small number of classes of existing  map even for produces with high resolution of 10 m. Indeed this is an issue, however, the authors only confirms this problem. After reading this manuscript I feel that the mapping product with 44 classes is still being praised even though its VERY WEAK mapping accuracy. Most researches are aware that mapping 44 or so often very heterogenous classes is pointless and this is why most large scale land cover products contain only between 10 to 20 classes. In this project the authors spent a lot of time (months) and worked on a project that provides simply qualitatively useless products. Besides that, this manuscript tries to persuade readers that the approach is very innovative and novel. The true is, however, that only the product with five classes is worth of attention considering its accuracy and quality. On the other hand, similar or higher accuracy to this low class product were achieved in other projects, still mapping more classes and providing the same or even higher spatial resolution. Because of this I think that the manuscript requires to be rearranged and the proper conclusions should be drawn. I have pointed this issue already in the first round. It is also quite strange that after receiving relatively low accuracy when mapping 33 classes as described in the first version of the manus, and after my comment on the weak performance of this approach, the authors decided to even extent the legend of their map to 44 classes. The authors addressed this point a bit in their rebuttal, but this only confused me even more. If producing reliable and accurate method for land cover mapping is not the point of the authors then I think discussion may be finished here. There are already too many poor methods available openly. If the authors want to add one more, which at the same time produce enormous amount of output data, this is a simply a dead end road. I cannot see any point to publish manuscript that describes a methodology or a framework resulting in less than 50% accuracy (different types of accuracy).
Additional comments: Some more comments:

- Line  - 416 – as pointed in the previous revision both ‘spatial’ and ‘spatiotemporal’ models use multi-temporal data. The fact that the former use data from a single year does not change the fact that it use multi-seasonal information. This in turn is multi-temporal data.
- Line 537 – very confusing: was the S2GLC data used for validation or classification?
- 553 – classes 323  and 322 were used in S2GLC (compare with table 6). BTW, what are the percentages in brackets?
- 574-578 – this sentences states that NEURAL NETWORK was better then the proposed meta-learner run on three different classifiers. This lines are not in agreement with lines 672-677
- 764 – 767 – a very nice idea, however the current study only confirm that the authors are far from this goal
- another important issue is the fact that the presented framework produce huge amount of output data, which in most cases is never used or is difficult to interpret. This concept need to be rethink.


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

Input:
Title: A SPATIOTEMPORAL ENSEMBLE MACHINE LEARNING FRAMEWORK FOR GENERATING LAND USE/LAND COVER TIME-SERIES MAPS FOR EUROPE (2000–2019) BASED ON LUCAS, CORINE AND GLAD LANDSAT
Review round: 3
Reviewer: 1

Output:
Basic reporting: See comments below
Experimental design: See comments below
Validity of the findings: See comments below
Additional comments: I think that the authors put a lot of effort into their study and this is fully appreciated. However, understanding the results is even more important, even if some of them shows that the mentioned effort did not bring expected output.

In my opinion there is no improvement in the presented methodology if the final results are much worse than other existing products and methodological approaches. As I pointed out previously, there are numerous methods that provide more reliable products. The authors claim that their aim is to produce a framework to map LULC changes instead of producing reliable products. I am not going to discuss with that point. But for me the quality of the product matter, not a complicated framework to process a very heavy set of data that produce output which cannot be trusted.

I have got fully confused reading the rebuttal letter. The authors admitted that their cross-validation resulted in a very low accuracy, but comparison with data coming from the S2GLC dataset showed promising results. But this is the point. Do the authors want to admit that their cross-validation is not as valuable as comparison with independent data. If so, there is no way to assess the products with 44 classes. So why there is so much emphasis put on the methodological approach that classify LC with so many classes if this dataset cannot be properly assessed. On the other hand, the authors incorrectly used the independent S2GLC dataset. According to Malinowski et al, (2020) the validation data was prepared for validating land cover product with resolution of 10 m, and in this current study it was used for validation of 30 m product. It is difficult to predict what difference this will make for the final result. Moreover, the good results indicated by validation with the S2GLC datasets are achieved for a product aggregated to 14 classes. This again confirms that achievement of relatively high accuracy is possible only when the legend is considerably reduced. It is valuable that the authors tried to map all CLC classes. But some of the CLC classes are so heterogenous that they my be mapped only by visual interpretation with use of ancillary data providing contextual meaning. This is how the CLC data was produced and is updated. Numerous studies showed this problem and in the current work the authors seems to be ignoring this issue completely.

Therefore, I want to emphasis that I appreciate the authors’ effort in preparing the whole methodology and processing all the data. I can see that there was a lot of work done. But in my opinion the work can be published only with right interpretation of the results. The work should highlight the multitemporal aspect of the study, but it should also consider all the limitations that were encountered and draw the right conclusions. The work cannot be, however, praised for delivering products that is difficult to trust. The level of quality that the product represents may be met with considerable reduced effort and less sophisticated methods.


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: VARIABILITY WITHIN A CLONAL POPULATION OF ERWINIA AMYLOVORA DISCLOSED BY PHENOTYPIC ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Manuscript writing is clear using unambiguous professional english.
2. Literature references of sufficient field background is, however, not exhaustive.
3. Article structure is professional, figures and tables are sufficient to support results.
Experimental design: Authors of the manuscript aimed to further disclose the genetic characterization and unveil the
phenotypic diversity of this E. amylovora population, resorting to MLSA, growth kinetics, biochemical characterization, and antibiotic susceptibility. This research question is not only well defined but also relevant and meaningful towards providing a more integrated approach in epidemiological surveillance of fireblight in Portugal.

However, the manuscript does not clearly state criteria used to select the chosen phenotypic evaluation (growth kinetics, carbon usage and antibiotic resistance).

For instance, carbon source usage is not directly related to virulence (the variable trait shown among isolates).

On the other hand, growth kinetics as a phenotypic trait to profile E. amylovora diversity, could be relevant if psychrotrophic adaptations were considered (Santander RD, Biosca EG. Erwinia amylovora psychrotrophic adaptations: evidence of pathogenic potential and survival at temperate and low environmental temperatures. PeerJ. 2017 Oct 26;5:e3931. doi: 10.7717/peerj.3931).

Additionally, "Biochemical characterization" as a title for a phenotypic evaluation might be misleading since the BIOLOG GEN III microplate analysis, while robust, is based only on carbon sources usage. This reviewer opinion is that authors should avoid the term "biochemical characterization" and instead use a more specific term (i.e. carbon source usage, or similar).

Carbon-source analysis does provide useful information for identification and fingerprinting at species level, but strains sample size is showing not to be enough to provide profiling data for Erwinia virulent isolates. Moreover, if molecular epidemiological characterization had shown clonality among isolates displaying diversity in virulence, this reviewer finds it hard to understand why phenotypic characterization based on virulence determinants (i.e. biofilm, motility, amylovoran production, siderophores) was not considered in the evaluation.

Attempting to find traits to further high-throuput profile E. amylovora highly clonal isolates, is of urgent relevance in the light of frequent pome-trees fire blight outbreaks. If this is the main driver underlying phenotypic analysis selection, it should be clearly stated early in a more comprehensive version of the manuscript.
Validity of the findings: Individually, experiments provide robust, statistically sound, and controlled data.

However, the manuscript lacks support in the selection and extent of phenotypic analyses. An improved manuscript should provide a benchmark against appropriate references or a suitable justification for the criteria behind growth kinetics and carbon source analysis in particular for these isolates.

Conclusions state that further phenotypic assessment of E. amylovora isolates is needed (due to isolates clonality); nevertheless, authors conclusions are vague and require a deeper analysis of provided results to fully reach their stated goal: "to assess growth kinetics, biochemical variability (carbon usage), antibiotics susceptibility, and phylogenetic markers, as features capable to discriminate E. amylovora strains within a genetically clonal population".

To link research question to conclusions, analysis could deepen on the phenotypic profiling that results display; i.e. are there phenotypes associated to phylogenetic markers? Does PCA (carbon usage) grouping relate to dendograms or phylogeny?

An improved manuscript containing a more coherent and straightforward conclusion, would explain the manuscript main contribution to knowledge in the area.
Additional comments: Minor corrections:
-- Line 279: PCA showed a clear separation between the seven strains of E. amylovora collected in Portugal, the type strain LMG 2024, and the reference strain CFBP 1430, regarding the
281 phenotypic characterization (Fig. 6).
R- is the word "between" correctly used in this sentence? aren´t authors comparing among groups?


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: VARIABILITY WITHIN A CLONAL POPULATION OF ERWINIA AMYLOVORA DISCLOSED BY PHENOTYPIC ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors report the variability within clonal population of Erwinia amylovora disclosed by phenotypic analysis like biochemical characterization and antibiotics susceptability.
The manuscript is really well written, in professional english. The background information and references provided are sufficient to clearly understand the topic and to perfectly follow the work.
Figures and tables well reports the experimental results.
Experimental design: The experimental design is straightforward and well planned. Starting from the basic phylogenetic analysis on housekeeping genes, the authors focused on phenotipic characterization of the clonal population, based on biochemical characterization and antibiotics susceptability. The results were statistically analyzed with the standard methods (Anova, Tukey's, PCA).
Validity of the findings: The results give new insights on the clonalality of Erwinia amylovora. Regardless the low genetic variability, the authors were able to identify some phenotipic differences.
The authors also conclude and state that the results may lead future analysis.
Additional comments: I found the manuscript really interesting and well written and I suggest to accept it in the present form.
However, I found some minor mistakes in the text that I suggest to correct before publication:

Lane 90: McManus et al. 2020 is reported in the References as McManus et al. 2002, which is the correct ublication year. Please correct the text.

Lane 380: remove the - in intro-duction.  (Maybe the formatting changed during the pdf creation).

Lane 429-430: as above, I see too many spaces in the senteces.

Lane 462: I do not find Donat et al. 2005 in the text. Please correct.

Lane 591: Prunus domestica goes in italic.

Figure 1 and Figure 5: Why are you using the same colors for Ea230 and Ea820? Or same orange and light blu for Ea320-LMG2024 and Ea390-CFBP1430? Do they represent same cluster? If yes, better state in the figure captions or use different colors for each of the strain.

The authors state that future analysis will probably performed based on MLVA analysis. What about whole genome sequencing? It is known that MLSA may not be sufficient to discern highly clonal population and MLVA may be labor and time consuming. Nowadays WGS is quite cheap and easy to perfomed for bacteria. Think about it.


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: VARIABILITY WITHIN A CLONAL POPULATION OF ERWINIA AMYLOVORA DISCLOSED BY PHENOTYPIC ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is written in clear unambiguous professional English.

Literature references and context is provided.

The article has a professional structure: Figures and tables are relevant to the content. Raw data are shared.

The article represents an appropriate "unit of publication".
Experimental design: The article contains original research within the scope of the journal.

Research questions are well-defined and meaningful. The aims of the study are clearly stated.

The investigations were performed at at a high technical standard.

The methods are described with sufficient detail to replicate the study.
Validity of the findings: All raw data have been provided, they are statistically sound.

Conclusions are well stated and linked to the original research question.
Additional comments: The article covers an important topic, i.e. the diversity within E. amylovora populations in Portugal. In general, the article is well-written and the methods used appropriate to solve the research question. I only have some minor comments on the Discussion section:

-L402: Why is whole genome sequencing not discussed as a method to uncover diversity within E. amylovora populations? Could this be added as a further research topic? Genome sequencing has been used previously in E. amylovora populations ( Sing&Kahn 2019, https://www.nature.com/articles/s41598-019-50589-z, and Parcey et al, 2020 https://www.sciencedirect.com/science/article/pii/S0888754320301117).

-L413: minor format error (E. amylovora not in italics)

I also have one comment regarding the Figure legends:

-Fig. 1 and Fig. 5: it would be helpful if the statistical test used to infer significant differences would also be stated in the legend (it is a bit bothersome to search again for it in the main text).


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: IDENTIFICATION AND VALIDATION OF IMMUNE RELATED CORE TRANSCRIPTION FACTORS GTF2I IN NAFLD
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors analyzed publicly available transcriptome datasets of NAFLD and identified GTF2I as a transcription factor associated with NAFLD. NAFLD is a common chronic liver disease. Understanding its biology is important for researchers and clinicians to develop new therapies and treat patients. The manuscript is properly written in a standard scientific format. All results are relevant to the main topic.

There is a MINOR point the author needs to revise:
In line 77: However, there are only a few studies on the involvement of TFs in 78 NAFLD by regulating immune cells.
The authors need to provide references for these "a few studies".
Experimental design: The experiments in the manuscript are well designed. Proper standards have been applied to analyses in the study. All methods are provided for other researchers to replicate this study,

However, there are two points that the author needs to clarify:
1.  Since the author used previously published datasets for this study, the criteria for sample inclusion or exclusion need to be stated in the manuscript.  This will also help the authors to rule out irrelevant information about patients that may contribute to gene expression. For example, are patients with a smoking history included?

2. All figures with heatmap need to state whether the clustering is using a supervised or unsupervised method.
Validity of the findings: All the data in the paper has been provided in a correct format with proper statistical analyses.

However, there is a MAJOR inconsistency between the title and the conclusion:
The title demonstrated that GTF2I is involved in regulating the activation of NK cells. However, this conclusion is from a bioinformatic prediction but not an experimental validation. Thus, the title is an overstated conclusion.

The authors should either provide NK cell activation markers staining in GTF2I high and low samples or clearly state that GTF2I-regulated NK cell activation is a bioinformatics-inferred conclusion in the title.
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: IDENTIFICATION AND VALIDATION OF IMMUNE RELATED CORE TRANSCRIPTION FACTORS GTF2I IN NAFLD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English of your manuscript must be improved before resubmission. I strongly suggest that you obtain assistance from a colleague who is well-versed in English.
Experimental design: The organization and subsections are also appropriate.
The survey methodology is consistent with comprehensive and unbiased coverage of the subject.
But some parts are confusing, such as 2. In lines 246 to 257, It is hard to understand what Figures 6A-C are. the figure legend and the context is a little different.
Validity of the findings: Technically solid, with no major concerns with respect to evaluation, resources, reproducibility, ethical considerations
Additional comments: In this study, based on the comprehensive bioinformatics analysis of healthy volunteers and 109 NAFLD patients’ liver samples, 39 DEGs of liver samples between NAFLD patients and healthy volunteers were identified. Furthermore, the authors identified two key DETFs from the 39 DEGs, both GTF2I and CHD2. To further verify whether the identified transcription factors GTF2I and CHD2 were correct, the authors performed western blot, qPCR, and immunohistochemical experiments and found that GTF2I was significantly higher in NAFLD tissue samples and no significant difference in CHD2 expression.

1.	In this study, the authors identified 39 differentially expressed genes, why focus on these two genes, GTF2I and CHD2？
2.	Figures 6a and 6d both show HE staining but are completely different.
3.	Figure 6G, In the right Panel (NAFLD) the scale is missing, please add. Please consider adding an arrow indicating the positive GTF21 in the right lower corner.
4.	The author showed GTF21 may be involved in NAFLD by regulating the proportion of activation of NK cells. It would be better if the authors could co-stain GTF21 with NK cell makers or immune cell markers.
5.	The primer sequence is confusing, please show it in the three-line table (do not draw vertical lines, horizontal lines only retain the top line, bottom line, and column line).


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: IDENTIFICATION AND VALIDATION OF IMMUNE RELATED CORE TRANSCRIPTION FACTORS GTF2I IN NAFLD
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Zhang M, and Zhang Y et.al has efficiently utilized bioinformatics and cell biology tools to identify a key transcription factor GTF2I associated with NK cells and highlight its putative role in NAFLD disease pathogenesis. The finding is quite interesting and based on the extensive analysis of a large dataset of 109 NAFLD patients.
The reporting of the manuscript is quite simple and conceptually easy to follow. The structure of the manuscript conforms to PeerJ guidelines. However, there is a need to improve the overall language of the manuscript to make it more understandable. There are few sentences which are incomplete, and there are quite a few grammatical and typographical errors throughout the manuscript. Certain words can be replaced to bring out the meaning clearly. I would suggest check for appropriate use of uppercase and lowercase letters where applicable. It is highly advised to take help of some appropriate online scientific editing and proofreading software, or the help of a colleague who is proficient in English to proofread the manuscript so that it is easy to understand by the readers. I have added few major edits in the additional comment section for the authors to re-check.
The manuscript is well-referenced throughout, and the references are correct. For Line 72 and 73 - For loss of the hepatic TF ChREBP in aggravating hepatic steatosis and insulin resistance, please cite the original articles instead of the review article.
The overall quality of the figures needs to be improved in resolution especially for Figure 2 and 4. Some of the color schemes needs to be changed (for example in Fig 4D) to enable reading the annotations. Some of the figures are not properly referenced or explained in the manuscript, which needs to be rechecked. I have elaborated more about results and figures in the Additional Comments section.
I thank the authors for sharing the Raw Data associated with the study. Only the Raw data for mRNA expression of CHD2 in BSA and FFA treated HepG2 cells is missing and should be added.
Experimental design: The experimental design of the study is scientifically sound and commendable and is based on extensive analysis of the public databases. The authors have also validated the bioinformatic finding nicely with in-vitro experiments. However, the manuscript would highly benefit from elaborate explanation of the figures in the results section and in the legends. The methods have been mostly well explained in detail. The dose/ concentration of FFA should be mentioned in the methods and the figure, and the reason for selecting that particular dose should be discussed or cited from literature.
Validity of the findings: The authors should explain the findings of the experiments as be lucidly and clearly as possible in the results section, with proper figure references. The authors have focused only on NK cells as a probable pathogenic immune cell for NAFLD, while their data also suggests possible role for T cells which also has high GTF2I expression.
Additional comments: There are several key concerns in the result and discussion section:
•	Figure1: Typographical error of “Gene Ontology”, can consider replacing “fraction of immune cells” with “immune cell subset/population”
•	Figure 2a and 3a) The color code legend in volcano plot to indicate healthy and NAFLD group is missing.
•	Figure 2b 3b and 5a) It will be useful to highlight (with a box) the DEGs and DETFs which were considered for downstream analysis. There is a nice and clear distinction between healthy and most of NAFLD patients for DEGs and DETFs. However, a part of the NAFLD patients is very similar in genes expression and transcription factors profile to the healthy group. Is there a subgroup within the NAFLD patient dataset?
•	Figure 2C-F: The annotations are missing words in all the figures. Especially in Figure 2F, it is not clear how the genes are associated with Huntington disease, Parkinson disease etc. because these diseases are not mentioned in the plot. I would suggest improving quality of the figures and adding legends wherever applicable.
•	Figure 3A and Figure 3B labelled wrongly in the manuscript, and Figure 3C is missing. Kindly provide the full form of TSD atleast once in the manuscript. There is lack of explanation of why T cells were not explored further although there were obvious differences in T cell subsets between NAFLD and non-NAFLD subjects.
•	There is no explanation or reference in the manuscript for Figure 4A, B and 4D.
•	It would be helpful to represent Figure 5B and 5D in a better way with clear explanation in the manuscript for easier understanding.
•	 Figure 6A has been improperly referenced in the manuscript and the figure legends. Figure 6A shows Oil red staining of NAFLD and Control tissues but has been mislabeled as HE staining. Figure 6D shows HE staining and should be properly referenced in the manuscript.
•	Kindly label the 2 plots of Figure 6B to indicate which cells (HepG2/human liver tissue) were analyzed for GTF2I in the 2 plots.
•	The statistical analysis in Fig 7A needs to be clearly represented for better understanding. The authors can consider breaking up the figure into 2 parts so that the figure is clear. A key observation is that memory T cells also shows high expression of GTF2I in NAFLD group, and maybe expressed more than NK cells. The authors should explain in the results and the discussion, why the role of T cells was not explored further.
•	The probable GTF2I involvement in ER stress is an interesting proposition and can be followed-up in future studies by the group. However, I do feel that the ER stress biology has been unnecessarily elaborated in the discussion and can be reduced. The possibility of T cells and macrophages association with NAFLD could be discussed based on the high expression of GTF2I in these cells, and also based on knowledge in current literatures.
•	An interesting observation is that free-fatty acids induces expression of GTF2I in HepG2 cells. The authors may discuss the probable mechanism of GTF2I upregulation on FFA exposure of cells in terms of what molecules/cell surface receptors/transcription factors might control GTF2I expression in liver cells and immune cells. This can also pave way for further analyzing the dataset for these GTF2I inducing markers.


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: IDENTIFICATION AND VALIDATION OF IMMUNE RELATED CORE TRANSCRIPTION FACTORS GTF2I IN NAFLD
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author clearly addressed the question from a reviewer.

One more thing needs to be double-checked:
In general, gene symbols need to be italicized while protein names do not. Since most data of the studies are from RNA-seq, the gene mentioned should be italicized.
Experimental design: No comments
Validity of the findings: The author has addressed all the questions from a reviewer.
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: IDENTIFICATION AND VALIDATION OF IMMUNE RELATED CORE TRANSCRIPTION FACTORS GTF2I IN NAFLD
Review round: 2
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: IDENTIFICATION AND VALIDATION OF IMMUNE RELATED CORE TRANSCRIPTION FACTORS GTF2I IN NAFLD
Review round: 2
Reviewer: 3

Output:
Basic reporting: The study has significantly improved after revision in terms of English language use and proficiency. The study is meaningful, well-structured and easy to follow. The conclusions have been explained sufficiently. The authors have added the necessary references as suggested in the previous comments. The quality of Figure 2 and 4 has been improved both in resolution and have been properly annotated and labelled.
Experimental design: The experimental design is good, and the figures have been properly reference in the manuscript as per suggestions. The dose of FFA has been added in the methods section and has been referenced. The methods have been appropriately discussed.
Validity of the findings: The results and conclusions have been logically explained. The authors have addressed the concern of focussing not only on NK cells, but also on other immune cells in the revised version
Additional comments: All the concerns raised previously have been addressed satisfactorily by the authors. There are no additional comments, except suggesting another round of proofreading for fixing some minor errors. For example, in Figure 1: The 'L' in liver RNA-SEQ should be in uppercase.


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 FUNCTION OF SMALL RNA IN PSEUDOMONAS AERUGINOSA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this review authors focused on the functions of small RNAs in Pseudomonas aeruginosa.
Overall the data are presented clearly and are analyzed in an appropriate manner.
However few spelling corrections are needed
Pseudomonas aeruginosa needs to be in italic throughout the manuscript.
The authors did not discuss sRNAs containing CRISPR sequences
I would  be nice if authors can include some figures to show the regulatory mechanism of the sRNAs
in line 18, it should be a gram-negative bacteria
In line 163: "The transcription of RsmY and RsmZ is positively regulated by RsmA and negatively
164 regulated by RsmY and RsmZ" not clear what the authors mean
Experimental design: n/a
Validity of the findings: n/a
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 FUNCTION OF SMALL RNA IN PSEUDOMONAS AERUGINOSA
Review round: 1
Reviewer: 2

Output:
Basic reporting: See attached
Experimental design: See attached
Validity of the findings: See 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: THE FUNCTION OF SMALL RNA IN PSEUDOMONAS AERUGINOSA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article has been fairly written stating the importance of small RNAs in P. aeruginosa. However there are quite a few questions/suggestions that has to be addressed before the article is considered for publication. These comments are mentioned below:

1. The English language usage used in the article should be assessed by a fluent English speaker. There are few areas in the text that need considerable improvement. For Instance, I would recommend in restructuring the abstract section. Lines 19-20: The sentence is not clear and the authors should consider restructuring.

2. The background should be elaborated. The Line 45: Here the authors should also mention about the co existence of P. aeruginosa with other bacteria in a biofilm.  There are references missing in different parts of the manuscript.
  (a) Line 49: reference missing
  (b) Line 96: reference missing
  (c) Line 149: Pseudomonas in italics
  (d) Line 212: E. coli in italics
  (e) Line 223: Pseudomonas in italics
  (f) Line 231 : Pseudomonas in italics
  (g)Line 313: reference missing
  (h) Line 329 : reference missing
  (i) Line 331: reference missing
  (j) Line 356: Pseudomonas in italics, Yushan et al (date????)
  (k) Line 362 : Pseudomonas in italics
  (L) Line 365 : Pseudomonas in italics: Line 366: Transporter in small letter
Experimental design: 1. In Survey methodology, Line 36: the authors have mentioned about some documents. They will have to elaborate on what type of documents did they search for.

2. Line 97: Here, the authors should describe AmrZ in 1-2 sentences with reference.
The section biofilm formation (Line 93) should be elaborated. Only one small RNA involved in biofilm formation has been stated here. The section should be elaborated to include almost all small RNAs involved in biofilm formation.

3. The same is applicable for sections including Quorum sensing (line 100) and Drug resistance (line 110). Both the sections should be elaborated with more references included. Additionally, I would suggest adding a picture/diagram or graphical representation of the sRNAs involved in quorum sensing.

4. Line 125, explain what is T3SS in few sentences. What is ExsA. Is it sRNA? If so explain. The construction of sentence is confusing.

5. Line 165: It is mentioned that the secondary structure of RsmY and RsmZ is similar. In this scenario, it would be better to include the picture or secondary structure of RsmY.
Validity of the findings: Nothing to add
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 FUNCTION OF SMALL RNA IN PSEUDOMONAS AERUGINOSA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all the comments. Overall the review now looks good. One minor comment is a figure showing how sRNAs in general function is missing.


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 FUNCTION OF SMALL RNA IN PSEUDOMONAS AERUGINOSA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Meets the standard.
Experimental design: Meets the standard.
Validity of the findings: Meets the standard.
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: GROWTH PARAMETERS AND RESPONSES OF GREEN ALGAE ACROSS A GRADIENT OF PHOTOTROPHIC, MIXOTROPHIC AND HETEROTROPHIC CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is very well written and the statistic is properly done. There is only one comment that needs to be taken into account. I have noticed in several places that the Authors use the wrong way of unit presentation such as g/L. This way is incorrect, especially since in other cases another way is applied.
Experimental design: It is correct.
Validity of the findings: In my opinion, this paper is a very important one as algal cultivation has practical application in CO2 capture and production of many important products. There are hundreds of possibilities to use the biomass. Concerning mixotrophy, it is not a totally new idea but the paper contains critical discussion and introduction that will be useful for many researchers and companies. The information is also important concerning the necessity to develop consortia of algae with other microbes to avoid problems with pathogens.
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: GROWTH PARAMETERS AND RESPONSES OF GREEN ALGAE ACROSS A GRADIENT OF PHOTOTROPHIC, MIXOTROPHIC AND HETEROTROPHIC CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Briefly explain what is the difference between PA,HT and MT cultivation system and pros and cons.
2. In the introductory part citation of latest references is required preferably last 5years including 2022.
3. some places authors used abbreviation, it is suggested to expand those abbreviations first and continue the abbreviation later in the text where ever required.
Experimental design: Line no: 159-162
Regarding acetate concentrations, authors mentioned they have used acetate ranges from 1-10g/l in the medium. is the same concentrations used for all trophic conditions(PA, HT, MT)? or they have used only 2g/l for HT condition?
it will give confusions to the readers, better clarification is required.
Table 1: update it with latest reports including 2022.
Validity of the findings: 1.Figure captions and legend, reduce the word count, too lengthy captions are not looking nice for a figure. modification is required for all figure captions.
2. What is the reason behind variations in cell size during PA,HT & MT conditions with acetate, present discussion is not convinced. proper explanation is required
how chl is reducing or bleaching in HT, or chl molecule is changing to other molecule during heterotrophic system or  other pigments are getting activated in HT, culture conditions. Include those  point in line no 445-485.
Additional comments: Text is not fluid, check the grammatical and typological errors and English language need to be improved further.


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: GROWTH PARAMETERS AND RESPONSES OF GREEN ALGAE ACROSS A GRADIENT OF PHOTOTROPHIC, MIXOTROPHIC AND HETEROTROPHIC CONDITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written, according with the jounral standards. It is well organized, structure is logic and coherent. Introduction fits to the main subject and justifies the problem undertaken. The cited literature is relevant to the scope of problem.
Graphical material clearly illustrates the experiment outcomes. I have found all of them necessary as they include results relevant to the hypotheses. Although the figures are readible, the overall quality (lines widths and fonts) needs improvement.
Experimental design: The manuscript is an interesting work on comparisons of cell culture parameters, and responses of cells over a gradient of mixotrophic, autotrophic and heterotrophic conditions for two species of green algae commonly used in biotechnology and for biofuels production. This is original primary research which falls in the scope of PeerJ journal. Reserch questions and problems have been clearly identified and justified.
Methods are described sufficiently  to be replicated. Cultures and growth parameters of both algae species were examined  according to the standards. Statistical methods (analysis of variance, mainly) seem  enough to justify achieved differences. Resuls are properly documented.
Validity of the findings: The authors showed different parameters for estimating growth and biomass production under photoautotrophic, mixotrophic and heterotrophic growth conditions  of  Chlorella vulgaris and Chlamydomonas reinhardtii, which varied  with a mode of energy supply. The results were a response to the formulated hypotheses. The results are statistically correct. The conclusions are relevant, supported by the results
Additional comments: It is a nice piece of work. Well organized and written.


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: GROWTH PARAMETERS AND RESPONSES OF GREEN ALGAE ACROSS A GRADIENT OF PHOTOTROPHIC, MIXOTROPHIC AND HETEROTROPHIC CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors are nicely revised the manuscript, this manuscript can be accepted for publications
Experimental design: The authors are revised the manuscript well and modifications are done as per the comments
Validity of the findings: manuscript can be accepted for publications
Additional comments: manuscript can be accepted for publication


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

Input:
Title: CROSS-SECTIONAL ASSOCIATIONS BETWEEN EFFORT-REWARD IMBALANCE AT WORK AND ORAL DISEASES IN JAPAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Minor problems exist in this section.
Experimental design: Significant improvement required in this section.
Validity of the findings: I think, the findings are valid.
Additional comments: Thanks for opportunity to review manuscript entitled ‘‘Cross-sectional associations between effort-reward imbalance at work and oral diseases in Japan’’ for Peerj Journal. The author/authors examined the association between effort-reward imbalance at work and oral diseases among Japon workers. The strength of the manuscript includes examining variables of interest in a country where such studies are scarce.  Overall,  although the article is generally well written and deserves to be published in this journal some necessary and minor revisions must be made before the publication of the article. Because my main philosophy  of reviewing a manuscript as reviewer and sometimes an editor to improve the manuscript and not punishing the authors, I provided very specific and detailed peer review of the manuscript to increase its quality and citation potential. I hope authors of the manuscript may benefit from my review. Necessary revisions reported section by section with the page and line number and when possible with suggestions.
Necessary Revisions
Abstract
1. Abstract, Line 15-16: Author need to revise following sentence ‘ ‘ However, evidence for association between work stress and oral diseases is lacking.’’ One revision may be ‘ ‘However, evidence for association between work stress and oral diseases is scarce.’’
2. Abstract, Line 19-23: Author must add used statistical analyses to Method section.
3. Abstract, Line 31: Discussion:   must revised as Conclusion:
4. Abstract, General: Keywords are completely missing and must be added.
Introduction
5. Introduction, Line 36:  There is a typo in the sentence. high e฀orts must be high efforts.
6. Introduction, Line 37-39: Following sentence need revision as when model predict these outcomes is unclear. ‘ ‘The model predicts increased risks for depression (Rugulies, Aust & Madsen, 2017), hypertension (Gilbert-Ouimet et al., 2014), coronary heart disease (Li et al., 2015), and type 2 diabetes (Kumari, Head & Marmot, 2004)’’ One revision may be that ‘ ‘The model predicts increased risks for depression (Rugulies, Aust & Madsen, 2017), hypertension (Gilbert-Ouimet et al., 2014), coronary heart disease (Li et al., 2015), and type 2 diabetes (Kumari, Head & Marmot, 2004) when effort-reward imbalance high.’’
7. The citation/citations needed for following sentence ‘ ‘Notably, most previous studies did not use the widely accepted measures for work stress assessment and did not include potential confounders, such as socioeconomic status and work-related variables’’
8. Introduction, General: Introduction section is very weak for a scientific article. Authors must firstly add information about previous studies, weakness of previous studies and what their study add to existing literature. Moreover, Authors need to answer Why it is important to examine effort-reward imbalance at work and oral diseases among Japon workers?  With convincing information.
9. Introduction, General: Author must move following paragraph to end of Introduction and synthesize with psychological stress paragraph. It illogical to first give information about independent then dependent variable.  ‘ ‘ Work stress has become more prevalent at the workplaces of industrialised countries and can negatively impact workers' health (Siegrist, 2016). The effort–reward imbalance model is a widely accepted model for assessment of work stress (Siegrist, 1996; Montano, Li & Siegrist,2016). It focuses on the failure in reciprocity in terms of high e฀orts spent and low rewards received. The model predicts increased risks for depression (Rugulies, Aust & Madsen, 2017), hypertension (Gilbert-Ouimet et al., 2014), coronary heart disease (Li et al., 2015), and type 2 diabetes (Kumari, Head & Marmot, 2004).
Method
10. Method, Line 71-91: Author must add mean and standard deviation of samples to Participants section. Moreover, author must add used sampling procedure (convenience sampling).
11. Method, Line 96: ranging between 3 and 13 must be ranging between 3 and 12
12. Method, Line 99: There is a typo in the sentence. high e฀orts must be high efforts.
13. Method, Line 155: In two-tailed significance, p must be small.
14. Method, General: Method section must rearrange using subtitles, Participants, Measures, Procedure, and Statistical Analyses and authors move all related information to related sections.
15.  Method, General: Author must give more information about established procedure for effort reward-imbalance. As I am unfamiliar this, it is difficult to understand.
Results
16. Results, General: All small n must be italic in Table 1 representing subsample or sample sizes.
17. Results, General: Authors must add Notes section under the Table 2 and report significance levels of significant findings.
18. Results, General: Authors must add Notes section under the Table 3 and report significance levels of significant findings.
19. Results, General: As a multivariate analyst, I think authors must add r square values for adjusted linear regression model and interpret effect size of this measures. Similarly, Int the table 1 mean difference statement is completely wrong for linear and negative binominal regression analyses. Expected mean difference may be correct. However, for negative binominal regression analyses, this have to be expected log odds of what ever measured. I think authors want to mean unstandardized regression coefficients for linear regression, and incidence rate ration for negative binominal regression analyses.
19. Results, General: Authors must add following information to tables. For linear regression analyses, unstandardized regression coefficients and standard errors with p values with three decimal, Moreover, r square value for final model. For negative binominal regression analyses and Poisson regression, IRR with its standard error and p value three decimal as well as pseuodo r square values.
20. Results, General: the reason for using negative binominal regression analyses and preference for posison regression unclear. Authors must give convincing information about using them. For example, overdispersion also may be modeled  zero-inflated negative binomial regression similarly, zero-inflated poisson regression also may be used to accommodate excessive zeros in teeth numbers.
Discussion
21. Discussion, General: Following information must remove before conclusion section ‘ ‘This study has two strengths. First, work stress was assessed using a widely accepted measure. Previous studies on work stress and oral diseases have often used nonspecific
questionnaires (Sato, Saijo & Yoshioka, 2021). This study captured work stress more accurately than previous studies. Second, this study included potential confounders, such as socioeconomic status and work-related variables. Our review revealed only two studies among the 11 studies included in the systematic review considered the potential confounders sufficiently (Sato, Saijo& Yoshioka, 2021). Our study is superior to previous studies in terms of these two points. However, our study had three limitations. First, periodontal status was assessed using a self- reported questionnaire which potentially has self-reported bias (Yamamoto et al., 2009; Beltrán-Aguilar et al., 2012). This questionnaire was not validated among young or female workers (Yamamoto et al., 2009). Furthermore, the cut-off point of this questionnaire was defined based on 7 mm or more of the clinical attachment loss, which differs from the 2018 periodontitis case definition by the European Federation of Periodontology and American Association of
Periodontology with Centers for Disease Control (Botelho et al., 2020). This limitation can
produce a bias. In addition, the participants with work stress may have had exaggerated
responses to questions on periodontitis screening. When interpreting the periodontal status
results, these limitations should be considered. In the second source population survey, DMFT was estimated using intraoral photographs with mobile phones. Molars had a relatively higher percentage of undiagnosable status than incisors and premolars, and some DT and FT were
missed. Therefore, DMFT could have been underestimated in the second source population. This unvalidated method can bring about a bias. However, the results in the second source population were consistent with those in the first source population, for which DMFT was assessed on the clinical examination. Second, in the two-source population, many participants had a high socioeconomic status. More than 70% of participants had university degrees or higher. The social gradients of oral diseases have been reported (Borrell & Crawford, 2012; Schwendicke etal., 2015). Additionally, blue-collar workers were also small in number. Participants in the two-source population had better oral conditions than those in the national survey (Ministry of Health, Labour and Welfare, 2016). The results of people with low socioeconomic status and blue-collar workers might have been underestimated in this study. Finally, this study was cross-sectional; therefore, a temporal association was not established. Further cohort studies are required.
22. Discussion, General: Clinical and practical implications of study findings are completely missing in the manuscript and must be added.


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: CROSS-SECTIONAL ASSOCIATIONS BETWEEN EFFORT-REWARD IMBALANCE AT WORK AND ORAL DISEASES IN JAPAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a carefully conducted study on a topic of scientific interest as few investigations are available, internationally, on this topic. Authors have presented the current state of art in convincing ways, adding their own systematic review (published 2021). The Introduction is well written, and the quality of study design, data analysis and interpretation meets the standard for publication in an international journal. Authors also critically address the limitations of their study, thus contributing to a process of knowledge acquisition. The main finding of a significant association of work stress, as measured by a theoretically grounded concept, with poor periodontal status is of practical relevance.
Experimental design: The study design deserves some comments. On the positive side, the procedure of data collection is clearly described. The statistical analyses reflect the current state of arts, and authors document their expertise e.g. by adopting most advanced statistical programs. The information presented in Tables is accurate, and a special strength is given by the inclusion of a substantial number of covariates into multivariable analysis.
On the negative side, one wonders what the effect of sample loss (e.g. from 527 to 184 participants in study 1) on the study results might be. If I understand correctly, work stress data were available from the larger sample (e.g. 527 in study 1) as well. In this case, authors could have checked whether the final sample differs in terms of the independent variable from the original sample. At least, in the Discussion section, authors should address the problem of sample loss.
No further comment.
Validity of the findings: Keeping the above mentioned critical comment (potential reduction of validity due to sample loss with uncontrolled bias) in mind, the validity of the reported findings is credible. Authors pointed to the methodological limitations of their study (especially cross-sectional design; underestimation of DMFT). As mentioned, the innovative message of this study should be considered a particular strength.
Additional comments: No additional comment


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: CROSS-SECTIONAL ASSOCIATIONS BETWEEN EFFORT-REWARD IMBALANCE AT WORK AND ORAL DISEASES IN JAPAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout
Experimental design: Rigorous investigation performed to a high technical & ethical standard.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound,
Additional comments: Thanks for opportunity review revised manuscript entitled ‘‘Cross-sectional associations between effort-reward imbalance at work and oral diseases in Japan’’. I would like the thanks to authors. They make a good job for improving quality of their manuscript. Authors revised the manuscript as I requested with a good will. In this form, Introduction reflects very well the previous studies and study aim, Method section and Result section is correct, and Discussion section adequately synthesis to previous study findings and current study results. Overall, I have no further comment regarding to manuscript. I congratulate to authors and wish them success on their future endeavors.


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

Input:
Title: EFFECTS OF GROWING COPTIS CHINENSIS FRANCH IN THE NATURAL UNDERSTORY VS. UNDER A MANMADE SCAFFOLD ON ITS GROWTH, ALKALOID CONTENTS, AND RHIZOSPHERE SOIL MICROENVIRONMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear authors, the manuscript Effects of growing Coptis chinensis Franch in natural understory vs. manmade scaffolding on its growth, alkaloid contents, and rhizosphere soil  microenvironment
presents a good contribution in the field, however, it is important that you consider the following:

In general, the spelling and writing of the manuscript must be checked.

Review the format of table 3.

Add some sentences of rationale to the beginning of your abstract.

The changes observed in the alkaloid profile are not discussed.
Experimental design: No comment
Validity of the findings: What do you conclude about the changes that were observed in the alkaloid profile?
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF GROWING COPTIS CHINENSIS FRANCH IN THE NATURAL UNDERSTORY VS. UNDER A MANMADE SCAFFOLD ON ITS GROWTH, ALKALOID CONTENTS, AND RHIZOSPHERE SOIL MICROENVIRONMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: a.	Some expressions are not concise and need to be refined. For example, line64-72 could be shortened.
b.	The authors need to include more background information and reference in “Introduction”. For example, line55-80 should at least discuss previous studies on alkaloids from Coptis chinensis Franch.
c.	Table/figure should be referred in your results part. Otherwise, it is hard to follow.
d.	Table 4-7: it is better to show RDA results with figures. Tables could be moved to supplementary data.
e.	Quality of Figure5-8 should be improved.
f.	Some figures could be combined. For example, Figure9 and Figure10 could be combined into one Figure X. Figure X-A refers to Figure9 and Figure X-B refers to Figure10.
Experimental design: a.	Reference is needed for some methods: line109-“cutting-ring method”; line112-“five-point method”; line141-“potassium dichromate oxidation-volumetric method”; line142- “Kjeldahl method”, etc.
b.	Line164-166: 515F/806R can only target 16S rRNA V4 region. What primers did you use to target all other regions? Primer sequence information should be included.
c.	ITS database reference should be mentioned.
d.	Line412: RDA analysis should be described in Materials and Methods part.
Validity of the findings: a.	Since fertilization was discussed as a key factor, data on fertilization should be provided to support your conclusion.
b.	Line530: species or class? Line532: species or class?
c.	Line589: soil pH could also be influenced by the microbial community.
d. Alkaloid contents were mentioned in the Title and Abstract part but barely mentioned in Discussion and Conclusion.
Additional comments: A lot of data was provided and analyzed in this study. I recommend the authors to refine the manuscript to make it concise and consistent.


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

Input:
Title: EFFECTS OF GROWING COPTIS CHINENSIS FRANCH IN THE NATURAL UNDERSTORY VS. UNDER A MANMADE SCAFFOLD ON ITS GROWTH, ALKALOID CONTENTS, AND RHIZOSPHERE SOIL MICROENVIRONMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. Spelling and grammar should be carefully checked. e.g. spelling of "Figure"; line452, extra period; line533 understory
2. Abbreviations should be spelled out when it is first used in the article. e.g. line65, TCM.
3. I would suggest the author label the significance level in Table2 like in Table3.
4. Two supplemental tables have the same title.
Experimental design: 1. Line176: "ITS1-5F region" should be "ITS1 region". 5F is the primer.
2. Canonical correspondence analysis should be described in methods part.
3. Fertilizer patterns should be moved from discussion to methods part. Also, fertilization rate data is missing.
Validity of the findings: In the discussion part, there are some redundant sentence repeating the results part. I would suggestion the author check carefully and remove the redundant part. e.g. line534-539, the results showed in Table2 was already described.
Additional comments: No additional comments


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

Input:
Title: EVALUATING THE READABILITY, QUALITY AND RELIABILITY OF ONLINE PATIENT EDUCATION MATERIALS ON POST-COVID PAIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: There is some unconventional language used, and although you can understand the point that the authors are trying to make, it is made in a slightly unorthodox way. I made some specific comments below.

Some specific comments.
Introduction
Line 57; late 2019 was when it was first detected, the medical and humanitarian crisis was later. It is emotive language, it would be really great to accompany such language with some numbers, for example, percentage of the population affected after 3 months or the number of deaths in the first month.

Line 71- It is evident that the correct treatment algorithm would help individuals with recovery given that pain symptoms adversely affect the quality of life during the post-COVID period.
This sentence seems a bit out of context, could you elaborate or clarify your point about pain symptoms. These symptoms are not mentioned earlier in the paragraph. I think this paragraph should focus more on post covid pain as that is a main aim of the manuscript that seems to get a bit lost.

Line 74; Patients can rapidly access the desired healthcare content using Internet-based patient educational materials (PEM), which have recently been used as an important tool for acquiring further information.
Could you add some references to this sentence?

Line 80 typo
Levelx

Line 80; In case the readability of online information posted on a website is above the said grade, it may be considered difficult to read and understand for an average reader. Therefore, it is important that
Suggest change to, “if the readability

Line 88 A study by Worrall et al. reported that the readability level of online information about COVID-19 90 was poor and difficult to read (Worrall et al., 2020).
Could you be more specific here? What do you mean by poor and difficult to read? Could you provide numbers?

Line 91; It is well established that patients furnished with information about the etiology, 92 pathophysiology, treatment, and prevention methods would more likely participate in and 93 comply with the disease prevention or treatment procedures.
Is it? Could you provide a reference here.

Line 93; It is evident that providing 94 individuals with reliable, high-quality, and readable online information about post-COVID-19 95 pain would help with the management of a condition that affects many people.
Is it? Again this needs a reference

Methods
Line 116; Could you clarify what you mean by a collective assessment?

Line 118;  What sector?

Line 139; Could you provide some more information on how it was decided to categorise the websites?

Line 191; Could you restate the aforementioned exclusions

Line 201; Content analyses

Line 208; For statistical analysis, the Mann–Whitney U or Kruskal 209 Wallis tests were used to compare groups with continuous values such as readability indices and 210 sixth class level. For comparison of frequency variables, the Chi-square or Fisher exact tests 211 were used. A p-value lower than 0.05 was accepted as a statistically significant difference.
I don’t understand the value of these statistical tests. It seems that they are to provide more weight/ impact or meaning to the study, but it seems a bit like manufacturing importance to me. I wonder if they are necessary, would it be just as informative to report numbers and percentages. Aren’t you just using different measures of readability, I don’t understand the value of knowing if there was a difference in the readability scores, isn’t it more important what the readability score was. Is there a gold standard readability score, perhaps it is best just to choose that and report the average readability score for the websites included?

Results
The authors do not seem to report on the content analysis outlined in the methods

I would expect the results to follow the same format as they are outlined in the methods.

Discussion
Line 285 this paragraph would be more useful in the introduction, as the information and rationale for investigating post-Covid pain information is not well-established in the introduction as it is.
Experimental design: The idea behind the design is good but I think the authors attempt to cover too much and in doing so make the analysis unnecessarily complicated. For example, they measure readability using 7 independent readability indices. They also add some statistical analysis of questionable value. I wonder if it would be just as informative to report numbers and percentages. For example, the authors report whether or not there is a statistical difference in the readability scores from the different indices. I don’t understand the value of knowing if there was a difference in the readability scores, that seems to be assessing the validity of the tool to assess readability rather than readability itself, isn’t it more important what the readability score was. Is there a gold standard readability score, perhaps it is best just to choose that and report the average readability score for the websites included?
There is insufficient detail on some aspects of how the analyses are conducted, for example, the authors report conducting a content analysis but with minimal information on how that was conducted.
Validity of the findings: There seems to be some value in the findings, but it is buried in a over-complicated analyses of association and statistical significance. I think the paper could be quite interesting if the authors restricted their analysis to simple measures of numbers and percentages, and illustrated their findings with box plots.
Additional comments: There is no doubt that the authors have worked hard on this paper but I think it would require significant revision before being accepted.


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

Input:
Title: EVALUATING THE READABILITY, QUALITY AND RELIABILITY OF ONLINE PATIENT EDUCATION MATERIALS ON POST-COVID PAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Thanks for inviting to review this master piece
This manuscript is well presented but lacks scientific writing at some points.
Suggesting proof reading, add literature references, improve rationale of study by stating research gap
Write COVID-19 instead of COVID throughout the manuscript
Experimental design: This study is well-designed but, there are a few things that should be considered in the method section.
How sample size for websites calculated??? kindly mention in manuscript
add more information about the study type in the method section.
Add heading on ethical considerations
heading on websites selection criteria
in Content Analyse, The websites were investigated and assessed , WHICH TYPE OF WEBSITES?? MENTION NAME OF WEBSITES AND ADD REFERENCES
Please write dependent and independent variables in the analysis section
Validity of the findings: The results of this study are well presented but author need to make headings of results like readability, quality, reliability, and sub-headings in an adequate manner.
If possible make bar chart of Figure 1
The discussion section is well explained

Add limitations of this study under separate heading.
Add strengths of this study under separate heading.
Additional comments: None


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

Input:
Title: EVALUATING THE READABILITY, QUALITY AND RELIABILITY OF ONLINE PATIENT EDUCATION MATERIALS ON POST-COVID PAIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: Thanks for the invitation to review this study. This is an interesting piece of work where the authors tried to estimate the validity and reliability of PEM which was used by the general population to address the post-covid pain. There are a few concerns that should be addressed before making any decision on this submission.
1. Please provide the rationale why post-covid-pain was selected as a topic of search or discussion, as the population was also more interested in seeking information on the treatment (either allopathic or herbal) of COVID-19.
Experimental design: Authors have used various criteria or tools to assess the reliability, popularity, readability, and quality of the online websites used to seek information on post-covid pain. It would be better and reader-friendly if the authors could provide a table with the name of these tools along with the parameters these tools are evaluating.
Why did the authors just limit the search to 100 websites? Google also provides results according to the region where the search is being performed. How the authors will exclude this bias to make the findings more generalizable.
Please provide the study flow diagram for a more detailed and comprehensive understanding of the study methodology.
Validity of the findings: The authors have presented the mean values with commas. The mean values and standard deviations are usually presented with a full stop for decimal values. Please consider the amendments.
I did not find that authors have performed the search and evaluation of the websites in duplicates or triplicates (two or more authors perform the process independently). If the search and evaluation are not performed in duplicates then how the researcher's bias can be omitted from the findings. There is a need for clarification.
Can authors provide the rank of reliability and quality of five groups presented in the current study in order to see which sources are more reliable and were of quality for healthcare-related information? This data is presented but not in a ranked way.
Additional comments: The limitation section should also discuss the involvement of bias as described in the above comments. Authors need to provide suggestions to authorities dealing with health and drug informatics.


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

Input:
Title: EVALUATING THE READABILITY, QUALITY AND RELIABILITY OF ONLINE PATIENT EDUCATION MATERIALS ON POST-COVID PAIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comments, well revised, accept in current form
Experimental design: No Comments, well revised, accept in current form
Validity of the findings: No Comments, well revised, accept in current form
Additional comments: No additional comments


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

Input:
Title: EVALUATING THE READABILITY, QUALITY AND RELIABILITY OF ONLINE PATIENT EDUCATION MATERIALS ON POST-COVID PAIN
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed my comments. I have no more concerns.
Experimental design: The authors have addressed my comments. I have no more concerns.
Validity of the findings: The authors have addressed my comments. I have no more concerns.
Additional comments: The authors have addressed my comments. I have no more concerns.


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: BALEEN WHALE INHALATION VARIABILITY REVEALED USING ANIMAL-BORNE VIDEO TAGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review for ms 73369

I feel this is a useful and timely study.  With the exceptions listed below, the basic reporting is good, as are the experimental design and presentation of results.  I must state that judging the validity of the statistical methods used should be done by one with a stronger statistical background than mine.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Specific comments:

Lines 97ff, 165ff, 181:  With the large number of variables discussed in this paper, some abbreviated, some not, this paper would benefit from the addition of a concise table of variables and their consistently used abbreviations (e.g. Fahlman et al, 2015; Sumich, 2021).

Line 142ff: Establishment of the beginning of each inspiration is critical to this study.  Although I find no problem with the approach stated here, are there associated audio tracks that might provide confirming acoustic ventilatory flow signals for the transition from expiration to inspiration.

Line 150: This seems an appropriate spot to include the terms inter-breath-interval (IBI) and extended dive times (EDT) and add a table of summary statistics of IBI and EDT values for each whale (See Butler and Jones, 1997 for background).
The selection of <5s duration and <10m depth to define surface sequences seems arbitrary.  Are there good reasons for choosing these values, and were alternative approaches considered, for example examining the bimodal distributions of log transformed values of all IBI and EDT dive durations of each whale and using the intermediate minimum (likely around 20-30s) to define the boundary between IBIs and EDTs.

Line 169: Does the video frame rate (30Hz) define the resolution of the time scale for determining expiration duration?

Line 181:  This is the first occurrence of IA as the abbreviation for the variable “integrated area’ although the variable name is used without abbreviating in the previous paragraph.  See first comment above.

Line 224: …duration.,

Lines 256, 322, 327-328:  Sumich, 2001 and Sumich and May, 2009 only measured expiration durations on gray whales, not inspirations of odontocetes.

Lines 273-287: Why is Fig. 5 on line 273 placed prior to Fig. 4 on line 287.

Line 294:  …confirmed that inhalation…

Line 364: …which could indicate suggests that…

Lines 414ff: excellent explanation

Line 425: Sumich et al, 2001
Line 444-445: delete entire first sentence of Conclusion, as that statement does not apply to all previous studies.
Line 455ff: Favilla and Costa, 2020 have shown that ADL of blue and fin whales is about 5x their typical foraging EDT values.  I suggest some discussion is needed here to explain why inspiration duration (as a proxy for O2 loading) should correlate with O2 requirements of anticipated upcoming dives when a whale’s upcoming typical dive durations only uses 1/5th of its actual aerobic dive capacity.
Fig. 1: inset graph needs units, otherwise very good.

Fig. 4.  This fig. is visually confusing to me. It seems to me to indicate that total inhale duration begins to decrease for upcoming dive durations longer than about 300s.  I don’t see that from the distribution of data points.  Why might that be and do you think it is physiologically meaningful?