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: ONLINE SEARCH TRENDS AND WORD-RELATED EMOTIONAL RESPONSE DURING COVID-19 LOCKDOWN IN ITALY: A CROSS-SECTIONAL ONLINE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overal the writing is relevant, concise and clear. The literature and introduction give a good description of the actual context of COVID-19 in Italy and insighful discussion of how this can be approached using classical approaches in emotion theory.  Most of my suggestions are fairly minor.  A list of presented below.

The results in the abstract (line 32-35) are compressed, making it difficult to parse without re-reading. “Lower valence and dominance judgments were given only to COVID-19-related words by individuals with less negative feelings and COVID-19 fear, but also to COVID-19-unrelated words by individuals with more negative feelings and COVID-19 fear..”

Instead, the following is clearer (verify that this interpretation is correct!)
“All individuals judged COVID-19 related words to be less positive and dominant.  However, individuals with more negative feelings and COVID-19 fear also judged COVID-19 unrelated words to be less positive and dominant.”

“Moreover, arousal judgments for all words decreased and increased, respectively, for individuals with less and more negative feelings and COVID-19 fear.”

This could become “Arousal ratings increased for all words among individuals with more negative feelings and COVID-19 fear, but decrease among individuals with less negative feelings and COVID-19 fear.

Line 59. Start new paragraph for “The dimension theory of emotions.

Line 85. “To soften the influence of the COVID-19 emergency on individuals’ mental health, it is thus compelling the need to evaluate their emotional state in response to COVID-19 pandemic.” Please edit to make this less wordy.

Line 86. “Indeed, it has been reported that the COVID-19 affected the content explored in internet by people (Effenberger et al., 2020), as internet is an essential channel where people convey their feelings and emotions and seek health-related information (Kalichman et al., 2003; Reeves, 2001)”

Suggest to change as follows:
“Indeed, it has been reported that the COVID-19 affected the content that people explored online (Effenberger et al., 2020), and online media and platforms offer an essential channels where people convey their feelings and emotions and seek health-related information (Kalichman et al., 2003; Reeves, 2001)”

Line 95. Can you more explicitly draw out the relation between online searches and emotional reaction you plan to study? It's clear that online searches and emotional reaction will change due to COVID, but it's not clear how both research questions inform each other.

Line 104. “Findings will inform about the real-time estimation of the COVID-19 pandemic impact on
participants’ emotional response”. This is perhaps a bit too strong. Change to “Findings might inform about the real-time estimation...”

Line 121. Describe the nature of the RSV data in more detail. Was there a datapoint for each day?

Line 126: “RSV data for one word were not available.” Please indicate which word.

Line 155 -1 158. “This number of stimul was the largest that can be reliably rated during a single online session” I’m not sure what this means. Can you please explain? Many online studies involve a much larger set of stimuli.

Line 158 – 165. Is it possible to be more concrete? It seems that in this case the correlation is used a a measure of similarity and framing it in this manner might make it more clear.

Line 174. “…and for exemplar stimuli” and four exemplar stimuli

Line 210 Consider adding a few sentences to signpost what the structure of the data analysis is to improve readability.

Line 244. Please add a table with simple descriptives (mean, sd,n) for the data collected in the study (scales, word ratings) supplemented with the descriptives for the same set of words from the Italian ANEW.

Line 186: “… basilar description” → “basic description”

Line 198. “… from May 4 to May 17” Perhaps add “2020” since there have been additional lockdowns since.

Line 280: Consider adding a table that lists the significant words with largest differences for valence, arousal and dominance.

Line 350: “Figure 2G”. Should this be Figure 2?

Line 362. Affected means “were related” Change accordingly.
Experimental design: Line 119. Can you explain why the first four months of 2020 were chosen? Presumably no cases were found until February 21,  so the preceeding period might be included as a baseline, but this wasn't clear at this stage.
Validity of the findings: Overal, I found the statistical analysis convincing, and commend the authors for taking a multiverse approach. I also found the power analysis useful.

The abstract mentions that the study involved healthy participants. How do you know whether the participants are healthy? Was this part of the questionnaire?

Line 155. Given the three measures, did they all provide te same outcomes?

Line 148. Can you indicate to what degree non-normality affected the data and whether a transformation reduced this issue (most likely skew)?

Line 237. In terms of analysis, it’s not clear what variables should be regarded as dependent and independent. In particular, treating PANAS as a DV would allow you a direct relation with fear, as a combination of (negative) valence and (high) arousal by adding those as interacting terms.
Additional comments: This work provides a nice demonstration of how emotional connotations of words is modulated by the emotional state of a person. It might be worth connection the findings reported here with previous literature that has manipulated the emotional state of participants who rate valence/arousal/dominance of words. Still, demonstrating this effect "in the wild" is something that is timely, especially in the context of COVID-19. Nice work!


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

Input:
Title: ONLINE SEARCH TRENDS AND WORD-RELATED EMOTIONAL RESPONSE DURING COVID-19 LOCKDOWN IN ITALY: A CROSS-SECTIONAL ONLINE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all my points and considerably improved their final work. I recommend publication of the revised manuscript.
Experimental design: The authors addressed all my points and considerably improved their final work. I recommend publication of the revised manuscript.
Validity of the findings: The authors addressed all my points and considerably improved their final work. I recommend publication of the revised manuscript.
Additional comments: The authors addressed all my points and considerably improved their final work.

I recommend publication of the revised manuscript.


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

Input:
Title: EFFECT OF ELEVATION, SEASON AND ACCELERATED SNOWMELT ON BIOGEOCHEMICAL PROCESSES DURING ISOLATED CONIFER NEEDLE LITTER DECOMPOSITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript reports on the influence of elevation, soil parameters (moisture, temperature), snowmelt timing, precipitations on coniferous leaf litter decomposition and soil microbial communities. As the authors note, in general, relevant (modelling) studies address soil C losses ignoring local environmental heterogeneity. Thus, this work has the potential to contribute to filling this knowledge gap.
The results of no elevation and snow-melting time effects on leaf litter decomposition are novel and surprising.
Unfortunately,
(1) the number of replicates was insufficient to carry out a consistent statistical assessment (in some measurements);
(2) FTIR analysis is not specific enough. For example, there are numerous peaks assigned to polysaccharides, and several of them need to be integrated to obtain a reliable value. How relevant is the peak at 1600 cm for amides when there are the NH2 stretches/scissors at 3370-3170/1620-1650, C=O at 1680-1630, C-N at 1430-1390 cm-1and several peaks for amide IV and V at 600-900? Commonly, a peak ratio is more informative (see an IR spectral decomposition index Margenot et al. 2015 SSSAJ).
(3) the eukaryotic (decomposing fungal) communities are not shown;
(4) a link between soil microbial communities and decomposition is missing. We have a microbial ecological response and an indirect interpretation through environmental effects. Still, no gas flux or other measurements were included (I’ve seen that you concomitantly collected samples for DNA and gas flux measurements on a few occasions).
The manuscript is clear, professional English language used throughout.
Introduction
-	The context could be better explained. How does snow melting affect the local environment? Which is the meaning of “high moisture”. Commonly, at high moisture content, microbial activity decreases because of low oxygen supply. I am not aware of any dramatic soil moisture effects on bacterial diversity or community composition, but their activity.
-	Provide the reasons for which litter type influence microbial decomposition processes.
-	At this stage, we do not know whether microbial communities are resilient or not to low moisture conditions, or do we know it from literature?
-	Line 73-75: This phrase is exciting. I read it a few times to understand it. Is it really necessary?
Experimental design: The investigation is novel and rigorously conducted. The authors acknowledged the limitation of the design that resulted in a low number of replicates.
-	Line 117: how large was the mesh size? Did soil fauna have access to the leaf litter?
-	L 171: which R function did you use?
I am not aware of the raw data supply.

Figures
-	The captions for supplementary figures are missing.
-	Fig. 2: please indicate that the arrows represent the sampling events.
-	Fig. 3: C:N ratio is essential, but discussing litter decomposition, changes in C and N are also relevant (maybe you can show it in the supplement, and discuss the N enrichment together with the observations in the FTIR amide peaks – just a suggestion).
Validity of the findings: No comment.
Additional comments: see above


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

Input:
Title: EFFECT OF ELEVATION, SEASON AND ACCELERATED SNOWMELT ON BIOGEOCHEMICAL PROCESSES DURING ISOLATED CONIFER NEEDLE LITTER DECOMPOSITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written in clear English language. Introduction part is well elaborated with relevant literature referenced and with clearly stated aims and hypothesis. Figures in main text are relevant and well described. Besides figures in main text authors provided many figures and tables in supplementary information which I appreciate, however the description to supplementary material is missing, which makes it difficult to evaluate presented results and data. Access to raw data is provided.
Experimental design: The manuscript deals with important and interesting topic that fits into the scope of journal. I appreciate the diversity and amount of data the authors collected in the sites that are difficult to access. However, material and method section contains some unclear steps and some of the methods are not described with sufficient detail- see below the specific comments.

line 89-90 Why was the litter collected in different location than the study site?

line 99 unclear what 25x18cm ring mean? What was the diameter of the ring?

line 102-103 unclear the calculation of 48 rings – you write you had 3 elevations, 4 replicates, 3 treatments (spruce, lodgepole, control) which gives together 36

line 109-110 Were the plants removed including the roots? If so this can affect soil microbial communities and respiration rates as rhizosphere microbiomes represent hotspots of microbial activity in the soil. If only above ground parts of plants were removed this can still affect the soil microbes as some of them may use photosythates as a source of nutrient. I find the removal of plants as very artificial treatment. Can you explain the reason for removing the plants?

line 111-113 can you be more specific about the dates of individual measurements and soil extractions?

line 118-119 what was the size of the mesh?

line 212-215 using such approach for soil sampling, how do you know that the collected volume of soil from each plot was the same, thus the results of microbial communities are comparable? Was the soil mixed and homogenized?

line 215 how many DNA extractions were done from each sample?

line 219 how many PCR reactions were done from each DNA sample?
Validity of the findings: The biggest problem I have with the manuscript is how the data was analyzed and presented. In several cases authors claim results that are not supported by statistical analysis or the used statistical analysis is not described- find below the specific examples. In general, I feel the manuscript lack the connection of collected datasets and the use of relevant statistical analysis that would allow to draw the conclusions.

line 280 statement: No clear differences.... Does that mean there were no significant differences? How was the significance for differences in needle decomposition calculated? I am unable to find it in material and methods.

line 283-285 Based on what analysis you concluded there were no pronounced differences in C/N needle ratios? In methods you write that possible significance between samples is indicated by non-overlapping error bars of box plots and in Figure 3 the error bars of spruce middle x lower and middle x upper do not overlap. Your statement of no differences is confusing, please explain.

line 300-301 differences were clear across elevation... based on what analysis you concluded this?

line 306-309 I presume that the significance is claimed based on box plot figures.... if so I feel such conclusion is a bit exaggerated and you should rather write possible significance.

line 325 and 332 unclear how was the significance assessed

line 339-342 how was this significance / non significance analyzed? I miss this information in material and methods.

line 344-346 In the text you mention the results of principal coordinate analyses but you refer to figure and table showing something else.

line 346-347 again not clear how was the significance tested

line 350-351 if you claim decrease or increase of microbial taxa you need to support it by statistical analysis, from the data I see in Figure S9 which represent I presume means of relative abundances you can not make such conclusion.

line 357 how was the significance calculated?
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: EFFECT OF ELEVATION, SEASON AND ACCELERATED SNOWMELT ON BIOGEOCHEMICAL PROCESSES DURING ISOLATED CONIFER NEEDLE LITTER DECOMPOSITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: The authors addressed all my critical points and specific comments.
The data suffer from a low number of replicates. Still, given that the study is under field conditions and the authors made all the efforts for sound statistical analysis, this study has its merit. The statistical analysis is explained in detail, and the scientific message is clear.
In my opinion, the manuscript is acceptable in its present form.


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

Input:
Title: EFFECT OF ELEVATION, SEASON AND ACCELERATED SNOWMELT ON BIOGEOCHEMICAL PROCESSES DURING ISOLATED CONIFER NEEDLE LITTER DECOMPOSITION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the revised version of manuscripts authors replied to reviewers comments and addressed most of the raised issues however there are still several things that needs to be revised. I still can not find the description of supplementary material, which makes it difficult to evaluate presented results and data. Find below the specific comments - line numbers correspond to the manuscript with tracked changes.

line 58.... add reference
line 61 ... delete dot before the reference
line 279-281... can you be more specific which particular analysis were conducted using the listed R packages? I am unable to find any particular results from DESeq2 analysis in the manuscript
line 310-311.... what data for soil moisture and soil temperature were exactly used? Was it average of some period of time?
line 383.... what is visual difference?
line 461 – 465... neither in figure S10 nor anywhere else in the text I am unable to find any results from differential abundance analysis that would show statistical significant differences in abundance of microbial taxa
line 465-466.... based on what analysis did you conclude that there was an overall increase in community richness in 2018? – I can not find any analysis that would support this conclusion


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: ICARITIN PROTECTS SH-SY5Y CELLS TRANSFECTED WITH TDP-43 BY ALLEVIATING MITOCHONDRIAL DAMAGE AND OXIDATIVE STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Using viral transduction, the authors have overexpressed TDP43 in SY5Y cells, and then tried to recover the defects observed using Icaritin.  This approach has been used previously to target mitochondrial function.

Overall, the hypothesis is sound and the data in general looks robust. However, the manuscript suffers from a chronic lack of detail, especially in the methodology section. Which is quite poor in terms of content, scientific clarity and presentation (constant switching between current and past tense, incorrect use of English). There also needs to be more clarity on the statistical methods used.

For the methods, there is little description in the figure legends, it is not clear what stats were used, the data should not be presented in block bars, individual data point should be shown and standard deviation not standard error should be used. The westerns contain no molecular weight markers and full length blots should be shown in the Supplementary data.

The conclusions are essentially a description of the results without historical literature contextualization and no discussion on what the results could mean in the context of ALS.

I suggest major revisions before being accepted.
Experimental design: The approach is valid but the lack of methodological detail is not acceptable especially around the use of the TDP virus the activity assays in figure 5 and the statistical approach.
Validity of the findings: The data need to be presented in a more transparent fashion, removing the use of block histograms and using individual data points, standard deviation, describing in detail the statistical approach used in every figure legend and putting markers on blots. Full length blots should be provided in the Supplementary section
Additional comments: Introduction
Please add more up to date TDP43 articles focusing on TDP43 mitochondrial dysfunction.
Correct numerous grammatical errors.
Re-write lines 49-52, they dont read well.

Methods- Please stick with past tense and correct numerous grammatical errors and use of incorrect English.
Please add detail on the following
Cell culture
Virus transduction-construct, cloning,  production, virus titration,
Western blot- 50g per lane were added? I assume this is a mistake, please amend. Pleas provide evidence that all blots were in the linear range. The GAPDH loading controls look saturated.
Add full detailed procedures for ATP/GSH/SOD/MDA/TAOC/JC-1
Stats- please add what normality testing was performed and what stats were performed for each assay. put this in the figure legends. For example most of the Figure Legends state stats were control vs TDP43 group or TDP vs Icaritin group which suggest two groups were compared only. But the methods state annova was performed which is performed on three or more groups. Please amend

Results- please present all data showing individual data points and standard deviation not standard error, please add descriptive text to westerns blots molecular weight, which bands represent TDP43. Figure 1/3 for example shows two TDP43 bands, what do each band represent. Please put full length westerns in the Supplementary data.
Figure 4B, please provide quantification of the JC-1 data
Figure 4C-the quantification  does not match the picture

Discussion, please provide detailed contextualized discussion of the data in terms of the existing TDP43 literature and what these results mean in the context of TDP43 diseases such as ALS. Listing the results is not a discussion.


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: ICARITIN PROTECTS SH-SY5Y CELLS TRANSFECTED WITH TDP-43 BY ALLEVIATING MITOCHONDRIAL DAMAGE AND OXIDATIVE STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The rationale of this study is straight forward, and the suppression of TDP-43 toxicity is an area of interest. However, data quality, data presentation and method details in several places need significant improvements to strengthen the manuscript. In addition to experimental results, original research articles are not sufficiently cited in the introduction. It is also strongly recommended that the manuscript should be extensively edited and proofread by authors and professional editors.
Experimental design: 1.	 (Figure 3) One key observation of this article is the reduction of TDP-43 after ICT treatment.
1a. Authors state that TDP-43 is significantly reduced in the TDP-43+ICT group. However, high quality western blot should be provided to support this conclusion. In current western blot, TDP-43 bands in the TDP-43 +ICT lane are fractured, which could lead to signal loss and underestimation of protein level. Also, the background signal of the blot is high. I would suggest the authors to optimize western blot procedures (e.g. antibody concentration, washing condition, amount of protein loaded, film exposure time) before performing bands quantification.

1b. More information about figure 3 data should be provided in the main text. It is unclear whether the exogenous, endogenous or both TDP-43 expression were reduced after ICT treatment? If the endogenous TDP-43 was reduced in TDP-43 + ICT, why did the endogenous TDP-43 level remain unchanged in Con + ICT? If only the exogenous TDP-43 was affected, authors should discuss whether ICT has any possible effects on the expression vector.

1c. Since TDP-43 repression by ICT is a main point of this article, readers would expect a bit more characterizations or in-depth discussion on how ICT could achieve it. For example, could ICT promote TDP-43 degradation through autophagy or ubiquitin-proteosome system?

2.	(Figure 4B) Authors observed reduced MMP after TDP-43 transfection, and ICT treatment prevented loss of MMP in TDP-43-transfected cells.
2a. Current image resolution is too low, and it is difficult to evaluate authors’ conclusion regarding changes in JC-1 color in different groups. To support authors’ conclusion, high resolution microscopic images should be provided.

2b. To strengthen the point that ICT protects cells from TDP-43-induced MMP loss, quantitative analysis showing MMP changes across treatment conditions is highly recommended. Specifically, the red/green ratio of JC-1 in each experimental condition should be presented.

2c. It would be of authors’ interest to include mitochondrial uncoupler treatment such as CCCP as a positive control in the JC-1 assay.

2d. Line 185: JC-1 can only exist in the cytoplasm as “monomers”, not as a “monocyte”.

3.	(Supplementary file 2) The corresponding films/bands of main figure 1E, 3 and 4C are not indicated in the uncropped western blots. I recommend that information such as protein names, experimental conditions and connections of these films to main figures should be included.

4.	(Materials and Methods) Some methods and experimental details need clarification.
Line 58: Please provide human TDP-43 vector information.
Line 104: I think “50 g” of protein loaded to each lane is a typo, please fix.
Line 108: Please provide the source and dilution of GAPDH, TDP-43 and cytochrome c antibodies.
Validity of the findings: Data quality improvement and more controls are required to support the main conclusion that ICT suppresses TDP-43 toxicity by reducing its level.
Additional comments: 1.	(Introduction) Basis of the hypothesis in the introduction needs to be clarified.
Line 46: The original research articles about Icaritin improving structure and function of brain mitochondria in transgenic AD mouse should be cited. The authors cite a review paper (Angeloni et al. 2019) about Icariin, but I couldn’t find the references in this review related to Icaritin improving mitochondrial functions in AD mouse. Please cite the original research here.
Line 48: The authors propose that TDP-43 reduction is a possible mechanism for ICT to improve mitochondrial functions, but this hypothesis seems irrelevant to the context of background. Relevant references should be cited in the introduction to elaborate why authors propose this hypothesis. For example, any evidence that show ICT could promote degradation of misfolded proteins?

2.	(Discussion)
Line 201: Please clarify “TDP-43 is a highly conserved ribosome”. I believe this is not the current knowledge about ribosome and TDP-43.


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: ICARITIN PROTECTS SH-SY5Y CELLS TRANSFECTED WITH TDP-43 BY ALLEVIATING MITOCHONDRIAL DAMAGE AND OXIDATIVE STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors in the most part have addressed all my concerns
Experimental design: The authors in the most part have addressed all my concerns. My remaining concern would be the western data with many of the bands looking saturated.
Validity of the findings: The authors in the most part have addressed all my concerns.
Additional comments: The authors in the most part have addressed all my concerns. My remaining concern would be the western data with many of the bands looking saturated.


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: ICARITIN PROTECTS SH-SY5Y CELLS TRANSFECTED WITH TDP-43 BY ALLEVIATING MITOCHONDRIAL DAMAGE AND OXIDATIVE STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been improved significantly with much better writing, data quality and presentation. The authors have also addressed most of the issues I raised.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for your response to my comments.


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

Input:
Title: DISINTEGRATION HALF-LIFE OF BIODEGRADABLE PLASTIC FILMS ON DIFFERENT MARINE BEACH SEDIMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The novelty of this study is suggested to restate. There are many papers addressing biodegradability of the biodegradable plastics under different conditions (e.g. composting, seawater, anaerobic digestion etc.). Did any other researchers investigate the material of Mater-Bi HF03V in the marine conditions? If done, what are their results? And also what is the research gap proposed to address in this study?
2. The background information of tested materials Mater-Bi HF03V is suggested to clearly introduce, including how it was made, what is the molecular weight and characteristics, and why it can replace the traditional plastics, etc.
3. The title of the paper is suggested to rewrite to reflect the specific topic studied in this paper, e.g. the new materials name, and the biodegradation conditions (e.g. grain size).
Experimental design: 1.Can the authors list the information of the tested materials in a table and briefly presented in the materials section? it is hard to read the comprehensive information in the text and putting the information in the tables could help the reader a lot understand it.
2. The description of methods including the experimental procedure is suggested to be more detailed, which helps the readers repeat the experiments. Also, the rationale of using such methods are also suggested to present.
Validity of the findings: 1. This paper lacks some in-depth discussion of the results, like compared to biodegradation of this material under other environments, the mechanism of biodegradation and involved microorganisms.
2. Why time of 50% degradation was specially investigated in this study? Usually, many researchers and standards look for the time of 90% degradation, like ASTM D6400.
Additional comments: 1. The supplemental materials are suggested to have more organization and all the tables and figures are suggested to combined in a file, instead of 14 individual files in this paper. In addition, the titles and oher necessary part were lacked in supplemental materials.


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: DISINTEGRATION HALF-LIFE OF BIODEGRADABLE PLASTIC FILMS ON DIFFERENT MARINE BEACH SEDIMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 80: May need a reference?

Supplementary tables: Need titles - slightly confusing when going back and forth without any information on the tables.

Raw data table for follow-up study: LDPE should be HDPE?

Fig. SI-5: Temperature log for Naregno missing?

This one might just be a personal preference, but perhaps p-values should be displayed explicitly when significant?
Experimental design: Line 162-179: Is there a particular reason why different sediment layer thickness and strip dimensions were used here compared to the pilot? Could these also be confounding factors impacting the resulting disintegration rates (e.g., due to SA:V ratio)? Considering the distinct difference in results between the pilot and follow-up study, a little justification for these differences may be warranted? Also, is it really just one (not two) replicate? From what I understand from the tables and figures, it seems n=3.

Line 163-165: What is the purpose of sealing with plastic film? Did the authors do the same for the pilot?

Line 180-182: Might want to justify why last sampling for one of the sites was different than the others.
Validity of the findings: Line 219-222: Interesting result but was not further discussed - authors should provide potential justification for this observation? Also, is there an issue of pseudoreplication?
Additional comments: Overall well written piece; very easy to follow and understand. Tables and figures succinctly presented, adequate amount of background information and references provided.

Research question and aim clearly defined. Adequate replication and novel results provided with relevant discussion and sound recommendation and conclusion. Experimental method sufficiently detailed for replication and understanding, but minor additions to explain certain decisions/results may be helpful.


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: DISINTEGRATION HALF-LIFE OF BIODEGRADABLE PLASTIC FILMS ON DIFFERENT MARINE BEACH SEDIMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The comments have been well addressed.
Experimental design: The comments about the experiment design have been well addressed.
Validity of the findings: The comments about the findings validity have been well addressed.
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: CEPHALOPOD SPECIES IDENTIFICATION USING INTEGRATED ANALYSIS OF MACHINE LEARNING AND DEEP LEARNING APPROACHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for opportunity of reviewing the paper entitled “Cephalopod species identification using integrated analysis of machine learning and deep learning”.

The manuscript deals with machine learning techniques applied to taxonomic identification of cephalopods from Malaysian waters, including octopus, squid and cuttlefish species. Following rigorous tests of both upper and lower beak images as possible tools for identification, results show that the use of latter yielded identification with ~ 91% of confidence.

The manuscript has merit to be published, but some limitations must be observed prior final acceptance. First, the manuscript would definitively benefit from a professional English language service. Second, the use of lower beaks as reliable tool for cephalopod identification is not new and these structures have been used at least since the 1960s (Clarke, 1962, 1980), so authors must acknowledge this in the Introduction and Discussion.

The discussion was very methodology-orientated, and failed in addressing the usefulness of the reported findings in the wider taxonomic context.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: You methods and results are clear and well-described. Thus, I focused my review on the Discussion.

Your discussion was very methodology-orientated, and I was really expecting you tell to the readers the importance of your findings in improving the use of lower beaks images for species identification. The use of lower beaks for cephalopod identification from stomach contents is not new and these structures are traditionally used for this purpose (please see Clarke, 1962, 1980 just for start – there are plenty of similar studies out there). Unless I missing something, I could not find any mention of these early (and recent) studies along your whole manuscript.

There were other important aspects you failed to mention. For instance, how your method performs in identifying correctly morphologically-similar species of the same family and genera as Uroteuthis chinensis and U. edulis? For the sake of exemplification: if one has a sample of 100 beaks of these two species mixed together in a stomach content sample, how good is your method in correctly identifying each species in that sample?

You should definitively expand the discussion regarding the performance comparison between your method and the results achieved by other authors (lines 395-397). It is neither informative nor useful (for the sake of discussion) just “calling” a comparative table (Table 7, in your case) and make a brief (and poor) comparison. This is not enough. You have sufficient and decent data to describe what is better/innovative in your results in relation to the published results from other authors. You also should recognize the limitaions of your method [for instance, the ANN method employed by Himabindu et al. (2017) had a higher accuracy than your results].

References

Clarke M (1962a) Significance of cephalopod beaks. Nature, 193,560-1
Clarke MR (1986) A handbook for the identification of cephalopod beaks. Clarendon Press, Oxford
Himabindu K, Jyothi S, Mamatha D (2017) Classification of squids using morphometric measurements. Gazi University Journal of Science, 30(2), 61-71.


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: CEPHALOPOD SPECIES IDENTIFICATION USING INTEGRATED ANALYSIS OF MACHINE LEARNING AND DEEP LEARNING APPROACHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript aim to compare eight machine learning methods for image classification of cephalopods. They argue that the comparison is relevant since this has not been done for cephalopods in Malaysia and that cephalopods are economically important for fisheries in the region as well as being an important prey in the marine food web.

Since the aim of the study is to determine which classification method is best, it would be relevant to include paragraphs in the introduction and discussion that address the literature that make similar comparisons of methods for species classification or more general image classifications. I.e. not limiting the comparison to classification of squid species as you did in Table 7.
Experimental design: No comment
Validity of the findings: Although it may be difficult to increase sample size, the limitations of a small dataset should be discussed. This might impact the both test results and the reliability of applications of the model to classify new material. I would suggest to perform some kind of sensitivity analysis that show the robustness.

For a biological material I also find it surprising that the dataset is not more unbalanced. I.e. more species with very few examples and a few number of species with most examples. Did you make additional steps to balance the dataset?

It is not clear from the manuscript if the number of species included are close to the number of known species in the region? You should also address how a relatively low number of species (classes) in the model affect the results.
Additional comments: No additional comments


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

Input:
Title: CEPHALOPOD SPECIES IDENTIFICATION USING INTEGRATED ANALYSIS OF MACHINE LEARNING AND DEEP LEARNING APPROACHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for the opportunity of (re-) reviewing this manuscript. The present version certainly improved on the early draft analysed, but I found an important taxonomic flaw, and I also made suggestions to improve the text presentation and clarity. These are described in the "General comments for the author" section below.

That said, I would strongly encourage the authors to seek a professional Academic English review service to improve their text. In my opinion, the English of this manuscript is not yet good enougth for publication in an international Journal such as PeerJ.
Experimental design: No comments here.
Validity of the findings: Their findings are relevant and certainly would improve cephalopod taxonomic resolution in poorly surveyed regions or in counries that lack taxonomic experts on the group.
Additional comments: ABSTRACT

Line 43:

Please, change “Besides, deep features were extracted (…)” to “In addition, deep features were extracted (…)”

INTRODUCTION

Lines 61–62:

Please, change “(…) which includes octopus, squid and cuttlefish.” to “(…) which includes octopus, squid, cuttlefish and nautiluses.”

MATERIALS & METHODS

You must briefly describe here the steps showed in the flowchart depicted in Figure 1. Something like: “This flowchart is described as follows: “Sample collection” show that squid, octopus and cuttlefish samples were acquired from local fisheries at two landing sites off western Malaysia. “Image Acquisition” depicts how images were acquired (…)”... And so on.

Lines 176–177:

Please make sure to use the appropriate “times” (×) symbol.

Thus, change “5312px x 2988px” to “5312px × 2988px” and “532px x 299px” to “532px × 299px”

Make sure to double-check this detail for the rest of your text.

Lines 189 and 190:

Please use the “end dash” (–) symbol to show value intervals.

Thus, change “(0 - 239)” to “(0–239)” and “(240 - 255)” to “(240–255)”.

Make sure to double-check this detail for the rest of your text.

Line 216:

Please, change “Besides (…)” to “In addition to (…)”

Line 246:

The “n” must be in italics here.

Make sure to double-check this detail for the rest of your text.

Line 278:

Please change “. 5-fold cross-validation (CV) (…)” to “Five-fold cross-validation (CV) (…)” (A number must no begin a sentence!).

Make sure to double-check this detail for the rest of your text.

RESULTS

Line 303:

Sepioteuthis lessoniana is NOT a cuttlefish but a true squid species belonging to the same family (Loliginidae) of the three squids you recognized as such (i.e. Loliolus uyii, Uroteuthis chinensis and U. edulis). That’s a flaw that certainly affected the interpretation of your results! You MUST reanalise your results under the ligth of this observation and modify whatever you find necessary accordingly.

Line 330:

The taxonomic nomenclature rules forbid abbreviate a species’ name. Thus, you must change “S. esc” to “S. esculenta” here and in your Figures 4 and 5. You must also write the complete species name of each one of the remaining cephalopod species listed in Figures 4 and 5.

Make sure to double-check this detail for the rest of your text.

TABLES

Table 1: You must include a brief description of each one of these ten features to the reader. For example, what exactly “Solidity / convexity” means?

Table 5: you must explain in the table caption what does means the values marked in bold.

Any table or figure in a scientific text must be self-informative! Please, improve your table captions!

FIGURES

You must explain in the figure caption how to interpret the precision recall-curve. I would also recommend you explain it briefly, when you call Figures 4 and 5 in your Results. Remember that not all readers are familiar with your statistical techniques/methods. Thus, these details must be explained for the sake of clarity.

In addition, please double-check the captions of all remaining figures. As I said before, any table or figure in a scientific text must be self-informative.


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: USING IN-SITU ENVIRONMENTAL DNA SAMPLING TO DETECT THE INVASIVE NEW ZEALAND MUD SNAIL (POTAMOPYRGUS ANTIPODARUM) IN FRESHWATERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is mostly clear, with a few sections missing potentially helpful details.
Most importantly, I worry about the literature references: Several milestone reviews on eDNA research such as Ruppert, Kline & Rahman (2019) or Huerlimann et al. (2020) have not been cited (the reviewer has not been involved in the mentioned publications); instead, some references do not contain the information they are supposed to. For example, in ll. 92-93, Lodge et al. do not refer to any cost-associated advantages of eDNA research. In addition, the citation of Machler et al. in ll. 83-85 is very close to a copy of the respective paragraph in the paper itself.
While the manuscript adheres to the professional article structure, there are worries about the depths of its analyses, what is reflected in the figures and tables (see below). The raw data is available. The hypotheses are self-contained, but results are interpreted wrongly.
Experimental design: While the research question is clearly stated, the experimental design suffers from:
(1) low sample size; (2) lack of replication per site (which is stated by the authors themselves and could have easily been included; spatial replication along a transect is not replication per site); (3) no impact of water volume on detectability is assessed (while many other eDNA studies use 1L, this manuscript focuses on the detection rate of one specific elusive species, so the volume could have been optimised); (4) while the design involves a river gradient, this spatial dependency is not being taken into account later on.
The methods are described in enough detail, whereas the co-author's previous papers were necessary to understand the entire context of the work.
Validity of the findings: The findings of the manuscript do not agree with the data.
The Fisher's exact test that is applied to compare traditional and eDNA surveying shows that there is NO significant difference between both methods based on the respective detection rates. The alternative hypothesis of a Fisher's exact test is that there is some sort of association between two different variables, which is the case in this study since for most of the sites eDNA and traditional surveys agree on the presence/absence of the species. In general, the authors describe that traditional methods detected 13 sites to be positive and eDNA methods detected 18 sites to be positive, so only 5 sites were only detected as positive by eDNA, but also one only by the traditional method. Just by thinking a bit about permutations and probabilities, one can see that this is not statistically significant.
The linear regression between number of snails and eDNA copies does not seem to have a uniform distribution of residuals, which is a prerequisite for applying linear regression. This results can therefore not be trusted either.
The water temperature mostly only takes three values - which makes other statistical tests more useful as well.
The authors talk about the problem of inhibition, but this has already been a problem in their previous paper; what has been done to tackle this problem since then?
Additional comments: In summary, I can only reject this paper. Sample size should be increased, replicates should be taken, followed by adequate statistical analysis. It would also be advantageous if the authors could trial different volumes of water, analyse the effect of up- and downstream sites, and include references to more recent eDNA work as well a short description of their previous work on mud snails.


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: USING IN-SITU ENVIRONMENTAL DNA SAMPLING TO DETECT THE INVASIVE NEW ZEALAND MUD SNAIL (POTAMOPYRGUS ANTIPODARUM) IN FRESHWATERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment; the paper meets all requirements for basic reporting.
Experimental design: Paper generally meets requirements. Any comments on the methods are provided in the overall comments to the authors
Validity of the findings: No comment; findings and results make sense.
Additional comments: Overall:
This paper sought to compare results from a rapid eDNA sampler with on-site results compared to traditional sampling for the invasive New Zealand mud snail. The authors report that on-site eDNA site detection was higher than traditional sampling. The paper is clearly written,  the results make sense and further add to ongoing eDNA research for conservation. Please see below I have provided more specific suggestions and comments.

Title: Minor issue, but consider replacing “for detecting” with “to detect”

Abstract:
L47: Minor suggestion to reword the end of the sentence to be “…compared to traditional visual survey methods.”
L50-51: Insert comma before “with five samples per site”

Introduction:
L66-72: Zebra mussels and red swamp crayfish are globally invasive, so not specific to Europe only.

L77: Revise “…, and then spreading” to read “and spread”

L80: Consider revising this sentence to reduce repetitiveness in use of “their”. Sentence could read, “…sampling methods due to small size, low population densities, patchy distribution, or complexity in use of habitat at different life stages.”

L85: Revise in-text citation to be Mächler et al. 2014

L92: Consider citation to Evans et al. 2017 as a more suitable study that directly examined the cost of eDNA vs. conventional sampling.

Evans NT, Shirey PD, Wieringa JG, Mahon AR, and GA Lamberti. 2017. Comparative cost and effort of fish distribution detection via environmental DNA analysis and electrofishing. Fisheries 42:90-99. https://doi.org/10.1080/03632415.2017.1276329

L98, L102: Minor formatting, revise in-text citations to use ;

L103: Minor comment to clarify that it is for freshwater or aquatic systems, given that terrestrial eDNA does not typically include collection of water. Maybe revise to read, “…there is not a standardized eDNA protocol for freshwater ecosystems, …”

L104-105: What does “with water collection stage showing to be a valuable step to increase detection rates” mean? Maybe revise this to read something like,

“Currently, there is not a standardized eDNA sampling protocol in freshwater ecosystems, but the overall sequence of steps includes water collection, preservation, and laboratory analysis. Further, research suggest that sampling decisions made during the water collection (i.e., volume of water) and handling step (i.e., on-site vs. lab filtration) may influence detection (add citation(s) here to support statements).”

Potential citations that authors may consider here:

Curtis AN, Larson ER, and MA Davis. 2021. Field storage of water samples affects measured environmental DNA concentration and detection. Limnology 22:1-4. https://doi.org/10.1007/s10201-020-00634-y

Mächler E, Deiner K, Spahn F, and F Altermatt. 2016. Fishing in the water: effect of sampled water volume on environmental dna-based detection of macroinvertebrates. Environmental Science Technology 50:305-312. https://doi.org/10.1021/acs.est.5b04188

Sepulveda AJ, Schabacker J, Smith S, Al-Chokhachy R, Luikart G, and SJ Amish. 2019. Improved detection of rare, endangered and invasive trout in using a new large-volume sampling method for eDNA capture. Environmental DNA 1:227-237. https://doi.org/10.1002/edn3.23

Materials & Methods:
L142-143: What does “positive detections” mean? Previous eDNA pilot study that confirmed eDNA detection here or that researchers had found mud snails here previously? I think it means snails have been recorded here previously, but use of positive detection made me think eDNA detection.

L149-152: Curious why samples were collected along the shoreline. Is this standard traditional sampling protocol?

L158-180: Consider moving this up to be the first section of the Materials and Methods to be before “Study Sites” section. This would allow the reader to have knowledge of the study organisms before reading the more specific information of the study sites and sampling.

L208-209: How many replicates per water sample were run? One qPCR replicate per water sample?

L210-211: Please provide information on the concentrations used to generate the standard curve. Additionally, it might be helpful to other researchers to know information about the gBlock, consider adding GenBank accession number or the sequence here or as supplemental.

Were plate blanks (NTCs) run?

Then if possible, provide estimates of the LOD, LOQ, R2, and % efficiency. Or are those values the same as reported in Thomas et al. 2018?

L231: I was confused here as to whether the sampling conducted was to confirm presence/absence of the snails or the eDNA. Please include a statement in this paragraph to state that snails were counted to get an estimate of site abundance, because this was not immediately clear to me.

L248: How many sites were removed?

Results:
L253-254: Clarify here what six sampling sites means. Does it mean stream or individual replicate along the 40 m transect? When I looked at the raw data that statement does not seem to be true. For example, neither eDNA or traditional sampling detected mud snails at the Columbia River/Kalama and only one sampling location had detection (eDNA only) at Capitol Lake. Then L255 states this, so consider deleting this sentence.

L256-257: I wonder if the eDNA was simply transported downstream from site 5 where the snail was at low abundance to the other sites (e.g. site 2) where eDNA was detected.

Discussion:
L279: Capitalize western (Western Washington) to match previous style

L282-283: Detection >1 snail/m2 is pretty good! Goldberg et al. 2013 can detect 1 snail/1.5 L of water, but in the field detection is at ~11 snails/m2. Also, as mentioned previously, this eDNA could simply be lower due to low abundance and transported downstream.

L299: What is mud snail DNA SQ? Starting quality of DNA? This abbreviation has not been referenced (also not explained in Figure 3A).

L301-302: Confused as to what “no formal analysis for this positive association” means. Isn’t this shown in Figure 3A?

L303: As a reader, I don’t understand what “which evaluated a limit of detection based on varying abundances of mud snails.” means here in context with the relationship between eDNA concentration and abundance of target organisms. Please revise to clarify this.

In this section about the relationship between species abundance and eDNA concentration, consider this reference which has found generally stronger relationships in laboratory studies and higher variability in field eDNA studies.

Yates MC, Fraser DJ, and AM Derry. 2019. Meta-analysis supports further refinement of eDNA for monitoring aquatic species-specific abundance in nature. Environmental DNA 1:5-13. https://doi.org/10.1002/edn3.7

L314-316: Is the relationship between temperature and eDNA maybe not significant because the methodology specifically targeted sampling during potential snail reproduction (as stated in L148-149) and maybe not due to degradation at relatively low temperature (14°C)? There are several eDNA papers that highlight the importance of reproduction (and increased temperature as a signal of reproduction) on eDNA detection. For example: Spear et al. 2015 or de Souza et al. 2016

de Souza LS, Godwin JC, Renshaw MA, and E Larson. 2016. Environmental DNA (eDNA) detection probability is influenced by seasonal activity of organisms. PLoS ONE 11:e0165273. https://doi.org/10.1371/journal.pone.0165273

Spear et al. 2015 is cited in-text at L310, but not in the reference list.

L318-319: The results do not show evidence of inhibition and from Figure 2 only one site was inhibited. Sample replication likely seems to be more important for detection, as stated in L324-327.

L341: In streams, transport, settling, and dilution can likely all influence detection.

References:
L499: Italicize Esox lucius

Tables/Figures:
Table 1 and 2: Nice work, these are very clear and easily to understand the results.

Figure 2: To me, presence/absence means detected/not-detected. But this figure is showing that and eDNA concentrations. Consider deleting the “Presence/Absence” from the first line of the figure legend.
Also, this figure is excellent! It very nicely displays the results of the study. Well done.

Figure 3: Consider moving the A, B, and C to the upper left of the figure.
Revise temperature units to read °C

Figure 4: The size of the figure legends and the yellow-red color here was difficult to read and is maybe not color-blind friendly. Consider either revising this or removing this figure because this information is mostly repetitive of Figure 2.


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: USING IN-SITU ENVIRONMENTAL DNA SAMPLING TO DETECT THE INVASIVE NEW ZEALAND MUD SNAIL (POTAMOPYRGUS ANTIPODARUM) IN FRESHWATERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a very well written, concise and interesting manuscript. The figures and tables were intuitive, and the written text was well polished.
Experimental design: The experimental design was sound - adequate replication and negative controls were included at all sites, and additional abiotic variables were measured which added an interesting component to the paper
Validity of the findings: The findings were discussed in relevant context, and the data provided in the supplemental file were sufficient to assess the validity of the study
Additional comments: I really enjoyed reading this paper, and was very impressed with the on-site collection, extraction and amplification method. I can see this being a useful method for monitoring a range of different species across different habitats, and the timely results could advise management decisions on the spot. A couple of the references could use some tidying up but other than that I'm happy to recommend the article be accepted in its current form


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

Input:
Title: USING IN-SITU ENVIRONMENTAL DNA SAMPLING TO DETECT THE INVASIVE NEW ZEALAND MUD SNAIL (POTAMOPYRGUS ANTIPODARUM) IN FRESHWATERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The comments have been incorporated.
Experimental design: I agree that a final monitoring approach should include as many samples as possible; the proposed research here is however a benchmarking approach of the method so I think it would have been essential to include at least one/a few replicates at the very same location to understand how much the results change by just sampling one second later at the very same spot. Understanding this variability will be important to understand the efficiency of the approach – i.e. how many samples should be taken by a practitioner to confirm presence.

Thank you for adding a figure about the spatial distribution. Ideally, this figure would include the raw number of read counts (why and how were the thresholds chosen that are currently being shown?) and would show a direct comparison with the traditional field sampling results. As the statistical results comparing the traditional and eDNA approach are still not correct (see below), this would be a minimum requirement for accepting the paper.

I agree that trialling different volumes of water was not a necessity for this study, but again, it would have given practitioners important information about the sensitivity of the approach.
Validity of the findings: As briefly mentioned above, the statistical results are still not correct. Table 2 now just shows the number of sites that did or did not detect snails, with the eDNA results in one column and the traditional results in the other column. This does not show any comparison between the two methods at all and a Fisher's Exact test on these values is meaningless. We are interested in the site-dependent overlap between the two approaches.
The authors really have to think about what they want to show. If they did a Fisher's exact test on the site-dependent overlap as just suggested, the test would not be significant. This just shows that we don't have proof that the approaches are different - it does not automatically mean that the approaches are the same since the power of such a test always depends on sample size. If the authors want to show that the approaches mainly overlap, how about just showing the number of overlaps and then argue why some of the predictions did not overlap? Especially the cases where eDNA was negative and the traditional approach positive are worrisome.
Additional comments: Thank you for incorporating some of my suggestions. I am sorry to say that more needs to be done for me to accept this paper. The statistical results are still not correct and there is no clear message of the paper - is eDNA now better than traditional monitoring? (Some sites were only positive using the traditional approach.)


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: USING IN-SITU ENVIRONMENTAL DNA SAMPLING TO DETECT THE INVASIVE NEW ZEALAND MUD SNAIL (POTAMOPYRGUS ANTIPODARUM) IN FRESHWATERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment. Paper appears to meet basic reporting standards.
Experimental design: No comment; see brief comments below
Validity of the findings: No comment
Additional comments: I commend the authors for doing a great job on thoughtfully and carefully revising their manuscript. I have some minor clarifying questions/comments below.

1. Still inconsistencies in "western Washington" and "Western Washington". Pick one and keep throughout. See Line 49 vs. 161

2. In the previous revision, I asked whether plate blanks were included and the response was that field blanks were included. Are machine/plate blanks not standard in field qPCR equipment? Field and plate blanks are standard now for eDNA research. I understand that the field blanks were confirmed negative and thus, it may be concluded that there was no contamination. Maybe I simply do not properly understand the Biomeme portable qPCR set-up and are reagents individually sealed to limit contamination?

3. On the limit of quantification: The authors cite to Thomas et al. 2018 S3, which lists LOQ at 50 copies and LOD at 10 copies. Did the authors then remove any samples that were <LOD/LOQ from analyses (Figure 3)? If not, what is the justification for not doing so?

4. L229-230: "...used nitrile gloves between each DNA extraction..." or changed gloves between extractions?

5. Literature Cited: Scientific names should be italicized. Peer J editorial staff may fix this for you.


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

Input:
Title: USING IN-SITU ENVIRONMENTAL DNA SAMPLING TO DETECT THE INVASIVE NEW ZEALAND MUD SNAIL (POTAMOPYRGUS ANTIPODARUM) IN FRESHWATERS
Review round: 2
Reviewer: 3

Output:
Basic reporting: This paper is well-written and provides useful background information. The article is structured well. Raw data is provided as supplementary information.
Experimental design: The methods are explained in adequate detail. There is an issue with pseudo-replication in the survey design, since the 30 sites are not all independent of each other – 5 samples were taken at 10m intervals within each site – therefore I would argue that there were only 6 sites, and 5 replicates at each. The reps are not true technical reps (as pointed out by Reviewer 1 in the first round), but I think it makes more sense to interpret them as reps rather than independent sites. This is especially relevant here because DNA from the upstream sampling points could be carrying down and affecting the results of the downstream points.

I don’t think this is a disqualifying issue, but it does affect how the statistical analysis can be carried out:

1)	The correct way to run the correlation analyses with a pseudo-replicated design is to take an average or median SQ value over the 5 reps at each site and correlate these with the conductivity/temperature readings and the visual counts. This would give only 6 data points rather than 30, but the log+1 of the mean SQ values still correlates nicely with the conductivity scores, and you could fit a trendline to the mean SQ vs the mean visual snail quantity – it’s probably not enough data for a p-value but the trend is evident.
2)	The fisher exact test will also be affected since there are only 6 data points rather than 30. The corrected layout of the original table (I don't think th table was set out right in the MS) is:
                             eDNA_positive eDNA_negative
visual_positive                         12                         1
visual_negative                         6                        11

this gives a fisher’s exact p-value of 0.0024 – I’m pretty sure this is right - the R code I used is:
x <- matrix(c(12, 1, 6, 11), byrow = TRUE, nrow = 2)
fisher.test(x)

Since the design is pseudo-replicated the corrected layout of the new contingency table will be
                             eDNA_positive eDNA_negative
visual_positive                          3                         0
visual_negative                         2                         1

Unfortunately we can’t get a fisher’s statistic on this since the sample size is too small and snails were only visually observed at 3 of the 6 sites.

I’m actually not convinced that a contingency table makes a lot of sense here anyway, since the idea is to show that the eDNA is more sensitive, not that there is an association between visual counts and eDNA SQ values (this would seem intuitive). The detection sensitivity is covered off well in the previous analysis, so I’d consider getting rid of the contingency table and Fisher test.
Validity of the findings: Aside from the technical issues outlined above, the findings show the method to be sensitive and effective at picking up the target species in the field.
Additional comments: As a proof of concept this study gives a good introduction to the method and explains why in-field eDNA analysis is a useful and promising tool. I’m looking forward to hearing more as the method is refined and optimised and applied across a broad range of environments.


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 REGULATING EFFECTS AND MECHANISM OF BIOCHAR AND MAIFANITE ON COPPER AND CADMIUM IN A POLLUTED SOIL-LOLIUM PERENNE L. SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper includes the valuable results obtained experimental studies to remove heavy metals from the polluted soil using different amendments.  In order to remove copper and cadmium from contaminated soil, biochar, iron modified biochar, maifanite have applied, and the mechanisms of copper and cadmium removal of soil particles have investigated. The effect of the application on the prevention of Cu and Cd accumulation in the shoots and roots of ryegrass has investigated. According to the results obtained experimental studies, the immobile effects of examined amendments on Cu has found better than Cd. In addition to the adsorption mechanism, complex formation occurred on biochar, iron modified biochar, maifanite. The results of experimental data have a great importance to the remediation of heavy metal polluted soils.
The paper is properly well organised and overall quality of the work is excellent. The paper is well within the scope of the journal it needs some minor improvements and revisions given below:
Avoid using words in first person, like: we, I.
In the Conclusions part at lines 374- 377 the sentence needs to be improved.


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 REGULATING EFFECTS AND MECHANISM OF BIOCHAR AND MAIFANITE ON COPPER AND CADMIUM IN A POLLUTED SOIL-LOLIUM PERENNE L. SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The theme of the manuscript is interesting and well organized.
The author has often used conjunctions in starting a sentence  which needs to be corrected and should also focus on grammatical errors.

For example:
1. lines 227-229: And for plants which enter the food chain, this indicator is much more
    important because it fully expresses the total amount of heavy metals uptaken from
    the soil.
2. Line 295-298: Few change was detected in the characteristic peaks of Fe
    modified/unmodified biochar after the adsorption of heavy metals (Fig. 5A), indicating
    limited damage on the structure of cellulose and hemicellulos

For more such comments, refer to the attached review document.

Some of the references cited in the paper are not adding any value or supporting the context.

For example:
1. In lines 299-303 it was stated that  "As for BC, the surface functional groups were
    apparently detected at 1700 cm-1, 1560 cm-1, 1420 cm-1, 1380 cm-1, and 617 cm-1;
    assigned to carbonyl (C=O) and the hydroxyl (-OH) groups, demonstrating that the
    surface of biochar from medicine residue included oxygen-containing functional
    groups, such as carboxyl, lactone, carbonyl, and hydroxyl (Foo & Hameed, 2010)."

but the cited reference is neither discussing about FT-IR nor about the functional group characterization.

For more such comments, refer to the attached review document.
Experimental design: no comment
Validity of the findings: In lines 305-308, author has stated that "The characteristic peak for –OH functional group of BC decreased at 1420 cm-1, indicating that the hydroxyl groups possibly contained on the surface of the biochar may participate in the adsorption reaction process of Cu and Cd."

but in reality a broad peak around 3500-3000 cm-1 confirms the presence of hydroxy functional group (-OH). This is visible in Fe-BC (3380 cm-1) whereas missing in BC.
Can the author explain the reason behind this?

In line 313-315, author has stated that "And absorption peak of Fe-OH at 796 cm-1 appeared, which is a characteristic peak for iron oxide, signaling Fe attaching on the surface of the biochar."

Author has to provide academic reference to support that a peak at 796 cm-1 characterizes the presence of Iron oxide.
Additional comments: The current paper is well organized. Through the abstract, the author clearly summarized major aspects of the paper such as the key purpose of the study, research problems, basic design of the study, and key findings. The introduction and background are reasonable given the premise of the manuscript submitted. All the tables and figures provided are comprehensive and clearly represent the experimental findings.

Please respond to the comments and questions regarding the FT-IR analysis that was conducted to find the variations in functional groups of amendments before and after adsorption of Cu and Cd.


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: DESIGNING OF THE N-ETHYL-4-(PYRIDIN-4-YL)BENZAMIDE BASED POTENT ROCK1 INHIBITORS USING DOCKING, MOLECULAR DYNAMICS, AND 3D-QSAR
Review round: 1
Reviewer: 1

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


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

Input:
Title: DESIGNING OF THE N-ETHYL-4-(PYRIDIN-4-YL)BENZAMIDE BASED POTENT ROCK1 INHIBITORS USING DOCKING, MOLECULAR DYNAMICS, AND 3D-QSAR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is written clearly and god to understand. Nevertheless, it should be checked to remove several typos and grammatical issues.

References are fine

Article structure is good. The format of Table 2 should be checked carefully. In caption of Figure 1, the second b) should read c).

At least the coordinates of the docked complexes should be available. I don't know about the journal's policy concerning input/output files for the other simulation techniques applied.
Experimental design: The workflow presented/applied to address the scientific question is clearly presented and state-of-the-art. All methord used are appropriate.

The scientific question is clearly defined.
Validity of the findings: As their general result, the authors present a series of new ROCK1 inhibitor candidates with promising properties. As in every drug discovery project, the ultimate proof will be experimental verification.
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 OF A CIRCRNA-MIRNA-MRNA REGULATORY NETWORK FOR EXPLORING NOVEL THERAPEUTIC OPTIONS FOR GLIOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: See General Comments
Experimental design: See General Comments
Validity of the findings: See General Comments
Additional comments: In this paper, He and colleagues constructed a circRNA–miRNA–mRNA network in glioma with GEO, GEPIA2, circBank, CSCD, CircInteractome, mirWalk 2.0, and mirDIP 4.1. The present study emphasizes the potential pathogenesis of the circRNA-miRNA-mRNA which may therapeutic options for glioma. In general, this manuscript was well written and well designed. Nevertheless, I have the following comments for the authors to address before accepting for publication.
1.	Dose sex difference was observed in the circRNA-miRNA-mRNA regulatory network on glioma?


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 OF A CIRCRNA-MIRNA-MRNA REGULATORY NETWORK FOR EXPLORING NOVEL THERAPEUTIC OPTIONS FOR GLIOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: HE and Chen et. al used multiple analysis methods constructed an interesting network in glioma, which provides potential therapeutic options for tackling glioma. The analyzes are rational and comprehensive and the results also make sense such as functional enrichment analyses revealed a lot of hallmarks of tumorigenesis. This study provides a good benefit to the glioma field. I only have the following minor issues:

1.	The biggest concern is that, also pointed out by the authors, there is no experimental verification. It is understandable that it is difficult to investigate glioma in human or even beyond the scope of this paper, but it would be really great to verify either the effect of the drugs using cultured cells or rodent models. Or it will be really interesting to take a look at the amount of the relevant circRNA and ceRNA in glioma tissue. Or is there any relevant reported data to verify the results?

2.	The paper is generally well written. The intro and background are clear. The results are written clearly but fluency should be improved a bit. Also some expressions need to be improved such as line 56 (‘that hope to’), grammar error such as line 67 (‘the’), and also please minimize the usage of line 297 ‘As we all know’.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION OF A CIRCRNA-MIRNA-MRNA REGULATORY NETWORK FOR EXPLORING NOVEL THERAPEUTIC OPTIONS FOR GLIOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study is well conducted and the manuscript is written in an unambiguously and professional way. However, there are a few questions/problems the authors need to solve:

1. The current study investigated 8 circRNAs but why only 6 of them are present in figure 2c?

2. The expression of RCN1 gene is not labeled as significant difference in figure 5.

3. Please give a brief introduction to explain the importance of assessing immune cell infiltration for the prognosis of glioma.

4. more references should be added in the introduction, results, and discussion for some sentences:
Introduction:
(1) “Gliomas comprise majority of primary intracranial neoplasms with high heterogeneity…”
(2) “Recent advances in precision medicine, genomics, immunology, and other disciplines have uncovered…”
(3) “Circular RNA (circRNA) is a type of non-coding RNA derived from the exon or intron…”
Results:
“Gene expression profiles from TCGA-GBMLGG dataset and immune cell signatures from a previous study …”
Discussion:
(1) “CircRNA can act as a molecular sponge for miRNAs to de-repress all target genes …”
(2) “Wnt signaling, commonly divided into β-catenin-dependent (canonical) and …”
(3) “The MAPK/ERK pathway is a chain of proteins that communicate …”
(4) “The current consensus is that the anti-tumor immune response in glioma is …”
(5) “Harmane is a tremorigenic β-carboline capable of inhibiting mitochondrial viability …”
(6) “Importantly, mifepristone and tretinoin were shown to inhibit cell cycle and DNA repair …”
Experimental design: Generally, the experiments are well designed but more details should be added to the methods in order to enhance the reproducibility:

Please provide a detailed description of how to integrate the 11 hub genes as a whole signature and compute the total risk score of each sample. Additionally, what are the criteria that separate the glioma patients into high and low-risk groups based on the risk score?
Validity of the findings: 1. The expression levels of the circRNAs and miRNAs are critical for their biological functions. Please provide the expression levels of the circRNAs and miRNAs in the tissues that are investigated in this study.

2. Please provide the exact ID numbers of the TCGA-GBMLGG dataset that is used for the assessment of immune cell infiltration.

3. Please provide the exact ID numbers of the CCGA dataset that is used for the assessment of hub genes.

4. The description in the discussion is not consistent with the finding in the current study:
“Our results showed that the selected genes were closely related to immune infiltration and had generally opposite correlations with macrophages …”
Additional comments: 1. Typo in the abstract (page 5 line 34): ceNRA > ceRNA


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 OF A CIRCRNA-MIRNA-MRNA REGULATORY NETWORK FOR EXPLORING NOVEL THERAPEUTIC OPTIONS FOR GLIOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I recommend this manuscript to be accepted for publication as all my concerns has been addressed.


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

Input:
Title: IDENTIFICATION OF A CIRCRNA-MIRNA-MRNA REGULATORY NETWORK FOR EXPLORING NOVEL THERAPEUTIC OPTIONS FOR GLIOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: He and colleagues substantially revised their manuscript according to reviewers' comments. I think it is now suitable for publishing in PeeJ now.
Experimental design: None
Validity of the findings: None
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 OF A CIRCRNA-MIRNA-MRNA REGULATORY NETWORK FOR EXPLORING NOVEL THERAPEUTIC OPTIONS FOR GLIOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have satisfactorily resolved all the questions I asked, and I think the manuscript is good to be published.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: IDENTPMP: IDENTIFICATION OF MOONLIGHTING PROTEINS IN PLANTS USING SEQUENCE-BASED LEARNING MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The manuscript needs to be checked for sentence construction and grammatical errors.
2. PMPIdent and IdentPMP has been interchangeably used in the figures and text.
3. Authors should make it more elaborate on how existing prediction tools (non-specific to plants) do not provide a suitable alternative for the IdentPMP.
Experimental design: 1. Research methodology is well elaborated and appropriate statistical methods have been employed in the study.
2. Is the prioritized feature combination the same for both the tools, IdentPMP, and MPFit.
3. It is appreciative that IdentPMP is the first prediction tool for the plant moonlighting proteins. As the authors state in the results and discussion that the existing variability between IdentPMP and MPFit is perhaps due to the dominance of proteins from animals and microorganisms in the case of MPFit, shouldn't IdentPMP be compared with some of the existing databases for more confidence in the data?
Validity of the findings: 1. How does the sensitivity and specificity curve comparison hold true between the two databases, IdentPMP and MPFit if the latter is predominant for animal proteins. Authors should mention in the text if the algorithm used to call for moonlighting function in the case of MPFit can be biased towards animal or plant proteins?
2. Authors should provide some information on comprehending the results obtained from the web interface. The user is provided with the score for each protein however, it will be beneficial to future users if information about the possible moonlighting roles can be integrated with the existing results.
Additional comments: Liu et al in their manuscript "IdentPMP: moonlighting proteins identification of plant using
sequence-based learning models" describes the first moonlighting function prediction tool for plant proteins. It is an important study that will not only shed light on the discovery of new moonlighting proteins but investigation into their functions will provide insights into their role in different cellular and organellar niches. The manuscript is suitable for publication provided the following comments are addressed by the authors.


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: IDENTPMP: IDENTIFICATION OF MOONLIGHTING PROTEINS IN PLANTS USING SEQUENCE-BASED LEARNING MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The paper designed a sequence-based model to identify moonlighting proteins. Although moonlighting protein plays several functions, the work presented in this paper is still far from satisfactory. I do not think the paper is worth publishing based on following comments.
1. The number of samples, especially for positive samples is too small to have statistical significance.
2. Redundant sequences were not removed from benchmark data. Thus, their proposed model is overestimated.
3. Although authors used ilearn to generate features, the description about features are very poor. Readers cannot repeat their work. Moreover, how many features were used?
4. Authors did not perform enough feature analysis to show why these features are validated.
5. No webserver or softpackage was established.


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: IDENTPMP: IDENTIFICATION OF MOONLIGHTING PROTEINS IN PLANTS USING SEQUENCE-BASED LEARNING MODELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic language is easy to follow however, the manuscript requires a serious revision to rectify errors related to the English language. I made some remarks in the abstract section below.

Line 22  “Hence, availability” should be “Hence, the availability”
Line 24 “data set that constructed by” should be “data set constructed by”
Line 25 “At the beginning” should be “in the beginning”.
Line 26 “plant protein,” incorrect use of punctuation “,”.
Line 37 “prediction tool were” should be “prediction tool was”
Line 29 & 30 “On the basis of again, ”,  What does the author want to say is not clear in this sentence.

Text is provided with sufficient field background and context. Article structure and other components such as figure table etc. look fine.
Experimental design: 1. The author included four ML algorithms to build models however there are several supervised methods such as decision trees, gradient boosting algorithms, etc were not included. The author did not provide any reasonable context to include these four and ignoring other algorithms.

2. MCC score produces a more informative and truthful score in evaluating binary classifications than accuracy. The author did not include MCC and  F1 scores in this study to assess the models.

3. In the present time, advanced algorithms such as recurrent neural network and other deep learning methods combined with Natural language processing based algorithms and features such as word2vector outperforming conventional machine learning models to classify protein and peptide sequences. However, the author did not use any advanced neural network-based method to compare the results.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: IDENTPMP: IDENTIFICATION OF MOONLIGHTING PROTEINS IN PLANTS USING SEQUENCE-BASED LEARNING MODELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript has greatly improved, addressing all the comments provided based on the clarity, unambiguous and professional English.
Experimental design: The revised manuscript has addressed all the concerns and greatly improved.
Validity of the findings: The revised manuscript has addressed all the concerns and greatly improved.
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: IDENTPMP: IDENTIFICATION OF MOONLIGHTING PROTEINS IN PLANTS USING SEQUENCE-BASED LEARNING MODELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors Liu et al developed IdentPMP- an SVM based predictor as a webserver, for predicting plant moonlighting proteins. The authors tried five different classification methods of which they claim to discover SVM to be working best.

The major strength is the right target of problem statement, that there are no plant moonlighting protein predictors. IdentPMP, being the first of its kind has merit and may benefit the plant community.

The limitation is of unbalanced data size for training and testing, with low number of training and test cases. The metrics of evaluation also do not show particularly great results.
Experimental design: 1.	Authors used several approaches but decided SVM is the best, while table 1 does not really support this inference. I think nearly all metrics being similar, overall RF seems to be the best. Deciding upon SVM based on a closely matching AUPRC -which again is a strange metric for evaluation instead of AUROC, unless the base positive ratios is known/clearly mentioned. To evaluate AUPRC, isn’t normal for all types of readers and authors did not mention it for clarity. There isn’t much to choose between RF and SVM. It appears that SVM was used and other methods were thrown into the mix as an afterthought.
2.	After the predictor was made, I expected some metrics to be shown for training set as well as test set. Then followed by a prediction to show its use case for some plant proteome, to demonstrate new findings, which is missing.
3.	Did not understand the feature engineering section as it is filled with too much jargon, at least for me. Too many abbreviations without context. The users may not be SVM researchers.
4.	Why were 80% features chosen? Rationale of many such choices isn’t very clear. Another is AUPRC, no cutoff description means users cannot decide what is good and why. AUPRC is not straight forward like AUC, why was it chosen if the rationale and interpretation cannot be given.
Validity of the findings: 1.	A gain of 13.89% in AUPRC and 10% in AUROC needs to be explained in context, over other methods as 0.41 (AUPRC) and 0.66 (AUROC) appear to be quite low values. This is not clear how these % improvements numbers are calculated. Also, the increase of 10% to get 0.66 may denote the previous best was 0.56 (?) for which other predictor (which is not plant moonlighting one) was used. It needs to be explained in better context because a method nto suited is bound to give such results (but 0.56 for a generic predictor is still bad and unexpected). How does MPFit perform for its own use case scenario?
2.	The paper requires a careful editing to correct for weak reasoning and arguments. It does not help the reader when they have to assume and fill in the interpretations and reasons behind the work, or interpretation of data properly. Flow of text from one argument to another seems to be missing.
Additional comments: 1.	How much time does the tool take to run? Mandating email address for every single sequence may not be prudent (even for example sequence?). As reviewer, I could not use the tool due to this reason.
2.	The language is a challenge to read with lots of grammatical mistakes. A careful editing is needed. Some examples are -
i.	Line 23-24, change “by ourselves” to “in house”
ii.	Line 45, change “overturned,” to “overturned by”
iii.	Line 52, delete “species’”
iv.	Line 55, perhaps authors meant “genomes” instead of “genes”
v.	Line 57, delete “very” , change “important,” to “important that”
vi.	Line 58, change “some” to “effective”
vii.	Line 59, change “some” to “the”
viii.	Line 70, delete “ tools regarding”
ix.	Line 71, delete “prediction tools to identify moonlighting proteins”
x.	Line 77, change “mainly identifying” to “ mainly to identify”
xi.	Line 78, change “organisms,” to “organisms, but” AND change “Considering that” to “ The”
xii.	Line 79, “microbial” to “microbes”

I gave up after this and may have certainly missed many places.
3.	Line 119, 2/3 rd data for each species is practically impossible for many species with 4, 2, 2, proteins.
4.	Features and feature class needs to be clearly defined, are they same?
5.	Sudden use of abbreviations at line 236?
6.	Results discussion /Conclusion needs proper thoughtful writing. Nothing is obvious what is claimed. 0.66 AUROC isn’t a great result.


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: IDENTPMP: IDENTIFICATION OF MOONLIGHTING PROTEINS IN PLANTS USING SEQUENCE-BASED LEARNING MODELS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has made necessary changes to improve the content, the flow has improved a lot and the errors have been corrected.
Experimental design: The revised manuscript has addressed the concerns and is improved a lot.
Validity of the findings: The revised manuscript has addressed the concerns and is improved a lot.
Additional comments: No further 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 NEW ANDEAN TREEFROG (AMPHIBIA: HYLOSCIRTUS BOGOTENSIS GROUP) FROM ECUADOR: AN EXAMPLE OF COMMUNITY INVOLVEMENT FOR CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, providing strong results that supports the recognition of a new species of Hyloscirtus. The figures have excellent quality, but some of them lacks scales. The tables with the call parameters could be improved including available data from published articles on the Hyloscirtus bogotensis species group.
Since I am not a native English speaker, I cannot evaluate the professional English along the manuscript.
There are several issues regarding the guidelines for the authors citation along the manuscript, missing references, and other references not cited in the text. I suggest reviewing all the authors guidelines regarding this subject.
Experimental design: The authors achieved all the aims proposed in the manuscript, and their finding are in the Scope of the journal. The methods are sufficient and can be replicated. I only suggest that they can include appendix providing all the genetic sequences information used in the phylogenetic analysis, and to provide a methodology of the uncorrected p genetic distance method employed.
Validity of the findings: The manuscript achieve conclusive and robust results and discussion. All my comments and suggestions are in the pdf.
Additional comments: The manuscript brings a description of a new species of Hyloscirtus from the Ecuadorian Andes, providing strong data (morphological, acoustic, genetic, biogeography) that supports its recognition. They also provide several new information regarding a new candidate species of the H. bogotensis group. I think the call description could be improved, providing more data about the call, and a better comparison with other species within the H. bogotensis group.
I inserted all my comments and suggestions in the pdf.
Overall, the manuscript brings an excellent contribution to the taxonomy, systematics and conservation of the Hyloscirtus bogotensis group, and incorporates a great example of community involvement in conservation.
I think the manuscript could be accepted after minor revision.


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

Input:
Title: A NEW ANDEAN TREEFROG (AMPHIBIA: HYLOSCIRTUS BOGOTENSIS GROUP) FROM ECUADOR: AN EXAMPLE OF COMMUNITY INVOLVEMENT FOR CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper presents a valuable set of data for describe a new species.
Experimental design: There are low  bootstrap support (68) between the new species and the sister species (the both species are very similar).
The phylogenetic position of the new species in the figure 1 is not clear. Phylogeny needs to be rebuilt with additional bootstraps.
Delete the localities of field work, is not the same where species occurs
the diagnosis need focus is the MAIN characters that distinguish the species. There is a lot "diagnosis" characters.
I strongly suggest to make a PCA using all morphometric data between species.
a t-student analysis are not enough to separate species
Validity of the findings: The findings are relevant to taxonomy of the group.
Additional comments: Phylogeny need rebuild
A PCA between species
Reduce the "diagnosis"
A clear and consistent figures
Discuss the limitation of your study (molecular markers)
Present the measurements of males and females
Present a table comparing "diagnostic characters" between species
There are other additional suggestions that are in the attachments


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 NEW ANDEAN TREEFROG (AMPHIBIA: HYLOSCIRTUS BOGOTENSIS GROUP) FROM ECUADOR: AN EXAMPLE OF COMMUNITY INVOLVEMENT FOR CONSERVATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The auhtors present a relevant data to describe a new species of Hyloscirtus. The MS was significantly improved in methodology and results.  The MS are well done and the description of the new species is clear.
Experimental design: Well done.
Validity of the findings: The MS presents a valuable set of data for describe a new species and improve the taxonomy of the genus.
Additional comments: not comments