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: DEVELOPMENT OF A HOMOGENEOUS TIME-RESOLVED FRET (HTRF) ASSAY FOR THE QUANTIFICATION OF SHIGA TOXIN 2 PRODUCED BY E. COLI
Review round: 1
Reviewer: 1

Output:
Basic reporting: This report by Nakamura et al, describes the development of a novel quantification method to quantify Shiga toxin 2, an important virulence factor of STEC strains associated with more severe diseases. This new methodology will be a valuable tool to better understand the virulence potential of specific strains in order to control STEC life-threatening complications.

Please find below some comments I suggest to help the readers understanding the general background and objective of this study.

1. I suggest to present a brief description in INTRODUCTION of the using of the HTRF assay for the quantification of toxins: explain that this is not the first report to apply this technology in bacterial toxin detection. Please give some examples, i.e., Cohen et al., DOI 10.1007/s10895-014-1354-7, for the detection of Bacillus anthracis PA.

2. Present also in the Introduction Section, a background of the methods available for Stx2 quantification including the VTEC-RPLA kit presented in results. Which is the disadvantage of the VTEC-RPLA kit? VTEC-RPLA seems to be also a simple method that does not require several steps to obtain the final data, thus, RPLA is applicable to a large number of samples. Please, explain why there is a need to develop a new method.

3. From the clinical and practical point of view, it will be important to investigate if the methodology present in this study can detect and quantify Stx2 in stool samples and/or in STEC culture supernatants (without any additional treatment, such as mitomycin C treatment and sonication). Have the authors consider this possibility?

4. In the paragraph of “Applicability of the HTRF assay to other Stx2 subtypes”, discuss the consequences of overlooking Stx2f by HTRF. Are Stx2f-producing STEC strains associated with less severe disease?

5. Conclusion, line 250: .... HTRF assay for quantifying “Stx2a”.... Should be “Stx2” or “several Stx2 subtypes”.
(In Table 1, the production level of all Stx2 subtypes, except Stx2f, are presented).

6. Conclusion, line 253. Please explain the disadvantage of “an endpoint assay to detect Stx2.”
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: DEVELOPMENT OF A HOMOGENEOUS TIME-RESOLVED FRET (HTRF) ASSAY FOR THE QUANTIFICATION OF SHIGA TOXIN 2 PRODUCED BY E. COLI
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Nakamura et al. describes a novel assay that relies on homogeneous time-resolved fluorescence resonance energy transfer (HTRF) for the quantitation of Shiga toxin 2 produced by E. coli.

The paper is well written and the methods employed are sufficiently clear; the results are also properly described and the figures are of an appropriate quality. The raw data is also accessible as per journal standards.

I noted a couple of typing/grammatical errors:

-Line 27: "The major virulence factor of STEC is Shiga toxin (Stx1 and Stx2)" should be "The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2)"

-Line 120: There is an additional parenthesis.
Experimental design: I have the following concerns about the experimental design:

1-The authors should have compared their quantitative data output from the HTRF technique to that from another quantitative assay, such as the widely available ELISA for these toxins. Comparisons to a semi-quantitative assay, with an output in 'units', such as reverse passive latex agglutination (RPLA) does not allow for proper evaluation.

2-There is another fluorescence resonance energy transfer-based assay for assessing levels of Shiga toxins, albeit with a narrower range (based on the literature), as indicated by the authors. How does this assay compare in a lab setting, in practice, to the one developed by the authors?

3-Would this assay work on non-purified protein (the authors tested the assay with purified proteins)? Would it work on patient samples or environmental samples such as water? The assay should be tested in practice.

4- In the introduction section, the authors make the argument that that there is a need for the novel assay since ELISA has many steps; however, the HTRF assay has an incubation time of 18 hours. Hence, it takes time more time than RPLA or ELISA. Hence, what is the relevance of developing the novel assay when an ELISA yields results faster, is a tried-and-true assay and seemingly has a comparable range?
Validity of the findings: The authors did not  provide statistical analyses and measures for the four pillars of a novel lab assay: accuracy, precision, specificity and sensitivity.
Additional comments: I recommend the authors evaluate their assay by comparing it other quantitative assays that are currently available. As a novel assay, it should be tested for its accuracy, precision, specificity and sensitivity as well with appropriate statistics being reported. On the other hand, I also recommend the authors clarify the relevance of developing the new assay particularly when "gold standards" of quantitative testing are available.


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: DEVELOPMENT OF A HOMOGENEOUS TIME-RESOLVED FRET (HTRF) ASSAY FOR THE QUANTIFICATION OF SHIGA TOXIN 2 PRODUCED BY E. COLI
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have generally addressed my comments pertaining to this section.
Experimental design: Although the authors have generally addressed my comments, I still believe that an established quantitative assay should be used for comparison purposes. The authors indicate that the ELISA is not available in Japan. Are there importing or financial restrictions that prevent the authors from obtaining this assay?

If there is a justifiable reason, then I suggest the authors at least acknowledge their awareness that accuracy, precision, specificity and sensitivity of their novel assay should be tested in comparison to an appropriate quantitative assay in their conclusions.
Validity of the findings: Same as the previous round of revision.
Additional comments: Nothing further to add.


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

Input:
Title: DISTRIBUTION AND ABUNDANCE OF THE LAND SNAIL POLLICARIA ELEPHAS (GASTROPODA: PUPINIDAE) IN LIMESTONE HABITATS IN PERAK, MALAYSIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is clear, and generally, the paper is clearly written.

Literature: I think the literature survey is not sufficient. The following sentence is simply not true: "Unfortunately, studies of individual species responses to its environmental variables are still lacking (Horsák et al., 2007)".
For example, species of the genus Vertigo have been studied in Europe with every imaginable methods, mostly in Poland and England and Scandinavia. The same is true for some clausiliid species such as Bulgarica cana and Ruthenica filograna, and Albinaria is the southern Balkans. I strongly advise the authors to perform a more detailed literature survey, because ecological studies focusing on single species are all over.
A land snail ecological paper without any citations of studies about Vertigo? Looks very strange.
Experimental design: Experimental design: Looks superficially good, and indeed provides good insights, but some parts are not described in sufficient details. For example, what about the size (age) of snails you collected? How many of them were juveniles? In this snail species the newborn babies are just a few mm large. How small were the smallest marked specimens?
Validity of the findings: The main result that the snail lives in the northern part of the hill but not on the southern part, and its explanation by limited dispersal ability sounds interesting, but I simply don't believe it. As a field person I known that collecting on the northern part of limestone hills will always provide more specimens and species than on the southern part, even if I cannot support this with published evidence. Literature survey, however, will surely provide some papers that state this. It is usually said that strong statements (like yours about the fine-scale distribution of P. elephas, and its explanation) need strong evidence.

The microchabitat survey was done only in a single month. What about the habitat characteristics during the entire year? The distribution of a species is determined by habitat characteristics throughout the whole year. I am not saying that your results are not publishable, just your conclusions should be smoothened since your field data are rather limited.
Additional comments: General comments: More thorough literature survey, and smoothen your conclusions please. Otherwise your data provide nice addition to our knowledge on habitat preferences of terrestrial 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: DISTRIBUTION AND ABUNDANCE OF THE LAND SNAIL POLLICARIA ELEPHAS (GASTROPODA: PUPINIDAE) IN LIMESTONE HABITATS IN PERAK, MALAYSIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is partly poor and terms used are not always clear.
(The authors will find some corrections in the pdf)
Experimental design: The research questions should be better defined.
(The authors should provide more background information on the study area, the taxon and on patchy distribution patterns of species in limestone habitats)

The methods are partly not described with sufficient detail and are partly difficult to follow.
(The authors will find specific comments on that in the 'general comments' and the pdf)
Validity of the findings: Some statements regarding the results of this study are very bold and partly too speculative. Limitations of this study are not discussed thoroughly.
(The authors will find specific comments on that in the 'general comments' and the pdf)
Additional comments: This study provides information on the small-scale distribution of a land snail species inhabiting a limestone hill in Perak, Malaysia, and gives valuable insights on the abundances, population sizes and distribution patterns as well as the association with environmental parameters. In the following you will find some more general comments on the study. Attached you will find a pdf, which includes more specific comments and corrections on the manuscript text as well as on some tables and figures.

The title (‘Some ecological aspects…’) is very unspecific. Consider changing it to fit it more to your specific results, e.g. by using ‘distribution and abundance’.

The introduction should me more focused on the aim of the study. E.g. the part about general species conservation in limestone habitats is too unspecific for this study and is not focus of the discussion later. In addition, this study focusses on patchy distribution patterns of species within an area. A section about this topic would strengthen the introduction and discussion.

A justification of the study area should be made. You studied only one limestone hill, but I guess this makes sense for the aims of your study. Please make this clearer (please see my comment in manuscript).

It would be helpful to say a few words about why you studied this species of land snails. For example, it is not known from the introduction that this species is ground-dwelling.

Your study consists of two sampling session events; the ‘pilot survey’ and the ‘CMR sessions’. It is quite difficult to follow, which plots were selected for each survey and where and when the environmental variables were recorded (see also my comments in the manuscript). It would be helpful to make this clearer. You could also think about giving a table with all study sites, showing which survey was done and when and which variables where recorded and when.

Also, I would recommend to rename the ‘pilot survey’, as this is not a preliminary study (as far as I understood), but a full survey of equivalent importance to the subsequent survey. You could think of a name which is based on the data you collected, such as ‘survey of presence/absence data’.

A statement about which survey was done to tackle which research question, would be very helpful.

More details about the marking of snails in the CMR session would be helpful (see comment). Also, it is not clear which snails were marked in Additional file 1 (see comment).

The topographic features, recorded in this study (e.g. aspect and ruggedness index) should be explained.

Terms are used in an unclear way throughout the manuscript. E.g. ‘population ecology’, ‘vegetation composition’. (please see my comments in manuscript). These terms should be explained and used in a comprehensible way.

Tables should include information about the respective units of the values given. For example, they are missing in table Additional File 4 (see comment in manuscript).

You state that soil temperature is explaining the occurrence of the studied snail. Although you tested other parameters for a correlation which snail abundance, you did not show a test for this parameter. I find this statement a bit speculative and you should be more careful with this interpretation. In addition, it might help to test for a correlation (same for air temperature and humidity).

In addition to my comment above, you could show a graph of the correlation of the plants that were identified as being correlated with the abundance of the snails.

Some statements in the discussion are relatively bold (e.g. that ‘all Malaysian land snails have been little studied…’) and should be made more careful. It would help here to focus more on the own study and on ecology, population densities, small-scale distributions or habitats.

Some statements about the results of this study are also very bold and partly speculative (see comments in the manuscript). For example, you say in the conclusions that the habitat, topography, microclimate, and vegetation variables affect the occurrence of the studied species. What you found, is a correlation with some of the variables. But for most you did not find any correlation. Try to make statements more careful here and be more specific about what your data show.

In another example you state that the leaf litter from some plants could be suitable food sources for the snails. Is there any evidence for that? You mentioned some (unpublished?) observations that the snails feed on leaf litter. Is this statement derived from these observations? Please make this part clearer.

I miss in the discussion a part about the statistics and the limitations of this study. E.g., you have a very high number of plant species in your plots. And species are differing a lot in different plots. Could the correlation with the abundance of snails you found for some plants just be caused by random effects?

Include a part about other variables you did not measure, but which are know to be important for land snails (e.g. pH or calcium availability). Is there a reason why you did not measure them? What about the kind of rock? Foon et al 2017 mentions that there are differences in the rock habitat in this area. You should discuss them. Are you sure that the environmental variables you recorded are important for the occurrence of your species or did you miss some parameters, which could also explain the absence of this species in some of the plots?

The statements in the conclusion are quite bold on one hand, and on the other they are repetitions of what was said earlier. You could strengthen the conclusion by including a part on species conservation. E.g. what do your results add to the knowledge about species (or population) conservation in limestone habitats? How useful is it to do species inventories based on only very few localities? What do your results specifically mean for conservation of the studied species; is it enough for this species to protect suitable habitats?

The English is relatively poor. I did some corrections in the text but it would be good if a native speaker would check the final text again.


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: DISTRIBUTION AND ABUNDANCE OF THE LAND SNAIL POLLICARIA ELEPHAS (GASTROPODA: PUPINIDAE) IN LIMESTONE HABITATS IN PERAK, MALAYSIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Advice: Publish after comments under C) and D) (see below) have been addressed by the authors, and the comments under A), B) and E) have been considered by the authors.
Experimental design: A) Suggestion to reorganize the paper a little. Starting at line 75 the initial observation is outlined (patchy occurrence of Pollicaria, and its absence on a part of the limestone hill). I suggest that the research question  is spelled out after line 79, and that this paragraph (starting at line 75) is included in a separate chapter. The paragraph starting at line 80 could be added as introduction to the chapter Material and methods.

Considering the somewhat inconclusive results, it may be useful to write in terms of the ‘initial hypothesis to be tested’ in this paper (‘Environmental parameters determine the patchy distribution of Pollicaria’) to which the answer is, in the chapter conclusions ‘well, eh, none that we measured, except perhaps soil temperature’. Then the authors could speculate about what else stops the animals from crowding the hill, such as homing behavior.
Validity of the findings: The paper is based on rigorous field work to explain an initial observation. The resulting dataset seems, unfortunately, to yield somewhat inconclusive results, as happens sometimes. This outcome is perfectly acceptable to the Journal, as stated under ‘validity of the findings’.
Additional comments: B) Title seems to general, too vague; it does not cover the contents of the paper. A suggestion, using a sentence in the summary: ‘Effect of environmental variables on the abundance of the land snail Pollicaria elephas (Gastropoda: Pupinidae) in a limestone habitat in Malaysia’. Or, even more in line with the initial hypothesis to be tested: ‘Do environmental parameters determine the patchy distribution of Pollicaria (Gastropoda: Pupinidae) in a limestone habitat in Malaysia?’

C) The text is often verbose. I suggest going through the text sentence by sentence and take out redundant words and rephrase sentences. Examples:
161: ‘before proceeding to statistical analysis’: ‘proceeding to’ can be deleted
177: ‘There were no living snails nor empty shells found in all 11 plots at the southern and the central 178 part of the limestone hill. On the other hand, we recorded living snails and empty shells in six of 179 the seven plots at the northern part of the hill’ (50 words): ‘Individuals were found in six out of seven plots on the northern part of the hill; none were found in plots on the central and southern part’ (27 words). The information that individuals include living animals as well as empty shells is given earlier.
227: ‘There are 43 species of vascular plant that…’ . ‘Altogether 43 vascular plant species… ’

D) Language needs to be checked for incorrect grammar and syntax.

E) There may be some glitches in logic. The manuscript should be checked throughout. Possible examples (Please note that I am a geologist and taxonomist, not an ecologist, and read this text as an outsider):
46: ‘Of the 64 critically endangered terrestrial animal species in Malaysia listed in IUCN Red List, 26 belong to land snail species’. Could it be that the high number of snails on the list is because other group of limestone invertebrates are less systematically studied? The suggestion here seems that limestone land snails are most affected, while the message the authors may have in mind is that limestone biodiversity is threatened, altogether thousands of species (including the tiniest arthropods etc.), and among these many narrow endemics.
200: Soil temperature: Measurements were done in July, the northern summer during which the North flank of the steep hill receives more sunlight. Could it be that in December the South flank generally receives more sunlight, and that measurements during that period would yield opposite results?
Fig. 4 seems not very conclusive to me as far as the relation between soil temp and # individuals is concerned.
276: ‘there were no obvious differences in environmental variables in terms of climate, topography, habitat, and vegetation between the northern and southern part’: but authors claim that soil temperature is generally higher on the northern flank.
284: Stringer et al study homing behavior of Placostylus, different mollusks. The most parsimonious explanation may be that the animals find it difficult to bridge areas with less favorable habitat to isolated spots with preferred habitat.
304 to 310: A food relation between abundance of Pollicaria and the plant species may be a step too far. The more parsimonious reasoning may be that both plants and snails prefer the same aspect of the local environment (an aspect not covered in this study, perhaps).
336: Fig. 4 seems to suggest, vaguely, the opposite


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: DISTRIBUTION AND ABUNDANCE OF THE LAND SNAIL POLLICARIA ELEPHAS (GASTROPODA: PUPINIDAE) IN LIMESTONE HABITATS IN PERAK, MALAYSIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English has improved, but in many cases (especially in the newly added text parts) the language is still not clear and needs further work. Most of my General Comments (see below) refer to language issues. Again, it would really help if a native speaker could check the text, especially any newly added text parts.
In addition, there are still many inconsistencies in the terms and names used.
Experimental design: The research question is much better defined now. The methods are clearer and more detailed, however, there are still some missing and ambiguous parts that need further improvement (see my General Comments).
Validity of the findings: I do think the results are worth to publish, but the number of plots, where snails were present, is low and the correlations found, are sometimes weak. Most statements concerning the correlations of variables with snail occurrences or abundances are thus far too bold and need to be scaled down considerably (see my General Comments for specific examples). In addition, all limitations need to be addressed very clearly (the changes on the manuscript that have been done are still not sufficient). The comparison of other studies on land snails with own data should be more specific and discussed in a meaningful way.
Additional comments: Please see the attached PDF file for the general 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: DISTRIBUTION AND ABUNDANCE OF THE LAND SNAIL POLLICARIA ELEPHAS (GASTROPODA: PUPINIDAE) IN LIMESTONE HABITATS IN PERAK, MALAYSIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English is, if not always strictly following grammar, clearly understandable. I suggest a number of small improvements in the text, and I advise a check by a native speaker to put the linguistic details right.
Generally, the reasoning in the manuscript is unambiguous; I left comments where I think that the text can be improved.
The structure of the manuscript follows professional standards. Figures and tables are of good quality and relevant to the text. The paper is self-contained.
Experimental design: The paper contains original research relevant to the Journal. The authors identify a gap in our knowledge, describe experiments to fill that gap and discuss the results, following prevailing standards and in sufficient detail for replication experiments.
Validity of the findings: Underlying data are provided. I cannot judge the statistical aspects of the experiment. Conclusions and speculations are separated.
Additional comments: I left comments and some improvements of the text with track changes in the manuscript, see file attached (attached to a separate mail, because the website refuses to accept the file as attachment). I think the paper can be accepted after the issues in the comments have been addressed, and after a language check-up of the text.


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

Input:
Title: DISTRIBUTION AND ABUNDANCE OF THE LAND SNAIL POLLICARIA ELEPHAS (GASTROPODA: PUPINIDAE) IN LIMESTONE HABITATS IN PERAK, MALAYSIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The English has clearly improved and the language is clear and unambiguous now.
Experimental design: The experimental design and all methods are described with sufficient detail now.
Validity of the findings: The results are well described and discussed. The limitations are addressed clearly now.
Additional comments: Please see attached document for my comments/corrections.


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: HONEY BEE (APIS MELLIFERA) COLONY STRENGTH AND ITS EFFECTS ON POLLINATION AND YIELD IN HIGHBUSH BLUEBERRIES (VACCINIUM CORYMBOSUM)
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are some clarity issues in text and minor changes that could be done to improve figure presentation.  Please see comments for authors below for details.

Raw data: items listed in metadata do not match the data sheets in the supplemental excel file?
Experimental design: There is no control treatment for brood disease evaluation.  This might be OK for the purpose of reporting the observed change over time but not to be presented as an effect of hive density or blueberry pollination.
Validity of the findings: Please check if your data meet the  assumptions for Pearson's correlation analysis. Some of the count-based data may be more compatible with non-parametric alternatives.
Additional comments: This paper presents a study in which the authors investigated whether similar benefits of employing more hives to pollinate blueberries could be achieved by increasing colony strength instead of increasing hive density.  The authors also compared three methods of evaluating colony strength (frame area coverage, cluster size determined by frame counts, and returning foragers at hive entrance) and concluded that the least invasive method could reliably predict colony strength.  Lastly, the authors reported increased EFB infestation in colonies at the end of the blueberry pollination season.
The manuscript for most part is well written with clear language although it feels less polished toward the end.
The experimental design is sound for an observational study.  I wish a “control” component had been included to the EFB evaluation, for example, hives placed at a lower density (to test if the increase infestation is associated with density) or hives not placed in blueberry fields.  Absence of the control treatment is not a deal breaker given the primary objective was about hive strength and yield, and the difficulty of conducting large scale field experiments with honey bees.  However, the authors need to be very careful about how they interpret the results.  What you found was an increased level of EFB in most colonies after a period of time.  Without a control treatment, you cannot conclude that the increase is related to aggregation of hives or blueberry pollination. It could simply be the phenology of the disease as well but there is no way to distinguish these causes in this study.
Selection of the statistical analysis— please check the underlying assumptions of Pearson’s correlation analysis to make sure the dataset meets the requirements. Otherwise use nonparametric alternatives.
Other minor issues:
Several brood diseases were checked during hive inspections, according to Methods. Why only EFB data is reported?  Although you’ve set monitoring EFB as one of the objectives, data on the other diseases would be equally valuable.  I would suggest summarizing the findings in text and/or including the data in the supplemental file.
There are some ambiguities that could benefit from additional clarification. Please see line-by-line comments below.

Line 40: Data or statistics of the 45,000 kg/ha increase is not presented in results?   Only an estimate of 4450 kg/ha increase for every 10 bees/min is presented (L258).  Then in Discussion you say there is a 70,000 kg/ha difference?
L 77-79: Please include citation for the recommendation of counting returning bees.
L 96-97: Arrington & DeVetter 2018 reports an average of 117 returning bees/minute in randomly selected colonies in their study.  But here you say none of the fields were stock with colonies with an average minimum 100 returning bees/min.  Did I miss something?
L97: If “this study” refers to Arrington & DeVetter 2018, their bee count data were recorded at air temperature above 18 degree C, according to their methods.
L98-100: at a quick glance, the message I get from the Arrington & DeVetter 2018 paper is that the colony foraging strength was above the 100 bees/min threshold and did not significantly differ between their treatments of 10 vs. 20 hive/ha, although the raw data of colony weight were not presented.  So they did validate colony strength in their study.  It sounds odd to speculate that the colonies in the cited study were consistently below grade without evidence.  Perhaps the authors have access to the unpublished data but readers would not know that. Either way, I think this paragraph could be revised to reflect the citation more accurately.
L125-127: It seems redundant to state again the three main objectives that you already explain clearly in this paragraph.  Also, you’ve only mentioned the assessment of EFB, which is only one of the many aspects of honey bee health.  Saying you are assessing “the effects of blueberry pollination on the health of honey bees” is an exaggeration.  “Define honey bee colony quality and strength parameters” does not make much sense as a sentence.  I suggest deleting these sentences in L125-127.
L137: Spell out OSU at the first mention.
L140-141: are n =166 and 161 the total number of colonies stocked in fields (so ~40 colonies were assessed each year) or the number of colonies (of the 25%) assessed?  Please clarify.
L148: Instead of “2-3 times per season, report the exact dates or information on the length of interval between assessments.
L148-151:  Conflicting information— in L148 you say frame-by-frame and cluster counts were done 2-3 times per season but then you say the assessment was done twice in 2019 and only once in 2020.  Could you clarify?
L168: just a thought related to the threshold temperature – has there been any studies on correlations between colony strength (bee population) and flight activities in cold weather?  I wonder if a strong colony is better at maintaining its cluster temperature than a weak colony, and therefore allows workers to start foraging at a lower ambient temperature threshold?  Perhaps this would further emphasis the importance of supplying strong colonies for pollination?

L190: Why only honey bees and bumble bees were recorded? Do other bees visit blueberries?
L209: Why did the analysis only compare EFB, but not the other diseases you’ve also recorded (AFB, chalkbrood, and sacbrood)?
L211-216:  some of the parameters are not continuous data (e.g., cluster counts and bee counts).  A non-parametric comparison such as Spearman’s rank-order or Kendall’s Tau may be more appropriate for these types of data.
L238: Provide descriptive statistics for incoming flight counts (e.g. means, range etc.)
L246 See comment for Figure 4 below.
Discussion:
L276, L279-280:  Could the increase in EFB infestation over time be explained by the phenology of the disease? In early spring honey bee colonies typically do not produce much brood, and therefore the absence of the brood disease symptoms could mean (1) absence of the pathogen in the colony or (2) the bacterial pathogen is still in the spore form due to a lack of brood that allows them to propagate.  Ideally the experiment would include a control group of bee colonies that were not moved to blueberry fields, and demonstrated that the control hives had less EFB.  Since there is no such control in this study, I feel the authors should at least discuss alternative explanations as well, and not fixate on the conclusion of hive aggregation in for crop pollination as the reason of increased EFB detection.
After reading the paragraph about EFB (L346-356), I would suggest deleting the sentence in L280 about reducing EFB as a potential benefit of reducing colony density and keep the EFB discussion in one place.  The single statement at the beginning of the discussion could be misleading.
L291-295.  This is a great discussion on predicting yield benefits based on number of returning bees at hive entrances, and would be a great tool for growers to evaluate whether their rental colonies meets their pollination target.  However, beekeepers at large still use frame counts to determined colony strength quality during inspection before delivering the hives to crop fields. Could you include a brief discussion on how the bee counts convert to frame numbers in respect of the projected yield benefits?
The final Discussion paragraph on the financial aspects belongs together with the paragraphs that calculates yield increase (L284-301) and hive rental policies (L311-323) to improve the flow of content.

L377.  The first sentence (colony strength increases blueberry yield) doesn’t make sense. I suggest “target yield could be achieved by increasing the strength of honey bee colonies without increasing colony density”.

L384: Delete “without aggravating brood diseases”.  Your experiments did not test if the prevalence of brood diseases was related to hive density.

Figure 1:  Change the Early box and whiskers to dark color lines.  Present both Early vs. late in the same color unless there is a reason to use different colors.

Figure 3. I think presenting scatter plots with P values might be more informative than the ellipses alone.  Also, check to make sure your date meet the linearity assumption for Pearson’s correlations.
Caption: Repetition of “adult (adult)” and “brood (brood)” is unnecessary.  Specify that these are frame measurements from the Liebefeld method.

Figure 4: Rotate the figure.  Present the equation as y = 0.8x + 3.
I think the trend line interpretation of every 13 bees = 1 increase in frames of adult bees is quite clever.  This would probably be better perceived than the very 10 bees = 3.8 frame in text (L245-247) because a whole frame is easier for a reader/beekeeper to visualize than fractions of frames.

Figure 5. Again, I think it would be more informative to also present the scatter plots.

Figure 6. Rotate figure and present equation as y = 445x + 14427

Supplemental Materials:
-	Some of the datasets listed in Metadata are apparently missing: total, alldata, diseasesplus2020, alldata2020, alldatabothyears, total2020
-	What is the difference between flight and newflight?
-	Does the “Disease” column present the number of different diseases recorded?
-	What are the measurement units for Adult (e.g. 14.25) and Brood (e.g 2.25)?


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: HONEY BEE (APIS MELLIFERA) COLONY STRENGTH AND ITS EFFECTS ON POLLINATION AND YIELD IN HIGHBUSH BLUEBERRIES (VACCINIUM CORYMBOSUM)
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: General comments
	This study investigated the blueberry pollination system with honey bees, in particular how colony strength impacts yield in the crop as well as disease (European foulbrood) in the colonies. Beekeepers have been noticing an uptick in disease when pollinating blueberry, and the hypothesis is that the stocking density may be too high leading to facilitated pathogen spread. The study fixed stocking density at 10 hives per hectare, but measured the variation in the colony strength by quantifying nest variables (bees and brood) as well as entrance flight counts (bees per minute). They found that EFB did indeed significantly increase in both years, and that colony strength varied significantly and was overall fairly low. They also found that yield was positively associated with colony strength, thus increasing colony strength rather than stocking density may help mitigate disease spread while increasing yield.
	Overall this is a nice study that constitutes quite a bit of work. My first read was a bit less generous, thinking that there was some circular reasoning in their hypothesis (stocking density and colony strength are both means to increase forager population) but in the end I think it’s a fair enough question that was able to be teased apart. My main criticism is how the pathogen confirmation results were largely dismissed, especially given how EFB can be difficult to diagnose in the field and overlaps symptomatically with several other conditions. Nonetheless, I believe that this study nicely adds to the literature in this area and helps reinforce some useful recommendations for both growers and beekeepers.

Specific comments
1.	Line 22-23: perhaps move “crop” from early in the sentence to just before “yield’ to make it more clear that the subject is the crop and not the colony? Also, consider changing “increasing” to “high” since the stocking density was actually not varied in this study (I understand that the densities are have been increased historically but that is out of context in the abstract).
2.	Lines 58-59: most wild bee species have been shown to be more efficient pollinators than honey bees ‘at the individual bee level,’ so please add the latter clause since native bee populations cannot be easily manipulated.
3.	Lines 69-70: I found this hard to believe, and even a quick Google search found several papers (The impact of honey bee colony quality on crop yield and farmers' profit in apples and pears; Geslin et al. (2017) AGRICULTURE ECOSYSTEMS & ENVIRONMENT; Comparison of Honey Bee (Hymenoptera: Apidae) Colony Units of Different Sizes as Pollinators of Hybrid Seed Canola; Ovinge & Hoover (2018) JOURNAL OF ECONOMIC ENTOMOLOGY). Change to “few” and avoid unnecessary hyperbole.
4.	Lines 90-92: there is a really nice recent meta-analysis on this topic that should be cited here: Rollin, O., & Garibaldi, L. A. (2019). Impacts of honeybee density on crop yield: A meta-analysis. Journal of Applied Ecology, 56(5), 1152-1163. doi: 10.1111/1365-2664.13355
5.	Lines 110-114: stocking rates, increased disease transmission, and competitive stress are all confounding variables that can lead to increased EFB rates. There is also another factor, exposure to fungicides, that is not discussed here but is highly pertinent to this system.
6.	Lines 177-180: would be better to briefly describe the molecular methods in Wood et al. 2020, including sampling and handling procedures in getting the samples to the lab. EFB is a particularly problematic disease to diagnose from symptoms, so it might be good to articulate those as well. Since one of the main findings from this study centers around these diagnoses, it is surprising that there is only one sentence in the methods devoted to their detection.
7.	Lines 195: here it states that  floral visitation measures were taken less than 15km/hr wind (also stated lines 166 during entrance counts) but in line 79 is states 4.5 m/sec when referring to the recommendations. These appear to be close but not equal (my calculation is 16.2km/hr), so you might wish to keep these units consistent.
8.	Lines 216-218: I understand the reasoning for dropping colonies that were not measured within 2 days from each other (although that is fairly conservative), but I don’t understand the reasons for dropping colonies without full data. If these were included, how would the correlations differ?
9.	Line 237: It really would be better to report the actual pathology data than cite a personal correspondence. Both the methods and the results on the laboratory confirmations seem quite dismissive to me.
10.	Figure 5: theoretically, one would expect a non-linear relationship here since the proportion of foragers increases as colony population increases, but it looks like there’s more scatter in the data to tease out that nuance. Might be interesting to at least test to see if it’s a better fit, but not a big deal if not found in my opinion.


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: HONEY BEE (APIS MELLIFERA) COLONY STRENGTH AND ITS EFFECTS ON POLLINATION AND YIELD IN HIGHBUSH BLUEBERRIES (VACCINIUM CORYMBOSUM)
Review round: 1
Reviewer: 3

Output:
Basic reporting: As detailed below in Comments there are omissions to literature context that must be addressed. There's also logic thread issues that fall under "basic reporting" shortfalls.
Experimental design: As detailed below, I have a question about "year" as a covariate.
Validity of the findings: As detailed below, your data and analyses are sound, but you need to be more careful and contextualize your paper with the larger literature.
Additional comments: PeerJ Review of Grant et al. 2021. Honey bee (Apis mellifera) colony strength and its effects on pollination and yield in highbush blueberries (Vaccinium corymbosum)

1.	Line 82 “perform” misspelt
2.	Lines 133, 134 and elsewhere, use of ‘Varietal’ name apostrophes inconsistent
3.	Line 211 sentence beginning “We used Pearson correlation . . .” makes “variation in true adult and sealed brood populations” the grammatic subject, then proceeds to list it along with dependent clauses (1), (2), etc. thus confusing subject with predicate.
4.	Lines 214-215 related to above: you need a (3) in front of “estimates of pollination activity . . .” and a (4) in front of “estimates of colony population.”
5.	Line 213 no “and” in front of (2)
6.	However, I would elevate your putative (4) predicate line 215 “estimates of colony population” to the subject of the sentence! As it stands, here and throughout the paper to this point there is a confusion of cause and effect. Essentially you want to say (1) entrance flight activity is (2) a reliable indicator of adult bee population which is (3) positively associated with bee field density which is (4) positively associated with bee flower visitation rate which (5) improves harvest metrics. This logic thread is vague and muddled.
7.	In fact this problematic sentence discussed in #s 3-6 strikes at my central critique of this paper. I’m puzzled why the authors have ignored, evaded, or understated the obvious – that colony population, whether measured by Liebefeled, Nasr, or their clever entrance flight metric, is simply a proxy for the biologically meaningful effect – bee flower visitation rate. From the abstract through the introduction, this central relationship isn’t even mentioned until methods on lines 185. The authors ignore papers like Aras et al. 1996, Dedej et al. 2003 and Danka et al. 2019 that clearly show this relationship, while foregrounded distractions like “hive density” which is probably least important of all. I strongly urge the authors to position their paper into this larger biological context, and clarify exactly what they are showing.
8.	Line 221 “the” misspelt
9.	Line 219 as year is a categorical, not continuous effect, I don't think it qualifies as a covariate; a block? A plot? Please clarify this language and what you did.
10.	Line 226 Finally demonstrating a relationship between EFB and blueberry pollination is very useful.
11.	As is your hive entrance population estimator.
12.	Lines 262-263 here and through the Discussion and Conclusions be clear that “strength translates to visitation translates to yield.” Be careful of leaping from 1 to 3!


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

Input:
Title: HONEY BEE (APIS MELLIFERA) COLONY STRENGTH AND ITS EFFECTS ON POLLINATION AND YIELD IN HIGHBUSH BLUEBERRIES (VACCINIUM CORYMBOSUM)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Satisfactory.
Experimental design: Satisfactory.
Validity of the findings: Satisfactory.
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: MSBOTS: A MULTIPLE SMALL BIOLOGICAL ORGANISM TRACKING SYSTEM ROBUST AGAINST NON-IDEAL DETECTION AND SEGMENTATION CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Figure captions should be improved and axis-labels clearly explained.
Experimental design: Methods description needs some improvement.
Validity of the findings: No comment
Additional comments: The authors present a fairly detailed study of the development of a tracking system for multiple small biological organism. There are some gaps in the study and/or presentation of the research. The comments below are separated as major or minor.

Major Comments:
1)	Line 139: what is a decaying envelope factor (alpha)? Also, it is not clear whether the method is suitable for detection and tracking if the organisms can stop at a location for a considerable amount of time before restarting their motion.
2)	Line 197-198: what is the underlying idea or reason for optimizing assignment based on the lowest summed distance within two successive frames?
3)	Line 202-209: to eliminate the false positives when constructing an individual trajectory, a method is described which removes points as segmentation noise based on a threshold distance criterion. Would this approach produce good results if the velocities are spread over a wide range (say, an order of magnitude), or would this method produce reliable results only when organisms are moving at similar values of velocities.
4)	Is the method robust in situations where an organism can move both forward and backward?
5)	Figure 4: The caption should be more descriptive (even though description is provided in the text when discussing this figure). What are (a), (b), and (c)? What is sequence? If sequence cannot have non-integer values, then why show them? Is the Simple Tracker the manual connection of location?
6)	The measure of MOTP is the number of pixels. This may not help readers fully understand the tracking performance if two important information are not known: (a) average number of pixels an organism occupies? and (b) the size of the video frame in pixels?
7)	The MOTA measure should be discussed in more detail to help the readers develop a better intuitive understand. Based on Eq. (10), the ideal value of MOTA is 1. What does a negative value of MOTA (as shown in Fig. 4a) imply?
8)	Fig. 6 and 7: Moe description in the figure caption is needed. What’s the difference between the 9 boxes in Fig. 6? What are the colors representing in Fig. 6? Why is the unit of acceleration shown as pixel and not pixel^2?

Minor Comments:
1)	MOTP and MOTA are used in the abstract without defining it first.
2)	For computation of organisms’ centroid, details should be provided about the recommended video resolution (i.e., minimum number of pixels for each organism), and how the centroid computation is handled when two or more organisms are in very close proximity.
3)	Details need to be added on the average speed of organisms being tracked and the recommendations for corresponding minimum frame rate.


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

Input:
Title: MSBOTS: A MULTIPLE SMALL BIOLOGICAL ORGANISM TRACKING SYSTEM ROBUST AGAINST NON-IDEAL DETECTION AND SEGMENTATION CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is not always clear, with a number of areas in the manuscript where the English grammar needs to be improved. I have attached an annotated pdf where I have highlighted the issues.

The figures should stand alone, and they do not - the figures legends barely provide any information. The reader is forced to read the text to make sense of the figures. Suggestions for improvement can be found in general comments below.
Experimental design: The authors compare their tracking software to three others, and I would think that this would involve some statistical comparison, but the authors only seem to report the % difference, which I presume is across all the video sequences? It's certainly possibly to report standard error or confidence intervals around this mean, but the video sequences are not technically replicates, as they increase in complexity. Perhaps this is OK for this type of study though.

I am not convinced that the study is repeatable, because there is not enough information on the complexity of each video (see further comments below).

I am also not sure that this manuscript meets ethical standards - the authors state that ethics were not required, but they used adult zebrafish, so I assume that would have needed ethical approval for this. Perhaps this just requires a bit more clarification. In general, it's not entirely clear if adult videos were used - they are mentioned several times int the manuscript, but I don't see any mention of adults on the github repository.
Validity of the findings: I was not able to find the zebrafish image frames on the github site, even though I was in the correct project folder.
Additional comments: This manuscript proposes a new organism tracking system, MSBOTS, and uses a series of videos to compare the performance of MSBOTS and three other tracking systems. Most of the methods contain a lot of technical jargon, so I did find it hard to follow, but I could understand the results and figures (once I deciphered what each figure was showing, as the figure legends provide almost no detail). MSBOTS appears to perform well, and better than other systems when the video frames have things like bubbles, shadows, debris, etc (i.e., later video sequences). That said, there are many aspects of the manuscript that can be improved, particularly the presentation of the figures. I also think there needs to be a clear justification on what this paper adds in terms of the zebrafish aspect of the study. Is this the same analysis as presented in the previous published study, or is this new? I also question the statement that ethical approval was not applicable because no chemicals or medicines were being tested. No ethics are required for the invertebrates that are used, and not the zebrafish larvae, but any manipulation of adults (even netting into a different tank) would require ethical approval in Australia.

Comments to help improve figures:
Figure 4. Why not label the blue ‘proposed’ as MSBOTS? The legend for this figure needs to define the axis. Otherwise, the reader needs to try and work out what MOTP and MOTA stand for – figures should stand alone. The symbol legend for panel b seems to have moved as well. It’s also a bit odd to put the a, b and c labels below the figures and the panels are not defined anywhere, except in the text. It is not clear why the x-axis includes 1.5, 2.5, 3.5, etc. for panels b and c. This represents the 5 and 5 videos sequences, so should be labelled with whole numbers, as in panel a. Also, there should be no connecting lines between the points – the sequences are different videos (if I understand the design correctly), so should be represented as point values only. While I recognize that the lines help to illustrate the patterns, it is technically misleading to include them.

Figure 4 appears well before it is ever mentioned in the text, and figure 5 appears before figure 4.

Figure 5 panel b and c are quite blurry, the artemia and rotifer within anyways. This figure would be more meaningful if it showed a frame from the least complex and most complex video for each species.

No idea what all the panels in Figure 6 are. I think I finally inferred that they are the zebrafish videos, 1-10, with 5 and 6 combined, as in Figure 7. But what are all the colours, and what scale is time on, seconds?

Figure 7, why are videos 5 and 6 labelled with video 5 and 6 on the x-axis? To clarify, video 1 had 4 objects, video 2 had 3, video 4, had 5? Some of the videos have very high velocities compared to others? What are all the red crosses in the figures? Are these outliers? If so, these should be a different colour or symbol (open circles work well for outliers), as it detracts from the red line used to illustrate the median.

For Figures 6 and 7, simplify the figures – remove the y and x labels from all except the far left and bottom panels and remove the O from the object labels on the x-axis for Figure 7 – simply label as 1, 2, 3, etc...

Figure 6 and 7 are zebrafish only? This should be indicated in the figure legend – again, figure legends need to stand alone. Not entirely clear what figures 6 and 7 are adding to the study, in terms of displaying the data for each figure separately? Would it be better to summarise the data in some way, or perhaps these be better suited to the supplement?

Other comments:
The description of the data on the github repository is a bit unclear. It says “this dataset consists of 10 video sequences....(including zebrafish larvae, Artemia and Daphnia)”, but it’s actually 5 artemia videos and 5 daphnia videos in this MSBOTS project folder, so the authors just need to remove zebrafish larvae from this first statement. They then detail where the zebrafish larvae (should this be adults too, as adults are mentioned several times in the manuscript text?) can be found, in the moving zebrafish larvae segmentation and tracking dataset. However, I can’t seem to access the zipped video files for the zebrafish though.

As for the artemia videos, video 1 appears to be the more complex (includes bubbles) in the sequences for artemia, but I infer that 5 should be most complex from the manuscript text, as that is how it is set up for the zebrafish? Not clear how the daphnia videos differ – video frames from video 1 and 5 look the same? Again, as mentioned above, more detail on the video settings is needed, in order to make this study repeatable.

Line 78. I have used idTracker in the past, and we didn’t find that the image conditions were strictly constrained any more than any other tracking program, so perhaps clarify this statement a bit further.

Not clear why there are so many methods in the results and discussion section, lines 268-312?

Line 279: drasophila? I think you mean artemia!!

If adult zebrafish are included in this study, then there needs to be more detail (i.e., fish age, sex, line, tank dimensions).

Definitions for MOTP and MOTA do not appear till the section that is at line 306.

Line 350 refers to supplementary table S2 and S3, but these are not included for review. There also is no reference to table S1?

The references are inserted incorrectly, without commas or brackets to separate them from the text, which made it quite tricky to read the manuscript.

Minor comments:
Figure legend 1 should include ‘biological’

Line 126. What is the unit for the 0.075 value

Line 131. ‘the’ should be removed

Line 133. Comma required after ‘approach’

Lines 302 to 305, not sure why these are presented as bullet point – incorporate into text. Note that the word examples in these should be example.

Line 410, depository should be repository


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: DEVELOPMENT OF THE BREASTFEEDING SUPPORT SCALE TO MEASURE BREASTFEEDING SUPPORT FROM LAY AND PROFESSIONAL PERSONS, AND ITS PREDICTIVE VALIDITY IN JAPAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Abstract:
Authors note the scale captures support from lay and professional persons in Japan, but some item questions relate to more institutional or cultural supportive factors rather than support from an individual. I would advise authors to think further about what aspect of support the items capture, to ensure the developed scale is accurately described.

Introduction:
Given the study aim is to develop a relevant measure of breastfeeding support provided to mothers in Japan, I believe authors should elaborate and clarify how support is conceptualised, and how the items relate to these conceptualisations of support in the introduction. While authors mention four types of support and the discussion does provide further details, some of the items are difficult to categorise as support (such as information about formula); rather, they seemed to be capturing broader ecological contexts. This is not a problem, but authors may need to reframe what their items were developed to capture – rather than support itself, perhaps the items relate to a broader measure a breastfeeding-supportive environment? (It might be helpful for authors to think about the measures in relation to the ecological systems theory, which clarifies different “layers” of the environment and how they may impact individuals. Note, this is not a suggestion to include ecological systems theory into the manuscript; just a tool to help authors think about support.) I recommend redevelopment of the introduction which would clarify the purpose and methods of the study. My specific suggestions are as follows, which I hope the authors find helpful:
•	In the first paragraph, the scale of the public health issue around breastfeeding duration could be clarified further by providing basic statistics on breastfeeding rates across the globe. Currently the issue is only verbally described, but this should be backed up by evidence.
•	In the first paragraph three reasons are given on why many mothers stop breastfeeding earlier, but this are not exclusive. I recommend rephrasing to “because of problems such as” which clarifies that there are many reasons.
•	Throughout the introduction, evidence is discussed with assumed universal applicability of the findings. However, the determinants of breastfeeding likely vary between populations, as evidenced from significant variations in breastfeeding initiation rates and average duration length within and between populations. I would recommend authors are more specific about the population where the evidence comes from. E.g., Studies from the US suggest…
•	Overall, support is not clearly defined: Authors outline various studies which found associations between various support measures and breastfeeding outcomes, but it is not clear how the different measures of support should be interpreted (i.e., are they all the same thing, or are they different?). Given the focus of the study is around support, I believe the paper requires a few sentences clarifying what support is.
•	Line 63-64: The importance of measuring support was not particularly clear in this sentence.  Further, promoting breastfeeding can conflict with support, therefore measuring support is not necessarily key for breastfeeding promotion. Better phrasing may be something like, “Therefore, accurately measuring the availability of support for mothers is a key step in identifying their support needs and providing effective breastfeeding support.”
•	Lines 70-71: “although quantitative information on support up to 6 months postpartum is also needed” – this is unclear; is this in regards to the scale, or is this a comment about something else? (I.e., needed by whom?).
•	Line 72-74: mothers in Uganda is specified, but it is not specified for scales among adolescents and working mothers. Were the scales for adolescents and working mothers therefore proposed to be cross-culturally applicable scales, but the one in Uganda for mothers in Uganda only? I am wondering whether, in fact, the support scale for adolescent mothers and working mothers are also population specific. If so, the population should be specified (i.e., USA). I am not confident we should assume studies in the US would automatically apply to the rest of the world!
•	Lines 75-77: Authors note, “Because the support needed to continue breastfeeding may differ according to social context, we considered that the existing scales may not be appropriate for measuring breastfeeding support among the majority of mothers in Japan.” This statement would be better evidenced if cross-cultural variations in how support influences breastfeeding was reviewed above. Up to this point, authors present a rather universal picture of how support measures associate with breastfeeding – but surely, this may vary across populations, not just in Japan?
•	Line 78: “Very few” is subjective – are there any national statistics available you could cite?
•	Line 79: “Majority” could vary from 51% to 99% - as above, are there any national statistics available you could cite?
•	Lines 83-84: “That gap between original intention and later practice might be due to a lack of support.” Many studies have reported the gap between breastfeeding intention/desire and practice and attributed this to the lack of support, so this hypothesis would be better supported by referencing existing studies which have also stated this.
•	Lines 86-87: “has not been quantified in a way that is relevant to Japan” requires more explanation. I recommend providing more detail on the existing scales in Lines 65-74, and raising specific issues with existing scales as an example in the final paragraph of the introduction to support this statement.

Other comments on basic reporting:
•	Line 107-114: A good definition of support is provided here, but this would be better moved to the introduction where concepts of support are first introduced.
•	Line 123: The reference here is unclear – it seems like Fitzner 2007 provides further details on the item review process? Perhaps better phrasing would be “As recommended by Fitzner (2007) to improve item validity, the items were reviewed by…“
•	Paragraph one and paragraph two of the methods should be combined as they relate to the same subject. Line 126 could be rephrased as: “Content validity was tested quantitatively by using the Content Validity Index (CVI).” Otherwise I think this section is very clear.
•	Line 115-116: This should be clarified in the SI; i.e., link each item to the listed types of support. This will help the reader better interpret the items.
•	Pilot testing: Please clarify roughly when and where this was carried out.
•	Line 240: “Not many” is subjective – please provide the number.
•	For Table 3, I think it would be helpful to include the item questions with the item number, so readers do not have to keep referring back to Table 2, and to include a brief description/label for each factor. This will help readers better understand/assess the factors.
•	Lines 281-285: This information is about deriving variables and provides descriptive statistics, and it should be placed in the methods (around Line 240?)

As a Japanese and English speaker, I also have some minor suggestions regarding the English translation of the Japanese items:

Item 1: Better translation may be “There is someone with whom I can speak to about breastfeeding” where kigaruni is implied. “Frankly” suggests some formality and assertiveness, so I am not sure this is a suitably translation.
Item 2: Emotional support may be better term rather than psychological support? As phycology is often linked to the “brain” and emotion to the “heart”
Item 8: Health care providers "such as" doctors, public health nurses, and midwives tell me about the benefits of breastfeeding.
Item 10: I can breastfeed comfortably when "I’m out and about" may be a better translation – as being away from home does not necessarily mean being out (you could be staying with your family at their house, for example).
Item 14: Just a point that this item seems to be a leading question with implicit judgement that not breastfeeding is negative and undesirable. A more neutral/less judgmental phrasing would have been something like “Sometimes I provide something other than breastmilk to my baby, because housework or parenting of an elder child gets in the way.” While this is not included in the final factor analysis, authors may want to mention this in the limitations.
Item 17: There is someone I can talk with whenever I have "issues" with breastfeeding. may be better
Item 18: There are discrepancies among what health care providers "such as" doctors, public health nurses, and midwives say about breastfeeding.
Item 24: I have trouble finding places to breastfeed when "I’m out and about."
Item 31: If necessary, I can consult with health care providers "such as" doctors, public health nurses, and midwives on how to breastfeed.
Experimental design: Overall I thought this was a well-executed study and the methods were clear, but I would have liked some more information on the methods regarding item development. In particular, further detail on the literature review process would help the reader understand the process of item development:

Firstly, what exactly were authors hoping to measure? I ask this, as some items seem to relate to perceived support, while others seem to relate to advertising of formula and it is not clear how this relates to support. (For example, how is an advert on formula related to support? Given that information on formula could be very supportive for mothers who are formula feeding, and an advert is not necessarily devised to be supportive, could this really be conceptualised as support, or is it to do with establishing norms?). Perhaps because the conceptualisations of support is relatively weak in the introduction, it is not clear how the items are intended to measure support.

Second, how were the literature reviewed identified? If all the reviewed papers were cited, it does not look like a comprehensive list of available studies and looks relatively dated. What kind of studies on support for working mothers were excluded? I note that the study was conducted in 2014, which would explain why some more recent papers are not included in the references. This is not an issue, but it would help the reader understand why more recent papers were not included if you provide the timeframe of when these steps were taken. I would recommend providing a more detailed outline of how the literature was identified/reviewed in the supplementary information.

Finally, each item in SI1 could be referenced to papers where it was derived from. Again, this will show the readers that the items are based on evidence & clarify the process of item development further.
Validity of the findings: Were any information on participant socioeconomic position collected, such as education level or household income? As this information will provide further detail on the generalisability of the findings. If not, this should be outlined in the limitations.

I would have liked more information on the Adachi Ward which will help with inference; i.e., how do the population/mothers in the Adachi Ward compare to the rest of Japan?

As mentioned under my comments on basic reporting, I recommend authors clarify the theories and concepts around what was measured so the findings are inferred appropriately.
Additional comments: Thank you for the opportunity to review this manuscript. Overall, I thought the paper was very interesting & the study was well-executed, but I recommend redevelopment of the introduction which would clarify the purpose of the study, what the items are actually measuring, and help the reader infer the study findings appropriately. I have provided a detailed review, and I hope this is useful for the authors in improving the 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: DEVELOPMENT OF THE BREASTFEEDING SUPPORT SCALE TO MEASURE BREASTFEEDING SUPPORT FROM LAY AND PROFESSIONAL PERSONS, AND ITS PREDICTIVE VALIDITY IN JAPAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This work is interesting. In order to present the need to develop new tools related to breastfeeding for Japanese women, a wide range of up-to-date literature needs to be reviewed. Especially in the introduction section, the reference should be changed to the latest.
Experimental design: no comment
Validity of the findings: The results in this study are clearly presented. However, the data used in the study was collected six years ago, which may not reflect changes over time. These limitations need to be described by adding them to the discussion as a limitation.
Additional comments: Introduction: Considering the cultural characteristics of Japan, it is necessary to present special matters that affect the support of breastfeeding.
Line 77-80: A reference should be presented in this sentence.
Line 119: S1 Table & Line 135: Table S2  - Please unify the way of the supplemental table numbers are written in the two parts.


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: DEVELOPMENT OF THE BREASTFEEDING SUPPORT SCALE TO MEASURE BREASTFEEDING SUPPORT FROM LAY AND PROFESSIONAL PERSONS, AND ITS PREDICTIVE VALIDITY IN JAPAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	This is a nicely structured, written and presented manuscript.
2.	The literature review cites the most important and relevant literature, which is used to identify the appropriate study design, and the introduction succinctly positions the study in the literature and well articulates the rationale for the study.
3.	The four tables are clear and informative. Data availability is indicated, and the file provided.
Experimental design: 4.	The survey design and statistical analysis is clearly explained, and the factor analysis appropriate for the research question.
5.	The statistical analysis appears to be conducted to a high technical standard, and is clearly and succinctly reported. The authors could consider providing a sentence or two explaining the choice of factor analysis and the Kruskal-Wallis test, and an example of how to interpret the statistics, for readers unfamiliar with factor analysis and non parametric tests.
6.	The authors could clarify in the abstract that the data is based on interview and maternal self-report of breastfeeding practice.
7.	Some information could be provided comparing the characteristics of participants lost to follow up to indicate possible bias in the final sample.
Validity of the findings: 8.	The authors conclusions appear well supported by the data and analysis, and the extent of generalisability appropriately acknowledged.
9.	Limitations could include reference to social desirability bias as a limitation in interpreting survey responses.
Additional comments: 10.	This is a well conducted and nicely presented study and addresses an important question that is potentially useful to clinical practice, and with wider social benefit to support mothers to breastfeed according to their goals and to recommended practice.
11.	The justification for conducting such a study is well made in the introduction. It could be useful for the authors to reflect further based on their results, on what may distinguish the Japanese cultural context from other studies of this kind, and how those other socio-cultural contexts differs from Japan. It seems to me that the low paid labour force participation rate of Japanese new mothers is a rather unique cultural factor that could explain why practical help is less important than in other cultural contexts where early maternal return to paid employment is more common but unpaid housework and childcare still remains predominantly a female role. A sentence or two with citations and relevant data could strengthen the wider insights from the study.
12.	The literature is clear that longer paid maternity leave is supportive of breastfeeding, and heavy unpaid work burdens involved in caring for an infant influence mothers’ infant feeding practices and ability to get sufficient rest and sleep etc.  Japan is no different in that regard to other countries, and it would be interesting in a future study to survey and validate measures of key support requirements (and tradeoffs) involved in breastfeeding for those Japanese women who would prefer to maintain paid work participation, as well as breastfeed.


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: DEVELOPMENT OF THE BREASTFEEDING SUPPORT SCALE TO MEASURE BREASTFEEDING SUPPORT FROM LAY AND PROFESSIONAL PERSONS, AND ITS PREDICTIVE VALIDITY IN JAPAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment - authors have addressed all my suggestions in the response/manuscript
Experimental design: no comment - authors have addressed all my suggestions in the response/manuscript
Validity of the findings: no comment - authors have addressed all my suggestions in the response/manuscript
Additional comments: Thank you to the authors for their clear responses. I have read through the response to authors and revised manuscript. I found the paper to be very clear and very interesting.

My only minor comment is that I am not sure the additional explanation of the statistical methods adds much to the paper, as they are quite basic methods and it may be more efficient to direct readers to textbooks/methods papers rather than outline the methods in more detail. However, I recognise this has been suggested by a reviewer, and it may be very useful for readers who are not familiar with the method, or those who hope to replicate/conduct a similar study.


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: RELATIONSHIP BETWEEN IMPULSIVITY AND SUICIDE AMONG THE RURAL ELDERLY IN CHINA: A CASE-CONTROL PSYCHOLOGICAL AUTOPSY STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript discussed the association between impulsivity and suicide death in elderly population in China. The paper is clear and well written. Authors reviewed related literatures sufficiently.
Experimental design: It's an excellent psychological autopsy case-control study. The hypothesis was well defined.
Validity of the findings: It's acceptable. The conclusions were clear.
Additional comments: Mental illness and prior suicide attempt are well-known risk factors of suicide death in China and other countries. Could authors verify why the two important variables were not included in the multivariate analysis?


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: RELATIONSHIP BETWEEN IMPULSIVITY AND SUICIDE AMONG THE RURAL ELDERLY IN CHINA: A CASE-CONTROL PSYCHOLOGICAL AUTOPSY STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although the paper is readable, I think it can be improved and written in a more concise way.
Line 63 TL. 2006 and line 70 Liu Z 2017. Please check the two references.
Line 76, "expression of suicide" is problematic.
Line 87, "negative" is inaccurate here, It could be "negatively associated with". But in the following example, it seems that the authors mean "no association".
Line 97-102 and line 106-107. The two paragraphs repeated the design of the study and objective as well.
Line 112, "died by suicides" is problematic here.
Line 138-139, the two terms, "marital stability" and "marital instability", seem problematic. I suggest the authors not to use the two to define two different marital status. The terms used in Tables are appropriate.
Line 138, please consider "the status of being left-behind".
Experimental design: See my general comments as below.
Validity of the findings: See my general comments as below.
Additional comments: 1. Abstract. Please specify how case and control groups were matched and how impulsivity was assessed. Please also add the conclusion of this study here. Please consider whether it is appropriate to say "impulsivity increased the risk of suicide". This is a case-control study and data were collected cross-sectionally, I do not think this study design can answer the causality question.
2. Line 87-96, I do not think the study "Wyder & De Leo 2007" can indicate no relationship between attempt and impulsivity, because no comparison between subjects with and without attempt. Please review studies on impulsivity and completed suicide here, because non-fatal suicidal behaviors, including attempted suicides, are different from completed suicide.
3. Measurements. For deaths by suicides, their data were collected from informants or proxies. Please specify this and consider whether these instruments are also valid and reliable when scales were not completed by themselves per se.
4. Table 2, means of suicide. I think it is too crude to dichotomize it into pesticides vs. others. The comparison results might be statistically significant if more subtypes were compared.


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: RELATIONSHIP BETWEEN IMPULSIVITY AND SUICIDE AMONG THE RURAL ELDERLY IN CHINA: A CASE-CONTROL PSYCHOLOGICAL AUTOPSY STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Need modifications on some English expressions. Overall clear messages can be seen.
Experimental design: Research question well defined, but would be good if with better justifications on research gap and significance.
Validity of the findings: Yes. The method and also the findings seems novel. But would be good if can be compared with other populations, maybe also consider some other factors, and show clearly the uniqueness and importance of the findings.
Additional comments: Title: Relationship between impulsivity and suicide among the rural elderly in China: A case–control psychological autopsy study

This is a useful study using psychological autopsy among rural older adults in China. The method is sound, however, the findings and the rationale of the study, as well as the discussion need further modifications.

Abstract:
1.	“The relationship between impulsivity and suicide is inconsistent in different populations. Hence, the relationship between impulsivity and suicide still needs to be studied among the elderly population.” ---This sentence looks less convincing as the rationale for the study?
2.	There is no conclusion in abstract? Please check if this complies with Journal requirement.
3.	A first impression is the OR is quite low 1.03 (95%CI: 1.01-10.6) though it is independent after controlling for the effects of marital status, education, family annual income, being left behind, social support, and negative life events. (Have other factors controlled as well, e.g. depression, anxiety?)
Background
4.	“Suicide, which is the act of intentionally taking one’s own life (TL 2006), has been considered an international public health problem. The global prevalence of suicide has remarkably dropped in recent years. However, suicide still has resulted in 828,000 global deaths in 2015(Mortality & Causes of Death 2016).” ---Maybe a better flow is : “Suicide, which is the act of intentionally taking one’s own life (TL 2006), has been considered an international public health problem. Suicide still has resulted in 828,000 global deaths in 2015(Mortality & Causes of Death 2016), although its global prevalence has remarkably dropped in recent years.”
5.	“Moreover, the suicide rates in rural areas are higher than those in urban areas in China (Liu Z 2017)” --- Is this referring to the older adults or general population in rural areas?
6.	Would the authors explain more why it is important to look into the relationship of impulsivity and suicide, especially among the older adults? The rationale of this study, in a clearer way?

Methods
7.	Please rewritten the sentence “Information on died by suicides among the elderly”---- “Information on death by suicides”?
8.	May need to clarify who are “We” in this sentence “We received training on psychological autopsy and interview methods before the start of the interviews.”
9.	It might be worthy mentioning that the interview for “the suicidal victims” group was conducted among the informants in methods, otherwise, a bit confusing, although a previous study covering the methods has been referred to.
10.	Maybe the authors want to describe a bit more on their statistical method: Conditioned multivariable logistic regression ---How it has been applied in their study.
11.	It may be better to also mention the results of the three subscales of BIS in text (since the authors have mentioned in the first paragraph). Were the three subscale scores also independently associated with suicide? (Were they significant in the final model)?

Discussion
12.	 “Impulsivity increases the risk of suicide among the rural Chinese elderly, which is consistent with previous studies (Fresan et al. 2016; Liu et al. 2018; Maser et al. 2015; Shelef et al. 2018).” –The authors may want to show this is consistent with some of the previous studies (as mentioned in the background).
13.	Overall the 3rd paragraph in Background, and the 2nd paragraph in the Discussion section seem showing similar information but it could be better if the background could show better rationale, and the discussion could show a better deep look at the results and comparisons?
14.	Limitations --- seems other important factors are not included and adjust in this study, e.g. mental disorders/depression. From the study by Zhou et al. in 2019, other factors are included and found to be significant (e.g. Independent risks of suicide included unstable marital status [odds ratio (OR) 4.19, 95% confidence interval (CI) 1.61–10.92], unemployed (compared with employed, OR 4.43, 95% CI 1.09–17.95), depressive symptoms (OR 1.34, 95% CI 1.21–1.48), and mental disorder (OR 6.28, 95% CI 1.75–22.54). Structural equation model indicated that the association between being left-behind and suicide was mediated by mental disorder, depressive symptoms, stressful life events, and social support.) Are there specific reasons?
15.	How would the results / significance of impulsivity among the rural older adults compared to other populations, e.g. younger / adult population, and urban areas?
16.	The impulsivity was not related to suicide time, place and means. Was this consistent with or different from previous findings? The authors mentioned that it was different from the youth? Are there any other reasons beyond age?
17.	“Our results indicate that suicides with different level of impulsivity may need different preventive strategy.” ---Can this be explained more?
18.	“In future research on suicide prevention, the family and society should pay increasing
attention to impulsivity and learn to handle them differently. People with impulsivity traits should be encouraged to consider and positively face life’s difficulties to effectively reduce or prevent the occurrence of suicide.” ---Can the authors explain more how the findings imply future services and practices?


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: RELATIONSHIP BETWEEN IMPULSIVITY AND SUICIDE AMONG THE RURAL ELDERLY IN CHINA: A CASE-CONTROL PSYCHOLOGICAL AUTOPSY STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is well written. Authors addressed reviewers' concerns well.
Experimental design: Good psychological autopsy case-control study.
Validity of the findings: Acceptable.
Additional comments: All my concerns has been well 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: RELATIONSHIP BETWEEN IMPULSIVITY AND SUICIDE AMONG THE RURAL ELDERLY IN CHINA: A CASE-CONTROL PSYCHOLOGICAL AUTOPSY STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No further comments
Experimental design: No further comments
Validity of the findings: No further comments
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: THE IMPORTANCE OF SPAWNING BEHAVIOR IN UNDERSTANDING THE VULNERABILITY OF EXPLOITED MARINE FISHES IN THE U.S. GULF OF MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: In reviewing the current manuscript, I found the reporting to be fairly straightforward and generally clear. The Introduction, Methods and Results were all well-structured and clear to the reader. However, I found that some sections of the Discussion contained very long sentences that in many instances could be made clearer by spitting them into two sentences. I also found a few sentences missing words or having a verb with a wrong tense. These minor errors need to be amended to clarify meaning. The conclusions were otherwise sound. References were appropriate, relevant and complete. Tables and figures, as well as supplemental materials were well-conceived and the data within them clearly presented. All the figures were of high quality and the figure and table texts adequately described the content, however I do not feel it is appropriate to have the reader have to go to a separate website to find references tied to tables, i.e. Table 3. I also observed some line issues with Table 3 that may be the result of the upload, regardless these need to be amended.
Experimental design: The research clearly fits within the scope of PeerJ. The manuscript provides a much-needed focus on reproductive behavior in fisheries management, as the formation of spawning aggregations, that has been heretofore largely missing. The authors clearly state the need for this information and provide rigor in testing how reproductive behavior impacts fishing vulnerability. Although the general concept of the vulnerability of aggregating species to fishing has been long known, this manuscript provides the first (potential) peer-reviewed publication of its inclusion during a vulnerability analysis. The design of the study is based on the utilization of previously published data containing relevant life history parameters, however I did not find how multiple values for life history parameters were dealt with. As an example, some species may have had multiple published values of, for example, k or maximum age. The authors do not provide clarity in the methods for how these were dealt with or what values were used. If multiple values were used then a mean and SE would be appropriately reported. If a single published value was used, the authors need to explain why and how the decision to use this value was attained. Additionally, sexual pattern is widely known to have an impact on vulnerability, however I only found one instance of its mention in the discussion. Many of the fishes have published information on sexual pattern and it would be useful for the authors to explain to the reader why this life history parameter was excluded as it may have had an impact on the results. Finally, the study would have been improved and the argument made stronger by the inclusion of a few species that do not aggregate to spawn. I found this in some ways unfortunate in that it may have provided even more evidence of the need to consider aggregating behavior as a highly vulnerable life history trait. While the authors do provide a range of aggregating types among the chosen species, it would have been insightful to see how these species compare to non-aggregating species and how it might otherwise impact management. Otherwise, the methods were well-described and the data and methodology are generally sufficient to allow replication.
Validity of the findings: The authors make the impact and novelty of their study clear and unambiguous. All of their findings appear to be conclusive and where findings were not significant, the authors make the case clear as to why. The statistics are appropriate and the methodology is robust. I did not find anywhere where speculation was given, indeed the authors clearly show how reproductive behavior affects the species listed and stick strongly to the findings in their discussion and conclusions.
Additional comments: The manuscript provides a much-needed focus on reproductive behavior, as the formation of spawning aggregations, that has been heretofore largely missing from fisheries management decision-making. Although knowledge of aggregating behavior to increased vulnerability to fishing has been widely known for decades, to my knowledge this is the first study to examine this analytically. I, therefore, applaud the authors for taking on this exercise and highlighting the vulnerability of aggregating fish to overfishing. Although the manuscript is well-designed and generally well-written, I found a few grammatical errors that need amending. I also found several sentences, particularly in the discussion, that need to be split or otherwise altered to provide clarity. I have made comments throughout the manuscript where attention is needed. Although I did not find any major issues with the study, I nonetheless had some concerns regarding the exclusion of sexual pattern as a parameter within the study, as this has been shown to increase vulnerability within aggregating species. I would like the authors to respond as to why this life history trait was not included. I would also like to have the authors clarify how they dealt with multiple life history parameter values, e.g. k or size at sexual maturity. I make this request as I saw nowhere in the manuscript on how this was dealt with. If multiple values exist, Table 3 should report means and standard errors, or alternative if only one of multiple values was used, then why? I also note that there was no discussion of how these results compared with the Redlist designations for these species (where available) or the US Endangered Species designations. It would be fruitful to have this discussed. My only major concern is the use of terminology. I note throughout that the authors state that these species are vulnerable during spawning, however this is a deceptive term in many ways because the vulnerability is actually during and due to aggregating, not spawning. As the authors state, fish may aggregate for many days when spawning is not taking place, yet these species are vulnerable during these times. Indeed, a number of species don’t bite during actual spawning so the vulnerability is in actuality decreased. Therefore, throughout the manuscript the word ‘spawning’ needs to be replaced with ‘aggregating’. The authors can look at the comments within the pdf and get an idea of where this is needed. I would also comment that the study and the manuscript itself could have benefitted from the inclusion of a few non-aggregating species. Such inclusion would have given both readers and management some greater idea of where aggregating fish fit comparatively in the range of vulnerability. Otherwise, I would like to congratulate the authors on conducting this study.