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 ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well written and clear. Even with substantial revisions, it remains well structured with no fatal flaws.  My previous concerns regarding references have been addressed
Experimental design: The design is generally consistent with the aims. This original primary research fits within the scope of the journal. The previously understated significance of this study has now been addressed. I am pleased the other reviewers have commented on the statistical analysis and these appear to have been adequately addressed.
Validity of the findings: This is markedly improved in this revision and the conclusions have been tempered to be more conservative.
Additional comments: I have 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 ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: 1. L226: the primary outcome should be the 'score change', but not the score itself. 'Prevalence of depression' should be revised as 'presence of depression'.
2. For table3 and table4, the coefficient and its 95% CI from univariate/multivariable linear regression should be reported instead of the mean change. For table2, please add 95%CI for each covariate.
Additional comments: Thanks the authors for addressing my questions.


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 ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have adequately addressed my questions. Thank you.


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

Input:
Title: FORBIDDEN LINKS, TRAIT MATCHING AND MODULARITY IN PLANT-HUMMINGBIRD NETWORKS: ARE SPECIALIZED MODULES CHARACTERIZED BY HIGHER PHENOTYPIC FLORAL INTEGRATION?
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript requires careful and extensive English language editing throughout. In its current state, the manuscript is difficult to follow and has no clear direction. The term "phenotypic floral integration" is used frequently, but is not explained. Many paragraphs are too long and have no clear objective.
Experimental design: While the methods used may be appropriate, I struggle to follow the manuscript in its current state.
Validity of the findings: Again, I cannot comment on the validity of the findings given the above.
Additional comments: I recommend that the authors either invite a native English speaker on as a co-author, or use an English editing service (these can be extremely helpful). The authors have conducted some excellent field work and made valid observations. However, it is unfortunate that these interesting observations are lost in poorly organized paragraphs and improper sentences. Consequently, I cannot recommend this manuscript for publication in PeerJ in its current state. Below is a list of comments I made before deciding to recommend reject:

L25-26: "allow the niche portioning and ultimately, modularity.". I don't understand what the authors are trying to say here. I believe they are referring to niche partitioning, which is the term used later in the manuscript. Please go through the manuscript and remove all excessive and particles such as "the" and "a/an" where they are not needed. Nouns do not always need to be associated with these articles, this is consistent difference between the Spanish and English languages.

L26-28: I don't understand this sentence. It has no clear direction. The term "floral integration" is used as if every reader is supposed to know it. This needs to be explicitly described in the introduction, and probably removed from the abstract. It should instead be described using plain English and no topic-specific jargon.

L39: "networks dynamics" should be "network dynamics"

L303-305: I'm unsure what constitutes "illegitimate" visits. Why were these Hummingbird species not included in the study? This is brushed over and not explained well.

L402: "phenotypic floral integration". Again, this term needs to be explicitly explained from the outset before it is used throughout.

L419: "sedentary species". This is probably not the term you want here. Birds are certainly not sedentary. Do you mean they do not migrate between habitats?

L429-430: remove "in this species rich tropical community".

L432: remove "in a diverse tropical community".

L437-439: I don't understand what the authors are trying to convey here. What is the point and how does it relate to what is described above (particularly nestedness)?

L452-503: this is an enormous paragraph that could easily be shortened with some careful editing. It is also poorly structured and hard to follow.

I find the authors arguments hard to follow. Does trait-matching promote specialization and modularity or the other way around?


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: FORBIDDEN LINKS, TRAIT MATCHING AND MODULARITY IN PLANT-HUMMINGBIRD NETWORKS: ARE SPECIALIZED MODULES CHARACTERIZED BY HIGHER PHENOTYPIC FLORAL INTEGRATION?
Review round: 2
Reviewer: 2

Output:
Basic reporting: In this manuscript, Palmas and colleagues present an association between morphological constraints in plant-hummingbird interaction networks from two distinct habitats in Mexico.
The main idea of associating network structure of the interaction network, with emphasis in modularity, with phenotypic integration of the flower assemblages is indeed very interesting. Data collection seem to be alright and overall, this is study that brings new ideas. Good job done.
I have two major concerns though related to the study, meaning that it would require major revising. One more conceptual and one methodological/choice of the analytical framework given their sampling of two interconnected habitats:
-	Morphological matching (trait complementarity) and forbidden links (understood as exploitation barrier) are not the same thing, albeit related. This needs to be properly understood by the authors, and the theoretical background of the manuscript should be thoroughly revised (see Santamaría & Rodrígues-Gironés 2007,
Experimental design: -	From the species composition and interactions in the two habitats, it is clear that the two studied habitats are highly interconnected by the hummingbirds. Although plant assemblages are distinct, this means that the two habitats form a single network of interaction. It could even be that two plants from different habitats are more intimately linked with each other through their shared hummingbird species than two plants of the same habitat with different hummingbird pollinators (see Bergamo et al. 2017, Ecology), and hence affect all consequences on floral integration that the authors are trying to show. Therefore, it makes more sense that an analysis considering one whole network and then contrasting the floral integration among modules should is conducted. At the very least, the authors need to show that these two habitat networks work as complete separate units. This may be the case as shown previously (Maruyama et al. 2014), but it seems not (modules do not reflect habitats when analyzing the whole network).
The English writing present many misspellings and inconsistencies, so a thorough revision is needed.
Validity of the findings: no general comment, see detailed comment below.
Additional comments: Detailed comments.
Abstract
Line 22. Plant-pollinator mutualistic networks, change throughout the text (also at line 62 and other places).
Line 23. “However, pairwise interactions show high variability in the number of interacting species” This sentence is strange, pairwise interactions should by definition, include two species each
Line 25. Niche partitioning.
Line 39. “The rainforest network showed higher levels of specialization and mainly composed of species of the Hermits clade…” How can this be if both habitats have the same hummingbird species?
Line 49. The conclusion presented refer to forbidden links and trait matching for one habitat, and agonistic behavior for other habitat as mechanisms structuring the interactions. But how this conclusion was reached is not clear and up to this point are it is not mentioned regarding the methods/results on what done and was found regarding this finding. Please explain and provide an abstract that more clearly present what you did and concluded based on what.
Line 52.What is “degree of specialization of pairwise interactions” To my understanding, degree and specialization are estimated for each species.
Line 84. What would be these upper and lower levels? Consumer and resources? Please clarify. What about other type of interactions? Do not we also find constraints on interactions?
Line 86-88. Trait matching and forbidden links (exploitation barrier) are two somewhat distinct concepts (e.g. Santamaría & Rodríguez-Gironés 2007; Sazatornil et al. 2016). Please check this and update it accordingly throughout.
Lines 103-107. This is not really related to your study, which is rather local scale.
Line 111. There is no idea of “opposition” as indicated by “Contrarily”.
Introduction. Maruyama et al. 2014 clearly linked modularity of a plant-hummingbird network with morphological traits and spatial distribution of species. Since your study touch upon this topic heavily, it could be explored better in Introduction to give context for your study.
Methods
Line 182-183. Why did you focus on understory plants only? Was this a limitation? Make this clear
Line 190. Were hummingbirds the main pollinators of all plant species? Make this clear.
Line 215-216. A citation for this one third addition?
Line 248. Bipartite should be in small case letters.
Line 255. It is somewhat affected, you could say “less affected” instead of “unaffected”
Line 277-282. This is a new approach used in this context, as such it needs to be better explained on what was done. At what scale was phenotypic integration calculated? For each species? For the entire assemblage (an average across species?). So that you could relate to the modules? It is important that we can understand this and that future studies can be based on yours.
Results
Line 293. “two belonged to the”
Line 296-297. “Two sub-networks with completely different plants without any shared species were clearly differentiated by habitat” How can you support this? Although plants are not, hummingbirds are the same between habitats. You should run a modularity analysis considering both habitats together since the two habitats form a single network of interaction connected by hummingbirds. It could even be that two plants from different habitats are more intimately linked with each other through their shared hummingbird species than two plants of the same habitat with different hummingbird pollinators (see Bergamo et al. 2017, Ecology), and hence affect all consequences on floral integration that the authors are trying to show. Therefore, it makes more sense that an analysis considering one whole network and then contrasting the floral integration among modules should is conducted.
Line 348-351. How was division of flowers determined? Can you support this without being arbitrary?
Line 356-357. Show the curvature of bill and corolla in the same scale
Line 360. “largest hummingbird”
Lines 369-384. Many of the info that is already present in the table, or it should be.
Line 390-392. Average across plant species? This needs to be clear.
Discussion
Lines 409. The patterns reported is also very similar with Maruyama et al. 2014.
Line 452-456. Needs to better define trait matching and forbidden links, their differences.
Line 484-488. The authors may also want to consider Maruyama et al. 2013 which specifically discuss this pattern quantitatively.
Lines 505-512. No biological process being discussed here. Suggest deleting.
Line 517. “where only plant traits differed from the modules” what do you mean?
Line 517-524. You did not analyze this in your study, so I do not think that is relevant discussion.
Line 527. “did not differed” please correct.
Lines 561-566. Too long and speculative.
Lines 569-570. Confusing sentence, please reformulate
The Discussion section is too long and could be definitely made more concise.
The Conclusion sections here seem unnecessary as the authors are only repeating their findings which are listed. The last paragraph of the Discussion already served as a conclusion (which was better written than the text in the Conclusion section).
Figure 1. Order the hummingbird species in the same order for the two networks.
Figure 3. Do you have similar images for the small flowers?
Table 1. Show this information in a figure, with the averages (boxplot? barplot?) and report the table as supplement.
References used in this review.
Bergamo et al. (2017). The potential indirect effects among plants via shared hummingbird pollinators are structured by phenotypic similarity. Ecology, 98(7), 1849-1858.
Maruyama et al. (2014). Morphological and spatio‐temporal mismatches shape a neotropical savanna plant‐hummingbird network. Biotropica, 46(6), 740-747.
Maruyama et al. (2013). Pollination syndromes ignored: importance of non-ornithophilous flowers to Neotropical savanna hummingbirds. Naturwissenschaften, 100(11), 1061-1068.
Santamaría & Rodríguez-Gironés (2007). Linkage rules for plant–pollinator networks: trait complementarity or exploitation barriers?. PLoS Biol, 5(2), e31.
Sazatornil et al. (2016). Beyond neutral and forbidden links: morphological matches and the assembly of mutualistic hawkmoth–plant networks. Journal of Animal Ecology, 85(6), 1586-1594.


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: FORBIDDEN LINKS, TRAIT MATCHING AND MODULARITY IN PLANT-HUMMINGBIRD NETWORKS: ARE SPECIALIZED MODULES CHARACTERIZED BY HIGHER PHENOTYPIC FLORAL INTEGRATION?
Review round: 3
Reviewer: 1

Output:
Basic reporting: I consider that all my previous considerations were incorporated.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I annotated in the attached manuscript pdf, some suggestions on small edits.
Good luck.

Pietro


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: FORBIDDEN LINKS, TRAIT MATCHING AND MODULARITY IN PLANT-HUMMINGBIRD NETWORKS: ARE SPECIALIZED MODULES CHARACTERIZED BY HIGHER PHENOTYPIC FLORAL INTEGRATION?
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment (see general comments)
Experimental design: no comment (see general comments)
Validity of the findings: not comment (see general comments)
Additional comments: “Forbidden links and trait matching promote modularity in
plant-hummingbird networks: the influence of floral
integration”

The present study aims to analyse processes related to the modular organization of plant-hummingbird networks in terms of morphological specialization and their implications on the phenotypic floral integration within modules. For this, authors selected two adjacent habitats (lowland rainforest and savanna-like vegetation) in Chajul Biological Station (Mexico) to record plant-bird interactions through two consecutive years. The obtained (and contrary to expectations) results show that floral integration within specialized modules were not significant higher that other modules, non-specialized.

The topic of this MS is potentially interesting. However, some aspects of the MS (see below) need to be considered in order to improve it. My principal concern is related to the number of modules obtained (just 3) because of small studied pollination network (26 plants and 4 bird species). It’s this value (n=3) good enough to test hypotheses about specialization and floral integration among modules? Besides, other aspects need to be considered through the MS. For example:

Title: Maybe authors need to change it. Its confuse. It would be more easily if authors add, in some way, the principal conclusion of this study: “specialized modules are not characterized by higher phenotypic floral integration”

Abstract. L23: Substitute “pairwise interactions” by “among species interactions”, or “mutualistic interactions”, or “plant-pollinator interactions”.

Abstract. L26-27: If not easy to understand the next sentence: “Thus, plants belonging to specialized modules should integrate their floral traits to optimize the pollination function” if all pollinator species (and plants) within each habitat were not included (just only understory plant and hummingbird species).

Abstract. L30: Maybe it is not necessary to explain the term “phenotypic floral integration” in the Abstract.

Abstract. L49-55: Maybe include this sentence from L47-48: “morphological specialization plays a minor role in the phenotypic floral integration” in Conclusion part.

Introduction. L 58-60: These two sentences need to be change. The first one is confuse and very general. In the second one, plant-pollinator networks just imply many species.

Introduction. L117: As previous specific terms, “modular structure” need to be explained briefly here.

Introduction. I miss some clear hypothesis to be tested. In general the Introduction is confuse. Need to be more focusing on “modularity and floral integration” and clarify the use of some terms such as “niche segregation by ecological specialization”. Besides, what about to remove sentences L102-108?

Methods. Authors need to consider if including partial plant-pollinator networks to include only understory species is good enough to study evolutionary trends (i.e. phenotypic integration) (L186). Besides, what about other pollinator species? Many plant species visited by hummingbirds are also visited by insects. Authors need to discuss in some way these aspects. It’s no clear at all if some selected plant species are also legitimate visited by insects (L241). Besides, I miss some information such as number of plant species inhabiting these habitats? And how many being visited by hummingbirds? How many hummingbird species? From these total values, how many were included in the study? What about sampling efforts for each plant species? It would be possible to include flower colour (L199)? How flower curvatures were measured (L203)? To know the accumulated nectar volume, how many hours each flower was excluded to pollinators, just 24h? The bipartite matrix included quantitative data (L253)? It’s really necessary to separate both adjacent habitats (L256)? For example, in savannah only 8 plant and 4 hummingbird species were included. What about the version of the bipartite package used (L289)?

Results. L311-325. Authors need to clarify if only birds visited these plant species. What about insects such as pollinators? What about to remove results by each habitat separately (L357-376).

Authors fail to find significant relationship between ecological specialization of modules and their phenotypic floral integration. This result could be related to the low number of plant species studied and number of obtained modules (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: FORBIDDEN LINKS, TRAIT MATCHING AND MODULARITY IN PLANT-HUMMINGBIRD NETWORKS: ARE SPECIALIZED MODULES CHARACTERIZED BY HIGHER PHENOTYPIC FLORAL INTEGRATION?
Review round: 4
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: not comment
Additional comments: In this new version of the MS authors have already included practically all my previous comments. The manuscript has been improved considerably, and authors have also included more information about. However, at time to analyse processes related to the modular organization of plant-hummingbird networks in terms of morphological specialization (i.e. phenotypic floral integration) I think that dataset are not good enough and its related to low number of obtained modules because of small studied pollination networks (26 plants and only 4 bird species). I mean, to analyse flora integration in just 3 modules its not good enough to conclude “specialized modules are not characterized by higher phenotypic floral integration”.

Instead I’m not sure about the difficult to obtain more data, and because I like the author’s novelty idea, I encourage them e.g. to review plant-hummingbird interactions in a wide region to obtain a higher network size. After, by using previous publications or just some flower pictures, maybe authors can obtain some of biometrical traits to analyse flower integration (maybe it is not necessary to obtain values from all traits in all plant species). Then, in this way authors will have the chance to increase number of specialized or generalist modules for a proper comparisons of their flower integration level. Authors could confirm their present results by using a smaller network size in order to generalize their principal conclusion. The MS is focusing to know relationship between ecological specialization of modules and their phenotypic floral integration.


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: ACTIVITY STRATEGY AND PATTERN OF THE SIBERIAN JERBOA (ORIENTALLACTAGA SIBIRICA) IN THE ALXA DESERT REGION, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript should be largely improved in terms of expression and scientific writing. Some expressions are ambiguous or even misleading, and the writing sometimes lacks preciseness. I suggest the authors seek for help from a native English speaker or a language service company. Introduction on background is poorly organized and seems to be unfocused.
Experimental design: This paper describes an interesting seasonal pattern of activity pattern in a mainly nocturnal rodent. The authors then seek to associate these changes with several environmental factors using RDA. This research should fit within the scope of PeerJ and may deserve publication, but not in its current form. First, the Introduction is not well organized and it is not clear why the authors choose this topic or why they decide to use this system. It is thus difficult to evaluate whether this research helps to fill some important knowledge gaps. Some important details of methods are not clearly described. Please see my specific comments for more information.
Validity of the findings: The validity of the findings needs to be strengthened, as I have some methodological concerns. Some procedures need to be validated and clarified (e.g. using vigilance behavior as a surrogate for predation risk, using lures to attract jerboas, individual identification, and performing ANOVAs without a check for the suitability of data).
Additional comments: Specific Comments

Line 2: The term “strategy” is unclear. What type of strategy do you refer to? Foraging strategy? Habitat selection? Life history strategy? Use an appropriate word throughout the manuscript.
Line 13: Seems to be an invalid affiliation.
Line 25: The term “activity time” is ambiguous. Do you mean the duration of active period or the time point when the rodent is active?
Line 48: Interspecific competition also plays a role in shaping activity patterns.
Lines 100-102: The logic here is problematic. I agree that this species can be viewed as a habitat generalist or a wide-ranged species as you mentioned, but could it represent other generalists in terms of activity patterns? Generalists can have various activity patterns (diurnal, nocturnal or crespucular, et al.).
Line 110: Energy supply is always needed as long as the animal is alive.
Line 117: Rewrite this part as “may encourage jerboas to withstand higher predation risk”.
Lines 135-137: What about the abundance and activity patterns of these sympatric rodents? Would interspecific competition also affect activity pattern of jerboas?
Line 138: What about the activity patterns of these predators? Are they active in the same period as jerboas? This is important for your hypothesis associated with predation risk.
Line 144: What does the “grid” mean? A site? A grid of cameras? A trapping grid? How many cameras are placed in each grid?
Lines 147-148: You need to add some support (e.g. citations) for this idea. In my experience, five-toed jerboas may occasionally be active during daytime, and some mainly nocturnal rodents may become more diurnal as the weather gets colder. In this sense, it would thus be somewhat risky to solely focus your investigation on nighttime. It does not mean that your analyses are invalid, but you do need to consider this potential limitation, at least in Discussion.
Line 151: When did you place the lure? How many peanuts are used for a lure? Would the lure significantly modify the behavior of jerboas and thus affect your results?
Line 162: Did you adopt some procedures to mark individuals? How could you tell whether it is the same animal or not?
Line 165: “Sentinel behavior” generally occurs in cooperative species. It means that some individuals act as sentinels while other group mates keep on foraging, as can be seen in meerkats. For jerboas, “vigilance behavior” may be a more appropriate word.
Lines 165-167: Please clarify how to define this behavior. What types of physical action response?
Lines 170-172: Where are the climatic data collected? Could they reflect the local environmental conditions in your study sites?
Lines 177-179: It is somewhat problematic to directly use vigilance behavior as an indicator of predation risk. Actually, vigilance reflects a trade-off among many aspects, such as predation risk, interference competition, individual energetic state, and so on. For instance, suppose you detect a jerboa to be less vigilant during the early night, it does not necessarily mean that the actual predation risk is low. Maybe it is simply because that at this stage the jerboa is rather hungry, so energetic intake rather than antipredation is more important for him.
Line 185: How do you estimate the biomass of the plants? Did you cut them off and measure their dry weight?
Lines 210-211: You need to test first whether your data meets the requirements (e.g. normality and homogeneity of variance) for ANOVAs. If not, data transformation, GLM, non-parametric tests or Monte Carlo methods should be used instead.
Line 227: Specify the two degrees of freedom for each F statistic, and hereafter.
Line 232: Where is the data supporting this point?
Line 312: Actually, animals often have to be active during less favorable periods, due to competition, predation risk or other environmental stressors.

Fig. 2-3: What do the bars represent? S. E. or S. D.?


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: ACTIVITY STRATEGY AND PATTERN OF THE SIBERIAN JERBOA (ORIENTALLACTAGA SIBIRICA) IN THE ALXA DESERT REGION, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: English should be improved. Some parts of the text are nor clear enough. Some terms are formally correct, but usually not used in professional literature.
Literature references are not sufficient.
The structure of the article is formally correct.
Theoretical background of the tested hypotheses is week. Results are interesting only as the natural history description of the species.
Experimental design: This is original research which is out of aims and scope of the journal.
Research questions are defined based on week theoretical background.
Data collection was performed to a high technical & ethical standard.
Methods described without sufficient details.
Validity of the findings: Presented results are interesting as the natural history description of the species.
Underlying data are provided; they are robust, statistically sound, & controlled.
Conclusions are well stated but not linked with original research question
Additional comments: The manuscript contains new data on seasonal patterns of daily activity of Siberian jerboa in Alashan desert of Inner Mongolia obtained with camera trapping technique. These data are interesting as the natural history description of the species. However, theoretical background of the paper is week in several aspects listed below:
1.	Authors used a bait (peanut) and thus, they measured mainly foraging activity.
2.	Authors does not describe in details what they considered as the “sentinel” behavior. It is necessary to provide description of the poses or the sequences of behavioral bouts that they attributed as alertness.
3.	Authors estimated abundance of foraging plant species based on the set of cafeteria experiments, but they did not record used parts of plants. It is well-known that Siberian jerboa feed on green parts of plants, underground parts of plants, seeds and insects in approximately equal proportion (Shenbrot et al., 2008 - Jerboas. Mammals of Russia and adjacent regions.  Science Publishers Inc, , Enfield, USA). Thus, their estimations of food abundance are not complete.
4.	Authors did not provide the data on abundance of predators in the study area. In the absence of such data, alertness can be explained also by searching the partners for mating and locating flying insects for foraging.
5.	Considering the time allocation for different types of activities, authors missed breeding activity, which is important at least in spring and beginning of summer.
6.	Finally, it is not correct to consider the time allocation for different types of activities out of the frames of the theory of optimal foraging.
Introduction is written in a chaotic style. Most references to studies of mammalian activity patterns are not related to rodents in desert environments, whereas rodent activity in deserts has very specific patterns.
In discussion, there are no references to publications on activity patterns of other species of jerboas (summarized in Shenbrot et al., 2008).
Incorrect English and Latin names of the studied species. Authors used the name “five-toed jerboa” and “Allactaga sibirica”. Genus Allactaga s.lato was found to be polyphyletic and because of this was divided into three separate genera; the species authors studied belong to the genus Orientallactaga (Lebedev et al., 2013-- Zoologica Scripta 42: 231-249; Michaux & Shenbrot  2017 - Family Dipodidae (Jerboas).  In: D.E. Wilson, T.E. Lacher, Jr. and R.A. Mittermeier (eds).  Handbook of the Mammals of the World. Vol 7. Rodents II.  Lynx Editions, Barcelona. Pp. 62-100). “Five-toed jerboa” is the name used for all species of the subfamily Allactaginae; correct English name for Orientallactaga sibirica is Siberian jerboa.


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: ACTIVITY STRATEGY AND PATTERN OF THE SIBERIAN JERBOA (ORIENTALLACTAGA SIBIRICA) IN THE ALXA DESERT REGION, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this revised manuscript, the authors have dealt with my opinions accordingly in most cases, which greatly improved the paper quality. However, in terms of language and grammar, this paper is still poorly written (I have listed some errors in specific comments). Moreover, many important data was absent, or not correctly stated. I strongly recommend the authors have the manuscript thoroughly checked and edited by a native English speaker, or (even better) a language editing company, to make this paper more readable.
Experimental design: This paper fits within Aims and Scope of PeerJ. The research question is interesting and relevant, and the methodology is appropriate in most cases.
Validity of the findings: Some important data is missing, or not correctly stated. Conclusions seem to be correct but some need to be identified as speculation.
Additional comments: General Comments

In this revised manuscript, the authors have dealt with my opinions accordingly in most cases, which greatly improved the paper quality. However, in terms of language and grammar, this paper is still poorly written (I have listed some errors below). Moreover, many important data was absent, or not correctly stated. I strongly recommend the authors have the manuscript thoroughly checked and edited by a native English speaker, or (even better) a language editing company, to make this paper more readable.

Specific Comments:

Line 16: It seems that a space was missing between “in” and “activity”.
Line 62: Add “main” before “factor”.
Line 73: Delete “been”.
Line 106: Add the Latin name after “jerboa”.
Line 174: Again, how could you tell whether they are different individuals of the same species, as you didn’t adopt any procedures to mark jerboas?
Lines 179-180: Grammatical errors.
Line 203: Add “the” before “three plots”. Check your manuscript carefully before resubmission, since such grammatical errors appear frequently throughout the paper.
Line 219: Write it as 95.80 ± 17.74 (Mean ± SD).
Line 292: The sentence “Considering…” is not a complete sentence.
Line 295: The sentence “the electronic chips…” should appear at Line 299. What type of chips did you use? PIT tags? The information on manufacturer and model is also needed. What about the weight of the chips?
Line 305: Delete “whether the leg rings are lost”.
Line 309: Use O. sibirica here and hereafter.
Line 337: Use “among” to replace “between”.
Lines 385-425: In this section, many important data is missing. For example, where is the data supporting the “negative correlations between activity time and relative humidity, temperature, wind speed…”? Lines 387-398 are also needed to be rephrased in a more concise way.
Line 438: Use “between” rather than “in”.
Line 503: Use “encourage” rather than “enable”.
Lines 507-508: Rephrase this sentence as “Why does it still choose to take risk in autumn, when food resources are abundant?”
Line 517: This sentence needs to be rephrased.


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: ACTIVITY STRATEGY AND PATTERN OF THE SIBERIAN JERBOA (ORIENTALLACTAGA SIBIRICA) IN THE ALXA DESERT REGION, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Dear Editor in Chief
This paper is talking about the activity pattern of the Siberian Jerboa. The overall structure of the paper is suitable but it needs some minor revisions. Meanwhile there is some excellent related papers on Jerboas activity patterns in desert areas from Iran, which I proposed to authors and I wish they add them to the citations and use them in the discussion.
Anyway, my detailed comments are embedded in the manuscript word file.
 Journal of Wildlife and Biodiversity 1(1): 33-36 (2017) by Arak University, Iran (http://www.wildlife-biodiversity.com/)
DOI: 10.22120/jwb.2017.27208
Nocturnal activity and habitat selection of Hotson Jerboa, Allactaga hotsoni Thomas, 1920 (Rodentia: Dipodidae)
I strongly suggest to use and cite these  papers:

 Journal of Wildlife and Biodiversity 1(1): 33-36 (2017) by Arak University, Iran (http://www.wildlife-biodiversity.com/)
DOI: 10.22120/jwb.2017.27208
Nocturnal activity and habitat selection of Hotson Jerboa, Allactaga hotsoni Thomas, 1920 (Rodentia: Dipodidae)
 Mastureh Darabi, Zohreh Zeini, Abdolreza Karami, Ali Kaveh

1. Notes on habitat affinities of the Hotson's Jerboa Allactaga hotsoni Thomas, 1920 (Rodentia: Dipodidae) from Isfahan province, Iran
Mansureh Khalatbari; Morteza Naderi
Volume 2, Issue 1, Winter 2018, Pages 41-43
10.22120/jwb.2018.30219
http://www.wildlife-biodiversity.com/article_30219.html

Nocturnal activity of Iranian jerboa, Allactaga firouzi (Mammalia: Rodentia: Dipodidae)
Mahmoud-Reza Hemami 1 , Gholamreza Naderi 2 , Mahmoud Karami 3 ,  and Saeed Mohammadi 4
•
Experimental design: The experimental design is based on camera trapping methods which seems suitable but the authors can explain how the similar projects used this manner as well.
Validity of the findings: In my idea, Jerboas are very interesting animals in desert areas which can be use an ideal model for investigating on small mammals activity patterns.
Additional comments: I suggested two related papers which Authors can use in the discussion part.


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: DETERMINING INSULIN SENSITIVITY FROM GLUCOSE TOLERANCE TESTS IN IBERIAN AND LANDRACE PIGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and with clear language despite minor english issues occurring throughtout the paper. Some examples that can improve its simplicity and readability are listed bellow:

line 10 - add "at" before 10 (also happens in line 198).
lines 42-43 - remove "compared with modern lean breeds".
line 61-64 - rewrite as follows: "After acclimatization and to subsequently avoid the stress of repeated blood sampling, each animal was surgically fitted with a chronic catheter (Tygon, i.d. 1.02 mm, o.d. 1.78 mm; Cole-Parmer, Vernon Hills, IL, USA) in the carotid artery following a procedure described previously (Rodríguez-López et al., 2013). "
line 68 - write the abreviation in full extense the first time it is used (also happens with the coefficent of variation, CV)
line 75 - rewrite to: (...) "and to expose the carotid artery."
lines 238-240 - This sentence doesn’t make sense as it is since fasting plasma insulin levels cannot be affected by something that didnt occur yet (IAGTT). I would suggest rewriting as follows: “In the current study we have shown that despite the higher fasting plasma insulin, Iberian pigs produce a higher insulin response after glucose infusion when compared to Landrace pigs.”
line 248 - i assume a "reduced" is missing before insulin sensitivity.
lines 289-290 - i would suggest changing to "be a consequence of the greater adipose tissue availability"
line 350 - add "the" before "values obtained for lean pigs.

The references used are somewhat old but fulfill the requirements for context in the topics discussed. All submitted tables and figures are well constructed and usefull but lack a title. I would also personally suggest for the fasting values to be added to table 1 so that comparing the effect of the before and after is easier on the reader perspective. I would also suggest "pigs" to be used in the title of the manuscript instead of "pig".
Experimental design: The aim of the study is to add new information about the previously suggested idea that insulin resistance or lower insulin sensitivity is occuring in Iberian pigs when compared with leaner breeds. The research methods used fulfill the basic requirements but as stated by the authors in the conclusions, the usage of an hyperinsulinemic euglycemic clamp would provide further knowledge regarding this topic. On the other hand, the authors also state that they didn't separate the LDL and HDL fractions which would help to have a better understanding to what is happening to the total cholesterol concentration. Furthermore isnt it also possible that the lower cholestol levels found in both breeds is related to the fact that they are still very young and could manifest the expected cholesterol changes at a later stage of their development? i think this aspect could have been more discussed...
Validity of the findings: The replication count is barely minimal to extract big conclusions and one of the breeds has one less replicate which i think is a good policy from the authors to explain how this occurred in the manuscript. The data provided are robust as they stand and the conclusions in line to what the results show.
Additional comments: no comment


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

Input:
Title: DETERMINING INSULIN SENSITIVITY FROM GLUCOSE TOLERANCE TESTS IN IBERIAN AND LANDRACE PIGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The current manuscript provides information on insulin sensitivity in two breeds of pigs, Iberian (a slow growing native breed of the Mediterranean basin) and Landrace (conventional) growing pigs. The authors hypothesized that Iberian pigs may be insulin resistant. The present paper is clearly written. Nevertheless, some methodological issues must be addressed and the result and discussion sections need to be improved significantly.

The current study is based on the investigation of 4 or 5 pigs/ breed which is quite low. In this context, we need more information on pig features. The authors should provide at least some data related to body composition (backfat thickness at least to assess adiposity). As the authors refer very often to obesity models in the discussion, we need to have more information on the 9 examined pigs.

Another major concern is dealing with result presentation and also with the analysis of the response to the glucose tolerance test. Data related to fasting plasma concentrations should be included in a table (table 2 or a new table). Similarly, other parameters (L199-201) should be also included in a table. It will facilitate the interpretation of data. There is redundant information on figures. Instead of showing Fig 4-6, it would be more relevant to determine the total AUC and to include it in a table. It should also shorten the result section.

With respect to the discussion, some parts are written like a review of the literature. For instance, it is unclear whether the authors refer to previous experiments or to their study (L247-250). The authors suggest the possibility of insulin resistance in Iberian pigs. To clarify the discussion, they should discuss together all the data assessing insulin sensitivity. There is also an issue with the age of pigs. This point needs to be considered in the discussion.

Other specific comments

Abstract
-	L12: we should read “than in Landrace…
-	L15: lactate only tended to differ between the two breeds. This point needs to be corrected.

Materials and Methods
- L113: the sensitivity (minimum amount or concentration detectable) of the assay should be provided.
- L114-116: it is necessary to give information on intra- and inter-assay variability.

Discussion
-	L227: what are the criteria used to define the obesity of Iberian pigs.
Experimental design: As indicated above, more information on the phenotype of animals is requested.
Validity of the findings: The findings are valid.
Additional comments: The current manuscript is clearly written but needs to be significantly improved.


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

Input:
Title: DETERMINING INSULIN SENSITIVITY FROM GLUCOSE TOLERANCE TESTS IN IBERIAN AND LANDRACE PIGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In the current revised manuscript, the authors incorporated the requested changes with one exception. As indicated previously, there was redundant information on figures. Instead of showing Fig 4-6, I indicated that it would be more relevant to determine the total AUC and to include it in a table. It should also shorten the result section. The authors replaced the 3 figures by 3 tables but did not calculate the total AUC to get one value/animal. This point should be considered.


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: T CELL MEDIATED IMMUNITY AGAINST INFLUENZA H5N1 NUCLEOPROTEIN, MATRIX AND HEMAGGLUTININ DERIVED EPITOPES IN H5N1 SURVIVORS AND NON-H5N1 SUBJECTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clearly written and well organized. The introduction and background are well justified.
Tables and figures are comprehensive and helpful to understand the manuscript.
Experimental design: The experiments were designed nicely.
Please mention the data analysis software/tests to generate the figures  in the "materials and methods" section.
Validity of the findings: No comments
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: T CELL MEDIATED IMMUNITY AGAINST INFLUENZA H5N1 NUCLEOPROTEIN, MATRIX AND HEMAGGLUTININ DERIVED EPITOPES IN H5N1 SURVIVORS AND NON-H5N1 SUBJECTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The study determined the T cell responses against the H5N1 HPAI virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals using IFN-γ ELISpot and microneutralization assays and flow cytometry. The study reported profound T cell responses mostly against selected H5N1 NP epitopes in PBMCs of two adult survivors which lasted over 4 years post infection. The study tested an array of H5N1-specific T cell epitopes located in HA, NA and NP peptides and came out with few unique peptides in H5N1 and non-H5N1 survivors. In addition, 60% of the H3N2 patients showed cross-reactive T cell responses against H5N1 NP peptides owing to sequence homology between these viruses.
The data on T cell responses in H5N1 human survivors are valuable. Identification of unique and conserved epitopes in NP gene showing high immunogenicity would guide in designing T cell based vaccines for broad subtype viral protection. The manuscript is well written with clear methodology.
However, there are few issues that authors need to discuss in the manuscript. For example:
a.	Size of the cohort: The study involves a small (only four H5N1 subjects) patient cohort from 2004.
b.	It was not mentioned in the text which clade of the H5N1 virus was detected in the four H5N1 survivors.
c.	Peptides generated from H5N1 virus of clade 1 was used for T cells response analysis. It is necessary to discuss how the T cell responses data of the study would correlate with the T cell responses against currently circulating H5N1 viruses in Thailand?
d.	The reason for higher immunomodulatory property of NP peptides needs discussion.
e.	Repetition of data should be avoided: Figure 2 and Table 1; and Figure 3 and Table 2.


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

Input:
Title: WHEN ECOLOGICAL MARGINALITY IS NOT GEOGRAPHICALLY PERIPHERAL: EXPLORING GENETIC PREDICTIONS OF THE CENTRE-PERIPHERY HYPOTHESIS IN THE ENDEMIC PLANT LILIUM POMPONIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The submission by Casazza et al. is for the most part written well and easy to follow. There are a few places where a series of sentences begin with phrases such as 'consequently' or 'therefore' that I found a bit distracting, but this is a stylistic issue. The scholarship here is first rate, with a detailed list of appropriate citations that support the main points that Casazza et al. seek to make. I found the structure of the manuscript to be appropriate, and figures and tables were easy to interpret.
Experimental design: The work here clearly fits into the scope of PeerJ. The design of Casazza et al is an effective exploration of the Center-periphery hypothesis. The CPH assumes that the 'optimal' environment is in the center of the geographic range, but this assumption should be tested. Casazza et al. design their study in an insightful manner by asking first if geographic and environmental centre-periphery gradients are correlated and second by asking how genetic differentiation and genetic diversity are associated with these gradients. Their study has a very good sample size. The genetic data (AFLPs) that they have collected are be limited for some research questions and applications but are perfectly appropriate here because they represent a quick survey of genomic diversity. The description of what they did was easy to interpret and follow, so I didn't have many questions about how or why the authors did what they did. I have one substantial concern that I'll describe in the next section.
Validity of the findings: Underlying data are provided, and this work is reproducible. My main critique concerns the climatic variables that they used. Climatic variables are potentially correlated with one another, which makes me question whether the authors should use all of the variables in the Chelsa database. I would suggest that  rather than using all of these data, Casazza et al. should identify and remove variables that are autocorrelated. This will likely make the spatial gradients in these variables more obvious and detectable, which in turn would provide a better estimate of the degree to which the climatic and genetic variation as associated.
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: WHEN ECOLOGICAL MARGINALITY IS NOT GEOGRAPHICALLY PERIPHERAL: EXPLORING GENETIC PREDICTIONS OF THE CENTRE-PERIPHERY HYPOTHESIS IN THE ENDEMIC PLANT LILIUM POMPONIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous language is used throughout. A few noted corrections and suggestions are noted in the comments to author. For the most part, the background, literature, etc. are sufficient. The formatting of the article is appropriate. I can't seem to find a statement indicating where the assembled datasets (climate, AFLP, etc.) are/will be available, so I have not viewed any raw data. Existing tables are appropriate but insufficient; I would add a table of accession information and/or a statement about population location. Were vouchers made, and if so, where were they deposited. If not, why? It is conceivable that this species may be the target of poaching. The results of the data analyses do address the questions stated at the end of the introduction.
Experimental design: This study is structured as an exploration 1) of the correlation between geographic and environmental gradients, 2) the genetic association between geo. and env. gradients and both genetic differentiation and genetic diversity. The research question is unfortunately poorly defined, mainly because of vagueness in the connection between the CPH in the introduction and the questions. Structuring the paper around subsidiary hypotheses might help guide the reader to a better understanding and interpretation of the results.

The study seems to have been conducted in a rigorous fashion and with appropriate ethical considerations (specifically, a scientific collection permit was obtained). I am concerned that the explanation of the AFLP protocol is misleading. The authors correctly note that the standard AFLP technique is inappropriate for Lilium because the large genome results in excessive fragments. The authors state that they use the method used by Sakazono et al. 2012 with Lilium longiflorum to reduce the number of fragments. Whereas conventional AFLP uses MSE and ECO primers with one additional base each in the preselective amplification and three additional bases in the second, selective amplification. This appears to be what was done in this study (lines 128-132). The Sakazono method differs in that they used THREE bases on the MSE primer for the preselective amplification and FIVE additional bases on it for selective amplification. This inconsistency needs to be resolved. I am not able to review the use of climatic variables.
Validity of the findings: It is not possible to fully gauge the validity of the results given the absence of the raw data and the concerns over the use of the AFLP technique. The statistical analysis seems appropriate for the genetic data. The patterns discovered in the data include an increase in genetic differentiation near the geographic periphery of the species range, and a reduction in genetic diversity in ecologically marginally habitats. The "refugia within refugia" hypothesis is consistent with the data for the former pattern, for the latter, the conclusions as stated are speculative. The data doesn't really allow for a measure of drift-based vs. selection-based explanations for the reduction in diversity in poorer habitats.
Additional comments: Line 39: "environmental" is misspelled.

Line 76 and onwards: This reviewer would appreciate a brief synopsis on studies that failed to find population parameters in line with the CPH hypothesis, and why genetic diversity does follow the predictions of the hypothesis. The following line, states that "Nevertheless, other studies suggest that the genetic diversity of populations is mainly affected by the environment." It is unclear how diversity can be affected by the environment if it is not mediated through the population parameters such as demography, population size, etc. that supposedly do not conform to predictions. Continuing in the introduction, the organization could be improved to more clearly connect the expected relationships of genetic differentiation and diversity with geographic and environmental gradients. It is unclear what findings are expected in this species. For instance, on line 94; the authors state that the "environment is therefore expected to have had a strong impact on... genetic variation": what would this look like? Increased heterozygosity? Deficiency in private alleles in populations? How would this differ from the effects of geographic isolation? A suggestion to clarify this would be to develop this part of the introduction explicitly in terms of the main forces of evolutionary change involved: selection, drift, mutation, migration, non-random mating.

Line 97: change "their" to "there".

Line 111: Table 1 is the AFLP primers, not a sample list. Table 2 lists population numbers but does not provide localities, so the statement that sampling covers the geographical range cannot be evaluated.

Line 116: The second sentence beginning on this line "The AFLP..." should be moved to a position after the description of DNA isolation- before "for the AFLP..." on Line 122.

Line 118: Muratovic (2010) is not in the lit cited.

Line 264: The statement that "genetically impoverished populations are more prone to genetic drift and consequently to divergence" is confusing cause and effect: both genetic impoverishment and divergence are expected consequences of genetic drift, as allele frequencies change stochasitcally in populations with small effective size. Note that this presents a problem for the idea that natural selection may be playing a role in reducing genetic variation (the "genetic draft" hypothesis discussed beginning on line 317) since populations subject to high levels of drift will be correspondingly unresponsive to selection.

Lines 290-300: the logic of this argument is contorted but I think it could be made clearer.

Line 311-313: was habitat quality specifically characterized? COULD it be characterized by working backward from the climatological analysis? (I don't know the answer to this.) If it could, then perhaps genetic diversity could be evaluated specifically against quality.

Line 329: species' (geographical/environmentalal) distribution

Line 332: PeerJ asks that speculation be identified as such. It seems that the relative contribution of drift-related effects and selection is not something the authors wish to take a position on, so the balance must remain speculative.


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: WHEN ECOLOGICAL MARGINALITY IS NOT GEOGRAPHICALLY PERIPHERAL: EXPLORING GENETIC PREDICTIONS OF THE CENTRE-PERIPHERY HYPOTHESIS IN THE ENDEMIC PLANT LILIUM POMPONIUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.English is not clear and concise enough. It is suggested to modify the language and expression.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the introduction, the author said that when plants encounter climate change, they will migrate to a new and more adaptive environment, and their traits will change. But the  CPH theory is that plant diversity and differentiation will gradually decrease and increase with the geographical center to the geographical edge. I don't think that the variation of plant traits from the geographical center to the edge of CPH theory seems to have much to do with the adaptation of plants to the new environment and the variation of  traits. It is suggested that the author should revise the introduction to meet the main idea of this 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: CHARACTERIZATION OF THE FIRST VAGINAL LACTOBACILLUS CRISPATUS GENOMES ISOLATED IN BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work presented by Marcelle Oliveira de Almeida and colleagues reports the sequencing of four strains previously isolated from individuals diagnosed as healthy or with bacterial vaginosis. Overall, the strains show genomic features that suggest can be safe for further use as a complementary therapy for bacterial vaginosis. Genomic analysis indicates that no antibiotic resistance genes are present in any of the four strains analyzed. Strains harbor Cas enzymes but they may also be inactive. Prophages are also present and may be active. Metabolic coding genes analysis shows that these strains may render protective benefits.
Overall, the manuscript is well written, methods and experimental design are sound, and the conclusions can be drawn from the data obtained.
Experimental design: This reviewer finds the experimental design sound and well-conducted. My only request regarding this is that authors add the accession numbers of the datasets deposited in GenBank and please add the statement the source of each strain as “from healthy patient” or “BV patient”.
Validity of the findings: In the general comments section, the authors will find the concerns rose by this reviewer and some suggestions that I think need to be addressed. The findings reported in this manuscript are relevant, although the prophage issue for this reviewer is important to complement this manuscript. Some analysis seems poorly discussed and that is highlighted in the General comments section. Overall, this reviewer thinks that the manuscript is of worth and can be published, but some modifications are needed to strengthen the manuscript.
Additional comments: Major comments:
In the methods section please add a statement of the GenBank data accession number, although given in Table 1, this is a must statement either at the end of the manuscript as data availability or in the methods section.
This reviewer finds that the phylogenomic tree is not fully discussed. I agree that the clustering between healthy and BV samples is not present but is interesting that the sequenced strains in this study belong to a cluster mostly represented by samples from the Netherlands. Does this have any meaning? Is there any relevant feature found in those strains? Also, most of the samples from the Netherlands are from BV samples, if the sequences from these samples are separately analyzed with the Brazil samples, any interesting feature surfaces? I suggest adding a line in the discussion section regarding this feature.
Line 308-310, to further support the safety of these strains, I encourage authors to provide at least as a supplementary figure the results for lytic phages present in CR14 and CRI17 strains, perhaps a plaque assay. This reviewer understands that authors may be preparing a report of these findings, but due to the statements in this manuscript regarding the safety of these strains, I encourage authors to provide the information requested, please read lines 332-333 in this regard.
Although interesting, the search for CRISPR loci and Cas coding genes seems out of place from the scope of the paper, I suggest authors revise the discussion (lines 311-322) to explain why this is important to BV and microbiome dynamics. Also, the authors emphasize that the CRISPR-Cas systems are not functional in these strains, I encourage authors to provide a line why this is relevant in their study and provide a line regarding the reading frame of these genes that fully explain that may be inactive.
Finally, regarding the probiotic effects of these strains, is there synteny between the lactic acid synthesis enzymes? Genomic rearrangements are found between strains?
Minot comments:
Please use in the abstract bacterial vaginosis instead of BV.
Line 69, please correct to “healthy microbiota”
Line 83 please correct to “BV microbiota were performed using samples isolated from the USA…”
Line 99 please change to: “with the approval of the ethics…”
In the methods section, please deposit or provide a legend to the in-house pipeline generated so readers can refer to the supplementary information provided and perhaps deposit the scripts used in a GNU or similar agreements.
Line 274: please correct to “health-related condition does not show clustering (Fig 1) as…”
Line 286, please correct to “a safety concern”
Line 302, please change to “carry a second gene, a …”
In line 345 please add a reference for the risk factors associated with vaginal homeostasis and dysbiosis.
Figure 1 legend, please correct to Filled squares, bacterial vaginosis samples, Empty squares, samples from healthy individuals.
In Table 4 the Prophages identified, please correct to questionable in full.


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: CHARACTERIZATION OF THE FIRST VAGINAL LACTOBACILLUS CRISPATUS GENOMES ISOLATED IN BRAZIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is mostly clear and unambiguous. I have noted several small grammatical errors that are completely understandable as the authors are not native English speakers. The issues listed below just illustrate these errors (I've only listed those in the Discussion, there are others throughout that can be easily fixed by a native speaker).
Several minor grammatical errors are present in the manuscript:
	Line 295: Fourteen known IS families and one new family (were) was predicted
Line 301: IS (ISs?) from families…. carry (a) second gene…
Line 312: ‘Type II and II-A are the most common type, and subtype in Lactobacillus, respectively, and…’ Is it rather ‘most common type and subtype in Lactobacillus, respectively…?
Line 331: ‘suggest that they attend the safety requirements (replace attend with ‘bear’ or something similar)?
Line 333: lysogeny (not lisogeny)
Line 361: change ‘has’ to ‘have’
Article structure is fine. I found figure 2 to be not so important and could be deleted or removed to supplementary.
Experimental design: The work is within the scope of the journal. It is not completely clear why an additional four genomes of L. crispatus are necessary, but the authors emphasize the need for coverage in South America. Hence, they publish these four sequences. The data is good, methods are well described.
Validity of the findings: Overall, this paper meets these criteria. I found the GWAS analysis the least useful and felt it could be deleted,
Additional comments: Suggest a proof by a native English speaker to correct minor grammatical errors. Overall, there is no concern about the data. The assemblies are good, if not completely closed. Some of the gaps could be associated with the broken genes identified and that could be examined more carefully. Clearly, the results are not 'earth-shattering', but should be relevant for people in the field. In addition to the previous comments, there are several minor issues:

1. Line 239: is it a mistake that subtype II-C Crispr system is only in CRI10? It seems to be present in CRI17 in Table 4?

2. Fig. 3. A legend with the colors identified would be better than the description (green, pink, red?).

3. Positive selection: one of the 8 protein genes was identified as secreted (was this identified with appropriate targeting sequences)? How was ‘cytoplasmic’ determined (lack of targeting sequence?).

4. Line 306-307: ‘ four to six incomplete prophages …. the genomes were not closed’. Were the phage genomes split by gaps in the assemblies?

I might suggest abbreviating the Discussion as it is rather extensive, and in particular, the GWAS section (see above comment).


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

Input:
Title: EXPRESSION ANALYSIS OF GENES RELATED TO COLD TOLERANCE IN DENDROCTONUS VALENS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors say "All raw sequence data were deposited in the NCBI Sequence Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/subs/) under BioProject accession no. PRJNA609406. However, I was unable to find this database on the NCBI BioProject database. Could the authors please check this. Perhaps the submission has not been fully finalized or has not yet been released to the public.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Overall, the manuscript was well written and the data are strong. Various comments are below. My greatest problem with the manuscript is that although many DEGs are identified and 112 candidate genes explored, the authors consistently fail to clearly state what they are comparing in both the Results and Discussion. We are told that genes are differentially regulated but not whether upregulation means that genes are expressed more strongly in January animals than in May animals or more strongly in May animals than in January ones (also what is the comparable definition for down-regulation). Even Table 4 does not define what the comparison is for “down” and “up”. Please make an effort to clarify sentences in the Results and Discussion so that readers can better appreciate which genes are apparently upregulated in support of winter survival vs those that are suppressed in the winter OR alternately which are not expressed in January but upregulated in the spring when eating and growth resume. This would help readers to understand the possible functionality of various differentially expressed genes with respect to cold hardiness versus spring resumption of growth.

1.	It would be helpful to include the common name of this beetle in the Abstract and/or the Introduction to attract readers who do not recognize the scientific name since, for example, pine beetles are well known in North America for their widespread devastation of pine forests.
For example, the second sentence of the Abstract could say “Adults and larvae of this bark beetle winter at….”
For example: the first line of the Introduction could be “Dendroctonus valens, the red turpentine beetle, is a species of bark beetle that mainly attacks the base of the trunk….”

2.	Line 65. The authors might also be interested in an early review by RW Salt: Principles of insect cold hardiness, Annual Review of Entomology, 1961, 6: 55-74.
3.	Line 123. Please indicate if the biological material was “whole animal” or whether a selected part of the animal was used. Also, did each sample consist of a single individual or were multiple animals combined per sample or per biological replicate.
4.	Line 125-6. Please define what CL, NL, CA and NA mean for readers. Clearly the L and A would be larvae and adults but C and N are not defined.
5.	Line 305. Is “degradation” the correct word when later in the sentence the authors include “21 genes involved in the synthesis of low molecular weight polyols”.  Perhaps the 44 genes are “related to cryoprotectant metabolism” including both synthesis and/or degradation.
6.	Lines 338-343. Please clarify the parameters of “up-regulated” and “down-regulated”. Are you comparing the May data to the winter January situation OR the January data with the later May data. This is important for data interpretation – for example, we would expect a reduction in transcripts coding for polyol synthesis genes in May, as compared with January, but not the reverse. This information is also needed for readers to be able to interpret and understand the results reported in lines 343-347 as well as other data mentioned later
7.	Lines 342-343. “…these results suggest that some physiological activities remain active or are initiated” – when? In May as compared with earlier in January? Or were active in January but no longer in May?

8.	Line 362-363.  Trehalose is NOT a protein, it’s the normal blood sugar of insects but is often elevated as part of the cryoprotectant system in winter. Do you mean “trehalase”, the enzyme that cleaves the disaccharide into 2 glucose molecules?
9.	Lines 388-395. Can the authors tell readers whether D. valens uses the freeze-avoiding or the freeze tolerance strategy for subzero survival? Freeze-avoiding insects that deeply supercool will have antifreeze proteins whereas those that are freeze tolerant are much less likely to have such proteins and instead have ice-nucleating proteins that help the insect to regulate ice formation in specific body compartments. If this has not been tested for D. valens, then perhaps this has been tested for D. ponderosae. Most beetles use the freeze avoidance strategy but it would be best to indicate what is known for D. valens or for pine beetles in general
10.	Line 388 is better stated as “Studies show that cold tolerance in many insect species is directly related to antifreeze proteins…”. Most of Duman’s work (as well as Kostal’s) has centered on freeze avoiding species that deeply supercool – hence, the interest in antifreeze proteins by these authors. For lots of other species, however, freeze tolerance is the adaptation used.
11.	Line 404-405 needs a reference citation for the Galeruca daurica statement.
12.	Line 444 and following. In these conclusions, please make it clear whether the enriched DEGs and the 112 screened genes were ones that were higher in January (ie. when cold hardiness is needed) or higher in May.
13.	For Table 4, please define what up and down are. Does UP mean the gene in was higher in January or higher in May? Same question for DOWN. Please define this in the legend of the table. This is crucial for readers to be able to understand the data. Also, I would suggest adding 2019 to the Table legend as in “collected in January 2019 or in May 2019” so that readers realize that the sampling timeline was January first and then May later.
14.	For the legends of Fig. 7 and 8, please define what the error bars are (SD or SEM) as well as the value for N (number of replicates). It would also be useful to restate that animals were sampled in January 2019 and then May 2019.


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: EXPRESSION ANALYSIS OF GENES RELATED TO COLD TOLERANCE IN DENDROCTONUS VALENS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript is generally clear, well-written, and well-organized. However, throughout spaces are sometimes missing between words, or when a word is followed by a parenthesis. Sometimes an extra space appears between the end of a word and a punctuation mark.

As described below in Comment to Authors:
-There are references lacking for some of the software used.
-The authors do not provide the relevant context to their work by omitting prior relevant research in other Dendroctonus beetles.
-Public deposit of transcriptome assembly and gene expression analyses should be done (NCBI TSA and GEO, respectively).
Experimental design: Although the experimental design is appropriate, I am not sure that the two timepoints chosen best reflect the changes that are happening to develop freezing resistance during the winter, as the winter timepoint would be after the insects are likely to have obtained their maximum freezing resistance and would have a reduced metabolic rate. As the authors suggest, perhaps proteomic (and metabolite) analyses would better answer what is happening at the January timepoints.
See General comments for the author
Validity of the findings: Authors should cite and incorporate their results with those previously reported in other Dendroctonus.
See General comments for the author
Additional comments: DNA data checks
Have you checked the authors data deposition statement?
Link does not exist
Can you access the deposited data? No
Has the data been deposited correctly? No
Is the deposition information noted in the manuscript?
Authors indicate that the raw reads have been submitted to NCBI SRA, but not the assembly to NCBI TSA, nor the gene expression results to NCBI GEO. These data should also be included as part of a complete set of data deposition of RNAseq transcriptomic data.
Field study
Have you checked the authors field study permits?
Link does not exist
Are the field study permits appropriate?
Cannot determine this

Comments:
Lines 1 and 451: I don’t think “screening” is appropriate here. It implies that they were assayed for function. For the title, remove “Screening and”. For line 451, change to “identified”.
Line 116: What instar were the larvae? How was this determined?
Line 118, modify as indicated for clarity: (Longitude: 118.43° E; Latitude: 41.41° N; Altitude: 1050 m)
Lines 118-119: How were these temperatures obtained?
Lines 133-134: What type of library was constructed? Manufacturer? Single or paired-end, stranded or un-stranded? What length of reads were generated?
Line 144: why “reassembled”? Was it previously assembled?
Line 175: Colin isn’t the last name of this author.
Line 208, modify as indicated for clarity: “…melting curves. Each reaction was performed for three biological and three technical replicates.”
Line 198: TUB is used as one of the reference genes (for larvae), yet in Table 4, it shows that two tubulin transcripts are differentially regulated in larvae. The transcript ID of TUB (and PRS) should be indicated, and there should be an explanation of which tubulin is used as TUB vs. those shown as DEG in Table 4.
Figure S1 needs a description included
Table S1: Amplicon sizes are lacking for TUB and PRS18. Primer-pair efficiencies are not shown at all nor mentioned in the M&M.
Line 150: What criteria was used for clustering with CD-HIT?
Lines 152 and 219 and Table S2: BUSCO information is not properly presented. What dataset was used? What does BUSCO score of 96.3%(16.7%) mean? Use the recommended format described here: https://busco.ezlab.org/busco_userguide.html#reporting-busco
Line 163: reference for DIAMOND?
Line 165: reference for BLAST2GO?
Line 167: reference for KOBAS?
Line 187: reference for goatools?
Line 199: reference for Primer 3Plus?
Line 209, modify as indicated for clarity: “…experimental data were normalized…”
Line 217, modify as indicated for clarity: “…After clustering and reducing redundancy…”
Line 238: This sentence is confusing because you mention FPKMs but DESeq2 uses counts for DEG analysis.
Lines 278-280: Cold tolerance is a process that likely begins in the fall with decreasing temperatures. By January, the beetles should already be well-adapted to the winter cold and their metabolic rate would be quite low, and thus I question whether the beetles are RAPIDLY synthesizing small molecules, such as sugars, as antifreeze protectants at this time.
Lines 291-293: Is there any information about this in other bark beetles?
Line 352: “…cytotoxic P450 enzymes…” implies that these enzymes are cytotoxic, when I think you really mean that these enzymes modify cytotoxic metabolites. Reword.
Line 353: “…cytochrome P450…” singular or plural?
Lines 345 and 354: “very long-chain fatty acids” are not proteins/genes. Do you mean proteins/genes in fatty acid biosynthesis?
Line 361: “…freezing tolerance.” Freezing tolerance is a completely different adaptive strategy to cold temperatures than freezing-resistance (avoidance). What information is available in this and other related bark beetles on these two mechanisms? Please be sure to use the correct terminology throughout.
Line 396: The metabolic rate of bark beetles in January at -12°C is probably going to be much lower than in July. Thus, could the abundance of down-regulated genes in winter insect merely reflect this decrease in metabolic rate, rather than genes inherently important to cold tolerance?
Lines 397-399: Maybe this has already been explored in another bark beetle:
Bonnett, T.R., Robert, J.A., Pitt, C., Fraser, J.D., Keeling, C.I., Bohlmann, J., and Huber, D.P. 2012. Global and comparative proteomic profiling of overwintering and developing mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae. Insect Biochemistry and Molecular Biology 42: 890-901. doi:10.1016/j.ibmb.2012.08.003.
Fraser, J.D., Bonnett, T.R., Keeling, C.I., and Huber, D.P.W. 2017. Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), larvae. PeerJ 5: e3284. doi:10.7717/peerj.3284.
Robert, J.A. et al. 2016. Gene expression analysis of overwintering mountain pine beetle larvae suggests multiple systems involved in overwintering stress, cold hardiness, and preparation for spring development. PeerJ 4: e2109. doi:10.7717/peerj.2109.
Line 438: -18.7°C Where does this temperature value come from?
Fig. 2C: Most of the non-Dendroctonus species are nematode, not insects at all. This suggests that there is significant contamination in the RNAseq data (>25%), most likely from nematodes in/on the beetles. It would seem that you already have the information from the BLAST and DIAMOND results to properly remove these sequences from your assembly.  These need to be removed from the assembly prior to DEG analysis. Transcripts could be incorrectly identified as differentially expressed merely because they are nematode transcripts, and perhaps only adults at a certain time carry these nematodes. You also loose statistical power for identifying differences in real beetle transcripts because of these spurious transcripts. You will find that NCBI will also flag these transcripts when you submit this assembly to NCBI TSA.
Throughout, spaces are sometimes missing between words, or when a word is followed by a parenthesis. Sometimes an extra space appears between the end of a word and a punctuation mark.


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: EXPRESSION ANALYSIS OF GENES RELATED TO COLD TOLERANCE IN DENDROCTONUS VALENS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The revised manuscript has addressed most of the prior concerns by the reviewers.
Experimental design: The revised manuscript and the rebuttal has addressed most of the prior concerns by the reviewers.
Validity of the findings: The validity of the findings may have been negatively affected by importing FPKMs into DESeq2, which it specifically warns against.
Additional comments: The revised manuscript has addressed most of the prior concerns by the reviewers. I identify the following:

Lines 62-63: This sentence doesn't have support. I would suggest deleting it. Freezing-tolerant and freezing-avoidant insects are not novel, so the mechanism is not as yet described, novel.

Line 128: mature -> late instar

Line 131: Inner Mongolia

Line 141: animal -> insect
Line 141: "Each sample consisted of..."
Line 141: repeats -> replicates

Lines 184 and 253, Table S2: There is no invertebrate dataset for V3 of BUSCO (https://busco-archive.ezlab.org/v3/frame_wget.html). What database and what version obd9(?) was used.
as per BUSCO's website, Reporting BUSCO:
Report results in simple BUSCO notation: C:89.0%[S:85.8%,D:3.2%],F:6.9%,M:4.1%,n:3023
Report the BUSCO set(s) you used for your assessments. Mention the creation date of the dataset, not only the name, e.g. archaea_odb10 (2019-01-04).
Report the BUSCO options you used.
Only the first 2 or 3 values are shown. Show them all as recommended, C:89.0%[S:85.8%,D:3.2%],F:6.9%,M:4.1%,n:3023.

Line 207: DESeq2 documentation specifically states that FPKMs should not be used as input to DESeq2, as mentioned in review of version 1 of this manuscript. Submitting normalized FPKMs will over-normalize your data and will likely negatively affect the statistical analysis of your data. The data from RSEM can be correctly imported into DESeq2 with the tximport package, which it seems you haven't used if you are incorrectly importing FPKMs into DESeq2.

Lines 264-5: "The sample" is confusing. Was only one sample contaminated? Do you mean that some sequences were contaminated? Was a sample deleted, or sequences deleted?

Line 403: accents missing from Koštál.

Other: It is concerning that the transcriptome is not acceptable to NCBI TSA due to errors. You do not specify why, so we cannot assess whether this is something that could affect the conclusions drawn, such as the prior nematode contamination you have now removed. Adapter or other contamination? The Sequin file included with the manuscript is not an appropriate alternative. I see that the transcriptome contigs are included with the GEO submission. Thus, interested readers in the future will be able to access the sequences that way. However, without submission to TSA, your data will be invisible to BLAST searches etc., which diminishes the value of your data generated, missing a wider consumer of the data.


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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article makes an interesting analysis based on genome mining and is has been written in correct English
In the background, please clearly indicate in rows 36 and 37 the abbreviation of polysaccharide lyases (PLs) and  glycoside hydrolases (GHs) to take into account in subsequent sections.
Since the main objective of the article is to verify the ability of the bacteria to degrade polysaccharides based on the presence of enzyme genes, the introduction should include a concise review of the corresponding Cazy PLs y GHs enzyme families. As it stands, it has only provided information on the structure of the polysaccharides used as a carbon source and the application of their corresponding oligosaccharides.
The structure of the article follows the standard format.
Regarding the figures, I believe that figure 1 is sufficient and it is necessary to graph it on a semilog scale.
Figure 2 does not have relevant data to show and may only be indicated in the text, in addition to that a different method of growth measurement has been used.
Files S3 and S4 of supplementary material should have clear titles and a brief description of the data displayed.
In file 4 an incomplete graph is observed.
Since the hypothesis is supported mainly by bioinformatic analysis, the other data referred to growth on polysaccharides based media (figure 1) should be strengthened, including, for example, a kinetic analysis with quantitative parameters (growth rate µ h-1)  (carried out by applying basic mathematical modelling).
Experimental design: The article highlights the ability of Catenovulum sp. CCB-QB4 to degrade polysaccharides (alginate, pectin ulvan, and gellan gum) and generate oligosaccharides with different applications.
The hypothesis deduces that the presence of genes for degradation and utilization of polysaccharides (alginate, pectin ulvan, and gellan gum)  in the genome of CCB-QB4  as well as  its  ability to grow in minimal media with these compunds as a carbon source can provide evidence of active metabolic pathways. Give that transcriptomics approaches offers direct evidence related to expression of the genes involved in the degradation of these polysaccharides, it is necessary to justify it because the proposed methodology may be sufficient to support the working hypothesis.
About the microorganism, Catenovulum sp. CCB-QB4, since some pectin utilization genes are present in the plasmid of the bacterium, indicate whether in all cases it has been verified that the bacterium has maintained its plasmid in all experiments.
Also please clarify if these genes are exclusive to the plasmid or are also present in the genome (this is not indicated in the Lau et a. 2019 reference).
About M&M: Please explain why two different growth measurement methods have been chosen. The data in figure 1 have been obtained by plate counting, while those in figure 2 result from measuring OD at 600nm. In both figures (1 and 2)  the shape of the growth curve on glucose as well as the growth times are different.
Validity of the findings: The article has a simple experimental approach, but the bioinformatic analysis contributes positively to the relevance of its results.
The conclusions are adequate although in some way future studies should be suggested for contribute to elucidate the metabolic potential of CCB-QB4.
Additional comments: The article is simple but interesting and can contribute to the knowledge about the metabolic capacities of CCB-QB4.


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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript investigated the growth conditions under different uronic acid-containing polysaccharides,  sequenced and annotated its genomic information. However, it is not suitable for publication in Peer J due to its preliminary experiment and insufficient works. It should be considered to be submitted to more professional journal such as Marine Genomics et al.
Experimental design: The experiment is preliminary and the results are insufficient to support the research.
Validity of the findings: The validity of the findings is sound and it need more results like protein expression and characterization to support the validity of the findings.
Additional comments: The manuscript investigated the growth conditions under different uronic acid-containing polysaccharides,  sequenced and annotated its genomic information. However, it is not suitable for publication in Peer J due to its preliminary experiment and insufficient works. It should be considered to be submitted to more professional journal such as Marine Genomics et al.


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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript provided by Furusawa et al. describes the characterization of genes involved in uronic acid degradation within the bacterium catenovulum strains QB4. In its current state, I felt that the manuscript was not focused making it hard to identify the main objective of this research, and several important data required to justify their claims were missing.

First of all, it is unclear overall on what the authors wish to convey to the readers. The authors presented the data by stating that strain QB4 harbors numerous genes involved in the depolymerization and utilization of uronic acids but did not state clearly on whether they wanted to say that QB4 is a strain that is unique and the first of its kind in comparison to currently reported strains or whether it is only among the catenovulum genus. Currently, based on the objectives and discussions provided, the authors wish to convey the later message but if this is so, the introduction at its current state is not appropriate. If they wish to convey the later message, the introduction should focus on catenovulum strains including a review on the use or ability of catenovulum strains in relation to uronic acid or polysaccharide degradation. If they wish to convey the former message, it is recommended that the authors provided a wider review covering the major bacterial strains in relation to uronic acid and polysaccharide degradation. Either way, it is recommended that the authors clarify the objective of their research and provide an introduction that supports the significance of their research. Please comment and if necessary, amend accordingly.
Experimental design: In addition to the overall message of the manuscript, several points also need to be addressed regarding the experimental design.

The authors provided data on the degradation of the various polysaccharides in Figure 1 and 2 by presenting the growth of strain QB4 in relation to each polysaccharide, but did not provide data quantitating the reduction or accumulation of the polysaccharides nor performed assays to identify the end-products of each utilized polysaccharide. It is hard to speculate on what genes or enzymes are being used by strain QB4 without this data. Why did the authors exclude these analyses as these data would allow readers to better understand the characteristics of strain QB4, its capability in uronic acid degradation, or what are the possible products that could be attained using this bacterium. Please comment. If there was a reason why they excluded the end-product assays and analyses, they should explain this in the manuscript as it is common in enzymology for researchers to perform analyses on the end-products of a given enzyme.

The authors also should include information on the sequence similarity (percentage) of their enzymes with those identified from databases in Table 1-3 as this would allow readers to determine the novelty of the proteins within QB4. Proteins that may have been annotated to a functional protein does not necessarily mean that the identified protein does function as the similar protein. It all depends on the similarity of these proteins and the presence of motifs and enzyme activity sites. Therefore, it is necessary for the authors to provide this information as it will support whether the discussion and speculations made by the authors are true or not. Please provide the necessary data.
Validity of the findings: In terms of the validity of the findings, in the results section, assuming that the authors did determine the function of their annotated genes based on the function regardless of the percentage of sequence similarity, the authors provided a unique way of describing their findings by speculating the role of strain QB4 where they used information of enzymes reported by other groups. This was observed in Line 200 – 214 and throughout the Results section of the manuscript. It is really dangerous to come up with such speculation without supporting experimental data. Terms like “indicated” were also used but again this is commonly used when referring to data or results attained from experiments. Please avoid presenting speculations or terms without supporting data. The authors should also not mix up the results and discussion together as they are meant to be separated. Please comment and amend accordingly.
Additional comments: Line 285, please amend “Qb4” to “QB4”.


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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: I think this sentence: "This study is first report to describe the degradation and utilization pathway of four different uronic acid-containing polysaccharides in the genus Catenovulum" is not fully supported by experimental data and are partially based on bioinformatic analysis.

Then it is suggested to replace as follow:

This study is first report to describe the degradation and probable metabolic pathways for utilization of four different uronic acid-containing polysaccharides in the  genus Catenovulum.
Additional comments: The introduction of the manuscript has been improved according to the recommendations made previously and more references have been included. Only one last suggestion has been included for the final paragraph.
The methods have been explained more clearly and some experimental calculations have been included that better analysis of the growth curves.
Figure 1 has also been corrected and the organization and presentation of supplementary material has been improved.
There are no more suggestions to improve 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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 2
Reviewer: 2

Output:
Basic reporting: Having reevaluated the contents of the revised manuscript, the authors have improved the introduction to suit the objective of their research. Therefore I feel that the manuscript is now sound and readability has improved.

Data was requested in relevance to the assay of the end-products upon polysaccharide degradation (Fig.1 and Fig. 2) but as commented by the authors, subsequent work to characterize the enzyme from strain QB4 is currently ongoing. Since the contents of the results and discussion are based on the characteristics of the enzymes which is degradation of uronic acid, I still feel that it is necessary for the authors to provide data to at least show that the growth of the cells is due to the utilization of the substrate. Cell growth does not fully justify that the polysaccharides are being utilized or degraded. Please comment or provide the necessary data.

Besides the point above, I see that sufficient data was provided to further justify their claims in this work.
Experimental design: No further comments.
Validity of the findings: As commented in the "Basic reporting" section, the authors just need to justify that strain QB4 does utilize the designated polysaccharides (Fig.1 and Fig. 2).
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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 4
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have improved the article according to reviewers recommendations. In my opinion it is ready for publication.