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 USE OF BAT HOUSES AS DAY ROOSTS IN MACADAMIA ORCHARDS, SOUTH AFRICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript could still use some English editing/grammar assistance to read more clearly - I make a few suggestions below (I stopped after the introduction unless the meaning of the sentence was ambiguous) but the entire manuscript would benefit from a thorough editorial review for a more concise manuscript, clearly emphasising the findings and implications of those findings. For example, the main results/key implications are not immediately apparent from the abstract; instead the reader has to re-read sentences and makes their own inferences to determine how the results from this study relate to roost site preferences of African bats generally. In addition, the manuscript could be structured in such a way to emphasise the main points - I recommend following the suggestions in this blog (https://dynamicecology.wordpress.com/2016/02/24/the-5-pivotal-paragraphs-in-a-paper/). Lastly, the decision of when to create a new paragraph seems strange to me. I strive to write so that each paragraph conveys one key message, but with all of the necessary information for that message. Perhaps it’s a formatting issue, but it seems odd to me that some sentences are pulled out as an entire paragraph when they fit with the preceding paragraph(s) - e.g., lines 74-76 comprise one sentence and one paragraph that seems to be floating between two paragraphs when all three could be reduced to one. Lastly, the reference formatting and use of spaces, or lack thereof, with “&” between references with two authors, should be checked. In some cases the reference list is unnecessarily long - e.g., lines 58-60 list 9 references when the most relevant 2-3 could be listed with the use of “e.g.”.
Line 24-26 - awkward sentence, should be teased into two sentences as I’m not sure it’s grammatically correct. Do you mean something like: “The loss of roost sites is one of the major drivers of this decline; roost site preferences, either natural or artificial, are not well known for bat species globally, or African species specifically.”
Line 28 - incorrect use of a preposition, should be “mounted on poles in six macadamia orchards”
Line 36-38 - could be more concise, perhaps “…to the environmental variables: distance to water, altitude, height, and difference in mean temperature…”
Line 37-39 - “Bat house occupancy was significantly higher in the central bat house, in the set of three, and the black bat house, in the set of two, compared to all other bat houses” - is this what you mean? Or do you mean that compared only to their neighbouring houses these two houses had higher bat occupancy? It’s not currently clear.
Line 39-41 - “Mean temperatures differed between houses in the set of three; the mean temperature of the central bat house was 0.46°C (± ? SE) higher than the first bat house, and no different than the third bat house.” - is this what you mean? It’s not immediately clear the importance of this finding - is it that mean temperatures were higher/lower in the bat house more/less exposed to sun and thus the implications for others installing bat houses is???? Also, missing some measure of precision, such as standard error, around the mean.
Line 42 - missing species “most recorded species and …”
Line 44 - “appear to positively influence bat house occupancy” can reduce for conciseness
Line 50 - missing comma “species, especially”
Line 52 - why is future research needed on the potential importance of altitude and distance to water? It’s not clear from your abstract - perhaps it’s worth noting that the only environmental variable that influenced bat house occupancy in your study was mean temperature, but that this may be an artefact of little variation in the other variables, and thus future studies are needed to directly test these potentially important factors - or something similar.
Line 70-73 - poor sentence structure; perhaps “Proactive management of bat populations requires filling existing knowledge gaps on roost site preferences for African bat species….” Etc
Line 80 - “built”, also this sentence is unnecessarily long
Line 84 - grammatically incorrect - “seem to be important factors”
Line 97 - what is meant by “warm” - can the hypothesis be more specific?
Line 98 - consider changing “do particularly well” to something more explicit, such as “will have higher bat occupancy”, otherwise the hypothesis is unclear and subjective.
Line 119 - “respectively” is out of place, revise the sentence to concisely state what is meant.
Line 121 - “were also mounted”, “one colony bat house”
Line 131 - poorly constructed sentence - including a misplaced modifier, “unless weather prevented access” reads like the bees were unable to access the house in poor weather
Line 136/137 - perhaps “to minimise disturbance”
Line 143 - delete the extra space between “one  set”
Line 145 - do you mean that temperature was recorded hourly or every 1.5 hours?
Line 148 - cardinal direction facing what?
Line 149 - is the height on poles the same as the height above the ground? Perhaps revise to clearly convey the importance of the measurement, as I imagine it is the height above the ground that was the interesting factor, not necessarily how high the pole was (i.e., what if the pole was mounted on something - is it the height above ground of on the pole that matters?).
Line 153 - “bat house”
Line 154 - “led”; is the point that all covariates were tested for collinearity and correlated variables (including VIF) above a certain level were removed. If so, what was the correlation threshold?
Line 156 - “were facing”
Line 217-218 - mean monthly bat counts? It’s not clear what is meant by average number of bats, was it the mean number of bats recorded in each bat house? Considering the start of the sentence is the total number of bats observed I am thinking it should be the highest total number of bats was recorded in March and May, and the lowest recorded in August and November.
Line 315  - “advise” rather than advice
Experimental design: Line 160 - fit a single model to analyze the relationship for each type of bat house? I appreciate the revision to better explain the model but still don’t think it’s completely clear on if it’s one single model or multiple models for each type of bat house. (Also it’s “fit” not “fitted” the model). If one model, then line 166 should read “final model”.
Lines 182-186 - the statistical methods are not clear and thus the results are not clear. I had concerns with the methods in my last review and while the comments provided were helpful, there is still a lack of clarity in the manuscript text. I am assuming that with the dredge function all possible combinations models were run using different combinations of the covariates. These models were ranked by AICc and the model with the lowest AICc was chosen as the top model, or multiple models within 2 AICc were chosen as per Burnham and Anderson (2002). It would be helpful if a table (perhaps the one referred to on line 184) was provided listing the top 5-10 models, the null model and the full model, all with their AICc values to provide the reader of an understanding of what covariates were included in the top models and their relative ranking. It’s unclear if the authors arbitrarily chose the final model because “type of bat house” was in each of the 5 “top” models within 2 AICc of each other or because it was the model with the lowest AICc. Also, it reads as though the authors chose the covariate with a p-value < 0.05, rather than ranking by AICc (without looking at p-values) to determine the covariate(s) in the “top” model.
Line 184 - Supplementary Table 2 was not made available, did the authors mean to refer to Table 1? If so, all five models and their respective AICc values were not listed but rather one AICc was listed for all variables, which I take to assume one model. Unclear regardless.
Line 187-189 - Because the previous paragraph was not clearly written I am not sure about these results. If multiple bat houses were part of a categorical variable, then what type of bat house is the reference category? If univariate models were run, please state this explicitly.
Lines 196-205 - Please clarify how hourly (or 1.5 hour) temperature data is independent and not auto-correlated. I am concerned that the significant p-values are an artefact of the high number of (correlated) samples and may (or may not, difficult to know) be spurious. If averaging min/max/mean values over a 24 hour period or each night (when bats are roosting) the worry of independence may be lessened. In any case, some measure of precision should be reported alongside the means (standard error or standard deviation). Perhaps using a time-series type of model and/or GAM to analyse the temperature data?
Table 1 - what is the reference category for bat house? Also, the caption has “bathouses” rather than “bat houses”
Validity of the findings: Line 220-221 - I don’t disagree that bats may be more likely to prefer a warm bat house but I am still not convinced that this conclusion/assumption comes from the findings. Could the authors more clearly make the connection? Perhaps it would be clearer if the results were presented more clearly.
The above comment could be made multiple times over in the discussion. The study either does or does not confirm previous assumptions (line 235) - I suggest explicitly stating if the study findings are consistent with previous studies, and if not, explain why there might be a difference or if yes, discuss how this study corroborates findings from other studies.
Line 283-284 - was altitude being considered as a proxy for temperature? This suggestion does not seem substantiated by findings in this study as this link has not been mentioned before, i.e., when altitude was introduced as a potentially important factor influencing bat house occupancy.
Additional comments: The objective of the study was to determine artificial roost preferences for insectivorous bats in macadamia orchards in South Africa. This is an important study, especially considering the global decline in bat populations and the need to find proactive management solutions to maintain and conserve bat populations. This is my second review of this manuscript - the authors have thoughtfully responded to the comments provided by all reviewers. However, I feel that the manuscript could use further revision (better explanation of the analysis and general re-writing to concisely convey the salient points) to more clearly and explicitly present the findings and provide implications beyond bat preferences for artificial roosts in macadamia orchards in South Africa.


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: CROSSMODAL CONGRUENCY EFFECT SCORES DECREASE WITH REPEAT TEST EXPOSURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally solid, if selective review of huge literature.

Would do better to compare the decline in magnitude of this effect with what one sees in other paradigms
Experimental design: Solid, borrowing well-established paradigm
Validity of the findings: I would have liked to see a more granular analysis of decline of CCE across trials.......
Additional comments: Review.
I guess my major issue with this manuscript would that the results may not surprise anyone. Most behavioural effects decline with practice.
These results would be more interesting/striking in the context of other results where interference effects declined more or did not decline at all for contrast.
In this regard, I seem to remember a paper from c. 20 years ago on object-based inhibition of return, that some authors found and other authors couldn’t replicate. The key turned out to be that the object based effect was only there for one block. One side of debate was researchers at University of Bangor from memory. Maybe that would be helpful to cite.
Plus, I guess it is useful to identify the magnitude of reduction in CCE over trials/blocks, but one other simple means of eliminating its influence is perfectly randomizing condition order, no? Not always possible, and not always done, but feels like the majority of studies may have counterbalanced /randomized order in which diff trial types presented so possibly eliminating problem, at least in terms of interpreting results. Surprisingly, this isn’t mentioned as a solution in abstract and elsewhere
The authors might also want to discuss other factors that have been shown to influence magnitude of CCE effect too? And perhaps consider which component of behavioural effect they think is/are declining: see Shore, D. I., Barnes, M. E., & Spence, C. (2006). The temporal evolution of the crossmodal congruency effect. Neuroscience Letters, 392, 96-100.
Perhaps also Shore, D., & Simic, N. (2005). Integration of visual and tactile stimuli: Top-down influences require time. Experimental Brain Research, 166, 509-517.
Is this a learning effect, a practice effect? Is there a meaningful difference between these terms

“42 representation of space in humans (Spence, Nicholls, Gillespie & Driver 1998;” From memory Spence et al 1998 was an exogenous spatial cuing study, and CCT only properly introduced by Pavani et al. 2000), or 1998 Psychonomics Society presentation by C Spence.

76 “time-intensive experimental sessions….” Meaning what exactly?

“77 score. Also,” Try not to start sentence with also…

78 “knowledge, there exist no studies to date
79 that have investigated modulation of CCE score due to repeated task exposures. If a task learning
80 effect exists for the CCE, results from previous studies that ignored such an effect may be
81 misinterpreted. Participants tested with previous CCE experience would be expected”
I guess here you could mention repeated testing of patient JW by Spence et al. 2001a, 2001b, as an example where this issue might be problematic?
Spence, C., Shore, D. I., Gazzaniga, M. S., Soto-Faraco, S., & Kingstone, A. (2001). Failure to remap visuotactile space across the midline in the split-brain. Canadian Journal of Experimental Psychology, 55, 54-61.

One thing that might deserve more mention is that most CCT experiments actually have a number of practice trials/blocks before proper data collection starts. Hence this should be factored in to any concern about practice/learning effects swaying result, and this point may be relevant to discussion about why diff studies seem to report diff magnitude effects.

“All participants had normal or corrected to 122 normal vision, no disorder of touch, were able to use both foot pedals, and were naïve to the CCE
123 task.” In what sense naïve, they had never heard of task, they had never performed it, or both?

Often CCE shows up in error rates, and in the patient studies just mentioned inverse efficiency (combining RT and accuracy into a single performance measure) was used. How does learning/practice influence errors rates seems an important question that is worth discussing in perhaps more detail, and also considering in review of other studies.

Given that the CCT has been used in many more studies than those reported here, perhaps it is worth stressing a little more that you are just mentioning a selective review of studies that have used the task?

Note inconsistent article capitalization between articles:

“355 Sengül, A., van Elk, M., Rognini, G., Aspell, J.E., Bleuler, H., & Blanke, O. (2012). Extending 356 the body to virtual tools using a robotic surgical interface: evidence from the crossmodal 357 congruency task. PLoS ONE, 7(12), e49473. doi:10.1371/journal.pone.0049473
358 Spence, C. (2015). The Cognitive Neuroscience of Incorporation: Body Image Adjustment and 359 Neuroprosthetics.

Why is volume in brackets?
“380 Zopf, R., Savage, G., & Williams, M.A. (2013). The Crossmodal Congruency Task as a Means 381 to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm.382 Journal of Visualized Experiments, (77), e50530.

A further review of CCT and findings =
Spence, C., Pavani, F., Maravita, A., & Holmes, N. P. (2008). Multi-sensory interactions. In M. C. Lin & M. A. Otaduy (Eds.), Haptic rendering: Foundations, algorithms, and applications (pp. 21-52). Wellesley, MA: AK Peters.

I didn’t find the x axes of figures talking about blocks and experiments very helpful. Surely what reader wants to know is how many trials does it take eg to reach steady performance.

Shading between bars in Fig 5 could be made much more distinct.


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: CROSSMODAL CONGRUENCY EFFECT SCORES DECREASE WITH REPEAT TEST EXPOSURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Gill et al. reported across 3 experiments to demonstrate that crossmodal congruency effect is subject to modulation due to (over-)exposure/learning effects in a visual-tactile interference task. They then suggest that use of CCE scores/protocols would be better off by taking exposure time into consideration.
Experimental design: I found the logic for including Exp 3 to be unclear. My understanding is that CCE will decrease as the subjects are exposed to more sessions (more trials), as it shows from Exposure 1 to 5 (see main results in Figure 1). It came with no surprise that fewer practice in the 4-block condition will produce a smaller CCE learning effect since the participants in the 4-block condition have had fewer trials for them to reach their asymptote than those participants in the 8-block condition. Therefore, the only valid comparison is to have both groups of participants to receive same numbers of trials in both 4-block (double number of trials per block) and 8-block conditions. Otherwise this suggested mitigation procedure is pretty invalid. All it says with Experiment 3 is that we should give the participants as little practice/exposure as possible but then if it is the case then it goes against the main argument/proposal of the manuscript that we should use CCE data only after the subjects reach their CCE asymptote.
Validity of the findings: The second criticism is that this study covers only 1 aspect of CCE where in the crossmodal literature there are several other classes crossmodal correspondence (e.g., visual-audio among others). So I pose the question to the authors that if they can provide more evidence to demonstrate some kind of “generalization of this CCE to other crossmodal correspondence domains”? This generalization across domains is theoretically very important to instantiate their arguments. Only if the authors can demonstrate (or refute) such domain-generality in CCE I would deem this paper to be worthy to be published in PeerJ. I suggest the authors pick a dataset from a domain (I suggest visual-audio correspondence) and look into whether the CCE will stabilize across exposure sessions to test for any analogous learning effect.
Additional comments: Overall the analyses are fairly elementary that only mean session-specific RTs are compared. Note that there has been a surge of interest in the research of trial-history in relation to current behaviors (e.g., Carlos Brody's recent Nature paper (https://www.nature.com/articles/nature25510; and also Merav Ahissar’s 2012 and 2014 papers in PLoS computational biology). Specifically, whether a preceding congruent trial will pose a differential effect on current congruent trial (also for an incongruent trial on a current incongruent trial, likewise for the other two possible combinations). It would be theoretical interesting to examine the trial-by-trial influence in each of the sessions using GLM analysis incorporating various (sets of) regressors.


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: CROSSMODAL CONGRUENCY EFFECT SCORES DECREASE WITH REPEAT TEST EXPOSURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: See comments
Experimental design: See comments
Validity of the findings: See comments
Additional comments: The authors investigate the effect of repeated exposure on CCE by presenting the task for five consecutive days. This could induce some kind of learning that decreases the CCE scores and that persists over six months. Moreover, the study shows the authors' effort to find a shortened length procedure of the task to mitigate possible participants’ fatigues and learning effects. Therefore, the overall aim of this study seems worth asking. However, my major concerns are about their conclusions and interpretations. Specifically, in my opinion the authors should be more careful and they cannot state that “…results from previous studies that ignored such an effect may be misinterpreted” and “A learning effect that has been ignored by the field may render inaccurate our current understanding of multisensory space representation in humans”. Indeed, to the best of my knowledge and based on the literature that they presented on the introduction, they have been the first that have presented the task for five consecutive days. The learning effect showed by their participants, after five exposures, could be not present in participants that have been exposed to a single session. So, the decrease in the CCE scores that they found, could be not relevant, or even not present, in the previous studies. Broadly speaking, I think that authors should enrich their introduction and should improve their experimental design (i.e. by using a more precise measure for the overall reaction time; or by measuring the overall reaction time before and after the sessions; or by measuring a possible learning/fatigue effect during a single session of 8 blocks; or by measuring if a learning effect persists after six months in participants tested with the short protocol).
Now, I will list several issues in more detail:
Abstract
Line 14: It seems that there is a confusion between the “effect” and the “task” labels. In my opinion, Crossmodal congruency task is the procedure and the implementation of the design (well described in the introduction, line 56-61) that has been used to quantify the Crossmodal congruency effect (CCE) score, namely, the difference between incongruent and congruent trials.
Introduction
In my opinion, authors should enrich the introduction by adding more studies that have investigated the Crossmodal congruency effect, especially studies that have used a repeated exposure procedure.
Line 39: The authors should introduce and explain the topic of “multisensory representation of human space” and incorporation of “information into body schema”.
Line 54: the first time that authors introduce the CCE they should explain what it is.
Line 56: the acronym CCE stands for Crossmodal congruency Effect, not for the task. To avoid ambiguity authors should consider to refer to the effect like “CCE” and to the task like “CCT”. It could be less confounding for the reader.
Line 65: CCE score is calculated by the difference between incongruent and congruent trials, not the reverse.
Line 70: CCE is the effect, not the task.
Line 78 - 85: The authors investigate the effect of repeated exposure by presenting the task for five consecutive days (line 78-79). This could induce some kind of learning that decreases the CCE scores and that persists over six months. To the best of my knowledge and on the basis of literature presented in the introduction, no one has exposed participants to a consecutive days procedure. Authors should explain better this point and should add some references about studies that have used a repeated exposure protocol, if they exist. Otherwise, they cannot state a possible misinterpretation of the CCE from the previous studies about the multisensory space representational (line 81-83). My point is that eight blocks or 60 minutes of a single session, might not imply a learning effect at all. Authors, to the best of my understanding, did not explore a possible learning effect within the 8 blocks.
Line 119: Authors should present the three experiments separately. That is, Experiment 1: participants (number, mean age and SD, left/right hand dominance), materials, experimental design/procedure, analysis and results. The same structure for experiments 2 and 3. CCE test implementation (line 90) and materials and apparatus (line 131), that are in common between the three experiments can be placed together in the description of experiment 1.
Line 150: “…CCE task..” I suggest to use a specific acronym for the task.
Line 155: Authors state that they have used a “rule drop task” to measure generalized reaction time. Authors should indicate a reference, and explain better why the need to measure the generalized reaction time.
Moreover, they should explain why they use the ruler drop task, usually used with children or adults with clinical disability, rather than another more common and, probably, more precise task, like a “simple rt task” (see, Elliot R., 1968 Simple visual and simple auditory reaction time: a comparison).

Line 160 and 165: design of exp 2 and exp 3 should be described after experiment 1 in their specific section.
Line 166: I wonder if 18 participants are sufficient to explore the exposure and learning effect. In the power analysis reported for experiment 1 is specified that with 2 sessions authors should need 45 participants. So, why they tested only 18 participants, furthermore divided into 2 groups? If the power analysis conducted for experiment 1 was not appropriate for experiment 3, why authors did not conduct a power analysis specifically for exp 3? Authors should disentangle this point.
Line 181-185: Analysis of exp2 and exp3 should be placed on their specific area.
Line 192: which specific exposure sessions differ from each other? Authors should run a post-hoc analysis to determine it.
Results
Exp 1
Line 198-199: what kind of “interacting factors” could intervene? Please provide examples.
Line 200: in figure 3 it seems that authors represent proportion correct instead of error rate. Please check the data. Moreover: why do authors do not represent the proportion correct divided for congruent and incongruent stimuli, like they do with the RT? I wonder if a learning effect should not emerge also with the participants’ hit responses, inducing participant to have pretty good performance also with incongruent stimuli and weakening the CCE scores. If a learning effect exists, authors should find some effect also in the hit responses, otherwise one could speculate that participants simply became faster. Authors should introduce these data and eventually explain the absence/presence of modifications in the hit responses.
Line 202 to 204: authors should present the post-hoc analysis to highlight which data point is different from the others. Once again, in figure 3b it seems that the participants are faster but the difference from incongruent and congruent seems to remain significant. So the CCE seems to persist over the learning effect.
Discussion
Line 250: Authors should explain better how they can exclude the “Fatigue effect”. Indeed, overall reaction times were always measured before the task. I think that, to evaluate the fatigue effect, authors should have measured the overall reaction times before and after the task, in each session.
Line 251: If authors had wanted to match the overall reaction times with the decrease in the CCE scores, probably they should have used a similar kind of response, i.e. authors could have used a simple reaction time task with a foot responses to measure overall reaction times task. Indeed, it could be that in five consecutive exposure the participants could have become faster with foot responses but not with their hand responses.
Line 251: in line 155 authors refer to “ruler drop task” here, in line 251, they refer to “ruler grasp task”. Please check for consistency.
Line 257: “the persistence of the learning effect… has stabilized”. My doubt persists: are there some studies that have used a consecutive five days procedure? Otherwise, authors should have had evaluated a possible learning effect after the eight blocks of the single procedure. Please, add some studies in the introduction which use a consecutive days procedure and, in the discussion, compare them with your results.
Line 260 - 261: It would be interesting to evaluate if the persistence over six months of this “learning effect” would manifest itself even after the short procedure.
Line 291 -304: “Additionally, different experimental setups could account for differences in the CCE scores… feedback incorporation”. If CCE scores are so sensitive to experimental setups, feedback modalities, etc., why authors decide to use these different experimental setups compared to other studies?
Line 299: please double check there is a typo in the reference.
Line 311: “… to determine if results reported elsewhere…” please add some references, results reported from which studies?
Figure 1:  If, on one hand, I appreciate the effort to recreate the design of the overall system setup, on the other hand, I think that a real picture of the device on the hand could be more explicative to understand the implementation. So, I suggest adding a picture to enrich the understanding of the procedure.
Figures 2, 3 and 4
In my opinion, labels in the x axes should be “1st session”, “2nd session”, etc… and not “exp.”since “exp.” might be misinterpreted as “experiment” and not for “exposure”.
Figure 2 and 3: in the caption authors should state that results come from experiment 1.
Figure 3a: please check the data, it seems that authors report proportion correct instead of error rate.
Figure 4: in the caption authors should state that they are CCE scores from experiment 2.
Figure 5: in the caption authors should state that results come experiment 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: PURIFICATION OF PLANT-DERIVED ANTI-VIRUS MAB THROUGH OPTIMIZED PH CONDITIONS FOR COUPLING BETWEEN PROTEIN A AND EPOXY-ACTIVATED BEADS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: 1. Optimize the time and temperature for the proteinA couple resin, Why choosing 18 h at 37°C ?
2. The antibody should be quantify by ELISA, not measure total protein. Because you need to compare using the same amount of the full assembly antibody
Validity of the findings: 1.The result showed clear conclusion that using pH8.5 to conjugate the proteinA to the bead will give good result in antibody purification. However, the authors should repeat the experiment to be able to do the statistical analysis. This will improve the conclusion. The significant finding of this study is to confirm that which condition is the best among all condition. Therefore, the statistical analysis is needed.
2.Provide the cost of protein A, to claim that the described resin will be cheaper than the commercial protein A resin.
3. In line 262, The virus-neutralizing efficacies of four mAbPSO57 purified from different resins (GE, pH 8.5, pH 9.5, pH 10.5) are similar and  approximately three times higher than that of anti-rabies human immunoglobulin.

What is the source of the anti-rabies human immunoglobulin using in this study. How to make sure that the amount of the antibody is equal to the antibody purified from different pH resin? To conclude that the antibody purified from different pH resin is higher than the anti-rabies human immunoglobulin, the authors have to use the same amount of the antibody.
Additional comments: 1. In line 250  the protein A with the different pH coupling conditions might hold both IgG Fc and IgG F(ab′)2,  compared to the GE protein A which holds only Fc …What is the evidence for this claim?
2. In line 250, It is speculated that  the protein A with the different pH coupling conditions might hold both IgG Fc and IgG F(ab′)2, compared to the GE protein A which holds only Fc. Why proteinA with different pH can hold Fab? Any evidence?
3. In line 257, The fusion of the ER retrieval modif, KDEL, to protein allow for their efficient retention in the ER. This is not related with the investigation at all. Because the authors used only one construct that contain KDEL.


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: PURIFICATION OF PLANT-DERIVED ANTI-VIRUS MAB THROUGH OPTIMIZED PH CONDITIONS FOR COUPLING BETWEEN PROTEIN A AND EPOXY-ACTIVATED BEADS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Article is well written, and is a stand-alone study on the relevance of pH on the coupling of Protein A to agarose resin for the purpose of chromatographic separation of mAbs. There are some small errors, but overall the article is written well.
Experimental design: The research question is defined, however, the pH of the running buffer was not well indicated. It appears as though the running buffer for chromatography was the same pH as that of the coupling buffer. The method indicates that the precipitated pellet was reconstituted into “an extraction buffer” (lines 137 and 138). Is this the same buffer at pH 7.5? If the pHs were different, I don’t see how the authors can draw any of their conclusions from the work. If they were all at the same pH, then that would be slightly different. Each run seems to have been performed multiple times, though the statistics on the in vitro assay are unclear.
Validity of the findings: - The impact of the research appears to be a better way to couple Protein A to agarose resin. However, there is no assessment of the coupling efficiency that I can see, meaning that the conclusion of better placement of the Protein A ligands may be called into question.
- The article states that the better pH for coupling is at 8.5, but ignores the fact that the 10.5 resin had a higher RFFIT value. Meaning, that though efficiency of total protein may be better for the 8.5 resin, the 10.5 resin appears to have a higher activity.
- There was no densitometry done on the gels to indicate a purity level. All calculations were done using absorbance, which is a measure of total protein (and DNA in many cases) only. There appears to be no purity advantage to the resin made at pH 8.5
- The in vitro assay performed does not indicate whether the fractions are pooled or not. If they are, then this would be a clear case to prefer the 10.5 pH resin.

Data is robust, conclusions (excepting the above) are sound.
Additional comments: The article is a well-written account of the effects of varying pH levels on the recovery of the mAb expressed in arabidopsis plants. There may need to be more characterization of the eluates before some of the conclusions can be drawn. I currently see no advantage to the pH 8.5 resin vs the 10.5. I would also appreciate a comparison of ligand density between the test resins and the positive control to determine if that might be the reason for increased efficiency.


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: PURIFICATION OF PLANT-DERIVED ANTI-VIRUS MAB THROUGH OPTIMIZED PH CONDITIONS FOR COUPLING BETWEEN PROTEIN A AND EPOXY-ACTIVATED BEADS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The quality of written English is good.

The article is appropriately structured and the figures are clearly reproduced. Raw data is provided.

There are a large number of references, perhaps too many. Some references are misformatted in the bibliography (e.g. Sharma and Sathishkumar – no journal, various journal names incorrectly capitalised, Hermanson GT 2013 – incomplete reference, no publisher)

Other minor issues:
L118 … based 5% skim milk. What does ‘based’ mean here?
L120 Supplier and product number of 2ndary antibody not stated
L150, 209, Figure 5 Nanodrop is a trademark of Thermo Scientific and should not be used to describe other spectrophotometers
L160 ADMEM supplier?
L183 ‘Repeating’ should read repeated, or sentence rephrased.
L244 ‘Epoxy-activated agarose coupled with protein A resin under pH conditions greater than 11 was not recommended for purification of monoclonal antibodies.’ Please provide reference.
Experimental design: This is a straightforward method development study comparing the ability of affinity matrices prepared under different conditions to bind mAb produced in transgenic Arabidopsis extracts. Samples are appropriate to allow comparisons between the yield and quality of antibodies eluted from different resins. The RFFIT test is an appropriate measure of functionality given the antibody’s specificity.
Validity of the findings: There are some weaknesses in the results presented for the antibody assays:

In Figure 4, it is not stated if these gels were run under reducing conditions (as they appear to be). Given the authors speculation that different protein A binding chemistries may predispose the resin to purifying F(ab’)2 fragments (L250) and also to better assess the purity intact mAb, non-reducing gel images should also be provided.

In Figure 5, error bars should be shown to illustrate the precision of the measurements. Is there a significant difference between yields under 10.5R, 9.5R and 8.5R conditions? Some calculations are made in the supplemental spreadsheet but only 2 measurements are provided instead of triplicates as stated in the Figure legend?

In Figure 6 again there are no error bars. Can the author say if there is any real difference between these samples on the RFFIT test? An appropriate statistical test should be performed.

L241 I feel the Groher & Suess 2016 reference is overinterpreted, the authors of that study simply say that binding via hydroxyl groups requires a highly basic pH which ‘not all ligands can withstand’ and do not mention protein A.

L256 -L261 the authors discuss the influence of including signal peptides and KDEL motifs on the antibody sequence but no connection is made to the results presented here, or even to protein A binding. This section should be removed.
Additional comments: Although manufacturers often provide instructions for performing immobilisations similar to that performed here to generate protein A affinity matrices, there is little practical information in the scientific literature to support these general protocols.

I believe this MS will be of practical use to a number of laboratories aiming to produce these columns as a tool for their research, and is suitable for publication if weaknesses in the assay data can be addressed. Addressing these concerns is important, as the data as currently presented does not fully support the conclusion that pH 8.5 is optimal.

Although the authors place importance on the production platform for the mAb (Arabidopis plants), I suspect these results will be applicable to other production methodologies. Perhaps the authors would like to speculate on this point?


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: PURIFICATION OF PLANT-DERIVED ANTI-VIRUS MAB THROUGH OPTIMIZED PH CONDITIONS FOR COUPLING BETWEEN PROTEIN A AND EPOXY-ACTIVATED BEADS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is clear with sufficient context provided.
Experimental design: The revised manuscript has good explanation on the experimental design.
Validity of the findings: The study showed the relevance of pH on the coupling of Protein A to agarose resin for the purpose of chromatographic separation of mAbs.
Additional comments: I think the revised manuscript is good to publish.


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: PURIFICATION OF PLANT-DERIVED ANTI-VIRUS MAB THROUGH OPTIMIZED PH CONDITIONS FOR COUPLING BETWEEN PROTEIN A AND EPOXY-ACTIVATED BEADS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Improvements have been made to raw data and the references as requested in revision. Other aspects to an acceptable standard, as before.

The minor missing details, etc, in my comments have been corrected.
Experimental design: Appropriate as before
Validity of the findings: Key improvements and clarifications have been made to Figures 4 and 5 and the discussion. These have largely addressed my concerns in my first review.

However, it struck me in a response the authors made to reviewer 2, that the recombinant protein A used in these experiments had in fact been engineered to contain a cysteine residue at the C terminus:

'...Therefore, we authors has optimized the cysteine residue at the C terminal of protein A to give a constant directionality. (Original protein A has no cysteine residue).'

This was not at all obvious from the original MS and clearly has implications for groups who may wish to produce equally effective columns with unmodified protein A. Please clarify this point in the MS and specify the modification(s) made.
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: DIETARY SUPPLEMENTATION OF BLACK SOLDIER FLY (HERMETICA ILLUCENS) MEAL MODULATES GUT MICROBIOTA, INNATE IMMUNE RESPONSE AND HEALTH STATUS OF MARRON (CHERAX CAINII, AUSTIN 2002) FED POULTRY-BY-PRODUCT AND FISHMEAL BASED DIETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The MS conforms to the standard of scientific reporting with clear language, structure, figures and tables, fully supporting the hypothesis made with relevant results and discussion.
Experimental design: The present experiment tries to generate information on marrons which are data deficient commercial species. At the same time the present paper explore the potential of alternate animal protein sources to replace fish meal in fish feed.
Experimental design has been well planned and carried out with ethical standard. The methods have been described clearly for to replicate. However another feed (treatment) comprising FM+PBM+BSF could have made interesting observations. Authors may also cite reasons why BSF has not been included beyond 12% in the diet.
Validity of the findings: The data presented are robust and statistically sound to represent the results.However few more observations are made here.

Rimoldi et al 2018 extracted DNA from faecal matter of the sampled fish, to study the microbial communities. In this experiment DNA was extracted from hindgut of the shellfish marrons. So how does it compare or how much of variation you may speculate in the gut microbial diversity.

It is desirable to discuss with more references related to shellfish like comparing gut microbiota of shellfish like L vannamei in which lot of work have been done.

The paper should shed some more light on nutritive values of BSF to correlate with the results.

Line 342 may be corrected by giving proper references or (53,54)  may be removed
Additional comments: It is a very well designed and elaborate work addressing many issues pertaining to sustainable aquaculture. Further studies may explore the large scale production potential of alternate protein sources to make commercially viable option to replace fishmeal in aquafeeds.


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: DIETARY SUPPLEMENTATION OF BLACK SOLDIER FLY (HERMETICA ILLUCENS) MEAL MODULATES GUT MICROBIOTA, INNATE IMMUNE RESPONSE AND HEALTH STATUS OF MARRON (CHERAX CAINII, AUSTIN 2002) FED POULTRY-BY-PRODUCT AND FISHMEAL BASED DIETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In their paper entitled Dietary supplementation of alternative protein sources modulate health status, gut microbiota and innate immune response of marron (Cherax cainii, Austin 2002), Foysal et al. tried to evaluate the effects of insect meal as a supplementary diet to fishmeal and poultry meal on the bacterial communtities of marron freshwater crayfish. Apart from bacterial communities diversity studied with illumine, growth indices were calculated as well as other factors such as lysozyme activity and total haemocyte counts, and the presence and abundance of cytocine genes.
Overall the study is interesting and well described. The English language used is clear although some mistakes are observed (e.g. genus, plural genera).
Introduction is well structured with sufficient references and raw data are supplied.
Experimental design: The research question is well defined and the methods are explicitly explained.
However the design lacks some information and can be misleading. For example nowhere is written how many samples (guts) were used for each treatment and method. I suppose that you used 16 samples for each treatment but as I also implied from your figures you used only three samples/ treatment for the 16S analysis (Figure 1). Am I wrong?
Again this is nowhere mentioned in the text and should be added somewhere.
Validity of the findings: Results are well explained and supported by statistical analysis, however in some cases they are hard to understand because of the bad description of samples (How many for what method?). I would suggest the authors to add one more table or provide more information in the manuscript.
The discussion for Bacterial taxonomy is very interesting, but I would suggest you add some more citations/information for the very interesting group of Ca. Bacilloplasma (Tennericutes) that is solely detected in animals gut systems. There are several citations (Wang et al. 2004,Kostanjsek et al. 2007, Meziti et al. 2010) for closely related species that show the importance of this group in gut systems and try to connect it with feeding.
Additional comments: As you will see I recommend the article for major revision since I believe that the addition of a better explanation for samples handling would make your article easier to read.


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: DIETARY SUPPLEMENTATION OF BLACK SOLDIER FLY (HERMETICA ILLUCENS) MEAL MODULATES GUT MICROBIOTA, INNATE IMMUNE RESPONSE AND HEALTH STATUS OF MARRON (CHERAX CAINII, AUSTIN 2002) FED POULTRY-BY-PRODUCT AND FISHMEAL BASED DIETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Foysal et al. replied to my major comments regarding experimental design. Also relevant references were added accordingly.
Experimental design: The authors made their methods more clear with the new additions.
Validity of the findings: Data is sufficient now with the changes authors did.
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: GENETIC VARIABILITY AND STRUCTURE OF THE OLIVE FIELD MOUSE: A SIGMODONTINE RODENT IN A BIODIVERSITY HOTSPOT OF SOUTHERN CHILE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a generally well-written piece. I am frustrated, after editing the entire ms. for English fluency, that my Abrobat failed and no text corrections could be saved. It needs minor attention throughout

Literature was generally present although two references to my own work were missed and would improve the authors inferences: one regarding the current taxonomy and distribution of Abrothrix olivacea (the authors cite Osgood 1943 and Mann F 1978, which both consider xanthorhinus a distinct species and brachiotis as the southernmost subspecies; in reality, hybridization across the brachiotis-canescens boundary is important and may account for the discovery of geographic structure). The current taxonomy of this SPECIES (i.e., Abrothrix olivacea, not A. o. brachiotis, which is a subspecies!) is given here--Patterson, B. D., P. Teta, and M F Smith. 2015  Genus Abrothrix Waterhouse, 1837. In Mammals of South America, Vol. 2: Rodents. J.L. Patton, U.F.J. Pardiñas, and G. D'Elía, eds. Pp. 109-126. Chicago: University of Chicago Press.

A critical part of the analysis was left unsaid: how is Abrothrix olicavea brachiotis distinguished? Species have reproductive and historic limits, but subspecies by definition intergrade. What is the effect of intergradation, or hybridization, at its boundaries with other Abrothrix olivacea subspecies affecting the patterns reported in this paper? This is not discussed anywhere in the paper.

Another major problem with the article as written is that Abrothrix olivacea would be among THE WORST species to study if anthropogenic deforestation was really a primary target of the article. Its presence in a host of habitats (they later cite Murua and Gonzalez 1982 but see also Patterson et al. 1990  Quantitative habitat associations of small mammals along an elevational transect in temperate rainforests of Chile. Journal of Mammalogy 71(4):620-633.) and throughout the offshore archipelagos (Guaitecas and Chonos) is testament to its superb colonizing ability and habitat tolerance. Only Oligoryzomys longicaudatus is more ubiquitous!! Why not just focus on understanding the population structure of this forest-associated species, without all the conservation hoopla of hotspots and deforestation, which is distracting and potentially even misleading?!
Experimental design: Appears strong. One could wish for longer sequences of a gene-encoding locus, but HV1 appears suitable for the present analysis.
Validity of the findings: Generally appear valid, but the authors need to reconcile their results, especially with respect to geographic subdivisions of the range, with the potential for hybridization with adjacent but unsampled subspecies of A. olivacea (see Fig 4 and the differentiation of the Aysen populations where A. o. brachiotis and A. o. canescens come together). The Geneland analyses apparently build topography into their predictions, but the extrapolations of distributions into Argentinian territory to the east of the Andes looks suspect

line 53: "Abrothrix olivacea brachiotis, one of the most common species of small mammals"--it is a subspecies, not a species
line 78: "second largest area of temperate forests in the world"--temperate rainforest (the taiga of Canada and Russia is far larger)
line 85: "overgrazing" -- isn't a far greater threat to native forest species clear-cutting for pasture?
line 106: citations: Both these authors recognized Abrothrix xanthorhinus in the south, and so their treatments of A. olivacea do not extend S of 45 degrees.  Better to cite here
Patterson, B. D., P. Teta, and M F Smith. 2015  Genus Abrothrix Waterhouse, 1837. In Mammals of South America, Vol. 2: Rodents. J.L. Patton, U.F.J. Pardiñas, and G. D'Elía, eds. Pp. 109-126. Chicago: University of Chicago Press.
which give range extensions and modern taxonomy
line 116: specify locus used? This statement of variability means different things for HV1 and Rag1?
line 119: Himes et al 2008.  see also
Monotypic status of the South American relictual marsupial Dromiciops gliroides (Microbiotheria) Elkin Y Suárez-Villota; Camila A Quercia; José J Nuñez; Milton H Gallardo; Christopher M Himes; G J Kenagy. Journal of Mammalogy, Volume 99, Issue 4, 13 August 2018, Pages 803–812, https://doi.org/10.1093/jmammal/gyy073
line 121: Although there has not been genetic confirmation of no differentiation, there is also the distribution of Loxodontomys micropus fumipes, known from Chiloé and Santa Cruz Patagonia
line 124: another Chiloé endemic is the nutria: Myocastor coypus melanops,
https://en.wikipedia.org/wiki/Coypu
line 140: The most critical question here is "how were individuals of "A. olivacea brachiotis" distinguished form the other 6 subspecies of A. olivacea recognized by Mann, Osgood, etc?" In other words, how do we know that the patterns of genetic variation you are describing do not result from hybridization with other adjacent subspecies, which have different habitat distributions and different population histories?
Line 215: choose-->chose
Line 311: isn't the geographic replacement of Abrothrix manni (northern Chiloé) and Abrothrix sanborni (southern Chiloé) worth mention here, to corroborate the compound history of the island?!
lines 324-326: the authors make this look like a conclusion from their analysis, whereas an even-handed examination of its wide distribution and generalized habitat in the Introduction would have made it expected
line 341: Anthropic effects=anthropogenic effects
line 347: role of hybridization?
line 357: A. sanborni relevant here
line 368: sea level lowering
line 380: the negligible effect of ...
line 383: this is where Patterson, Bruce D., Peter L. Meserve, and Brian K. Lang.
 1990  Quantitative habitat associations of small mammals along an elevational transect in temperate rainforests of Chile. Journal of Mammalogy 71(4):620-633. could be cited
line 394: heterozygosity
Additional comments: This is a useful analysis that should be published. There is no need to argue beyond the analysis (e.g., conservation importance of its results). I would instead argue that A. o. brachiotis is co-distributed with a majority of species inhabiting the south temperate rain forests and that factors affecting its population structure may be general and applicable to many of them.


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

Input:
Title: GENETIC VARIABILITY AND STRUCTURE OF THE OLIVE FIELD MOUSE: A SIGMODONTINE RODENT IN A BIODIVERSITY HOTSPOT OF SOUTHERN CHILE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some sentences need to revise to improve the flow. I have made some suggestions in general comments.
Experimental design: The authors of this work investigate through mtDNA sequences and 12 microsatellite loci the genetic diversity and structure across 11 sampling sites of a sigmodontine rodent in the temperate forest in Chile. The assumption is that historical and anthropogenic factors have shaped the genetics of this species, which is found in continental and island locations. The authors state that historical factors rather than ecological have shaped the genetic patterns observed in this study. The study is within an interesting and important biodiversity hotspot in South America, as such it contributes with valuable genetic information for an area where more studies are needed. However, I found the introduction and the abstract (last sentence) misleading. The introduction mentions important likely effects of habitat fragmentation on genetic diversity and structure due to human activities in this region, however, there is no explicit test to address the effects of landscape change such as habitat fragmentation. Also, there are assumptions on geographic barriers (island vs mainland; the Cordillera de Piuchén) and geographic isolation on genetic patterns, but no explicit test for those were carried, such as an IBD or Barrier test. Thus, the abstract and introduction should be restructured to make clear the objectives that were explicitly tested.
Second, methods also need restructuring, some test were not mentioned in methods, but mentioned in results, or information is missing such as details on sampling, laboratory procedures and data analyses.
Validity of the findings: The discussion also needs to be changed as the authors conclude on factors that were not tested, such as anthropogenic effects, or geographical isolation (this last one, which can be easily addressed with IBD test). The conclusion thus lack data support based on the analyses carried.
Additional comments: Ln48-51: This sentence is long and not much clear, divide the ideas.

Ln52; loci to evaluate the genetic variability across several populations of the sigmodontine rodent

Ln53: Abrothrix olivacea brachiotis, a common small mammal species of these disrupted forests

Ln54-56: Here again separate ideas in two shorter sentences.

Ln58: latter island? I don’t understand this.

Introduction

Ln63: the word disruption can mean disturbance, interruption, modification, etc. After reading the first paragraph I think the authors ideas are more specific to habitat fragmentation, which affects connectivity. So, I suggest to better change the term to habitat fragmentation since this is widely used in the scientific literature

Ln64: of the genetic diversity. I would say genetic diversity of populations rather than a species
Ln65: Since the sentence refers to loss of genetic diversity due to habitat fragmentation, I suggest to better include reference that directly assessed these effects such as meta-analyses carried in animals and plant taxa (see Aguilar et al. 2008 Mol Ecol Dec;17(24):5177-88)

Ln65: Loss of genetic variation

Ln69: I would say better environmental change

Ln88: So, readers know Chiloe is an island: the north population of the Chiloe island is located.

Ln93: Among these, the Pleistocene glacial cycles affected an important area of the landscape in the southern portion of South America

Ln96: higher than? Otherwise just use high

Ln104: important species

Ln107: remove former

Ln116: Be more specific of the type of DNA marker used (mtDNA sequences)

Ln125-127: Why is this interesting?

Ln127: however, microsatellite data showed genetic differentiation between the Continent and the Chiloe island, which implies that the Chacao channel is a recent barrier to gene flow (about 8,000 years BP) (Napolitano et al. 2014).

Ln130-136: Be more specific about the aims of the manuscript. State in which way this work is different from that of Rodríguez-Serrano et al. 2006 not only in the type of molecular markers used but, on the question, asked (e.g. contemporary patterns of gene flow). Also, a large portion of the introduction states the effects of habitat fragmentation due to human activities (including the abstract), but the Chacao channel as a barrier to gene flow (when the comparison is made with the Leopardus guigna study) is not a human-made factor, but a geographic factor. From the information presented I can see this is a fragmented landscape, both natural and human-made, but the methods do not explicitly test any anthropogenic landscape effect on genetic diversity or structure, such as habitat fragmentation, agriculture, or urbanization. The introduction should be restructured to make clear this is ms is not about investigating anthropogenic effects on genetic structure.

Methods

Sampling. Explain better the distribution of the subspecies and if the sampling covers mostly its distribution or if gaps exist and where. Why for instance, are some sampling sites very nearby (Senda Darwin, Valdivia, Panguipulli), and others separated by more than 50km (Alto Rio, Mininco)? I understand the limitations of an extensive sampling, but information on this should be provided so the reader has an idea of the scope of the results.

Year of sampling collection?
Why if 187 samples were collected, 92 individuals were DNA sequenced? And how many samples for microsatellites were processed?

For DNA sequences and microsatellites how many samples were repeated to calculate sequencing and genotyping error rate, in particular for microsatellites?
Since the 12 micros are new, it is important and valuable to other readers to show the table not within appendix but within the main text with information such as bp size, motif, and genetic diversity parameters.

Does the alleles for the micros were observed through fragment analyses (platform)?
Which software was used for microsatellite genotyping?

The data analyses would be clearer if they are split by molecular marker, there are some methods specifically for each marker type and some for both.

Ln193: which parameters were used to calculate Tajima´s D. If a population is in equilibrium which value is expected? If positive?

Ln199: usually the standard is at least 10 runs per K

Ln201-202: This is a popular method, so this info can be omitted (these models assume..)

Ln204: How the output (barplots) were observed and created?

Ln209: genetic populations

Ln219: Again, four replicate runs may be few. And how the output maps were created?

Ln225: This paragraph more than statistical analyses is on genetic diversity parameters and it goes first than any other method.

For the microsatellites, since they are new, other important test should be conducted such as Linkage disequilibrium. Are some loci that show LD more frequently? If so, then some loci should be discarded if STRUCTURE analyses are done. Otherwise, if LD and HWE are present, other multivariate methods such as PCA or DAPC can be performed which are free of LD and HWE assumptions.

Also, is there evidence of null alleles, allele drop out or other genotyping bias such as stutter peaks? if so corrections should be done for genetic diversity and differentiation parameters.

Ln231-232: How pairwise FSTs were calculated (software)? And only for microsatellites? Both pairwise FSTs for mtDNA and micros across the 12 localities can be estimated and compared.

Since there are some regions that show genetic structure with microsatellites and that later these are discussed to be related to some geographical features, it can be performed other test such as AMOVA or SAMOVA, to test how much genetic variation can be explained by the different geographic regions. Other test that specifically test for barriers are BARRIER, which uses a Monmonier algorithm to spatially detect barriers across a geographic area. This can be a test to provide support to the assertions on the discussion, which were not directly tested.

 On the other hand, is there evidence of isolation by geographic distance?

Results.

Would be more informative to show genetic diversity parameters for both markers within a table per sampling locality.

Ln240: that populations show neutrality? But the Tajima D was calculated for the total of populations and not for each population or K genetic cluster, which may have a different history?

Ln246: Is not very clear the description of this result. Usually will start stating which is the central haplotype(s) that has the highest frequency and from which regions are. When it is stated a haplotype has low mutational steps, respect to which another haplotype? Referring to low or high mutational steps is always in a pairwise comparison, otherwise is not clear what this means.

Ln259-265: Although the most probable number of K is 5 according to the Evanno method, from the barplot it can be seen is 3 or 4, what about barplot k=4? There is high admixture across locations 1 to 6.

Ln278-295: move this to the beginning of the results. Also, all methods reported in 290-295 were not mentioned in methods. Every method should be stated in methods first. These results are the first one to be reported in the section.

Table 1 and 2: Why not to calculate pairwise FST and genetic diversity across the 12 localities. The FST values obtained would support either the results from structure or not.

Fig. 1 The blue shade can be confusing at first glance as it gives the impression that the blue is actual land. I think the best is to send it back to the map or with transparency so the lines with the limits of the continent and the archipelago looks more marked. However, I don´t have much clear how this LGM representation is specifically used in the ms to make a point to discuss.

Also in the larger map of whole Chile, spot the distribution of the temperate forest (Maule and Aysen) so the reader can see this data on the map.

Maybe fig 1 and 2 can be combined, the dots if sampling localities with the same color of the haplotype network to better have a visualization of the results of fig. 2.

Fig 3. Which are the probability of membership of these 3 clusters? If not provided is not possible to judge if they are likely true clusters

Discussion

Ln316-326: there is no real evidence on this, as it was not explicitly tested: I don’t agree it is safe to state there is no effect of habitat loss as either the sampling nor the analyses performed allow to conclude this.

Ln327-336: I don’t agree there is five genetic clusters, that’s what one analyses showed, but the membership probability states other thing. These ideas are redundant with results, I think this paragraph explain the result better that the one presented in results.

Ln335-339; But what about the Tajima D result? It does not indicate population expansion.

Ln340-345: There is no real evidence for this statement. Besides, there is no data collected on the size of populations, neither on the migration routes. Contrary, I think there is evidence of gene flow as it is seen on Structure and GENELAND from locations 1 to 7.

Ln349-360. But also, which is the explanation locations Panguipulli and Cucao are highly genetically related to be in one cluster (there is no explanation to this)? And that instead there is two clear genetic clusters within the island?

Ln363-365: But not isolation by distance was tested, and it is a very easy test to do.

Ln367-368: I don’t understand how this cluster sustain the hypothesis the channel would be a barrier, if these localities are within one single cluster? Is not the opposite, not a barrier?

Ln380-382. Same to previous comment, no factual evidence.

Ln399: But no IBD test performed.


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: GENETIC VARIABILITY AND STRUCTURE OF THE OLIVE FIELD MOUSE: A SIGMODONTINE RODENT IN A BIODIVERSITY HOTSPOT OF SOUTHERN CHILE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article should be revised to better communicate hypotheses, results, and interpretation of results. Some of the language used was overly vague. More suggestions related to Basic Reporting are included in my General Comments to Authors, below.
Experimental design: Yes, the project appears to have been designed reasonably well. Additional analyses would be helpful, however, including the inclusion of a phylogeny. More suggestions related to experimental are included in my General Comments to Authors, below.
Validity of the findings: All of the analyses appear to have been done correctly. Some of the conclusions are obvious and well supported by the data. Some conclusions regarding the impact of humans is especially speculative and not tied to a specific hypothesis. More details are provided below.
Additional comments: In this study, the authors investigate the population structure within a portion of the range of Abrothrix olivacea in Chile, with an emphasis on understanding the differentiation between populations on Chiloe Island versus mainland Chile. They sequence part of the mitochondrial control region and 12 microsatellite loci for nearly 200 individuals. They analyzed these datasets using various summary statistics and the programs Structure and GENELAND. A haplotype network of control region sequences shows relatively high genetic diversity, with some haplotypes being spread across wide geographic distances. I recommend supplementing this analysis with a phylogeny of control region sequences where geographic details are illustrated at the tips of the tree, which might demonstrate this pattern more effectively. The Structure analysis that assumes 5 populations shows a great deal of mixed ancestry for coastal temperate forest populations. Chiloe Island and Northern Patagonia populations are somewhat more consistent in population assignment. Overall, I agree with the authors that their Structure results do not show deep population structure. The GENELAND results confirm that there is no significant population structure between Chiloe and mainland populations.

Overall, I think this study relies on a solid dataset with good geographic and genetic sampling. It is modest in scope and, disappointingly, somewhat unfocused. The Introduction should be reframed around specific, testable hypotheses with clear predictions. It should also more clearly explain the geography of the region and the recent geological history. Some sections of the paper are clearly written (e.g., Methods and, for the most part, Results) but others (e.g. Discussion) seem incomplete or haphazardly reasoned.

For example, the focus on human impacts and conservation seems quite separate from the question of evolutionary drivers of population size changes and movements. The paragraph that starts at Line 340 discusses potential human impacts on population structure, and I found it difficult to parse. What, exactly, are the specific predictions we would expect to observe if human modification of the landscape affected the population structure of this species vs. more ancient drivers like glacial recession? Human impacts would necessarily be quite recent in evolutionary time, so it’s possible any impact would not be detected from control region or microsatellite data. This, of course, needs to be discussed.

The paper should be carefully proofread again and many sentences need to be revised because there were numerous small errors throughout. Typically, this did not prevent me from understanding the main results. Vague language, however, especially in the Discussion and Conclusion, did prevent me from taking away any clear message beyond the finding that there is little population structure between Chiloe and the mainland. One example of this vague, confusing language comes from Line 397 of the Conclusion, where the species is said to “reacts [sic] more to historical than to ecological events regarding its genetic population dynamics”. It’s not clear to me what evidence is used to justify this claim.

Other issues and suggestions are provided below:

Abstract

*Line 45: First sentence should be rephrased; not clear what “their impacts” is referring to.

Introduction

*Line 73: list is not consistently structured; “livestock” and “highways” are not activities
*Line 75: temperate rain forests, not just temperate forest, correct?
*Line 88: North population of what? No organism has been introduced yet. Should introduce the island of Chiloe here, too. What makes it distinctive or important?
*Line 98: “associated to” should be “associated with”; be sure to check throughout the text for that minor English language idioms like this are corrected.
*Line 101: how were these refuges recognized? And where are they?
*Line 104: misuse of “being” here and throughout. Rephrase.

Methods

*Line 196: which dataset was used for the Structure analysis? Just microsatellites?

Results

*Line 248: Authors state “there is an haplogroup (“light blue”) spread all over the network that corresponded to the locality of Valdivia”. I don’t know what this means. How are you defining “haplogroup”? By definition, a given haplotype can only be present in one place on a haplotype network.
*Line 279: Four clusters? This is explained confusingly. The Structure results suggest 5 clusters.
*Line 290: what is Micro-checker? Needs to be explained in the Methods

Discussion

*Line 299: The first sentence is not clear to me. Yes, it appears that there is high genetic diversity at this one locus across the species’ range. But how does the fact that there are few mutational steps between haplotypes translate into low nucleotide diversity? And then the next sentence starts with “thus” but I do not logically connect the first sentence to the second. Certainly, the haplotype network does not show clear division between Chiloe and the mainland—emphasize that point.
*Paragraph at Line 340: Discussion of potential human impacts on population structure is confusing. Human impacts would necessarily be quite recent in evolutionary time, so it’s possible any impact would not be detected from control region or microsatellite data. This needs to be discussed.

Figures

*Figure 1: light green lines are difficult to see. Draw map with black lines.

*Figure 2: this network is difficult to interpret on its own. Consider placing it on a map, or somehow denoting geography here.


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: GENETIC VARIABILITY AND STRUCTURE OF THE OLIVE FIELD MOUSE: A SIGMODONTINE RODENT IN A BIODIVERSITY HOTSPOT OF SOUTHERN CHILE
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have read the revision version of this manuscript, and I found that the authors have addressed my comments adequately. I found the aims, analyses and results adequate, so I have no major comments. I only noticed some minor grammatical and small issues that should be addressed before its publication.

Ln56: clusters
Ln57: showed a genetic barrier in those areas, while the Chacao channel was not a significant barrier for this rodent.
Ln128: type of markers,…..may not be
Ln217: Add a comma after population structure
Ln230: Omit the comma after genetic differentiation
Ln232: Isolation by Distance (IBD) were calculated through Mantel test with GenAIEx
Ln234: Simple Euclidean algorithm?
Ln267: Delaunay
Ln305: test for IBD was positive and significant? .
Ln359: Microsatellite genetic diversity indices showed…
Ln388: an
Ln395: has been recognized
Ln407: Omit the comma after historical factors
Ln441-443: I would omit these sentences of results here, as this data was already mentioned. Just focus on giving general conclusions.
Experimental design: Adequate
Validity of the findings: Adequate
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: GENETIC VARIABILITY AND STRUCTURE OF THE OLIVE FIELD MOUSE: A SIGMODONTINE RODENT IN A BIODIVERSITY HOTSPOT OF SOUTHERN CHILE
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript has significant and persistent grammar and spelling issues. Almost every paragraph needs to be revised. Often, multiple sentences within a given paragraph contain typos, comma mistakes, or non-standard constructions. I noted this in my last review and see no attempt to fix these issues in the revision. I hope the authors are able to fix this because it is not publishable in its current form.

In general, the literature references are fine.

Figure 1 is much improved over the last version. The colors are helpful.
Experimental design: This is improved over the last version. I'm glad the authors eliminated the confusing focus on human impacts. The authors did not heed my recommendation from my previous review to include a phylogeny of control region sequences. I still think this would be helpful, even as a supplement. Phylogenies are routinely included in phylogeographic studies. It would be especially helpful to include sequences from related outgroup populations. This would put the subspecies in a phylogenetic context.
Validity of the findings: The dataset is solid and the conclusions are mostly reasonable. The paper is modest in scope.
Additional comments: This is my second review of this manuscript. The quality and clarity of the writing is the last major issue that must be resolved in my opinion. The addition of a phylogeny would also be helpful and very easy to do.


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: EVIDENCE FOR LOW PREVALENCE OF RANAVIRUSES IN ONTARIO, CANADA’S FRESHWATER TURTLE POPULATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the author used clear and concise English throughout the text. The references provided throughout the text are sufficient and provide the necessary background information to the reader. The article is structured appropriately for the target journal. The figures and tables referenced in the paper are included in the submission, however Figure 1 and Table 1 referenced in the Materials and Methods section are incorrectly labeled at the end of the text. Additional comments on wording, cited literature, figures and tables can be found in the comments to author portion of this review.
Experimental design: The article has a clear, well defined objective of determining the prevalence of ranavirus in free-ranging turtles in Ontario, Canada. The research is novel in that no such survey has been conducted previously meaning little is known regarding the impact ranavirus has on chelonians in Ontario, Canada. The methods used are described well enough that the study could be replicated.
Validity of the findings: The results are clearly presented. I commend the author for writing this article, despite not detecting ranavirus in any of the samples tested.  Surveys which result in no detection are likely under-reported in amphibian disease literature.
Additional comments: Line 40-42: The conclusions sentences found on these lines are very similar to what is repeated in line 43 of the Methods portion of the abstract. Re-wording the last two sentences of the Background section of the abstract might remove some redundant text.
Line 74: Please provide a citation for the statement regarding FV3 occurrence in reptiles.
Line 82: Brenes et al. 2015 should be Brenes et al. 2014
Line 82: I understand why you mention amphibians as reservoirs here, but you might want to add a statement suggesting turtles are also a potential reservoir for ranaviruses since they can tolerate infection. The Brenes et al. 2014 paper cited in the previous sentence suggest turtles and fish as reservoir species.
Line 83-84: Suggest changing “susceptibility for infection” to “host susceptibility”
Line 87: common snapping turtle
Line 89: eastern box turtle (Terrapene carolina carolina)
Line 91: rephrase ; ranavirus prevalence was determined to be 4.8%-31.6%  and 0% for eastern painted turtles (Chrysemys picta) and common musk turtles (Sternotherus odoratus) respectively in Virginia, US.
Line 95: consider spelling all numbers <10 throughout the text (e.g. 8 to eight)
Line 110: Turtles are admitted to the OTCC hospital
Line 116: Consider changing: Sixty-three turtles submitted to OTCC from Ontario, Canada (Figure 1) were tested for ranaviral DNA using PCR;
Line 120: Consider rephrasing: During necropsy or evaluation each turtle was observed for clinical signs of disease (Table 1).
Line 123: Consider adding a Cat# for the swabs
Line 129: Consider changing “viral testing” to “PCR testing”
Line 130-134: The only section of the methods which needs more description is found in these lines. Please provide what kit was used to extract the DNA from your samples. Also, please describe whether positive and negative controls were run on each gel.
Line 140: Rephrase:  “liver and kidney pooled samples” to “liver and kidney homogenate from deceased turtles”
Line 151: “Ontario Turtle Conservation Centre” should be OTCC after the first mention.
Line 158: A citation needed at the end of this sentence. You could possibly cite the “Comparative Pathology of Ranaviruses and Diagnostic Techniques” section of the “Ranaviruses” book published in 2015. DOI: https://doi.org/10.1007/978-3-319-13755-1
Line 159: Consider changing “mimic” to “appear similar to”. Using mimic would be an anthropomorphism
Line 164-166: Either ignore my previous comment for Line 91 or remove this section.
Line 168: (Terrapene Carolina Carolina) to (Terrapene carolina carolina)
Line 171: (Goperus Polyphemus)  to (Gopherus polyphemus)
Line 174: “may die before surveyed” to “may die prior to being surveyed” ; “,so prevalence may be underestimated.” to “resulting in an underestimated prevalence.”
Line 208: “Our facility” to “The OTCC”
Table 1: is actually Figure 1; make sure all scientific names are italicized and spelled appropriately.
Table 2: is actually Table 1; caption needs more description. (e.g. “Species, sex, case number, clinical signs and case outcome for turtles submitted to the OTCC which were tested for ranavirus via PCR.”


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: EVIDENCE FOR LOW PREVALENCE OF RANAVIRUSES IN ONTARIO, CANADA’S FRESHWATER TURTLE POPULATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: I find the paper to be well written, however I have some comments on the reporting of the work.

I think the title of the article does not do a good job of encapsulating the results that follow. I would consider changing the title to something like "Evidence for low prevalence of ranaviruses in Ontario's freshwater turtle populations."

I believe that given the likely readership and the narrow scope of this paper, the amount of detail afforded to the general biology and pathology of ranaviruses during the introduction could be deemed excessive. I would consider tightening up the introduction to focus primarily of ranavirus prevalence and the important of understanding pathogen prevalence in wildlife diseases.

My personal opinion is that this work would be better suited for presentation as a shorter format communication in a reputable journal specialising in wildlife diseases. Given that this is essentially a survey, the work does not seem to be based on any particular testable hypothesis. If the authors do wish to publish in PeerJ then I would like to see the introduction reworked to include some sort of hypothesis declaration and clearer rationale for the study.
Experimental design: The experimental design is straightforward and appears to be sufficient for the purposes of this study.

However, there is a lot of missing information regarding the molecular diagnostics employed for this study.

Specifically:

How was DNA extracted from each sample type?

How was DNA subsequently handled?

Why did the authors decide to use conventional PCR rather than one of the available quantitative PCR assays?

How was the positivity, or not, of a PCR test deduced? I assume this was by electrophoresis, however it would be useful to state this for clarification.

Were positive controls included in each PCR / Gel?

Were samples extracted / amplified in duplicate or triplicate, as is standard in diagnosis the presence of a pathogen using PCR of qPCR.

At present the molecular portion of the work is not replicable and as such this section requires significant attention before this work can be published in its current form.
Validity of the findings: The current finding of the paper is zero prevalence of ranavirus in the turtles sampled.

I believe that this paper would be strengthened by incorporating the methods alluded to in lines 195 - 197 of the discussion into the paper methods so that paper can include the finding of a suggested prevalence less than 5% in turtles in Ontario. This is a potentially more impactful statement to make.

Whilst there is nothing wrong with presenting a negative detection story, in order for it to be interesting of a generalist journal such as PeerJ, the authors should take care to present the results in a positive framework.
Additional comments: I find the paper to be well written. However, I feel that the methodologies are seriously lacking in detail and that the presentation of the papers results lacks ambition.

It is my personal opinion that this work would find a more suitable home in a wildlife diseases journal rather than a generalist journal like PeerJ.


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

Input:
Title: EVIDENCE FOR LOW PREVALENCE OF RANAVIRUSES IN ONTARIO, CANADA’S FRESHWATER TURTLE POPULATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Minor grammatical errors within sections - please see the annotated PDF for suggested changes for strength and clarity. Otherwise, the article meets the standards for literature references, background, article structure, and relevant results for a general survey (not hypothesis-driven, but important for prevalence and disease studies".
Experimental design: Minor additions would be helpful within the methods section, specifically concerning PCR protocol. The research fits within the Aims and Scope of the journal, with a meaningful research question.
Validity of the findings: Statistical results were first reported in the discussion section- they should be moved appropriately, but they are justified and helpful to the reader. Negative results are important for prevalence studies, and are well-supported in the discussion and conclusion section.
Additional comments: Overall, I think this paper is useful for the epidemiology of ranavirus in chelonians. With minor edits as suggested in the attached PDF, it will be a good piece to have available, especially if ranavirus should become problematic in the region in the future and to enhance our evaluation of the disease dynamics at a population and geographic scale.


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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 1
Reviewer: 1

Output:
Basic reporting: L 88. What do risk factors mean in this line? I recommend that the authors describe more specific.
L 97. Finally, how many samples (patients) were used as a candiduria marker? The authors should explain exact numbers which you used for analysis.
L 100. I cannot understand why the authors used a total of 330 YLC samples. Do authors have any reasons?
L 126. I suggest that the authors explain the meaning of numbers after a plus-minus sign.
L 228. Do the authors have any references for the connection between candiduria and candidemia? I cannot find any references about that in the line.

Table 2. I recommend that the authors put “Gender” above “Age, Year, n (%)” in table 2 if you mention “Gender” first in the manuscript (L139). It is easy to understand the content of the table.
Table 3. I propose that the authors use the asterisk for the results on the table to show the significance (P < 0.05) of analysis.
Experimental design: L 108. I guess that the authors assumed that the data was a normal distribution. I would like the authors to explain the reasons why you used t-test for the analysis in this manuscript. In addition, I recommend the authors do the test for normality of the distribution of the data if you could.
Validity of the findings: L 194. Do the authors have any reasons why you mentioned “significantly higher” in the line? It is better to write the reasons like p-value of the test.
L 199. I suggest the authors describe the consideration of the reason why the candiduria incidence was significantly higher in females than in males.
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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author described the performance of Sysmex UF-1000i in detecting candiduria and possible risk factors for candiduria using clinical data in China. Overall, the article was clear throughout and structured in an easy to read format. However, there are few additions that I think would improve the paper;

1. Could you add the approved Institutional Review Board (IRB) number in manuscript (P9L84 or P15L239-242)?

2. I would like to check the total number of hospitalized patients again. I think it should be 31,648, not 31,684 (P9L85).

3. Current Table 2 shows distribution of candiduria patients among each age and gender group. However, as the purpose of this table is showing age and gender distribution of the candiduria patients, it would be nice to see percentage of each age and gender group among candiduria patients (P23L1).
In addition, I would like to know statistical significance in candiduria incidence rate between candiduria group and non-candiduria group like Table 3.

4. In addition, It may be worth implementing the limitations of present study in discussion part.
Experimental design: I agree that the accessing risk factors for ICU candiduria patients through statistical method is very important and unsettled question.
However, unpaired t-test can determines only whether the population means related to two independent samples are equal or not. Thus, I am not convinced that presence of a catheter, renal failure and high APACHE score can be risk factors of candiduria through the only unpaired t-test. It would at least be nice to see that the odds ratios adjusted with variables used in this study.
Validity of the findings: No further comments. See above for some concerns about the methodology which would cause concerns for the validity of the findings.
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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 1
Reviewer: 3

Output:
Basic reporting: Background: To collect large set of data efficiently would improve the understanding about the epidemiology of candiduria as intended by authors.
The authors also insist that the clinical significance of candiduria is limited because the sample size is small. But as the authors referred in line 57-58, the limitation comes from the difficulty to differ infections from colonization or contamination.  To affect clinical decision making, more direct evidences are needed.
So, it would be better that the authors clarify the difference between what did they intend to prove and what is the unmet needs to be solved.
Experimental design: The authors wrote the aim of this study was to collect epidemiological data in line 76-78.  They are providing 1. the performance of the Sysmex system. 2. the characteristics of candiduria patients.
The authors should define what the performance of  the Sysmax system would explain about epidemiological data about candiduria. They may compare the epidemiological data obtained from the Sysmex system with from the culture method.
Validity of the findings: The authors provided some reason for low accuracy compared with study from Gutierrez-Fernandez et al. But it is not adequate to use the system with a significantly low performance as screening test. It would make the discussion part of this article better if author would provide what could be the best role of the test.
Because the epidemiological data used in the analysis are obtained from the test of very low performance, the authors might be required to show the results of the analysis based on data obtained from other method and that two results are similar.
Additional comments: Your study was impressing. It required a large number of samples and a lot of resources. To make idea clear and prove with your own 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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 2
Reviewer: 1

Output:
Basic reporting: For figure 1, I recommend that the authors make error bars visible and explain what they are i.e., standard deviance or confidence interval.
Experimental design: The manuscript has well revised.
Validity of the findings: I agree that urinary characterization and renal failure are statistically significant risk factors, though, "APACHE Ⅱ score" is not a risk factor. I recommend the authors carefully consider it again.
Although higher risk APACHE Ⅱ score showed a statistical significance between candiduria and non-candiduria, I do not know the range of the actual APACHE Ⅱ score. Therefore, I cannot compare them and tell how big impacts the result has. In addition, SD seems to vary widely but there is no explanation. I would be grateful if the authors could have a deeper discussion about those things.
Additional comments: Although the manuscript has revised well, there are some equivocal expressions and insufficient explanations in the contents of the manuscript. I would like authors to revise those points carefully.


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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for reflecting my comments in revised manuscript.
Experimental design: The statistical methods used in the present study were well described in method part. However, still I am not convinced that presence of a catheter, renal failure and high APACHE score can be risk factors of candiduria. By using the Fisher’s exact test and Mann–Whitney U test, we can determine whether two independent samples were selected from populations having the same distribution, but not adjusted effects with potential confounders. Also, I think odds ratios in revised table 4 are crude odds ratios, not adjusted one. As study population in this study is hospitalized patients, they may have several risk factors at the same time. Therefore, because of the high possibility of presence of confounding variables due to characteristics of study population, I think matching or adjustment is highly needed in this study.
In addition, I cannot understand why odds ratio for APACHE score is not estimated in this table. Even though it is continuous variable, we can calculate adjusted odds ratio through logistic regression. Furthermore, as one of the main results in present study is that high APACHE score can be a risk factor of candiduria in ICU patients, showing adjusted odds ratio for APACHE score is required.
Validity of the findings: Conclusions and limitations are well described in this paper
Additional comments: I strongly agree that the accessing risk factors for ICU candiduria patients through statistical method is very important question and data used in this paper is quite valuable.
However, it would be really nice to see that the adjusted odds ratios to avoid the effect of confounding variables and I strongly believe that this additional analysis can improve the strength of results in this paper. 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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment.
Validity of the findings: The statistical methods were improved.
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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 3
Reviewer: 1

Output:
Basic reporting: well designed
Experimental design: well revised
Validity of the findings: I suggest that authors consider the conclusions of your manuscript again. Because the authors mentioned that the use of catheter was an only independent risk factor in the abstract, the conclusion from line 254 should be changed.
Additional comments: I would like authors to revise points mentioned above carefully.


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: CANDIDURIA IN HOSPITALIZED PATIENTS: AN INVESTIGATION WITH THE SYSMEX UF-1000I URINE ANALYZER
Review round: 3
Reviewer: 2

Output:
Basic reporting: 1. P15L246: I think only the presence of catheter was found as statistically significant risk factor.
Experimental design: 1. In Table 4, why death was considered as a risk factor for candiduria? In addition, if there is logical reason for this one, that variable should be used as adjustment in multi-factor logistic regression.
2. Also, I would recommend to state which variables were used as adjustment in Method part (statistical analysis) and also legend of Table 4.
Validity of the findings: no comment
Additional comments: No additional comments