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

Input:
Title: A RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA?
Review round: 1
Reviewer: 2

Output:
Basic reporting: The background needs to demonstrate the importance of this work.
Experimental design: The method of research needs to be detailed.
Validity of the findings: no comment
Additional comments: This study is to investigate the differences in upper airway morphology between positional (POSA) and non-positional (NPOSA) in 75 patients with obstructive sleep apnea. But please consider the following questions:

1.What is the significance of exploring this difference in the progress and treatment of the patients with OSA?

2.How to judge POSA and NPOSA? You should provide for the method of analysis of these concepts.

3.How to consider the relationship between nocturnal rostral fluid shift and the severity of OSA? How to avoid this kind of confounding factors?

4.Why all of these patients only have OSA? How about MSA or CSA these patients?


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

Input:
Title: A RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA?
Review round: 1
Reviewer: 3

Output:
Basic reporting: Jiao and colleagues retrospectively described the differences in upper airway morphology between 30 patients with positional and 45 with non-positional OSA. Various measurements and diameters calculated on CT scan are compared between the 2 groups.
The paper is overall well written and shows some significant results, however there are some major and minor revisions to address. In particular, the authors use too many abbreviations, and some of them are not so easily interpreted, which makes the article sometimes difficult to read, since the reader is obliged to check the meaning of the abbreviation quite often. Furthermore, The discussion is poorly structured, I suggest to briefly report the main results and then discuss each of them comparing with prior literature.

Major revisions:
- Materials and Methods: the authors state that they performed a PSG, but they did not include electroencephalographic data. I suggest to add this among the study limitations.
- Results: Mean age of study population was 39 years. Differently from other studies, The study population is very young, can the authors justify and comment on this?
- in the Results, line 119, the authors do not write if BMI was different between groups. This data is written in table 1, however, given its importance, I suggest to add it in the text, too.
- discussion, lines 154-157: the meaning of this sentence is not clear, I suggest rephrasing.
- discussion, Lines 159-166: I suggest to deepen this point.
- discussion, line 178-179: 'specifically...35.41mm'. This result is not present in the result section. I suggest to add it to the section and then discuss.
- discussion, line 196-199: ' while there... the two groups': I suggest to better explain this point.
- discussion: add to the limits of the study the retrospective nature.
Experimental design: Major revisions:
- CT test is not routinely asked in patients with OSA. The authors should explain why the exam was requested in this group of patients. If it was requested for research purposes, patients should have signed an informed consent.
Validity of the findings: no comment
Additional comments: Minor revisions:
- introduction line 59: few studies on the Chinese population. Line 60: Chinese patients using...
- Materials and Methods line 68: treated in the hospital
- Materials and Methods line 93: would have had calming
- Results line 133: GP was significantly smaller.
- conclusions, line 217: were in SPL, and the CD...
- figure 1: the position of the head during CT was not in the correct position
- figure 1: >25% of all.


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

Input:
Title: A RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA?
Review round: 2
Reviewer: 1

Output:
Basic reporting: I acknowledge that the authors have made some corrections and improvements to the original manuscript, however most of the statements that were not clear in the first version are still not clear. The manuscript absolutely needs to be revised by an English translator, in fact, many statements sound incomplete and without the main verb.

Minor comment:
- Materials and Methods: the authors state that the 105 patients underwent surgery. I suggest to add the reason why they required surgery (OSAS treatment)
Experimental design: no further comments
Validity of the findings: no further comments
Additional comments: No additional comments


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

Input:
Title: MULTIGENE PHYLOGENY OF THE SCYPHOZOAN JELLYFISH FAMILY PELAGIIDAE REVEALS THAT THE COMMON U.S. ATLANTIC SEA NETTLE COMPRISES TWO DISTINCT SPECIES (CHRYSAORA QUINQUECIRRHA AND C. CHESAPEAKEI)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text presents an important contribution to the understanding of the diversity of jellyfishes from US east coast, by presenting new and detailed data on a well-known thought to be a single species (until now named Chrysaora quinquecirrha). I am not a native English-speaker, but the manuscript is well written, and the language used is very clear throughout the text. “Introduction” section provides background on the topic and included traditional and updated literature. The structure of the text is in accordance with the standards, but I suggest the authors to move the “Systematics” section before the “Discussion”. Figures and tables are of high quality and essential for helping to clarify the results, unless when noted.
Experimental design: The methods were clearly described and are updated with the available literature.
Conclusions are well supported by the data presented.
Validity of the findings: Although there was an indication in the literature about the occurrence of two varieties of the jellyfish studied, the authors provided robust data to identify and sort them out. The nomenclatural issues were carefully followed and the main result (species name validation) is sustained based on the articles of the Code. Summing up, my evaluation of the manuscript is that it corresponds to an important contribution to Marine Biology in general as it provides advances in the field, especially unravelling the identity of a significant jellyfish species of the Atlantic US coast.
Additional comments: In general the authors were cautious in not proposing new names for the different groups found among the genus Chrysaora. I think that additional data must be gathered to perform such big changes in the classification of the family. There are some minor corrections and suggestions performed directly on the PDF file, summarized as follows in order of importance: 1) holotype specimen must be deposited in a well-known collection, as well as other comparison individuals; 2) provide further data on the Senegalese specimens (images as supplementary material would be welcome); 3) correct some references regarding format and some 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: MULTIGENE PHYLOGENY OF THE SCYPHOZOAN JELLYFISH FAMILY PELAGIIDAE REVEALS THAT THE COMMON U.S. ATLANTIC SEA NETTLE COMPRISES TWO DISTINCT SPECIES (CHRYSAORA QUINQUECIRRHA AND C. CHESAPEAKEI)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No Comment
Experimental design: No comment
Validity of the findings: No Comment
Additional comments: PeerJ
Multigene phylogeny of the scyphozoan jellyfish
family Pelagiidae reveals that the common U.S.
Atlantic sea nettle comprises two distinct species
(Chrysaora quinquecirrha and C. chesapeakei)
(#17919)

Brief summary

This study seeks to reconstruct the phylogenetic relationships among 4 pelagiid genera and 11 of 13 species of Chrysaora. In addition collections were made along the US Atlantic and Gulf coast of C. quinquicirrha, from which molecular, gross anatomical and cnidomic data were collected. The stated goals of the authors was to clarify taxonomic status among genera and species of this group of nuisance species. The molecular approach used nuclear 28S and mtDNA COI and 16S. Morphological analyses focused on tentacle and lappet number, oral arm length and nematocyst dimensions. Results suggest that C. quinquicirrha is polyphyletic, and comprises two distinct lineages one from the Gulf and the other from the Atlantic. This conclusion is supported by molecular as well as morphological evidence.

This paper is certainly interesting scientifically as well as provides an important contribution to applied resource management. The phylogenetic analysis presented is technically sound, and the resulting systematic claims are convincing. Figures are of sufficient quality, including maps, photos and trees.

The systematics sections is about 11 pages, raising the editorial question of whether this section is appropriate for the journal PeerJ, or might be included as an appendix, otherwise this could be more appropriate for a taxonomic journal.

I have made a number of minor grammatical suggestions and minor corrections of typos.

Specific Comments
L 122
Please italicize “Sanderia”

L153
Use of the small unprimed italicized letter implies that the heme prosthetic group is in a hemochrome linkage, and a lower-case italicized letter, e.g., c', should be used.
L184
Italicize “c”

L201 and L203
Please write out the NIH DNA database as “GenBank”

L219
“jModelTest”

L328
I’d suggest rewording “two distinct clades that were highly diverged” “two distinct, highly divergent clades”

L332 & 335 and throughout
Please be consistent: “Figs” vs. “Figures”

L337
Replace “highly-supported” with “well-supported”

L344
Replace “observed” with more appropriate term, such as “evaluated” or “examined”

L345-6
“were observed for 20 quantitative and qualitative macromorphological characters either taken from Gershwin and Collins (2002) or new to this study (maximum oral arm length)”

This sentence mentions 20 traits, and lists a single trait that was unique to this study.  It remains unclear to the reader why a single character is listed, this could lead to confusion as to whether this single trait listed is the only trait used that was not one from the Gershwin and Collins 2002 paper, whereas if those traits that fall into both categories are indicated in a table, perhaps simply referring to the table would be more clear at this point.

L361
In: “<~1.5%” inclusion of the symbol “~” seems awkward, does removing this symbol drastically alter the meaning?  If not I’d suggest deleting it.

L374
“all those sequenced from” this is admittedly a bit of a pet peeve of mine, but to state that “individuals” are sequenced is inaccurate, strikes me a slang, since DNA, or gene fragments are sequenced rather than jellyfish, or individuals being sequenced.  I’d suggest rewording to something like “jellyfish included in the molecular analysis” or something along these lines that more precisely reflects the author’s intended meaning.

L423
Replace “a paraphyletic Chrysaora.” with “paraphyly of Chrysaora.”
L434
Should a question mark be added following “limited”

L475
“therefore, conclusions based on these sequences should be made with care.” Why?  Perhaps consider explaining, for instance “because the geographic provenance of these individuals is unclear”, or what ever the reasoning.

L484
The following sentence: “C. quinquecirrha occurred in two well-differentiated” opens with an abbreviation of the genus, whereas starting a sentence with an abbreviation should be avoided.  This is an editorial, and grammatical decision.

L596
“and” not italicized

L604 and 608
Include “(MBA)” after first mention of “Monterey Bay Aquarium”.

L634
“(Figure 4 and 6)” please follow journal guidelines and be consistent in the manner in which two figures are referred to, there are instances where the following is used “Figures 4, 6”


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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors test the suitability of a macrobenthic ecological observation due to salinity gradients, on sediment microbial community structure and function. The authors provide sufficient background on the original ecological concept, their study system, and their methods. Raw data are available. Data and figure reformatting is suggested. Minor grammar and word changes in the manuscript are recommended.
Experimental design: The authors use 16S rRNA gene sequencing of sediments from a fresh-to-marine transect, and typical metrics to analyze the role of habitat and environmental influences on microbial communities. The study seems to build off a previous publication from the same group, that evaluates the influence of salinity on bacterial community structure in lagoons in the same sampling location (http://www.sciencedirect.com.proxy.lib.umich.edu/science/article/pii/S1874778716300058), but the current study incorporates a broader salinity range than previously evaluated. The authors infer functional characteristics of observed bacterial communities, but functions for 38-75% of taxa in each sample cannot be obtained. The authors also tentatively evaluate archaeal community composition, of which this study design is not optimal.
Validity of the findings: The authors conclude that Remane’s “species minimum” concept does not translate to sediment microbial community structure and function. Their main supports are 1). A decrease in species richness over the salinity gradient; and 2). Strong correlation between salinity and community composition, and salinity and function. The authors rightly suggest caution when interpreting archaeal relative abundances and functional profiles from bacterial 16S rRNA gene data. The authors use a paragraph in the introduction discussing the unknown impacts of sea level rise and salinity changes on freshwater systems. Their study system could represent a microbial community in transition from fresh to saline conditions, and functional signatures could be evaluated in that context; the authors could address this compelling ecological consequence in their discussion.
Additional comments: Please review paragraph breaks for clarity and cohesion. There are examples of unexpected paragraph breaks at lines 114-115, the methods section for “DNA extraction, PCR amplification and 16S rRNA sequencing”, 340-341, and 369-370.
Please check grammar and flow. For example, lines 224-225 could be clearer: “The number of sequences assigned to an OTU in each sample represents the relative abundance of the OTU in a sample.” Suggestion for lines 201-203: “The raw sequence reads retrieved from all the sediment samples were quality trimmed using sickle (Joshi & Fass, 2011), to where the average quality score dropped below 20 (-q 20) as well as where read length was below 10bp (-l 10).”
Methods: Lines 133-139 detail two different instruments to measure dissolved oxygen, salinity, and temperature. Were these instruments cross-calibrated? Please see comment about Supplementary Table 6.
Lines 169-172: This statement implies that the authors used multiple primer sets on different samples, and compared resulting community profiles. However, primers impart community biases, and the use of different primer sets in the same study is puzzling. Please provide background research on or results comparing bias from the selected primer pairs.
Lines 224-226: Are your analyses on each station using averaged representations of OTUs across the replicates? If so, do you have tests on the similarity of the replicates?
Figure 2: Are there any significance values attributed to the ellipses, or are they just encircling the station points?
Figures 3, S2, and S3: Please clarify if each bar represents the relative abundance of phyla in one sample at each station, or if the relative abundance profiles of samples at each station were averaged to get a mean representation of phyla at each station. If they were averaged, see above comment about methods. Graph axes need labels.
Figure 4: Vertical lines separating each triplet per KEGG would make interpreting the graph easier. Axes need labels.
Figure 6: Would error bars on OTU abundances be visible on this graph, or be obscured by the station points?
Supplementary Table 1: Please change “paired-end reads” to “read pairs”. One overlapped read is the result of two paired-end reads.
Supplementary Table 6: Chlorophyll a is reported for all stations, however the methods state that fluorescence ~ chlorophyll a was measured at only Arachthos delta and Kalamitsi stations. How was fluorescence or chlorophyll a measured at all the other stations? Also, the chlorophyll a values for those latter locations have a different format than the rest.
Discussion: Your study has a clean and simple design, of using the sediment microbial communities and relevant environmental parameters to test Remane’s species minimum in other systems. I think the results will have broader significance if you can relate them to benthic ecological succession due to rising sea level and climate change.


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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: -Data is robust, but statistical analysis could be improved to specifically test the role of salinity in driving community patterns.
-Conclusion are well stated, but not linked to original research question as novel aspects (community variation and functional profiles) were added and not justified at the first place.
Additional comments: I praise the authors for their extensive data set and for their thorough statistical analyses. The manuscript is clearly written and there are only minor points to be checked for improved clarity. Yet, some points should be addressed to make the study stronger as follows:

1) The study would gain from being more focused on what the title and abstract described, i.e. the testing of the theory that "taxonomic diversity of microbenthic organisms is lowest within the horohalinicum". The manuscript should have thus consisted of testing local microbial richness (alpha diversity) as a function of the salinity gradient. The authors extended their analysis to community turnover and to the functional relevance of their observed diversity patterns, which in my opinion, analyses that dilute the main message of the study or at least have not been justified with respect to Remane’s theory.
I would suggest that the authors either change their title and abstract to be broader in scope, or fit their text content to the testing of Remane’s theory as main objective.
2) Some technical points are not clear:
2a. Line 213: why did the reads have to be mapped back to the OTUs? AT this stage of the procedure, all reads should be clustered into OTUs. Please clarify.
2b. Line 209 the singletons were removed, and few lines later (line 213), they were added back. Is this a normal procedure (please provide references then)? It seems that this procedure may add sequences that will not fit to any OTUs because those reads were not considered in building the OTU matrix at the first place.
3) One main point that the authors touched upon is whether salinity is the main driver of community patterns or not. The current analysis based on correlations does not account for factor confounding and for the amount of variation each factor might explain. To address this point, I suggest that the authors perform a variation partitioning analysis (see vegan package varpart function for instance) in order to quantify the amount of variation in community that is due to salinity uniquely while taking other environmental and spatial factors into consideration. Hence, factor confounding on community variation can be disentangled. The same approach can be applied to changes in richness and would be a strong point to be made in this study.
4) The salinity gradient (e.g. Figure 6) does not include samples at the 5-8 psu that are mentioned in Remane’s concept. This would suggest that the authors should reinforce the idea that the authors should refocus their study on the effect of salinity and biogeography on community variation, instead of addressing Remane’s concept.
Minor comments
1) Line 197: The reference Leinonen et al., 2011 could be removed or put after "ENA" on line 196.
2) Figure 2. The legend "nMDS of the microbial OTUs" is not a valid legend, as nMDS applies to a distance matrix. Please rephrase.
3) Lines 325-326: please rephrase as the sentence is intrinsically redundant.


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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT
Review round: 1
Reviewer: 3

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

1) Lines 41-42:
In the Abstract, the authors provide an important statement that “the sediment microbial OTUs in this study area do not follow Remane’s concept which could be attributed to the different life strategies of micro- and macroorganisms”. Although the idea of differences in life strategies between unicellular plankton and large multicellular bottom-dwelling organisms is highly relevant to the results of this study, it is, unfortunately, neither mentioned in the Introduction nor discussed elsewhere in the manuscript. Moreover, this idea was originally published by Telesh et al. (2013) and then referred to in several other articles, e.g. by Herlemann et al. (2016), who wrote: “Telesh et al. (2013) suggested that the life strategies of unicellular planktonic organisms differ substantially from those of large multicellular bottom-dwelling organisms, resulting in deviations from the species-minimum concept, which is supported by the results from our study of bacterioplankton” (Herlemann et al., 2016, page 7). Therefore, I definitely recommend considering this viewpoint in the manuscript and discussing it, providing the appropriate citation(s). Alternatively, this statement should be deleted from the Abstract since it is not a result obtained by the authors of this submission.

2) Lines 101-103:
It is very difficult to agree with the authors’ statement that “…confirmation of Remane’s concept for prokaryotes would mean that small-bodied, fast-developing and rapidly evolving microbes respond in a similar way as benthic macroorganisms in a salinity gradient”, mainly for the following reasons.
First, this is likely not so because of specific morphological, physiological and ecological characteristics of these groups of organisms. Thus, the differences in their reaction to salinity gradient will always be present due to dissimilarities in size, mode of life, osmoregulation etc. These issues are discussed in many details in Telesh et al. (2011, 2013, 2015 and 2016).
Second, even if diversity of sediment prokaryotes happen to follow the Remane’s curve, one should look for more appropriate (maybe even unknown yet) reasons for this pattern. In this respect, a good example was provided in the paper by Schubert et al. (2011, Mar. Pollut. Bull., Vol. 62) where the authors revealed the Remane’s curve-like pattern in the diversity of macrophytes in the salinity gradient in certain cases; however, the reasons for the species minimum of those macroalgae were the lack of suitable hard substrates but not at all the critical salinity (horohalinicum) in those habitats.
Therefore, I suggest that the authors provide deeper analysis/explanations of the obtained results and a broader discussion of the possible mechanisms that underpin those findings.

3) Lines 313-321:
The result which showed that for certain microbial OTUs a KEGG profile could not be retrieved, thus these OTUs constituted the fraction of unexplained taxonomic units (FTU), and that this fraction was the highest in the lagoon samples (67.51 %) and lowest in the marine samples (38.91 %) is of special interest. I agree with the authors that this issue needs further investigation which might shed more light on the reasons and mechanisms behind the natural prokaryote diversity distribution pattern in the salinity gradient. However, I believe that the existing knowledge on microbial diversity in the other large water bodies with salinity gradient (e.g., the San Francisco Bay, or the Chesapeake Bay and its sub-estuaries) allows for certain generalizations already now. The paper will benefit from the enlarged discussion of these important issues.

Specific suggestions:

1) Line 69: peak (not “pick”).
2) Lines 280-281 and elsewhere in the text: I suggest to change “it seems” to “it is likely” or “we believe that” etc.
3) Legend to Figure 4:
Please consider salinity gradient while compiling the legend and change the order of symbols in Fig. 4 to follow the gradient: river -> lagoon -> sea.


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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I accept all authors' responses to my critical comments and suggestions.

One minor 'technical' correction should be made: in Line 725 of the MS, the format is corrupted (two references are not separated by the line break).


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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: 1  pdf  line 41  “ist (No authors listed,   1985; Kinyoun et al., 1989). Screening for diabetic retinopHY “
This is not  a correct refeence.  Why not use numbers as is now almost invariable in reference list and put the numbers in the text?   The actual rerence ( pdf line261  is missing the year
2:  lines38-41  grammar poor  The reviewer re-writese:
….is indirect stereoscopic biomicroscopy using a (60)D lens.after mydriasis. ( ETDRS report –
3:   line 43   ? Retinography?
4:    line 46   “ due to “ implies a causal relationship.  Has this really been established?
5:   lines 49-50  “in the   ophthalmological consultations. “ replace by a full stiop – cut it out
6: Line 54 “. In other words, the influence of intraretinal fluid has not been considered.”  This is not logical and also it is not “ in other words”. If there is intraretinal fluid then  either the retina is thicker or retinal cells must  have vanished.   I think this requires much more explanation. Much of what is referred to here is usually thought of as retinal cysts.  Thickening without cyst formation would be  an important variable
7:  Foveal thickness. The foveal   is a very small area, and the macula is much bigger.  The authors should make a distinction between clinically non significant macular oedema and clinically significant macular oedema.. I think a figure illustrating OCT findings in the  various categories they propose   would make things clear
8: Line 60- I do not know what is meant here by bootstrapping.  The area under the ROC curve is a method of obtaining specificity and sensitivity values It is not a calibration. Its setting a criterion. Is this the word the authors wanted?  It permits one to obtain optimal values for dividing categories. These depend upon  several factors and one is the proportion of patients who have the condition.  Its no use having high specificity if very  numerous patients have t be examined-  the number of false positives wil be so large that the criterion cannot be used-the service will be overwhelmed.  I think up to line 68  should be revised.
9: Line 72 The “who”  implies that after “Elche”   there should be the word  “were”
10: Line 76   an area of 169555  inhabitants”  Catchment area perhaps?  Wrong power!
   inhabitants number   =x1   while              area =x2
11:  line 89  But early Alzheimer causes reduction in the nfl  layer!  The authors should check the  liturarture.
12 :lines 91-96  I have already commented that a very similar sentence is poor English.
13:  lines 105 107  The authors must understand  “ an area of 1000 cetrnal microns “ is not normal scientific  use.    “valuation” is used incorrectly.
14:  line 124  What does accuracy mean here? Given the strange way s quantitative  data have been handled,  the reviewer suggest that that  the authors state the number of false positives and false negatives  per 1000 referrals,
15:  line 147  The statistical package  may have worked but what is written is slightly  nonsensical.  Putting in decimal points  when dealing with individuals  is illlogical.  See table1
Table 1 caption  What does this mean?
“n(%), absolute frequency (relative frequency)”; Why not insert words into the table ? The revewer has used  have used medical statistics for over 50 years, and have never seen S used as an indicator of standard deviation.  If the figures given aree mean values, don’t we want to know the standard error of the mean?  This is usually indicated by s.e. or s.e.m.   The variables important to diagnosis include age and weight Surely these were recorded?  And nephropathy? And crdiac nsfficiency ? Other interesting differences found in our work is country of origin. Do migrant workers form part of the sample?
16: line 169  The reviewer feels that given what the authors describe as intraretinal fluid is what he would call a cyst
17:  The reviewer  cannot comment on this bootstrap technique . Figs  1-5 are meaningless to him. He would like to see the ROC curves
18:line 181   “However, we must bear in mind that we are  validating a diagnostic test through the calculation of sensitivity and specificity, independent  parameters of disease prevalence (Lalkhen & McCluskey, 2008). In other words, the use of a  sample obtained in specialised care consultations is not a selection bias “ The reviewer does not agree The  optimal specificity and sensitivity of a test does depend upon the severity and frequency of a condition  in the population which hs been recruited,
19: line 185   “ In addition, our sample  was selected completely at random.”  How can this assertion be made without providing evidence?  Screening of populations is tricky. And the present sample of patients is so different clinically to the one provided by  table 1  column 1  that small differences between optimal results is not surprising or even noteworthy.
20:   for all the references  If DOIs  or other unique codings are not given (and they are not) then the year of publication must bre inserted.


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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript is well written and the scientific english language is adequate. References, figures and structure of the article are correct and meet the objective of the authors and the journal's recommendations. In the discussion section, I recommend to clarify the clinical benefit that can have an ophthalmologist reading this paper and to implement the introduction section analyzing in more detail the purpose of the article. Data and statistical analysis are correct, and the value of the research may have globally a good level of impact.


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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Innovative
Experimental design: Well conceived
Validity of the findings: Good
Additional comments: Over all a worthwhile manyscript.
One major spelling error in abstract: Optimal Coherence Tomography should read as Optical Coherence Tomography


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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: the sample size was 134 eyes. To estimate whether this sample was sufficient to validate the diagnostic test, the Authors computed the specificity estimation using the following parameters: an accuracy of 6.36% was obtained. However, since the sample is not big, I suggest discussing it as a potential, not strict, limit.
I agree with the conclusion that given that the validation was performed in the health area covered by their hospital, the Authors encourage other researchers to perform validation in their own communities and only if similar results are found, these cut-off points for foveal thickness can be used in routine clinical practice
Additional comments: This study contains  interesting observations


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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: General
It seems to this reviewer that what the authors have done is to analyse their OCT results in their diabetic patients, and correlated these with clinical opinion derived from slit-lamp microscopy. From this analysis they have obtained values for OCT -derived measures of maximal retinal thickness which can occur in diabetics who do not have clinically significant diabetic macular oedema.   The  work seems well done.  There are a number of mostly linguistic problems in the writing.  There is no way  of knowing whether these results are applicable to any other hospital  or to other maker’s instruments.   The MS refers to previous work (2015) published by the same group which seems to the reviewer very similar to the present MS.  What is new is that the 2015 results are reproduced by the data of 2017

In detail
Title    Would “independent” be better than External?   Better word order is “ ….for foveal thickness using optical coherence tomography in the presence or absence of intraretinal fluid  in patients with diabetic macular oedema”
Line 41  delete “by the ophthalmologist”
Line 41  Wrong method of citation  Possibly  “clinical manual  Boston “  This is a matter for house style to decide
Line 49    Better word order   would be “ Optical coherence tomography (OCT), which has been available for several years, allows us to perform a quantitative and qualitative study of diabetic macular oedema by  providing a cross-sectional image of the retina, at high magnification, and automatically measures the thickness of various retinal layers. The OCT also shows the abnrormalities in structures such as the presence of cysts snd the accumulation of fluid “.
Line 53 “ two parameters”   The reviewer does not understand what is meant.   DMO most often begins extrafoveally, and the retina near the macula may contain cysts etc  while the fovea  is unaffected. Do the authors mean “ macula”  where thy write “fovea”?
Line 54-57 “trying to determine a cut-off point from which we can say that there is diabetic macular oedema. In other words, the influence of  intraretinal fluid in the OCT has not been considered, as when evaluating a diagnostic test it is  possible that the presence of intraretinal fluid on OCT does not agree with the Gold standard”   The reviewer dos not really understand what is meant. In assessing an individual, OCT is so much more powerful than slit lamp with a70D lens that there is  no point raising this.   We differentiate between sight-threatening and non-sight threatening macula oedema.  Is this what the authors are attempting to discuss?
Line 62   The reviewer suggests that an illustration showing retinas with and without intraretinal fluid would make things plain!
Line 63-64  The fovea is the region where the OCT shows a pit, because there are only photoreceptors, and a thickness of the retina at this point of 90 microns is  reasonable. But what is this 310 micron figure?  Does this mean that if the fovea is not involved a thickness of 310 microns in extra-foveal retina  indicates clinical significance? ( sight-threatening)
Line 66  Why use AUC for the Receiver Operating Charcteristic?  Its almost invariably referred to as ROC  for obvious reasons
Line 66 The sensitivity and specificity of a diagnostic test depends  on the ROC but also on the values taken.  This is in no sense a “calibration”  The cut off points are arbitrary. If one wants to include almost all patients, ( that is, to have a high sensitivity )  then the specificity will decrease.  The value finally adopted depends on various factors which include the prevalence of the condition, the  rate of progress and the severity  and also the resources available to track and treat the patients after the intial examination.
Line 67.  The reviewer is going to accept the statement  that there is only one publication giving statistical data about OCT measurements  in patients with DMO , and he agrees that confirmatory data in different centres  with different patient populations  is required.  Thus the results presented below are publishable. But a quick search of PUBMed gives 321 papers with some information about this subject ( even if not formalised )  since 2005!
Line 72  “ in our community”   It is certain if you have a new instrument-of any sort- it is a good idea to collect your own normal values, even if the instrument has been well tested and comes with normative results.  But such data are not necessarily for publication!
Line 88  The authors can determine whether their results are in agreement with those of Hernadez-Martinez : to call this ‘externally validating’ is a bit presumptious!  What would the authors have done if their analysis did not agree with this previous paper?
Line 92  “ wanted to participate”  -the reviewer rather worries about this.  It is conceivable that this could result which was biased and was no generally applicable. .
Line 93 Ot is usual to say “exclusion criteria were  : dementia. Recent cataract  surgery…..”,   This is a linguistic point-  the conditions cannot be criteria ( in English)
Line 100  dilated pupil indirect ophthalmoscopy   would read better
Line 103  These examinations were carried out by expert…
Line 104  Does this mean that the same doctors were used as in the previous cited publications?If so this whole project is scarcely independent!  This is important!
Line 123  Was renal function measured?
Statistical methods  as described seem standard, but they cannot produce what is referred to ss  a calibration   ( line 142)

Line 158  This data has been given previously  see line 124  There are other such repetitions which shouldbe cleaned up.
Line 193  If the patient population  equipment and operators were  exactly the same as those in  the 215 paper, then there would be no point in doing the work at all because it would not establish any generality: all that could be said would be that for these limited conditions  th test was reliable.
Line 198  Earlier it was stated that randomness was not achieved.
Line 218  “externally validated”  see  reviewers previous comments
Line 223   This suggests to the reviewer that this MS deals with patients passing through the same hospital, seen by the same doctors, and examined with the same equipment, as in the 2015 project: and despite there being minor differences between this patient group and the one in the 2015  project, the results are very similar.  Bujt what information    in  this paper would be of immediate use to a hospital in, for example, Venezuela?
Figures  The reviwer suggests an additional figure with OCT images   It would be nice to see an ROC graph. Are any of the other figures really required?


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 COMPLETE CHLOROPLAST GENOME SEQUENCE OF STRAWBERRY (FRAGARIA  × ANANASSA DUCH.) AND COMPARISON WITH RELATED SPECIES OF ROSACEAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Sufficient field background information was provided. In general, writing was clear.
Experimental design: This work will provide valuable information through the complete sequence of the chloroplast of the cultivated octoploid. This will allow to improve chloroplast genetic engineering and to discard this genome in whole genome sequencing done on nuclear DNA or RNA. The research question was clear, relevant and performed accordingly. The applied methods were well described and detailed.
Validity of the findings: Data was well analyzed, statistically sound. Conclusion were well stated, and linked to original research question.
Additional comments: The cultivated strawberry (Fragaria x ananassa Duch.) is one of the youngest domesticated plants, developed from chance hybridization between two western hemisphere octoploid species (F. chiloensis and F. virginiana), which becomes one of an important fruit crop in the world to date. However, little is known about the chloroplast genome of the species that gave rise to this important fruit crop.
In the manuscript, the authors present the complete sequence of the chloroplast genome of the cultivated strawberry. For generating the complete genome, they performed a combination of de novo assembly and reference-guided mapping of contigs. The complete genome sequence provides a new reference for the evolution and genetic diversity of plants.
Then, they compared the chloroplast genome structure and organisation of F. × ananassa cv. Benihoppe with ones of other Rosaceae species. Finally, they performed a cpDNA markers and sequence polymorphisms analysis using data from ten species belonging to Fragaria genus.
This work will provide valuable information through the complete sequence of the chloroplast of the cultivated octoploid. This will allow to improve chloroplast genetic engineering and to discard this genome in whole genome sequencing done on nuclear DNA or RNA. The research question was clear, relevant and performed accordingly. The applied methods were well described and detailed. The article is in the scope of the journal Peer J. Minor version is required to considered the following comments.
Compared with the nuclear genome, the chloroplast genome is small, and the rate of nucleotide substitutions is so low that the chloroplast genome is considered to be an ideal system for studies on phylogeny and population genetics (Wei et al., 2005). I do not think low rate of substitution is good for population study, definitely is good for phylogenetic analysis.

There are 130 genes in the chloroplast genome of F. × ananassa cv. Benihoppe, 112 of which are unique. What do you  mean by the “unique”?

Six fast-evolving DNA regions were individually and combined to construct phylogeny of ten Fragaria species. More discussion on the phylogenetic analysis with previous ones is suggested to be expended.

Please explore the basis for disagreement between some of their own findings and a highly relevant prior study involving Benihoppe.


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 COMPLETE CHLOROPLAST GENOME SEQUENCE OF STRAWBERRY (FRAGARIA  × ANANASSA DUCH.) AND COMPARISON WITH RELATED SPECIES OF ROSACEAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: An additional chloroplast assembly exists that is not mentioned which provides results of a partial 130 kb chloroplast assembly of F. vesca as submitted GenBank GU363535.1 and described in Davis TM, Shields ME, Reinhard AE, Reavey PA, Lin J, Zhang H, Mahoney LL, Bassil NV, Martin R. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae 859: 221-228.

Line 13 Fragaria chloroplast exhibits low variation on what basis? What is the evidence? Or is this based on work of Wei and al 2005 for chloroplast genomes in general or based on Zhang and al 2017 for Rosasceae?  The statement is not clear to this reviewer.
Line 24 What did the comparative analysis consist of briefly?
Line 29. How were F. chiloensis and F.virginiana compared to cv Benihoppe and which F. chiloensis and F. virginiana?  For ease, the PI# should be used to indicate each of the accessions used in the study for Fragaria and for all of the Rosaceae species.
Introduction
Line 57 Hirtula rose as distinct from rose?
Line 58 Assembly of five Fragaria species (Shulaev et al., 2011; Hirakawa et al., 2014).  There should be more such as Tennessen et al  including octoploid and F. vesca ssp bracteata publications.
Line 60 Assembly of nuclear genomes are still a work in progress and not finished.  Maybe state that drafts assemblies have been released?
Line 78-79 Provide references to support that Fragaria has limited variation in chloroplast sequences.
Line 86 cp genomes of F. vesca by TMD. The following reference also provides results of a partial 130 kb chloroplast assembly of F. vesca and reference to a GenBank submission. Davis TM, Shields ME, Reinhard AE, Reavey PA, Lin J, Zhang H, Mahoney LL, Bassil NV, Martin R. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae 859: 221-228.
Line 98 Transgenic Benihoppe nuclear genome released versus reported?
Line 239 - 244.  Sampling of 8x and 2x species is too limited to make broad statements on the species, statement and comparisons should be made to those specific accessions that were compared.
Line 334 comparison to how many F. virginiana and F. chiloensis and which ones?
Experimental design: Inclusion of additional accessions of especially F. virginiana and of F. chiloensis to represent the diversity of the species would permit the kind of conclusions drawn in the study.  As only one cultivar was sequenced, the ancestry of the cultivar might be useful as insight into the possible similarity to cultivars in use other countries.
Validity of the findings: Any plant accessions used in the study should have Plant Introduction numbers and if Plant Introduction numbers do not already exist, the authors should submit them to a germplasm repository and obtain plant introduction numbers. There are subspecies of both F. virginiana and F. chiloensis and variation within the subspecies, so the specifics of what material was used is critical.  There is also variation in F. vesca and F. iinumae and so conclusions should be limited to the comparison made with the specific accessions used in this study and not be drawn to encompass the entire species.
Additional comments: If the above comments are addressed, I recommend the article for publication.


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

Input:
Title: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clear, comprehensible written. Despite the literature was well referenced and relevant, I consider that the author should refer to the 2010 version of The World Health Organization, and not to the one that was published in 1999.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The present study is an interesting work applying geometric morphometric to analyze sperm head contours within a population and between mouse strains and/or fertilization stages. The authors identified morphological indicators of highly fertile spermatozoa, and developed a pipeline that might also be applicable to human normal sperm. However, there are some aspects that the authors should take into consideration.

- The authors said that their method would greatly facilitate and enhance current reproductive technologies in an objective, high-throughput manner. It is not clear how this method can be used in the clinic laboratory to select a better sperm to perform ICSI. Authors should explain with more details this aspect.

- The percentage of sperm hyperactivation correlates with high fertilization rates and is different depending on the fertilization stage (cauda sperm vs PVS sperm). It is possible that the difference in fertilization rates between the 2 strains is due to differences in the percentage of hyperactivation. The author should also perform analyses between motility patterns and aspect ratio in BDF1 (in different fertilization stages) and B6N sperm populations.

- In Figure 4, there are only 3 animals analyzed, I recommend at least 4 to increase the number of cells per groups.

- It would be interesting if the authors perform what they proposed in the discussion: to test dithiothreitol on caput/cauda sperm and analyze head minor axis and aspect ratio of sperm. These results will explain the changes in these parameters during sperm maturation.

- Supplemental Figures: there is not any Figure legend and they are necessary to understand those figures.

- Supplemental Figure 2: It is not clear if they stimulated the acrosome reaction such as using progesterone or ZP or if the authors evaluated spontaneous acrosome reaction. It is possible that acrosome reaction by zona pellucida change the sperm aspect ratio.

- Line 97, authors wrote Fig.S4C, and there is not such panel in Fig.S4.

- Line 165, the authors should define also PC4, PC5.


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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript's premise is that the fertilizing potential of spermatozoa is correlated with their overall morphology. The authors focus in subtle differences between sperm, not in dramatic differences in morphology. The manuscript is well written and the assumptions made to compare fertilizing potential are sound. From the biology point of view, the results are interested and have the potential to be relevant in clinical practice. However,  this reviewer's expertise in cell imaging is not sufficient to give an opinion on the imaging strategies as well as on the mathematics used to produce the data. I hope other reviewers of this manuscript are experts in cell imaging.
Experimental design: As said, assumptions made to choose sperm for analysis are sound. However, the conclusions heavily rely on the validity of the cell imaging methodologies used. Another reviewer should comment on this.
Validity of the findings: Nothing to add
Additional comments: Nothing to add


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

Input:
Title: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors presented a clear introduction with citations relevant to the topic and a clear research question.

1)	In the line 83 of the introduction, the authors state that the sperm head contours of spermatozoa at different “fertilization stages” was analyzed. This is confusing to the reader, because the authors are using a broader definition of fertilization than the classical (the ability of a spermatozoa to penetrate and fuse with an egg (Yanagimachi, 1994)). In their definition, they are including sperm maturation in the epididymis and sperm capacitation in the female tract as part of fertilization (Okabe, 2013) instead of as required steps to acquire fertilizing-capacity. Therefore, it would be useful if the authors clarify this point by adding a sentence in which this broader definition of fertilization is stated.
Experimental design: The experimental design is not completely clear.

2) About the in vitro fertilization experiments:
a)	Line 105: The authors state that the eggs were placed in TYH medium drops. Were the cumuls cells previously removed? If not, this line should state that “Cumulus-enclosed oocytes were placed in 100 µl drops of TYH medium”.

b)	For this reviewer is not clear why the authors chose 8 hours of co-insemination between spermatozoa and oocytes before collecting sperm in the perivitelline space. It has been shown that after half an hour fertilization sperm has already fused with the oocyte  (Tateno et al, 2013). In addition, Storey et al (1984) reported that mouse capacitated spermatozoa penetrated the zona of cumulus intact eggs in less than two hours after insemination. Therefore, after 8 hours of fertilization it seems difficult to find sperm in the perivitelline space unless fertilization and the cortical granule enzymatic reaction was inactivated to avoid polyspermy blockage and allow more spermatozoa to penetrate the zona.  Is this the case? Please clarify this point.

As a minor comment, in the line 116: the world “eliminated” should be replaced by “removed”.

3)	As this work is based in imaging analysis, it is important in the section of imaging and analysis to specify the camera and the settings that were used for taking the images. The specifications of the objective lens used are also important (for example, numerical aperture).

4)	Minor comment. In the section of statistics, please state if when applying parametric tests, the assumptions of normality and homogeneity of variances were evaluated, and if any transformation of data was required.
Validity of the findings: The data is robust, statistically sound and controlled.

Some minor changes would improve the quality of the work.

5)	Figure 4 a. The axis Fertilization stage is misleading because the graph is including different stages of maturation in the epididymis together with spermatozoa recovered from the female reproductive tract.

6)	As a minor comment, in line 218 would be useful to disclose the fertilization rates of BDF1 and B6N cauda epididymis-isolated spermatozoa obtained in your laboratory.
Additional comments: The paper entitled “Mouse spermatozoa with higher fertilization rates have thinner nuclei” proposes a new way of morphometric and statistical analysis of mouse sperm heads as fertility indicator. The work presented combines imaging analysis with a multivariate statistics method that is intended to develop a new way of classifying sperm and determine fertility potential.
The data presented is good however the results section flow was not the best. The text guides the reader to start from supplementary figure 2 , then jumps to Figure 1 A, jumps again to supplementary figure 3, before going back to Figure1B and then continues with all the supplemental information before describing figure 2. I suggest to re-evaluate which information to include in the main figures and which one in the supplementary materials, in order to make the reading and understanding easier for the readers.
Finally, it is important to remark that the manuscript shows that the Principal Components Analysis was useful to identify the main variables that represent the morphological variability of the sperm head nucleus: width, and the hook shape of the tip.


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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors took care to answer to my questions and make this revised version of the manuscript easier to follow. I apologize for not finding the supplementary figure legends in the first round of revision.


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

Input:
Title: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed all comments from reviewers.
Experimental design: No Comment.
Validity of the findings: No comment.
Additional comments: The authors have addressed all 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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have carefully addressed the issues that were pointed out by this reviewer. The revised version of the manuscript is improved and no further corrections are needed.
Experimental design: No further comments.
Validity of the findings: No further comments
Additional comments: No further comments


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

Input:
Title: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript was well and clearly written (with very little grammatical errors), and was backed up by relevant literature references
Experimental design: Experimental design was suitable and robust for the research objectives spelt out.
Validity of the findings: Results and findings obtained were substantiated by raw data, and conclusions drawn were in accordance with the objectives outlined. However, results on biochemical characterization of the purified CbhB should also be highlighted in the conclusion as an answer to one of the objectives set out (line 115).
Additional comments: 1. The English language can be improved, especially for lines 127 (growth of the yeast were performed), line 246 (incongruent sentence), line 273, 279, 296, 300, line 336- mentioned, not mention, line 353- Insensitivity of CbhB rather than CbhB insensitivity, line 370, line 379, line 382, line 384,
2.  Please include a close bracket after mL-1, in line 144
3. line190: sugar produced, not product
4. Line 230- state the equipment used
5. Line 346- use of "of course" not quite appropriate
6. Line 353- was statistical test carried out? Cu and Ba seemed to affect at 10mM concentrations, as seen by the reduction of relative activity to less than 80%.
7. line 358- Gurgu and Barby, not Gurgu et al.
8. line 367- rephrase. The loadings were optimised, not the hydrolysis.
9. line 386- was, not is.  Also line 391, 396
10. line 390. Use primary reference
11. line 416. please add a qualifier
12. References: please spell out all the authors's names to replace the et al. (lines 441, 466, 475, 481, 524
13. line 521: italicise genus and species names
14. Table 1, line 2- specific activity,  not spesific
15. Table 2: table should be self-explanatory. Please write the names of the substrates in full in the notes below the table
16. Figure 4, Lane 2. IMAC- purified CbhB deglycosylated using PNGaseF, rather than  deglycosylated IMAC- purified CbhB using PNGaseF
17. Figure 8. Range of Cellic CTec2 in the title was 1.5-30%, but in the actual figure was 5.50 -25%


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

Input:
Title: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: None
Experimental design: The procedures were efficient to produce and purify an Aspergilllus niger CBH in P. pastoris. Produced enzyme was properly evaluated and used as an additional enzyme during hydrolysis of a model substrate hydrolysis (pretreated oil palm empty bunch). Hydrolysis intervals in this substrate were shorter than usual (5h), but results were adequate for evaluating the recombinant enzyme mixed with commercial CellicCtec.

Experimental design was adequate
Validity of the findings: Experimental data are well interpreted and discussed
Additional comments: The work is a general contribution to the area related to enzymes used in biomass conversion. Production and purification of a recombinant CBH was developed.
Over glycosylation of the recombinant enzyme could be one of the causes of non-action on complex and insoluble substrates such as crystalline cellulose.

In general, the results are sound and merit publication


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

Input:
Title: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Woon et al., describes the heterologous expression and characterization of a CBH from A.niger, as well as its ability to increase the saccharification yields of oil palm empty fruit bunches when added to a commercial preparation.

The manuscript is well-written and literature references and scientific background are provided.
Experimental design: This study is within the Aims and Scope of the journal.

In general, the experiments are described with sufficient detail, were performed correctly and the conclusions follow the results.

Some parts of the experiments and results needs more clarifications, such as the cloning of the cbh2 gene (PCR amplification, primers and polymerase used) and the experimental design by response surface methodology (enzyme supplementation, choice of upper and lower limits).
Validity of the findings: In general, conclusions are well stated and supported.

There are some parts that need clarification, such as the low recovery yields of CBH2 and the imprortance of ATR-FTIR data on evaluation of CellicCtec2 supplementation with CBH2 and the changes on substrate morphology.
Additional comments: - Lines 62-63: Keywords need to be more specific and representative of this work, for example bioethanol and lignin are not within the scope of this study.
- Line 81: “Aspergillus niger is a filamentous fungus well-known for its ability to produce copious amount of lignocellulose degrading enzymes”: this paper by Zoglowek et al., 2015 mainly describes how A.niger has been used as a host for heterologous production of CBHs and other cellulases.
- Line 89: T.reesei has low extracellular β-gucosidase activity, but this fungus expresses many intracellular and cell-bound CBHs.
- Lines 90-92: Supplementation is also used because different types of biomass have different properties, therefore they require a set of enzymes that has to be tailor-made. For example, endoglucanases are needed for amorphous substrates, whereas CBH supplementation is preferred for substrates with high crystallinity.
- Lines 93-95: cellulases consortium has been reported much earlier than 2016, so another reference is suggested. The same for line 103, for CBHs acting on reducing and non-reducing cellulose chain end.
- Line 106-107: Apart from glycosylation, there are other post-translational modifications that affect the enzyme performance and activity, ex. N-terminal pyroglutamate fo. rmation that is commonly observed in crystal structures of Cel7A enzymes and is related to enzyme stability, is not possible to occur in the Pichia pastoris expression system. This makes the recombinant enzymes less active and less stable than their native counterparts (Biotechnol. Bioeng. 2014;111: 842–847).
- Line 130-131: A full description of the cloning procedure is missing, i.e. information about the gene (exons, introns), the primers that were used and the PCR conditions, as well as the polymerase used.  This part is also missing from the Results and Discussion Section (Line 262-263), where cbh gene should be analyzed for glycosylation sites, signal peptide and homology with characterized CBHs and an accession number for Genbank should be given. There is some information on Lines 295-305, but this part should be re-written again carefully. Line 302, “molecular mass of CbhB to ~ 70 to 100 kDa”: it is not clear what authors mean.
- Line 170-172: After the digestion with PNGaseF, how was the extent of N-glycosylation estimated? Did authors perform an SDS to compare the molecular weight of the enzyme before and after the deglycosylation? If so, this should be mentioned here.
- Lines 197-198: Did authors checked the stability/autohydrolysis of MUF at high temperatures, such as 70-80οC? Did they run control reactions without the addition of enzyme?
- Line 206: What was the CBH concentration that was used for calculating the kinetic parameters? How was the end product inhibition estimated?
- Line 217: Authors should choose another title, more specific than “complementary effects”. Same for Line 366.
- Line 217-220: When a monoenzyme (for example a CBH) is added to a commercial mixture, then there are two types: one is when the enzyme loading of the commercial preparation remains the same and CBH is added, so the final enzyme loading increases (this is called addition/ supplementation), and the other one is when one part of the commercial preparation is replaced by the CBH and the final enzyme loading is constant (this is called replacement/complementation). It should be mentioned which type is being used here. 0
- Line 226: Did authors check the composition of the material (glucose, xylose, lignin) before and after the pretreatment?
- Line 233-235: Apart from %TRS rate, the %hydrolysis/saccharification rate could be also estimated by taking into account the glucose and/or xylose content of OPEFB according to the analysis of solid fiber fraction from the following equation (WIREs Energy Environ. 2013; 2, 633-654):
% hydrolysis rate=[TRS(mg/mL)/DM(mg/mL)*glucose content*1.111] *100
Did authors check glucose or xylose yields after enzymatic reactions?
- Line 245-247: How did the authors chose the limits of the enzyme and Cellic®CTec2 relative abundances? In general, it should be avoided not only working within a wide domain, as this may impact the reliability of predictions, but also limiting in a narrow domain, since extrapolation outside the borders is impossible. The lower and upper limits of each component should be chosen by combining data from the literature with rational consideration.
- Line 284-285 and Table 2 data: it is obvious that the enzyme is quite unstable and this can be attributed to many reasons, including the absence of post-translational modifications, such as the pyroglutamate formation ect. CBH is degraded and gradually loses its His-tag and this is the reason why it is not possible to be purified by IMAC and the purification fold decreases. Authors should give an explanation. Did they perform an SDS during the cultivation in order to check whether the molecular weight of the protein remains stable?  What is there on the lowest part of the SDS-PAGE shown at Figure 2 (it is cut and not shown)?
- In order to check whether the addition of CBH reduced the substrate crystallinity or not, study of the X-ray diffraction spectra could give more accurate results than ATR-FTIR.
- According to gene/protein nomenclature, genes are denoted by three italicized lower case letters, while Protein names are the same as the gene names, but the protein names are not italicized, and the first letter is upper-case (see "Guidelines for Formatting Gene and Protein Names", http://www.biosciencewriters.com/Guidelines-for-Formatting-Gene-and-Protein-Names.aspx), so it should be cbhB gene and CBHB protein.
- The authors should also check carefully for references' format, as well as typing/editing and punctuation errors in the text and in the figure captions. All organism names should be written in italics.


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: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comments
Additional comments: The manuscript was greatly imporoved after revision and now is accepted for publication in 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: NESTS OF RED WOOD ANTS (FORMICA RUFA-GROUP) ARE POSITIVELY ASSOCIATED WITH TECTONIC FAULTS: A DOUBLE-BLIND TEST
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have conducted a double-blind test to test the hypothesis that red wood ants occur in proximity to active tectonic faults. Former publications of some of the co-authors have shown this already. I have to admit – I was not aware that this hypothesis or the published results were criticized by the myrmecologist community. In this case I welcome this study with its rigorous experimental approach if it helps to corroborate former results in an unbiased way.
Experimental design: A potential weakness of the study is the polydomous colony structure of one of the ant species studied here (see comment 1). I have two points that I think the authors should clarify in this otherwise well-performed and well-presented study.
1. The authors state that the RWA nests were spatially clustered. Especially for F. polyctena this is no surprise as this ant is known to be highly polydomous (and polygynous) (e.g. in Seifert 2007 or the edited book on RWA ecology and conservation by Stockan and Robinson). Thus, the clustering of nests can to my mind be considered a pseudoreplication as nests are (at least in part) not independent of each other. What are the results for F. rufa alone in comparison to F. polyctena? Here this problem should not be as pronounced. Are the results comparable? Could you potentially do the analysis with the grid cells themselves (RWA present or absent)?
2. One point that is to my mind not made clear enough in the methods section is how random points in the same region were generated for comparison. Please add one or two sentences.
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: NESTS OF RED WOOD ANTS (FORMICA RUFA-GROUP) ARE POSITIVELY ASSOCIATED WITH TECTONIC FAULTS: A DOUBLE-BLIND TEST
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Line 20-22: How conducted study in this manuscript does help us to apply (design) double-blind ecological studies in “distribution” and “abundance” studies. As far as I understand the presented study is not related to any of these groups in their classical definition, at least. An example in the discussion/conclusion can help to understand the point authors try to address.
Validity of the findings: Line 94-100: How do authors deal with spatial autocorrelation in this analyze.

Line 125-149: I miss any discussion/conclusion on why ant nests are significantly closer to faults?
Additional comments: The manuscript has overall been written well. It seems authors have not been able to decide if the main topic is testing double-blind approach in the Ecology or an ecological experiment on relationship of ant nests and tectonic faults. Authors can the re-organize the structure in a new way that readers can follow the paper better.


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: PULMONARY TRANSCRIPTOMIC RESPONSES INDICATE A DUAL ROLE OF INFLAMMATION IN PNEUMONIA DEVELOPMENT AND VIRAL CLEARANCE DURING 2009 PANDEMIC INFLUENZA INFECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a descriptive study examining the transcriptome changes that occur during a mild H1N1 2009 influenza infection in mice.  The manuscript is coherently written and laid out in a logical and cogent manner.  However there are several weaknesses to the manuscript that reduce enthusiasm for acceptance.

1)  Although the authors claim no research has been done to examine the  non-fatal 2009 flu model, this is untrue.  There have been multiple studies performed on H1N1 2009 (Itoh et al Nature 460, 1021-1025,  Rowe et al 2010 Virology. Jun 5; 401(2): 257–265 ). to name a few.  These should be referenced and more emphasis should be placed on what the purpose of this study is and how it differs from the other studies (CA2009 vs Cat2009 etc).

2)  There needs to be more thorough editing performed on the paper, for instance:
  line 51-  "this severe" should be changed to "these severe"
  line 56 "severe patients infected" should read "severely infected patients"
  line 64-  "previously put in relationship" would read better as "previously correlated with"
Experimental design: The experimental procedures seem well defined and the authors demonstrate a previously undescribed mouse influenza model.  The numbers of mice seem appropriate and the statistical methods are fine.  There is a little confusion in the materials and methods section that need to be clarified as well as some figures that need improvement.

1)  Figure 2 demonstrates severe pathology at day 1 and 5.  It is unclear what the differences are between day 1 and 5 though.  This figure would greatly benefit from histopathological scoring.  It is stated in the results section, but as the figure stands, it is showing very severe pathology at day 1 and not explaining that this was only from 2 of the mice.

2)  it is confusing why they state "data not shown" on line 102 yet, they seem to have shown the data.
3)  line 128 "the to"  should be changed.
line 135:  the authors say "as described elsewhere"  that is fine, but give a citation showing where.
4)  Line 176:  Change "significantly higher body weight loss" to significantly greater weight loss"
5)  line 182:  "one day after mice infection" remove the word mice
6)  line 183:  Please use scientific notation for the standard deviation.  same for line 184.
7)  Line 225:  change "induced also" to "also induced".
Validity of the findings: 1)  Since the 2009 H1N1 outbreak there has been extensive studies into this viral strain and the effects in multiple models (in vitro, mouse, ferret, pig) as well as in patients themselves.  The discussion would greatly benefit from a better explanation of how this study ties in to the already abundant data that is out there.

2)  The authors use a human isolate and it is unclear whether it has been passaged in mice already.  The discussion would benefit with a comparison of well characterized fatal (PR8 or multiple mouse passaged 2009) infections.

3)  It is unclear why the authors focused solely on day 5 when they have data from 3 different time points.  The manuscript would benefit in a comparison of the changes from day 1, to day 5 to day 10.
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: PULMONARY TRANSCRIPTOMIC RESPONSES INDICATE A DUAL ROLE OF INFLAMMATION IN PNEUMONIA DEVELOPMENT AND VIRAL CLEARANCE DURING 2009 PANDEMIC INFLUENZA INFECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is reported in clear and unambiguous manner. There are some grammatical errors and I hope I have highlighted them all in my review.

There is an exhaustive list of references and an acknowledgement of an article (Pommerenke et al., 2012), which is quite similar in some aspects. The cited literature provides a good background and context to the study.

Figures and tables are provided in the main text and as supplementary data. There are clear and well structured. In the legend of Figure 1, statistics are presented in part B, but there is no asterisk on the viral load graph (B). I would recommend that statistics be presented out with the A and B description.

In figure 2 would be it possible to use arrows to indicate the infiltrate in the viral infected lungs

Figure 3 is a clear representation of differences in GEP, it would be beneficial to relate this figure to a table of genes that are a different within the figure legend. In this way it would be easier for reader to follow the process.

Does Table 1 represent only genes that are unregulated? could examples of down regulated genes also be included?
Experimental design: This study represents primary research within the aims and scope of the journal. The research question is clear, but I would record highlighting how it advances knowledge in this area and expand on lines 239 and 240 to explain what this study offers.

It is good to see microarray data accompanied by histological data. Methods and ethics are clearly described.
Validity of the findings: Data is robust and in line with previous literature.

Conclusion is well stated and linked to the research question.
Additional comments: This study is clear, easy to read and a good example of the importance of microarray data to illustrate a global view of immunological responses in a specific tissue.

I would recommend clarifying some of the figure legends to ensure that figures are fully explained in regards to the complexity of microarray data. For example in figure 3, I understand that the GEP profile is changing, but my immediate question is, what is changing? Can the figure 3 legend be connected to table 1 or supplementary table? Can table 1 be coloured to represent how it refers to figure 3 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: PULMONARY TRANSCRIPTOMIC RESPONSES INDICATE A DUAL ROLE OF INFLAMMATION IN PNEUMONIA DEVELOPMENT AND VIRAL CLEARANCE DURING 2009 PANDEMIC INFLUENZA INFECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is vastly improved from the last version and the authors were very responsive to the critiques.  The discussion would benefit from a more thorough editing for proper english.
Experimental design: the experimental design is well defined and meaningful.
Validity of the findings: the data is robust and well stated.
Additional comments: No additional comments


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

Input:
Title: THE EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: All good
Experimental design: All good, but see suggestions in general comments
Validity of the findings: All good, but see suggestions in general comments
Additional comments: Overall this is a good study. My only issue is that the expectations are a little naive. There is every reason to suspect that the magnitude of response is largely due to inherent properties of the fish (size, size of the mauthner neuron etc) thus I fail to see any reason why the authors would expect to see familiarity effects on these traits.   It makes sense for other parts of the avoidance strategy (eg time to first detection, reaction distance etc) as these are modified by social interactions. With this in mind I would alter the introduction a little.
Secondly it is hardly surprising that size swamps all other aspects of the escape mechanics. I would use some kind of residual to remove size effects completely. It’s unlikely you’ll find anything, but worth a try. Be sure to show us that familiar and unfamiliar groups do not differ in size.
*Note I have marked the pdf for editorial suggestions

Specific comments:
L63: familiarity has other benefits as well, eg enhanced social learning (swaney et al 2001).
L108: I can understand the reasoning behind the idea that familiar groups might respond in greater synchrony, perhaps even respond more quickly at longer distance, but I cant imagine how it would effect things like the magnitude of the response. This is largely innate and controlled by the mauthner neuron.
You might even argue that familiar fish feel safer in their shoals and therefore respond later. Hard to say.
L115: You need to tell us how long ago. Anti-predator behaviour declines rapidly in captivity.
L143: what did you do with the fish afterwards? Did you put them back in the home tanks? Reuse them? Was each fish only tested once?  We need these details here.
L216: when it comes down to it the Ns are pretty low
L231: where length is a covariate it will be important to show that familiar and unfamiliar groups did not differ in length.  Note you really need to remove length. Perhaps use residuals.
L247: If you ask me this is not unexpected at all. See reasons stated above.
L273: The authors might like to read Brown 2002 (Journal of Ethology 20, 89-94)


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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: In investigating the role of familiarity in influencing group and individual level anti-predator responses the authors seek to understand what details of responses may be influenced by group composition. Overall the manuscript is clearly written and their justification for their study is well supported. However there are two areas in particular where rewording could help better reflect the study they conducted and results they found.

1) In the abstract the authors state that “Using the Trinidadian guppy we examine the effect of different early social conditions in the three main components of predator evasion”, implying that they manipulate early life experience. Instead the paper manipulates recent social experience (i.e. the two weeks prior to the experiments; lines 136-137), which is a different line of study. I would therefore suggest the authors revisit all mention of “early social experience”.

2) In the discussion, the authors state quite strongly that they “unambiguously demonstrated that familiarity plays a significant role in shaping how groups of fish respond to a stimulus”. Since familiarity only predicted the number of individuals that responded, but did not predict several of the other variables they analyzed, this tone appears unwarranted. The statement they include later on in the conclusion, namely that “In combination, our study suggests that, while familiarity affects how groups respond to a visual stimulus, it plays a less meaningful role in determining the quality of the escape response” is a much more measured and appropriate summation of the study. I would suggest that they revisit the tone in the discussion, especially since PeerJ does not require results to be “flashy”, and thus there is no reason for overstating their case.

Finally, and on a more minor note, the (Strodl & Schausberger 2012) paper that they cite does not investigate responses to predators, and I think it may have been cited in error (e.g. line 284, 293). The authors of the paper in question wrote other papers that year which the authors of this ms may have instead intended to cite. I would suggest they double check all references they list.
Experimental design: While most of the experimental design was easy to understand (Figure 1 helped especially), there are a few minor areas where more detail would be helpful.

Line 128-129: Without knowing the origin of the stock in the different tanks, it is hard to assess whether the individuals were related. A little more detail here, such as how long the stock had been separated and from where it was originally sourced would be useful.

Additionally, it would be nice to know if the source and experimental tanks were featureless or did individuals have access to cover? (which could influence anti-predator behavior)

Finally, were the size differences between individuals (one big, one medium, one small) also maintained in the unfamiliar trials?
Validity of the findings: Since the authors only looked at the details of the anti-predator behavior in the first responding fish of any group (as stated lines 189-190), they were potentially sampling a non-random individual from the group. Given that the size of the individual, as opposed to the familiarity of the group, appeared to predict much of their response behavior, it would be very informative to know whether the size of the first responding individual was similar across the familiar and unfamiliar groups. I would suggest that the authors consider this addition to their analysis because it could mask the effects of familiarity on anti-predator behavior. If there are differences between the groups in the size of the first responding individual, then the authors need to consider analyzing the behavior of other group members.
Additional comments: (see comments in previous three sections)


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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clearly written, but the overall presentation can be improved in quite a few places, either by rewording or shortening of the prose. Below, I've made specific suggestions to the author. The background and context of the study, as laid out in the introduction, are strong and well-supported by the literature cited. Structure is fine and overall the results speak to the hypothesis (predictions). I believe the authors have generally met the basic reporting criteria.
Experimental design: The design is good, but there are analysis issues that need to be addressed. Specifics outlined in comments to author
Validity of the findings: no comment
Additional comments: Analysis. I would strongly suggest choosing to use either a strict hypothesis-testing approach or a modeling approach, but not both.  My inclination would be the former, because you have set your study up as a test of a hypothesis (predictions). Also, you have relatively few parameters and a relatively small sample size (adequate, but small for this kind of analysis).

If you choose the AIC approach, I would recommend calculating a AICc for small sample size. I would also provide the variance inflation factor, c, calculated as X2/df, based on the full model, not the reduced models. Check Burnham and Anderson.

Make clear on line 201 that you are using a generalized linear model framework..  Yours is a fixed effects model, so although obvious, spell it out for the reader. Also, need to include the link function used, which I assume was a normal log link, but again, need to make these particulars clear.

I believe you may have a mistake in calculating K. If your analysis (generalized linear models) used the log likelihood method of model selection, which I’m guessing it did, then K = no of explanatory variables, + intercept + variance.

Finally, with the AIC approach you would not be reporting p-values, except possibly for the Wilcoxon test (line 198).

Table 1. treatment + length should be “treatment x length” since it is an interaction. The order of model effects (factors) column seems unconventional to me. I would check this against other published tables. Possibly start with the full model that contains all of the factors, including the interaction, then all remaining combinations thereof, putting them in increasing order of the ΔAIC. You could choose to report just those with an AIC < 4.0. That would be one way to do it.

Results/Discussion. You might consider the effect of group size in your discussion. It would have been useful, in hind sight, to test for this, because group size could interact with familiarity. Contrary to your predictions, groups composed of familiar individuals were less responsive than groups of unfamiliar individuals, suggesting that groups size may be an important factor. Group size could combine with safety in numbers, so two curves of response plotted against group size may start the same (n = 1), separate around 3 or 4 ( your result) but then converge at higher groups, like around 8 with very low responses. Do you think this is possible? If so, perhaps work it into the abstract in place of 31-32?

Additional comments
Abstract.
19  please! driver = cause
19-20: Suggestion: Traits that contribute to the avoidance/amelioration of potential threats should be under strong selection during these stages, along with the process of familiarization.
22: “Less, however, is known” suggest “Yet, gaps in our knowledge remain”.
27: suggest: …we compared the number of individuals in each test group that responded to an artificial stimulus, their reactive distance and magnitude of their response (acceleration and distance) in groups composed of…”
30-32. Consider revising, perhaps bringing in the idea of group size, assuming you think it could be playing a role here.
34.  suggest: Our experimental approach revealed specific aspects of the escape response that are more likely to be influenced by variable(?) early social conditions.
53 for = to
57 between individuals within a group
64 Ward and Hart (2003) found….
66 Groups composed of…individuals have been found to be more…
73 “will have an idea” = “may remember”
80 take out “fairly”
81 furthermore = for example
91 “…responses. We used a tropical fish, the Trinidad guppy ( ), because its behavioral ecology and predatory regimes are well known (Magurran 2005).”   (This is a suggestion, and you might be able to write it better, but I would try to come up with an explanation along these lines)
105 hypothesis = prediction
127 size?
134 I would take out these sorts of personal observations unless you are prepared to provide some data. A critical reader might be skeptical that really observed this.
139-143 It took me a while to figure out that each test group has 3 fish. You should state here in the methods, clearly, so that the reader has it in mind going forward.
154-155. Again, I don’t think you need to say this. You already have a very artificial set up and the reader knows and appreciates that you are not necessarily trying to exactly duplicate nature. That’s not your point.
162. Take out “gently”. You didn’t do it “roughly”, right?
163. Take out “so no …required”
164  standard
166 escape
166 and = or
167 insert “were”
256 “, escaping may not always be the best strategy” As written, this doesn’t make sense to me if the alternative is not to escape and be eaten. How about writing “responses may be highly variable”?
283-284. I suggest shortening the Discussion, and statements like this could easily come out.
296-307: Good. Keep this paragraph!

Overall, Yours is a nice contribution that will be of interest to the many researchers who are studying predator avoidance behavior.. I believe that if you go with the hypothesis testing approach, deleting the AIC material, you will have a successful paper. I would also recommend shortening the Discussion, and possibly even the Introduction. It could be tighten a bit too.


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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have satisfactorily addressed all of the issues that I raised in my previous review. They agreed with my comments in almost all cases with the exceptions being only suggestions, and in those cases I understand their reasoning.

Their combination of the hypothesis-testing with information theoretic approaches was a mistake in my view and so I was very relieved to see that they had agreed and omitted the latter.

I read the other reviews, which also raised important questions, and again, it appears to me that the authors have addressed them, although it is possible that the reviewers may not agree.
Experimental design: I believe that the article meets the PeerJ standards with respect to experimental design.
Validity of the findings: no additional comments
Additional comments: No additional comments