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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper reports expression patterns of mouse and zebra fish a-crystallin promoter constructs in zebra fish embryos and examines native gene and protein expression using PCR and mass spec. There is some nice data here. The GFP reporter results are clear. They confirm that crystallin promoters can work across species, as has long been known, and also define functional fragments of the zebra fish promoters. Mass spec detected aA and aBa peptides and provided evidence for eye/non-eye regulation of aBa in development. aBb was not detected.

Unfortunately the PCR results are not so convincing. Fig 2, shows aA expression throughout development quite well. Fig 5, however, has poor image quality and, unlike tubulin, the aBa,b primers seem to be generating smaller non-specific bands. These might be solvent front, but the aA and tubulin gels are clear in this region. Were these products confirmed by sequencing? Expression in adult is obvious, but the inconsistent results between panels A and B for embryonic expression are hard to explain. Both show strong bands on single developmental days, followed by almost nothing, which by itself seems biologically unlikely. The difference in timing in different samples is also strange and looks artefactual. I think it would help if this figure was repeated using different primers. The lack of detectable peptides for aBb also raises questions about the PCR. As for the control genes, is tubulin really a good control? Might tubulin be developmentally regulated?

Although the authors cite the Hou et al 2006 paper on expression of the human bB1promoter in zebra fish in the introduction, they don’t compare their results in the Discussion. Some mention seems appropriate.

Minor points:
Line 32: Specify chaperone activity refers to a-crystallin.
Line 102: aB? Seems to have symbol character.
Line 398: Specify a-crystallin.
Figs 1,2,3: In legends – state ages of embryos for GFP expression.
Experimental design: Experimental design is fine in general, but the paper is let down by the execution of the PCR.
Validity of the findings: No further comment
Additional comments: No additional comments


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

Input:
Title: THE ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, M. Posner and coworkers conducted a series of gene reporter, expression, and proteomic studies to determine expression of zebrafish and mouse -crystallin genes in zebrafish. The A- and B-crystallins are key structural proteins of vertebrate lenses. Although some insights into transcriptional control of these genes are known for mammalian crystallins, understanding of their temporal and spatial control both in and outside of the lens requires additional studies. Zebrafish is an excellent cost effective experimental model to accelerate these studies. The findings are novel and address a number of questions in the field. The present studies thus fill a critical gap in our understanding of lens differentiation and crystalline gene expression.

The manuscript is well-written and within the scope of PeerJ. The data are of excellent technical quality. There are several minor issues to be addressed in the revision:
1)	Introduction: Lens development in zebrafish and mouse differs as the mouse lens placode invaginates into the lens vesicle, prior the formation of lens fibers. Lens fiber cells in zebrafish are generated through the delamination from lens placode. This should be mentioned as both zebrafish and mouse models are discussed.
2)	Methods: the coordinates of DNAs used to generate eGFP reporters should be included.
3)	The paper by Zou et al. 2015, Exp. Eye Rees. 138:104-13 should be quoted here.
Experimental design: Some technical details regarding the DNAs have to be addressed (see above).
Validity of the findings: Addition of supplemental files including three video is highly valuable.
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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present an excellent review of the literature. Clear and well written manuscript.
Experimental design: 1. The overall purpose is unclear. Why functionally assess mouse lens promoters in zebrafish? And how would the authors determine whether negative expression in fish generated from a potentially new mouse lens enhancer would be due to species differences or merely that the enhancer was not a true enhancer in mice either?

See Intro: “This conservation suggests that mammalian a-crystallin promoters could be functionally assessed in the zebrafish, providing a faster and less expensive system than traditional mouse transgenic approaches.”

Furthermore, the authors spend a lot of effort to simply confirm that EXISTING, KNOWN regions of the mouse gene promoter function in the fish. What new is gained here, since these regions were already known to control expression in the mouse as such?
See Results: “Our results indicate that the mouse aB-crystallin promoter drives GFP expression in zebrafish embryos, and that the resulting spatial patterns reflect the functional regions first identified in mouse.”

2. The authors attempt to use promoter driven GFP to assess endogenous onset of gene expression. If an enhancer or modifier is missing in their cloning, then their entire conclusions are in error. The characterization of the developmental patterns produced by the promoters should be perform by analyzing the expression of the endogenous products.

See Results: “However, no study has characterized developmental patterns produced by the two respective promoter regions. We produced a GFP-linked 3 kb fragment of the zebrafish aBa-crystallin promoter and a series of GFP-linked fragments… We found no difference between the timing of onset for any of these zebrafish promoters… Overall these data suggest that the divergent expression of the two zebrafish aB-crystallin promoters…”

They then follow this up with RT-PCR and show that it is essentially present at all timepoints analyzed (no timepoints prior to 1 dpf were analyzed). Since this wasn’t qPCR, assessment of abundance should not be given. I am not sure that the following statement in the Discussion is valid.

See Discussion: “Lastly, by examining promoter activity, mRNA expression and protein abundance for zebrafish aBa-crystallin we resolve remaining questions about the timing of expression of this gene.”
Validity of the findings: Data are robust and controlled. See point 2 above regarding experimental design issues leading to over interpretation of findings.
Additional comments: Annotation of Figure 5 would be appreciated. Legend for colors. A-B: aA, C-D: aB.


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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the manuscript is much improved. Indeed the authors have made several important corrections.
I only noticed two minor points:

I would drop the last sentence of the Abstract. It is speculative and doesn’t relate to these results.
Line 144 in the PDF has a square symbol character that probably should be a roman B.
Experimental design: No further comment
Validity of the findings: No further comment
Additional comments: No additional comments


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

Input:
Title: THE ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Satisfactory.
Experimental design: Meet standards.
Validity of the findings: No comment.
Additional comments: The authors have address all main minor points raised in the earlier review.


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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION
Review round: 2
Reviewer: 3

Output:
Basic reporting: No problems here. Clear and well written manuscript.
Experimental design: 1. The authors have done a better job addressing the significance of the research to the field.
2. The authors attempt have resolved the issue with qPCR.
Validity of the findings: Data are robust and controlled.
Additional comments: A genuine and sincere effort was made to improve the manuscript.


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

Input:
Title: THE RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Strong. See below for some points to consider.
Experimental design: Strong. See below for some points to consider.
Validity of the findings: Revisions are needed to bring the conclusions in line with the results.
Additional comments: This is an interesting paper that presents a novel meta-analysis of whether egg size correlates with helper number in cooperative breeders. This relationship would be predicted if, for example, mothers plastically adjusted their egg size according to helper numbers. This is an issue of broad current interest in the cooperative breeding and maternal investment literature. While the study struggles with sample size limitations it does provide a novel contribution to knowledge in this area. Revisions could be usefully made, however, to better align the overall conclusions with the findings, increase transparency regarding the use of repeated measures of species, clarify a few points, and provide some more inclusive coverage of the relevant literature. I cover these points below.

KEY POINTS

1. Greater caution is needed with interpretation, as the meta-analytical findings do not currently provide direct evidence of maternal plasticity or helper effects.

Evidence that egg size varies with helper number does not necessarily reveal (i) that mothers actually show plasticity in egg size (as the observed variation in egg size could arise from among- rather than within-female variation in egg size, and while some studies considered this many did not), or (ii) that this variation, whether plastic or not, is causally attributable to variation in helper number (as there could well be confounds of helper number; e.g. territory quality).

The manuscript would therefore benefit from…
- Acknowledgement of both points in the discussion
- Attention to the potential alternative explanations for the findings that recognition of these points highlights
- Adjustments to the phrasing of arguments and conclusions throughout the manuscript regarding plasticity and effects of helper number (including the abstract and title, see point 2).

2. The title needs to be revised to more acurately reflect the findings.

The title “Cooperatively breeding mothers reduce their investment in eggs in larger groups: a meta-analysis across species” currently seems misleading for two reasons:
(i) the statistical evidence for such a correlation across all species falls shy of the significance threshold once repeated measures of species are controlled
(ii) the meta-analysis is not based exclusively on within-female relationships between helper number and egg size (and so the meta-analytical result could be a product principally of among-female variation in egg size rather than within-female plasticity; see point 1).

A more general title based on the question rather than the answer would resolve this easily. E.g. “Investigating egg size variation with helper number in cooperative breeders: a meta-analysis across species”

3. Please clarify the classification of species.
In Supp Table 1, it is not currently clear why, when using the data for Malurus elegans from Lejeune et al 2016, the species has been classified as showing compensatory care for the within-mother effect sizes but additive care for the among-mother effect sizes. Surely the species either shows compensatory or additive care? Indeed, why use among-female effect sizes at all where within-female effect sizes are available, if the goal is to study plasticity? Please clarify / resolve.

4. Please adjust the figures to increase transparency regarding the repeated measures of species.
In the funnel plots for Figures 2 and 3 it would be great if the authors could place a number next to each data point indicating which species it comes from (there aren’t many so this should work well), and then have the numbered list of species in the figure legend. This will allow the reader to consider the extent to which any apparent pattern is influenced by the inclusion of repeated measures of species.

SPECIFIC POINTS

INTRODUCTION

5. It would be good to see the introduction better address two relevant areas of the literature:
(i) The introduction currently skates over the rather intensive research effort to date on load-lightening of post-natal maternal investment in cooperative breeders. Given the parallels with the questions asked here (re the impact on pre-natal maternal investment) it would be good to more fully explain the advances in this area to date (in particular the relevant insights from the comparative work in Hatchwell 1999 – currently only cited in passing).
(ii) It also seems odd to cite virtually none of the empirical literature addressing maternal plasticity in egg size in cooperative breeders, when this is the focus of the manuscript.

6. Lines 78-81 – please clarify the argument here

7. Lines 102-105 - It seems odd to justify the study’s focus on pre-natal rather than post-natal maternal investment on this basis when …
- Its not clear why maternal investment in egg size “cannot be directly influenced by helpers”, as helpers do have the potential to impact maternal condition at laying (e.g. via effects of group size on vigilance/foraging trade-offs etc.), and
- Presumably the main reason for focussing a meta-analysis on pre-natal investment in the egg is novelty (much work has already been done on post-natal maternal load-lightening)?

8. Lines 105-109 and in the paper’s Summary
Please clarify the definition of ‘additive’ and ‘compensatory’ care. As set up by Hatchwell 1999, doesn’t ‘additive care’ refer to a scenario where the mother does not compensate for the contributions of helpers (and ‘compensatory care’ refers to a scenario where she does)? The wording in this manuscript suggests that the distinction between additive and compensatory care here is made on the basis of whether the helpers (rather than the mother) compensate for each others’ contributions. This is subtly different, and presumably has implications for both the logic and the classification of species. Please clarify.

9. Lines 196-197 – please remove ‘broad taxonomic spread of species (fish to birds) in our sample’ from this argument or rephrase, as this doesn't really help to justify not controlling for phylogeny (there is only 1 fish).

METHODS

10. Lines 119-122 – The definition of ‘cooperative breeder’ needs a clearer definition of ‘helper’. The inclusion of dunnocks (presumably polyandrous?) in the analysis, for example, suggests that the authors are comfortable with including species in which the helpers are actually breeders. What then is a ‘helper’? To increase transparency and clarify the logic it would also be good to explicitly acknowledge here and (briefly) justify the inclusion of cooperatively polyandrous species.

11. Conducting a statistical test for a difference between species with additive and compensatory care strategies seems to me to be pushing the data too far - there are data for only 3 species with compensatory care (perhaps 2 depending on the answer to point 3 above), so such a comparison at present seems pretty meaningless and potentially misleading. Wouldn’t it be more appropriate to simply plot the current figures and discuss the patterns by inspection? It would still be good to follow this up with an analysis of the overall data set restricted to species with additive care only (as there are at least 8+ species of this kind), which yields an interesting result, but then also conducting this analysis on the (just 2 or 3) compensatory species alone also seems to be pushing the analysis too far.

RESULTS

12. To enhance transparency regarding the data structure throughout, whenever quoting sample sizes it would be good to break down the sample size in to the number of effect sizes and the number of species. So rather than reporting “n = 20” report “n = 20 effect sizes from 10 species”.

DISCUSSION

13. Line 290 – rephrase – this isn’t ‘demonstrated’.

14. lines 291-293 – it seems a little late to acknowledge this critical point. To maximise transparency it would be good to include this point in the 1st paragraph of the discussion where the findings are summarised.

15. The final paragraph of the discussion goes a little hyperbolic – please bring the claims in line with the findings (greater caution needed throughout).
e.g. Rephrase “The prevalence of LL indicates adaptive plasticity”. A prevalence of LL would indicate plasticity but such a prevalence has not been demonstrated here (as the analyses haven’t focussed on within-female relationships / experimental work) so a more cautious phrasing is needed.
e.g. “our findings demonstrate the pervasive influence of the social environment on life-history” – that would be nice, but they don’t - the effects, if they do reflect plastic responses, could presumably be driven instead by correlates of the social environment (see point 1).
e.g. “Our results demonstrate the importance of plasticity in maternal reproductive investment in cooperative breeders” – same issue.


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 RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study on an important topic that is gaining increased interest. I have a lot of comments, mainly editorial, and so easy to deal with, but important nonetheless. The key issues are: (1) Many references from studies of non-cooperative breeders are used in inappropriate places to support points that are particular to cooperative breeders; (2) In many cases were references to cooperative breeding studies are made, they pertain to provisioning and not egg investment, when in this case the point is not necessarily transferable from nestling to egg investment; and (3) As a consequence of each, key information and or references are missing on the theory of egg investment in cooperative breeders (see Savage et al. 2013a, b and 2015) (these references are provided, but key theory presented within is generally missing (e.g. Carranza et al. 2008; Russell & Lummaa 2009). I have provided as much help as I can to address these issues. You might not agree with all, and that is fine of course, but the overarching point is that more theory on egg investment in cooperative breeders needs to be introduced to put your study in context and acknowledge the growing background in this area, and I personally think you need to be quite careful with many of the references you use as they are not necessary transferrable to the point being made, and so are either confusing or mis-leading.

Line 65: Other possible references include Metcalfe and Monaghan 2001 and Lummaa and Clutton-Brock 2002, both in TREE. I mention these because they really deal with either other aspects of fitness (the former) or long-term, intergenerational effects (the latter). As such, I feel that both add to your reference list, while at the moment, I feel that the references are really saying the same thing.

Line 66-67: I think you are making an empirical point, but mix this up with references of a theoretical nature (e.g. Maynard Smith 1977). To me, what is really needed here is some clear experimental evidence of a cost of not just reproduction, but increased effort.

Line 71: Few is subjective, so I would suggest you use Fewer instead.

Line 73-74: The word excellent is subjective (maybe switch to apt or appropriate), and the subject of the sentence needs to be made clear. At the moment, I think you are trying to carry the subject from the previous paragraph, but it is not so clear.

Line 75: Delete in a single location as this is not true. Meerkat pups following the groups are not in a single location, and nor are the offspring in the numerous plural cooperative species in which helpers provide care to offspring from multiple nests.

Line 78: The Hatchwell 1999 reference is fine for this, as it is a review, but the komdeur 1994 reference is a bit random. First it is subsumed within Hatchwell. Second, it is not the first evidence. Third, it is not the best evidence. Either you want to provide the first suggestion and/or the best, latest experimental evidence (preferably both) followed by a review, but not a random paper.

Lines 78-81: Several lines are missing here, as you go from something super-general to suddenly in the next sentence talking about eggs in birds. In line 78, all we know is that you are talking about cooperative breeders, maybe in birds, and something to do with investment. But in line 79 you are on eggs in birds. You need several intervening sentences. First, you need to say that theory suggests that mothers should change investment according to workforce (e.g. Crick 1992; Carranza et al. 2008; Johnstone 2011; Savage et al. 2013, 2015; McAuliffe et al. 2015). Second, you need to say that experimental tests have been largely conducted during provisioning (e.g. Hatchwell & Russell 1996; Wright et al. 2008; Russell et al. 2008). Finally, you need to raise the question about whether or not such effects should be also manifest at the egg stage (Savage et al. 2015 deals with this directly).

Line 84: Not sure you need to call this load-lightening or maternal load-lightening, this is kind of the same thing. Koenig et al. 2008 termed the specific hypothesis at hand as the concealed helper hypothesis.

Line 85 and Lines 88-89: Not sure either Crick or Heinsohn dealt with egg investment, the more appropriate references here are Russell and Lummaa 2009 and Savage et al. 2015.

Line 87: Lessells, has 2 l’s

Line 91: Again, do any of the references deal with egg investment, again the more appropriate references as outlined above. Personally, I think there is a very important distinction between reducing investment at egg versus chick stages, and the references used throughout should acknowledge this difference.

Line 94: You need to be careful here. The Burley hypothesis is distinct from the Sheldon one, and both are distinct from the Russell and Lummaa one. The Burley reference is actually more akin to load-lightening (in a weird way because she allows her mate to load-lighten when high quality), than to Sheldon’s definition of differential allocation, and Russell and Lummaa 2009 expand on the Sheldon definition to make it appropriate to cooperative breeders.

Line 96-97: Maybe not here, but you should acknowledge in the discussion that it is conceivable that reduced egg investment with increasing group size is non-adaptive, and results from increased foraging competition. There are some nice studies that can rule this out, e.g. Canestrari et al. 2011.

Line 101: Personally, I would set this up as a direct test between the concealed helper hypothesis/load-lightening and differential allocation, rather than a simple description of how mothers react to increasing helper numbers. Russell and Lummaa 2009 as well as Savage et al. 2015 clearly set out some predictions. The former suggest that DA might be predicted in plural cooperative breeders, or other cooperative breeding social structures that generates competition among reproductive females within groups. Savage et al. suggested that DA versus LL is expected as a function of the costs off egg laying, with LL occurring when the costs of egg production are high, and DA when it is low. In other papers, Savage et al. 2013 (BES) also suggested that it will vary as a function of helper relatedness. Further, Carranza et al. 2008 also set out circumstances in which DA can arise. In saying all this, I am not suggesting that you can provide a full test of these hypotheses, but it is important to at least provide the background theory, and the point of your study. I think it would be fine to say that the primary is to shed light on these hypotheses by testing whether helpers generally cause LL or DA effects.

Line 103: I would not say that egg size cannot be affected by helper number. You can have evolved responses, and in meerkats, helpers influence maternal condition, through allowing her to reduce investment in pup feeding, which in turn positively influences investment in neonates (see Russell et al. 2003 BE). In some bird species, helpers feed females before egg-laying, which might also impact their condition. I think would be okay to say that they presumably have reduced direct effects in egg compared with offspring, but whether or not direct effects are not yet known.

Lines 107-109: I don’t understand this, nor the reference to Langmore et al. 2016.

Lines 118-119: This is not strictly true, usually only the female incubates the eggs, I suppose you mean provisioning, but need to be more specific.

Lines 122-126: There are plenty of non-eusocial insects where the above constraints are lifted. The main problem with insects for your purposes is that there are almost no studies of egg size across helper numbers. Russell & Lummaa 2009 could not find any non-eusocial cooperatively breeding insects in which maternal investment in egg size as a function of helper number was assessed, but it would be worthwhile checking to see if there are any since this publication, which would help bolster your sample size.

Lines 154-158: Sorry about this, I was in the middle of a high load of teaching. I can confirm to you and to the other reviewers and editor, that categorisation of helper effects according to the presence versus absence of helpers is valid in wrens as the number of groups with greater than one helper were very limited. Nevertheless, categorisation will inevitable inflate your effect size estimate. I will provide you with the necessary details, although there will be no qualitative change to your results.

I am not sure Figure 1 is necessary, perhaps it could go online?

Line 268: I think it would be nice here to re-iterate that this results provides general support for the concealed helper hypothesis.

Line 271: It might be nice here to re-iterate that this evidence complements the hypothesis that additive care by helpers should lead to LL in females in terms of provisioning (Hatchwell 1999).

Line 280: You need to be a wee bit careful here, as Russell et al. 2007 found the evidence and came up with the idea, but Koenig et al. which did not find any supporting evidence, coined the hypothesis. At the moment, you use the two citations to different ends, but this is not clear, making it suggest that the Koenig et al study is also supportive.

Line 286: At the moment, you use the term In contrast, to contrast with LL, but the subject of the previous sentence is about support not LL per se. It therefore sounds, as written, that you are going to provide contrary evidence for LL, but in fact go onto provide contrary evidence for DA. I think I would suggest starting a new paragraph, and making the subject of the sentence clear.

I also think it would be appropriate to discuss the suggestion by Russell and Lummaa that DA should be more likely when reproductive females compete within groups, since their fitness will be impacted by the relative success of their offspring. It is at least noteworthy, that the majority of the species that you studied were singular breeders, meaning females do not compete reproductively amongst each other within groups.

Line 311: I think that this Savage et al. 2015 reference should be cited and discussed in line with your results, but this is the wrong citation here, and should be Carranza et al. 2008.
Experimental design: No comments
Validity of the findings: No comment
Additional comments: I have added everything above under basic 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 RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: I am very sorry to say that i am disappointed by this latest version of the MS, and again have spent at least a full day refereeing it.

In particular, I still have quite a few significant issues with clarity and citation use, which to me are very linked in this MS. I said this last time, but perhaps I was not clear. For example, there are many examples of using the wrong citations or using a mixture of citations that confuse rather than clarify. When this occurs, it is unclear whether the point being made is wrong and the citation correct, or vice versa. Either way, citations should help clarify a point, not confuse it.

A key issue is the use of citations based on theory of post-natal care to support a point made about pre-natal investment (e.g Crick 1992, Hatchwell 1999 or even Brown 1987 or Stacey & Koenig 1990). There are many reasons why one should not do this. Most notably, mothers might reduce investment in offspring provisioning because chicks are less hungry due to the contributions of other group members and consequential effects on offspring begging. By contrast, given that mother must by definition lay the eggs before the contributions of any others, there are obvious reasons why load-lightening at this stage might not be expected, despite showing post-natal LL. This is why LL at the egg stage was not thought of prior to 2007. It is incorrect, presumptuous and mis-leading to use Crick 1992 or Hatchwell 1999, to support the load-lightening hypothesis at the egg stage.

Using theory provided to understand post-natal LL as a means to understanding pre-natal maternal strategies (e.g. by citing Hatchwell 1999) leads one inevitably to conclude that it is the post-natal responses to variation in helper number that drive pre-natal maternal investment strategies. By contrast, theory on maternal investment pre-natally make no such assumptions (see Russell et al. 2007, Taborsky et al. 2007, Russell & Lummaa 2009, Savage et al. 2013, 2015). Instead, in the vast majority of the hypotheses or theory provided in the above papers, the point concerning LL are more simple: an increase in helper numbers leads to an expectation of more food (or protection) for offspring, which allows mothers to save resources at the egg stage without losing fitness. The authors are right that if all co-carers fully compensated for each other’s presence, this assumption falls away. But there is next to no evidence that this is the case. Instead, any compensation is partial, and so increases in helper number (or helper presence versus absence) generate additive care. Further, more detailed theoretical attention, particularly by Savage et al., shows that post-natal care responses, are themselves linked with pre-maternal investment. This means that it is much safer to use helper presence/number as a predictor of maternal pre-natal investment (as is typical) rather than helper behaviour (this study). Thus, I am happy with the first analysis on helper effects on maternal investment, but less happy with the suggestion that helper behaviour post-natally drives this pre-natal investment (since it could be the other way around).

My worry about the second analysis is exacerbated by the lack of clear definition of compensatory versus additive care. For example, dunnocks appear to be used as an example of compensatory care (Discussion), but increasing group sizes are associated with additive care in this species, because compensations are incomplete. I would like to see a table inset in this MS, which outlines the species used as compensatory or additive with a clear justification for each one. I think that what we will find is that there is a continuum, from rarely fully compensatory to highly, or even rarely, super-additive care. Finally, on this point, I worry that because of the rarity of fully compensatory systems (perhaps absence) the question of whether additive care post-natally is collinear with the first question?

Below I outline my numerous specific comments

Line 72-73. As currently written this statement is untrue. Not only are there ca. 100 studies in cooperative breeders looking at parental responses to variation in the social environment, but variation in the social environment includes variation in co-carer contributions and so includes bi-parental care systems. There are over 100 more studies in this care system. I am not sure this is fewer than the number of studies looking at variation in response to other traits, and even if it is, the point is moot.

Lines 74-75. As written, this includes bi-parental care. I think it is fine to include bi-parental care at this point, but to get to cooperative breeders you need to say how they differ from bi-parental care systems, and the answer here is that they have variation in the number of carers. This allows maternal variation in investment to be linked to variation in the social environment, without direct manipulation of that environment (ie in cooperative breeders the social environment varies among mothers, which is not so obviously the case in bi-parental care systems).

Lines 75-79. The point that needs to come across here is not so much what cooperative breeders are, but that there is substantial variation in helper number within and among species, allowing tests of maternal responses. This is one of the key points made by Russell and Lummaa 2009, which formed the basis for their predictions about how this variation might be expected to affect maternal investment at the egg stage.

Lines 80-84. You need to be careful here. As the other Reviewer pointed out last time, you cannot use words like plasticity, unless you show within-female variation, and the only way to show this is to do an experiment or to use long-term data which allows sufficient contrasts. This is a classic case of where your message might be correct, but most of your citations are inappropriate, or whether you citations are appropriate but that the point being made need not be restricted to plasticity. Given the sentence before, I am assuming that the statement is correct, but most of the citations are not, including Hatchwell et al. 2004, Heinsohn 2004, Koenig et al. 2011, as none of them use experiments to specifically detail within-parent responses. Cockburn et al. 2008 provides comparisons of long-term data to show adaptive female, but not male responses in superb fairy wrens. One of the Macdonald et al. bell minor papers also uses nice removal and chick begging experiments to test for plasticity in bell minors, and one of these should also be cited.

Lines 84-89. This is a good example where multiple points are made, but all the references are added to the end – making it impossible to know which substantiates which point. To make matters worse here, the points are incorrect, and all of the references are inappropriate. First, you are talking about responses here, and so by definition the consequences of experimental manipulation or very carefully detailed statistical analyses based on longitudinal data. There are only a handful of such studies, listed above (none cited in this section), but I am not sure they are sufficiently numerous to talk about a typical response. So, instead. I would suggest you say…One such response is…..

Second, if you are somehow trying to talk more generally, you cannot use the word response, it would not follow your previous sentence and again you cannot really talk about a typical response (see Hatchwell 1999). In this case, the Reyer and Manica references are irrelevant, but at least Hatchwell and Kingma are good. Either way, you need to be more careful in your use of words, and more precise in both what you say and how you cite the work.
Line 82: Manica and Mirani reference is a poor choice, there are many confounding factors in that paper and compensation cannot be inferred. Compensation can only be shown by experiment and these have been done rarely, e.g. Hatchwell & Russell 1996, Wright 1998, Macdonald et al. in a series of bell minor papers; Russell et al. 2008

Line 92: Neither Brown 1987 nor Koenig and Stacey have anything to do with eggs and should be deleted. Canestrari et al. should also be removed unless you want to cite every single paper that is in your meta-analysis. The only paper that outlined the idea and made any theoretical predictions is Tabosrky et al. 2007 (for protection) or Russell et al. 2007 (provisioning) and Russell & Lummaa 2009 (inclusive of both and other helper effects); and then the formal models of Savage et al.

Lies 89 & 96: You have Savage et al. 2015, then Savage, Russell and Johnstone 2015, these formats need to be reversed (full authors first and then et al. 2nd)?

Line 101. Crick 1992 makes no mention of eggs. I said this in my last review: one cannot extend theory on responses to offspring provisioning with responses on eggs. They are many reasons why load-lightening at the egg stage is very different, including the fact that no other group member in real time can respond. The only theoretical treatment of helper effects on egg investment are Taborsky et al. 2007, Russell et al. 2007, Russell & Lummaa 2009, and the series of Savage et al. papers; particularly his 2013 and 2015 papers. No other paper is relevant that I know, and certainly not those cited.

Line 103-104: Remove Canestrari et al. since this is entirely empirical and does not extend any of the points made previously. And Santos 2016 is a thesis, which should be removed completely.

Line 111. Savage et al. 2015 should probably cited here which squarely considers how mothers should invest with increasing helper numbers.

Line 116. Remove the word “exists”

Lines 117-119. Not sure what is trying to be said here. If I understand correctly, does a meta-analyses really indicate whether detailed studies are required to understand selection and the biology of individual species?

Lines 119-122. I am not sure of your logic here. I am not sure you need to justify why you look at pre-hatching rather than post-hatching investment. They are totally different questions. I think the important point is more because it is a stull relatively under-studied area of research, with demonstrable implications for understanding selection on cooperative systems (e.g. Russell and Taborsky 2007 refs).

Lines 125-128. Sorry, I have no idea of the logic here. I suggest that the authors check out Russell & Lummaa 2009 and Savage et al. 2015, for logic in when we expect LL of DH at the egg stage. Again the authors cite a paper that has nothing to do with egg investment, and so confuse the point being made. The problem with understanding post-natal investment is that maternal, paternal and helper contributions are dynamic and potentially negotiated as chicks age (McNamara et al. 1999). Post-natally - one cannot say categorically that mothers are more likely to LL when helper effects are additive, because the reverse might be true, helper effects might be additive because mothers load-lighten. The interesting thing about the egg investment stage, is that mothers need to invest before partners or helpers are able to respond. Indeed, the response of co-carers, when it occurs, is itself a part-function of maternal investment at the egg stage (Savage et al. papers). Thus, it is circular, mis-leading and potentially wrong, to use co-carer investment strategies at the chick stage to predict maternal investment strategies at the egg stage. Predictions about LL versus DA at the egg stage need to be articulated from papers that deal specifically with these points at the egg stage.

Line 130. Need to say at the egg stage

Line 131. Following my comments above, you need to be careful to define what you mean by additive care. You mean at the chick stage, and you mean of all co-carers other than the mother? Or do you mean overall, irrespective of LL by the mother at the chick stage. You state in the methods that you simply used what the authors had reported, but 1-2 lines summary of this is required in the Intro and a table in the methods. For example, in chestnut-crowned babblers, mothers reduce their provisioning contribution to almost zero in large groups. But, because the contributions of male carers are unaffected by the number of co-carers, and because the number of co-carers can be numerous, there is a positive association between carer number and overall provisioning rate of the brood. I am guessing that you would call this additive care, and I would too, but serves to illustrate the point that not all individuals in the group need to show additive care for care to be additive. Indeed, you could also have a situation where all group members LL, but care is still additive, because LL is only partial. Finally, it is unclear whether you are saying that because care is additive, we expect maternal LL at the egg stage (which is what I think you are saying), but I would say that opposite is also possible, additive care at the nestling stage arises because of maternal LL at the egg stage. You do not need to make all these points of course, but I hope you can see that some clarity is required, and you need to be clear that any detected association between LL at the egg stage and additive care at the nestling stage could arises as a cause or effect.

Lines 142-146. I fundamentally disagree with this comment, and have predicted elsewhere (Russell & Lummaa 2009) that maternal effects in social insects should be much stronger than in vertebrates. All you need to say here is that the data is lacking.

Lines 146-149. This is also incorrect. Broods often receive more incubation and more food in communal birds because the number of nest attendants is far great than the usual 2. The problem here is that you cannot identify which egg came from which mother, and so it is difficult to know what causes any changes in egg size in communal nesters.

Line 209, were they not also inclusive of different data, which I would have thought would have been more important. For example, Langmore et al., published 9 years later, used far more years of data than Russell et al..

Line 273. Helper effects can be additive even if they are compensatory. Please can you clarify if these species show additive care, albeit perhaps to a lower level than others species.

Lines 288-289. Need to specify that less means reduced egg size not clutch size

Lines 293-294. Is this association not just an epiphenomenon of the fact that there is an association between helper number and maternal egg investment across species, and almost all of the species included showed additive care?

Lines 294-298. I don’t understand what is being said here. Dunnocks show additive care, females with 2 males receive more food for their young than those with only 1 male. This additive are arises from incomplete compensation, and females increase clutch size as a consequence (Davies and Hatchwell 1992). As a consequence, I do not follow the logic of your point – females increase egg investment and decrease provisioning when breeding polyandroulsy; so is the decrease in provisioning because of the extra male or because of their increased egg investment, or a bit of both?

Lines 311-315. Please at least acknowledge that causality is unknown. We do not know whether maternal LL at the egg stage generates additive effects at the chick stage, or not. I do not necessarily disagree, but we do not know the direction of causality at this time.

Line 317. Did Woxwold and Magrath not suggest that the relationship between group size and breeding success might be weak because not all group member contribute? If so, this is a very different argument to the one you present here, which was really the foundation of Russell et al. 2007, and then furthered by Koenig et al. when he coined the term concealed helper effects.

Lines 340-345. I do not disagree with any of this, but it is at least noteworthy that this analysis does not control for maternal egg investment, and so any role of maternal egg investment (particularly clutch size) in generating differential nestling starvation (Liebl et al. 2016 Anim Behav).


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 RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES
Review round: 3
Reviewer: 1

Output:
Basic reporting: Great
Experimental design: Great
Validity of the findings: Great
Additional comments: Very well done for dealing with all of my previous comments, I do think the MS is now far clearer and more informative than before.


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 IMPACT OF SELF-INCOMPATIBILITY SYSTEMS ON THE PREVENTION OF BIPARENTAL INBREEDING
Review round: 1
Reviewer: 1

Output:
Basic reporting: On the whole, the paper is well written, and the background literature is well reviewed. The figures are clear and well presented.

That said, there are times where the literature is incorrectly explained, and the relationship between the major hypothesis and the results is not particularly solid (see below).

Another major shortcoming of the manuscript is that the computer code is not provided. It should be uploaded with the next submission, and must be stored permanently on dryad conditional on acceptance.

However, all of these concerns can be addressed relatively easily.
Experimental design: I believe the research is well executed and suitable for this journal. As said in '1. Basic reporting' it is not repeatable without computer code, but I get the sense of their approach.

The investigation is rigorous and honest.
Validity of the findings: I believe that the work as stated was well executed.

A concern I had is that results were described as significant or insignificant without the use of p-values. I would suggest a different approach (if computationally possible) - that the authors increase their sample size such that differences are apparent without the need to invoke null hypothesis significance testing. If that degree of replication is not feasible, than the authors should provide p-values with their statistical statements.

My most pressing concern is the relationship between the motivating question and the results. I describe this below.
Additional comments: This is an interesting paper. That said I have a significant concern about the framing of this paper with regard to the major motivating hypothesis.

1. The authors claim to investigate if differences in the extent of biparental inbreeding associated with alternative incompatibility systems can favor chemical based self-rejection, and claim to reject this hypothesis. However, the simulation does not address this problem. Rather, the authors investigate differences in the genetic load and the extent of biparental inbreeding as a function of alternative self-incompatibility systems, and never directly test their proposed hypothesis. I therefore would like to see the authors ask directly if different modes of self-incompatibility change the extent of autozygosity, allozygosity, and inbreeding depression (the question they asked). The question of selection for this form of self-rejection should be relegated to speculation in the discussion.

2. In fact it is not clear what such a test of this hypothesis would be. The authors do not make clear wether they imagine such incompatibility systems replacing one another in natural populations, or if they expect higher level species selection to act. This later hypothesis is often invoked to explain the distribution of plant mating systems (e.g. Goldberg et al 2010). These levels of selection should be clarified in the introduction (lines 34-38).

3. I am somewhat worried about the sensitivity of results to a few somewhat idiosyncratic decisions concerning the nature of the genetic load that was simulated (modest selection for a small number of loci with high mutation rates)  and the expression of this load (individuals with high inbreeding depression take up space in the lattice). I would be curious to see results with more, rarer deleterious recessives with more extreme fitness effects (as we generally assume). I would also be interested to see how early-acting inbreeding depression (such that unfit inbreds did not take up space) would impact the results.

4. There are a few (relatively minor) misstatements in the introduction. As the authors' know being that 95% of plants are hermaphrodites does not mean "that a single plant is capable of self-fertilizing and reproducing without a mating partner," but rather that plants have male and female parts. The authors should clarify this for their audience.   I also think that Fisher's automatic advantage could be explained better.  The advantage is that selfers can fertilize themselves and someone else not that they pass a higher fraction of their genes to their offspring (if all pollen that self-fertilized an individual fertilized a different individual instead, fitness would be identical). Another minor point is that heteromorphic incompatibility systems often have genetic based rejections, however, I don't think this would impact the modelling or the results.


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 IMPACT OF SELF-INCOMPATIBILITY SYSTEMS ON THE PREVENTION OF BIPARENTAL INBREEDING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is nicely written, with professional language and clear figures.  Ideas and relevant literature frame the context well (except for the specific concerns noted below).  There are no data to be archived, but the code is available.
Experimental design: This is a primary research manuscript with a clear motivating question.  Investigation of the question is rigorous (except for the specific concerns noted below).  The code provided and settings described are enough to replicate the research.
Validity of the findings: The findings are summarized appropriately.  Conclusions are mostly stated clearly, though some improvements are noted below.  My concerns about the overall biological interpretation are noted below.
Additional comments: This manuscript is focused on the general question of how different systems of self-incompatibility affect biparental inbreeding.  This is an interesting question to ask for both evolutionary and ecological reasons.  The authors have done an excellent job of clearly and concisely presenting the results from many simulations.  Before this work is published, however, it is essential to clear up some issues about the interpretation of the SI systems and conclusions.

(1) My one big concern is about "heteromorphic SI." The model implements a system called PSI, in which selfing is prohibited but there are no additional mating incompatibilities.  This system is useful as a control, in that it does nothing to reduce biparental inbreeding.  However, it bears no resemblance to real heteromorphic SI systems, in which intra-morph matings (not only selfs) are prohibited by an S-locus that is linked to one or more morphological loci.  The authors only admit that PSI is an entirely artificial system near the end (lines 375-378), and the entire introductory framing, as well as much of the discussion, is cast as heteromorphic versus homomorphic SI.  This is extremely confusing and misleading.  Please either completely remove the discussion of heteromorphic SI, or simulate new results using a genetic system that reflects how heteromorphic SI works in nature.

(2) My other main concern is about the central conclusion: that homomorphic SI systems do not do much to prevent biparental inbreeding.  For one thing, some of the statements about angiosperm evolution are far too sweeping to be based on this one small simulation study (e.g., lines 27-28, 322-323).  Additionally, I suspect this finding from the model may in part be an artifact of the large number of S-alleles (Table 1).  I think these are at least twice as large as what's known from natural systems, which would greatly reduce the potential to avoid biparental inbreeding.  It would actually be quite interesting to see how the inbreeding avoidance scales with the number of S-alleles, at least a comparison between "few" (say, 10) and "many" (say, 40).  Alternatively, the rate of generating new S-alleles could be lowered.  Either way, please provide some references to support that the number of S-alleles in the model is biologically realistic.

(3)  My remaining comments are much smaller, ordered by appearance rather than importance.

throughout: "biparental" is more usual than "bi-parental"

throughout: "Mating system" typically refers to the selfing rate rather than the type of SI.  But admittedly the literature is frustratingly inconsistent with this kind of terminology.

throughout: SI does not always act in the stigma.  For most or all GSI, it acts in the style.

30: Renner (2014) says that 5-6% of species are dioecious, but there are lots of other sexual systems (monoecy, gynodioecy, etc.) besides hermaphroditic.

30-31: Just because an individual is hermaphroditic doesn't mean it is capable of uniparental reproduction (e.g., SI).

35: The logic of "consequently" is not clear.

52-54: Using "haplotype" here means it isn't correct for sporophytic SI.

67-68: Can an empirical citation be provided?

81: Purging hasn't been explained.

91: It's not clear what "continuous" means here.

115: "if they share *only* the same recessive"

129-143: I think of inbreeding as defined at the level of which *individuals* mate with which, so it sounds a bit odd to say that inbreeding is reduced at particular parts of the genome.  Maybe homozygosity would be clearer?

148: The statements here are true (but see a comment below), but "for this reason" is not their logical connection.

158: For isolation by distance, a toroid doesn't seem appropriate.  Better to allow dispersal off the edges?

209-211: Why is autozygosity defined by grandparents rather than by parents?  If the latter, it seems like selfing would automatically be separated from biparental inbreeding.  I'm wondering if the current "proportion autozygous" results (Fig 3) might be dominanted by selfing, obscuring differences between the SI systems.

220-227: This would make more sense if there were some indication of what quantities will be compared.

Results: It would help if each subsection began with an indication of what is being checked or what questions are being answered, rather than just jumping into descriptive results.  Lines 259-262 do a good job of this.

The Discussion leads off with a small matter that is not the main point and sounds like an artifact.  It would be more effective to start with big questions and perspective.

313-314: I don't follow this sentence.  Several generations from when?

341, 346, 348, and 148-149: The claim is that there are many S-alleles in a population *because* that's what's necessary for enough compatible matings.  This reads like population-level group selection in the sense that real populations or simulation runs without enough S-alleles preferentially went extinct.  Is this the intent?  Natural SI populations with few S-alleles do exist, e.g., after a bottleneck.

373-375: This seems like an extremely aggressive claim with no foundation in logic or data.

386-389: SI can also weaken in older flowers simply because proteins denature, rather than because it's a beneficial strategy.

Table 1: The s^2 column doesn't seem necessary, or it could at least be placed right next to the sigma column.

Figure 1: This is a nice depiction. One small adjustment is that the pollen's inner red circle looks orange (on my screen, at least).


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: FORELIMB MUSCLE AND JOINT ACTIONS IN ARCHOSAURIA: INSIGHTS FROM CROCODYLUS JOHNSTONI (PSEUDOSUCHIA) AND MUSSAURUS PATAGONICUS (SAUROPODOMORPHA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-written and well-executed paper examining muscle moment arms and joint ranges of motion in the forelimb muscles of an extant ‘sprawling’ archosaur and an extinct ‘parasagittal’ archosaur.

Basic reporting

The English is generally clear and unambiguous throughout.  There is some confusion of UK vs. US English throughout; e.g. meters (US), but modelling and centre (UK). I have identified a few typos in the text and picked them on the attached PDF. The paper is set in an appropriate context, the literature is appropriately referenced, and the structure is appropriate.

The figures containing moment arm graphs are far too small. I was unable to read the text in the figures, and while they might be reproduced slightly larger in final print, they are still way too small. I suggest breaking the moment arm figures up and making the text at least 10 points at final size.

The manuscript states in one place that models are available upon request, but in another that they are included with the study. My preference would be to see the models included with the study, but that is the prerogative of the authors.

You state at the beginning that extension is negative, but throughout there is confusion about flexion and extension and which is negative.

Maybe I missed something but I can’t see any figure captions for the supplementary information figures.
Experimental design: The research question is clearly framed, well defined, relevant and meaningful. It has been performed to a high standard and the methods have been described in sufficient detail.
Validity of the findings: The data appears robust, is clearly described, and is appropriately interpreted.

Conclusions drawn are linked to the original research questions and supported by the results.

I have some comments regarding your interpretations about quadrupedality.

1)	You state that not being able to flex the humerus anterior of vertical would preclude quadrupedalism. I don’t think that’s the case. In obligate quadrupedal ornithischians, I don’t think the humerus could have been protracted past vertical because of the location of the humeral head on the caudal surface of the humerus and the morphology of the glenoid. Because the elbow is strongly flexed, however, this doesn’t prevent quadrupedalism. I’ve said this somewhere in print and can probably dig it out for you. But these animals were clearly quadrupedal. It must have meant they had a very short stride, admittedly, but I don’t think it precludes quadrupedality;

2)	A pronated manus isn’t necessarily necessary for quadrupedality. Trackways of iguanodontids show quadrupedal locomotion with an apparently supinated manus;

3)	In sauropodomorphs, quadrupedality seems to be considered to only have occurred once. I’m not sure we necessarily have to assume this. It’s possible that there was mosaic evolution of the forelimb in sauropodomorph evolution and that facultative quadrupedality could have been present in some forms and not in others. Thus contradictory findings might be expected on the sauropodomorph ‘stem’ when examining different basal sauropodomorphs.
Additional comments: There are many minor comments on the attached pdf.


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: FORELIMB MUSCLE AND JOINT ACTIONS IN ARCHOSAURIA: INSIGHTS FROM CROCODYLUS JOHNSTONI (PSEUDOSUCHIA) AND MUSSAURUS PATAGONICUS (SAUROPODOMORPHA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Clear, unambiguous, professional English language used throughout.
Excellent.  Just some minor typographical errors were noted with sticky notes.

2) Intro & background to show context. Literature well referenced & relevant.
The introduction and background are good, but the section on pronation appears to have overlooked the most recently published tests of the references that they rely on most heavily in their results and discussion.  I provided detailed comments, explanations, and references to ask for some clarifications in terminology and their interpretations of pronative ability in their study specimens.

3) The structure of the submitted article should conform to one of the templates.
No comments.

4) Figures should be relevant to the content of the article.
The figures are good, and I trust that the published version will indicate which side is Crocodylus, and which is Mussaurus.

5) The submission should be 'self-contained,' should represent an appropriate 'unit of publication,' and should include all results relevant to the hypothesis.
No comments

6) All appropriate raw data has been made available in accordance with our Data Sharing policy.
No comments
Experimental design: 1) Original primary research.
- No comments.

2) Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
- In regards to their stated knowledge gap for dinosaurian pronation, I inserted comments as needed to help identify what some of the newest studies have tested in relation to their research questions.  Hopefully these comments can help identify how their pronation study fits in with the latest research.

3) The investigation must have been conducted rigorously and to a high technical standard.
- Excellent.

4) Methods described with sufficient detail & information to replicate.
- Excellent.

5) The research must have been conducted in conformity with the prevailing ethical standards in the field.
- No comments
Validity of the findings: 1) The data should be robust, statistically sound, and controlled.
- No comments

2) The data on which the conclusions are based must be provided or made available in an acceptable discipline-specific repository.
- No comments

3) The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results.
- Excellent, but with some inserted concerns as to their support for their pronation results.

4) Speculation is welcomed, but should be identified as such.
- No comments

5) Decisions are not made based on any subjective determination of impact, degree of advance, novelty, being of interest to only a niche audience, etc. Replication experiments are encouraged (provided the rationale for the replication, and how it adds value to the literature, is clearly described); however, we do not allow the ‘pointless’ repetition of well known, widely accepted results.
- No comments.

6) Negative / inconclusive results are acceptable.
- No comments
Additional comments: Very nice piece of writing - This paper is carefully written, with thoughtful consideration to different points of view and terminologies so common to myological/arthrological studies.  The layout is very organized and flows nicely; the figures are easy to interpret.  I am impressed with the support for your interpretations of how to categorize movements based off of your moment arms; this is the type of quantitative support needed to help standardize some of the terminology for sprawling versus parasagittal clades.  I inserted a number of sticky notes and highlighted text throughout the pdf markup.  Most of them are minor suggestions, typographical error notations, and questions.  My only concern was with the pronation aspect of the study.  My latest research is testing some of your primary references on forearm pronation in dinosaurs, and I noticed that your arguments could benefit by incorporating them.  I tried to outline how your interpretations of forearm pronation could be affected by this research.


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: FORELIMB MUSCLE AND JOINT ACTIONS IN ARCHOSAURIA: INSIGHTS FROM CROCODYLUS JOHNSTONI (PSEUDOSUCHIA) AND MUSSAURUS PATAGONICUS (SAUROPODOMORPHA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I believe that the changes in discussion and interpretation are more than satisfactory in helping identify how your study fits in with the latest pronation research.


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: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK
Review round: 1
Reviewer: 1

Output:
Basic reporting: English writing still need to be improved, because there are some issues on the wording, spelling, and sentence transition and connection. For example, you should pay attention on the consistent italic style for gene names, in your manuscript, some names are italicized but some are not, please correct them.
Line 74, "Identified" change to "identified"; Line 75, changed "guide" to "reference"; Line 79, " please write full name of "dH2O".
Line 80, "3 min pre-denaturation at 94ºC" change to "pre-denaturation at 94ºC for 3 min"
Line 96, delete " mRNA" and "present", change "quantity" to "expression".
Line 106, change "sequined" to "sequenced".
Line 107, delete "using"
Line 110, change "using" to "in".
Line 116, change "egg traits' to "egg production traits".
Line 119, add "Difference with" before "P value".
Line 124-128, gene sequence of Muscovy duck is identical to "that of" Anas and Anser.
Line 137, add "also" after "other tissues".
Line 169, add "However" before "we only locate..."
Line 173, change "IGF2 high expression" to "high expression of IGF2"
Line 189, " However" change to "In addition"
Line 202, change "with" to "on".

In addition, there are some statements that lack important information for proper understanding. For example, line 32-33, IGF2 in the dominant follicles support function of which hormone? Line 70-71, cDNA was used as template to amplify what region of IGF2 and DRD2 genes? Line 110, how many females are genotyped through sequencing? Line 146, 2 SNPs of IGF2 and 11 SNPs of DRD2 were selected for further association study based on what? Line 173, IGF2 high expression in the ovary many be related to whose development? Line 181, IGF2 might regulate cell proliferation during follicular development in what animal? Line 212-213, are the two SNPs with high linkage? You never mentioned in the results part.

Literature references should also be selected carefully and properly based on the purpose of your study. In this study, your aim is to investigate association of IGF2 and DRD2 with egg production in Muscovy ducks. In this case, you should focus more on the literature about reproduction of Muscovy ducks and research of the two gene in the reproduction system of avian species, and suggest how your research is important to fill the current knowledge gaps. Why do you choose some literature on their relationship with human and fish diseases (line 35-38, line 195-199, line 204-206)? In addition, are you sure you understand the reference paper correctly? In line 197, it is DRD2 agonist used as a reagent  for the treatment of the disease rather than DRD2 itself used as a agonist for the disease. I totally doubt whether your corresponding advisor has read it or not.
Experimental design: This study is a primary research within aims and scope of the PeerJ journal. However, the authors did not make enough justification for their experimental design in the introduction part. For example, why do they study Muscovy ducks instead of other birds? why do they choose IGF2 and DRD2 to study? how did they find these important genes? why do they choose E300D and E59W instead of egg production at other time points as important traits in their study?

In addition, the method is also not described sufficiently. For example, how are  the primers designed for sequence cloning, qPCR and SNP detection? Although the authors listed all the primers in Table 1, they did not describe how are the primers used in each step.You should cite the primer names in the methods section and also put a note under Table 1 to explain the meaning of primer names.  In addition, I suggest you to put table 1 to supplementary materials and add a figure to explain the structure of the two genes and the location of each primer in the gene sequence.

In Figure 1, you made a phylogenetic tree of muscovy duck DRD2. But you never mentioned in your method section how the tree was generated and how the sequence alignment was performed. Why do you only made phylogenetic tree for DRD2 but not for IGF2?

In Figure 2 and 3, how do the gene expressions differ among different tissues? Please label letters above each bar to denote the significant differences.

In Figure 4, you should include the whole heatmap in the figure rather than the significant block you are interested in.

In Table 2, I suggest you to add frequency of each mutation as another column.

Finally, in the animal experiment, you did not give enough information on how the animal were obtained from the company. How many ducks were offer? How were they were bred before the study? What feed were they provided?
Validity of the findings: The authors did not discussed enough to assess the impact and novelty of their study. Although the data is statistically sound and some significant SNPs were identified to be associated with egg production traits, they failed to make logical connection with current knowledge in the field, and make further biological speculations on how the SNPs may affect function of the genes and egg production traits. I think they still need work hard to improve their introduction and discussion part. Simply listing findings of previous studies in human, mammal and fish but lack of necessary rationale and linkage does not make a sound discussion at all.
Additional comments: This article described two genes and their polymorphisms, specifically high expressions in ovary, and significant SNPs related to the reproduction traits in Muscovy ducks. The study is important and interesting. However, you still need to do more work to improve English writing make it smooth to read, add more information in the methods part for sufficient description, and read more and think more to make a logical discussion to speculate possible reasons and convince the importance of your discovery. Based on the reading, I doubt whether the corresponding author has proofread and revised it or not.


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: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is well organized, but sustains grammatical errors.

The introduction is informative, but does not speak to the importance to this study, nor the relevance of this study to industry.  Inclusion of this information would make this a more successful manuscript.

The images used in Figures 4 and 5 are of poor resolution.  Using a higher quality version of this image would be advantageous.

According to the instructions for authors: multiple references listed within parenthesis should be listed chronologically; in the reference section, there should be a period between the first initial of the last author’s name and the year; the full title should be used in each of the references (see Lines 242, 250, 260, 265, among others); references with 3 authors should all be spelled out, while 4 and more use et al. and the year (these references on these lines are not cited this way in the manuscript: 255, 298, 303, 312).

The following are a sample of grammatical errors which should be addressed:
Line 32 – Especially is not the correct word to use in this sentence.  It should be removed.
Line 58 – First egg age acronym not consistent as compared with the rest of the manuscript.
Line 117 – statement should state “effect of each genotype”; genotype should be singular
Line 120 – a space should appear before and after the less than or equal sign.
Line 149 – should read “C-1704G SNP of the IGF2 gene”
Lines 154 & 159 &160– “Highly significantly” should be replaced with “indicate highly significant associations”
Line 157 – Should read “highly associated with E59W”
Line 172 – “Was” should be replaced with “is”
Line 172 – “but predominantly” should be replaced with “with the highest expression found”
Line 181 – add a comma before “while”
Line 183 – should read “highly productive”
Line 183 – change “were” to “are”
Line 184 – change “ones” to “chickens”
Line 266 – remove extra space
Line 352 – Should read “or are highly significant”
Experimental design: Answering the following questions would provide clarity to the manuscript:
Why were egg collection data taken specifically at 300 days and 59 weeks?
Where the ducks all raised in the same run, or were portions raised at different time points/seasons/locations?
What age were the ducks euthanized?
Where the same ducks used to gather production data and tissue data for expression analysis?
Why were some SNPs chosen over others for association studies?
Validity of the findings: Be specific when defining the tissues expression of IGF2 occurs in in the Tissue Expression of IGF2 and DRD2 Genes section.

The explanation of Table 3 in the Results section should be re-assessed to make it more clear.  The superscripts differentiating AA, AG, and GG of A-1864G for the E59W trait should be listed as a, ab, and b, respectively.  The p-values reported in the text, and those in the tables are different.  Clarifying the reasoning behind this would make this paragraph much more understandable.

The presence or absence of associations in T+3024C and A+3183C for DRD2 should be addressed in the Results section.

After reading this manuscript in its entirety, it remains unclear the purpose of this study, and implications of its findings relevant to the duck industry.  This information would make this paper much stronger.  Additionally, the conclusion does not explore reasonings behind association of certain SNPs to specific production parameters measured, more than a just a repeat of basic findings from the Results section.  This manuscript would greatly benefit from a re-written conclusion that goes further into interpreting results from this study.

Concerns should be raised about the validity of the findings in the expression analysis, since age of tissue collection is not explained. The information does not appear to help add to the findings of the manuscript.  At which point in the duck’s production timeline tissues are collected could play a major role in the expression levels. This information would be more useful if gathered at the same time points the production traits were collected (AFE, E300D, E59W). Having both SNP association data and expression data would present a more informative discussion as to the results in this manuscript.
Additional comments: Information regarding functions of IGF2 are reported in mammals, but would be more compelling if linked to poultry, or a statement should reflect that the author could not find any such evidence in poultry to clarify.

Table 4 superscripts are not consistent in capitalization.

Listing the table number within the first sentence of the explanation of the results would improve the flow of the findings.

In two cases (Zhang on lines 38 and 42; Ferrero on lines 198 and 200) one of each of these author’s references were not correctly cited in the text, therefore, there is no way to distinguish which reference is being referred to.


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: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK
Review round: 2
Reviewer: 1

Output:
Basic reporting: English writing can still be improved.
In introduction section:
Line 2: change "prized" to "priced".
Line 5: change "pay" to "paid"
Line 8: E300D and E59W are both egg production traits at certain time age, how can they be peak time and end time for laying. Please revise this sentence.
Line 9: add "Therefore," before "we"

In sample collection part of Material and Methods section:
Line 1: change "800" to "Eight hundred"
Line 2: add "were" between "which" and "in"

change "distillation-distillation" to "double distilled"

In Polymorphisms of IGF2 and DRD2 Genes part:
change "S" to "s" in "sequencing"

In Association of IGF2 and DRD2 with egg production traits part:
Line 2: Change "Linkage disequilibrium" to "linkage disequilibrium analysis"
Line 8: change "were" to "had", delete "indicate"
Line 13: change "were" to "was"

In discussion section:
Line 1: delete space in "poultry", change "it" to "which"
Line 2: change "i" to "I" in "improving"
Line 5: change "I" to "We"
Line 14: delete "found", change "suggest" to "suggests"
Line 22: add "that" after "than", change "low productivity" to "lowly productive"
Line 34: It is "DRD2 agonist" rather than "DRD2" that "the production and secretion of vascular endothelial growth factor protein in granulosa cells"
Experimental design: The experiment design is good and enough to answer the research question. Methods is also described with sufficient details and information.
Validity of the findings: Data in this study is robust and statistically sound. Reasonable conclusion and speculations are also made based on the results.
Additional comments: Generally, the manuscript have been greatly improved after your revision. But English writing still needs to be improved. I believe it is a good study that would be published soon.


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: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK
Review round: 2
Reviewer: 2

Output:
Basic reporting: Content added by the authors in the Introduction, Methods, and Discussion sections contain substantial grammatical and syntax errors in the revised manuscript.  The information included did not aid in clarifying the relevance of this study.  This manuscript contains potentially important information for the duck industry, but the authors have not adequately conveyed this message in these sections.  Additionally, without appropriate citations to back up their claims which are used to substantiate the significance of this study, overall the argument is less compelling.

Table 3 remains hard to understand.  The current title is not sufficient in allowing the reader to understand everything going on, and does not make clear how to differentiate the meaning between lower case and capitol letter associations in the LEM.

Despite mention of the correct formatting for references in the initial review comments, two-authored references are not cited correctly in the text of the manuscript.  Finally, sentences ending in the citation of multiple references are not listed chronologically.
Experimental design: A brief statement as to the rationale of eggs at day 300 and week 59 is warranted, even if widely known in the duck production community.  PeerJ readers are likely to not know of the specifics of duck egg laying cycles.

The sampling methods indicate in the manuscript that all ducks were run together, while in the rebuttal, the author states that ducks sampled for production and tissue analysis were from a “different batch”.

The rationale behind selecting 2 out of the 5 SNP identified in IGF2, and 11 out of 28 SNP identified in DRD2 needs to be explained in the text.
Validity of the findings: The impact of this study was not sufficiently addressed in the revision.  The discussion includes statement in reference to non-avian species, but fails to acknowledge whether any information is known within birds, or if positive associations of the data are being backed up by the authors for different taxa.

The authors did a good job of noting that only positive associations were identified, but that further investigation is warranted in regards to causation due to the SNPs
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: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX
Review round: 1
Reviewer: 1

Output:
Basic reporting: The title of the review article is not reflecting at all the content of the work, since only sulfonamides are trated, and more precisely the few sulfonamides described by Matulis group. Although the authors mentioned the five types of small molecule CA inhiibtors targeting thismisoform, then only a very limited number of compounds is considered.
Experimental design: There are no experiments presented. The various methods of inhibitor assay are not very meaningful in a revie article on such a specialized area
Validity of the findings: Two structures of CA IX inhiibtors are presented which are of little interest since such compounds did not ptogress even to preclinical work. The part on antibodies is very badly written and antibody-drug conjugates not even mentioned
Additional comments: Overall this is a badly written ms which has a mixture of diverse topics whoch do not for, a solid ms, being too heterogeneous. For example it is difficult to understand what the zebra fish model did for the discovery of CA IX inhibitors.


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: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is clear, but the selection of the articles for this review is absolutely authors-oriented!
Experimental design: not applicable
Validity of the findings: Table 1: I could not understand or quantitate the differences between "minor effect on pH" and "significant effect on pH".
is CA IX expressed in Xenopus transmembrane (as in humans) or cytosolic? This could be detrimental for the significance of these data. And maybe this part, dealing with only two CA inhibitors could be deleted because it is not validated for this target in the literature.
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: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX
Review round: 1
Reviewer: 3

Output:
Basic reporting: See comments to the authors
Experimental design: See comments to the authors
Validity of the findings: See comments to the authors
Additional comments: This review was written by an expert team who has developed new molecules to target carbonic anhydrase IX and has contributed significantly to the field. However, I think it is highly focussed on their own work rather than a general review, for example, only focussing on one subclass of inhibitors, the aromatic sulfonamides, so it is more of a report of their work only, where they, for example, comment “for the first time we emphasised the use of mammalian models”. However, they have already published this in 2016 and 2017, so anyone looking at the literature would find this.

They also make several claims, for example, “the first review to combine information about animal models and other methods used in the field”, whereas there are many reviews on carbonic anhydrase IX as a target. In fact, it would be useful to include other recent reviews in the references. Essentially, it is partly a technical description of work they have done in terms of the sorts of assays that have been around or published already. They also cover the zebra fish work already published and is well recognised as a useful phenotypic screen.

CA-targeted strategies focused mainly on preclinical work and the clinical studies are poorly reported. For example, the carbonic anhydrase IX inhibitor in clinical study was indisulam, yet it has been discontinued from clinical study a couple of years ago now. Similarly, the Willex antibody was actually effective at predefined set points of expression of CA9, yet has not been commercially developed. Finally, SLC0111 has gone through phase I but there is no information on the pharmacodynamics used, toxicity, or the dosing or any other details. So for someone who is interested in the clinical development, there is out of date information, not enough discussion as to why apparently successful compounds are counted as failures or update on the most recent compounds or how they are proven or not to have an effect on carbonic anhydrase IX.

Overall, therefore, I felt it is mainly concerned with their own publications in the last two years and summarising those data without an adequate discussion of the clinical problems and why with over 1000 compounds reported by Supura, onlya single compound has progressed to phase II studies in over 15 years since it was recognised an important therapeutic target. A much more detailed discussion of these problems would be of more interest.


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: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX
Review round: 1
Reviewer: 4

Output:
Basic reporting: Kazokaité et al. have submitted a review article summarazing the current status of development of anti-tumor agents targeting carbonic anhydrase IX (CA IX). CA IX acts in control of tumor pH, modulates cell adhesion and invasion and contributes to tumor progression. Therefore, CA IX is ranked among promising anti-tumor drug targets with many new developed CA IX-targeting molecules every year. For that reason the topic of submitted review is actual and beneficial.
Professional English is used throughout the review, but in some parts is the text more complex than necessary. The number of references is fully sufficient for this type of a review, some of the references that I would recommend to add will be mentioned later There are no figures or charts present in publication, I would suggest adding at least one or two charts to improve readability. The review contains two helpful tables, but Table 2 has incorrect text wrapping in several columns.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: 1.	Line 55: „The unregulated pH is a common feature of many aggressive tumors to adapt to hypoxia.“ I lack the clarification of the relationship between hypoxia and the problem with pH regulation. Explanation of the extracellular acidification of  tumor microenvironment is given below, but there is no link to hypoxia.
2.	In  text, the terms „antitumor“ „anti-tumor“ „anticancer“ „anti-cancer“ are used extensively. It is necessary to unify the writing of this term, with or without a dash.
3.	I suggest that the text from lines 157-166 is not thematic in the chapter. The division of inhibitors according to CA inhibition mechanisms is not important in describing the SFA method.
4.	Lines 240-242: I wonder what resulted from discovery of a compound that is so tight bound to CA IX that the binding can not be measured by the SPR method? Is such compound a good candidate for CA IX-targeting agent? And is the SPR method appropriate for searching for suitable ligands?
5.	Lines 291 and 297: It is necessary to add the number of carbonic anhydrase IX to headings. There is only „CA-targeted“ but the meaning is „CA IX-targeted“.
6.	Line 305: I think that in the phrase "who are at risk of relapse" it would be appropriate to add „who are at high risk of relapse“, because unfortunately there is a risk of relapse for all patients.
7.	Capture 4.1: In this part you do not mention the monoclonal antibody VII/20, which also has the therapeutic potential. I would recommend adding data from the publications Zatovicova et al. 2003 and Zatovicova et al. 2010.
8.	Line 340: I would recommend changing the word order in the sentence to: „To demonstrate the anti-tumor effect  of CA IX inhibition in vivo,...“
9.	Line 359: There is no specification which „compounds may be suitable...“
10.	Line 430: The use of the term "hypoxic cancers" does not seem to me to be appropriate. In my opinion, it would be better to use, for example, "hypoxic tumor areas".
11.	Line 442: You note that antibodies binding does not affect the catalytic activity of CA IX. Could it be possible that it is affected by antibodies with the ability to internalize CA IX protein?


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: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX
Review round: 2
Reviewer: 1

Output:
Basic reporting: the manuscript is now suitable for publication
Experimental design: not applicable
Validity of the findings: suitable
Additional comments: none


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: ANTIBODIES TO BORDETELLA PERTUSSIS ANTIGENS IN MATERNAL AND CORD BLOOD PAIRS: A THAI COHORT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article meets all basic reporting criteria.
Experimental design: The work is original and the study design is appropriate and clearly explained. The methods are well described and the analyses and presentation of results are adequate.
Validity of the findings: The investigators establish the poor level of protection against pertussis in both mothers and infants in a Thai population, setting the stage for additional work in support of maternal immunization with Tdap. Interestingly, this work in a location where whole cell pertussis vaccine is given to infants provides an opportunity to understand the role of adolescent and maternal vaccination with acellular pertussis vaccine followed by whole cell vaccine in infants.
The discussion and conclusions of this study are well stated and supported by the data presented.
Additional comments: Well written manuscript, with relevant and up to date references, clear description of methods, analyses and results. The figures are illustrative of the key findings of the study.


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

Input:
Title: ANTIBODIES TO BORDETELLA PERTUSSIS ANTIGENS IN MATERNAL AND CORD BLOOD PAIRS: A THAI COHORT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figure 1, showing the distribution of declared pertussis cases according to age from 2011 up to 2016 is not very informative, as only for 2015 (n=51) and 2016 (n=72) the figures can be split  in reasonable numbers. I would suggest to put the data of Figure 1 in a Table and also indicate how many of the cases were laboratory defined and by what method.
Experimental design: No comment
Validity of the findings: One minor comment on the conclusions:
-Line 149 states: There were no statistically significant differences in the proportion of recently infected rates in women born before or after the EPI program: statistics on 1 vs 2 cases? This can not be used for a statistical analysis
Additional comments: A general comment:
Line 227: Our results explained the susceptibility for pertussis among newborn infants in Thailand and supported the requirement for a pertussis booster vaccine during pregnancy. It is clear that pertussis booster vaccination during pregnancy will increase the antibodies in newborns, but it is also  clear there are no actual correlates of protection for pertussis.  Although antibodies to PT and Prn certainly play a role in protection, there is also evidence that other factors such as local IgA levels and cell-mediated immune responses are important.


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: ANTIBODIES TO BORDETELLA PERTUSSIS ANTIGENS IN MATERNAL AND CORD BLOOD PAIRS: A THAI COHORT STUDY
Review round: 2
Reviewer: 1

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