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: NON-LINEAR CURVE ADJUSTMENTS WIDEN BIOLOGICAL INTERPRETATION OF RELATIVE GROWTH ANALYSES OF THE CLAM TIVELA MACTROIDES (BIVALVIA, VENERIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: MS Ref: PeerJ_24725
Title: “Non-linear curve adjustments widen biological interpretation of relative growth analysis of the harvested clam Tivela mactroides (Bivalvia, Veneridae)"
Authors: Alexander Turra, Guilherme N. Corte, A. Cecília Z. Amaral, Leonardo Q. Yokoyama, Márcia R. Denadai

REVIEWER COMMENTS:

The present manuscript submitted to “PeerJ” compares different types of models (simple linear, power and polynomial models) to assess allometric growth of Tivela mactroides, aiming to improve the biological information gathered in studies of relative growth.

Overall, the manuscript is well written, organised and presented, providing interesting and valuable information on this research topic. Accordingly, I support the acceptance of the manuscript for publication in “PeerJ” following major revision (please see the general and specific comments listed below).

General comments:

Since the trigonal clam (T. mactroides) is a locally important fishing resource along the Brazilian coast, targeted by fishermen and recreational harvesters, the number of specimens analysed in the study is surprisingly low. In fact, after one-year of monthly sampling (Nov 2003 - Oct 2004) only 187 individuals were sampled (≈ 15 individuals per month). Nevertheless, the samples comprised individuals with a broad size range (3.0–39.0 mm in shell length) and the adjustment of the models presented invariably high determination coefficients and statistical significance levels. Still, I invite the authors to explain why such a “limited” data-set was used in the present study.

The results revealed that “the power function was more suitable to describe the relationships involving shell length and weight parameters”, i.e. the “classic” model (Y = aXb) for analysing relative growth remained the best approach for assessing isometry vs. allometry in relationships involving both linear and ponderal variables. On the opposite, “morphometric relationships between shell parameters… were better represented by polynomial functions”. In this context, I invite the authors to improve their discussion on this subject, by explaining why relationships between shell measurements (linear variables) required more complex models (polynomial functions), whereas in relationships between shell measurements (linear variables) and weighings (ponderal variables), the “classic” power function was the most suitable model and more complex models did not provide any added-value.

In the Discussion section, authors limit their explanations to the results obtained in the present study and do not provide comparisons with analogous studies performed with this species. For this reason, if available, I invite the authors to add and compare data gathered in previous studies on morphometric / allometric relationships in T. mactroides. Ideally, authors should highlight eventual differences resulting from the adjustment of different models (“classic” vs. “complex”) and the main advantages of adopting the present approach in terms of the information obtained about morphometric trends during growth (i.e. ontogenetic shifts) related to changes in the species biology / ecology.

Specific comments:

Title:
- I suggest removing the word “harvested” from the title, because it does not add relevant information about the objective and main conclusions of the study.

Abstract:
- line 18: Please replace “economically exploited” by “fishery-exploited”.
- line 20: Please replace “Nevertheless” by “However”.
- line 22: Please replace “overcome poor and misleading results” by “overcome poor results and misleading interpretations”.
- line 26: Please replace “fishery resource” by “fishing resource”.
- lines 27-28: Please replace “to each other and to soft parts (dry weight (DW)” by “among each other and with soft parts of the organism (dry weight (DW)”.
- line 31 (and throughout the entire text): Please replace “(i.e.,) by “(i.e.).
- line 33: Please replace “represented” by “described”.
- lines 35-36: Please replace “benthic invertebrates” by “marine bivalves”.
- line 38: Please replace “benthic invertebrates” by “molluscs”.

Introduction:
- line 44: Please replace “permits” by “allows”.
- line 47: Please replace “allows” by “provides”.
- line 50 (and throughout the entire text): Please replace “(e.g.,) by “(e.g.).
- line 50: Please replace “shortage of food” by “food availability”.
- line 50: Please replace “spawning periods” by “gonad maturation and spawning periods”.
- line 52: Please replace “in the ontogeny” by “during the ontogeny”.
- line 54: Please replace “selectivity of dredges and meshes” by “size-selectivity of fishing gears”.
- line 59: Please replace “Gaspar et al. 2001; Gaspar et al. 2002;” by “Gaspar et al. 2001, 2002;”.
- line 61: Please replace “another part x” by “another body part x”.
- line 64: Please replace “the two body parts” by “the body parts x and y”.
- line 65: Please replace “Nevertheless” by “However”.
- lines 66-67: Please replace “resulting from marked changes in the environment or physiology, for example” by “for instance resulting from marked changes in the environment or physiology”.
- line 73: Please replace “to find out which one better fits” by “to ascertain which better fits”.
- line 79: Please replace “Therefore, the variation” by “Therefore, variation”.
- line 81-82: Please replace: “growth of five different populations of the bivalve Pinna nobilis, for example, registered significant variation in the growth among the populations” by “growth of the bivalve Pinna nobilis for example, registered significant variation in growth among five populations”.
- line 85: Please replace “development of gonads” by “development and maturation of gonads”.
- line 86: Please replace “than to the shell” by “than to shell deposition”.
- line 88: Please replace “are done with a large” by “are performed on a wider”.
- line 93: Please replace “Tivela mactroides,” by “Tivela mactroides (Born, 1778),”.
- line 93: Please replace “fishery resource” by “fishing resource”.
- line 94: Maybe “eastern South American coast” is better described as “Southeastern America coast”. Please clarify.
- lines 96-97: Please replace “investigated if alternate models” by “investigated whether alternative models”.
- line 97: Please replace “more biological” by “additional biological”.

Materials and Methods:
- line 102: Please replace “Tivela mactroides” by “T. mactroides”.
- line 104: Please replace “food resource” by “feeding source”.
- line 106: Please replace “tourists” by “recreational harvesters”.
- line 110: Please replace “done” by “performed”.
- line 111: Please replace “contains” by “comprises”.
- line 117: Please replace “the dry weight of the soft parts” by “the soft parts dry weight”.
- lines 119-120: Please replace “(Vasconcelos et al. 2008; Vasconcelos et al. 2016)” by “(Vasconcelos et al. 2008, 2016).”
- lines 123-124: Please replace “dry weight of the soft parts” by “soft parts dry weight”.
- line 124: Please replace “calculated” by “recorded”.
- line 127: Please replace “that related” by “that relate”.
- lines 128, 130, 131: Please replace “(reference or independent variable)” by “(independent variable)”.
- lines 128-129: Please replace “dry weight of the soft parts” by “soft parts dry weight”.
- line 131: Please replace “the dry weight of the soft parts” by “the soft parts dry weight”.
- line 137: Please replace “fitted curves” by “fitted equations”.
- line 151: Please replace “case of more than one having” by “multiple models having”.
- lines 152-153: Please replace “which was the one with the smallest number of parameters to be estimated.” by “i.e. the model with fewer estimated parameters”.
- line 156: Please replace “from the” by “issued by”.

Results:
- line 162: Please replace “High values of r2 (>0.842) were found in all the models” by “High determination coefficients (r2 > 0.842) were obtained in all models”.
- line 172: Please replace “20 mm; after this size,” by “20 mm L; above this size,”.
- line 172: Please replace “faster in relation to the shell height” by “faster compared to shell height”.
- line 174: Please replace “after maturity” by “after reaching maturity”.
- line 176: Please replace “(with shell length less than 20 mm)” by “(below 20 mm L)”.
- line 177: Please replace “in weight of soft parts” by “in soft parts weight”.
- line 177: Please replace “after maturity” by “after reaching maturity”.
- line 179: Please replace “during the development of individuals” by “during growth”.
- line 180: Please replace “dry weight of soft parts” by “soft parts dry weight”.
- line 181: Please replace “in relation to the dry weight of soft parts” by “in relation to soft parts dry weight”.

Discussion:
- line 189: Please replace “true pattern” by “real growth pattern”.
- line 192: Please replace “growth of the trigonal clam T. mactroides.” by “growth of T. mactroides.”.
- line 195: Please replace “prevents” by “prevent”.
- line 198: Please replace “as dry weight, shell weight, and” by “as shell weight, dry weight and”.
- line 201: Please replace “for many of the morphometric relationships in marine invertebrates” by “for many morphometric relationships in some marine invertebrates”.
- line 204: Please replace “among the individuals” by “among individuals”.
- lines 205-206: Please replace “between soft tissues and shell parameters” by “between shell parameters and soft tissues”.
- lines 208-210: Please replace “inhabited by T. mactroides. This species occurs in relatively exposed sandy beaches with an abundant quantity of suspended material (Narchi 1972), which may supply the
species with enough food” by “inhabited by T. mactroides, which occurs in relatively exposed sandy beaches with abundant suspended material (Narchi 1972) that supplies enough food”.
- line 211: Please replace “constant supply of food” by “constant food supply”.
- line 214: Please replace “consequence of living in disturbed waters” by “consequence of inhabiting disturbed waters”.
- line 219: Please replace “By using polynomial functions” by “By adjusting polynomial functions”.
- line 223: Maybe by “migration” authors mention a depth segregation between juveniles in the subtidal and adults in the intertidal. In this case, maybe authors could replace “migration” by “subtidal-intertidal migration throughout ontogeny”. Please clarify.
- line 225: Please replace “tide level” by “tidal level”.
- line 226: Please replace “(burrowing behavior, for example)” by “(e.g. burrowing behaviour)”.
- line 227: Please replace “Alterations in the morphometric relationships of bivalves” by “Alterations in bivalves morphometric relationships”.
- line 228: Please replace “the environmental conditions” by “the prevailing environmental conditions”.
- lines 229-230: Please replace “The shell of the pill clam Pisidium subtruncatum (Malm 1855), for example, change” by “For instance, the shell of the pill clam Pisidium subtruncatum (Malm 1855) changes”.
- lines 231-232: Please replace “This change enables the individuals to burrow in coarse sediments easier.” By “, which enables the individuals to burrow easier in coarse sediments.”
- line 233: Please replace “species which change” by “species that change”.
- line 235: Please replace “abrupt change in the shell shape,” by “abrupt change in shell shape during growth,”.
- lines 235-236: Please replace “from squarer to more elongated, as the individuals grow (Barón et al. 2004).” by “from squarer to more elongated (Barón et al. 2004).”
- line 238: Please see the comment provided above regarding the “migration”. Maybe authors could replace “migration process done by this species” by “subtidal-intertidal migration undertaken by this species”. Please clarify.
- lines 242-243: Please replace “Maturity of individuals is also a” by “Maturity is a”.
- line 244: Please replace “than to the shell” by “than to shell growth”.
- lines 246-247: Please replace “in their allometric growth during their life cycle” by “in allometric growth during their life cycles”.
- line 247: Please replace “utmost importance to be able to detect” by “utmost importance to detect”.
- line 248: Please replace “adequate represent” by “adequately represent”.
- line 250: Please replace “to locate the discontinuities” by “to discern discontinuities”.
- line 255: Please replace “bringing important knowledge for the understanding of the life history” by “bringing further information for understanding the life history”.
- line 256: Please replace “living resources” by “fishing resources”.
- line 259: Please replace “show that more” by “indicate that more”.
- line 260: Please replace “maximizes its shell growth” by “maximizes shell deposition”.
- line 262: Please replace “Only after maturity, a faster increase in somatic elements” by “Only after reaching maturity, a faster increase in somatic tissues”.
- line 263: Please replace “should include individuals with shell length between 25-30 mm length preferably.” by “should target individuals with shell length between 25-30 mm length.”.
- line 264: Please replace “individuals were able” by “individuals are already able”.
- line 265: Please replace “local clammers since a better ratio meat/shell would be achieved.” by “local harvesters since a higher meat yield (ratio meat weight/shell weight) would be achieved.”

Conclusions:
- line 269: Please replace “of the harvested clam Tivela mactroides” by “of T. mactroides”.
- line 270: Please replace “environmental or physiological process.” by “environmental or physiological processes.”.
- line 271-72: Please replace “we perceived modifications” by “we perceived subtle modifications”.
- line 275: Please replace “more informative results” by “more informative conclusions”.

Tables:
- Tables 1 and 2: Please standardise the notation / abbreviation used for the third-order polynomial equation, since it is presented as “(Cb)” in Table 1 and as “(Cub)” in Table 2. Please revise and correct accordingly.
- Table 2 - line 1: Please replace “coefficient of determination” by “determination coefficient”.
- Table 2 - line 3: Please replace “of all the models” by “of all models”.
- Table 2 - line 4: Please replace “DW, dry weight of the soft parts” by “DW, soft parts dry weight”.
- Table 2 - line 5: Please revise whether it should be “(Cb)” as Table 1 or “(Cub)” as in Table 2.
- Table 2 - line 7: Please replace “are in bold” by “are highlighted in bold”.
- Table 2 - Column “SE”: Whenever possible, please use only 3 decimal places in the SE values (just like in all other columns presented in Table 2).

Figures:
- Figure 1 - Map: Maybe authors could improve the map by indicating the main areas where the specimens were collected.
- Figure 2 - Legend: Please replace: “Scheme indicating the measurements” by “Schematic representation of the measurements”.
- Figure 3 - Legend: Four graphs display b values (“b>1*” and “b<3***”) to highlight the type of relative growth between variables (i.e. “b>1” = positive allometry and “b<3” = negative allometry) in the models adjusted using the power function (Y = aXb). In addition, I presume that the asterisks denote the statistical significance (* = p < 0.05 and *** = p < 0.001) of those allometry coeeficients. This information (b values and respective asterisks) must be added in the legend of Figure 3, duly described in the Results section and explained in the Discussion section.
- Figure 3 - Models equations and SE values: Whenever possible, please use only 3 decimal places (just like in the r2 values and p values displayed in Figure 3 and in almost all columns presented in Table 2).
- Figure 3 - Graphs Y-axes: In the five graphs with values above 1, please remove the unnecessary decimal places.
- Figure 3 - Graph DW vs. SW: In order to keep conformity with all the other graphs, I suggest changing the order of the variables (i.e. X = shell weight; Y = dry weight) and accordingly revise the parameters of the adjusted model.


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: NON-LINEAR CURVE ADJUSTMENTS WIDEN BIOLOGICAL INTERPRETATION OF RELATIVE GROWTH ANALYSES OF THE CLAM TIVELA MACTROIDES (BIVALVIA, VENERIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article reports the findings of an assessment of non-linear curve adjustments of relative growth analyses of Tivela mactroide. The paper is interesting and revealed the importance that classical and more complex models in studies of allometric growth of benthic invertebrates.

The article is clearly laid and all the key elements are present (abstract, introduction, methodology, results and discussion).

Please carefully read through the manuscript and correct inconsistencies and issues with orthography - grammar and style improvement (especially space between words). Overall, the paper is writen in clear English, mistakes are comparatively rare.
Experimental design: This is a original primary research within aims and scope of the PeerJ. The research question well defined, relevant and meaningful. However, some questions about methods and experimental design need to be clearer.

The sampling design and sampling area were not described and contain no information about replicates. It is not possible to understand the sites where the sampling was made neither by the map or by the succinct description of the study area.
The authors mentioned in the introduction that the growth and shape of the shell are normally influenced by environmental factors, but there is nothing about characterization of the physical variables in the study area. Although the physical variables characterization was not one of the aims, it is important to characterize the environment to strengthen the hypotheses addressed in the discussion.

The number of individuals was extremely low for allometric growth analysis. It was used approximately 15 individuals per month and only 5 individuals per size class. In one of the cited studies (Gaspar et al 2002), the shell morphometric was performed on 25 species with a measured N of approximately 300 individuals per species.
Validity of the findings: The authors used the small-sample bias-corrected form of the Akaike Information Criteria, AICc (Hurvich & Tsai 1989) of the AIC (Burnham & Anderson 2002a) for the model selection, but why not to use Akaike (AICw)? Weights measure the relative likelihood of a model being the best for the given data. This is a straight-forward procedure in a multimodel inference routine (Burnham & Anderson, 2002).

The discussion, in general, is suggestive and repetitive. The discussion still needs some work on the flow of logic and clarity of ideas. Example, in first paragraph Ln 199-202: “The usual approach when studying allometric growth in marine species is to a priori adopt the classical linear or allometric model (power function), which may have implications in the accuracy and precision of the estimated parameters (Katsanevakis et al. 2006; Rabaoui et al. 2007)”.  The authors can not state this based on their own results. Depends on the selected parameters, in its study the power function was more suitable to describe all the relationships involving shell length and weight parameters.
Additional comments: Maturity of individuals is also a frequent cause of a distinct change in morphology, given that more energy is allocated to the development of gonads than to the shell (besides considerable changes in weight). Would not it be more interesting to consider sex in the analysis?

The authors mentioned that allometric growth analyses are performed using the linear equation and power function; and that these equations do not consider discontinuities in growth, besides may mask important biological information, however, it was the best model selected for half of the relations evaluated, especially relationships involving shell length and weight parameters. What do you suggest about it?

Time is really an important factor in growth analysis. Do you consider a year as the ideal period for this kind of study? In addition to the allometric growth, individuals were separated by size class for the one of the novelties of the study: evaluate relationships between the dry weight of soft parts and the shell weight and ash-free dry weight. Is an N of 5 individuals sufficient to characterize the population standard?


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: NON-LINEAR CURVE ADJUSTMENTS WIDEN BIOLOGICAL INTERPRETATION OF RELATIVE GROWTH ANALYSES OF THE CLAM TIVELA MACTROIDES (BIVALVIA, VENERIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well-written; language is concise, unambiguous and technically correct. It also conforms to technical standards of courtesy and expression.

The manuscript includes a sufficient literature review to provide adequate introduction and background relative to recent approaches in the study of allometric growth in bivalves. Relevant literature seems appropriate referenced; however, the titles of the articles in the list of references appear either as sentences (e.g. Corte 2015) or as titles (i.e., capital as the first letter of each word, see Caill-Milly et al. 2014).

The three figures are relevant to the content of the article, they are appropriately described and labeled and their quality and resolution are good though could be improved; for example, equations in Fig. 3 would be clearer using a larger font and fewer decimals. Both tables are also relevant and clear. The provided raw data file is complete and clear.

I believe that the article is self-contained in the sense that all results relevant to its hypothesis are included.
Experimental design: The research question, i.e. the potential shortcomings of “traditional” allometric growth models, is well within the scope of PeerJ. The topic is well defined, relevant and meaningful, and the results of this case of study are valuable. The investigation was conducted rigorously, and the methods are described with sufficient information and clarity to be replicated.
Validity of the findings: Data used in this article are statistically robust; the number of examined organisms is large, and the size-classes are well represented within the sample. The methods and criteria used for selecting the best model in each case are also adequate. In particular, it is commendable that the principle of parsimony was applied even though the alternative models that the authors are proposing are the less parsimonious. Even though, the authors were able to identify minor, subtle changes in growth which could be meaningful, and which are not detectable by the traditional approach. Up to this point the conclusions are properly stated and supported.
Additional comments: This submission is a good example that the scope and decision criteria of PeerJ are adequate. For the most part the results were negative in the sense that the “traditional” power function model showed the best fit to the data. If negative results were not accepted, this article could be rejected based on a subjective determination of low impact or not enough novelty. Though the conclusions are properly stated and supported, and since PeerJ welcomes speculation as long as it is clearly identified as such, I would have liked to see a more extensive discussion on the meaning and possible causes of the subtle changes in allometric growth.


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: NON-LINEAR CURVE ADJUSTMENTS WIDEN BIOLOGICAL INTERPRETATION OF RELATIVE GROWTH ANALYSES OF THE CLAM TIVELA MACTROIDES (BIVALVIA, VENERIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see "General comments for the authors"
Experimental design: Please see "General comments for the authors"
Validity of the findings: Please see "General comments for the authors"
Additional comments: MS Ref: PeerJ_24725_v1
Title: “Non-linear curve adjustments widen biological interpretation of relative growth analyses of the clam Tivela mactroides (Bivalvia, Veneridae)"
Authors: Alexander Turra, Guilherme N. Corte, A. Cecília Z. Amaral, Leonardo Q. Yokoyama, Márcia R. Denadai

GENERAL EVALUATION:

After another careful revision of the manuscript, I can confirm that the authors adopted most changes and improvements suggested by the reviewers in the previous evaluation of the manuscript. Accordingly, I recommend the acceptance of the manuscript for publication in PeerJ following “minor revision” (please see the specific / detailed comments provided below).

Specific comments:

Running head:
- line 13: I suggest replacing “Allometric growth of Tivela mactroides” by “Relative growth in Tivela mactroides”.

Abstract:
- line 22: Please replace “have been suggesting” by “have suggested”.
- line 24: Please replace “different models” by “diverse models”.
- line 31: Please replace “soft part weight parameters” by “soft parts weight parameters”.

Introduction:
- line 46: Please replace “Vasconcelos et al. 2016” by “Vasconcelos et al. 2018”.
- line 53 (and throughout the entire manuscript): Please replace “e.g.,” by “e.g.”.
- line 54: Please replace “Vasconcelos et al. 2016” by “Vasconcelos et al. 2018”.
- line 59: Please replace “Vasconcelos et al. 2016” by “Vasconcelos et al. 2018”.
- line 64 (and throughout the entire manuscript): Please replace “i.e.,” by “i.e.”.
- line 66: Please remove: “, for example”.
- line 67: Please replace “models which consider” by “models that consider”.
- line 75: Please replace “Vasconcelos et al. 2016” by “Vasconcelos et al. 2018”.
- line 79: Please replace “patterns on the shell” by “patterns of the shell”.

Materials and Methods:
- line 110: Please replace “16 Km” by “16 km”.
- line 115: Please replace “sea temperature” by “seawater temperature”.
- line 117: Please replace “variations are only found near the rivers” by “variations only occur near the rivers”.
- line 122: Please replace “through October 2004” by “until October 2004”.
- line 123: Please replace “Fig 1” by “Fig. 1”.
- line 127: Please replace “At the intertidal zone” by “In the intertidal zone”.
- line 131: Please replace “at the intertidal area” by “in the intertidal area”.
- line 135: Please replace “30 samples (dredgings) were collected per month” by “30 samples were dredged per month”.
- line 139: Please replace “had their shell length measured” by “measured for shell length”.
- line 146: Please replace “the soft parts dry weight and the shell weight” by “the shell weight and the soft parts dry weight”.
- line 148: Please replace “(Vasconcelos et al. 2008; 2016)” by “(Vasconcelos et al. 2008, 2018)”.
- line 169: Please replace “estimeted” by “estimated”.
- line 170: Please replace “rather than being from only one” by “rather than from one”.

Results:
- line 199: Please replace “relations” by “relationship”.
- line 204: Please replace “The allometric growth” by “The relative growth”.
- line 206: Please replace “allometric growth between shell length and width was always positive” by “relative growth between shell length and width was always positive allometric”.
- lines 207-208: Please replace “20 mm in length,” by “20 mm in L,”.
- line 208: Please replace “show that” by “showed that”.
- line 209: Please replace “; however, a rapid” by “followed by a rapid”.
- line 214: Please replace “observed” by “established”.

Discussion:
- line 225: Please replace “show that” by “showed that”.
- line 242: Please replace “allometric regressions models” by “allometric regression models”.
- line 253: Please replace “sea temperature” by “seawater temperature”.
- line 254: Please replace “also contributes” by “probably also contributes”.
- line 258: Please replace “between shell measure parameters” by “between shell parameters”.
- line 259: Please replace “in the morphometric relationships” by “in morphometric relationships”.
- line 260: Please replace “Alterations” by “Changes”.
- line 261: Please replace “normally” by “usually”.
- line 268: Please replace “which shows” by “that shows”.
- lines 279-280: Please replace “burrowing in this sediment.” by “burrowing in this type of sediment.”.
- line 283: Please replace “Besides bringing” by “Besides providing”.
- line 285: Please replace “relationships between soft parts and shell” by “relationships between shell size and soft parts weight”.
- line 289: Please replace “was observed” by “was recorded”.
- lines 289-290: Please replace “should target individuals with shell length between 25-30 mm preferably.” by “should preferably target individuals with shell length between 25-30 mm.”.
- line 291: Please replace “, and also provide” by “, thus providing”.

Conclusions:
- line 300: Please replace “recommendations” by “recommendation”.

References:
- lines 436-439: Please replace:
“Vasconcelos P, Moura P, Pereira F, Pereira AM, and Gaspar MB. 2016. Morphometric relationships and relative growth of 20 uncommon bivalve species from the Algarve coast (southern Portugal). Journal of the Marine Biological Association of the United Kingdom:1-12.
10.1017/S002531541600165X”
By:
“Vasconcelos P, Moura P, Pereira F, Pereira AM, and Gaspar MB. 2018. Morphometric relationships and relative growth of 20 uncommon bivalve species from the Algarve coast (southern Portugal). Journal of the Marine Biological Association of the United Kingdom:98(3):463-474.
https://doi.org/10.1017/S002531541600165X”

Tables:
- Table 2 - line 6: Please replace “suitable model” by “suitable models”.

Figures:
- Figure 3 - Legend: Please replace “describing the relationship” by “describing the morphometric relationship”.
- Figure 3 - Models equations and descriptive statistics: Please use 3 decimal places in all “b” and “SE” values (please check figures c, d, e and f).


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: ONE-POT MULTICOMPONENT NITRO-MANNICH REACTION USING A HETEROGENEOUS CATALYST UNDER SOLVENT-FREE CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comments
Validity of the findings: no comments
Additional comments: Manuscript id: Peer J -23415
Title: One-pot multicomponent nitro-Mannich reaction using a
 heterogeneous catalyst under solvent-free conditions

Authors: Giovanna Bosica and Ramon Zammit

An environmentally-friendly, one-pot multicomponent reaction of various aldehydes, amines and nitroalkanes for the synthesis of β-nitroamines is described using Amberlyst A-21 supported CuI catalyst.

The Ms can be accepted for publication after minor revision.

1.One of the minor problems of this manuscript is English, which needs through editing.

2.  Delete Fig 2.   as advantages are summarized in conclusion.


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: ONE-POT MULTICOMPONENT NITRO-MANNICH REACTION USING A HETEROGENEOUS CATALYST UNDER SOLVENT-FREE CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article meets the standards for being published in PeerJ.
Experimental design: The article meets the standards for being published in PeerJ.
Validity of the findings: The article generally meets the standards for being published in PeerJ.

However, there is one experiment that should be carried out and reported to fully support the statement that the catalyst is truly heterogeneous. Indeed, the only way to acertain that the catalytic activity is strictly and solely the effect of the solid (and not for example from the 1% Cu that is leaching) is to perform a "hot filtration test". The reaction should be stopped at an early stage, when conversion has started but did not reach high values. Then the catalyst is removed (e.g. by filtration) and the filtrate is further allowed to react in the exact same conditions. If conversion value do not evolve at all, then the heterogeneous nature of the catalysis is confirmed.

When mechanistic insights are provided, it is not always clear what is speculation and what is known from the litterature (e.g. 153-155). In the latter case, a reference would be needed.
Additional comments: Though the paper is clearly of high quality (and interestingly includes valuable insights on the "greenness" of the process), I would like to make some suggestions.
1. abstract "catalysts can be reused up to 8 times". This statement is misleading. Authors have tested 8 recycling and found only minor deactivation (10%). But it is for the next user to decide if they want to recycle the cat further. Statement should be something linke "The catalyst has been tested up to 8 times with only a minor activity loss".
2. a genera Scheme of the Mannich reaction would nicely complement the first paragraph of the intro
3. Line 45-46. It is not correct to state that stoechimetric amount of catalyst is used. A catalyst is by definition not stoechiometric.
4. Line 54 "have substituted". What do authors mean? Industrially, everywhere, heterogeneous catalysts are used and not homogeneous cats? Only 1 specific paper is cited to support the sentence... Or maybe authors meant "have been shown to also catalyse"...
5. 68. Please clarify the sentence "which could be immobilized on a solid". Indeed, i the table, I see highly soluble species like KOH, Na2CO3, etc.
6. line 241. What does "nano-sized copper salt" means?


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: ONE-POT MULTICOMPONENT NITRO-MANNICH REACTION USING A HETEROGENEOUS CATALYST UNDER SOLVENT-FREE CONDITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Comments for the authors

The authors describe an environmentally-friendly one pot multicomponent reaction of aldehydes, amines and nitroalkanes using the Amberlyst A-21 supported copper (I) iodide.  The present nitro-Mannich type reaction tolerated versatile aldehydes and amines and regioselectively afforded the beta-nitroaminoalkanes in high yields.  Furthermore, the reaction was achieved in a solvent-free condition.  The authors also show the environmental acceptability of the nitro-Mannich reaction by calculating both the atom economy and the E-factor (as shown in eq 1 and 2).  The catalyst seems to be practical and useful according to the investigation of Table 8 (Comparison of E-factore of the catalytic systems) and Table 9 (which shows the percentage of copper ions that leached into the solution from the heterogeneous catalyst). The manuscript seems to be suitable for the article of the PeerJ after minor revisions.

1)	Text: line 33: synthesis via umpolung chemistry ----- synthesis of umpolung chemistry
2)	Experimental Section: please check the 1H NMR.
4e: 1H NMR: 2H is missing.
4g: 1H NMR: 1H is too much.
4j: 1H NMR: 1H is too much.
4k: 1H NMR: 2H is missing.
3)	The authors should cite the reference on the Aza-Henry reaction of nitriles with copper iodide/cesium carbonate/DBU in nitromethane (Org. Lett. 2018, 20, 1130).
Experimental design: good
Validity of the findings: good
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: SEASONAL DYNAMICS OF TERRESTRIAL VERTEBRATE ABUNDANCE BETWEEN AMAZONIAN FLOODED AND UNFLOODED FORESTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: While the authors repeatedly state on the investigation of the hypothesis of seasonal movements of terrestrial invertebrates between the investigated forest types, their research design does not entirely allow for a true test of this hypothesis. The camera trap stations monitor abundance and richness data allowing for spatial and temporal comparison, but do not allow for the detection of movements.  While I acknowledge that the results can be interpreted in that way, I suggest rewording the related statements in the abstract and the Introduction (i.e., L10-22, L113-114), which could make the general scope of the manuscript much stronger.  In the end, vertebrates can move within the single forest types, entering the inventoried region from up- or downriver, etc., and these movements are not considered nor discussed.
Experimental design: As the primary scope of the manuscript is crystal-clear (investigate spatial and temporal differences in abundance and richness, and relate them to abiotic variables such as elevation and flood pulse), I wonder why the authors tried to incorporate anthropogenic disturbance as an additional factor in in their analyses (L118-120). Anthropogenic disturbance is difficult to assess and quantify, and I cannot see the benefits of including it here.  Rather, I think simply excluding it would give more emphasis to the main result and message of the paper. If the authors decide to leave it in, they should also describe the results on that in more detail and include some discussion on it.

In the second sample design (L164-169), it is not clear how many CTS were established in flooded and how many CTS were established in terra firme forest. It would also be good to know if CTS established in varzea were located at the same or different elevations (= flood levels), as I expect huge floristic differences (=resource differences) between highly flooded and less flooded forest types within varzea. For example, phenological peaks during the high water season is mostly concentrated on highly inundated trees (low-varzea trees), while fruiting of less inundated (high-varzea) trees is much less synchronized with the flood level and season (i.e. Parolin et al. 2010, book chapter ed. by Junk et al. 2010, Springer).
Validity of the findings: I found the section on conservation implications a bit long and wordy. In addition, not all information given here is directly related to your results. I think this topic could be significantly shortened.
Additional comments: Some minor comments:
L120-124: A good reference here would be from Sabo et al. (2005, Ecology 86:56-62): “Thus conservation planners can easily increase the number of species protected in a regional portfolio by simply including a river within terrestrial biodiversity reserves”.

L133-134: The statement about the climate classification can be deleted.

L135-137: Not sure about the numbers you present here “…peak water levels of 8-12 m”. You mean: flood amplitude is 8-12 m? Please state exact numbers from the water gauge.

L137: 65-170 m above sea level includes terra firme, right? Please state the elevational range for varzea and terra firme separately here.

L139: Not sure about “small-scale selective logging”.  In fact, all varzea forest is (intensely) selectively logged even if far away from bigger cities.  You may better state that logging is impeded since the establishment of the RDS.

L147-149: I would place this info in the acknowledgements, or in a separate section at the end of the manuscript.

L321: …and consumption by fish and other aquatic foragers.

L394ff: “…communities with greater access to terra firme are more agricultural….” Don’t agree. Just the crops change. Usually, agriculture is much more intense in varzea than it is in terra firme because of the nutrient-rich soils that allow for several crops per year.  See Junk et al. (2000), or McGrath et al. (2005, PLEC News and Views).

L398: Heavea spp. should be Hevea spp.

Fig.1:  Instead of low and high elevations, you should give the exact altitudinal range for the colors used in the map.


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: SEASONAL DYNAMICS OF TERRESTRIAL VERTEBRATE ABUNDANCE BETWEEN AMAZONIAN FLOODED AND UNFLOODED FORESTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reporting is clear and professional. However, a quick check by a good proof reader could take out some typos, such as choose for chose, exploitating for exploiting, and at for on.

The derived data is shared, but in common with most camera-trap studies, the images are not. These could be deposited somewhere.
Experimental design: Within the logistical limits, the design is good.
Validity of the findings: The findings are straight forward and the conclusions well stated, with caveats when necessary.
Additional comments: The study involved a great deal of work and is well done. You might like to look at the paper by Lyn Branch (Primates 24(3):424-431), which indicated that some species move into the várea at high water.


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: SEASONAL DYNAMICS OF TERRESTRIAL VERTEBRATE ABUNDANCE BETWEEN AMAZONIAN FLOODED AND UNFLOODED FORESTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: - Clear and unambiguous, professional English used throughout: no comments.
- Literature references, sufficient field background/context provided: Yes
- Professional article structure, figs, tables. Raw data shared: Yes
- Self-contained with relevant results to hypotheses: Yes
Experimental design: - Original primary research within Aims and Scope of the journal: Yes
- Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap: No. This point presents some problems. See General Comments to the authors.
- Rigorous investigation performed to a high technical & ethical standard: Yes.
- Methods described with sufficient detail & information to replicate: Yes.
Validity of the findings: - Impact and novelty not assessed: This point should be improved (See comments)
- Data is robust, statistically sound, & controlled: Yes.
- Conclusion are well stated, linked to original research question & limited to supporting results: There are some problems with this point (See comments to the author).
- Speculation is welcome, but should be identified as such: Some problems with this point and for implications for conservation (See comments to the authors).
Additional comments: I have carefully read the manuscript #25773. The authors carried out an evaluation of the migration of the fauna in a forest system that is under the stress of seasonal flooding. I believe the general aim of the manuscript is in line with the objectives of the journal. Moreover, the study area deserves special attention, given its important biodiversity and accelerated environmental degradation.

However, I recommended a reorganization of the structure and content of the paper in order to facilitate its understanding. For example, authors must add clear mentions of (1) the problem or gap of knowledge (Introduction), (2) the hypothesis/questions (Introduction), (3) the different predictions (Introduction), (4) the usefulness of each analysis or sampling design to answer each question. Moreover, a straightforward discussion about the implication of the results to conservation is needed.

I addressed major comments in this text, and some minor comments in the PDF.  (English is not my mother tongue; please excuse any errors on my part).

Abstract
1. Line 20. "We examined the hypothesis of seasonal movements between two adjacent forest..". Whose movements?
2. Lines 23-24. "Species richness differed in neighboring terra firme forests between the high-and low-water phases of the flood pulse". Please, specify what the difference was (higher richness during the high-water phase?).

Introduction
1. The second paragraph (about the hunting problem of migratory species) felt very disconnected from the previous one (which talks about the importance of wetlands) and from the next (which talks about the flooded systems in the Amazon).
2. The gap of knowledge, problem statement or justification of the importance of this study, is not well addressed in the Introduction. For example, the two paragraphs preceding the Objective are basically an explanation (with facts and citations) of the lateral movements of the fauna between varzea and terra firme. Curiously, the abstract did mention (although imprecisely) the existence of a gap on knowledge.
3. Lines 105-109. The authors mentioned the prediction, but not the hypothesis. I believe the hypothesis is hide in this paragraph, but it is important to make it clear.
4. Lines 115-117 "To test this hypothesis we conducted camera-trapping surveys in both terra firme and várzea..." Again, what hypothesis? The existence of lateral movement?
Methods
1. I think that a clearer structure and explanation of the link between the questions/predictions/hypothesis and the design/analysis is needed. In general, the Methods felt like a great amount of tests, whose purposes are difficult to detect.
For example, the Design 1 (160-162), which compares terra firme fauna during both periods and uses a paired t-test, makes sense to me. With this analysis we can test if the prediction was correct (i.e. high richness in terra firme during the high-water phase). However, it is not clear why did the authors use the distance intervals in the sampling. It was for evaluating the effect of anthropogenic threats (a secondary objective)? Or did they expect high richness in sites closer/farther from the varzea interface?
On the other hand, the Design 2 only compares terra firme and varzea during the low-water phase (Lines 164-166). Thus, I am not sure about how this design can test the prediction of "a higher richness in terra firme (only) during flooding than during low water level" or to test the hypothesis of lateral movement (if this is the hypothesis). What were the expected results from this design? What if the richness in terra firme were lower than in varzea during the low-water phase? I suspect that this design is more useful to understand how the species migrate among forests. In any case, it would be helpful to describe the purpose of this analysis in Methods or to write the specific question that this design will answer.
2. Lines 218-220. "We tested the hypothesis of seasonal faunal movement between forest types and investigated the effects of landscape context and anthropogenic disturbance employing Generalized Linear Models (GLMs)". What are the response variables? How did the authors test the hypothesis of the existence of lateral movement (if this is the hypothesis) by doing these GLMs? By testing if the water level is selected as predictor of species richness in terra firme? Please, clarify this.

Discussion
1. Line 218. Here is the first description of a hypothesis.
2. Lines 290-291: "Salvador, Clavero & Leite Pitman (2011) reported that floodplain forests in the Peruvian Amazon are more species-rich than terra firme forests during the dry season". This result is contrary to yours (lower richness in flooded forests). Why? Is this difference important?
3. Line 305-307. "These results imply that forest fauna can exhibit ephemeral occupancy of várzea sites during the dry season...". With this sentence the purpose of Design 2 is a little clearer. I don't know if I am right, but for me the Design 2 is more related to testing if species from terra firme forests are also present in varzea, which may support (although not confirm) the existence of species migration. In other words, I believe the importance of Design 2 is more about species absence/presence than richness.
4. The discussion about Conservation Implications is quite messy, with several distinct ideas mixed within the same paragraph, and without clear statement sentences or concluding sentences. Moreover, there is an extensive literature review, and it is difficult to distinguish which arguments are derived from such review or from the results. I recommend a more straightforward discussion about the conservation implications. For example, given the result that várzea and terra firme forests are interconnected habitats that are seasonally utilized by a suite of mobile species, the authors might organize the discussion in this way (it is just a suggestion): (1) the synergistic effect of having threats from connected terrestrial and folded systems, (2) the importance of protecting such forests in a spatially connected way (and also in management), (3) the current way in which these systems are being protected (pros and cons), and (4) the challenges of protecting such systems.
5. Line 358. This (and others) paragraph presents a mix of different ideas. The statement sentence is about the anthropogenic threats, but the rest of the paragraph is an extensive revision about hunting (who relevance or relation to the results is not clear).
6.  Lines 375-376. "The existing protected area network and management policies were created principally to protect terrestrial ecosystems and are therefore not adequate to mitigate the aforementioned impacts". Please clarify which are the aforementioned impacts (only hunting?). Moreover, why protected areas that are designed to protect terrestrial ecosystems cannot mitigate hunting? I think the big problem of Amazonian reserves is more related to the challenge of monitoring remote and big areas, and the limited budget.
7. Line 375. I do not understand the main idea of this paragraph. Is about the current problems of Amazonian protected areas? The authors seem to criticize the establishment of protected areas with sustainable-use because their limited effectiveness. But, what about the importance of such type management for local populations and their livelihood? There is a lot of literature focused on understanding the complex effect of management types. Be careful.
8. Line 389. What is the main conclusion or idea of this paragraph? That varzea and terra firme have different threats and... ? It feels like just a description of threats, without a conclusion or implications.
9. Lines 406-408: "Different Amazonian forest types exhibiting staggered resource pulses must be included within the same or neighboring sustainable-use protected areas". This sounds contradictory, because I got the impression that the authors criticize the protected area with sustainable use. Moreover, creating large areas that include terra firme, varzea, etc has some important challenges / implications.


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

Input:
Title: A NEW, THREE-DIMENSIONAL GEOMETRIC MORPHOMETRIC APPROACH TO ASSESS EGG SHAPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Clear, unambiguous, professional English language used throughout.

OK, some minor suggestions made throughout the text.

•	Intro & background to show context. Literature well referenced & relevant.

I really liked introduction section. All background showing the broad importance of this study (from comparative analyses to poultry industry) is in this section.
Experimental design: •	Original primary research within Scope of the journal.

Yes, the study is within PeerJ Scope. It presents a new method for accessing egg shape variation. Knowing how egg shape varies from within clutches to across species is very important for comparative analyses (it is also relevant for poultry industry) but has not received enough attention due to difficulties in evaluating it. The method presented in this paper for accessing egg shape variation is potentially important for future studies, but need improvements (see general comments, specially those of lines 129-130 and 392-393).

•	Methods described with sufficient detail & information to replicate.

OK. More than that, scripts for analysis routines are also provided.

•	Conclusion well stated, linked to original research question & limited to supporting results.

The manuscript lacks a general conclusion. The last paragraph of discussion only concludes about the relevance of this method for poultry industry.
Authors need to include a paragraph summarizing the importance of their method for future studies, based on the last paragraph of the introduction.
Validity of the findings: •	Data is robust, statistically sound, & controlled.
It is, but see General Comment of Introduction (lines 129-130).
Additional comments: The manuscript is well written, designed, and organized, presents a new and important methodology for accessing egg shape variation, but need some improvements. The most important are listed below. Small ones are in the annotated pdf (please ignore annotations in blue).

Abstract:
The authors state (abstract, line 40) that: “The approach developed in this study to assess variation in shape is freely accessible…”
Although the method is freely available, this statement may be misleading since the costs for acquiring a 3D scanner can be prohibitively high. Authors should substitute “approach” for “routine” or something similar or compare the costs of their method with other methods for obtaining egg parameters.

Introduction:
In the last paragraph (lines 129-130) authors state: “The aim of this study is to investigate within- and between-clutch variation of avian egg shell shape”, whereas the manuscript title is: “A new, three-dimensional geometric morphometric approach to assess egg shape variation within and between clutches”.
These statements must agree. For me it seems that the study is more about a new method, as stated in the title, but using a “case-study” with a few selected species to present the use of their method. If the focus is on investigating variation in egg shape within- and between-clutch, it seems that data is not robust enough and analyses would require including phylogenetic information. If the focus is on a new method, comparing (e.g. cost, error, etc) with previous 2D methods is lacking in the manuscript and is absolutely necessary.

Methods:
Unable to know the costs and how easy to implement this method.

Lines 207-208: There are other methods, using mathematical equations that should be mentioned here and even implemented in your study:

Preston, F. W. 1969. Shapes of birds’ eggs: extant North American families. - The Auk: 246–264.
Troscianko, J. 2014. A simple tool for calculating egg shape, volume and surface area from digital images. - Ibis 156: 874–878.

Lines 249-256: Authors criticize using 2D egg contours due to possible imperfections on radial symmetry of eggs. I think they should show how much error is associated with “subtle differences in curvature around the entire eggshell” to show the reader how much improvement their 3D method brings compared to 2D methods. Is really misleading assuming radial symmetry?

Lines 281-283: See comment at lines 391-392.

Results:

Lines 391-392: Troscianko (2014) must be cited here. I am sure that it is possible to quantify the same kind of differences in egg shape using his approach, especially because the main sources of variation (PC1 and PC2) were related to elongation and tapering, respectively. Possibly, by using Micro-CT you can find even finer-scale differences, but according to the results they were unimportant in describing egg shape.
I also do not like using Principal Components to describe egg shapes because of repeatability of results (interpretation of PC) in future studies. PCs are used to reduce dimensionality of multivariate data, but their interpretation is usually subjective and depends on the correlations among variables and variable loadings on each PC (Cadima and Jolliffe 1995). Because egg shape may be easily decomposed in specific parameters, I do not see any reason for using PCs. Using specific parameters (see Troscianko 2014, Preston 1969) will make egg shape results comparable across studies.
Given that PC1 and PC2 were related to elongation and tapering, I strongly suggest using: 1) the same (or equivalent) parameters/indexes used in Troscianko (2014), Preston (1969), Havlíček et al. (2008), and/or Narushin (2001); 2) using directly elongation, tapering, radial symmetry imperfections, etc. instead of Principal Components.
It is a shame that Troscianko’s did not implement reporting the value of egg shape parameters, because it does not allow comparing results among studies. Would be very helpful for future studies if you could do that using your approach.

Cadima, J. and Jolliffe, I. T. 1995. Loading and correlations in the interpretation of principle compenents. - Journal of Applied Statistics 22: 203–214.
Preston, F. W. 1969. Shapes of birds’ eggs: extant North American families. - The Auk: 246–264.
Troscianko, J. 2014. A simple tool for calculating egg shape, volume and surface area from digital images. - Ibis 156: 874–878.

Table 1:
Most of table 1 columns (i.e. Order, Clade, Body length, Body mass, Primary diet, Clutch size average, Clutch size range, Nest material, Nest shape, Nest site, Incubation, Incubation period, Young development) are unnecessary since all this information was not was not evaluated in the analyses.

Order: not needed since all species are Passeriformes

Table 3:
Headings are incomplete. It is not possible to know if clutch refers to a unique clutch/set id or to clutch size of that set. Also it is not possible to know what “variation in shape” refers to. Is it a PC1, PC2, or another parameter?


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

Input:
Title: A NEW, THREE-DIMENSIONAL GEOMETRIC MORPHOMETRIC APPROACH TO ASSESS EGG SHAPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No Comments.
Experimental design: No Comments.
Validity of the findings: In this paper, the authors present a new way of quantifying egg shape based on 3D geometric morphometrics derived from micro-CT data. This method offers some advantages over traditional methods of shape description because it incorporates details about curvature around the entire eggshell. Due to the novelty of this approach, I think this paper works well as a methods paper and for demonstrating proof-of-concept.

Next the authors ask the following:

“With these data, we investigate whether there is greater shape variation within a clutch than between clutches for a given species, and ask if egg shape be used to distinguish between bird species? According to general assumptions in evolutionary comparative approaches across species or taxa, intraspecific variation in egg shape should be lower than interspecific variation (e.g., Felsenstein, 1985). If natural selection is acting on egg shape, we would expect differences in egg shell shape will reflect adaptations that will optimise incubation and/or hatchability (Hoyt, 1976).”

My main concern here is that the authors have a low sample size: 4 clutches only for each of their 4 target species, for a total of 16 clutches. To me, it seems critical to include an analysis that shows the point at which adding additional clutches per species no longer increases the occupied morphospace for that species. In other words, in Figure 3, what happens if you add 1 or 2 (or 3, 4, 5…) more clutches of Grey shrike-thrush? Does morphospace increase or stay the same in size? What is the effect of your sample size on your analyses, and how might low sample sizes lead to bias? Without addressing this, it’s difficult to know whether the observed differences within and between species are unbiased. Do the authors have access to more than 4 clutches per species? Finally, how do the authors intend to test the prediction that “if natural selection is acting on egg shape, we would expect differences in egg shell shape will reflect adaptations that will optimize incubation and/or hatchability”?

The authors write in the discussion that:

“Surprisingly, we find no evidence of selective pressure in the form of a constraint on egg shape for the species investigated. Overlap in shape independent of size across species suggests that either there is little constraint or selection on shape across these diverse taxa, or that shared selective influences have led to convergence.”

How have you demonstrated this? What specific hypotheses were you testing, and how did your tests lead to this conclusion? There might be some "hidden" constraints. All four species are similar in development, incubation and even average clutch size. Perhaps altricial species are, in fact, constrained to your observed egg shape morphospace. What if egg shape is related to clutch size, and selection for eggs of a particular shape is, in fact, strong for clutches of these sizes? With so few species (4) and no phylogenetically controlled statistical models, these statements are a bit of stretch -- and require more explanation.
Additional comments: I think this study would be improved if the authors could address the two points raised above: issues with sample size and treatment of ecological/life history traits in assessing adaptive hypotheses for egg shape. The solution might lie in focusing on the benefits of the shape analysis approach (which is already done to great effect, and could be improved upon by direct comparison to other shape quantification methods) rather than on the study’s ability to resolve questions about adaptive explanations for egg shape variation.


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

Input:
Title: A NEW, THREE-DIMENSIONAL GEOMETRIC MORPHOMETRIC APPROACH TO ASSESS EGG SHAPE
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: Understanding egg shape variation is an important and timely topic, and in this revision the authors argue that 3D geometric morphometrics is the way forward, providing “a more precise representation of eggshell curvature.” However, the data provided in this study do not show that the method provides a more precise representation. As the authors write in their response “We recognize the need to evaluate the degree of error for different 3D and 2D approaches to measure egg shape...but unfortunately [that] is beyond the scope of this study.” Therefore, I think the authors can present their method as a promising new tool which could be more precise – but statements like these in the abstract are, in my mind, not supported by the current work.

 In fact, as Reviewer 1 noted, the variation detected by PCA still reveals the classic axes of egg shape variation: elongation and asymmetry. It is therefore difficult to know what the advantages of micro-CT/ 3D morphometrics may be over cheaper methods (photography). Because the authors no longer have access to the physical egg clutches used in this study, they cannot provide a comparison of different methods. An additional challenge, raised also by Reviewer 1, is that principal components can be difficult to interpret. In particular, using PCs to characterize a morphospace (see Figure 3) can be problematic, because PCs change depending on the data used. Choosing 4 different species would likely reveal different PCs, so the space is not robust to additional datasets or studies. Why not use specific parameters to define your space, as suggested by Reviewer 1? The authors defend PCA but it seems that nothing would be lost by using the rich morphometrics data to also calculate specific parameters (like elongation and asymmetry).

  “As such, we find no evidence of selective pressure in the form of a constraint on egg shape for the species investigated.” I am still not sure what you mean here. The fact that there is egg variation between and within clutches does not show that there are no constraints on egg shape. There likely are real biophysical constraints on egg shape (eggs are not cubes, for example).

 Looking at Figure 4, I am surprised to see that the turquoise bars in B (presumably representing Superb fairy-wren) show considerable among-clutch variation, but this does not correspond to the low species-level variance shown for SFW in A. Then I looked at Table 4 and I must confess I am very confused. There seems to be no correspondence between the color of the clutches in B and the clutches shown in Table 4. For example, looking at SFW clutches (which I thought were in turquoise in B), the clutches listed here in Table 4 are E10499 (red in Fig. 4 B), E12643 (red in Fig. 4B), E13865 (blue in Fig. 4B), and E14555 (orange in Fig. 4B). What is going on here? I am not sure how to interpret these data any more.

 In sum, I think this paper still has potential, but the focus should be on the possible merits of the new method.  I am excited about the new method and I think the case study works well here for demonstrating that it can work well for showing subtle intra-clutch variation. Statements about how this method is more accurate should be tempered, however, given the current data. I think discussing the need for investigating inter- vs. intra-clutch variation is an important one, but I am no longer sure what is being shown in Figure 4B and how that relates to Table 4. Finally, I would remove any discussion of selection pressure on egg shape. With only 16 clutches representing 4 species, your data seem too limited to comment on this.


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: PATHWAY ANALYSIS IDENTIFIES ALTERED MITOCHONDRIAL METABOLISM, NEUROTRANSMISSION, STRUCTURAL PATHWAYS AND COMPLEMENT CASCADE IN RETINA/RPE/ CHOROID IN CHICK MODEL OF FORM-DEPRIVATION MYOPIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Title: Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/choroid in chick model of form-deprivation myopia (#24645)

Giummarra L, et al., present an interesting manuscript investigating the mechanisms of form-deprivation myopia and recovery from FD myopia using chick model, and identified the involvement of mitochondrial metabolism, neurotransmission, structural pathways, complement cascade, and bile acid and bile salt metabolism pathways in retina/RPE/choroid. This manuscript is well written and structured. Authors employed genome-wide gene expression profiling and various analysis methods to investigate the role of different signaling pathways in myopia development. This study provides interesting insight into the role of retina/RPE/choroid in FD myopia model, but some questions need to be addressed for the consideration of publication.
1.	One of the major concerns for this study is the pooling of samples, or pooling n=5 for each conditions to n=1 for Affymetrix chips. Pooling samples together from a couple of individuals in general is not preferred if researcher can obtain enough sample or RNA from each eye. Collecting enough RNA from each eye should not be a problem for this study since combined retina/RPE/choroid tissues are being collected, and more than 1 ug of RNA can be easily collected from each eye for microarray assay. Authors please give the reason of pooling samples together. Please also give explanation that n=1 for each condition works.

In addition, authors emphasized the similarity between retina/RPE/choroid and retina/RPE (McGlinn et al. study) (Line 153-154), as well as pooled and individual samples (Line 190-197). They also discussed the limitation of this study at the end of discussion. However, the difference between these situations should be discussed more. First, every tissue has unique anatomical properties and functions. For example, choroid is a very complex tissue which include large amount of vessels, endothelium cells and other cell types that are very different from retina and RPE. Retina also contains so many different cell types including neurons that may respond to FD or defocus differently with different period of treatment. While RPE in nature is epithelial cell. These tissues are so different and they may act differently in myopia development. Second, experiments using combined tissues are a kind of practice of pooling sample in nature. Third, pooling samples together from different animals can be problematic in many ways, and should be only considered when no enough sample is available under certain conditions (L190-197).

2.	Figure 1 is very confusing from Introduction to Materials and Methods, as well as Results:
Line 69: Is Figure 1 the result of current study or from references? I checked references listed here but did not find anything related to Figure 1. Legend of Figure 1 indicated that it is curtesy of Egan, G. If this is true, then no figure is needed in this manuscript (just add reference), and no need to describe MRI in Materials and Methods. Please revise manuscript to make it clear.
If Figure 1 is the result of this study as described in Materials and Methods – MRI imaging section, then please describe MRI methods and results as appropriate.
Please label Figure 1, including retina, choroid, and sclera in both panels if this is your original research. It is very confusing that authors indicated 24 h of recovery after FD treatment in this study, but MRI figure (1B) was taken 72 h of recovery.

3.	Results for ocular biometrics and Figure 2:
It is appreciated that authors collected extra data to compliment McGlinn et al. study, but these data should be plotted separately. The reason is that these data were collected from 7 days old chicks which is different from current study. Plot these data together can be misleading (Figure 2A & 2B).

I suggest plotting Figure 2B AL and VCD in separate bars. The reasons are: 1) it is hard to compare ALs when they are stacked on top of VCDs which ends at different levels. 2) Readers may misinterpreted black + gray bar as the axial length of the eye, and adding AL and VCD in one bar is not meaningful anyway.

4.	Enrichment analysis for short-term FDM induction and Table 1
Summarize genes identified from McGlinn et al. study to 13 enriched pathways and Table 1 is not informative. It will be better to provide gene list for each identified pathway.

Minor points include
1.	Line 80, line 135, line 156, line 163-164, etc.: Please be consistence with form-deprivation and form deprivation used in manuscript, as well as 6hrs, 72hrs, 6 hr, 24 hr; short term, short-term, long term, long-term;
2.	Line 129: Is this study using 10 days FD treatment? Typo here for “following 7 days of translucent occlusion”?
3.	Line 187: Typo “n=65”
Experimental design: Authors need to make clear sample number for each experiments
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: PATHWAY ANALYSIS IDENTIFIES ALTERED MITOCHONDRIAL METABOLISM, NEUROTRANSMISSION, STRUCTURAL PATHWAYS AND COMPLEMENT CASCADE IN RETINA/RPE/ CHOROID IN CHICK MODEL OF FORM-DEPRIVATION MYOPIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/choroid in chick model of formdeprivation myopia” by Giummarra et. al. has profiled transcriptome wide gene expression changes in during the induction and recovery from form-deprivation myopia (FDM) in chick. The GSEA analysis revealed important pathways in the pathophysiological process. Overall the manuscript is well executed with clear background knowledge and experimental approaches. However, the rationale/hypothesis were not well described in the result section for the experiment/analysis performed. The quality of this manuscript could be improved by addressing the following concerns. At the present stage of the manuscript, I suggest considering for publication in PeerJ following a revision.
Experimental design: 1.	I suggest validating core gene expression changes, at least one gene of each of the enriched pathways by an independent method (for instance, quantitative PCR method).
Validity of the findings: 2. The title of a figure legend should describe the figure succinctly, not the experiment. I suggest using conclusions of the result as title of the result sections as well as Figure legend.
3.	I suggest including rational of the experiments, preceding the actual experiment and conclusions in each “Result” sections.
4.	Figure1; please move the MRI imaging method to the “Materials and Methods” section.
5.	Figure 2 a, b, c, supplementary figure 1; include statistical significance test p and r value on the figures and wherever applicable on other figures.
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: PATHWAY ANALYSIS IDENTIFIES ALTERED MITOCHONDRIAL METABOLISM, NEUROTRANSMISSION, STRUCTURAL PATHWAYS AND COMPLEMENT CASCADE IN RETINA/RPE/ CHOROID IN CHICK MODEL OF FORM-DEPRIVATION MYOPIA
Review round: 2
Reviewer: 1

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


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

Input:
Title: PATHWAY ANALYSIS IDENTIFIES ALTERED MITOCHONDRIAL METABOLISM, NEUROTRANSMISSION, STRUCTURAL PATHWAYS AND COMPLEMENT CASCADE IN RETINA/RPE/ CHOROID IN CHICK MODEL OF FORM-DEPRIVATION MYOPIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have significantly improved the clarity of the manuscript. I recommend to consider the manuscript for publication in PeerJ after resolving the concerns raised by the first reviewer.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: OSTEOHISTOLOGY OF LATE TRIASSIC PROZOSTRODONTIAN CYNODONTS FROM BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The fossils studied are really important to understand life history and physiology around mammal origins. It is original and important what the authors have done.
This paper treats Brasilodon and Brasilotherium as separate taxa – the standard way – although it hints from unpublished data from some of the authors that they may represent different ontogenetic stages of the same species. The histological data presented supports the latter hypothesis.

The discussion of several taxa in lines 375-382 is in part confusing. I think the ‘However’ in line 378 misleads, as the finding in Irajatherium does no contradict what is reported just before – it just leads to interpretations of some kind, etc. Please revise this section.

In terms of bone compactness and small, burrowing mammals, the authors may wish to consult this work on bone compactness in talpids:
Meier PS, et al (2013) Evolution of bone compactness in extant and extinct moles (Talpidae): exploring humeral microstructure in small fossorial mammals. BMC Evolutionary Biology 13:55

Figure 5 is very useful. It would be good to add in the caption the source of some of those events mapped other than those treated in the manuscript (for example, reference for point 5 on vibrissae). This reference attempted something similar but at a much more general and rigorous level – it may be outdated but the authors may wish to check if there is information there to be integrated or commented upon.
Sánchez-Villagra MR (2010) Developmental palaeontology in synapsids: the rock record of ontogeny in mammals and their closest relatives. Proc Roy Soc London, B. 277: 1139-1147.

The authors may consider adding a table summarizing the histological features described for the different taxa, also those cited from the literature. I do not see this as essential.

In several places of the manuscript, the word ‘interestingly’ is used (e.g., lines 382, 397, 446) – this is a stylistic aspect criticized in many writing manuals with which I concur – I invite the authors to consider this. Here I quote from Letzman 1995: ‘Rather than telling a reader that a result is significant or interesting, show them how it is significant or interesting’. The authors do already the second, so they can just delete the ‘Interestingly’. (Check if you wish a dialogue in ‘Captain fantastic’ which refers to ‘interesting’, www.youtube.com/watch?v=YXimxT2iE8I)
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Line 53 – ‘in its definition’ – it is unclear what is meant here. This sentence presents terms most readers won’t be familiar with – I think you mean that crown mammalia is part of mammaliaformes – correct? Maybe the second sentence of the Introduction, to start, adding the stratigraphic occurrence, is more clear?
Line 56: I would delete ‘unranked’
Line 70 – being this a new paragraph, I would not start with ‘However’. See also line 600.
line 165: …were so small THAT only one or two…
lines 294-295: something missing and comma misplaced.
Line 324: I would rephrase to:..and provides an example of the predominant type of growth… - is this what you mean?
Line 325: …well vascularized, COMMA
Line 341: …all of which express…
Line 347 …continued, COMMA
Line 352 …growing, COMMA
Line 703-794 – format italics journal.

There is a paper by Felder et al. 2018 that just came out on animal’s size and secondary osteons which may be relevant to consider in the discussion in this work on scaling.
Felder AA, Phillips C, Cornish H, Cooke M, Hutchinson JR, Doube M. 2017 Secondary osteons scale allometrically in mammalian humerus and femur. R. Soc. Open sci. 4: 170431. http://dx.doi.org/10.1098/rsos.170431


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: OSTEOHISTOLOGY OF LATE TRIASSIC PROZOSTRODONTIAN CYNODONTS FROM BRAZIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is overall well written; the language is clear and unambiguous. The authors take great care in explaining terms which is very helpful for following the results and discussion. I only noticed a few typos in line 165 “than” should be replaced with “that”; line 242 “there” should be replaced with “it”, line 607 “Morganucodon (Fig. 5(6)), that which is typical of extant placental” delete that or which.

The figures are relevant and of high quality. The raw data in form of images is already included in the manuscript. However, I would suggest the following minor edits

1. Figures 1–4: to better relate the structures described in the text with the images it would be helpful to label examples of e.g., Fig. 1 large resorption cavities, vascular canals, primary osteons; Fig. 2 primary osteons, radiating canals osteocyte lacunae and so on.

2. Figure 5: Please explain the color pattern (change from yellow to red, why are some taxa white and others black) in the figure caption. I would further suggest replacing clade names with the names of the species mention in the discussion (for example Procynosuchus, Triarachodon, Massetognathus). Though the figure might take up a bit more space it would make it easier to relate the section on implications for cyndodont physiology to the figure

3. Figure 2: LZB is not listed in the abbreviations
Experimental design: no comment. The manuscript meets the standards listed: the research question is well defined, the authors specifically point to the gap in knowledge that this research is filling, the bone histology is described in great detail, the methods are sufficiently detailed.
Validity of the findings: no comment. The manuscript meets the standards
Additional comments: 1. The authors are in few instances vague in their use of derived prozostrodontians and mammaliaform. For example:

line 58 “the Tritylodontidae and Tritheledontidae, both closely related to the Mammalia”
it would be more correct to use Mammaliaformes here as the sister clade.

line 427 “because the only mammaliaform that has been sectioned is the...” as all extant mammals are mammaliaforms this statement is not correct. I do see what the authors mean; but it would be more appropriate to use non-mammalian mammaliaform or basal mammaliaform

line 48, 630 “small derived prozostrodontians” I suggest changing this to non-mammaliaform prozostrodontians. As derived prozostrodontians could refer to any derived mammal today as well.

There might be other instances and I believe it would be good to check the manuscript carefully. It is all too easy to slip into using mammaliaforms or prozostrodontians as if it would only refer to the basal forms within this group.

2. Growths and life span of Morganucodon. New, but unpublished data (presented for example at the International Symposium on Paleohistology 2015 by Newham et al.) on cementum suggest that Morganucodon might have grown for much longer time periods. Even Chinsamy and Hurum (2006) indicated that several LAG’s were visible in the ulna of Morganucodon though they do not mention or indicate how many. I see that this will be fairly difficult to address as the cementum data is not published yet, but it might be possible to include Chinsamy and Hurum’s (2006:p.328) statement on Morganucodon: “This area includes several rest lines, which indicate pauses in the rate of bone formation, and hence, pauses in growth.” Though the authors note that brasilodontids had a relatively more protracted growth period, it seems possible that it might have been even longer for Morganucodon.

3. line 43 Late Triassic/Early Jurassic mammaliaform Morganucodon and Late Cretaceous multituberculate mammals. I would suggest to change “and Late Cretaceous multituberculate mammals” to “and the Late Cretaceous multituberculates Nemegtbaatar and Kryptobaatar” to be more specific and consistent with the reference to Morganucodon


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: OSTEOHISTOLOGY OF LATE TRIASSIC PROZOSTRODONTIAN CYNODONTS FROM BRAZIL
Review round: 1
Reviewer: 3

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: Botha Brink and her co-workers are the leaders when it comes to bone histology of therapsids.

This paper gives a detailed account of the histology of Late Triassic forms that are mammals closest relatives. The account given of the histology is a valuable contribution and I recommend publication of this portion of the paper. However the authors expand far beyond the important  descriptive part  and provide a rather confusing account of the relationship of bone histology to endothermy.

I think it would be improved if they clarify the ontogeny of different bone types in these advanced brazilosdontids and compare this to mammals of the same size.

As understand their conclusion the first type of bone formed is FLB indicating rapid growth this is followed by PFB that includes a lag line and this in turn by FLB and always lack a EFS and lack definitive growth? They interpret the single lag to mean that after one year only a slight amount growth occurs. It would be helpful if more attention was paid to the meaning of these layers. The authors suggest that FLB = rapid growth, PFB = slow growth. Is the latter due to seasonal changes? And if so, does the lag line occur sometime during this season when adverse environmental conditions prevent growth completely and there may be some bone resorption? Following this does some slow growth occur as conditions improve? It would be helpful if this could be compared with  the same detail in  a small mammal. Lag lines are common in marsupials, but do the small mammals they are comparing their specimens with show a single lag line followed by FLB and EFS. They conclude that many of the attributes of homoeothermic endothermic was present in the forms the authors studied and claim they possibly possesed  elevated maximum and basal metabolic rates. I suggest a few of these should be included .  Definitive growth occurs in small mammals; this is associated with endothermy  but I appreciate the authors caution in  not claiming  this for prozostrodonts.

The value of this paper is the detailed description of the bone histology of prozostrodontis and the conclusion on their growth patterns. I think the long sections on the origin of endothermy detract rather than add to the papers importance. For example the relative size of the olfactory chamber in Thrinaxodon is similar to all the non-mamalian cynodonts, I agree that some reptiles use limited endogenous heat production during parental, but   there is no credible basis for the claim that Lumkuia or other advanced forms, before mammals had extended parental care. Also there is no evidence that Morganucodon possessed ossified turnbinals. Some of these may be true but without some justification they add little to what I consider to be an excellent paper.


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: HUPERZINE A ATTENUATES NONALCOHOLIC FATTY LIVER DISEASE BY REGULATING HEPATOCYTE SENESCENCE AND APOPTOSIS: AN IN VITRO STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript addressed an important issue of huperzine A on oxidative stress and hepatocyte senescence in nonalcoholic fatty liver disease. Xiao-na Hu et al. used an in vitro fatty liver model. Data were collected on inflammatory factors, oxidation product and hepatocyte senescence. However, the manuscript cannot be accepted in its current form because it needs to be improved.

1. There are repetitions in the manuscript that needs to be addressed.
2. Figure1. The cell viability was lower in L02 cells treated with different ratio and concentrations fatty acid compared with control. What explains this? Is the concentration of fatty acids too high? Need to clarify.
3. How do you treat with fatty acids? It needs to be described in the Materials and Methods.
4. Which fatty acid concentration is used in Figure 2, 3, 4. It needs to be described in the Figure Legends.
5. Figure 2A needs to add scale.
6. Figure 2D. It's better to show the protein expression pattern.
7. GAPDH was as reference gene in your experiment, whether is suitable or not for hepatocytes. Do you have basis of some scientific researches?


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: HUPERZINE A ATTENUATES NONALCOHOLIC FATTY LIVER DISEASE BY REGULATING HEPATOCYTE SENESCENCE AND APOPTOSIS: AN IN VITRO STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was not written in professional English. There are various grammatical and non-scientific terms used throughout the manuscript. Authors should consult professional academic writing service.

The authors did not show sufficient background in the field.

Figures are in poor quality and really confusing. Be more informative in legends and state clearly what the units are for each measurements.
Experimental design: Methods are extremely ill defined. It is questionable how authors obtained their results.

Research question is not meaningful due to poor experimental design. Authors should consider re-run the experiment with better experimental design.
Validity of the findings: Data generated are not trustworthy due to problematic methods.
Additional comments: The study is poorly designed and the manuscript is not well written. There are major flaws in this study in addition to academic writting and therefore should not be considered for publication.
Major issues:
1. The author used fatty acid without conjugating to BSA, which do not represent in vivo scenario.
2. Authors used a certain ratio of fatty acids without justification of the composition and ratio or its physiological relevance. Yet the authors claimed their model was successful just because there is more lipid accumulation in the cell compared with Control.
3. Also, the author used a fetal hepatic cell line to study adult fatty liver disease.
4. The methods are poorly defined.
5. Extremely casual results interpretation without scientific rigor and thus not trustworthy. For instance, authors claimed cell senescence are altered in response to Huperzine A treatment, from Fig2, it was clear that the cell density are different between treatments. The authors disregard such pitfalls and just interpret the result to their favor.


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: HUPERZINE A ATTENUATES NONALCOHOLIC FATTY LIVER DISEASE BY REGULATING HEPATOCYTE SENESCENCE AND APOPTOSIS: AN IN VITRO STUDY
Review round: 2
Reviewer: 1

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


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

Input:
Title: HUPERZINE A ATTENUATES NONALCOHOLIC FATTY LIVER DISEASE BY REGULATING HEPATOCYTE SENESCENCE AND APOPTOSIS: AN IN VITRO STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: English has been improved grammatically, but still not academic writing level.
Experimental design: See specific comments
Validity of the findings: See specific comments
Additional comments: General comments:
The authors need to write up to scientific standard. When reporting results, every time the author claims significance or difference, statistical proof is required. Also, Materials and methods were mixed in the results section. The authors need to restrain from making general statements. Only suggest and conclude based on what your results tell you, not generalize.

Ln65-66: duplicated reference
Ln67: studies? Only 1 reference is shown.
Ln83: What is a super net?
Ln170: If authors chose to use 2- Ct method, PCR efficiency needs to be included.
Ln200-203: Describe the model used. Including fixed and random effects.
Ln209: Where’s statistical proof of difference?
Ln210: where’s your statistical proof of your claimed significance?
Ln216-218: This is not results. Move to Materials and methods section.
Ln225: avoid using words such as “obvious” in here throughout the manuscript.
Ln290-294: repetition of Ln 220-224.
Ln301: The author’s model seemed to be able to imitate some aspects of the steatosis, but authors cannot claim that the model is well established. Two-hit model is already outdated, authors should acknowledge the fact that multiple-hit model better reflect NAFLD. Therefore, authors should instead state here that the model in this study represents some aspects (lipid accumulation, inflammation) of hepatic steatosis.
Ln 304-305: This study or further studies? If further studies, where are your references?
Ln310: Which studies? Where are your references?
Ln 320: This is your objective of this study? If so, relocate it to the end of the introduction section.
Ln322: new? Specify what new anti-inflammation and antioxidative effects?
Ln323: studies? Why is there only 1 reference?
Ln329: The authors need to restrain from making general statements. Only suggest and conclude based on what your results tell you, not generalize. You only tried Huperzine A here, you do NOT have evidence to suggest other antioxidative agents’ effect.


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

Input:
Title: A REFERENCE CYTOCHROME C OXIDASE SUBUNIT I DATABASE CURATED FOR HIERARCHICAL CLASSIFICATION OF ARTHROPOD METABARCODING DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The manuscript is written in good English and is well understandable.
- The use of references/literature is appropriate and sufficient background/context is provided
-The manuscript is well structured and raw data is available.
-Results appropriatley show the performance of the used software/pipeline.
Experimental design: - The manuscript fits the aims and scope of the journal
- The reason for developing the described pipeline for taxonomic assignemnt and the need for this kind of research becomes clear.
- The described pipeline has been sufficiently tested and methods are described with sufficient detail.
Validity of the findings: -The findings are valid and the benefits of using the described approach/pipeline for building reference databases are evident.
-Conclusions are well stated and support the results.
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: A REFERENCE CYTOCHROME C OXIDASE SUBUNIT I DATABASE CURATED FOR HIERARCHICAL CLASSIFICATION OF ARTHROPOD METABARCODING DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. l. 92 Sickel et al. 2015
2. l. 93 Why do you cite Sayers et al. 2013 specifically? The "Database Resources of the National Center for Biotechnology Information" is published yearly with multiple newer iterations, e.g. https://doi.org/10.1093/nar/gkx1095 and https://doi.org/10.1093/nar/gkw1071
3. l. 138-142 This is a good way to make sure that there are no other taxonomic groups that are lost for similar reasons as Strepsiptera and Embioptera. Other groups might be lacking not entirely but in large fractions this analysis should reveal them. I can't find the results of this inventory at the three stages anywhere in the paper. It would be nice to present them in some way (at least as supplementary tables).
4. l. 222 Figure 3B
Experimental design: 1. l. 87 Why use 2500bp as the cutoff?
2. l. 88 What was the exact search term used?
3. l. 99 How was this archetypical sequence selected? Is it an actual sequence or a reconstructed ancient sequence?
4. l. 104 How does splitting the original sequences into multiple files increase the accuracy of this process?
5. l. 160 Why use 5000 instead of all (~20000) binary classification outcomes?
Validity of the findings: no comment
Additional comments: The authors describe the construction and evaluation of a COI database for hierarchical classification.
The construction and curation process is well documented and reproducible.
The evaluation of the classification performance of this database with metaxa2 is thorough.
In combination this work is useful for researchers in need of a COI classification database, as well as researchers wanting to create, curate, or evaluate a database themselves.
I raised some minor points that I would like to see addressed by the authors above.
In addition I have the following general comments:
1. It is a nice idea to publish the data and code via Github. You could consider additionally archiving it via Zenodo https://guides.github.com/activities/citable-code/ This way it will be available in the exact version with a unique doi independent of Github.
2. Using a single split into test/training set gives limited information about the robustness of estimates. The whole process could be repeated multiple times.
3. Does the final data included in metaxa2 also contain the test data?