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: GENOME-WIDE ANALYSIS OF MAIZE OSCA FAMILY MEMBERS AND THEIR INVOLVEMENT IN DROUGHT STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a very nice study on identifying and characterizing maize OSCA gene family and their involvement in drought stress of maize. The authors have done a wide range of research works including phylogenetic analysis, synteny with the rice, maize, and sorghum genomes, expression profiles, et al. More importantly, a significant association between the natural variation in ZmOSCA4.1 and drought tolerance at the seedling stage was identified. Combine with it's expression changes in response to drought stress, the results suggest that this gene might play an important role in drought stress tolerance in maize. This paper provides a solide foundation for future study the functions of ZmOSCA genes in response to water deficit stress, as well as understanding of the natural variation in drought tolerance in maize. I can't find any technical problems in the work, it seems impressive. I recommend to publish in Peer J.


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: GENOME-WIDE ANALYSIS OF MAIZE OSCA FAMILY MEMBERS AND THEIR INVOLVEMENT IN DROUGHT STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: In the paper entitled “Genome-wide analysis of maize OSCA family members and their involvement in drought stress”, Ding et al. identified 11 ZmOSCA genes from Maize based on the protein homology with Arabidopsis thaliana. The authors also identify the OSCA genes that upregulated or downregulated in response to drought stress at the deferent developmental stages.    They also showed the difference homology between of the OCSA gene families from rice, sorghum, maize and Arabidopsis.
These findings advance our understanding of the homology between OSCA genes and would be importance for future functional and physiological studies.

However, few major and minor points should be addressed in the study:

1-	The authors showed the protein domain and transmembrane regions of ZmOSCA based on the TMHMM Server V2 and they predicted OSCA has 9 transmembranes. Recently 4 different research articles were published on the structure of Arabidopsis OSCA1.1, OSCA1.2 OSCA3.1 and rice OSCA1.2 (Zhang et al., Jojoa-Cruz et al., Murthy et al and Maity et al). All those articles demonstrated that OSCA protein has 11 transmembranes. The authors did not mentioned those studies or make protein domain based on the known structure of OSCA. The authors should update figure 3 based on the structure of those 4 papers and include the new results and udpdate the discussion.
2-	There have been many reported microarrays and RNA-Seq under drought stress on Arabidopsis and other plants and the data available publicly. The authors should  compare their finding to the other plant.

Minor comments
1.	In figure 1, the ZmOSCA 1.1a and ZmOSCA3.1 is small protein. Authors should make sure whether it is a real protein. Since, ZmOSCA 1.1a does not have full sequence in compare to others OSCA from maize or Arabidopsis, the change of expression level in figure 5 and 6 of that gene might not be real.
2.	The scale bar of the heat map figure in 5A is not clear. Should be explained.
3.	The relation between OSCA genes and drought were mentioned in the discussion. However, no literature have been cited.


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: LEAF ANATOMY AND ULTRASTRUCTURE IN SENESCING ANCIENT TREE, PLATYCLADUS ORIENTALIS L. (CUPRESSACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript follows the form of a scientific paper. The English language is reasonably good but needs to be improved. Use of tense is incorrect throughout the manuscript making it difficult for the reader to understand when the authors are describing the results from the current manuscript and results from published papers. There are also minor mistakes or different forms of spelling, or mixing nouns and verbs, which need to be corrected (e.g. ageing – aging, identify – identity, crop – corp).

Introduction describes the context but it is rather long; it can be shortened. For example, it is not necessary to compare human/animal ageing to plant ageing, whereas examples between different type of plants, such as conifers and deciduous trees, are relevant. There is repetition in Introduction, especially when discussing senescence of leaves – part of this could be removed or used in Discussion.

I’d suggest to start the introduction by definition of ancient trees and describing the Mausoleum of Yellow Emperor forest, followed by description of Platycladus orientalis (conifer, evergreen, scale leaves). Then moving into senescense of trees and leaves and the aim of the study.

There are a number of relevant references. It is always the question which references should be left out; one could for example take out the papers referring to human ageing.

Figures and tables are relevant to the study and correctly numbered and referred to in a systematic way throughout the text. However, the image quality is not optimal regarding optimal illumination and resolution (Fig. 2-4).
Experimental design: The manuscript fits in the scope of the journal. The research as such is not very original as senescence of leaves have been studied intensively in different crop plants – but it is justified as a study focusing on the senescence of ancient trees. However, the authors should state more clearly the need of the study. For example, why would you expect that senescence of ancient tree leaves would differ from other plants? Are there emerging problems with vitality of P. orientalis, possibly related to climate change or other environmental factors? The authors could underline even more the importance of the tree species and thus, more detailed knowledge of its anatomy and physiology would gain understanding its life cycle and future management.

Sample collection, preparation and analyses have been performed systematically.

Materials and methods are quite well described, but repetition must be avoided. Some improvement is needed: 1. State more clearly that senescent degree evaluation was performed according to the government standards. 2. State more clearly how the one-year-old leaves were chosen as the right time point is crucial for the study. State also what time of the day the samples were collected – as the amount of starch in chloroplasts depends on the timepoint. 3. I don’t understand the sentence starting on line 154. 4. Line 160-162: State more clearly the composition of FAA. Write ‘graded series of alcohol’ as a mixture of alcohols was used. 5. Is there a reference for staining with Safranin and Fast Green? Otherwise describe the staining. Were they used as counterstains only or as histochemical stains? 6. Line 175: repetition, just write ‘One-year-old leaves were collected…’. 7. Line176: ‘width with 4 mm long’? Line 178-179: ‘0.1 mol l-l phosphate…’? pH 7.3. Line 181: ‘dehydration was performed…’ Line 183: ‘semithin’ rather than ‘half-thin’. Line 189: ‘300 and 10.000’ without mikrometers.
Validity of the findings: The findings are not unexpected – the level of senescence has proceeded from healthy to senescent trees.

It would be good to state more clearly how the one-year-old leaves were identified as the whole study depends on the correct sampling. It would be informative to show the visual morphology of the leaves by adding images of healthy, sub-healthy and senescent shoots to Fig. 2, and pointing out the one-year-old leaves. Does the colour differ between the groups? Also, it would be important to know which time of the day the samples were collected – as you have compared starch granules between the leaf samples.

About light microscopy (Fig. 2): the large opening in the left (Fig. 2b) looks like a resin duct as it is surrounded by parenchymal cells. Are you sure the opening marked with ‘rc’ is a resin duct and not air space between the mesophyll cells? Please, re-check. The same for Fig. 2c – how can you tell which is resin duct and air space? I have not, of course, looked at the samples in microscope where it may be easier to see the difference.

I would prefer changing the order between TEM images, showing Fig. 4 before Fig. 3 as it would help to look at the low-magnification images of the cells before going into details.

In connection of ultrastuctural studies, one should always take to account the possibility (risk) of artefacts due to the extensive sample preparation. For example, the ultrastructure of healthy leaves is not as good as one could expect: cytoplasm is not well preserved and the thylacoid membranes, though clearly having the layered structure, appear as negative (white) towards the background – when they should be dark as both osmium and lead citrate were used in sample preparation. Possibly the initial fixation with a strong 4% glutaraldehyde has blocked the effect of following 2.5% glutaraldehyde and osmium fixation. The internal membranes (cristae) in mitochondria are even less well preserved. Having said this, it is of course important for comparisons that all samples were treated in a similar way.

The comparisons are based on statistics which is fine. However, it may be that it is to some extent overdone. When counting different parametres it is important to remember that you are looking at 2-D sections of cells – you do not have the information for the whole 3-D cells unless keeping track of serial sections. For example, I am hesitant about measuring mitochondria length and width, as they can bend and it is random in which angle and position mitochondria happen to be in a section. I am also hesitant about measuring and counting starch granules as they are also depending on the random sections. Some compensation comes from having a number of cells analyzed.

You should check how you write about the ultrastructure in Results and Discussion – I’d say it is not correct to write about for example ‘number of mitochondria per cell’. Line 289-290: I don’t understand ‘…stacks composed mostly of chloroplast lumen’. Or line 291-292 about electron density and rapid rate of photosynthesis. Line 306-307: osmiophilic granules are not formed during the sample fixation.

In Discussion, there is quite a lot repetition of the results, which could be diminished. Some statements seem opposite to each other such line 389 ‘The senescence of whole trees…’ and line 404 ‘Leaves are a vital organ…’. Discussion has many good elements such as references to the literature about differences between deciduous and conifer trees, or importance of cuticle, but the text needs rewriting. References to the influence of environmental factors on ancient tree anatomy and physiology, and as a cue to protection, might add to the quality of discussion.

Conclusions can be written more clearly. I am hesitant about the last sentence ‘Leaf ultrastucture can be regarded as an efficient way to characterize the degree of senescence of trees.’ The study rather confirmed that the visual evaluation of tree vitality is sufficient. Besides, performing TEM is time-consuming; it can not be an efficient standard method.
Additional comments: It is good to see an anatomical study as understanding changes in anatomy is the key for understanding changes in physiology. The study is performed in a systematic way and the plants studied –ancient trees– are really interesting.

Think about the title: it is not unexpected that the anatomy becomes worse with senescence. I’d suggest a neutral title, for example: Leaf ultrastructure in senescing ancient tree, Platycladus orientalis L. (Cupressaceae). Important to check the abstract and underline the relevance of the study for future management of the ancient tree forest. I would not recommend using the word ‘artificial’ about an ancient planted forest. Fig. 5, a schematic drawing about senescence is usable in oral presentations or in a review article, but I am not convinced it is suitable for the present manuscript. If used, the senescent cell should be clearly plasmolytic.

I’d also recommend to write the whole name of the university in author’s addresses.


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: LEAF ANATOMY AND ULTRASTRUCTURE IN SENESCING ANCIENT TREE, PLATYCLADUS ORIENTALIS L. (CUPRESSACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The topic of the manuscript is interesting and shows that the authors understand the difficulties of this type of work, and hence have embarked on resolving the problem of senescence degree in the ancient trees with appropriate and many methods, and have tackled the obvious difficulty of tree sampling adequately. However, the manuscript is suffering of poor English language: the tense is wrong in many places in the text and the authors use subjective terms, such as in the title 'leaf anatomical structures become worse'. 'Worse' is not an objective description and hence I would suggest a better formulation of the title in this way 'Advancing senescence is shown in leaf anatomical structure and ultrastructure in ancient Platycladus orientalis L. (Cupressaceae)'. Also, the authors should check the use of subjective terms elsewhere in the text.
Experimental design: Experimental design is of utmost importance in work of this nature. The authors have taken samples of the trees from several experimental plots and several trees in each plot, which seems adequate. In addition, it is stated that the selected branches had no obvious damage by insects or diseases, and the sample scales were taken from mid-crown of the trees, so lower braches were avoided where natural senescence would probably be prevalent.
Validity of the findings: The different degrees of senescence are adequately described, which is vital, and the data seems statistically sound.
Additional comments: The work behind this manuscript is well executed and the data seems sound. I would strongly encourage the authors to have the English language checked by a native speaker.


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: LEAF ANATOMY AND ULTRASTRUCTURE IN SENESCING ANCIENT TREE, PLATYCLADUS ORIENTALIS L. (CUPRESSACEAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have carefully followed the comments in the previous review. The English language is much improved. However, there is still some fine tuning left to be done.

 Instead of ‘morphological structure’ and ‘anatomical structure’ it would be fine to use ‘morphology’ and ‘anatomy’ throughout the manuscript. These terms as such refer to the structure.

The authors have consequently written ‘senescent degree of ancient trees’. I wonder if ‘senescence degree’ would be more correct. Note that ‘senescent tree’ is fine.

Abstract

Line 26-27: I’d suggest ‘…morphology, anatomy and ultrastructure were conducted with one-year leaves of ancient P. orientalis…’

Line 32-33: I’d remove the sentence ‘The recession…’. Then ‘Leaf ultrastructure clearly reflected the senescence degree of ancient trees, confirming the visual evaluation from above-ground parts of trees.’

Line 36-38: Suggestion: ‘Understanding… senescence can support government (decisionmakers?) in planning protection of ancient trees more promptly and effectively, by adopting the timely rejuvenation techniques before the whole tree irreversibly recesses.’

Introduction
Line 69: ‘of’ instead of ‘which’
Line 91: ‘shares’ instead of ‘shared’
Line 113: possibly ‘abaxial and adaxial’ instead of ‘front and back’?
Line 114: ‘from’ instead of ‘with’
Line 115: ‘unique’ instead of ‘unique typical’
Line 119: ‘have been’ instead of ‘are’
Line 122: ‘senescence in ancient trees’
Experimental design: The authors have carefully followed the suggested improvements. Just some details to clarify even better:

Material and methods
Line 141-143: why not to say ‘..conducted an age investigation… of every single P. orientalis tree according to the government standards (Table 1)’.

Line 155: Transfer the meaning ‘Leaf samples were collected in June 2014.’ to the line 151.

Lines 152-154: I don’t understand the meaning. Please, correct.

Line 168: ‘calculate’ instead of ‘calculated’

Lines 173-174: I still don’t understand. Do you mean ‘One-year-old leaves of approximate size 4 x 4 mm were collected…’?

Line 178: ‘was performed’ instead of ‘effected’
Validity of the findings: Again, the authors have carefully taken to account the previous suggestions. Some comments:

Results

Light microscopy

Fig. 2 is much better now, informative and easy to understand. Figure legend have to be improved. For example a) shows a branch and the red arrow one-year-old leaves.

Line 204: I suggest to take off this first meaning.

Line 211: ‘…P. orientalis representing the three experimental groups…’

Line 235: Is it correct to write ‘upper and lower epidermis’ of this type of leaves?

Line 238: ‘vascular cells’ instead of ‘vein cells’

Line 238: You could write ‘The resin channels were located on opposite sides of the central vein.’

Line 242: ‘…smooth and round in shape.’

Line 246: ‘In senescent tree group (Fig. 2g),…’ would be more correct than ‘In senescent tree groups of different tree ages…’.

Line 247: ‘…the resin ducts broken…’

Line 248: ‘…a small number of palisade cells was identified; most spongy tissue cells had lost…’

Line 249: ‘The vascular system became disintegrated and fragmented…’

TEM

The order of TEM images is easier to follow now, good.

Figure 3 legend: ‘nucleus’ instead of ‘nuclear’
Figure 4 legend: reconsider if using ‘osmiophilic granules’ (see below for explanation)

Line 254: not necessary to repeat ‘parameters’

Line 263: ‘Crossections of mesophyll cells…’

Lines: 268-271 Better to write ‘Starch is an accumulation product of photosynthesis in chloroplasts.’ Starch accumulation should be described directly after chloroplast ultrastucture. Mitochondria after that.

Lines 273-274: ‘…cell walls, chloroplasts, mitochondria and vacuoles.’

Lines 281: ‘…bicuspid shaped. … distribution of chloroplasts had changed.’

Line 282: ‘…chloroplasts had moved…’.

Line 287-288: Maybe easier to write ‘Sub-healthy trees had many vacuoles of irregular shape distributed near the plasma membrane.’

Line 289: The sentence ‘Situation…’ can be removed or modified. Do not use ‘worse’.

Line 293: ‘Mitochondria had lost…’

Lines 294-295: Remove ‘Mitochondrial membranes showed unclear conditions…’

Line 296: ‘mitochondria had lost…’

Line 299: ‘oval shape’

Lines 301-302: Modify. ‘Chloroplast ultrastructure’ is not the organelle.

Line 307: ‘..had collapsed’

Line 309-311: ‘…chloroplasts had lost… contents had leaked out… stacks had decreased … had increased…’

Line 311: ‘Thus, the thylakoid lamellae were re-arrenged from order to disorder..’

Line 317-318: Modify the sentence ‘Chloroplast inclusion…’

Line 319-320: You would need a reference to the sentence ‘Osmiophilic granules… osmium tetroxide’. Though at the current study, the osmiophilic granules do not show as black as they should when treated with osmium. Here the granules are shown as white against the background, which is fine as they can be seen. Maybe the authors should consider using some other word instead of ‘osmiophilic granules’, such as ‘lipid droplets’.

Line 322: ‘..sizes..’

Line 329: ‘Strach grains in chloroplasts changed significantly…??’

Line 339: avoid using ‘amazing’

Discussion

Line 343: ‘studied’ instead of ‘detected’

Line 353-354: ‘renewed and disappeared.. and the leaves still… Since leaves from previous years can not…’

Line 357: ‘Leaves are the largest organ…’

Line 369: ‘The leaf anatomy…’

Line 371: ‘..increased leaf thickness..’ instead of ‘thick leaf thickness’

Note: when you discuss about chloroplast structure and photosynthesis, you could mention that less starch in senescent tree leaves suggest less effective photosynthesis compared to healthy trees.

Line 373: together with Yao et al. 2001 you could already refer to Riederer & Schreiber 2001 here, instead of later

Lines 387-390: I don’t understand, modify or remove the two sentences: ‘The ratio…shape changes.’

Line 394-397: Modify (shorten) the sentence ‘In ancient senescent trees…’

Line 397: ‘Chloroplasts are…’

Line 400: ‘In a study…’ you have to refer directly to Vlckova et al. 2006

Lines 406-408: Modify, better not to write Hörtensteiner & Feller twice. What do you mean by ‘Toren and Rober’? Is it a reference? In case, why do you mention Balaban et al. 2005?

Line 418: Arabidopsis thaliana in italics

Line 420: ‘senescence’ instead of ‘senescent’

Line 431: ‘longer’ instead of ‘older’

Line 432: you should consider some other word than ‘worst’

Lines 435-443 are repeating things written before; try to combine and shorten the text at p. 11

Line 450-451: You could remove the sentence ‘The recession degree…’

Line 452: Maybe you could state something like ‘The relationship… structure can support the decision making of when and how to apply rejuvenation techniques…’

Line 455: remove ‘etc.’

Line 457: I’m not sure why you say ‘Senescence is the final external performance…’

Line 460: ‘future research’ or ‘further research’ instead of ‘additional research’

Lines 462-464: I’m not sure if you need to take up details in denied sampling.

Lines 464-465: I don’t understand the meaning of ‘The accurate age… internationally.’

Lines 465-466: I don’t understand the meaning of ‘Some areas… lack relevant data records.’ In China? Or in general – would not it be ‘many areas’ or ‘most areas’?

Compare lines 468-474 and Conclusions (lines 477-482): you could combine and shorten these two parts into Conclusions.

Check Figure 5. Minor changes are needed:
‘Firm cellular structure’ instead of ‘Good..’?
‘Anatomy’ instead of ‘anatomical structure’
‘thinner epidermis’ instead of ‘…epidermal thickness’
‘increasing leaf thickness’ instead of ‘thick…’
‘plasmolyzed mesophyll cells’ instead of ‘irregular…’
‘irregular resin ducts’ instead of ‘wide & irregular resin cavities’

Ultrastructure
‘loose’ or ‘lost’ instead of ‘lose’ and ‘lack’
‘breakdown’ or ‘breakage’ instead of ‘break’

‘quick death’ instead of ‘quick to death’
Additional comments: The suggestions above are of minor characteristics to improve the manuscript even more. They should not take long for the authors to check. I trust the authors have double-checked that all references used in the text, and only them, are included in the reference list.


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

Input:
Title: GENETIC DATA OF MUSEUM SPECIMENS ALLOW FOR INFERRING EVOLUTIONARY HISTORY OF THE COSMOPOLITAN GENUS SIRTHENEA (HETEROPTERA: REDUVIIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper reports substantial new research and analysis of a major group of true bugs with very good taxonomic diversity represented in the analyses.
The manuscript is presented in an intelligible fashion and written in standard English. The background of the performed study is clearly presented with the special emphasis of the gaps in the present knowledge of reduviids’ phylogeny and evolution. The literature cited is well-chosen. All morphological characters, presented in the data set matrix (raw data), are very well figured (Figs. 1-3) and clearly described.
Experimental design: The Chapter Material and Methods is very well organized with distinct subdivision for three main tasks: Molecular data analyses with the divergence time estimation and phylogenetic analysis of the taxa studied as well as Morphological analyses with attaching very detailed data set matrix. Therefore, the analysis can be repeated by other researchers and the matrix can be used for other studies on phylogeny of true bugs. All analysis are based on strong data obtained.
Validity of the findings: Very few reports have attempted to present phylogenetic analyses of both molecular and morphological datasets in a single paper - therefore presented manuscript is an important contribution in systematics of this group of insects. Complement in the form of a molecular clock completes the work well (Fig. 4). In the manuscript, the background of the ecological conditions (i.e. the presence of the available niches), sheds new light on Sirthenea global distribution. This aspect is thoroughly discussed. Conclusions are well stated. It is worthy to underline, that the work is well connected with earlier studies of authors on the genus Sirthenea.
Additional comments: The presented manuscript is a significant contribution to understanding the relationships within interesting assasin bugs genus of unique global distribution. It is an important study because it combines molecular, morphological and biological data with broad taxonomic sampling. The paper is nicely organized overall. The data analysis is precise, appropriate and thorough. The results and figures are clear. The discussion of the results and the implications for further study are very clearly stated.
Comment on strengths of the manuscript
-	Important group of insect with a stable taxonomy (the revision of the genus Sirthenea has already been done) and unique global distribution;
-     extensive data set compiled from numerous worldwide entomological collections (all known species of the genus Sirthenea have been analyzed);
-     an innovative method of DNA extraction (the ability to analyze very old material);
-  well defined ecological and biogeographical background to the obtained results, especially “the fossula spongiosa theory” .

Comment on weaknesses  of the manuscript
- Poorly presented abstract which definitely should be improved
- Information if the Morphological matrix is an original ones, created by the authors should be added.

My only reservation is to do with the abstract improving. This part needs more information on materials and methods and results obtained. This is important work and deserves to be much better presented.


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

Input:
Title: GENETIC DATA OF MUSEUM SPECIMENS ALLOW FOR INFERRING EVOLUTIONARY HISTORY OF THE COSMOPOLITAN GENUS SIRTHENEA (HETEROPTERA: REDUVIIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: I feel that the Discussion section not sufficiently elaborates the phylogeographic aspect of the study. From the presented results it seems to me, that the genus is of South origin, and was undivided untill African-Madagascan-Indian plates separated from Antarctica-Australian-South American plate, which gives the perfect match to the molecular phylogeography of the genus. It is relatively easy to find corresponding papers and cite them and discuss the potential routes of divergence and migration. In this respect some more discussion on the potential habitat of divergence would be also appriciated.
Additional comments: I would also recommend transfer of detailed list of morphological traits studied to Supplementary Files. They are very important but very long, being only specialists intrested in, and for general public they are of lesser importance. I would keep the table with features in the text, just to give the reader the opportunity to see the general morphology of the genus. The tables may be described as they are, only with refference to supplementary material for detailed information.


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: GERMINATION CHARACTERISTICS AMONG DIFFERENT SHEEPGRASS (LEYMUS CHINENSIS) GERMPLASM DURING THE SEED DEVELOPMENT AND AFTER-RIPENING STAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting parameters are okay, but there are a few issues.

The authors need to define exactly what is meant by "genotypes", which is a term used in the title, abstract, and throughout the manuscript. Most or maybe all Leymus species are self-incompatible meaning that every plant and every seed is genetically unique. Thus, it is impossible to say that "All the genotypes of the seeds were obtained and harvested in both 2016 and 2017." (see lines 133-134). What exactly are the 20 genotypes mentioned on lines 131-132? Are these different collections from different locations or different plants from one population? Can the authors provide GPS coordinates for the origin of these population(s)?

I do not agree or understand the sentence on lines 38-29 "genotypes with typical germination patterns become ideal candidates for studying seed germination".

The word "great" was overused (three times) in the last two sentences of the first pargraph in Introduction (lines 51-54). The sentence on lines 50-53 has two redundant parts, it could be simplified "...grassland restoration."
Experimental design: The Materials and Methods for the after-ripening experiment was somewhat unclear. I understand that the authors measured germination of seeds from 20 genotypes harvested over six different stages of development, measured by days after pollination (DAP), and four different stages of after-ripening, with 3 replicates. Were there 6 DAP x 4 DAH treatments or 6 DAP + 4 DAH? I think it was 6 DAP + 4 DAH, where all DAH treatments were harvested at 42 DAP? If so, sentence 147-149 needs to be clarified and the authors should not say "each state of seed development as well as at during after-ripening process" (lines 51-152). Figure 5 shows four stages of after-ripening (0, 15, 30, and 60 DHA) but Table 4 only shows 2 df for after-ripening? I don't understand how the authors got 29 df for genotype in Table 4. It should be absolutely clear from the text in the Materials and Methods  or Results how to explain df in all ANOVA tables.
Validity of the findings: The main conclusions of the study (lines 337-342) concern the importance of "genotype". However, the genotypes used in this study are not well-defined. In fact, the are not really defined at all. These genotypes could be different plants from the same population, different populations, or different plants from different populations? The readers really need more information about the "genotypes" used in this study.
Additional comments: Seed germination and harvest time are important topics for utilization of native perennial grasses, and I think that this Yang et al. PeerJ submission provides useful and practical information for Leymus chinensis.

Genotypic variation and QTLs controlling seed germination have also been documented in other Leymus species and we continue working on issues related to seed production and seed quality of native Leymus species of North America.

Larson, S.R., and E.A. Kellogg. Genetic dissection of seed production traits and identification of a major-effect seed retention QTL in hybrid Leymus (Triticeae) wildryes. Crop Sci. 49:29-40. 2009.

Best wishes.


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: GERMINATION CHARACTERISTICS AMONG DIFFERENT SHEEPGRASS (LEYMUS CHINENSIS) GERMPLASM DURING THE SEED DEVELOPMENT AND AFTER-RIPENING STAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: .•	The manuscript is well organized. Due to large number of datasets used, some places it became difficult to understand the correct explanation given by the authors.
•	Besides some places; clear, unambiguous, professional English language used throughout.
•	The introduction is reasonable and shows the context.
•	Literature well referenced & relevant.
•	Though the Figures are relevant, labelled & described but vertical axis mismatched in different sub-figures. The captions of the Figures need to be checked again.
•	Data is robust, statistically sound, & controlled.
•	Discussion and conclusion are linked to the original research question.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: •	Line number 16 and 44: Remove unnecessary brackets from (Leymus chinensis ((Trin.) Tzvel)) and change to (Leymus chinensis (Trin.) Tzvel).
•	The following paragraph cited in the abstract is very long and creating confusion. This paragraph needs to be rephrased into two sentences for clear sound.
Line number 19-23. To clarify the appropriate seed harvest time and the effects of genotype, 20 seed development and after-ripening on seed germination, we selected 20 genotypes of 21 sheepgrass to analyze the seed morphological and physical changes as well as the seed 22 germination and dormancy characteristics of sheepgrass during seed development stages using a 23 7 - d gradient after pollination (DAP).
•	Line number 23: Full form of DAP is not clear in the sentence.
•	Line number 26-28: Furthermore, our results indicated that there were 6 types of germination patterns, including genotypes with increasing germination rates in the developing seed, such as S19 and Z37……………..in this sentence authors mentioned Z37 which is not correct as per results/Figure 3.
Explanation of Z37- the germination rate continued to increase at the early developmental stages until 35 DAP and then began to decrease.
•	Line number 30: add the years before 2016 and 2017; result change to results.
•	Line number 131: coded asK8; add space in between as and K8.
•	Line number 134: add the years before 2016 and 2017.
•	Line number 136: Why only 4 Genotypes i.e. K4, S19, S10, and N14 have been mentioned under heading Morphological and physical observations of the developing seed? Not clear from supplemental excel file too?
•	Line number 161: Date analysis should be change as Data analysis.
•	Line number 277: approximately15% add space in between.
•	Figure 1: Authors are advised to provide a habit/population photo of Leymus chinensis which ensure a clear image of the plant to the international audience.
•	Figure 3: Since the horizontal axis value are same for all the sub-figures, if possible try to keep the vertical axis value same for all sub-figures or in two groups such as A, C, E up to 100 and B, D, F up to 80.
•	Figure 3: Authors missed caption (C) and in place of (C) authors have mentioned D. In same; caption D is not in the bracket. In the caption of Figure 3A, Genotype N14 is missing. In the caption of Figure 3B, Genotype C18 is missing.
•	Figure 4A: Vertical axis value mismatched and giving wrong interpretation; keep the vertical axis value up to 100 as mentioned in 4B and 4C. In vertical axis add 14 DAP, 28 DAP and 42 DAP in sub-figures A, B, C respectively for the easy explanation as given in the caption.
•	Figure 5: Not in a uniform way. Authors are advised to keep the vertical axis value same for all the figures such as 0 to 100. In Figure 5C the vertical axis value 100 is missing.
•	Since the paper is characterizing seed germination, authors are advised to add types of germination (epigeal/hypogeal) in sheepgrass in the text.
•	How the authors are confident that pollination has occurred in different Genotypes of sheepgrass? How the authors are confirmed about the exact date of pollination such as a first day or first few Days After Pollination?


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: GERMINATION CHARACTERISTICS AMONG DIFFERENT SHEEPGRASS (LEYMUS CHINENSIS) GERMPLASM DURING THE SEED DEVELOPMENT AND AFTER-RIPENING STAGES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. The paper is an outcome of a very simple experiment, the finding of which are worth a research note.The only information generated is that germination of seeds of different genotypes differ; the underlying reason for this behavior has not been investigated/explained in terms of, seed viability, type of seed dormancy etc.
This information will be of practical importance only if the results (days after pollination) are interpreted in terms of the physiological stages of the crop (leaves become straw colored, seeds turn cream to brown etc.) as pollination that spreads over a few days can not be made a criteria for deciding the harvesting time.

2. In the introduction, there is a mention of pre-sowing treatments that promote germination of seeds. But in the methodology there is no mention of the use of pre-sowing treatments.

4. References are absent in 'Materials and Methods'. For example ISTA manual is an important reference material in seed testing; this not mentioned.

5. The term  '7-d gradient'  in the Abstract, is ambiguous.
Experimental design: There is no mention of any design (Simple Random Design, Randomised Block Design etc)
Validity of the findings: No comments
Additional comments: No 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: GERMINATION CHARACTERISTICS AMONG DIFFERENT SHEEPGRASS (LEYMUS CHINENSIS) GERMPLASM DURING THE SEED DEVELOPMENT AND AFTER-RIPENING STAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: All basic reporting criteria are satisfactory.
Experimental design: Experimental design criteria are satisfactory.
Validity of the findings: good
Additional comments: good


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: GERMINATION CHARACTERISTICS AMONG DIFFERENT SHEEPGRASS (LEYMUS CHINENSIS) GERMPLASM DURING THE SEED DEVELOPMENT AND AFTER-RIPENING STAGES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Acceptable
Experimental design: Sound
Validity of the findings: Valid
Additional comments: This revised draft is acceptable


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: VIRMINE: AUTOMATED DETECTION OF VIRAL SEQUENCES FROM COMPLEX METAGENOMIC SAMPLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper describes the software virMine and its application to metagenomics. The work is well written. There is a comprehensive introduction to the subject of viral metagenomics and software that can be used to identify viral contigs within metagenomes. There are two pieces of software that should be included which aren’t, these are MARVEL and DeepVir.
The software takes a different approach to identify viral contigs compared to other software, using the extensive datasets that are not viral to classify viral contigs, which is an interesting approach. The software is an all in one approach, taking raw reads and running through QC, assembly, gene prediction and classification. This in its self is a useful tool.

It would help to have the tools that can be used, to be labelled in Figure 1, allowing an nonexpert to see what tools are used at each step
Experimental design: The paper then goes onto described a number of test cases
The first test case uses a phage and bacterial genome sequence, whereby they demonstrate that virMine can assemble these genomes and identify the phage contigs. Thereby showing as a proof of principle the pipeline works on a simple synthetic system.

Case study 2 then focuses on using the tools on metagenomics datasets that have previously been analysed. Here new viruses were discovered that had not previously been reported in the original studies, which is an interesting result. However, if this a result of the approach taken in this study or simply better databases –the original analyses are between 5 and 8 years old. With significant increases in database sizes since the original publication. It is not clear if these viruses would be identified if the original analysis was carried out with updated databases. Furthermore, it is not clear if virMine is a better tool at identifying viral contigs than any other tool, such as DeepVir, VirSorter etc

Case study 3, the tools are used to analyse urinary viromes, where it identifies numerous contigs that are viral. Again it is not clear if in this example the tool is better than other methods for identifying viral contigs .

Case study 4 uses the tool to analysis viromes from a freshwater lake, which has previously been produced by the same group. They also compare the analysis to results produced by VirSorter and highlight some differences.

At the end of this analysis, it is not clear if the all in one pipeline is better than any tools that are currently available for virome analysis. In terms of a one-stop shop, then the pipeline is easy to use as it takes reads and outputs contigs that maybe viral.
Undoubtedly, the initial part of the pipeline is useful to be able to QC and assemble reads. However, there is no evidence that the sorting approach is better than current tools such as VirSorter DeepVir etc at identifying contigs or an equal to them. There is also do data presented on the speed and accuracy of the tool. Without this information it hard to make a decision on whether to use this tool rather than any other tool
I would recommend a more thorough comparison of the viral comparison tool against other leading tools such as VirSoter, VirFinder. This could be done using a similar approach as was carried out by  Ren J et al  2017 or provide data on how accurate the tools is on more complex synthetic datasets to allow  accuracy, speed to be compared to other tools.
Clearly, a large amount of work has gone into this tool and there is much to be commended in terms of how the paper is clearly written and the provision of docker images. However, without this crucial data on how the tool performs against other tools, it is not possible to assess its usefulness.

Minor points
Line 76- The use of “bin” is ambiguous in this sentence. The Virfinder tools gives each contig a probability of being viral or not, it is not binned as such. Please clarify what is meant
Line 83- MARVEL should be included in this list of software

Line 133
The premise of the paper makes much of the comparison against databases and the approach is innovative. However, much will depend on the databases used.  Looking at the script that creates the viral database  in   https://github.com/thatzopoulos/virMine/blob/master/virmine_make_dbs.py
Line 10- the script downloads the NCBI viral database from the FTP site. Whilst a good starting point the database only includes a small number of complete bacteriophage genomes that are available in Genbank, missing ~ 8000 phage genomes
Having a more comprehensive set of phage genomes as the starting point would improve the use of the tool – The authors do state in the text this can be manually curated set (Line 141-143) that would allow users to add in more phage genomes. Based on the GitHub repository it is not immediately clear how this can be done. Instructions on how to do this would significantly improve the usefulness of this software.
Line 168- as stated above the RefSeq database excludes 8000 complete phage genomes that are available
Line 249 –it is not clear when the database was created. But the sequence of the highlighted phage: MH617627 is a clear example of why a more up to date viral database is needed
Line 257 – whilst there is a clear underestimation of the vital data in the Qin dataset, it is not clear if this is due to better performance of virMine or simply increased database size in 2018 than in 2010. The  way the analysis is carried out do not allow this to be determined
Validity of the findings: The data does support that conclusions, that the tool can be used to identify viral contigs from raw reads in a single pipeline. However, there are other tools that do this - not in single pipeline admittedly. I would argue the identified viral contigs should be predicted as accurately as other tools can ,which currently cannot be assessed
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: VIRMINE: AUTOMATED DETECTION OF VIRAL SEQUENCES FROM COMPLEX METAGENOMIC SAMPLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reporting is clear. Context was developed early in Intro, and the figures are well presented.
Experimental design: The experiments were well designed with sufficient details provided.
Validity of the findings: Conclusions are supported by the results.
Additional comments: Work reported:
virMine is presented as a new software to identify viral genomes by omitting non-viral sequences from raw reads. Currently available tools such as VIP, VirFinder, Phage Eco-Locator, VIROME, MetaVir, VirSorter, MetaPhinder, VirusSeeker and FastViromeExplorer rely upon homologies to known viral sequences, and generally require manual curation. virMine was used as a single software to distinguish viral sequences in four case studies presented. virMine performed well with synthetic data set, it reconstructed viral genome when more than half of the set included specific viral sequences. The software identified two transposases as viral sequences because these were categorized as mobilomes (COG category X) and were excluded from non-viral set. Second case study was performed with gut data sets. In the first, virMine was able to assemble crAssphage, and identified transposases and integrases, and other complete viral genomes. In the second gut data set, expected to be mainly bacterial, virMine found higher %-age of viral sequences than previously reported. The authors suggest these to be prophage sequences. In the third case study urinary viromes from individuals with urinary tract infections were examined. Several unknown category sequences were found with similarities to viral sequences. For case study 4, two freshwater lake samples were examined. VirSorter was used for comparison with virMine. Manual curation was performed with both methods. virMine predicted higher number of sequences as viral (60 vs. 20), however,  both software assembled the same two contig sequences belonging to VirSorter category 2. Both software picked sequences that belonged to bacterial rRNA and tRNA, and identified two each (different sequences) with similarity to phages. Manual curation was necessary for both tools. With the last dataset, virMine found 9 contigs likely to represent new viral sequences. VirSorter identified one of the contigs and previous work had picked 5 contigs. Overall, virMine performed better in identifying more unknown contigs.

Critical comment:
1. virMine utilizes a clever approach to subtract known (non-viral) sequences. As demonstrated by the authors, this approach works well with datasets with higher representation of viral sequences. When the datasets are more complex and have lower representation of viral sequences, this approach requires manual curation, particularly to confirm that the identified sequences are indeed of viral origin. Several statements should be toned down about virMine as a fully automated software tool. For example “virMine automates sequence read quality control, assembly, and annotation” and “virMine is a single tool to perform raw read quality control, assembly, and analysis”. The data shows virMine is not fully automated, it requires an essential manual curation and interpretation step. This should be made clear throughout the manuscript. It does not take anything away from the ability of virMine to distinguish between viral and non-viral sequences, it is important to set the right expectation early on. This reviewer was disappointed that in the end the software did not perform fully as was promised earlier in the text.
2. As expected from the approach, virMine identified sequences that were not well identified or annotated in the databases, including transposases, integrases, prophage sequences, and even bacterial rRNA and tRNA sequences. virMine uses SPAdes or metaSPAdes for assembly, contigs are queried using blastx. Modified GLIMMER script is used for ORF prediction and compared to viral and non-viral datasets. It might be of great advantage to integrate one more step, borrowing from other software such as VIROME and VirSorter, to search for recognizable homologies to known viral sequences after the non-viral sequences have been differentiated. This step will add value in increasing the confidence of viral origin prior to the manual step, and could be useful for the end users to categorize their database searches. The authors should comment if they intend to integrate an additional “vral recognition” step, or if not, why it may not be necessary. I believe this will be of interest to the readers.
3. All in all, virMine is a good approach for identifying new viral sequences. However, only one software “VirSorter” was compared against virMine and only for case study 4. The comparison showed that while virMine predicted higher number of viral sequence, both software performed similarly in assembling contig sequences and both required manual curation. It should be of great interest to the authors to test the performance of virMine with VirSorter with other case studies, such as the second gut database and the infected urinary viromes. In addition, performance of other software should be compared, such as VirFinder to compare a broader set of approaches (nucleotide usage based approach). These direct comparisons are likely to illuminate the utility of virMine further, and will add significantly to this manuscript.

Minor comments:
Noticed (but not noted) minor typographical errors. The manuscript should be screened carefully for extra brackets and other minor errors.


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: AUTOMATED ANALYSIS OF SMALL RNA DATASETS WITH RAPID
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment'
Additional comments: The tool fills a gap but its current stage of the deployment is in my opinion not optimal. While the author offer an installation via the package manager conda, the package seems to be not properly set up. I personally have not created a conda package for a Shell or R script so far but I would imagine that it also for those possible to generate proper callable versions. In the current implementation the user has to set the path to the rapid file (in the documentation suggested as "rapid=/home/<username>/miniconda2/envs/<environment_name>/bin/ (or) rapid=/Users/<username>/miniconda2/envs/<environment_name>/bin/") and has to provide the command-line parameter "-r" with that path. In my opinion this is not really user-friendly and should be improved.

Further issues suggestions:

- I would recommend to submit the code also to Zenodo to ensure it   availability (https://guides.github.com/activities/citable-code/) and to add a CFF file (https://citation-file-format.github.io/) for better citability.

- Please write in the manuscript that the tool is for eukaryotic sRNA. Bacterial sRNA are as far as I understand not supported by the tool but the distinction is not explicitly done.

- The Docker image does not work.
  '''
  $ docker pull quay.io/biocontainers/rapid
  Using default tag: latest
  Error response from daemon: manifest for quay.io/biocontainers/rapid:latest not found
  '''

- The generated heatmaps use a red-green colormap that are not
  red-green-blindness safe.

- The documentation page has no proper title tag and the file
  "Installation.rst" is missing a newline. Due to this the code block
  "conda search rapid" is not properly formated.


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: AUTOMATED ANALYSIS OF SMALL RNA DATASETS WITH RAPID
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Karunanithi et. al describes a pipeline to visualize specific aspects of RNA sequencing data for pre-defined genomic loci. I am not an expert in the field of eukaryotic sRNAs and can only provide an outsiders view on the topic.
The manuscript is clearly written and appropriate literature is cited. The existing figures are justified, however the majority of the RAPID visualization features is not covered by a figure and not explained based on examples. It would be also informative for the reader to give more examples and guidance of how a specific RAPID output facilitate the analysis of sRNAs. For example, what is the benefit of the read length distributions? Is it possible to draw conclusions for sRNA synthesis or mode of action?  As a general question; how meaningful is the length distribution for reads which are pre-filtered for a given length?
Due to the completely different scopes of the tools I think the comparison in Table 1 is kind of misleading. Nearly all of the displayed tools are miRNA or piRNA specific with many additional dedicated features not mentioned in the table, while besides in principle generic, RAPID seems to be primarily tailored for siRNAs.
Experimental design: As mentioned above the method seems to be tailored regarding to the needs of an siRNA analysis and maybe other cis-acting RNAs. This should be clearly stated within abstract and introduction.
The presented data reflect only a subset of RAPID functions. If the missing functions are not relevant they should be omitted, otherwise they should be explained in the paper. I would highly appreciate a complete example analysis based on a given locus including all visualizations and a reference discussion/interpretation of the results.
In general I am struggling to understand the term “modified bases” in this context. In my understanding modified bases are chemically modified by e.g. methylation. How can this be detected by Ilumina sequencing of the cDNAs, or does "modified bases" refer to another aspect?
Validity of the findings: The comparison of the normalization methods in Figure 3 lacks the results for the DESeq2 method.
Additional comments: Minor points:
Random searching for the tools in Table 1 showed that "sRNAanalyazer" is misspelled (sRNAnalyzer).


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: AUTOMATED ANALYSIS OF SMALL RNA DATASETS WITH RAPID
Review round: 1
Reviewer: 3

Output:
Basic reporting: The introduction provides sufficient background and motivation for the software. There is clearly plenty of software available for small RNA analyses, and at least some of the literature on this is referenced, and a comparative table of their features has also been provided. Though I’ve only examined these aspects cursorily: the manuscript seems to be correctly structured and formatted according to the PeerJ guidelines. The language used in the manuscript is quite acceptable too. The figures are generally clear, with the exception of Figure 2's axis labels being extremely tiny - way too small to be legible (at least in the printed version of the manuscript to be reviewed). Raw data is available.
Experimental design: Software installation, testing and reproducibility:

I think experimental design/rigorous investigation isn't the primary concern in this manuscript. However, functioning of the software is certainly worth considering. So, I'm using this section to address this. Generally I would state that the motivation for the manuscript and software are sufficient and that the methods are described adequately. Regarding replication, see the comment towards the end of this section.

I attempted to install RAPID using conda as described in the documentation. I had error messages using this installation approach that caused the pipeline to abort (and also incidental warning messages too - though these may be platform specific).

bash runTest.sh
ERROR: unknown parameter "-p"
 ________________________________________________________________
|_______               ________       _____                          |
|  |    |                 |    |   I    |  \                        |
|  |    |      /\         |    |   I    |   \                      |
|  |____|     /  \        |____|   I    |    |                      |
|  |\        /____\       |        I    |    |                     |
|  | \      /      \      |        I    |    |                     |
|  |  \    /        \     |        I    |   /                      |
|  |   \  /          \    |        I    |__/                       |
|                                                                    |
| -Read Alignment, Analysis, and Differential Pipeline- V 0.1   |
|____________________________________________________________________|
Usage:

./rapidStats.sh -o=/path_to_output_directory/ -f=reads.bam -ft=BAM --remove=no --annot=file.bed --index=/path_to_index
Parameters:
-h|--help
-o|--out=/path_to_output_directory/ : path to the output directory, directory will be created if non-existent
-f|--file=filename : the input file
-ft|--filetype = BAM/SAM/fq : Mention either BAM/SAM or FASTQ. Default FASTQ
-a|--annot=file.bed : bed file with regions that should be annotated with read alignments (Multiple Bed files should be separated by commas)
-r|--rapid=PATH/ : set location of the rapid installation bin folder (e.g. /home/software/RAPID/bin/) or put into PATH variable
-i|--index=PATH/ : set location of the bowtie2 index for alignment
--contamin=yes : use a double alignment step first aligning to a contamination file (default no)
--indexco=PATH/ set location of the contamination bowtie2 index for alignment (only with contamin=yes)
--remove=yes : remove unecessary intermediate files (default yes)
Run comparative analysis using config file test.config
During startup - Warning messages:
1: Setting LC_COLLATE failed, using "C"
2: Setting LC_TIME failed, using "C"
3: Setting LC_MESSAGES failed, using "C"
4: Setting LC_MONETARY failed, using "C"

Attaching package: ‘gplots’

The following object is masked from ‘package:stats’:

    lowess

Error in file(file, "rt") : cannot open the connection
Calls: read.table -> file
In addition: Warning message:
In file(file, "rt") :
  cannot open file './TestRapid/Regions/alignedReads.sub.compact': No such file or directory
Execution halted
During startup - Warning messages:
1: Setting LC_COLLATE failed, using "C"
2: Setting LC_TIME failed, using "C"
3: Setting LC_MESSAGES failed, using "C"
4: Setting LC_MONETARY failed, using "C"

Attaching package: ‘gplots’

The following object is masked from ‘package:stats’:

    lowess

Error in file(file, "rt") : cannot open the connection
Calls: read.table -> file
In addition: Warning message:
In file(file, "rt") :
  cannot open file './TestCompare/NormalizedValues.dat': No such file or directory
Execution halted

I was wondering if I had the latest version installed, since the banner from the test command indicates version 0.1? The command “conda search rapid” gave the following:
rapid                       0.7     pl526r341_0  bioconda

Can the authors fix this issue with the info I provided? My main reason for suggesting a "minor revision" is to request that this can be sorted out, as the conda installation should be the preferred option.

Due to the problems above, I switched to installation from the GitHub master branch source for RAPID. It took a while to get all the prerequisites installed, but when I did so, I believe the pipeline ran to completion, as I was able to see graphs for all the analyses. I discovered that in addition to the dependencies noted in the documentation online, RMarkdown needs to be installed too (and was described as being used in the methods).

I was not able to install pandoc as described in the software documentation (I looked around briefly on the internet to figure out where it's available, but gave up trying to determine how to get it installed in R):

> install.packages(c("pandoc"))
Warning message:
package ‘pandoc’ is not available (for R version 3.5.1)

The PeerJ standards for bioinformatics software tools (see relevant section at: https://peerj.com/about/policies-and-procedures/#discipline-standards), recommends: “If available as a package (e.g. Python, R, Matlab, Octave), it should be accompanied by a minimal script that downloads the data (if not included in the package), loads it, and performs the analysis to reproduce the results (tables, plots, visualizations etc.) in the manuscript.” Would it be too onerous to include at least some of this? This might assist in making the software more robust, and would, I think, inspire more confidence that everything is working correctly than the provided test scripts. (Alternatively, it would be helpful to have the output of the test scripts downloadable from GitHub to compare to).
Validity of the findings: Since this is primarily a software article, I think the results demonstrate the utility of the tool as necessary. Reporting on the capabilities of the software is balanced. Two relevant biological data sets were analysed. I don’t think any of the interpretations of the analyses were unduly speculative.
Additional comments: I think this software will be of utility to researchers interested in analyzing small RNAs, and look forward to the potential expansion to include sequence level analyses in future.

The normalisation for sRNA counts will be useful in cases where primary (knockdown) and secondary siRNAs comprise a large fraction of the total sRNA population. I think it would be useful if the normalisation took into account not only siRNAs against the gene to be knocked down, but also those generated from transcription of the vector backbone (both could be included in the quantity “K”). One of the common vector backbones used in RNAi (L4440) has significant amounts of non-insert RNA production due to the lack of transcription terminators (see, e.g., Sturm et al. 2018: https://academic.oup.com/nar/article/46/17/e105/5040046). A note about this issue may be useful to users of the software.

Minor comment:

Pg. 6, line 192: What is “goodness” of the normalisation method? Maybe “performance”?


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: PITUITARY TISSUE-SPECIFIC MIR-7A-5P REGULATES FSH EXPRESSION IN RAT ANTERIOR ADENOHYPOPHYSEAL CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and organized.
A few minor comments are:
1.	It would be helpful to readers if the authors provided additional details around the miR-7a-5p mimic and inhibitor that were used.
2.	All prior studies around FSH regulation by miRNAs discussed in the Discussion have been conducted only in the context of cell culture. It would interesting if the authors could discuss work, if any, that has been done in vivo (e.g., in rats), or comment on this limitation.
Experimental design: The manuscript presents robust data to support its conclusions.
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: PITUITARY TISSUE-SPECIFIC MIR-7A-5P REGULATES FSH EXPRESSION IN RAT ANTERIOR ADENOHYPOPHYSEAL CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Wang CJ et al., aim to demonstrate the role of miR-7a-5p in regulating the expression of follicle-stimulating hormone (FSH). Overall, the manuscript is well structured, and provides evidence to suggest a role for miR-7a-5p in down regulating expression of FSH. A few minor revisions could help strengthen the quality of the manuscript:

There are several issues with figure 2. Firstly, the method used to determine miR-7a-5p expression (qPCR?) is not stated in the figure legend or main text. This also applies to figure 3b & 4a, and needs to be clarified. Secondly, it appears expression in figure 2A was normalized to immature animals (by setting this value to 1). If so, it should be explicitly stated (also applies to figure 3b).

The overall design of the luciferase reporter used in figure 1 isn’t immediately obvious from reading the figure legend. Is only the miRNA seed sequence fused to luciferse, or is a larger fragment of the gene included? Perhaps, adding a cartoon or visual graphic of the overall structure of the reporter construct would be helpful.

In several figures, the precise cell line used is not clearly indicated in the figure legend (e.g. fig. 4).  Clearly indicating such information in the figure legend will help improve the readability of the manuscript

There are a few instances of grammatically incorrect sentences. For example, line 158 (“was been acquired”), line 170 (changes in expression over development not “discrepancies”), etc.
Experimental design: In line 167, the authors claim that “mir-7a-5p directly regulates FSHb expression by directly targeting the FSHb gene”, based on a reporter assay where presumably the 3’UTR of FSHb (or a mutated sequence) is place upstream of luciferase reporter. While this proves that the FSHb 3’UTR has a potential binding site for mir-7a-5p, direct targeting cannot be concluded from this experiment. Similarly, the experiment in figure 4b certainly proves that mir-7a-5p expression can regulate FSHb levels, but once again, direct engagement is possible, but not the only explanation. To conclude direct targeting, an in vitro cleveage assay with mir-7a-5p, recombinant AGO2 protein and full-length synthetic FSHb mRNA should be used. If such an experiment is not feasible, the authors should consider adjusting the language to reflect alternate hypothesis.

In the experiment in figure 2A it is unclear what tissues from the immature and mature rats were used in determining miR-7a-5p expression. This is important to define as it also relates to the data presented in figure 2B.
Validity of the findings: The authors find that expression of mir-7a-5p is highest in rat pituitary glands. However, the pituitary is also where FSH expression is highest. Obviously, a direct anti-correlation is unreasonable to expect, but perhaps a brief discussion as to other positive regulators of FSH in the pituitary would help address this point.

Similarly to the above, the expression of mir-7a-5p is also significant in the rat brain. Assuming this isn’t merely a contamination associated with separation of pituitary from other brain tissue, this finding could be interesting as it might suggest a function of FSH in the brain. The authors should consider adding a few sentences (even if speculative) addressing this in the discussion.
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: PITUITARY TISSUE-SPECIFIC MIR-7A-5P REGULATES FSH EXPRESSION IN RAT ANTERIOR ADENOHYPOPHYSEAL CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made a good faith effort to address both my comments. I do not have any additional feedback.
Experimental design: The authors have made a good faith effort to address both my comments. I do not have any additional feedback.
Validity of the findings: The authors have made a good faith effort to address both my comments. I do not have any additional feedback.
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: PITUITARY TISSUE-SPECIFIC MIR-7A-5P REGULATES FSH EXPRESSION IN RAT ANTERIOR ADENOHYPOPHYSEAL CELLS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have clearly taken my previous comments in a constructive manner and implemented significant changes through the manuscript, and I commend them for their effort.

My only minor remaining comments are:
1) In response to comment#3, "In several figures, the precise cell line used is not clearly indicated in the figure legend (e.g. fig. 4). Clearly indicating such information in the figure legend will help improve the readability of the manuscript", the authors respond with "I have added a detailed description in the figure legend". Yet I do not see any significant change to figure 4 legend, and this information is still missing from the legend.

2) There are still a few instances of grammatical errors. I would recommend a native English speaker review the manuscript in detail to fix these.
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: TRANSCRIPTIONAL PROFILING IN THE LIVERS OF RATS AFTER HYPOBARIC HYPOXIA EXPOSURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I have two questions for the authors regarding the experimental design of the current manuscript:

1) It wasn’t clear what were the conditions for the experimental groups, e.g. were they submitted to simulated altitude? What was the altitude tested? Any particular devices were used? Also, what are the genders of the rats used? Could there be gender difference?

2) It wasn’t clear to me how was the 2-week time point chosen. This is one of my major concern of the manuscript. Was any shorter period of hypobaric exposure tested? In my humble opinion acute effect should be measured at much shorter time points, such as 24-72 h. Would the authors explain their rational in choosing the 2-week time point? I would also like to see if the results would be different for a shorter period such as 48h.
Validity of the findings: Have the authors validated the results of the RNA-Seq?
Additional comments: In the manuscript "Transcriptional profiling in the livers of rats after hypobaric hypoxia exposure", the authors explored the gene expression in the livers of rats after 2 weeks or 4 weeks of hypobaric hypoxia exposure. In addition to my questions above, there are a few other things I would like to ask the authors:

1) In figure 6, it is interesting to see that all the pathways selected were enriched only in the first cluster, but no one showed up in the other two clusters. I am curious if any other pathways showed up in the other two clusters in this pathway analysis?
2) Since one of the major function of liver is metabolism, I am curious if the signal pathways/genes involved in metabolism are changed. The authors mentioned very briefly that the galactose metabolism pathway was enriched, but it seems no other information was provided or discussed.
3) Also, did the authors explore, or have plan to examine, the gene expression pattern in other organs under similar conditions?
4) There is a paper published on Physiol Genomics in 2010 titled “gene expression of the liver in response to chronic hypoxia” by Baze et al. (PMID 20103700), would the authors cite and discuss that paper?
5) Most of the figure legends are unreadable in the manuscript. Please correct them.
6) In the Abstract, the third sentence “The liver must physiologically and ….” and the fourth sentence “Since the liver is the largest metabolic organ…” should switch order.


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: TRANSCRIPTIONAL PROFILING IN THE LIVERS OF RATS AFTER HYPOBARIC HYPOXIA EXPOSURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	In introduction, the language is not cohesive.  Make sure to use connective words in between sentences to make introduction easier to read.

2.	In introduction, a sentence starting at line 60 is very long. It fails to get the message across. I suggest authors break down that sentence into few small sentences to get the message across
Experimental design: 1.	In materials and method section, authors indicate that “the liver tissue was snap-frozen…”(line 92). What part of the liver was utilized for this study?  Indicate if similar sections of the liver was isolated and utilized consistently throughout the study.
Validity of the findings: 1.	In introduction (line 57), authors suggest that energy metabolism of heart and skeletal muscle is altered.  Based on the results of energy metabolism changes in liver, would there be extrapolation for anticipated changes (or literature evidence) in heart and skeletal muscle?  Suggest include that in discussion.

2.	One of the major finding for this study is that immune-related pathways play a key role in hypobaric hypoxia exposure.  It would have been ideal to include certain functional end points in the study to have the evidence of translation of gene expression to functional end points.  However, can authors suggest the evidence of the functional end points in literature in discussion to support major finding?
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: TRANSCRIPTIONAL PROFILING IN THE LIVERS OF RATS AFTER HYPOBARIC HYPOXIA EXPOSURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: Authors have addressed my concerns.  No further comments.


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

Input:
Title: MATURATION TRADE-OFFS IN OCTOPUS FEMALES AND THEIR PROGENY: ENERGY, DIGESTION AND DEFENCE INDICATORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The MS requires much work to present clearly both methods and results for several reasons detailed below and in the pdf. Also, the introduction and discussion need to be improved.

There is  objectives defined.
The presentation of the paper looks like a draft. The format of the figures, abbreviation, spelling errors, etc. ... that should be reviewed deeply, there are many errors that makes it very difficult to review the work.

Authors should also revise all the MS, and the English should be improved, please see some examples in the detailed comments in the pdf.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study performs analyses the biochemical changes during sexual maturation and its effects on embryonic development of O. mimus, an important octopus specie in the Pacific Ocean. It examines the levels of metabolites, digestive enzymes and antioxidant defences system in females during gonadal maturation, and during embryo development.

Although this is not the first study showing changes in biochemical composition related with sexual maturation, and/or during embryos development in cephalopods, including octopus, this paper is interesting because provides new information on for this octopus species, about how energy and antioxidant defences can change during sexual maturation (in specific tissues) and embryonic development.

But the MS requires much work to present clearly both methods and results for several reasons detailed below and in the pdf. Also, the introduction and discussion need to be improved.

The introduction would benefit from expanding the general ideas given in several paragraph (ej. Lines 69-- “ A mixed maternal diet (what do you mean?)  resulted in more hatchlings (what do you mean? ) from O. vulgaris females than from females fed with mono-diet (only crabs) (Márquez et al. 2013. Lines 78-84 “Caamal-Monsreal et al., (2016) and Sánchez-Garcia et al., (2017) showed that from stage XV onwards………these studies are related to Tª, then the paragraph must refer to this abiotic factor.).  Also, it is important to state the objectives.

  Another concern is the format of the figures, abbreviation, spelling errors, etc. ... that should be reviewed deeply, there are many errors that makes it very difficult to review the work.

The discussion would greatly benefit if the author explains why some enzymatic activities in particular the AChE, CbE and GST, are determined and discussed together with antioxidant enzymes.

 Another concern is the format of the figures, use of different abbreviations or names to describe the same thing, spelling errors, etc. ... that should be reviewed deeply, there are many errors that makes it very difficult to review the work.

Authors should also revise all the MS, and the English should be improved, please see some examples in the detailed comments in the pdf.

Title:

The title does not reflect properly the content of the paper, which is manly biochemistry analysis. Rewrite a title that better fits the study.


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

Input:
Title: MATURATION TRADE-OFFS IN OCTOPUS FEMALES AND THEIR PROGENY: ENERGY, DIGESTION AND DEFENCE INDICATORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I’m not a native speaker but I think that the English language is clear and technically correct. The authors have shown a good knowledge of the previous researches. They have a wide experience in this field with several papers in nutrition and metabolism in cephalopods.
In general terms, introduction is well focused, although some aspect should be improved:  Other comments about the introduction: Line 75-76: I think the introduction arguments about females and eggs should be split in two different paragraphs. How did you select the antioxidant mechanisms and ROS to be analyzed? A paragraph about the role of Ache, CbE, ORP, etc should be included.  Authors should consider to put the paragraph 93-116 at the beginging of the introduction. The last paragraph of the introduction is not well connected with the previous paragraph. Other references to be considered on this field
Steer, M.A., Moltschaniwskyj, N.A., Nichols, D.S., Miller, M., 2004. The role of temperature
and maternal ration in embryo survival: using the dumpling squid Euprymna
tasmanica as a model. J. Exp. Mar. Biol. Ecol. 307, 73–89.

Farías, A., Navarro, J.C., Cerna, V., Pino, C., Uriarte, I., 2011. Effect of broodstock diet on the
fecundity and biochemical composition of eggs of the Patagonian red octopus
(Enteroctopus megalocyathus Gould 1852). Cienc. Mar. 37 (1), 11–21.
Cagneta, P., Sublimi, A., 1999. Productive performance of the common octopus (Octopus
vulgaris C.) when fed on amonodiet. Seminar of the CIHEAMNetwork on Technology
of Aquaculture in the Mediterraean on “Recent Advances in Mediterranean Aquaculture
Finfish Species Diversification”.
Quintana, D., Márquez, L., Arévalo, J. R., Lorenzo, A., & Almansa, E. (2015). Relationships between spawn quality and biochemical composition of eggs and hatchlings of Octopus vulgaris under different parental diets. Aquaculture, 446, 206-216.
Zuniga, Oscar & Olivares, Alberto & Rosas, C. (2014). Octopus mimus. Cephalopod Culture. 397-413. 10.1007/978-94-017-8648-5_21.
The structure present a standard format and the manuscript could be considered as an "unit of publication" including all the relevant results according to the hypothesis proposed. From my point of view, most of the figures are relevant, although a deep review of the details should be made.   There is an important lack of description in the figures as well as heterogeneity of information among different figures.  The legend should include a general title or description and then a detailed description of each figure (A, B, etc.). On the contrary, detailed description of some parameters (ex. maturation stages in Fig 1 and 2 or embryonic stages in Fig 8) should be avoided, because has been described in Mat and Met.   All legends should include the meaning of all abbreviations. You must use the same denomination of parameters (Example to avoid: reproductive system weight in Mat and Met vs Reproductive complex system in Results vs  reproductive tract in fig 1). Fig 1 and 2, Cortes or Cortez? The statistical letter are not always well allocated Ex. Fig 4). The different statistical comparisons should be indicated in the footnote (Ex . a vs a’ vs a’’ in Fig 4).  Legend of Fig 3 doesn’t include information about the three graphs (A, B and C). Fig 3 (B). uncorrected values in the glucose axis. Embryonic stage should be named as roman numbers ( I, II, IV…). Fig 4. Axis legend only mention acid protease. In summary authors should revise and homogenise the information among material and methods, figures and results because  in the current form is confusing and with a lot of misinformation.
Regarding the Figure 9 and 10, I agree with the aim of the authors to summarize the results. However, in the present form, the real contribution of these figures is questionable, particularly for Fig 9. The pathways proposed by the authors (Ex. Protein to glycogen, TG to Cho, etc.) are not the only option and further studies are needed to confirm the prevalence of these pathways. I agree to propose a theoretical model but the authors should highlight that it is just a hypothetical model. If Fig 9 cannot be improved according to these comment, its should be eliminated. The theoretical model could be including in the text. Regarding Fig 10, I’m not sure if it is worthy to be include. In any case, I would change the “XXX” by “+++”. I think that the development of each parameter should be include in the text instead of the figure legend.
Experimental design: The research is within the aims and scope of the journal. The knowledge gaps are correctly identified and the hypothesis and methodology to fill the gap are well elaborated. Both ethical and technical standards are high. The methodology description is enough to be reproducible although a clearer scheme of the sample analysis is necessary. There are many different analyses in this manuscript and the current exposition is confusing about this aspect. For future studies, I recommend to use MgCl2 or ethanol for humanitarian end-point according to the Fiorito et al., 2015 and Butler-Struben et al., 2018.
Fiorito, G., Affuso, A., Basil, J., Cole, A., De Girolamo, P., D’angelo, L., ... & Mark, F. (2015). Guidelines for the Care and Welfare of Cephalopods in Research–A consensus based on an initiative by CephRes, FELASA and the Boyd Group. Laboratory animals, 49(2_suppl), 1-90.
Butler-Struben, H. M., Brophy, S. M., Johnson, N. A., & Crook, R. J. (2018). In vivo recording of neural and behavioral correlates of anesthesia induction, reversal, and euthanasia in cephalopod molluscs. Frontiers in physiology, 9, 109.
Some minor aspects should be improved in Material and methods section. All the devices should include name, country and city of the company.Line 141. Eviscerated weight is the fifth weight instead of four. 143. Oviductal gland weight is separated in Fig 1. 174. I understand that no food was supplied to the paralarvae but this aspect should be mention.  “mL” instead of “ml”. Acylglicerides are not exactly TG (Include MG and DG). It can be assumed that most of them are TG and you can use both terms but always the same. Anyway you cannot use acylglicerides levels as lipid levels because phospholipids has not been measured. In similar ways Bradford is a technique for soluble protein, It is not a good indicator for several structural protein such as collagen. 193-196. How did you decide the Ph of acid and alkaline proteases? Previous studies should be mentioned. Please include details about “ Universal buffer”. There are not trypsin and lipase data at the first egg stages and 2 days-old paralarvae. Pkease, explain the reason. 219-223. Samples were frozen in liquid nitrogen, lyophilized,  stored at -20ºC and later, supernatant  stored at -80ºC. Are you sure that enzyme activity was maintain at -20ºC despite of the lyophylized process? 274-276. Why don’t you name initial (Ini) organogenesis instead of Pre organogenesis? 278-280: abbreviations should be explained. Lipase and trypsine should also be mentioned
Validity of the findings: No previous data about this subject have been published in Octopus mimus. Since paralarval viability is the most important bottleneck in octopus aquaculture, to improve the hatchling quality through broodstock nutrition and embryonic development is a very useful research line in this field. The data obtained are robust and statistically sound. There is an adequate number of replicates  and the sample timing is correct to study the metabolism development in the digestive gland, ovarium and eggs.
However, several aspects of the results should be revised. 309-312. Please revised this text according with Fig 2. 316. The beginning of the maturation process is the “ Phys Mat or “Ea FuncMat”?  What is the difference between this sentences and the next sentence (2.4 times higher).  The data and the name (HI%) are different between them. 320-321: A reduction in 38% of glycogen. When?  365: What about the CHO in the eggs? 369-379: please review carefully this paragraph according to the Fig 5 there are several mistakes. 385. C to E instead of B to D. 385. Why don’t you consider CbE as antioxidant enzymes instead of oxidative damage? 401. Which differences? 405: 19.7 according to the table. 420: a negative correlation. 426-427. Tese results are different from the results described in Fig 5 and line 374-376. It should be eliminated. 437-438. Glu, Cho and TG are similar in all egg stages (see figure). This text 436-441 should be eliminated. 447. Similarly “lower acid protease than embryos” is not correct.
Regarding the discussion and the conclusion, the authors should make a deeply review of this section on the basis of capacity of the techniques used in the present study. As I mentioned before, I agree with these techniques to obtain a general overview of the nutrient metabolism, but to go beyond, additional techniques such as radiolabelled studies, gene expressions or intermediate metabolic enzymatic profile should be used. In addition some relevant nutrients such as phospholipids, free aminoacids or non soluble proteins have not been determined. My point of view is that  several assessment about  nutrient sysnthesis or metabolism are highly speculative because are based just in the nutrients contents in different tissues along the development. Therefore the conclusions should be adapted to the methodology limitations. Authors have include new data/references in Conclusion section (ex. 623-626). I think that these references belong to the discussion section. Conclusion should be a general overview without detailed comments. Maybe a brief summary of the hypothetical models explained in the figures 9 and 10.
Minor changes: 475-479. Both sentences appear to be in contradiction between them. Please review them. 559-561. I cannot understand this sentence
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: MATURATION TRADE-OFFS IN OCTOPUS FEMALES AND THEIR PROGENY: ENERGY, DIGESTION AND DEFENCE INDICATORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no commnet
Experimental design: The experimental design is adequate and the methods described are in general with sufficient detail, except some questions explained below. However, the study of additional ages of post-hatching paralarvae would also be included helping to reinforce the results and discussion described.
Validity of the findings: no comment
Additional comments: The study reports the biochemical composition changes that take place during gonadal maturation of Octopus mimus females and its consequences in embryo and hatching paralarvae. This physiologic study is very interesting and helpful for the improvement of the rearing of paralarvae of O. mimus, a species with a high potencial for aquaculture in the Pacific Ocean area. The results obtained are adequate and very interesting. However, as main comment, the conclusions concerning the biochemical and physiological dynamics that occurs in female maturation and through embryo development and post-hatching are quite speculative. Authors should perform additional analysis at biochemical and even at molecular level to study additional enzymes and molecules involved in the referred signaling pathways in order to ascertain the activation of that biochemical pathways.
Some specific suggested changes and comments are reported below:
-Line 101, 105: Please, change "68 d old" by "68 days old". Check it all around the manuscript.
-Line 173: Please, specify the protocol of sampling method of embryos. Was the whole embryo frozen?, or do the authors proceed to dechorionation of eggs before freeze the samples for posterior biochemical analysis?. It is also important to know if the results obtained are from embryos with or without yolk sac.
- Line 174: Please, explain this sentence. "Paralarvae of 1 or 2 days post-hatching were sampled and analyzed at biochemical level". What is the reason that authors decided to sampling at that sampling points so close?. No big differences are described between 1 and 2 days post hatching in paralarvae, and in fact, the statistical results reported detected significant differences among stages, but no differences among 1day and 2 days post hatching paralarvae were observed. However, a sampling point at more advanced age would be of great interest.
- Line 315 and Fig. 3A. The standard deviation of the data of glycogen obtained in female DG (and in some other data presented in the manuscript) are very wide and therefore the results and conclusions obtained should be checked.
-Line 317: Please, change "Fig. 1A" by "Fig. 3A".
-Line 320: Please, rephrase or explain in better way: "A reduction in 38% in digestive... values was registered".
-Line 359: Data of cholesterol in embryo and paralarvae corresponding to Fig. 4B were not included in the results section. Please, add it.
-Figure 3: Please, add "3A", "3B" and "3C" and indicate it with the corresponding letters in the graph.
-Figure 4: Same thing than Fig.3
-Figure 5: Please, add "5A" an "5B" to the figure legend.
-Figure 8. This figure is not clear in the current format. Letters are overlapping and are not read properly


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: MATURATION TRADE-OFFS IN OCTOPUS FEMALES AND THEIR PROGENY: ENERGY, DIGESTION AND DEFENCE INDICATORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The qulity of the manuscript has been clearly improved, however, there are still some minor changes

Abstract:
Line 26: A list of metabolites (as in line 31) should be included
33-49: This paragraph include some statements that cannot be not fully proved with the techniques carried out in the present study (I will give further details onwards).  I consider that authors should start with a sentence like this: “ Based on the data obtained, we hypothesised that….”. In general terms, I agree with the interpretation of the data, but it should be clear that  several aspects of this paragraph are speculation  or are not fully proved (ex. Cho storing, gluconeogenic pathway, energetic cost of the organogenesis, maternal origin of the ROS in the egg or ROS production during planktonic life). This comment is also valid for the Conclusion of the manuscript.
Introduction:
72-73: Marquez et al., 2013 used three mono-diets instead of mixed diets. Crab and squid produced better results than fish. Please review the paper and Quintana et al., 2015 Aquaculture 446: 206-216.
Results:
Several statements should be revised because there is no enough statistical support  to maintain them. These statements are mainly related with the changes in metabolites or enzyme activity along the  development. Most of the time, there are too much variability to obtain clear conclusions. See details below:
Section 3.1.2.  Line 368: Given the SD, there is no a clear reduction of  Gly between “Imm” and “Phys” (Fig 3A). This sentence should be eliminated. Line 373: Cho levels of “La Func” are very similar to “Imm”. Line 384: Authors cannot say that “Imm” are higher in Cho and lower in TG than  “La Func” . See Fig 3.
Section 3.1.3: Lines 392-394: Protein is the only metabolite that seems to reduce in the “La Func” stage and despite of that it is not clear if this difference are significative. In similar ways (line 396). Alkaline proteases don’t seem to increase in a significant way. I think that the sentence between 408-410 ( variability in “Phys” stage) should be located among the first sentences of this section. Line 411 and 412: I don’ agree about these comments regarding “Prot” and alkaline protease.
Section 3.2: This section should include a initial paragraph similar to section 3.1 with details about the number of the samples for the analysis, as well as the specific stages  of the development used. For example, Which specific stages were selected for “Ini Organ” or “Post” groups? How many samples from each stages?
Section 3.2.1: Line 420-422, 427-429 and 423: I cannot see a clear tendency in the Cho levels. This data should be rewrite taking into account the high variability of the Cho. Line 427: “…high in TG and Glu..”
Section 3.2.3: Line 463: “ (Fig. 6G).” Line 464: Review the % data. Are different from the figure. Line 465: “among”
Discussion:
Line 487: A reference for this study is needed
Line 545-554: The high variability of the Cho should be mention in this paragraph. I agree with the interpretation, but in the present study there is no enough data for this statement. This is a speculative hypothesis.
Line 576-582: I don’t understand how the authors obtain this conclusion. Please include more details and argument to justify this paragraph.
Line 589 and 593: Usually, I understood that embryos used the yolk from the beginning of the development. Why do you use the word “Yolk” just from organogenesis onwards?
Conclusion: As I mentioned before some statements are not fully proved. You cannot use some words as “This study demonstrate” (line 643) , “indicate” (line 651) or “these process were key” (line 656) among others. Please, focus this aspects as speculative arguments.
Table 2:  Section 3. “Metabolites and digestive enzyme activity  in female…”