Round 1

Reviewer 1 Report

Overall: The concept of this work was clearly defined. The experiments were well designed, and the manuscript was nicely written. In brief, the authors performed gene expression analysis through a transcriptomic platform. This finding helps to understand the differences between gonad development in male and female prawns at molecular levels. A whole concept contains novelty and scientific merit which will be of interest to researchers in this field. However, there are some revisions required before getting published.

Materials&Methods

1. Based on what the authors mentioned that mature males and females (5 each) were used for gonad tissue collection – can you please further specify their reproductive stages (i.e., early or late stage of gonad development/maturation, or using their average gonadosomatic indices) in the main text? Were their gonad stages conformed among 5 individuals of same sex or it was a mixture of different stages?
2. Analysis of DEGs and Functional Enrichment – how was the DEGs between 2 datasets compared? Like testis against ovary, or vice versa? Please include this detail in the main text so that it will be clear whether ‘483 were up-regulated and 1,666 were down-regulated’ (line 184) means that those genes were up-/down-regulated genes in the testis when compared to the ovary (or vice versa)?
3. In the Supplementary Table S6 – please check for missing information (e.g., cells with texts ‘#NAME?’, ‘inf’ etc). Furthermore, please recheck the fold-change expression of insulin-like growth factor-binding domain protein (IAGBP), I assume based on ‘baseMean_Sample_jingchao=13.43 FPKM’ vs ‘baseMean_Sample_luanchao=3921.61 FPKM’ (could you also revise these sample names properly? E.g., jingchao means?) that this gene should be considered as ‘up-regulated’?
4. Based on Supplementary Table S4 - a list of all DEGs. There were only 503 genes out of 2149 genes that are annotated, which is not surprising as rosenbergii is a non-model species. This therefore required further manual search of important genes based on previous literature, and the authors have done a great job in this case, showing a result in Supplementary Table S6. However, I have noticed that there are a few reproductive hormones that were not included in Supplementary Table S6, for examples, androgenic gland hormone-like protein (MAL) (NCBI accession number: FJ595507), crustacean female sex hormones, neuroparsin etc., with noted that the latter hormones appeared to be down-regulated in the current study. Hence, I am wondering if there were hormones/sex-related genes that the authors had looked at and found that they were not significantly changes (and hence not included in Suppl. Table 6)? If there were, could you please include their names/description (together with their expression) in Suppl. Table 6 so that it will be more informative to the readers?

Discussion

1. Line 293: ‘Furthermore, MRPINK has a regulatory effect on M. rosenbergii fertilization.’ – will need a citation for this statement.
2. Line 333-337: Based on literature, I assumed that Dmrt gene is a testis-specific gene, both in insects and crustaceans. However, the evidence from the current study showed that this is not the case. I recommended the authors to elaborate more discussion on this finding. Would it be possible that there are other forms of Dmrt genes (which is/are testis-specific) presented in the transcriptome data generated but they were not differentially expressed like Dmrt99B? Would this be an indicator of a specific function of 99B form, that is opposite to a testis-specific form?

Author Response

Hello dear reviewer, here is your questions and my answers to your question 

Comments and Suggestions for Authors

Overall: The concept of this work was clearly defined. The experiments were well designed, and the manuscript was nicely written. In brief, the authors performed gene expression analysis through a transcriptomic platform. This finding helps to understand the differences between gonad development in male and female prawns at molecular levels. A whole concept contains novelty and scientific merit which will be of interest to researchers in this field. However, there are some revisions required before getting published.

Materials&Methods

1. Based on what the authors mentioned that mature males and females (5 each) were used for gonad tissue collection – can you please further specify their reproductive stages (i.e., early or late stage of gonad development/maturation, or using their average gonadosomatic indices) in the main text? Were their gonad stages conformed among 5 individuals of same sex or it was a mixture of different stages?

Thank you for your comment, in fact we used late mature gonads of Macrobrachium rosenbergii in this study, and the developmental stages of the gonads in the same group of five Macrobrachium rosenbergii were the same. We have made corresponding changes and added references in the article, see line 79 for details.

2. Analysis of DEGs and Functional Enrichment – how was the DEGs between 2 datasets compared? Like testis against ovary, or vice versa? Please include this detail in the main text so that it will be clear whether ‘483 were up-regulated and 1,666 were down-regulated’ (line 184) means that those genes were up-/down-regulated genes in the testis when compared to the ovary (or vice versa)?

Thank you for your comment. The purpose of differential expression analysis is to find out the differentially expressed genes among different samples. After obtaining the differentially expressed genes, we do GO functional significance and KEGG Pathway significance analysis on them. The differential expression of genes was calculated according to the negative binomial distribution test in the DESeq software (http://bioconductor.org/packages/release/bioc/html/DESeq.html). The NB (negative binomial distribution test) was used to test the significance of the difference in the number of reads, and the basemean value was used to estimate the expression of the gene expression. When using RNA-seq data to compare and analyze the differential expression of the same gene in two samples, two criteria can be selected: one is FoldChange, which is the fold change of the expression level of the same gene in the two samples; the other is pvalue or FDR (adjusted pvalue), the calculation method of FDR value is to calculate the p-value of each gene first, and then use the FDR error control method to perform multiple hypothesis test correction on the p-value. The default criteria for screening differences were p<0.05 and a fold difference greater than 2. If the basemean value of the testis of a gene of Macrobrachium rosenbergii is higher than the basemean value of the ovary, it means that the gene is down-regulated and on the contrary up-regulated.

3. In the Supplementary Table S6 – please check for missing information (e.g., cells with texts ‘#NAME?’, ‘inf’ etc). Furthermore, please recheck the fold-change expression of insulin-like growth factor-binding domain protein (IAGBP), I assume based on ‘baseMean_Sample_jingchao=13.43 FPKM’ vs ‘baseMean_Sample_luanchao=3921.61 FPKM’ (could you also revise these sample names properly? E.g., jingchao means?) that this gene should be considered as ‘up-regulated’?

Thank you for your comment, I am very sorry, we made this mistake, we have revised the content in Supplementary Table S6, in fact, the fold-change expression of insulin-like growth factor-binding domain protein (IAGBP) is correct, It's just that the values of baseMean_Sample_testis and baseMean_Sample_ovary were accidentally reversed by us. In addition, we have changed the name. Please see Supplementary Table S6 for details. Thank you for your attention.

4. Based on Supplementary Table S4 - a list of all DEGs. There were only 503 genes out of 2149 genes that are annotated, which is not surprising as rosenbergii is a non-model species. This therefore required further manual search of important genes based on previous literature, and the authors have done a great job in this case, showing a result in Supplementary Table S6. However, I have noticed that there are a few reproductive hormones that were not included in Supplementary Table S6, for examples, androgenic gland hormone-like protein (MAL) (NCBI accession number: FJ595507), crustacean female sex hormones, neuroparsin etc., with noted that the latter hormones appeared to be down-regulated in the current study. Hence, I am wondering if there were hormones/sex-related genes that the authors had looked at and found that they were not significantly changes (and hence not included in Suppl. Table 6)? If there were, could you please include their names/description (together with their expression) in Suppl. Table 6 so that it will be more informative to the readers?

Thank you very much for your comment, it can be seen that you are very professional, this opinion is very professional, thank you. We have supplemented the information on genes androgenic gland hormone-like protein (MAL) , crustacean female sex hormones (CFSH), neuroparsin in Supplementary Table S6. In addition, we have added a discussion of these genes to the manuscript. In the Discussion section of the paper, we have added some references, see the revised manuscript for details. As you mentioned, these genes may play an important role in the process of sex differentiation of Macrobrachium rosenbergii, and it is worth doing further indepth research. This is a good direction, and this may be our future research direction. Thank you again.

Discussion

1. Line 293: ‘Furthermore, MRPINK has a regulatory effect on M. rosenbergii fertilization.’ – will need a citation for this statement.

Thank you for your comment, sorry for that, we made this mistake, we have added the reference, see line 293 for details.

2. Line 333-337: Based on literature, I assumed that Dmrt gene is a testis-specific gene, both in insects and crustaceans. However, the evidence from the current study showed that this is not the case. I recommended the authors to elaborate more discussion on this finding. Would it be possible that there are other forms of Dmrt genes (which is/are testis-specific) presented in the transcriptome data generated but they were not differentially expressed like Dmrt99B? Would this be an indicator of a specific function of 99B form, that is opposite to a testis-specific form?

Thank you for your comment, as you said, the Dmrt gene family is an important sex-related gene family in invertebrate and vertebrate sex development, which plays an important role in both sex differentiation and maintenance of sex characteristics. Most members of the Dmrt gene family are testis-specific, but some members are not testis-specific, that is, they are distributed in other biological tissues, even contrary to testis-specific, in our findings, dmrt99B is the ovary Specifically, we reasoned that it is less likely that dmrt99B is involved in the mechanisms by which male reproductive system differentiation and development is involved, or that this role is through indirect effects. In addition, unfortunately, we did not find other Dmrt gene family members in the differential genes reported in the transcriptome, which may be related to the species-specific expression of other Dmrt gene family members.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study deals with the sexual differences of gonadal mRNA expression levels in giant freshwater prawn Macrobrachium rosenbergii. This study attempts to provide an important contribution to elucidate prawn biology. While the experimental design and obtained data sound well, I have concerns about the content of this paper. This study is overlapped previous studies such as Jiang et al (2019). Moreover, most of the genes which authors highlighted in Fig.4 and Fig.5 have already been addressed in prior publication. Therefore, I found it a little difficult to decide what to make of this work. I suggest that newly identified genes in Table S6 should be highlighted and applied to the further experiment.

Suggestions are listed in below, which I believe will improve the MS.

Major point

This manuscript is lack of novelty. Most of selected genes focusing on this paper has been confirmed in the same species. In figure S6, many genes which has not been evaluated in M. rosenbergii are listed. I suggest that authors select those genes for highlights for this MS.

Minor points

Overall: the abbreviations of genes name should be listed in the first appearance.

Line 58: References are missed regarding “MRPINK”.

Line 79: Please provide the information of the gonadal maturation stage of prawn.  

Reference 18 (line 102): I suggest changing the cited reference regarding Trinity.

Line 105: “Swissprot” should be “Uniprot”.

Section 2.5: How many prawns did author used for qPCR.

Line 127: Please give the information of Promega. Here is the first appearance in MS.

Line 130: Please remove the locational information of Takara. Here is not the first appearance.

Line 132: This program would be dissociation curve method. The expression of this sentence should be corrected. For example, then 15 s at 95°C and then 10 seconds each at 0.2°C increments between 60°C and 95°C.   

Section 2.7: Usually, one-way ANOVA is cited for the comparison among the groups (more than three groups). Two groups were compared in this experiment.

Line 146: Please remove the locational information. Here is not the first appearance.

Lines 148-149: Is the 30 Gb correct?

Line 163: not pulex, should be magna.

Line 170: binding cell?

Line 186: 187 DEGs in female, 90 DEGs in male?

         Authors have already chosen 483 up-regulated (highly expressed in male) and 1,666 down-regulated (highly expressed in female).

         What is the differences between these two data?

Table S4: The header contains Chinese, such as jingchao. Please correct.

Line 196: P should be expressed in a small letter.

Lines 223-226: Please provide the reason why authors chose these 7 kegg pathways only?

Lines 226 and 233: Why authors concluded that these pathway related to metabolic pathway?

Line 236: For male, only top 12 were selected.

         The title should be corrected.

Lines 242-243: Please remove the full names of genes. Here is not the first appearance.

Table S6: How authors chose these listed genes?

         Blank columns were found in Column A.

Figure 5: The label of Y axis should be log2FC?

References: Many references should be corrected and unify the styles.

Please check the authors name (Reference No.3)

  Page number is missing or wrong (Reference No.10, 14, 22, 27, 29, 36, 40, 44, 46, 47, 51)

Parenthesis should be removed (Reference No.11, 24, 26, 34)

The publish year appears twice (Reference No.37)

The journal name is missing (Reference No.41)

Author Response

Hello dear reviewer, here is your question and my answer to your question (marked in red)

This manuscript is lack of novelty. Most of selected genes focusing on this paper has been confirmed in the same species. In figure S6, many genes which has not been evaluated in M. rosenbergii are listed. I suggest that authors select those genes for highlights for this MS.

Thank you for your suggestion. Our work has identified 12 genes related to the development of the gonads and sex differentiation of Macrobrachium rosenbergii, and we also divided these genes into male-related genes and female-related genes. This is an extension of previous studies. In addition, at the same time, these genes include not only the three genes of Mrr, MRPINK, and IR, but also IAGBP, TESK1, and dsx, ERR, Sxl3, cyclinB, Dmrt99B, PPP2A, and ADCY9 genes. The work can be seen as a complement and extension of the work of previous studies. At the same time, compared with the work of previous studies, our innovation is that we have also discovered some signal transduction pathways related to the maturation, spermatogenesis and oogenesis of Macrobrachium rosenbergii, including mitogen activated protein kinase (MAPK) , Wnt, Gonadotropin Releasing Hormone (GnRH) and Vascular Endothelial Growth Factor (VEGF). In addition, we also found some reproductive hormone-related DEGs in the testis and ovary of M. rosenbergii, such as androgenic gland hormone-like protein (MAL), crustacean female sex hormones (CFSH) and neuroparsin (NP). We believe that the data of our study provides a very useful genome resource for future research on sex differentiation and actual aquaculture of Macrobrachium rosenbergii. At the same time, we also discuss the genes in Table S6 (the red part in the table), please check the latest revised version for details.

Minor points

Overall: the abbreviations of genes name should be listed in the first appearance.

Thank you for your comments. We have made corresponding revisions to the full text. We have added the full names and abbreviations of all gene names that appear for the first time. Due to the word limit of the abstract, we use abbreviations for the genes in the abstract. We have revised all the other parts of the article, please check.

Line 58: References are missed regarding “MRPINK”.

Thank you for your comments, we are very sorry that we made this mistake, we have inserted the relevant literature, please see lines 56-57 for details.

Line 79: Please provide the information of the gonadal maturation stage of prawn.

Thanks for your comments, we have added information and references on the stages of gonadal maturation of Macrobrachium rosenbergii, please see line 79 for details.

Reference 18 (line 102): I suggest changing the cited reference regarding Trinity.

Thank you for your comments, sorry for the mistake, we have changed the relevant references, see line 101 for details.

Line 105: “Swissprot” should be “Uniprot”.

Thank you for your opinion. Your understanding is very correct. In fact, UniProt is the English abbreviation of Universal Protein, which is the most informative and resourceful protein database. It is formed by integrating data from three major databases, SwissProt, TrEMBL and PIR-PSD. Its data mainly comes from the protein sequences obtained after the completion of the genome sequencing project. It contains a wealth of information on the biological functions of proteins from the literature. Therefore, we think it is more appropriate to use the SwissProt writing method.

Section 2.5: How many prawns did author used for qPCR.

Thank you for your comments. We used a total of five male M. rosenbergii and five female M. rosenbergii in our experiments. For validation of the sequencing data, DEGs were chosen for qRT-PCR analysis. The total RNA of qRT-PCR was obtained from residual gonad samples used in RNA-seq experiments and then cDNA was synthesized from RNA (n = 5), which was used for qRT-PCR quantification, with the support of GoScriptTM Reverse Transcription System.

Line 127: Please give the information of Promega. Here is the first appearance in MS.

Thank you for your comments, we have added relevant information, see line 127 for details.

Line 130: Please remove the locational information of Takara. Here is not the first appearance.

Thank you for your comments, we have deleted the relevant content, see line 130 for details.

Line 132: This program would be dissociation curve method. The expression of this sentence should be corrected. For example, then 15 s at 95°C and then 10 seconds each at 0.2°C increments between 60°C and 95°C.

Thank you for your comments, we have changed the expression of the PCR reaction program, see line 132 for details.

Section 2.7: Usually, one-way ANOVA is cited for the comparison among the groups (more than three groups). Two groups were compared in this experiment.

Thank you for your opinion, we used one-way ANOVA when processing qRT-PCR data.

Line 146: Please remove the locational information. Here is not the first appearance.

Thank you for your comments, we have removed this location, you are very attentive, thank you.

Lines 148-149: Is the 30 Gb correct?

Thanks for your interest, 30GB is correct.

Line 163: not pulex, should be magna.

Thank you for your suggestion, we have changed pulex to magna in the article, see line 163 for details.

Line 170: binding cell?

Sorry, we made this mistake, but what I meant to say was the major subcategories were “cellular process” and “cellular component”, we have made corresponding changes, please see line 170 for details.

Line 186: 187 DEGs in female, 90 DEGs in male?

         Authors have already chosen 483 up-regulated (highly expressed in male) and 1,666 down-regulated (highly expressed in female).

         What is the differences between these two data?

Thanks for your interest. In our report, 483 genes were up-regulated (highly expressed in male Macrobrachium rosenbergii, of which 90 genes were exclusively expressed in male Macrobrachium rosenbergii) and 1666 down-regulated (highly expressed in female Macrobrachium rosenbergii). expression, of which 187 genes were exclusively expressed in female Macrobrachium rosenbergii).

Table S4: The header contains Chinese, such as jingchao. Please correct.

Thank you for your suggestion. We have changed Chinese to English in table S4. For details, see table S4. In addition, we have also changed similar errors in all attachments.

Line 196: P should be expressed in a small letter.

Thanks for your suggestion, we have changed the letter P to uppercase, see line 196 for details.

Lines 223-226: Please provide the reason why authors chose these 7 kegg pathways only?

Lines 226 and 233: Why authors concluded that these pathway related to metabolic pathway?

Thank you for your interest, in fact, because these 7 metabolic pathways have the highest Enrichment score in the testis of male Macrobrachium rosenbergii, their scores are in the top seven. Therefore, we have reason to speculate that these seven metabolic pathways may be related to the masculinization characteristics of Macrobrachium rosenbergii.

Line 236: For male, only top 12 were selected.

         The title should be corrected.

Thank you for your comments. We have revised the title of the accordingly. For details, please see the title of the revised manuscript.

Lines 242-243: Please remove the full names of genes. Here is not the first appearance.

Thank you very much for your suggestion, we have removed the full name of the gene that is not the first occurrence, see lines 242-243 for details.

Table S6: How authors chose these listed genes?

We carefully studied all the differentially expressed genes reported in the transcriptome, and selected these genes that may be related to sex differentiation and gonadal development of Macrobrachium rosenbergii in combination with previous articles related to sex differentiation and gonadal development.

Blank columns were found in Column A.

Your findings are correct. Because a gene often corresponds to multiple transcripts in an organism.

Figure 5: The label of Y axis should be log₂FC?

Your understanding is correct, the label of the Y-axis is log2FC, we have modified the relevant expression, see the content of lines 266-268 for details.

References: Many references should be corrected and unify the styles.

Please check the authors name (Reference No.3)

Thanks for your comments, we have revised the authors' names in the reference, see reference 3 for details

Page number is missing or wrong (Reference No.10, 14, 22, 27, 29, 36, 40, 44, 46, 47, 51)

Thanks for your comments, we have revised the page number.

Parenthesis should be removed (Reference No.11, 24, 26, 34)

Thank you for your comments. The extra brackets in these references have been removed by us. For details, see references 11, 24, 26, and 34.

The publish year appears twice (Reference No.37)

We are very sorry, we made this mistake, we have removed the redundant content, see lines 498--500 for details.

The journal name is missing (Reference No.41)

Your comments are greatly appreciated, we have made revisions in the manuscript and added references as detailed in lines 508-510.

Reviewer 3 Report

Author Response

Thank you for taking the time to review my manuscript.

Round 2

Reviewer 2 Report

The manuscript has been revised well and much improved; however, I cannot recommend the acceptance of the present paper in this form for publication. As I mentioned before, the main part of genes and statements of this papers overlapped previous studies. Newly identified genes should be highlighted.  

Regarding discussion about pathways ( Lines 348-358 and Fig. S7), first of all, the title of this figure in MS (Line 403) and  the title of excel sheet (containing Chinese) are not identical.  Authors chose some signaling pathways; however, only few genes were identified in some pathways. I guess the enrichment scores is low. Moreover, some genes (such as comp119876_C0_seq1 etc.) are overlapped in many pathways. It would not be the pathway specific genes. Authors should carefully check.

Lines 38-39: Compared to previous version, the PCR cycles has been reduced to 40 cycles. However, I recommended to change the sentence regarding the method of dissociation curve.

Line 154: please correct “30 Gb of raw reads”.   The unit of reads is not Gb. If authors indicate raw bases, it should be 20 Gb (testis  11 Gb, ovary 9 Gb in table1)?

Line 176: Fig. S5 should be S4

Line 176: In Fig. S4, “cellular component” does not exist.

Lines 181-182: Please rewrite this sentence.   “genetic information process” should be “ribosome”.   “human diseases” should be “pathway in cancer”.

Figure 3B: It is difficult to find the dots.

Lines 226-240 and Fig.3: Previously, I asked the reasons of the 7 pathways selection. Authors replied “Thank you for your interest, in fact, because these 7 metabolic pathways have the highest Enrichment score in the testis of male Macrobrachium rosenbergii, their scores are in the top seven.” How about ovary? Moreover, I recommend to add the reason and/or explanation to MS.

Lines 226-240 : As I mentioned before, it is too rough to conclude that all selected pathways were related to “metabolic pathway”. As long as I read the MS, “reproduction” and “some other terms” seems to prefer for female.

Overall: Please unified the styles of the company’s location. Some parts include state.

References

No.14: Page number is missing

No.15: Page number is missing

No.20: The format is different compared to others. A colon should be a comma.

No.36: Please remove the parenthesis.

No.38: Please remove the parenthesis.

No.51: Is this a doctoral dissertation? The page numbers do not exist?

Author Response

The manuscript has been revised well and much improved; however, I cannot recommend the acceptance of the present paper in this form for publication. As I mentioned before, the main part of genes and statements of this papers overlapped previous studies. Newly identified genes should be highlighted.

Response: Thanks for your advice. Our work has identified 12 genes related to gonadal development and sex differentiation in M. rosenbergii and divided these genes into male-related genes and female-related genes. This is an extension of previous research. In addition, these genes include not only the 3 genes (Mrr, MRPINK, and IR) but also the other 9 genes (IAGBP, TESK1, and dsx, ERR, Sxl3, cycling, Dmrt99B, PPP2A, and ADCY9). This work can be seen as a complement and extension to previous research work. In addition, we also newly discovered some new genes (MAL, CFSH, VgR, and NP) that may be related to gonadal development and sex differentiation of M. rosenbergii. We add the discussion of these new genes (MAL, CFSH, VgR, and NP) in the last paragraph of the manuscript. At the same time, compared with previous research work, our innovation is that we also discovered some signal transduction pathways related to the maturation, spermatogenesis, and oogenesis of M. rosenbergii, including mitogen-activated protein kinase (MAPK), Wnt, Gonadotropin-Releasing Hormone (GnRH) and Vascular Endothelial Growth Factor (VEGF). Our study data provide a valuable genomic resource for future studies on sex differentiation and practical aquaculture of M. rosenbergii. At the same time, we have replaced the pictures with the higher definition in the latest manuscript; please check the newest revision for details.

Regarding discussion about pathways ( Lines 348-358 and Fig. S7), first of all, the title of this figure in MS (Line 403) and the title of excel sheet (containing Chinese) are not identical. Authors chose some signaling pathways; however, only few genes were identified in some pathways. I guess the enrichment scores is low. Moreover, some genes (such as comp119876_C0_seq1 etc.) are overlapped in many pathways. It would not be the pathway specific genes. Authors should carefully check.

Response: Thank you for your comment. Sorry for the mistake. We have revised the titles of Table S4, Table S5, and Table S7, but some titles are slightly different from those in the manuscript due to the word limit. We have made corresponding changes. Please see Table S4, Table S5, and Table S7 for details. In addition, we have changed the Chinese to English in Table S4, Table S5, and Table S7. You are very professional. As you said, only a few genes were identified in some pathways. For example, there is only one gene in the VEGF signaling pathway. Although its Enrichment score is relatively low, this does not mean that its significance in the sex differentiation of M. rosenbergii is relatively tiny. Although the Enrichment scores on some signaling pathways are relatively low, they are of great importance. We should not ignore these signaling pathways with relatively low Enrichment scores in scientific research.Furthermore, we have carefully checked the manuscript. Although some genes (such as comp119876_C0_seq1, etc.) are distributed in many pathways, they are indeed DEGs. You can find the details of comp119876_C0_seq1 on line 756 of Table S4.

Lines 138-139: Compared to previous version, the PCR cycles has been reduced to 40 cycles. However, I recommended to change the sentence regarding the method of dissociation curve.

Response: Thanks for your comments. We have changed the sentence regarding the method of dissociation curve. The qRT-PCR was performed under the following conditions: denaturation at 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 25 s, and 95°C for 15 s, finally 60°C for 1 min.

Line 154: please correct “30 Gb of raw reads”. The unit of reads is not Gb. If authors indicate raw bases, it should be 20 Gb (testis 11 Gb, ovary 9 Gb in table1)?

Response: Thanks for your comments. Sorry for the mistake. The correct statement should be “A total of 78,074,612, and 89,598,146 raw reads were created from the ovary and testis samples. After removing low-quality reads, 20 Gb of clean data were retained for subsequent de novo assembly” We have made changes in the article; see line 154 for details

Line 176: Fig. S5 should be S4

Response: Thanks for your comment. We have changed Fig. S5 to Fig. S4.

Line 176: In Fig. S4, “cellular component” does not exist.

Response: Thanks for your comments. We have replaced Fig. S4 with a clearer picture. In addition, The significant subcategories were “cellular process”, “cell” and “cell part”. We have made corresponding changes in the article, see line 176 for details.

Lines 181-182: Please rewrite this sentence. “genetic information process” should be “ribosome”.“human diseases” should be “pathway in cancer”.

Response: Thanks for your comments. We have changed "genetic information process" and "human diseases" to "ribosome" and "pathway in cancer".

Figure 3B: It is difficult to find the dots.

Response: Thank you for your comment. We have replaced Figure 3B, please see Figure 3B for details.

Lines 226-240 and Fig.3: Previously, I asked the reasons of the 7 pathways selection. Authors replied “Thank you for your interest, in fact, because these 7 metabolic pathways have the highest Enrichment score in the testis of male Macrobrachium rosenbergii, their scores are in the top seven.” How about ovary? Moreover, I recommend to add the reason and/or explanation to MS.

Lines 226-240 : As I mentioned before, it is too rough to conclude that all selected pathways were related to “metabolic pathway”. As long as I read the MS, “reproduction” and “some other terms” seems to prefer for female.

Response: Thank you for your comments. The 7 metabolic pathways (ko04212, ko04142, ko04914, ko04114, ko04113, ko04140, and ko05166) had the highest enrichment scores in the ovaries of M. rosenbergii. Therefore, we have reason to speculate that these 7 metabolic pathways may be closely related to the ovarian development and maintenance of feminization characterizations of M. rosenbergii. These 10 metabolic pathways (ko00600, ko04924, ko04745, ko04970, ko04512, ko05418, ko04670, ko04270, ko04520 and ko04611) had the highest enrichment scores in the testis of male M. rosenbergii. Therefore, these 7 metabolic pathways may be closely related to the testis development and maintenance of masculinization characteristics of M. rosenbergii.

Overall: Please unified the styles of the company’s location. Some parts include state.

Response: Thank you for your comment. We have unified the style of the company's location in the article. The revised part is marked in red, and please check it in the revised manuscript.

References

No.14: Page number is missing

Response: Thank you for your comment, this is a dissertation, so there is no specific page number.

No.15: Page number is missing

Response: Thank you for your comment, this is a dissertation, so there is no specific page number.

No.20: The format is different compared to others. A colon should be a comma.

Response: Thank you for your comment. We have changed the colon to a comma. Please see reference 20 for details.

No.36: Please remove the parenthesis.

Response: Thank you for your comments. We have removed the parenthesis here. Please see reference 36 for details.

No.38: Please remove the parenthesis.

Response: Thank you for your comment. We have removed the parenthesis here. Please see reference 38 for details.

No.51: Is this a doctoral dissertation? The page numbers do not exist?

Response: Thank you for your comments. As you said, the reference here is a dissertation, so there is no specific page number.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

While I appreciate the reversion of the manuscript, I think there have little expectation to improve in the further. Previously, I pointed out several points to review such as the pathway analysis and its discussion etc. However, as far as I read the new version, I could not find the convincing answer.

As I mentioned regarding pathway analysis before, authors should reconsider the discussion. I understand GnRH signaling pathway are important for gonadal maturation and I understand comp119876_C0_seq1(cell division control protein 42) are highly expressed in female. However, cdc42 are ubiquitously expressed in many pathways like a second messenger. The score of GnRH signaling pathway is low and cdc42 are not only specific to GnRH signaling pathway. Furthermore, the second genes (comp125266_c0_seq6: Troponin C) of GnRH signaling pathway are neither pathway specific genes nor highly expressed in males (not female).

Wnt signaling pathway also reconsider. The score is low. The first gene (comp91391_c0_seq1: Wnt5a ligand) are highly expressed in female. However, the second gene (comp117864_c0_seq1: Serine/Threonine kinase domain protein) is ubiquitously expressed, which means not Wnt specific gene. Moreover, this gene is highly expressed in male. Authors should not highlight these results of pathway analysis.

Lines 26-29, 189-194: As I asked about DEGs before, I don’t still understand how authors selected DEGs. DEGs means the genes were “exclusively” expressed in males and/or females? Or just differently expressed genes? What is “exclusively”?  Moreover, how authors carried out the multiple tests for N=1? (lines 121-122)?

Lines 137-139: A dissociation curve method are now missing. How did authors confirm the amplified products were derived from the correct coding sequences?

Lines 234-237, 245-247: As I asked before, how authors define these 7 and 10 pathways relate to metabolic pathways? I also check the KEGG pathway database; however, I could not find the words for “metabolic pathway” in some pathways. Could you show me the evidence?

Table S7: Please change the title of excel sheet. Please use English words.

Lines 358-360: In reference No.54, I could not find the supporting sentences. Nor “VEGF” word.

Lines 366-368: In reference No.58, I could not find the supporting sentences. This article relates to molting. Not gametogenesis.

Line 378 and table S6: The MAL stands for “androgenic gland hormone-like protein (line 378)” or “anchored androgenic gland specific factor (table S6)”?

References
・No.14 and 15: The volume and page number are granted. These are the published journals.
・ No.23:　 Please unify the style. Authors use the abbreviations for the journal name, excepting for this.
・No.36：　Please unify the style. Authors use parentheses for Pt 1
・No.38(line 540) : Remove “Basel” and use a parenthesis for issue.

Author Response

Comments and Suggestions for Authors

While I appreciate the reversion of the manuscript, I think there have little expectation to improve in the further. Previously, I pointed out several points to review such as the pathway analysis and its discussion etc. However, as far as I read the new version, I could not find the convincing answer.

Response: Thank you very much for your patience in reviewing our article. In response to your comments, we have responded one by one below, and the content of the reply is highlighted in red.

As I mentioned regarding pathway analysis before, authors should reconsider the discussion. I understand GnRH signaling pathway are important for gonadal maturation and I understand comp119876_C0_seq1(cell division control protein 42) are highly expressed in female. However, cdc42 are ubiquitously expressed in many pathways like a second messenger. The score of GnRH signaling pathway is low and cdc42 are not only specific to GnRH signaling pathway. Furthermore, the second genes (comp125266_c0_seq6: Troponin C) of GnRH signaling pathway are neither pathway specific genes nor highly expressed in males (not female).

Response: Thank you for your comment. Sorry for the mistake. We noticed a relatively low enrichment score for the Wnt signaling pathway, so we have removed the reference to the Wnt signaling pathway

Wnt signaling pathway also reconsider. The score is low. The first gene (comp91391_c0_seq1:Wnt5a ligand) are highly expressed in female. However, the second gene (comp117864_c0_seq1:Serine/Threonine kinase domain protein) is ubiquitously expressed, which means not Wnt specific gene. Moreover, this gene is highly expressed in male. Authors should not highlight these results of pathway analysis.

Response: Thank you for your comment. Sorry for the mistake. We noticed a relatively low enrichment score for the Wnt signaling pathway, so we have removed the reference to the Wnt signaling pathway

Lines 26-29, 189-194: As I asked about DEGs before, I don’t still understand how authors selected DEGs. DEGs means the genes were “exclusively” expressed in males and/or females? Or just differently expressed genes? What is “exclusively”? Moreover, how authors carried out the multiple tests for N=1? (lines 121-122)?

Response: Thank you for your comment. Sorry for the mistake. The purpose of differential expression analysis is to find out the differentially expressed genes among different samples. After obtaining the differentially expressed genes, we performed GO functional significance and KEGG Pathway significance analysis on them. The differential expression of genes was calculated according to the negative binomial distribution test in the DESeq software (http://bioconductor.org/packages/release/bioc/html/DESeq.html). The NB (negative binomial distribution test) was used to test the significance of the difference in the number of reads, and the basemean value was used to estimate the expression of the gene expression. When using RNA-seq data to compare and analyze the differential expression of the same gene in two samples, two criteria can be selected: one is FoldChange, which is the fold change of the expression level of the same gene in the two samples; the other is pvalue or FDR (adjusted pvalue), the calculation method of FDR value is to calculate the p-value of each gene first, and then use the FDR error control method to perform multiple hypothesis test correction on the p-value. The default criteria for screening differences were p<0.05 and a fold difference greater than 2. If the basemean value of the testis of a gene of Macrobrachium rosenbergii is higher than the basemean value of the ovary, it means that the gene is down-regulated and on the contrary up-regulated. 484 DEGs (down-regulated) were significantly highly expressed in the testis of M. rosenbergii, while 1,665 DEGs (up-regulated) were significantly highly expressed in the ovary of M. rosenbergii. Figures 1A and 1B show the distribution of these DEGs, among which 699 DEGs were expressed only in the ovaries of M. rosenbergii (These DEGs were not expressed in the testis of M. rosenbergii), and 191 DEGs were only expressed in the testes of M. rosenbergii (These DEGs are not expressed in M. rosenbergii ovaries).

Lines 137-139: A dissociation curve method are now missing. How did authors confirm the amplified products were derived from the correct coding sequences?

Response: Thank you for your comment. Sorry for the mistake. The sequences of all our target genes are obtained from the transcriptome, and then we set upstream and downstream primers according to the open reading frames of each gene to amplify the sequences of these genes. The qRT-PCR was performed under the following conditions: denaturation at 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 25 s, and 95°C for 15 s, finally 60°C for 1 min. Further melting curve analysis was then performed to confirm whether only one PCR product was amplified. All samples were analyzed with three biological replicates and the relative expression levels of target genes were normalized to β-actin and calculated by the 2^−ΔΔCT method. Based on this, we believe that the gene we amplified is the target gene. In fact, the method of obtaining target gene sequence based on transcriptome sequencing and then using it for subsequent research has been widely used.

Lines 234-237, 245-247: As I asked before, how authors define these 7 and 10 pathways relate to metabolic pathways? I also check the KEGG pathway database; however, I could not find the words for “metabolic pathway” in some pathways. Could you show me the evidence?

Response: Thank you for your comment. Sorry for the mistake. Although the seven metabolic pathways (ko04212, ko04142, ko04914, ko04114, ko04113, ko04140, and ko05166) and the ten metabolic pathways (ko00600, ko04924, ko04745, ko04970, ko04512, ko05418, ko04670, ko04270, and ko045270) had the highest enrichment scores in the ovaries and testes of M. rosenbergii, but it is inaccurate to infer that they are closely related to the gonadal development of M. rosenbergii. Because their direct relationship remains to be proven, we have removed these less rigorous inferences from the manuscript. However, these metabolic pathways do have the highest enrichment scores in the gonads of M. rosenbergii, which is worthy of our attention, and we will pay more attention to these metabolic pathways in future studies.

Table S7: Please change the title of excel sheet. Please use English words.

Response: Thank you for your comment. Sorry for the mistake. We have changed the relevant content in table S7 to English, please see the table S7 title for details.

Lines 358-360: In reference No.54, I could not find the supporting sentences. Nor “VEGF” word.

Response: Thank you for your comment. Sorry for the mistake. As you said the enrichment score for the VEGF signaling pathway is relatively low, so we removed the content about the VEGF signaling pathway.

Lines 366-368: In reference No.58, I could not find the supporting sentences. This article relates to molting. Not gametogenesis.

Response: Thank you for your comment. Sorry for the mistake. We have changed references, please see reference 55 for details

Line 378 and table S6: The MAL stands for “androgenic gland hormone-like protein (line 378)” or “anchored androgenic gland specific factor (table S6)”?

Response: Thank you for your comment. Sorry for the mistake. The MAL stands for “androgenic gland hormone-like protein”. In addition, we also changed the “anchored androgenic gland specific factor” in table S6 to “androgenic gland hormone-like protein”, please see table S6 for details.

References
No.14 and 15: The volume and page number are granted. These are the published journals.

Response: Thank you for your comment. Sorry for the mistake. We have added volume and page numbers to references, please see references 14 and 15 for details

No.23:Please unify the style. Authors use the abbreviations for the journal name, excepting for this.

Response: Thank you for your comment. Sorry for the mistake. We have changed the journal name to an abbreviated form, please see reference 23 for details.

No.36：Please unify the style. Authors use parentheses for Pt 1

Response: Thank you for your comment. Sorry for the mistake. We have made corresponding changes in the manuscript, please see reference 36 for details.

No.38(line 540) : Remove “Basel” and use a parenthesis for issue.

Response: Thank you for your comment. Sorry for the mistake. We have made corresponding changes in the manuscript, please see reference 38 for details.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.