Potential Functions of Gem-Associated Protein 2-Like Isoform X1 in the Oriental River Prawn Macrobrachium nipponense: Cloning, qPCR, In Situ Hybridization, and RNAi Analysis

Round 1

Reviewer 1 Report

The article submitted by Jin et al., seeks to investigate the function of the GEM gene in the gonads of the oriental river prawn M. nipponese. I found the study interesting, as it presents novel information about the expression of the GEM gene in the prawn’s reproductive tissue. Additionally, this study is of potential significance for prawn farming and aquacultures. However, the manuscript needs some improvement in order to become suitable for publication in IJMS. My comments are outlined below:

Major concerns:

1. I strongly recommend to the authors to revise the English language and style throughout the manuscript as in its current form it is not easy to navigate through.  

2. At any place of the text the authors describe the type of protein GEM is (Transcription factor, receptor, enzyme…?). It is not clear what the supposed cellular/molecular function is. If this information exists, the authors should include it in the introduction and take it into account while discussing their results.

3. Figures are not referred at any place in the text. I strongly recommend to the authors to cite the figures in the appropriate results sections. 

4. From the in situ hybridization experiments it is not clear what the n number of examined individuals is. The authors should state how many individuals they have examined and also say if the labeling is consistent between the independent biological replicates. 

5. Based on the RNAi experiments, the authors examine if GEM functions to regulate the expression of another gene: IAG,as well as testosterone levels. To my opinion this part of the manuscript is incomplete and requires further explanations, if not experiments. The significance of increased testosterone levels is not discussed at all. Also, does GEM RNAi lead to any anatomical or physiological changes of the gonads in RNAi-treated individuals in comparison to controls? Maybe some measurements or an H&E staining experiment on tissues from RNAi-treated vs vehicle-treated animals could address that. Overall here, I think that some additional information on whether any changes in reproductive tissue appearance or function is observed following GEM RNAi would improve the quality of the manuscript.

The least authors could do is to discuss what effects the increase in IAG expression and testosterone levels could have on prawn’s reproductive tissue and physiology.

6. Along the same lines, if CHH, GIH and MIH (not sure what these refer to exactly; genes I assume) also have regulatory roles on male differenciation in the androgenic gland as IAG does, the authors might consider comparing their expression in the GEM RNAi vs vehicle-treated animals to gain further insights in GEM functions.  

7. Fig. 2: statistical relationships need to be explained in the figure legend.

Minor concerns:

8. Italicize in situand M. Nipponesethroughout the text (e.g. in figure legends).

9. Adding a schematic of the GEM gene sequence would be helpful. It can go as Fig. 1A and the phylogenetic tree can become Fig. 1B.

10. Legend of Fig. 4 says “scale bar: 50 μm”, however no scale bars are shown on the images. Please, show the scale bars. Additionally, if possible, the authors could provide higher magnification images for Figures 4 and 5 to better see the cell types (e.g. spermatid), in which the mRNA expression is detected. 

Author Response

Major concerns:

1. Thank you for your comments. The language has been revised by a professional English editing company. We hope the language meet the journal’s demand.  

2. Thank you for your comments. I have re-investigated the importance to analyse the functions of GEM in introduction at Line 53-57 and Line 58-61.

3. Thank you for your reminding. The figures have been cited in the manuscript.

4. The samples for in situ hybridization experiments were collected from 3 different prawns. All of these 3 samples were performed the in situ hybridization experiments. The labelling is consistent between the independent biological replicates. We have provided this in the manuscript at Line 124-125 in “Results” section, and at Line 259-261 in “Materials and Methods” section.

5. We agreed with your comment. We have discussed the significant increase of testosterone after the injection of GEM dsRNA at Line 202-204. In addition, we provided more details about the important roles of testosterone in aquatic species, especially for that in crustacean species at Line 209-213.

Actually, we performed H&E staining experiment on tissues from RNAi-treated vs vehicle-treated animals, in order to analyse whether GEM RNAi resulted in any anatomical or physiological changes of the gonads in RNAi-treated individuals in comparison to controls. However, we did not observe any difference. A reasonable explanation for this is that we only injected dsRNA of GEM for once. The interference period is too short that cannot result in any changes. So we did not put the histological observation in the manuscript. In this study, we have identified the potentially novel roles of GEM in male sexual differentiation and development through supressing the expression of IAG and the secretion of testosterone. In further M. nipponense aquaculture, we will perform the long-term GEM RNAi experiment, in order to observe whether GEM RNAi can result in any anatomical or physiological changes of the gonads.

Lastly, we discussed the effect of the increase in IAG expression and testosterone levels on male sexual differentiation and development in M. nipponense at Line 214-217.

6. Actually, CHH (Crustacean hyperglycemic hormone), GIH (Gonad-inhibiting hormone) and MIH (Molting-inhibiting hormone) are secreted by eyestalk, which have been proven to promote ovarian development and molting in M. nipponense. No evidence showed that they involved in the sexual differentiation. I have removed the sentence with these three genes.

7. I have edited the statistical relationship in Figure legend as: Data are shown as mean +SD (standard deviation) of tissues from three separate individuals. Capital letters indicate expression difference between different samples. We hope it is clear now.

Minor concerns:

8. Thank you for your reminding. In situ and M. nipponese had been italic in the whole manuscript.

9. A schematic of the GEM gene sequence has been provided as Fig. 1.

10. Firstly, we added the scale bars on Figure 4 and 5. Secondly, we have made the pictures as clear as possible in order to better see the cell types.

Reviewer 2 Report

In this manuscript, Jin et al. analyzed the function of a novel crustacean protein, GEM, using real-time PCR, in situ hybridization, and RNAi. This protein was originally identified by proteomic profiling and demonstrated to be highly expresses in the androgenic gland, an important regulator of male sex differentiation of this species. Although mammals have GEM orthologues, their function has not been analyzed in detail according to the authors. I did not find any literature either. I therefore found this study highly novel and interesting.

The major flaw in this manuscript includes the lack of detailed amino acid sequence analysis and insufficient representation of data/methods, as pointed out below. Manuscript is logically written in acceptable English.

Major comments:

1. Title: Please correct the spelling of Macrobrachium nipponense.

2. I would recommend the authors to perform detailed amino acid sequence analysis of GEM, not only BLAST comparison of amino acid sequences. This will add more useful information how this novel protein promotes male sexual differentiation. First, please add an alignment of some GEM proteins with information of mammalian orthologues. Then, please consider following questions: what domains does GEM have? Signal peptide, transmembrane, DNA- or lipid binding domains?  Putative glycosylation sites, phosphorylation sites? Molecular weight and pI?  Many online tools are available to predict the function from amino acid sequence.

3. Fig. 1. Phylogenetic tree should be revised because it does not reflect the known phylogenetic relationship between animal phyla (unless the authors want to challenge that). Especially, the poor bootstrap support of two maajor branches (28 and 32) makes this tree unreliable. I would suggest to: 1) use maximum-likelihood and Bayesian method instead of the neighbor-joining method, 2) try several alignment programs such as Clustal Omega, MUSCLE and T-COFFEE, 3) Use latest version of MEGA for ML analysis.

4. Fig 2 and others: Please provide exact number of samples for all data, not like "N > 3". 

5. Figs. 4 and 5. Make panels larger. It is difficult to see which cells are stained. Scale bars are not shown in these figures although it is mentioned in captions.

6. Figs. 6 and 7. Please change patterns of bar graphs. Control and RNAi look too similar (actually identical to me). 

7. Statistics: In L344-346, the authors described ANOVA followed by Duncan's test. This could be correct for several figures, but for Figs. 3B, 6, and 7, the authors must have conducted t-test (or other two group comparison statistics). Furthermore, Duncan's multiple range test is now widely criticized by the risk of high type I error, which is not very appropriate in the case of this paper. Tukey-Kramer would be a better option in the post-hoc test.

Specific comments:

8. L35-36: Remove double quotation?

9. L42: Change "Macrobrachium" to "M." ?

10. L56: Check the sentence again. I did not understand the meaning of "including [16]."

11. L74: Are species in Table 2 used for BLAST? I suppose that the authors used M. nipponense GEM sequence as a query, found outhologues from species in Table 2, and then used the sequences for alignment and phylogenetic analysis. Check the word usage carefully. Also, Clustal Omega would be a better choice to calculate amino acid identities compared to BLAST since identity is greatly affected by query coverage.

12. Section 2.2 and others: Please make sure to cite Figures in text.

13. L116 and others: "in situ" is sometimes italic, sometimes not. Please make it consistent.

14. Bar graphs: Only positive SDs are shown although the authors write "± SD".

15. L238: The insulin-like androgenic gland hormone (IAG) is abbreviated here, but this molecule should be mentioned with abbreviation in Introduction. 

Author Response

In this manuscript, Jin et al. analyzed the function of a novel crustacean protein, GEM, using real-time PCR, in situ hybridization, and RNAi. This protein was originally identified by proteomic profiling and demonstrated to be highly expresses in the androgenic gland, an important regulator of male sex differentiation of this species. Although mammals have GEM orthologues, their function has not been analyzed in detail according to the authors. I did not find any literature either. I therefore found this study highly novel and interesting.

The major flaw in this manuscript includes the lack of detailed amino acid sequence analysis and insufficient representation of data/methods, as pointed out below. Manuscript is logically written in acceptable English.

Major comments:

1. Thank you for your reminding. The spelling of Macrobrachium nipponense has been correct in the title.

2. The alignment of some GEM protein sequences has been provided as Fig. 2-A. The signal peptide, phosphorylation sites, molecular weight and pI have been predicted in the manuscript at Line 74-76.

3. We agreed with your comment. Firstly, we used Clustal Omega to align the amino acid sequence of GEM. Then Mega5.1 was used to construct the phylogenetic tree, followed the maximum-likelihood and Bayesian method to instead the neighbor-joining method. The results and discussion can be seen at Line 83-86 and Line 153-155. We hope the new phylogenetic tree was much better now.

4. We agreed with you comment. The exact number of samples has been provided in the manuscript.

5. Firstly, we added the scale bars on Figure 4 and 5. Secondly, we have made the pictures as clear as possible in order to better see the cell types.

6. We agreed with you comment. The patterns of bar graphs have been changed. We hope they are easy to distinguish now.

7. We agreed with your comments. We used “Tukey-Kramer” to perform the difference analysis for the data of Fig.2 and Fig.3A. In addition, according to your comment, T-test was used to perform the difference analysis for the data of Fig. 3B, Fig. 6 and Fig. 7. I also revised the analysis method in “Materials and Methods” section at Line 284-289.

Specific comments:

8. The quotation at Line 38 has been removed.

9. Macrobrachium rosenbergii was appeared in the manuscript for the first time. So I think the full-name should be used as here.

10. Thank you for your reminding. The “including” has been removed. It is typing errors when I wrote the manuscript.

11. We agree with your comments. Clustal Omega has been used to calculate amino acid identities between different well-defined amino acid sequences of GEM. Then the new alignment results was used to construct the phylogenetic tree.

12. Thank you for your reminding. Figures have been cited in the manuscript.

13. Thank you for your reminding. We have made it consistent as italic in the whole manuscript.

14. Thank you for your reminding. We have revised it as “+SD”.

15. The abbreviation of IAG has been provided in Introduction.

Round 2

Reviewer 1 Report

I appreciate the effort of the authors to improve the quality of their manuscript by addressing the comments from my initial evaluation.

I have only few additional minor recommendations regarding the style/language and discussion of results. They are listed below:

1. There are several places in the text, where the authors refer to genes (Mn-GEM, Mn-IAG), in which the gene names are not italicized. Please, go over the manuscript again and italicize gene names.

2. Lines 72 and 73: “The open reading frame was 777 bp long and contained 258 amino acids”. To my opinion as the ORF corresponds to a nucleic acid sequence, technically it can’t contain amino acids. I recommend to replace  “and contained” with “encoding for”.

3. Lines 74 and 75: this sentence refers to the protein, not the DNA sequence, so please add “of the protein” after “isoelectric point”. It sounds a little confusing otherwise, as the previous sentence refers to the DNA sequence. 

4. Line 79: Please add “GEM of” before Panaeus vannamei.

5. Line 89: Please add mRNA before “(Fig.3A)”. You refer to the expression of the gene, not the protein.

6. Lines 92-101: at several places in sections 2.2 and 2.3 the authors write “relatively” or “dramatically” (e.g. lines 92, 93, 95, 100, 101) when they describe the results from their gene expression analysis. To my opinion, this is not an accurate way of discussing the data and I recommend to the authors to use the actual quantitative measures (e.g fold change) when describing the results. In the text describing the results presented in Fig. 4A and B the authors refer to the actual fold change. They should similarly discuss the other qPCR results as well.

7. Line 157: Add “expression” after “GEM”.

8. Lines 188: I’m not sure if it is appropriate to cite reference [13] here as the authors refer to the actual finding described in this manuscript. Reference [13] corresponds to the characterization of another gene. 

9. Line 219: Here, instead of “We confirmed”, I recommend to the authors to use a more conservative statement such as "Our data strongly suggest that", as there is no functional validation of this prediction (e.g. RNAi experiment). 

10. Line 261: Please replace “consistent” with “consistency”.

11. Line 264: Please replace “analysis” with “analyze”.

Author Response

1.  Thank for your reminding. All gene names have been italicized.

2. We agreed with your comment. We have revised it in the manuscript at Line 72-73.

3. We agreed with your comment. We have added “of the protein” in the manuscript at Line 74.

4. We agreed with your comment. We have added “GEM of” before Panaeus vannamei at Line 80.

5. We agreed with your comment. We have added “mRNA” before “(Fig.3A)” at Line 90.

6. We agreed with your comment. “Relatively” and “dramatically” were not accurate. We have deleted these words. Instead, we used the actual fold change to show the expression change in result section at Line 93-101.

7. We agreed with your comment. “expression” has been added after “GEM” at Line 169.

8. We agreed with your comment. Reference 13 has been removed from the manuscript.

9. We agreed with your comment. We used “Our data strongly suggest that” to instead “We confirmed” in the manuscript at Line 229-230.

10. We agreed with your comment. “consistency” has been used in the manuscript at Line 272.

11. Thank you for your reminding. “analyze” has been used in the manuscript at Line 275.

Reviewer 2 Report

The authors partly improved the manuscript, but their revision was superficial and far from satisfactory. 

1. Phylogenetic tree:

Maximum-likelihood and Bayesian are different methods and therefore it is not correct to write "we used MEGA 5.1 followed the maximum-likelihood and Bayesian methods (L82)" when they have a single tree. While it is my fault to have dropped an "s" from my previous comment ("use maximum-likelihood and Bayesian method"s" instead of the neighbor-joining method"), is it too much to expect the authors to check the name of the method they just used for this revision?

Also, the latest version of MEGA is X for most platform. MEGA 5.1 is not the latest in any platform (see https://www.megasoftware.net). If they want to use an older version of the software, the authors are expected to provide certain scientific rationale. The interpretation of the tree is poor (L153-155).  

2. Sequence alignment:

My previous comment is: "I would recommend the authors to perform detailed amino acid sequence analysis of GEM, not only BLAST comparison of amino acid sequences. This will add more useful information how this novel protein promotes male sexual differentiation. First, please add an alignment of some GEM proteins with information of mammalian orthologues. Then, please consider following questions: what domains does GEM have? Signal peptide, transmembrane, DNA- or lipid binding domains? Putative glycosylation sites, phosphorylation sites? Molecular weight and pI? Many online tools are available to predict the function from amino acid sequence."

The authors added an alignment figure and only two sentences in L74-76. But which amino acids were predicted to be the O-GlcNAc?  Where is the membrane helix? When the authors make a figure, they are supposed to study how the figure should be by reading other papers. I have rarely seen this style (i.e., describe domains in text without showing them in the alignment).

Furthermore, when I BLASTed their sequence, I found that Macrobrachium nipponense GEM contains a signature sequence of the SIP1 superfamily. Their analysis lacks even this very basic information and failed to address one of my questions, "what domeins does GEM have?"

Overall, scientific quality of their revised data was far below the publication level, which makes me suspicious of the overall quality of other data in this paper.

Author Response

1. According to your comment, MEGA X was used to re-construct the phylogenetic tree, followed the maximum-likelihood method. More details of the tree were provided at Line 161-167 and Line 252-253.
2. According to your comment, we have re-analysed the characterizations of Mn-GEM sequence at Line 75-77 and Line 155-159. The domains, membrane helix and strand, SIP1 superfamily were indicated in the alignment figure.

Round 3

Reviewer 1 Report

Line 71: Please, refer to Fig.2 at the end of this sentence. 

Please, make a final check for English grammar and style.

Reviewer 2 Report

Authors addressed my concern in this second revision. I would recommend publication.