Molecular Characterization of a Male-Specific SoxE Gene in the Swimming Crab, Portunus trituberculatus, and Transcriptional Interaction with Insulin-like Androgenic Gland Hormone
Round 1
Reviewer 1 Report
The manuscript "Molecular characterization of a male-specific SoxE gene in the swimming crab, Portunus trituberculatus, and transcriptional interaction with insulin-like androgenic gland hormone" aims to describe the SoxE gene in the male swimming crab.Their findings show a male-specific correlation regarding the PtSoxE expression, and they argue that their results show an association between androgenic gland (AG) and IAG hormone.
This study has adequate scientific soundness, and it will be interesting to the readers. However, there are some methodological issues that the authors should address, before publication.
Please follow the comments below
line 48-50: Reference is missing
line 51-52: Reference is missing
M&Ms
line 89-104: I guess the authors bought the specimens alive and killed them immediately while they collected the tissues, otherwise the RNA would be degraded. They should mention the method of anesthesia, the regulations of killing, and the well-being procedure of the specimens. Also, the authors should mention the year of sampling. Please be explicit.
line 114-115: It is not clear if either the primers were taken from the literature or designed by the authors. Please specify. In the case that the primers were designed by the authors, they should also mention Tm, GC%, and ΔG values.
line 127-137: Same as before (Tm, GC%, ΔG)
line 140: Please be more specific on the type of tissue you used.
line 158 - 163: Please specify the Taq (manufacturer) you used.
line 166-168: Please specify the Real-Time machine, and the software you used. Also very important, the authors should describe the protocol in terms of biological and technical replicates of each sample, and the dilution scheme they followed.
Results
lines 193-194: At Figure 2A, the ladder scale is missing. Also, the authors should use the whole gel-photo to show that there are not any secondary products.
Did you run a multiplex PCR, so you could test the amplification of β-actin and PtSoxE in the same run per individual? Also, in the PtSoxE of females, a positive PCR control is missing. The positive PCR-control is crucial to certify the smooth PCR running. The absence, maybe due to malfunctioning of the PCR machine.
lines 196 - 198: At the Figure 2, the significance among comparison is missing. The authors could indicate the significance between certain comparisons with an "asterisk".
lines 199-200: This is a discussion, so please delete it from here and transfer it to the relevant chapter.
lines 202-205: At Figure 3, the significance among comparison is missing. The authors could indicate the significance between certain comparisons with an "asterisk".
lines 207-211: The actual legend of the Figure is missing. This is a legend of a supplementary material. Please provide an explained legend of the two materials.
lines 220-231: Please provide the p-values. Also, please have in mind that you MUST correct your alpha-value using either Bonferroni or False Discovery Rate. Discussion
line 275: To the best of our knowledge
lines 285-286: The authors should run specific analytical statistical tests to refer to that. GLM analysis could give some insight regarding the correlation between AG and IAG expression. One major issue I can see is that, a time-series is missing. The profound difference of AG and IAG within a year it could be attributed to other factors. The authors should discuss their findings with respect to potential pitfalls or limitations of the study.
Minor English editing
Author Response
Thanks for your suggestion.
Point 1
line 48-50: Reference is missing.
line 51-52: Reference is missing.
Responds 1: Sorry for the negligence, the following references were added to according to locations.
[6] Hu, Y.; Wang, B.; Du, H. A review on Sox genes in fish. Reviews in Aquaculture 2021, 13, 1986-2003. https://doi.org/10.1111/raq.12554.
[8] Nitzan, G.; Chris, R. F.; Sophie, W.; S. Alexandra, G.M.; Isabella, M.S.; Shiela, C.S.; Ryohei, S.; Francis, P.; Danielle, M.M.; Robin, L.B. Sex reversal following deletion of a single distal enhancer of Sox9. Science 2018, 360, 1469-1473. https://doi.org/10.1126/science.aas9408.
[9] Takehana, Y.; Matsuda, M.; Myosho, T.; Suster, M.L.; Kawakami, K.; Shin, I.T. et al. Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena. Nature Communication 2014, 5: 4157. https://doi.org/10.1038/ncomms5157.
[10] Schartl, M.; Schories, S.; Wakamatsu, Y. et al. Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements. BMC Biology 2018, 16:1-17. https://doi.org/10.1186/s12915-018-0485-8.
Point 2
M&Ms
line 89-104: I guess the authors bought the specimens alive and killed them immediately while they collected the tissues, otherwise the RNA would be degraded. They should mention the method of anesthesia, the regulations of killing, and the well-being procedure of the specimens. Also, the authors should mention the year of sampling. Please be explicit.
Responds 2: Thanks for the suggestion, such information was added in revised manuscript, see line 91-100.
Point 3
line 114-115: It is not clear if either the primers were taken from the literature or designed by the authors. Please specify. In the case that the primers were designed by the authors, they should also mention Tm, GC%, and ΔG values.
line 127-137: Same as before (Tm, GC%, ΔG)
Responds 3: Thanks for the suggestion, we have added the information about Tm, GC%, and ΔG values in Table 1. As for line 127-137 (now see line 131-141), the siRNAs were designed and synthesized by GenePharma (Shanghai, China), and such information was not provided.
Point 4
line 140: Please be more specific on the type of tissue you used.
Responds 4: The androgenic gland (AG) was used for obtaining the homogenate, we have now specified in line 143 in revised manuscript.
Point 5
line 158 - 163: Please specify the Taq (manufacturer) you used.
Responds 5: The information has been added in line 117-119 and in line 164-166 in revised manuscript.
Point 6
line 166-168: Please specify the Real-Time machine, and the software you used. Also very important, the authors should describe the protocol in terms of biological and technical replicates of each sample, and the dilution scheme they followed.
Responds 6: The biological replicates for this study are indicated in the figure legends, while in qPCR, each sample was carried out in three technical replicates. We have added steps such as melting curves and gradient dilution to detect amplification efficiency. Please see in line 174-179 in the revised manuscript.
Point 7
Results
lines 193-194: At Figure 2A, the ladder scale is missing. Also, the authors should use the whole gel-photo to show that there are not any secondary products.
Did you run a multiplex PCR, so you could test the amplification of β-actin and PtSoxE in the same run per individual? Also, in the PtSoxE of females, a positive PCR control is missing. The positive PCR-control is crucial to certify the smooth PCR running. The absence, maybe due to malfunctioning of the PCR machine.
Responds 7: Thanks for your instructive suggestions. There are some primer dimer bands in the whole gel photo that we think may affect the aesthetics of figures, so we put it in the supplementary material. We did not run a multiplex PCR, but we agree that it is a good way for testing both control gene and target genes in the same run, which we will conduct in future studies. We did not introduce a positive PCR control either, but we tested duplicates of the results, so the absence shouldn't be due to malfunctioning of the PCR machine.
Point 8
lines 196 - 198: At the Figure 2, the significance among comparison is missing. The authors could indicate the significance between certain comparisons with an "asterisk".
Responds 8: It is not clear that we fully understood the meaning of the reviewer，but the statistically significance was tested using multiple-group comparisons, which the differences were indicated by different letters, we thought this may be a better way to see the dynamic changes. Also, there was no comparison between expression in Te and AG, because they used different controls when calculating using comparative Ct method.
Point 9
lines 199-200: This is a discussion, so please delete it from here and transfer it to the relevant chapter.
Responds 9: The sentence has been deleted.
Point 10
lines 202-205: At Figure 3, the significance among comparison is missing. The authors could indicate the significance between certain comparisons with an "asterisk".
Responds 10: The same as above, we thought that the multiple-group comparisons which use different letters to indicate significantly differences may be a better way to show the dynamic changes.
Point 11
lines 207-211: The actual legend of the Figure is missing. This is a legend of a supplementary material. Please provide an explained legend of the two materials.
Responds 11: Sorry for the negligence. The legend for figure 2 has been added.
Point 12
lines 220-231: Please provide the p-values. Also, please have in mind that you MUST correct your alpha-value using either Bonferroni or False Discovery Rate.
Responds 12: Thanks for the suggestion. That's probably we didn't explain these maps well. This map was the relative mRNA expression level of every gene in AG and Te, and made by GraphPad Prism 8 software. The used data for making the maps that were using the comparative Ct (2−ΔΔCt) method to calculate, and the different colors (in figure 4B, 4C and figure 5B) represent these data. The color calipers on the right are the relative expression level reference, the higher the relative expression of the tested genes had the darker the color, and vice versa. Every row in the map is a parallel comparison of each gene in different group, that all had subjected to a one-way analysis of variance (ANOVA), followed by Student’s t-test or Tukey’s multiple-group comparison test (SPSS 24.0 software), and significant differences were accepted at P < 0.05.
Point 13
Discussion. line 275: To the best of our knowledge
Responds 13: We have changed according to your suggestions.
Point 14
lines 285-286: The authors should run specific analytical statistical tests to refer to that. GLM analysis could give some insight regarding the correlation between AG and IAG expression. One major issue I can see is that, a time-series is missing. The profound difference of AG and IAG within a year it could be attributed to other factors. The authors should discuss their findings with respect to potential pitfalls or limitations of the study.
Responds 14: In crustaceans, IAG is extremely highly expressed in the androgenic gland (AG), and the gene is related to sex differentiation. PtSoxE, being male-specific expressed, also showed a potential in regulating sexual development. The relationship we speculated between PtSoxE and IAG was not statistically. It is true that we should discuss the potential pitfalls or limitations of our findings. We have added such discussion in conclusion section.
Reviewer 2 Report
Mechanisms of sex determination in crustaceans are not well known and may vary among species. Jiang et al. characterized the structure and function of the SoxE gene in swimming crab, Portunus trituberculatus, showing its structure, phylogenetic variation, tissue specificity of expression, time specificity of expression, and interactions with insulin-like androgenic gland hormone. The results are of interest to aquaculture and to science more generally. The authors used a variety of methods, and to my estimation, appropriately. I have no major issues about methodology or interpretation, although the presentation can and should be sharpened by attention to the points below. I also have marked the manuscript, especially to help sharpen the prose.
Some issues came up repeatedly. I encourage the use of italics to denote gene names and regular font for proteins, and have marked the manuscript appropriately. Species’ Latin names should be italicized. Context-dependent comments follow.
Introduction. – At line 59, the fish mentioned should be described as the sequentially hermaphroditic orange-spotted grouper Epinephelus coioides.
Methods. – At line 110, who manufactured the gDNA Remover Mix?
At line 137, what is DEPC-H2O?
At line 145, we need a manufacturer or supporting citation for pET-28a-sumo.
At line 146, who manufactured the HisTrap HP column?
Results. – Both panels of Figure 1 are too small to be understood by the reader. Can they be presented larger?
The caption for Figure 2 is the wrong one. This is the caption for Supplementary Figure 1. The authors or journal staff must provide the correct caption.
This being a fisheries journal, most readers will not know what RNAi is, so that should be spelled out at line 220.
At line 226, shouldn’t “banding” be binding?
Figures 4 and 5 would be better in color than in grey-scale? Can’t we get that in color?
Discussion. – I suggest many prose fixes to strengthen the Discussion.
References. – The literature citations have many departures from journal citation stylistics, including italicization of gene names and species’ Latin names. Key words of article titles should be capitalized or not, but consistently. I ask the authors to consult a recent issue of the journal to see the stylistic used.
Comments for author File: 
Comments.pdf
The English prose is rough. I have marked the manuscript to assist with revision.
Author Response
Thanks for your suggestion.
Point 1
Introduction. – At line 59, the fish mentioned should be described as the sequentially hermaphroditic orange-spotted grouper Epinephelus coioides.
Responds 1: We have changed according to your suggestions.
Point 2
Methods. – At line 110, who manufactured the gDNA Remover Mix?
Responds 2: Sorry for the negligence. We have added at line 112.
Point 3
At line 137, what is DEPC-H2O?
Responds 3: Sorry for the negligence. We have changed the wording at line 141.
Point 4
At line 145, we need a manufacturer or supporting citation for pET-28a-sumo.
Responds 4: Sorry for the negligence. We have added at line 149.
Point 5
At line 146, who manufactured the HisTrap HP column?
Responds 5: Sorry for the negligence. We have added at line 151.
Point 6
Results. – Both panels of Figure 1 are too small to be understood by the reader. Can they be presented larger?
Responds 6: Sorry for this problem. This is not the original HD image, so the picture is not clear enough after printing. We had uploaded a new one.
Point 7
The caption for Figure 2 is the wrong one. This is the caption for Supplementary Figure 1. The authors or journal staff must provide the correct caption.
Responds 7: Sorry for the negligence. The legend for figure 2 has been added.
Point 8
This being a fisheries journal, most readers will not know what RNAi is, so that should be spelled out at line 220.
Responds 8: We have add the full spell at line 234.
Point 9
At line 226, shouldn’t “banding” be binding?
Responds 9: We have changed according to your suggestions.
Point 10
Figures 4 and 5 would be better in color than in grey-scale? Can’t we get that in color?
Responds 10: We have changed according to your suggestions.
Point 11
Discussion. – I suggest many prose fixes to strengthen the Discussion.
Responds 11: We have added relevant content of the Discussion.
Point 12
References. – The literature citations have many departures from journal citation stylistics, including italicization of gene names and species’ Latin names. Key words of article titles should be capitalized or not, but consistently. I ask the authors to consult a recent issue of the journal to see the stylistic used.
Responds 12: Thank you very much for your corrections and for embellishing the text. We have revised the article according to your marked manuscript, but we are not sure that all of them are corrected accordingly.
Round 2
Reviewer 1 Report
Thank you for the revised manuscript. I suggest the publication of the ms.
