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  • Jisheng Wang1,†,
  • Mengying Zhang1,† and
  • Sufei Jiang1,2,*
  • et al.

Reviewer 1: Shang Mei Reviewer 2: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The author studied the JHEH gene in female Macrobrachium nipponense: sequenced the gene; did similarity comparison and phylogenetic analysis; did gene expression in tissue level; finally, use RNAi study the JHEH function in ovarian maturation. The whole study is comprehensive and detailed.

Here are some details that need correction:

Line 148 GFP gene was used as the control gene. But there is no result or figure show any result of this control gene’s expression or location.

Line 163-166 Were the samples collected at day 1, 7, 14 twice? Or just once at day 1, 7, 14 and 20?

Line 208 stop codon is TGA, not TAG

Figure 5’s resolution is too low, cannot read the scale bars. The magnification of picture O Negative is much higher than O HE and O Positive.

 

Figure 7 A the percentage of pass stage III should increase for all groups. For the control group which progressed to the next round show also indicated in the figure.

Author Response

Dear Reviewer 1,

First of all, we are very grateful for your recognition of our research work. Thank you very much for your comments and suggestions. The valuable comments from you not only helped us with the improvement of our manuscript, but suggested some ideas for future studies.

Below you will find our responses to your comments:

  1. Line 148 GFP gene was used as the control gene. But there is no result or figure show any result of this control gene’s expression or location.

Response: Thanks for your kindly reminding. We used the green fluorescent protein (GFP) gene as the control gene. Prawns in the control and experimental groups were injected with ds-GFP and ds-JHEH, at a dose of 4 µg/g body weight (b.w.), respectively. The control group in Figure 6 is the expression of Mn-JHEH in prawn ovaries after ds-GFP injection.

  1. Line 163-166 Were the samples collected at day 1, 7, 14 twice? Or just once at day 1, 7, 14 and 20?

Response: Thanks for your kind comment. At 1, 4, and 7 days after injection, prawns were randomly collected from each group and ovaries were dissected and analyzed for interference efficiency. At days 1, 7, 14, and 20 after injection, prawns were randomly collected from each group, ovaries were dissected, and weights and gonad weights were recorded to determine GSI. Ovarian samples on days 1 and 7 were shared, and GSI was determined and then immediately frozen in liquid nitrogen to analyze interference efficiency. Ovaries for interference analysis were collected on day 4. Ovaries for GSI determination were collected on days 14 and 20.

  1. Line 208 stop codon is TGA, not TAG

Response: Thanks for your kind comment. We have amended the error as follows: " The start codon ATG and the stop codon TGA are marked in red. "

  1. Figure 5’s resolution is too low, cannot read the scale bars. The magnification of picture O Negative is much higher than O HE and O Positive.

Response: Thank you for your kind reminder. We have made the changes you suggested and recreated Figure 5.

  1. Figure 7 A the percentage of pass stage III should increase for all groups. For the control group which progressed to the next round show also indicated in the figure.

Response: Thanks for your valuable comment. We may not have described the results in Figure 7A clearly enough. Based on your suggestions, we have made further changes in the revised manuscript as follows: " At the beginning of the experiment, female prawns at the stage IV of ovarian development were selected, and the proportion of ovarian stage O-III was 100%. Most of the ovaries of the prawn developed to stage II after 7 days in both the experimental and control groups. On day14, most of the prawn ovaries of had developed to stage IV in the control group, while the experimental group mostly developed to stage III. The ovaries of the control group began to empty gradually after the 16th day and started to enter the next round of development, and almost all of them were emptied by the 20th day. In the experimental group, the ovaries began to empty gradually after day 18, while most of the ovaries were still in the previous round of development. "

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The MS is very interesting. The authors investigated the role of the JHEH gene in the reproductive process of female shrimp Macrobrachium nipponense, showing that its expression is related to ovarian maturation and moulting frequency. The results indicate that downregulation of Mn-JHEH significantly reduces the rate of ovarian maturation and moulting. This study may contribute to the development of more precise methods for controlling reproduction in shrimp farming, which may support the optimization of production under controlled conditions. However, before the final publication of the MS, the authors should improve the MS. MS contains many errors, but everything can be supplemented and corrected. I noticed that there are histological photos in the work, but there is no thread in the Materials and methods section that an histological analysis was performed. My detailed comments are below:

Line 2: Change MS title to: Regulation roles of Juvenile Hormone Epoxide Hydrolase Gene 2 in female river Prawn Macrobrachium nipponense Reproductive process.

Line 27: Delete: "Macrobrachium nipponense", "JHEH". Please add other keywords for example: endocrine hormone, enzyme JHEH, 

Line 33. There is a dot ".This" at the beginning of the sentence, you need to add a space after the dot and move it to line 32.

Line 40: Please correct to: Many genes were found with sexual rapid maturation [7].

Line 42. Remove "we"

Lines 51-53: Please correct to: JHEH is a key enzyme in JH metabolism, catalyzing the opening of the epoxide ring in JH to form juvenile hormone diol (JHD). Additionally, it can degrade juvenile hormone acid (JHA) to juvenile hormone acid diol (JHAD) [14]

Line 53. Reference number missing after: Touhara et al [...]

Lines 92-93: Please correct to: Tissues collected for analysis included the eyestalk (E), brain (BR), heart (H), hepatopancreas (He), gills (G), ovary (O), and muscle (M) , with six samples per tissue (n=6).

Line 98 -100: Please correct to: Larval (L) and post-larval (PL) developmental stages were sampled every 5 days across 9 time points: L1, L5, L10, L15, PL1, PL5, PL10, PL15, and PL25. All samples were stored in liquid nitrogen.

Line 120: This table title is unacceptable. Please change it to something like this: Primers involved in studying gene expression in female shrimp Macrobrachium nipponense in reproductive process.

Line 148: The sentence "We used the green fluorescent protein (GFP) gene as the control gene." replace with: "The green fluorescent protein (GFP) gene was used as the control gene."

Lines 158 – 160: Please correct to: "Three hundred healthy female (n=300) prawns (0.58 ± 0.16 g) in ovary stage IV were selected and randomly assigned to either the experimental or control groups, with 3 replicates of 50 individuals (n = 50) in each group."

Line 176 – 178: Please correct to: "At the end of the experiment, one prawn at stage O-IV was randomly selected from each of the experimental and control groups; their ovaries were dissected and prepared as paraffin sections for comparison."

Line 181 – 182: Replace with: "The relative expression of the genes was calculated using the 2^−∆∆Ct method [24].

Line 228: Please deleted a dot before: “. (Figure 3)”

Line 272: Fig. 5. It is poorly described. All abbreviations should be explained. Photo 3 is not visible. Use the letters A-F to describe the photos more clearly. In addition, it seems to me that these are histological images and, in the Material, and methods section I have nothing written about histological analysis, how the preparations were stained, how the samples were preserved, what analysis program was used. All this should be supplemented

Line 285: Please add the species name to the title of Fig.

Line 288-290: Please correct to:  "To determine the effect of JHEH gene knockout on ovarian maturation in M. nipponense, the cumulative proportion of ovaries reaching stage O-III (Figure 7A) and the gonadosomatic index (GSI) were also recorded for the two groups after injection (Figure 7B)."

Line 319-321: Please correct to: "In this study, the JHEH gene was identified from the hepatopancreas transcriptome of M. nipponense [5], and a cDNA containing a full open reading frame of 1383 bp was obtained."

Line 345-347: Correct the sentence to: "Given the high expression levels of Mn-JHEH in the hepatopancreas, examined its expression patterns in both the hepatopancreas and ovaries at different stages of ovarian development."

Lines 375-376: Correct the sentence to: "In addition, during the experiment, the amount of molting was recorded, and the frequency of molting was counted, revealing that molting was significantly inhibited after successful silencing of Mn-JHEH."

Line 385-388: Further studies will investigate the presence of JH hormone in M. nipponense and its regulatory relationship with JHEH, aiming to enrich the molecular mechanism of ovarian maturation in crustaceans."

Author Response

Dear Reviewer 2,

First of all, we are very grateful for your recognition of our research work. Thank you very much for your comments and suggestions. The valuable comments from you not only helped us with the improvement of our manuscript, but suggested some ideas for future studies.

Below you will find our responses to your comments:

  1. Line 2: Change MS title to: Regulation roles of Juvenile Hormone Epoxide Hydrolase Gene 2 in female river Prawn Macrobrachium nipponense Reproductive process.

 Response: Thanks for your kind suggestion. We have made the change in the revised version.

  1. Line 27: Delete: "Macrobrachium nipponense", "JHEH". Please add other keywords for example: endocrine hormone, enzyme JHEH.

Response: Thanks for your kind suggestion. We have made the following changes in the revised version in response to your suggestion: “ovarian development; molting; endocrine hormone; enzyme JHEH”.

  1. Line 33. There is a dot ".This" at the beginning of the sentence, you need to add a space after the dot and move it to line 32.

Response: Thanks for your kind reminder. We have made the change in the revised version in response to your suggestion.

  1. Line 40: Please correct to: Many genes were found with sexual rapid maturation [7].

Response: Thanks for your kind suggestion. Based on your good suggestion, we have made the change in the revised version.

  1. Line 42. Remove "we"

Response: Thanks for your kind reminder. We have made the following change in the revised version based on your suggestion: "Through an analysis of the hepatopancreas transcriptome in female M.nipponense adult ovaries during stages O-I to O-V,  identified that the “insect hormone synthesis” signaling pathway was closely linked to ovarian development based on KEGG enrichment results [5]."

  1. Lines 51-53: Please correct to: JHEH is a key enzyme in JH metabolism, catalyzing the opening of the epoxide ring in JH to form juvenile hormone diol (JHD). Additionally, it can degrade juvenile hormone acid (JHA) to juvenile hormone acid diol (JHAD) [14]

Response: Thanks for your kind suggestion. We have made the change in the revised version in response to your suggestion.

  1. Line 53. Reference number missing after: Touhara et al [...]

Response: Thanks for your kind reminder. We have made the following change in the revised version in response to your suggestion: " Touhara et al [15]"

  1. Lines 92-93: Please correct to: Tissues collected for analysis included the eyestalk (E), brain (BR), heart (H), hepatopancreas (He), gills (G), ovary (O), and muscle (M), with six samples per tissue (n=6).

Response: Thanks for your kind suggestion. We have made corresponding change in the revised manuscript.

  1. Line 98 -100: Please correct to: Larval (L) and post-larval (PL) developmental stages were sampled every 5 days across 9 time points: L1, L5, L10, L15, PL1, PL5, PL10, PL15, and PL25. All samples were stored in liquid nitrogen.

Response: Thanks for your kind suggestions. We have made corresponding change in the revised manuscript based on your suggestion: " Larval (L) and post-larval (PL) developmental stages were sampled every 5 days across 9 time points: L1, L5, L10, L15, PL1, PL5, PL10, PL15, and PL25. All samples were stored in liquid nitrogen."

  1. Line 120: This table title is unacceptable. Please change it to something like this: Primers involved in studying gene expression in female shrimp Macrobrachium nipponense in reproductive process.

Response: Thanks for your kind reminder. We have made corresponding change in the revised manuscript based on your suggestion: "Primers involved in studying gene expression in female prawn Macrobrachium nipponense in reproductive process."

  1. Line 148: The sentence "We used the green fluorescent protein (GFP) gene as the control gene." replace with: "The green fluorescent protein (GFP) gene was used as the control gene."

Response: Thanks for your kind suggestion. We have made corresponding change in the revised manuscript.

  1. Lines 158 – 160: Please correct to: "Three hundred healthy female (n=300) prawns (0.58 ± 0.16 g) in ovary stage IV were selected and randomly assigned to either the experimental or control groups, with 3 replicates of 50 individuals (n = 50) in each group."

Response: Thanks for your kind suggestion. We have made the following change in the revised version in response to your suggestion: " Three hundred healthy female (n=300) prawns (0.58 ± 0.16 g) in ovary stage IV were selected and randomly assigned to either the experimental or control groups, with 3 replicates of 50 individuals (n = 50) in each group."

  1. Line 176 – 178: Please correct to: "At the end of the experiment, one prawn at stage O-IV was randomly selected from each of the experimental and control groups; their ovaries were dissected and prepared as paraffin sections for comparison."

Response: Thanks for your kind suggestion. We have made the change in the revised manuscript in response to your suggestion.

  1. Line 181 – 182: Replace with: "The relative expression of the genes was calculated using the 2^−∆∆Ct method [24].

Response: Thanks for your kind reminder. We have made corresponding change in the revised manuscript.

  1. Line 228: Please deleted a dot before: “. (Figure 3)”

Response: Thanks for your kind suggestion. We have made the following consequential changes in the revised version: " Phylogenetic tree analysis showed that the JHEH of M.nipponensis clustering with the JHEH of Macrobrachium rosenbergii in the crustacean clade (Figure 3)."

  1. Line 272: Fig. 5. It is poorly described. All abbreviations should be explained. Photo 3 is not visible. Use the letters A-F to describe the photos more clearly. In addition, it seems to me that these are histological images and, in the Material, and methods section I have nothing written about histological analysis, how the preparations were stained, how the samples were preserved, what analysis program was used. All this should be supplemented

Response: Thanks for your kind reminder. We have added the missing explanation of the abbreviations in the revised version and explained the pictures with the letters “A-F” as follows: " (A) Blank control for the ovary group; (B) positive experimental group for the ovary group; (C) negative control for the ovary group; (D) blank control for the hepatopancreas group; (E) positive experimental group for the hepatopancreas group; (F) negative control for the hepatopancreas group. OC: oocyte; N: nucleus; CM: cytoplasmic membrane; Y: yolk granule; FC: follicle cell; LG: lipid granules; HC: hepatocytes; Ct: connective tissues. Scale bars: 100×(O); 400×(He)."And recreated Figure 5. Staining preparation, sample preservation, and procedural analysis have been supplemented in the “Materials and Methods” section.

  1. Line 285: Please add the species name to the title of Fig.

Response: Thanks for your kind reminder. Regarding line 285, we have added the following supplement: “Expression levels of Mn-JHEH in the ovaries of the Macrobrachium nipponense after injection of ds-GFP and ds-JHEH.”

  1. Line 288-290: Please correct to: "To determine the effect of JHEH gene knockout on ovarian maturation in M. nipponense, the cumulative proportion of ovaries reaching stage O-III (Figure 7A) and the gonadosomatic index (GSI) were also recorded for the two groups after injection (Figure 7B)."

Response: Thanks for your kind suggestion. We have made the change in the revised manuscript in response to your suggestion.

  1. Line 319-321: Please correct to: "In this study, the JHEH gene was identified from the hepatopancreas transcriptome of M. nipponense [5], and a cDNA containing a full open reading frame of 1383 bp was obtained."

Response: Thanks for your kind suggestion. Regarding line 319-321, we have made the change in the revised manuscript in response to your suggestion.

  1. Line 345-347: Correct the sentence to: "Given the high expression levels of Mn-JHEH in the hepatopancreas, examined its expression patterns in both the hepatopancreas and ovaries at different stages of ovarian development."

Response: Thanks for your kind suggestion. Regarding line 345-347, we have made the change in the revised manuscript in response to your suggestion: “Given the high expression levels of Mn-JHEH in the hepatopancreas, examined its expression patterns in both the hepatopancreas and ovaries at different stages of ovarian development.”

  1. Lines 375-376: Correct the sentence to: "In addition, during the experiment, the amount of molting was recorded, and the frequency of molting was counted, revealing that molting was significantly inhibited after successful silencing of Mn-JHEH."

Response: Thanks for your kind suggestion. We have made the change in the revised manuscript in response to your suggestion.

  1. Line 385-388: Further studies will investigate the presence of JH hormone in M. nipponense and its regulatory relationship with JHEH, aiming to enrich the molecular mechanism of ovarian maturation in crustaceans."

Response: Thanks for your kind suggestion. Regarding line 385-388, we have made the change in the revised manuscript in response to your suggestion: “Further studies will investigate the presence of JH hormone in M. nipponense and its regulatory relationship with JHEH, aiming to enrich the molecular mechanism of ovarian maturation in crustaceans.”

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for improving the article according to my suggestions. MS is really very interesting.