Molecular Characterization and Dietary Regulation of Glutaminase 1 (gls1) in Triploid Crucian Carp (Carassius auratus)

Round 1

Reviewer 1 Report

Generally speaking, this paper was well designed and carefully conducted. And some interesting results were reported, which would benefit the flesh quality control, especially the umami amino acids. However, there are some flaws which should be corrected or clarified before its acceptance for publication. Specific comments could be found in the annotated pdf file.

Comments for author File: Comments.pdf

Author Response

Generally speaking, this paper was well designed and carefully conducted. And some interesting results were reported, which would benefit the flesh quality control, especially the umami amino acids. However, there are some flaws which should be corrected or clarified before its acceptance for publication. Specific comments could be found in the annotated pdf file.

Line 27: What are the protein sources and which one is the control? Please specify these.

Response: Thanks for this comment. Following the requirements, authors have re-described the results of different protein source experiments. Please see Line 28-30.

Line 52: Maybe the free glutamate and IMP are determinant to the umami flavor of fish, not total glutamate and IMP. You should identify them.

Response: Thank you for pointing out this problem. According to your suggestion, authors have revised the manuscript. Please see Line 56 and 59.

Line 86: Write the full name when the abbreviation firstly appears.

Response: Thanks. Authors have added the full name of TCC when it appears for the first time in the Introduction. Please see Line 87.

Line 95: How about the fish size?

Response: Thanks for this comment. Because this study includes several experiments, and the specifications of fish used in each experiment are different, the fish size are not marked on line 95. According to your suggestion, authors have supplemented the size of fish used in each experiment. Please see Line 115, 123, 139, 164 and 182.

Line 108: When you collected the fish, did you fast the fish for 24h? or just sample the fish after feeding?

Response: Thanks. During sampling of the diurnal variation experiment, the fish was fed twice daily at 9:00 and 15:00. In other experiment, fishes were fasted for 24 h before sampling. Authors have revised the manuscript. Please see Line 115, 118, 144, 171 and 188.

Table 2: The total for fishmeal group was not 100%. Please check your data.

Response: Thanks for your kind reminder. Authors have revised the manuscript. Please see Table 2.

Figure 2: Please add a * to indicate it is from your study.

Response: Following the requirements, authors have added a “*” behind the Carassius auratus. Please see Figure 2. Thanks.

Author Response File: Author Response.pdf

Reviewer 2 Report

General comment: Major drawbacks of the current study found to be related with wrong statistical analysis and missing important information in M&M and INTRODUCTION sections as well as the abstract section should be rewritten again. The manuscript in its form cannot supported for revision process and should be rejected.

Detailed comments:

Q1: why the author had been identified the brain and liver, heart, gut, kidney, spleen and muscle, for the expression of gls1? This information should be described in the introduction part?

Q2: the structure of abstract section is vague and not provide any scientific information. It should be completely rewritten again?

Q3: the author should include more details about the main mode of action of glutamate into fish body, the main biological effects and its relation with fish production and tasty in introduction part?

Q4: the author detailed all information about mice as experimental animal, how could we predict the same biological effects in fish or aquatic creatures? The main hypothesis of the current study must be convinced the readers.

Q5: the material and method section missed so many important information about experimental groups and rearing conditions. I did not agree with the author for choosing one Way ANOVA for analysis this study. There are at least two main effects (different time and glutamate levels) how could we assessed the gene expression during different times under another effects of protein diet resource with one way ANOVA? Thus, the obtained results should be completely rewritten again and the statics should be reanalyzed.

Q6: the discussion of the current study not defined the interaction effects between time and other study variable. The discussion should be rewritten again for supported new results obtained from the corrected statistical analysis?

L 18 why the author did not mention the main objective in more concise manner? What is the importance of estimating the glutamate production and glutamine utilization in fish?

L 18-21 the author failed to described their treated groups or experimental design in shortened? This is not scientific structure of abstract?

L 29 I can not define any related information about how the author had been examined there in vitro or in vivo experiment?

L 31 we cannot supported general conclusion, it should be specified in more details?

L 45 and L 49 missing cited references for this mentioned information?

L 54 "animals" the example here should be related with fish or aquatic species.

L 57 repeated information

L 99 the all-water quality measurements should be described during the acclimation period?

L 101 anesthesia dose should be defined?

L 123 the fiberglass tanks dimensions should be described?

L 122 the experimental group not defined?

L 149 how could the author avoid the mRNA contamination?

Author Response

General comment: Major drawbacks of the current study found to be related with wrong statistical analysis and missing important information in M&M and INTRODUCTION sections as well as the abstract section should be rewritten again. The manuscript in its form cannot supported for revision process and should be rejected.

1. Why the author had been identified the brain and liver, heart, gut, kidney, spleen and muscle, for the expression of gls1? This information should be described in the introduction part?

Response: Thanks for your comments. Because brain, liver, heart, gut, kidney, spleen and muscle are the main tissues of fish body, they are often selected for tissue expression analysis of genes, such as papers by Hu et al. (2017) and Luo et al. (2020).

[1] Luo, W.J.; Song, P.; He, Z.M.; Cao, S.P.; Tang, J.Z.; Xu, W.Q.; Xiong, D.; Qu, F.F.; Zhao, D.F.; Liu, Z.; Li, J.Z.; Yin, Y.L. JAK2 Mediates the Regulation of Pept1 Expression by Leptin in the Grass Carp (Ctenopharyngodon idella) Intestine. Front Physiol, 2020, 11, 79.

[2] Hu, R.; Wang, Y.; Qu, F.; Tang, J.; Zhou, Y.; Lu, S.; Zhang, J.; Liu, Z.; Zhou, Z.; Guo, X. Molecular Characterization and Dietary Regulation of the AlaSerCys Transporter 2 in Grass Carp, Ctenopharyngodon idella. Journal of the World Aquaculture Society, 2017, 48, 333-341.

2. The structure of abstract section is vague and not provide any scientific information. It should be completely rewritten again?

Response: Thanks. Following the requirement, authors have revised the abstract sections by a wide margin. Please see Line 17-38.

3. The author should include more details about the main mode of action of glutamate into fish body, the main biological effects and its relation with fish production and tasty in introduction part?

Response: Thanks for this comment. Following the requirement, authors have supplemented content related to glutamate in the introduction. Please see Line 57-58.

4. The author detailed all information about mice as experimental animal, how could we predict the same biological effects in fish or aquatic creatures? The main hypothesis of the current study must be convinced the readers.

Response: Thank you for pointing out this problem. Just as you said, a reference about fish or aquatic species would be a better choice. However, there is poor research related to gls1 in aquatic fish, so we have to use the gls1 research of other animals. Just as the papers of Jiang et al. (2014) and Liu et al. (2020).

[1] Jiang, J.; Feng, L.; Liu, Y.; Jiang, W.D.; Hu, K.; Li, S.H.; Zhou, X.Q. Molecular cloning and expression of kidney-type glutaminase from common carp (Cyprinus carpio) and its up-regulation by glutamine in primary culture enterocyte. Aquaculture Nutrition, 2014, 20, 731-740.

[2] Liu, S.; Li, N.; Lin, Q.; Liu, L.; Niu, Y.; Liang, H.; Huang, Z.; Fu, X. Glutaminase 1 in mandarin fish Siniperca chuatsi: Molecular characterization, expression pattern and function involving in virus replication. Aquaculture, 2020, 519.

5. The material and method section missed so many important information about experimental groups and rearing conditions. I did not agree with the author for choosing one Way ANOVA for analysis this study. There are at least two main effects (different time and glutamate levels) how could we assessed the gene expression during different times under another effects of protein diet resource with one way ANOVA? Thus, the obtained results should be completely rewritten again and the statics should be reanalyzed.

Response: Thanks for your excellent comments. Following the requirement, authors have supplemented the information about experimental groups and rearing conditions in materials and methods. Please see Line 104-108, 119, 124-134, 141-144, 168-172, 183-188. In the section of muscle cell culture and treatment with glutamate, cells were treated for the same time in each group. The concentration of glutamate in the medium is the only main effect, so the impact of time does not need to be considered. It is also the reason why we adopted one-way ANOVA for data analysis.

6. The discussion of the current study not defined the interaction effects between time and other study variable. The discussion should be rewritten again for supported new results obtained from the corrected statistical analysis?

Response: Thanks for this comment. We are sorry for our inaccurate description and authors have revised the description of the method section. In the present study, there was only one variable in each experiment, so we did not consider the interaction effects between time and other study variable.

7. L 18 why the author did not mention the main objective in more concise manner? What is the importance of estimating the glutamate production and glutamine utilization in fish?

Response: Thanks. Following the requirements, authors have re-described the research object. Please see Line 17-18. The free glutamate is one of the major umami substances in fish meat, which is synthesized from glutamine by glutaminase. And glutamine is known as one of the most abundant free amino acids in fish plasma and muscle. From this perspective, gls1, the gene encoding kidney-type glutaminase, could be a good entry point to improve the concentration of free glutamate in muscle and the quality of fish fillet.

8. L 18-21 the author failed to described their treated groups or experimental design in shortened? This is not scientific structure of abstract?

Response: Thank you for pointing out this problem. Following the requirements, authors have complemented the experimental design. Please see Line 26-27. We also want to describe the treatment groups, experimental design, and even experimental methods in this study, just like the abstract of most papers. Unfortunately, as you see, this study is composed of several sections, including several feeding trials. If we described treatment groups and experimental design in the abstract, the content of abstract would become redundant, which is contrary to the characteristics of concise and brief of abstract. Therefore, we have referred to the writing methods of previous papers, and the references are attached below.

[1] Jiang, J.; Feng, L.; Liu, Y.; Jiang, W.D.; Hu, K.; Li, S.H.; Zhou, X.Q. Molecular cloning and expression of kidney-type glutaminase from common carp (Cyprinus carpio) and its up-regulation by glutamine in primary culture enterocyte. Aquaculture Nutrition, 2014, 20, 731-740.

[2] Liu, S.; Li, N.; Lin, Q.; Liu, L.; Niu, Y.; Liang, H.; Huang, Z.; Fu, X. Glutaminase 1 in mandarin fish Siniperca chuatsi: Molecular characterization, expression pattern and function involving in virus replication. Aquaculture, 2020, 519.

9. L 29 I can not define any related information about how the author had been examined there in vitro or in vivo experiment?

Response: Thanks for this comment. Following the requirements, authors have revised the manuscript. Please see Line 31-32.

10. L 31 we cannot supported general conclusion, it should be specified in more details?

Response: Thank you for pointing out this problem. Authors have re-described the conclusion of the abstract. Please see Line 33-38.

11. L 45 and L 49 missing cited references for this mentioned information?

Response: Thanks. References have been cited separately. Please see Line 52 and 55.

12. L 54 "animals" the example here should be related with fish or aquatic species.

Response: Thank you for pointing out this problem. Just as you said, a reference about fish or aquatic species would be a better choice. However, there is poor research related to glutaminase in aquatic fish, so we have to use the gls1 research of other animals. Just as the papers of Jiang et al (2014). and Liu et al (2020).

[1] Jiang, J.; Feng, L.; Liu, Y.; Jiang, W.D.; Hu, K.; Li, S.H.; Zhou, X.Q. Molecular cloning and expression of kidney-type glutaminase from common carp (Cyprinus carpio) and its up-regulation by glutamine in primary culture enterocyte. Aquaculture Nutrition, 2014, 20, 731-740.

[2] Liu, S.; Li, N.; Lin, Q.; Liu, L.; Niu, Y.; Liang, H.; Huang, Z.; Fu, X. Glutaminase 1 in mandarin fish Siniperca chuatsi: Molecular characterization, expression pattern and function involving in virus replication. Aquaculture, 2020, 519.

13. L 57 repeated information.

Response: Thanks for this comment. Authors have deleted the repeated information and re-described the sentence. Please see Line 61-64.

14. L 99 the all-water quality measurements should be described during the acclimation period?

Response: Thanks for this comment. Following the requirements, authors have supplemented the culture conditions during the acclimation period. Please see Line 104-108.

15. L 101 anesthesia dose should be defined?

Response: Thanks for your kind reminder. The information of anesthesia dose has been supplemented. Please see Line 109.

16. L 123 the fiberglass tanks dimensions should be described?

Response: Thanks. Because the fiberglass tank is cylindrical, we have not marked the dimensions of tanks. However, the volume (90 L) of the fiberglass tank has been described. Please see Line 140.

17. L 122 the experimental group not defined?

Response: Thanks for this comment. In this section, the type of feeds is equivalent to the type of treatment groups. Please see Line 136-138 for feed design. There are 25 fishes in each tank, and each treatment group is composed of 3 tanks. Please see Line 140-141.

18. L 149 how could the author avoid the mRNA contamination?

Response: Thanks. Fish was anesthetized before dissection. The whole dissection operation was carried out on the disposable sterile gloves which padded on the ice box. The samples were frozen in liquid nitrogen rapidly for temporary storage after collection, and then stored at -80°C until analysis. Following the requirements, authors have revised the manuscript. Please see Line 172.

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper entitled “Molecular characterization and dietary regulation of Glutaminase 1 (gls1) in triploid crucian carp (Carassius auratus)” with manuscript ID 2003018 by Xiao et al., performed an elaborate analysis on the molecular characterization, tissue distribution and diurnal expression profile and dietary regulation of the glutaminase 1 gene in triploid crucian carp.

Overall, the study is multileveled and provides useful information regarding the regulation of gls1 expression in a triploid fish species. However, there a few aspects that should be addressed in order to make this paper reproducible and clarify the necessity of some the performed analysis.

Major comments

First, the authors have not provided any information regarding the rearing conditions, e.g. temperature, stocking density, tank sizes, initial and final body weight of fish in the first experiment, and have provided only a part of them in the dietary experiment.

Second, no information on the feeding – standard ratio or ad libitum ­–, as well as on the growth of fish has been provided. Therefore, it is impossible to know whether fish fed with the experimental feed consumed the food equally well with the control group.

Third, the authors have performed the vast majority of their analysis is muscles. However, the choice of this tissue should be justified in order to clarify the necessity of this analysis. The choice of muscle comes as a surprise since according to Figure 3, the expression of gls1 in the muscle in very low compared to the brain and the liver. Especially the liver is a major site for metabolic function and it would seem a more integrated choice.

Moreover, how were fish dissected? Where they euthanized prior to dissection? What was the dosage of MS-222 used and how the anesthesia performed?

Finally, what type of ANOVA was used and how were the replicate tanks treated? Was it a simple or a nested ANOVA? Why have the authors reported their results as SEM and not SD? Have their checked the assumptions of the ANOVA?

Minor comments

L82-85: Please rephrase this sentence, it is hard to understand.

L87-92: I believe that the manuscript could benefit from the rephrasing of the objectives of the study. In their current form they are not very clear.

In the discussion, care should be taken when using references from other animal taxa, as for example chicks (L340) and mice (L348-350) in order to explain results obtained in fish.

Author Response

The paper entitled “Molecular characterization and dietary regulation of Glutaminase 1 (gls1) in triploid crucian carp (Carassius auratus)” with manuscript ID 2003018 by Xiao et al., performed an elaborate analysis on the molecular characterization, tissue distribution and diurnal expression profile and dietary regulation of the glutaminase 1 gene in triploid crucian carp.

Overall, the study is multileveled and provides useful information regarding the regulation of gls1 expression in a triploid fish species. However, there a few aspects that should be addressed in order to make this paper reproducible and clarify the necessity of some the performed analysis.

1. First, the authors have not provided any information regarding the rearing conditions, e.g. temperature, stocking density, tank sizes, initial and final body weight of fish in the first experiment, and have provided only a part of them in the dietary experiment.

Response: Thanks for this comment. According to your suggestion, authors have supplemented the rearing conditions (including water temperature, stocking density, tank volume, dissolved oxygen content, ammonia nitrogen concentration, pH and light period) in the section 2.1. Please see L104-108, L142-145, L169-172 and 186-188. Because this study includes several experiments, and the initial body weight of fish used in each experiment are different, initial body weight of fish for each experiment has been given separately, please see Line 116, 124, 140, 165 and 183 respectively.

2. Second, no information on the feeding – standard ratio or ad libitum ­–, as well as on the growth of fish has been provided. Therefore, it is impossible to know whether fish fed with the experimental feed consumed the food equally well with the control group.

Response: Thanks. In this study, fishes in all groups were fed to apparent satiation in each feeding trial, twice daily at 9: 00 and 15: 00. And all the fish can well accept the control and experimental diets. Authors have revised the manuscript, please see Line 141, 168 and 185 respectively.

3. Third, the authors have performed the vast majority of their analysis is muscles. However, the choice of this tissue should be justified in order to clarify the necessity of this analysis. The choice of muscle comes as a surprise since according to Figure 3, the expression of gls1 in the muscle in very low compared to the brain and the liver. Especially the liver is a major site for metabolic function and it would seem a more integrated choice.

Response: Thanks for your excellent comments. gls1 is the encoding gene of kidney-type glutaminase, the primary limiting enzyme for glutamate production, and free glutamate is one of the major umami substances in fish meat. Therefore, we chose muscle as the research object, aiming to provide reference data for the optimization of fish meat quality, even though the gls1 expression in the muscle of triploid crucian carp is lower than that in the brain and liver. Meanwhile, muscle tissue was also selected as the research object in the study of Watford and Wu (2005).

[1] Watford, M.; Wu, G. Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis. Comp Biochem Physiol B Biochem Mol Biol, 2005, 140, 607-14.

4. Moreover, how were fish dissected? Where they euthanized prior to dissection? What was the dosage of MS-222 used and how the anesthesia performed?

Response: Thanks for your comments. In this study, all fishes were immersed in 50 mg/L MS-222 for anesthesia before dissection. The whole dissection operation was carried out on the disposable sterile gloves which padded on the ice box. The samples were frozen in liquid nitrogen rapidly for temporary storage after collection, and then stored at -80°C until analysis. Following the advice, authors have revised the manuscript. Please see Line 109-112.

5. Finally, what type of ANOVA was used and how were the replicate tanks treated? Was it a simple or a nested ANOVA? Why have the authors reported their results as SEM and not SD? Have their checked the assumptions of the ANOVA?

Response: Thanks for this comment. From our study, there is only one variable in each experiment, such as dietary protein levels, dietary protein sources, dietary glutamate levels, etc. So, we choose one-way ANOVA as the main statistical method during each experiment. Meanwhile, each treatment group contains three repeated tanks in the feeding trial. The samples of each experiment conform to the normal distribution, and every group was randomly sampled for testing, so we used SEM instead of SD. It is consistent with the statistical analysis method used by Jiang et al (2014) and Liu et al (2012). In our study, homogeneity test of variance has been checked before one-way ANOVA, and the p-value is >0.05.

[1] Jiang, J.; Feng, L.; Liu, Y.; Jiang, W.D.; Hu, K.; Li, S.H.; Zhou, X.Q. Molecular cloning and expression of kidney-type glutaminase from common carp (Cyprinus carpio) and its up-regulation by glutamine in primary culture enterocyte. Aquaculture Nutrition, 2014, 20, 731-740.

[2] Liu, Z.; Zhou, Y.; Liu S.J.; Zhong, H.; Zhang, C.; Kang, X.; Liu, Y. Characterization and dietary regulation of glutamate dehydrogenase in different ploidy fishes. Amino Acids, 2012, 43, 2339-2348.

6. L82-85: Please rephrase this sentence, it is hard to understand.

Response: Thanks. Following the requirement, authors have revised the manuscript. Please see Line 88-89.

7. L87-92: I believe that the manuscript could benefit from the rephrasing of the objectives of the study. In their current form they are not very clear.

Response: Thanks for this comment. Following the advice, authors have redescribed the research objectives. Please see Line 91-97.

8. In the discussion, care should be taken when using references from other animal taxa, as for example chicks (L340) and mice (L348-350) in order to explain results obtained in fish.

Response: Thanks for your comments. Just as you said, a reference about fish or aquatic species would be a better choice. However, there is poor research related to gls1 in aquatic fish, so we have to use the gls1 research of other animals. Just as the only research on aquatic animals conducted by Jiang et al. (2014) and Liu et al. (2020).

[1] Jiang, J.; Feng, L.; Liu, Y.; Jiang, W.D.; Hu, K.; Li, S.H.; Zhou, X.Q. Molecular cloning and expression of kidney-type glutaminase from common carp (Cyprinus carpio) and its up-regulation by glutamine in primary culture enterocyte. Aquaculture Nutrition, 2014, 20, 731-740.

[2] Liu, S.; Li, N.; Lin, Q.; Liu, L.; Niu, Y.; Liang, H.; Huang, Z.; Fu, X. Glutaminase 1 in mandarin fish Siniperca chuatsi: Molecular characterization, expression pattern and function involving in virus replication. Aquaculture, 2020, 519.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors asked to extensively revised English language to avoid any grammatical errors 

Author Response

The authors asked to extensively revised English language to avoid any grammatical errors.

Response: Thanks for your comment. Authors have revised English language of manuscript. The modification was marked to highlight. Please refer to the revised manuscript.