Optimizing Nitrogen Fertilizer Application for Synergistic Enhancement of Economic and Ecological Benefits in Rice–Crab Co-Culture Systems

Response to Reviewer 1 Comments

1. Summary

We are grateful to you for your positive comments on the study! In the revised manuscript, all issues raised in the review process have been addressed point by point. We hope the revised manuscript can be endorsed.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Thank you very much for recognizing our work.

Is the research design appropriate?

Yes

Thank you very much for recognizing our work.

Are the methods adequately described?

Yes

Thank you very much for recognizing our work.

Are the results clearly presented?

Yes

Thank you very much for recognizing our work.

Are the conclusions supported by the results?

Yes

Thank you very much for recognizing our work.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Material and methods were nicely written but experimental design need to rewritten including weather parameter.

Response 1: We have accepted the suggestion. The experimental design section has been rewritten. In particular, the fertilization method has been described more clearly, the basis for the irrigation method and the depth of the surface water have been described more specifically, and the inappropriate writing order has been corrected. All changes are marked in red for ease of viewing. In addition, the weather parameters were described in the study site section.

Comments 2: Presentations of the results are very impressive. However, discussion section needs to be improved by writing more precisely.

Response 2: We think it’s a helpful suggestion. We overhauled the discussion section to add comparisons of data, such as “The increase in rice yield was attributed to improved agronomic traits and N use efficiency, which were enhanced in the RC system (Table 1), leading to an average of 4.2% higher rice yields in the RC system compared to the RM system (Figure 3a)”. Also, all discussions were rechecked for references to increase comparisons with other studies.

Comments 3: Reference of the manuscript should be checked thoroughly and it should be uniform as per the author’s guidelines.

Response 3: We agree with your suggestion. We checked all references, replaced inappropriate references, and organized all in-text numbering. Number of references expanded from 41 to 66. It was confirmed that all references were necessary and appropriate.

Comments 4: The style and use of English in the manuscript need to be improved without the loss of message.

Response 4: Thanks for your suggestion. We performed a detailed grammar and spelling check throughout the text and corrected all errors. All changes are marked in red.

Comments 5: The conclusion of the manuscript need to be revised with significant results of the study.

Response 5: We have rewritten the conclusion section, and all important results from this study are presented, including rice and crab yields, agronomic traits, N use efficiency, water quality, GHG emissions, N application rates for NEEB and RC system.

4. Response to Comments on the Quality of English Language

Point 1: The style and use of English in the manuscript need to be improved without the loss of message.

Response 1: Thank you very much for recognizing the methods section, we have rechecked and improved the grammar and vocabulary in order to improve the quality of the article!

For research article

Response to Reviewer 3 Comments

1. Summary

We are grateful to you for your positive comments on the study! In the revised manuscript, all issues raised in the review process have been addressed point by point. We hope the revised manuscript can be endorsed.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Thank you for your suggestion. We have expanded the introduction section. The additions included the current status of the rice-animal co-culture systems in China, the ecological potential of the RC system and the trend of N application in the RC system increasing year by year. The number of references was increased from 20 to 32. It was confirmed that all references were necessary and appropriate.

Is the research design appropriate?

Yes

Thank you very much for recognizing our work.

Are the methods adequately described?

Yes

Thank you very much for recognizing our work.

Are the results clearly presented?

Yes

Thank you very much for recognizing our work.

Are the conclusions supported by the results?

Yes

Thank you very much for recognizing our work.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Introduction can present some data on the amount of fertilizer (N source) that could be reduced by adopting the co-culture system and more details on the positive impacts it would offer to the environment and human health.

Response 1: We have accepted the suggestion. We have added information in the introduction section to show the positive impacts of the RC system on the environment and human health: “In the RC system, crabs can effectively control weeds and reduce the use of fertilizers and pesticides [18,19], thus helping to mitigate the risk of non-point source pollution and pesticide residues [20,21]”.

Comments 2: In Discussion, I believe it can be explored the fact that analysis on N metabolism in rice and crab organisms are also interesting to be performed to verify the impact the co-culture may offer, specially on the synthesis of substances containing nitrogen, which could significantly impact nutritional aspects. I understand that it is not the aim of this work, but I believe it deserves to be mentioned.

Response 2: We think it’s a helpful suggestion. We did not add relevant data and analyses to this study due to limitations on the length and thrust of the article. We have added relevant notes in the discussion section to suggest future research: “However, N application may affect the metabolism of nitrogen in both rice and crabs, thus altering the quality and taste; therefore, further research is needed [63,64]”.

Comments 3: Conclusion section could benefit from the addition of future perspectives.

Response 3: Thanks for your suggestion. We believe that the conclusions on optimizing N application in RC systems are very important for farmers, and we have not added future perspectives in order to highlight the final conclusions. However, we have added relevant content in the discussion section. If it is necessary to add the future perspectives, we will modify it.

4. Response to Comments on the Quality of English Language

Point 1: I am not qualified to assess the quality of English in this paper.

Response 1: We performed a detailed grammar and spelling check throughout the text and corrected all errors. All changes are marked in red.

5. Additional clarifications

[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

Response to Reviewer 4 Comments

1. Summary

I have gone through the manuscript entitled “Optimizing nitrogen fertilizer application for synergistic enhancement of economic and ecological benefits in rice-crab co-culture systems” and concluded the following:

The manuscript was prepared professionally. It includes a well-drafted abstract and an exhaustive introduction that justifies the research undertaken. The introduction points to the deficiencies in the literature on the subject. The aim is clearly defined. The discussion of the results is well prepared. The conclusions are well-defined. The illustrative material methods are appropriate. In my opinion, the manuscript after the corrections that are following will be suitable for publication in the Journal of Agronomy.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Thank you very much for recognizing our work.

Is the research design appropriate?

Can be improved

Thank you for your suggestion. The experimental design section has been rewritten. In particular, the fertilization method has been described more clearly, the basis for the irrigation method and the depth of the surface water have been described more specifically, and the inappropriate writing order has been corrected. All changes are marked in red for ease of viewing. In addition, the weather parameters were described in the study site section.

Are the methods adequately described?

Yes

Thank you very much for recognizing our work.

Are the results clearly presented?

Can be improved

We agree with your suggestion. We checked all results for accuracy. Grammar and vocabulary in the results section are checked for clarity.

Are the conclusions supported by the results?

Yes

Thank you very much for recognizing our work.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In Figure 1 "right side": Rm270 treatment was not found, while Rm210 treatment presented six times. Check Figure 1.

Response 1: We have accepted the suggestion. Thank you very much for careful checking of Figure 1, in which we have corrected the labeling of RM270.

Comments 2: In 2.2 Experimental design:  the experimental design used in the experiment was not mentioned according to the study factors (random, fixed).

Response 2: We think it’s a helpful suggestion. We modified the distribution of treatments: “Each treatment was replicated three times and randomly distributed in 24 experimental plots measuring 15 m × 8 m (Figure 1)”.

Comments 3: In line 199, the Shapiro-Wilk test was used to evaluate the normality of the data distribution. However, no table containing the test statistic and significance value was given.

Response 3: We agree with your suggestion. We assessed the normality of the data using the Shapiro-Wilk test, which showed that the normality of the data was verified and all p-values were greater than 0.05, therefore no separate tables for test statistics and significance values were presented. We think it is clear enough to mention this test in the text, but if you think it is necessary, we can consider adding the corresponding tables in further revisions.

Comments 4: It is better to test homogeneity between the two seasons (years) and use combined analysis if homogeneity is achieved and analyze each season alone when the seasons (years) are not homogeneous.

Response 4: Thank you very much for your suggestion! The main components of this study, including rice and crab yields, CH4 and N2O emissions, and NEEB were less homogeneous in two years. In order to reflect the fact that although there were differences in the above parameters in both years, the optimal N application rates for the RC and RM monoculture systems were 210 kg ha-1 and 270 kg ha-1, the data for each year were analyzed separately.

Comments 5: It was not explained which of the methods of comparing the means were used.

Response 5: Thanks for your suggestion. We added the method of comparing the means in the Statistical analysis section: “One-way analysis of variance (ANOVA), Duncan's post-hoc test and t-test were used to examine the differences between treatments.”.

Comments 6: In tables (1-3) containing the mean ± standard deviation, it is clear that the value of F for the three-way interaction) Y × S × N) or S × N interaction are not significant, while the letters following each mean indicate the presence of a significant difference between the values, which leads to a conflict in the results obtained from the F test, the method of comparing the means used. As well as for figures 3a and 5.

Response 6: Thanks for your suggestion. We should like to point out that as in the response to your comment 4, we would like to analyze each year's treatments separately, so the letters labeled after all means in Tables 1, 2, and 3 to represent variability indicate variability in only 8 treatments in a one-year experiment, as do Figures 3a and 5.

Comments 7: Line 227: "50.5%-95.6%" correct to 50.3%-95.8%.

Response 7: Thank you for your suggestion. Thank you for your careful checking of the data in the manuscript, we have corrected the deviating data.

4. Response to Comments on the Quality of English Language

Point 1: I am not qualified to assess the quality of English in this paper.

Response 1: The style and use of English in the manuscript need to be improved without the loss of message.

Response to Reviewer 5 Comments

1. Summary

We are grateful to you for your positive comments on the study! In the revised manuscript, all issues raised in the review process have been addressed point by point. We hope the revised manuscript can be endorsed.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Thank you for your suggestion. We have expanded the introduction section. The additions included the current status of the rice-animal co-culture systems in China, the ecological potential of the RC system and the trend of N application in the RC system increasing year by year. The number of references was increased from 20 to 32. It was confirmed that all references were necessary and appropriate.

Is the research design appropriate?

Can be improved

Thank you for your suggestion. The experimental design section has been rewritten. In particular, the fertilization method has been described more clearly, the basis for the irrigation method and the depth of the surface water have been described more specifically, and the inappropriate writing order has been corrected. All changes are marked in red for ease of viewing. In addition, the weather parameters were described in the study site section.

Are the methods adequately described?

Can be improved

We agree with your suggestion. We modified the methods by replacing more appropriate references and checking the soundness of the methods. All references are correctly numbered.

Are the results clearly presented?

Can be improved

We agree with your suggestion. We checked all results for accuracy. Grammar and vocabulary in the results section are checked for clarity.

Are the conclusions supported by the results?

Yes

We accepted your suggestion. We have rewritten the conclusions section to ensure that all results are derived from our findings.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The introduction is sufficient, nevertheless, it is also important to mention the previous successful attempts or normal application of rice cultivation with animal farming.

Response 1: We have accepted the suggestion. We have expanded the introduction section. The additions included the current status of the rice-animal co-culture systems in China: “In China, the adoption of animal co-culture systems has reached 2.9 million ha [12], accounting for 9.5% of the rice planting area, providing 21.5 × 10⁶ t of rice and 3.9 × 10⁶ t of aquaculture products [13-15]”

Comments 2: How is the height of the water level in the area? It is important parameters to counteract the methane production, productivity of the cultivations, as well as water footprint.

Response 2: We think it’s a helpful suggestion. We describe in the experimental design section that the RC system used in the experiment referenced local management practices: “For irrigation water requirements of rice and crabs, and with reference to local water management experience, all treatments were kept flooded with surface water depth maintained at 5 cm to 10 cm throughout the rice growing season [25,30]”.

Comments 3: what would happen if the concentration of N was increased more than 270 kg/Ha? does the productivity remain the same or it would increase the productivity as well as CH4 emission?

Response 3: We agree with your suggestion. We added a basis for the selection of N application rate in the experimental design section: “The rice variety of Yanfeng 47 was used in the experiments, planted at a spacing of 12 cm × 30 cm. Since the N application rate over 270 kg ha-1 reduces the yield of this rice variety, the N application rates used in this study were 0, 150, 210, and 270 kg ha-1 for RC and RM systems, respectively [33]”. Excessive N application rates are not enforceable in practice.

Comments 4: How many harvests in this experiments? It is important to note that there should be more than one harvest in two years as rice is not a perennial crop.

Response 4: Thanks for your suggestion. We added instructions in the experimental design section: “Rice and crab are cultivated in a single season in each year, starting in May and ending in October, respectively.” Local temperatures are not conducive to agricultural activities in winter, hence single-season cultivation.

Comments 5: Interesting fact from Fig 6 a and b shall be elaborated more. the N2O usually produced at the beginning of the fertilized addition, and later on it is moving down while the CH4 peaked. What was the process inside the soil/water during these periods?

Response 5: We have added relevant information to the discussion section: “During the early stages of the planting season, the application of N resulted in elevated concentrations of N in surface water and soil (Figure S1e, S1f, S2e, S2f), along with high levels of DO (Figure S1e and S2e), which enhanced nitrification and denitrification [50,51], leading to a surge in N2O emissions (Figure 6b). In contrast, N2O emission fluxes after the tillering stage were suppressed due to the intensification of anaerobic conditions in the soil as a result of persistent flooding, as well as the lack of N [52]. Anaerobic conditions favored CH4 production [53, 54], and the increase in carbon substrates from root secretions and decaying leaves of growing rice [55], along with the formation of advanced aerenchyma [56], led to a gradual increase in CH4 emissions after the tillering stage (Figure 6a). CH4 emission fluxes gradually decreased in the later stages of rice growth as temperature decreased and the ventilation tissues aging and breaking [57]. Similar results were observed in other studies [30,58].” And we added references to compare with other studies.

Comments 6: NEB and NEEB showed exactly similar results in terms of differences in each experiment. elaborate more in the discussion? It also important to mention the investment difference in each treatments?

Response 6: Thanks for your suggestion. In practice, farmers generally focus only on the NEB. However greenhouse gas emissions should also be considered because of the potential impacts of the sustainability of the system on all farmers, although farmers are rarely concerned about these impacts. In the supplementary material, we provide information about the inputs. However, it is worth noting that the price of nitrogen fertilizer is low relative to other inputs.

Comments 7: 7. It is interesting to see that the data showed reliability in two years cultivation period. The authors shall mention this and emphasize as the advantages of the combination.

Response 7: We couldn't agree with you suggestion more. In the Materials and Methods section we have added instructions: “To increase the reliability of this study, the field experiments were carried out for two years, in 2020 and 2021, respectively.”

Comments 8: The authors shall mention the constraints between monoculture and combination of them. The improvement that shall be done also need to be mentioned, and the limitation of the experiment/study also worth scrutinized at the end of the discussion. Currently, authors only focus on the data and the literal findings without translation of these findings for future application and comparison with other similar studies or approaches.

Response 8: We accepted your suggestions. Firstly, we suggest the factors limiting RC system in discussion: “Compared to other optimization strategies, adjusting the N application rate is the most feasible and easy-to-implement improvement for farmers, as the main constraints to the development of RC systems are the aging labor force and the technical capacity of available technology”. Secondly, we added the limitations of this study in the last paragraph of the discussion. Finally, we compared other innovative RC systems to elucidate the strengths of this study and future research prospects.

Comments 9: Overall the manuscript showed an interesting idea with well data supports. The key findings shall be mentioned and emphasize more based on the comments.

Response 9: We have rewritten the conclusion section, and all important results from this study are presented, including rice and crab yields, agronomic traits, N use efficiency, water quality, GHG emissions, N application rates for NEEB and RC system.

4. Response to Comments on the Quality of English Language

Point 1: I am not qualified to assess the quality of English in this paper.

Response 1: The style and use of English in the manuscript need to be improved without the loss of message.