Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript Title: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in Zhangzhou's Xixi River

General comments

The manuscript assesses a comprehensive study of stock enhancement efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi River. The authors are integrating traditional fishery survey methods with environmental DNA (eDNA) analysis for this assessment. The authors compare the resources including the abundance, mortality and survival of the species using both traditional and eDNA-based approaches that strengthens the study and provides valuable data for aquatic resource restoration and ecological management in the Jiulong River basin. However, some content is necessary to be addressed. The manuscript could be reconsidered after carefully addressing the following issues.

 

Title: The title could be "Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in Jiulong River"

 

Abstract:

Line 17-25: Redundant. The introductory statement should be limited to one to two sentences. After that, clearly state the objectives of the study.

 

Introduction:

Line 116: Stocking should be in small letters.

Line 123: Basin should be in small letters.

Line 138: Delete “analyzing their growth characteristics” as no growth characteristics were measured.

Line 143: Delete the repetitive words “in the”.

 

Materials and Methods:

Line 147: Delete the repetitive words “in the”.

Line 150: Mention the names of the sampling sites.

Line 169: In the traditional survey method, you sample seven times, while in the case of eDNA sampling only two times in October 2023 and April 2024. You should discuss the reasons for this.

Line 203: In Pauly’s model, only temperature is considered, but there are also other parameters like dissolved oxygen, pH, turbidity, etc., which might influence the mortality and survival of the fish species. You should discuss this.

 

Results:

Line 250-252: Prepare a different graph with error bars and explain the graph with more clarification.

Line 282: Table 6: Add confidence interval for mortality and survival contribution.

Line 285: Table 7: Use the source of the data.

 

Discussion:

Line 312: Previous should be in small letters.

Line 311-313: Sentence meaning is not clear.

Line 314: Mention the species name. Do you mean “Carassius auratus”? Please check.

Line 315: Check the citation.

Line 319: Use the abbreviated form of environmental DNA.

Line 340-349: Better to move this paragraph to the introduction.

 Line 393: Replace ‘Stream’ with ‘basin’.

Line 416-429: Conclusions should be more concise and specific to the findings.

 

Reference:

Reference 13 and 18 are the same. Please cross-check throughout the text and reference list.

 

Author Response

Ms. Ref. No.: water-3810940

Title: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin

Reviewer #1:

1. The manuscript assesses a comprehensive study of stock enhancement efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi River. The authors are integrating traditional fishery survey methods with environmental DNA (eDNA) analysis for this assessment. The authors compare the resources including the abundance, mortality and survival of the species using both traditional and eDNA-based approaches that strengthens the study and provides valuable data for aquatic resource restoration and ecological management in the Jiulong River basin. However, some content is necessary to be addressed. The manuscript could be reconsidered after carefully addressing the following issues.

Answer: We are much grateful for your careful reading of our manuscript and your valuable comments and suggestions to help improve the paper.

 

2. 2. The title could be "Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in Jiulong River".

Answer: Based on your suggestions, we have changed the title to “Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin”, because it is mainly a research and discussion on the Xixi River Basin.

 

3. Line 17-25: Redundant. The introductory statement should be limited to one to two sentences. After that, clearly state the objectives of the study.

Answer: We are very grateful for your suggestion and have made revisions to make it more reasonable and concise

 

4. Line 116: Stocking should be in small letters.

Answer: We have followed your comments, and corrected it.

 

5. Line 123: Basin should be in small letters.

Answer: We have followed your comments, and corrected it.

 

6. Line 138: Delete “analyzing their growth characteristics” as no growth characteristics were measured.

Answer: We have followed your comments, and corrected it.

 

7. Line 143: Delete the repetitive words “in the”.

Answer: We have followed your comments, and corrected it.

 

8. Line 147: Delete the repetitive words “in the”.

Answer: We have followed your comments, and corrected it.

 

9. Line 150: Mention the names of the sampling sites.

Answer: We have followed your comments. We changed station A, B, and C to upstream A, midstream B, and downstream C, respectively, and provided explanations for the sample point selection.

 

10. Line 169: In the traditional survey method, you sample seven times, while in the case of eDNA sampling only two times in October 2023 and April 2024. You should discuss the reasons for this.

Answer: We have followed your comments. Our primary analytical approach remains centered on conventional fishing methods, with eDNA methodology serving as a supplementary tool for species composition analysis. Sampling was initiated in October 2023, with subsequent collections in October 2023 and April 2024. These specific months were selected to align with the beginning and end of the reproductive cycle, providing optimal conditions for biomass analysis.

 

11. Line 203: In Pauly’s model, only temperature is considered, but there are also other parameters like dissolved oxygen, pH, turbidity, etc., which might influence the mortality and survival of the fish species. You should discuss this.

Answer: We have followed your comments. Pauly's formula provides a baseline estimation under general environmental conditions, reflecting both the original intent of the model construction and the data availability at the time. Temperature, compared to other factors, is more stable, reliable, data-rich, and readily accessible, making it a suitable predictor variable. Furthermore, all physiological and biochemical processes in fish—including respiration, feeding, and reproduction—are dependent on ambient temperature. It directly and comprehensively drives metabolic rate, which serves as the primary intrinsic driver of natural mortality.

 

12. Line 250-252: Prepare a different graph with error bars and explain the graph with more clarification.

Answer: We have followed your comments, and corrected it. As shown in Figure 3.

 

13. Line 282: Table 6: Add confidence interval for mortality and survival contribution.

Answer: We have followed your comments, and corrected it.

 

14. Line 285: Table 7: Use the source of the data.

Answer: We have followed your comments. We have made revisions to our methodology, incorporating economic value estimation through recapture yield analysis.

 

15. Line 312: Previous should be in small letters.

Answer: We have followed your comments, and corrected it.

 

16. Line 311-313: Sentence meaning is not clear.

Answer: We have followed your comments. We wrote this section to explain that eDNA technology can detect fish species that cannot be caught using traditional methods, and to demonstrate that eDNA technology is feasible for monitoring purposes. In addition, we revised any unreasonable statements.

 

17. Line 314: Mention the species name. Do you mean “Carassius auratus”? Please check.

Answer: We are very grateful that you have carefully read our manuscript and provided valuable opinions and suggestions. We have already checked and revised it.

 

18. Line 315: Check the citation.

Answer: We have followed your comments. We have checked and revised it.

 

19. Line 319: Use the abbreviated form of environmental DNA.

Answer: We have followed your comments, and corrected it.

 

20. Line 340-349: Better to move this paragraph to the introduction.

Answer: We have followed your comments. We have checked and revised it.

 

21. Line 393: Replace ‘Stream’ with ‘basin’.

Answer: We have followed your comments, and corrected it.

 

22. Line 416-429: Conclusions should be more concise and specific to the findings.

Answer: We are very grateful for your suggestion and have made revisions to make it more reasonable and concise.

 

23. Reference 13 and 18 are the same. Please cross-check throughout the text and reference list.

Answer: We have followed your comments, and corrected it.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the manuscript: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in Zhangzhou's Xixi River. This is an interesting analysis of stocking a river and attempts to monitor subsequent biomass by traditional and eDNA methodologies.

My overall comment is that the manuscript needs a major revision before publishing. The authors have produced evidence of the continued presence of the stocked species, but there is a lot of confusing and missing information around the background, the methodologies and the comparison with eDNA.

 

Specific comments:

Lines 26-31- the results suggest that the surveys have been taking place since 2017, the catch results are only discussed from the one year period?  But the average survival rate is used from all the years? Will need to clarify the timeframe, methodology and results.

Lines 31 – 33 – there is no evidence that the environment is more suitable for H molitrix relative to A nobilis. The reverse could be stated since A nobilis had greater survival – the subsequent population relative to H molitrix was a greater ratio than the stock ratio.

Line 42 – Chinese national government?

Lines 78 – 96 – would be good to see more discussion on how eDNA techniques in the context of stocking could be compared with traditional catching/marking techniques to understand biomass. How do they compare side by side? How could this be applied in this research project?

Lines 97 – 134 – this needs a rewrite, combining these two very large paragraphs into some clear smaller paragraphs. It is confusing and hard to follow. Need to separate out parts and explain clearly:

– how do stocked species fit into the ecology of the river in the past, what changed in the river, how has the life cycle been broken.

-why are these species used for stocking, what is their ecological or commercial value? Are they artisanally, commercially or recreationally fished? What kinds of quantities? Good to compare with mortality rate later on. A nobilis is not a dominant species – why used at all?

                - what makes the species good for stocking versus other species?

Lines 135 – 143 – what is the approach for comparing the traditional capture versus eDNA technique? Why? What benefit? You suggest that eDNA could substitute for traditional capture but how could this happen an how are you testing this?

Introduction/methods – would be good to have photos of stocked fish and sampling locations.

Introduction – good to have some details on how big these fish grow, it appears only small fish are being caught in the nets?

Lines 150 – 151 – explain the sampling locations. Why were these chosen? Describe the environment for each? Do  the sampling locations cover the different life cycle locations and habitat needs for the species? Juvenile, spawning, adult normal habitat, etc...

Figure 1 – is very confusing. I cannot understand what the inset maps are and the location of the research area in China. There should be a small map of China in the corner, with a small box in it showing the location of the research area. The two rivers just stop – do they combine or are they at the coast?

Lines 155 – 161- the net arrangement is not clear – the authors talk about a three layer net, but then talk about 4 mesh sizes. Perhaps a drawing would help to understand the arrangement. 50 m apart seems very close to each other, why?

Traditional methodology – looks like capture surveys have been taking place since 2017. What is historical data and what is part of the study? How do the methods compare over those years? Why is only one year of sampling results being discussed but survival discussed for all years? Should combine all the years into one big study?  I can see how you might want to compare the capture studies with the eDNA for just the one study period, but this manuscript is not focussing on the comparison of the two techniques?

eDNA methodology – the methodology is not clear. How many samples are taken and in what quantities? I see 5l then 3 x 1l. What is the purpose of replicates – to generate confidence limits around the measured values or are they all combined into one sample for the whole river?

Lines 177 – 196 – too many details. Can you provide basic approach and provide reference to a methodology that you followed?

Lines 188 – 196 – not clear what is going on here. There appears to be comparison with a database? How is this done?

Data analysis – if a prior published methodology was followed, can provide basic approach and then refer reader that paper rather than providing all the details.

Some of the parameters are not explained or evident in the equations– VGBE, dti, K – others?

Line 210 – resource volume? Perhaps biomass or number?

Line 199 – mention of use of Microsoft excel unnecessary

Data analysis – how is actual catch factored into these calculations? Is there an estimate of fishing mortality separate from natural mortality. These are a relatively long living fish, so 50% mortality is pretty high.

Lines 224 – 232 – what do the percentages mean? Weight? Proportion of species?

Lines 229 – 232 – good to understand the differences between sampling sites

Table 3 – Red Tilapia?

eDNA results –

-not clear what percentages are

-O mossambicus not caught manually, but clearly very dominant presence

- A nobilis, a stocked species not detected at all

 

Line 247 – Order level?

Table 4 – sort by dominance like table 3?

Table 4 – Coila greyii repeated under different taxa, any others like this?

Table 5 – Fishing mortality came from which data source? Natural mortality from where, especially given the unnatural state of environment for those fish

Lines 270 – 281 – keep mixing the ongoing annual surveys with this one research project, clearly explain how the fit together

Table 7 – how did these economic values get calculated? There must be estimates of fisher catch value? From where?

Lines 308 – 310 – skipping the 2^nd most dominant species O mossambicus

Lines 310 -312 – why is there a “good” effect?

Line 313 – insert full stop between Carassius and Information

Line 314 – “auratus”?

Lines 315 – 319 – discussion of non-native species seems out of place. Provide more explanation

Lines 325 – 334 – need a discussion about comparison of eDNA versus capture methodologies for practical purposes. Is there any reason to use eDNA? Not clear from this manuscript.

Lines 360 – 265 – difference in survival? Both caught and eaten? A nobilis has higher survival, why?

Lines 365 – 366 – need to provide more information behind economic yields -how are these calculated?

Line 367 – A nobilis does not really have a “significant” population, not even detected by eDNA...

Line 368 – how is stocking “optimised”?

Lines 371 – 372 – provide reference and context for social surveys.

Lines 376 – 379 – paragraph out of place here

Lines 383 – 387 – how is this demonstrated? What is the evidence for the enhancement, substantial ecological contributions, and tangible benefits for restoration?

Author Response

Ms. Ref. No.: water-3810940

Title: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin

Reviewer #2:

1.Thank you for the opportunity to review the manuscript: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in Zhangzhou's Xixi River. This is an interesting analysis of stocking a river and attempts to monitor subsequent biomass by traditional and eDNA methodologies.

My overall comment is that the manuscript needs a major revision before publishing. The authors have produced evidence of the continued presence of the stocked species, but there is a lot of confusing and missing information around the background, the methodologies and the comparison with eDNA.

Answer: We are much grateful for your careful reading of our manuscript and your valuable comments and suggestions to help improve the paper.

 

2. Lines 26-31- the results suggest that the surveys have been taking place since 2017, the catch results are only discussed from the one-year period? But the average survival rate is used from all the years? Will need to clarify the timeframe, methodology and results.

Answer: We have followed your comments. The mortality coefficients were not averaged across all years. Although stock enhancement initiatives began in the Xixi River basin in 2017, the absence of annual catch analyses precluded baseline data availability. Mortality rates and survival numbers were estimated using length-frequency data collected from October 2023 to October 2024, processed through FiSAT II for mortality coefficient calculation. This analysis encompassed seven sampling events conducted over a precise one-year cycle.

 

3. Lines 31 – 33 – there is no evidence that the environment is more suitable for H. molitrix relative to A. nobilis. The reverse could be stated since A. nobilis had greater survival – the subsequent population relative to H. molitrix was a greater ratio than the stock ratio.

Answer: We have followed your comments. We have adjusted this section to focus more on explaining that the survival rate of H. molitrix is higher than that of A. nobilis, rather than that the environment is more suitable for H. molitrix growth. In addition, based on an analysis of feeding habits, H. molitrix prefer phytoplankton, and water bodies with more phytoplankton are more suitable for H. molitrix growth. Therefore, the analysis that the growth environment of H. molitrix is better is one-sided.

 

4. Line 42 – Chinese national government?

Answer: We have followed your comments, and corrected it.

 

5. Lines 78 – 96 – would be good to see more discussion on how eDNA techniques in the context of stocking could be compared with traditional catching/marking techniques to understand biomass. How do they compare side by side? How could this be applied in this research project?

Answer: We have followed your comments. We have expanded the comparative framework to include multiple dimensions such as biomass, population composition, dominant species, temporal resolution, and sample analysis. This allows for a comprehensive evaluation of the respective advantages of each method across different aspects. In this study, eDNA methodology was employed as a supplementary tool to analyze species composition and dominant species, demonstrating its feasibility and applicability for informing future stock enhancement strategies.

 

6. Lines 97 – 134 – this needs a rewrite, combining these two very large paragraphs into some clear smaller paragraphs. It is confusing and hard to follow. Need to separate out parts and explain clearly: how do stocked species fit into the ecology of the river in the past, what changed in the river, how has the life cycle been broken. why are these species used for stocking, what is their ecological or commercial value? Are they artisanally, commercially or recreationally fished? What kinds of quantities? Good to compare with mortality rate later on. A nobilis is not a dominant species – why used at all? what makes the species good for stocking versus other species?

Answer: We appreciate the reviewer's feedback. We have revised and reorganized this section to elaborate on the changes in the river and their impacts, the reasons for selecting H. molitrix and A. nobilis, their reproductive conditions, and the advantages they possess compared to other fish species. Due to economic development along the river, intensive human fishing activities, and the construction of cascaded hydropower stations, the aquatic ecosystem has been disrupted, affecting the survival and reproductive conditions of fish and leading to a decline in their populations. H. molitrix and A. nobilis exhibit characteristics such as rapid growth, strong adaptability, and high aquaculture yields.

 

7. Lines 135 – 143 – what is the approach for comparing the traditional capture versus eDNA technique? Why? What benefit? You suggest that eDNA could substitute for traditional capture but how could this happen an how are you testing this?

Answer: We appreciate the reviewers' comments and suggestions. Our objective is to demonstrate the usefulness of the eDNA method in detection, not to replace traditional trawling methods with eDNA technology. In future work, this method can be considered for inclusion to achieve greater efficiency and speed. By comparing the species composition, biomass, and dominance of the two methods, we analyzed their respective advantages. However, eDNA technology still has many shortcomings that need to be further improved.

 

8. Introduction/methods – would be good to have photos of stocked fish and sampling locations.

Answer: We have followed your comments. We have supplemented them in the materials and methods section.

 

9. Introduction – good to have some details on how big these fish grow, it appears only small fish are being caught in the nets?

Answer: We thank the reviewer for their valuable suggestions. We did not include specific data on the body length and weight of the captured fish in the manuscript because these measurements were directly incorporated into the formulas for calculation and were not separately highlighted. The catch composition varied in size, and the fishing gear used was capable of capturing individuals across a range of specifications. Notably, both H. molitrix and A. nobilis with body lengths exceeding 30 cm were present, as can be referenced in the provided photographs of the catch. We hope this explanation adequately addresses the question.

 

10. Lines 150 – 151 – explain the sampling locations. Why were these chosen? Describe the environment for each? Do the sampling locations cover the different life cycle locations and habitat needs for the species? Juvenile, spawning, adult normal habitat, etc...

Answer: We thank the reviewers for their suggestions. We have provided additional explanations in section 2.1(Study Area and Sampling Stations) and clarification. The Xixi River is one of the tributaries of the Jiulong River. It converges into the Jiulong River after sampling station C, and eventually flows into the sea. Considering the overall habitat of this fish species, the distribution of the fish population after release, and the actual fishing areas of fishermen, and since H. molitrix and A. nobilis are mid-to-upper layer fish species that are more commonly found in areas with river bends and slow-moving water, station A is located at a bend in the Xixi River basin, station B is the core release area with calm water surfaces, site C is located before the estuary. Since H. molitrix and A. nobilis are freshwater fish, selecting a more upstream location as the site is more appropriate.

 

11. Figure 1 – is very confusing. I cannot understand what the inset maps are and the location of the research area in China. There should be a small map of China in the corner, with a small box in it showing the location of the research area. The two rivers just stop – do they combine or are they at the coast?

Answer: We have followed your comments. We have made certain changes. Xixi is a tributary of the Jiulong River, located on the coast. Xixi will flow into the Jiulong River at the downstream estuary, and then the Jiulong River will flow into the sea.

 

12. Lines 155 – 161- the net arrangement is not clear – the authors talk about a three layer net, but then talk about 4 mesh sizes. Perhaps a drawing would help to understand the arrangement. 50 m apart seems very close to each other, why?

Answer: We thank the reviewers for their suggestions. We have addressed the issue of mesh size by adopting three-layer gill nets with mesh sizes of 4 cm, 7 cm, and 10 cm respectively. Each net is 100 m in length and 2–5 m in height for sampling. The 50-meter spacing between nets was chosen based on deploying three nets per sampling station to ensure catch efficiency. However, we acknowledge that this distance may be too short, and we will improve this aspect in future studies

 

13. Traditional methodology – looks like capture surveys have been taking place since 2017. What is historical data and what is part of the study? How do the methods compare over those years? Why is only one year of sampling results being discussed but survival discussed for all years? Should combine all the years into one big study? I can see how you might want to compare the capture studies with the eDNA for just the one study period, but this manuscript is not focussing on the comparison of the two techniques?

Answer: We thank the reviewers for their suggestions. Traditional capture surveys were not conducted starting from 2017; rather, stocking activities were initiated that year. Historical data in this study refers specifically to past stocking records used for current analysis. Due to the absence of previous fishing activities, no comparative data or methodological benchmarks from prior years were available. The application of an average mortality rate was adopted to enhance data reliability, facilitating the calculation of annual survival rates to support subsequent economic yield estimations, thereby enabling a comprehensive evaluation of actual stocking efficacy. This study's comparison was limited to methodological differences in species composition and dominance analyses, with the primary analytical focus remaining on data derived from traditional fishing methods.

 

14. eDNA methodology – the methodology is not clear. How many samples are taken and in what quantities? I see 5l then 3 x 1l. What is the purpose of replicates – to generate confidence limits around the measured values or are they all combined into one sample for the whole river?

Answer: We have received your suggestions from the reviewer and provided supplementary explanations. We selected three sample collection stations, taking three parallel samples from each station, and collected a total of 18 samples. The purpose of repeating the experiment is to conduct an overall analysis of the data of a river.

 

15. Lines 177 – 196 – too many details. Can you provide basic approach and provide reference to a methodology that you followed?

Answer: We have followed your comments. Regarding the use of primers, we have supplemented the references. The specific methods used in this study are as described in the text and are different from the PCR amplification conditions and kits in the reference primer papers.

 

16. Lines 188 – 196 – not clear what is going on here. There appears to be comparison with a database? How is this done?

Answer: We have followed your comments. Our eDNA samples were sent to Bio-Van Biological Technology Co., Ltd. in Wuhan, China, for processing and analysis. The company conducted the comparative analysis against databases uniformly.

 

17. Data analysis – if a prior published methodology was followed, can provide basic approach and then refer reader that paper rather than providing all the details.

Answer: We have followed your comments. Regarding the use of primers, we have supplemented the references. The specific methods used in this study are different from the PCR amplification conditions and kits in the reference papers. Therefore, we will still provide a detailed explanation of this part.

 

18. Some of the parameters are not explained or evident in the equations– VGBE, dti, K – others?

Answer: We have followed your comments. We have already checked and revised it.

 

19. Line 210 – resource volume? Perhaps biomass or number?

Answer: We have followed your comments, and corrected it.

 

20. Line 199 – mention of use of Microsoft excel unnecessary.

Answer: We have followed your comments, and corrected it.

 

22. Data analysis – how is actual catch factored into these calculations? Is there an estimate of fishing mortality separate from natural mortality. These are a relatively long living fish, so 50% mortality is pretty high.

Answer: We have followed your comments. The fishing coefficient was calculated to provide supplementary context for the data. In the study, estimates were made for both fishing mortality and natural mortality. The fishing mortality rates for H. molitrix and A. nobilis were 12.6% and 11.85%, respectively, while the natural mortality rate for both species was 47.15%.

 

23. Lines 224 – 232 – what do the percentages mean? Weight? Proportion of species?

Answer: We have followed your comments. These percentages refer to the proportion of the number of species.

 

24. Lines 229 – 232 – good to understand the differences between sampling sites.

Answer: We have followed your comments. We mentioned in the text: species distribution across sampling sites showed: station A contained 24 species from 6 orders and 9 families; station B hosted 28 species across 6 orders and 12 families; station C supported 32 species representing 7 orders and 15 families. Overall, station C has the most diverse distribution.

 

25. Table 3 – Red Tilapia?

Answer: We have followed your comments. We made the correction and changed it to Oreochromis.

 

eDNA results

26. not clear what percentages are.

Answer: We have followed your comments, and corrected it.

 

27. O mossambicus not caught manually, but clearly very dominant presence.

Answer: We have followed your comments. O mossambicusz is widely distributed in the Xixi River and is classified as an invasive alien species. Its growth characteristics, local climate, and hydrological conditions have led to its proliferation, giving it a dominant position in the river.

 

28. A nobilis, a stocked species not detected at all.

Answer: We have followed your comments. The failure to detect target species may be attributed to limitations in primer design, DNA degradation, as well as issues related to DNA extraction and preservation. This also reflects that eDNA technology has certain limitations

 

29. Line 247 – Order level?

Answer: We have followed your comments. We commonly use "order" as the taxonomic rank for proportional description in classification. Therefore, this study continues to employ the order-level category for hierarchical explanation.

 

30. Table 4 – sort by dominance like table 3?

Answer: We have followed your comments. We directly filled in the data based on the distribution in the checklist and calculated the numerical proportion of fish species.

 

31. Table 4 – Coila greyii repeated under different taxa, any others like this?

Answer: We have received your suggestions from the reviewer and carefully checked and verified them.

 

32. Table 5 – Fishing mortality came from which data source? Natural mortality from where, especially given the unnatural state of environment for those fish.

Answer: We have followed your comments. The length-converted catch curve method in FiSAT I yielded total mortality coefficients (Z) of 0.91 and 0.73 for H. molitrix and A. nobilis, respectively. Applying the mortality formul, total mortality rates were calculated as 59.75% and 51.81%. By incorporating the mean habitat water temperature T (24.7°C) from Xixi River basin sampling, along with parameters L∞, K, and T into Pauly's empirical equation, natural mortality coefficients (M) and fishing mortality coefficients (F) were derived for both species.

 

33. Lines 270 – 281 – keep mixing the ongoing annual surveys with this one research project, clearly explain how the fit together.

Answer: We have followed your comments. The core of our research is based on historical stock release data from 2017 to 2023 and current fishing catch data, aiming to evaluate the effectiveness of stock release—including population survival numbers, annual contribution rates, and economic output—analyze long-term trends in contribution rates and economic benefits, and determine whether the long-term outcomes of release strategies remain stable or show new changes, thereby providing a scientific basis for fisheries resource management. The integration of historical data and annual surveys forms a closed-loop management framework of "assessment–adjustment–reassessment." Based on the findings of this study (for example, the current estimated population ratio of silver carp to bighead carp is approximately 1.97:1), management authorities can formulate or adjust future stock release plans. Subsequent annual surveys will then be used to monitor whether these adjusted management measures yield the expected positive outcomes, thus enabling scientific and precise management of enhancement release activities.

 

34. Table 7 – how did these economic values get calculated? There must be estimates of fisher catch value? From where?

Answer: We have followed your comments. The recapture yield was estimated based on stocking quantity, mortality rate, and market price parameters. The calculation was performed cumulatively using: (estimated mortality rate × number of stocked individuals) + (residual population from the previous year × mortality rate). As shown in Table 7 and historical stocking data, the year with the highest stocking quantity yielded the highest estimated economic value, demonstrating a positive correlation between stocking quantity and economic output.

 

35. Lines 308 – 310 – skipping the 2nd most dominant species O mossambicus.

Answer: We have followed your comments，and it has been supplemented and modified.

 

36. Lines 310 -312 – why is there a “good” effect?

Answer: We have followed your comments. We believe that eDNA technology detects species that traditional methods cannot monitor, demonstrating its broad detection range and rapid response time. Therefore, we believe that eDNA technology is effective.

 

37. Line 313 – insert full stop between Carassius and Information.

Answer: We have followed your comments, and corrected it.

 

38. Line 314 – “auratus”?

Answer: We have followed your comments. We have corrected the spelling error that appeared here.

 

39. Lines 315 – 319 – discussion of non-native species seems out of place. Provide more explanation.

Answer: We have followed your comments. We included this section to demonstrate that eDNA technology can detect fish species missed by traditional fishing methods.  This comparison highlights the feasibility and applicability of eDNA-based monitoring. In addition, we have revised the unreasonable sentences.

 

40. Lines 325 – 334 – need a discussion about comparison of eDNA versus capture methodologies for practical purposes. Is there any reason to use eDNA? Not clear from this manuscript.

Answer: We have followed your comments. We have made partial revisions to this section. Traditional fish resource monitoring primarily relies on physical capture gear, which has significant limitations. During our sampling, we observed low operational efficiency, time-consuming and labor-intensive processes, and incomplete species coverage. The method shows insufficient capture efficiency for small-sized individuals, low-density populations, and rare species, all of which can easily lead to biased assessments of community structure. Furthermore, fishing gear can cause physical harm to fish.

In contrast, the eDNA method adopted in this study is more convenient, faster, highly sensitive, non-invasive, and time-efficient, thereby reducing sampling time and labor costs. We employed eDNA methodology to demonstrate its utility in detecting species composition and biomass. Future resource surveys could integrate both approaches to supplement data, enabling a more comprehensive and efficient assessment of biodiversity within the basin and providing a broader perspective on species composition in aquatic ecosystems.

 

41. Lines 360 – 265 – difference in survival? Both caught and eaten? A nobilis has higher survival, why?

Answer: We have followed your comments. We have supplemented some of this content, but the two are different, with fishing being more prevalent. The higher survival rate of A. nobilis compared to H. molitrix may be attributed to differences in stocking quantity, fishing pressure, and behavioral traits.  Based on our findings, H. molitrix were captured more frequently than A. nobilis. H. molitrix tend to be more active and easily startled, making them more vulnerable to capture. Additionally, their feeding habits primarily involve foraging on phytoplankton in upper water layers, where heightened photosynthetic activity promotes plankton growth, further increasing their exposure to fishing efforts.

 

42. Line 367 – A nobilis does not really have a “significant” population, not even detected by eDNA...

Answer: We have followed your comments. The catch volume of the A. nobilis is lower than that of other species, so it is not a dominant species. Possible reasons include its life habits and the impact of environmental changes. Most other fish species are mid-to-upper layer fish. The reasons why the common carp was not detected include limitations of the primers, DNA degradation, and issues with extraction and preservation, which also indirectly highlight the limitations of eDNA technology.

 

43. Line 368 – how is stocking “optimised”?

Answer: We have followed your comments. It has increased the overall fishery resources in the Xixi River basin, improved the population status, and played a role in adjusting the ecology between different fish species in the entire basin. Therefore, it has optimised the population structure in fishery waters and played a positive role in maintaining the stability of fishery resources and ecosystems in the discharge waters

 

44. Lines 371 – 372 – provide reference and context for social surveys.

Answer: We have followed your comments. We conducted a questionnaire survey and interviewed local residents to understand the public's views on stock enhancement and restocking, which we have also supplemented in this article.

 

45. Lines 376 – 379 – paragraph out of place here

Answer: We have followed your comments. We have taken your advice and deleted the inappropriate content.

 

46. Lines 383 – 387 – how is this demonstrated? What is the evidence for the enhancement, substantial ecological contributions, and tangible benefits for restoration?

Answer: We have followed your comments. The supplementation of resources has facilitated the regulation of population niches, leading to ecological improvements in the Xixi River basin. The feeding habits of H. molitrix and A. nobilis have contributed to the reduction of plankton biomass, thereby helping to prevent eutrophication. Thus, it is demonstrated that stock enhancement of these species has made tangible contributions to the remediation and ecological benefits of the Xixi River basin aquatic ecosystem.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Comments

This manuscript has improved over the original version and is found to be a solid contribution to the field.  The authors also note their response to most of the changes. Thank you! However, some attention should be given before accepting the manuscript. Addressing them will help polish the presentation and ensure a smoother reading experience.

1. Objectives should be more specific.
2. In objectives, delete growth parameters as there is a lack of growth parameters. Better use survival parameters instead of growth parameters.
3. Figure 3 caption is redundant. Change the caption to a precise and self-explanatory caption.

Author Response

Ms. Ref. No.: water-3810940

Title: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin

Reviewer #1:

1. This manuscript has improved over the original version and is found to be a solid contribution to the field. The authors also note their response to most of the changes. Thank you! However, some attention should be given before accepting the manuscript. Addressing them will help polish the presentation and ensure a smoother reading experience.

Answer: Thank you very much for reading our first revision and providing valuable comments and suggestions to help us improve the paper. We have made a second revision based on your comments.

 

2. Objectives should be more specific.

Answer: Thank you to the reviewers for their suggestions. We have detailed this part of the content.

 

3. In objectives, delete growth parameters as there is a lack of growth parameters. Better use survival parameters instead of growth parameters.

Answer: We appreciate the reviewer pointing out this issue, and we have made the necessary revisions.

 

4. Figure 3 caption is redundant. Change the caption to a precise and self-explanatory caption.

Answer: We greatly appreciate your suggestions. We have acknowledged the error in this section and revised the caption.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review version 2. While this version has improved, several of the major edits were not evident in the document despite the authors claiming to have made changes. The manuscript still requires many minor revisions in my opinion.

Lines 48-49 – I think you mean population is higher. The survival of H molitrix after restocking is lower than A nobilis as per previous sentence.

Lines 122-124 – need to rewrite this sentence, not clear what you are talking about here. Are you comparing eDNA with traditional netting/population? Explaining the value of eDNA in contrast to traditional capture methods?  This would be a great place to explain clearly what each provides – capture methods estimate biomass, passive eDNA collection provides presence absence information but not quantitative. How do they then fit together?

Lines 110-131 Still have not explained how eDNA and capture methods fit together. Your text suggests eDNA can replace capture methods (lines 124-127). But it cannot unless you can accurately measure populations/biomass with eDNA. Has anyone done this accurately? Are you aiming for this? If not, then need to simply state that eDNA provides a simple non-invasive way to gather additional information, but cannot measure biomass yet(?).

Line 132 – you would need to provide references and explanation of the “ecological importance”, which refers to broad biological value to the natural environment. Perhaps you just mean large and economically important in the region?

Line 135 – perhaps use different words from “ecological buffer zone”, what do you mean by those words?

Line 139 – need reference for decrease in fishery resources

Line 150 – delete “its”, use “their”

Line 152 – delete “it”, use “They”

Lines 154 – 156 – do these fish still have access to rapid water movement at the right time of year or not? Please state clearly.

Line 199 – Delete “Therefore”, start paragraph with “The main objective...”

Lines 202 – 206 – this text is good.  As stated earlier, just need to clarify earlier in the introduction about relative value of capture versus eDNA methods.

Line 218 – need to show stocking location on the map, also need to show boundaries of Westlake Ecological Park if you feel it is important to include. You could just show stocking location.

Section 2.1 – would be good to explain quickly if any rapid water movement is available in the stocked region and, if so, whether it was sampled for mature fish with developed/ripening gonads. This is important to support your comment about no reproduction taking place.

Secion 2.2 – need more explanation of historical stocking, whether samples were collected in the past or is this first time, purpose of sampling, date/time of sampling, frequency etc....  

Figure 1 map – the little square in the corner of the small China map has no purpose and unclear what it shows. The larger regional map has Zhanzhou city taking up the whole red area – is that correct? Please add lines to show the coastline around there – the blue lake at the bottom right of the river map – is that a lake or is it supposed to be the estuary? If estuary, then do not enclose in blue and connect with coastline.

Line 242 – barbed wire!? Really?

Line 258 – what is “parallel” sample?

Lines 257 – 264 – methods confusing – you mention 5l bottles but then 1l triplicates taken from surface and bottom? Are all these taken from the river, or perhaps the 1l samples were taken from the 5l samples?

Section 2.3 – total number of eDNA water samples? Were subsamples combined? Were replicates used to understand variation in results?

Section 2.3 – This section was not changed by authors. Still left all the eDNA details of methodology. Is there a reference for this methodology or do you have to provide all the details? Better to use reference, and briefly explain any varaiations.

Section 2.4 – there must be a basis for fishing mortality or natural mortality for those species – what are the references and then need discussion on how appropriate they are.

Line 325-326 – what do you mean by “diverse distribution”?

Lines – 325 – 326 – you presented Order/Family numbers for A and B but not C.

Table 1 – Red tilapia  Oreochromis – is that O sp., O spp or O mossambicus?

Table 1 – O mossambicus should be in this list, it is very abundant. Was it caught?

Lines 280 – 288 – authors claimed to change this section but it was not done. This section is confusing with too much detail. Please simplify on how eDNA was compared.

Figure 3 – Header is not clear, poor English. Rewrite.

Figure 3 – were you able to pick up similar seasonal changes in capture methodology? This might provide some amount of biomass comparison for some species

Table 4 – Authors did not make change, still not sorted by dominance like table 3

Lines 381 – 382 – this stocking information needs to be discussed in Introduction

Lines 390 – 391 – clarify what you mean by contribution rates – by restocking? Or survival from one year to the next?

Lines 391 – 392 – where did the average come from? Average of what?

Lines 393 – 397 – still no explanation of size and market price information for the value of the fish, what is the value you are calculating? Value to fishers? Value in markets? Artisanal value – how calculated?

Table 6 – confidence limits in wrong columns, mortality rates?

Results section – need to explain the results clearly and simply. You have plugged information into fisheries statistics – is that appropriate? Were your choices of parameters (e.g. M) correct and reflect that species in an altered environment? What do the results mean?

Discussion –What does the contribution rate indicate  about the stocking? These should be right at the beginning of the discussion.

Line 419 – strengths of eDNA were not clear in this study, results not directly comparable with capture, reliability not clear, cost factor not clear relative to capture methods,....

Line 425 – 429 – you have not proven that these results provide similar results to other studies. You are not able to provide any indication of biomass and your eDNA techniques did not pick up your stocked species. The best you can state is that your eDNA methodology for this river is developing, needs improvement for identification of species present, need to compare eDNA results with accurate biomass estimates to find a ratio for comparison and provide suggestions for improving the methodology.

Line 432 – “This demonstrates...”

Lines 488-489 –  What do you mean by “differential stocking proportions”? Suggest delete this whole sentence.

Lines 489 – 494 – Authors don’t need to address why one species is caught more often than the other....and the logic and comments are incorrect in these sentences. A nobilis has higher survival rate, or is more likely to get caught.   The key point is that one species (A nobilis) appears to have a higher than expected proportion in the population given the original stocking ratio. So, either A nobilis has a higher survival rate (which authors data suggest it does) or A nobilis is easier to catch in these studies (which the authors suggest is not the case). Perhaps, A nobilis is harder to catch by fishers?

Lines 502 – 503 – you need to provide proof that fishery resources have increased. You need a baseline before stocking, or at some time in the past, to compare with the current measurement of biomass.

Lines 504 -505 –  I suggest deleting “optimising”. The use of the word “optimising” suggests that you are changing the population for some optimal goal? What is that? How do you know it is optimal? Provide the proof.  I expect that the purpose of stocking those fish is presumably because people like to eat them. Maybe they are also good for cleaning up the river.

Lines 510 – 514 – need reference for the survey results.

Line 511 – delete “riparian”, use “local”

Lines 523 – 525 – you discussed this already above

Lines 525 – 527 – I cannot understand what is meant in this sentence

Lines 523-530 -to me this should have been discussed earlier on why these species are stocked in the Xixi.

Any benefits of the stocking program? You state there are but provide no proof. More fish for people? Cleaner water for consumption, swimming? Reduced algal blooms? 

Author Response

Ms. Ref. No.: water-3810940

Title: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin

Reviewer #2:

1. Thank you for the opportunity to review version 2. While this version has improved, several of the major edits were not evident in the document despite the authors claiming to have made changes. The manuscript still requires many minor revisions in my opinion.

Answer: Thank you very much for reading our first revision and providing valuable comments and suggestions, which have helped us improve the paper. We have made a second revision based on your comments.

 

2. Lines 48-49: I think you mean population is higher. The survival of H molitrix after restocking is lower than A nobilis as per previous sentence.

Answer: We have received your valuable comments, and we agree with your perspective.

 

3. Lines 122-124: Need to rewrite this sentence, not clear what you are talking about here. Are you comparing eDNA with traditional netting/population? Explaining the value of eDNA in contrast to traditional capture methods? This would be a great place to explain clearly what each provides – capture methods estimate biomass, passive eDNA collection provides presence absence information but not quantitative. How do they then fit together?

Answer: Thank you for your valuable questions and suggestions. We sincerely apologize for the lack of clarity and precision in our original expression. We have provided further clarification and explanation. This study employs eDNA as a simple, non-invasive method to gather additional information. eDNA samples were collected from release areas using the same sampling locations as fish catches. Relative quantitative analysis was performed on the number of sequences obtained via eDNA technology. Data from the eDNA method was used to supplement traditional analyses, comparing it with biomass from traditionally caught species to understand the distribution of dominant species. This approach was integrated with mortality rates and contribution rates derived from traditional analyses for comprehensive evaluation.

 

4. Lines 110-131: Still have not explained how eDNA and capture methods fit together. Your text suggests eDNA can replace capture methods (lines 124-127). But it cannot unless you can accurately measure populations/biomass with eDNA. Has anyone done this accurately? Are you aiming for this? If not, then need to simply state that eDNA provides a simple non-invasive way to gather additional information, but cannot measure biomass yet(?).

Answer: Thank you for your valuable questions and suggestions. We have incorporated your suggestions. We have provided a concise explanation that eDNA offers a simpler method for information collection, and have optimized it in light of the issues you previously highlighted.

 

5. Line 132: You would need to provide references and explanation of the “ecological importance”, which refers to broad biological value to the natural environment. Perhaps you just mean large and economically important in the region?

Answer: We have received your valuable comments. We fully agree your suggestions and have added supplementary explanations regarding its ecological significance. The Jiulong River is a vital ecological waterway in southern Fujian Province. Designated as a key mangrove biodiversity conservation area and a Grade III water conservation functional zone in the national ecological zoning plan for the southeast coastal region, it serves as a crucial ecological barrier along China's southeastern coast. Its complex estuarine environment provides spawning and nursery grounds for numerous fish species, playing an indispensable role in maintaining biodiversity. Simultaneously, it functions as a major fishing base for areas including Xiamen and Longhai, boasting abundant fish resources.

 

6. Line 135: Perhaps use different words from “ecological buffer zone”, what do you mean by those words?

Answer: Thank you for your valuable questions and suggestions. We agree with your comment and have revised the term “ecological buffer zone” by reintroducing the original expressions “fisheries resource reservoir and ecological barrier” in the context of the Xixi watershed. This adjustment was made based on our understanding that the Xixi area not only serves as a major drinking water source for Zhangzhou but also contributes to the restoration of fishery populations through long-term aquatic enhancement and release programs. These efforts help maintain biodiversity and improve the aquatic ecological environment. Furthermore, initiatives such as the construction of safe ecological water systems, small river management, and soil erosion control have established a robust ecological water system characterized by sufficient water volume, natural flow, and good water quality. Together, these measures form an effective ecological barrier and regulatory mechanism. Based on these comprehensive functions, we have chosen these terms to more accurately reflect the integral role of the Xixi watershed.

 

7. Line 139: Need reference for decrease in fishery resources

Answer: Thank you for your valuable questions and suggestions. This section does indeed require supplementation, and we have added additional content accordingly. Due to surrounding economic development, reservoir construction, hydropower projects, and overfishing, the river has undergone severe “lake-like transformation” and water quality deterioration. This has altered the integrity of the river ecosystem and its biodiversity, placing pressure on fishery resources. Some economically important species, such as Konosirus punctatus and Mugil cephalus, are already overexploited.

 

8. Line 150: Delete “its”, use “their”

Answer: Thank you for your careful review of the manuscript. We have made revisions in accordance with your suggestions.

 

9. Line 152: Delete “it”, use “They”

Answer: Thank you for your careful review of the manuscript. We have made revisions in accordance with your suggestions.

 

10. Lines 154 – 156: Do these fish still have access to rapid water movement at the right time of year or not? Please state clearly.

Answer: We have received your valuable comments. The construction of hydropower stations and flood control projects in the Xixi River basin has altered its original hydrological conditions, resulting in slower water flow. It is no longer allowed to enter waters with a faster flow rate. Consequently, during the breeding season, the fish cannot receive the necessary hydrological stimuli, leading to poor reproductive outcomes and low hatching rates.

 

11. Line 199: Delete “Therefore”, start paragraph with “The main objective...”

Answer: Thank you for your careful review of the manuscript. We have made revisions in accordance with your suggestions.

 

12. Lines 202 – 206: This text is good. As stated earlier, just need to clarify earlier in the introduction about relative value of capture versus eDNA methods.

Answer: Thank you for your agreement with this section. We have made further revisions to the preceding content.

 

13. Line 218: Need to show stocking location on the map, also need to show boundaries of Westlake Ecological Park if you feel it is important to include. You could just show stocking location.

Answer: We have received your valuable comments, and we have marked the coordinates on the figure.

 

14. Section 2.1: Would be good to explain quickly if any rapid water movement is available in the stocked region and, if so, whether it was sampled for mature fish with developed/ripening gonads. This is important to support your comment about no reproduction taking place.

Answer: We have received your valuable comments. We have explained this content. The water flow in this stocking area is gentle and cannot generate rapid currents, preventing effective stimulation of gonadal development. Consequently, no mature fish specimens with well-developed or mature gonads were collected.

 

15. Secion 2.2: Need more explanation of historical stocking, whether samples were collected in the past or is this first time, purpose of sampling, date/time of sampling, frequency etc....

Answer: We have received your valuable comments. We will provide an explanation for this section. The Xixi River basin has conducted continuous stocking and release operations since February 2017. This sampling represents the first sampling effort, primarily targeting fish species. Sampling commenced in October 2023, with intervals of two months between each sampling session. A total of seven sampling sessions were conducted, with the final sampling date occurring in October 2024.

 

16. Figure 1 map: The little square in the corner of the small China map has no purpose and unclear what it shows. The larger regional map has Zhanzhou city taking up the whole red area – is that correct? Please add lines to show the coastline around there – the blue lake at the bottom right of the river map – is that a lake or is it supposed to be the estuary? If estuary, then do not enclose in blue and connect with coastline.

Answer: Thank you for your valuable suggestions. In the small grid on the China map, we intended to indicate Zhangzhou's specific location within China's territory and show that this area borders the sea. On the larger regional map, we will use red lines to delineate the entire Zhangzhou area and depict the distribution of the Jiulong River and Xixi River within it. We acknowledge that the lower right corner of the river map was not handled well—this area is not a lake but an estuary. After flowing into the Jiulong River, the Xixi River flows into the ocean. We have made some modifications to this part of the content, adding the release site, Xihu Ecological Park, and adjusting the enclosed area. We have used a black line to mark the boundary of the land, which indicates the estuary. The area within the line is the inner sea, and the water of the Jiulong River flows into it from here.

 

17. Line 242: Barbed wire!? Really?

Answer: Thank you for your careful review of the manuscript. We have made revisions in accordance with your suggestions.

 

18. Line 258: What is “parallel” sample?

Answer: Thank you for your valuable questions. Three sampling points were selected in the Xixi River section in October 2023 and April 2024 (Figure 1). At each sampling station (A, B, and C), three parallel eDNA samples were collected using a water sampler to independently obtain three replicate water bottles at the same location and time. This replication was designed to evaluate the spatial heterogeneity of eDNA distribution in the environment, identify and exclude potential contamination outliers, enhance the reliability of the data, and improve the detection rates of target species.

 

19. Lines 257 – 264: Methods confusing – you mention 5l bottles but then 1l triplicates taken from surface and bottom? Are all these taken from the river, or perhaps the 1l samples were taken from the 5l samples?

Answer: We have received your valuable comments. We sincerely apologize for any misunderstanding caused by our unclear expression. For each sample, 5 L of surface water and 5 L of bottom water were collected, thoroughly mixed, and then subsampled to obtain a final volume of 1 L. This composite sample was stored in a pretreated 1 L wide-mouth bottle for subsequent processing.

 

20. Section 2.3: Total number of eDNA water samples? Were subsamples combined? Were replicates used to understand variation in results?

Answer: We have received your valuable comments. We did combine the three subsamples for the overall monthly analysis, and we also performed the analysis with replicates.

 

21. Section 2.3: This section was not changed by authors. Still left all the eDNA details of methodology. Is there a reference for this methodology or do you have to provide all the details? Better to use reference, and briefly explain any varaiations.

Answer: We have received your valuable comments. We had not fully grasped your point earlier, but we have now revised the content to make it appear more concise and reference water temperature literature was introduced. Amplification was performed using primers and methods adapted from Ji et al., with the reaction volume increased to 30μL. Subsequent data processing included quality control, assembly, and removal of chimeric sequences. Using the UPARSE algorithm, refer to Gayanilo et al., operational taxonomic units (OTUs) were clustered at a 97% similarity threshold. Non-fish sequences were excluded, and non-redundant sequences were extracted after removing singletons.

 

22. Section 2.4: There must be a basis for fishing mortality or natural mortality for those species – what are the references and then need discussion on how appropriate they are.

Answer: Thank you for your valuable questions and suggestions. In the revised manuscript, we have clarified the basis for estimating natural mortality (M) and fishing mortality (F). Specifically, we added the following information:

(1)       References for the mortality estimation methods

The natural mortality coefficient (M) and the total mortality coefficient (Z) were estimated using the formula proposed by Pauly [64]. This method was developed based on data from 175 populations of 84 fish species, incorporating parameters such as the asymptotic length L∞ (cm), the growth coefficient K, and the mean habitat water temperature T. Pauly's analysis revealed that M is negatively correlated with growth parameters but positively correlated with both mean annual water temperature and the growth coefficient. This approach has been widely applied in fisheries research.

The total mortality coefficient (Z) and fishing mortality (F) were estimated using the Length-Converted Catch Curve (LCCC) method in FiSAT II, with reference to the approach described by Gayanilo et al. [65]. 

(2)       Discussion of appropriateness

We have already discussed it, pauly's formula provides a baseline estimation under general environmental conditions, reflecting both the original intent of the model construction and the data availability at the time. Temperature, compared to other factors, is more stable, reliable, data-rich, and readily accessible, making it a suitable predictor variable. Furthermore, all physiological and biochemical processes in fish—including respiration, feeding, and reproduction—are dependent on ambient temperature. It directly and comprehensively drives metabolic rate, which serves as the primary intrinsic driver of natural mortality. Moreover, the area we studied belongs to the subtropical climate zone with stable water temperature. It is appropriate to incorporate water temperature into the M estimation.

However, we acknowledge that this approach provides an indirect estimation, and thus, the values may carry uncertainty. We have now added this limitation in the Discussion, suggesting that future studies may validate mortality parameters through age-based methods, long-term mark–recapture studies, or direct experimental approaches.

 

23. Line 325-326: What do you mean by “diverse distribution”?

Answer: We have received your valuable comments. We apologize for any confusion caused by our unclear description. “Diverse distribution” refers to the fact that station C collected the highest number of fish species among the three stations, indicating greater species diversity compared to the other stations.

 

24. Lines – 325 – 326: You presented Order/Family numbers for A and B but not C.

Answer: We have received your valuable comments. We have separately described the species composition at the family and order levels for the three sites in the text. tation A contained 24 species from 6 orders and 9 families; station B hosted 28 species across 6 orders and 12 families; station C supported 32 species representing 7 orders and 15 families.

 

25. Table 1: Red tilapia Oreochromis – is that O sp., O spp or O mossambicus?

Answer: Thank you for your valuable questions and suggestions. We acknowledge the shortcomings in species classification and will address them in future work. It should be O sp.

 

26. Table 1: O mossambicus should be in this list, it is very abundant. Was it caught?

Answer: We have received the question you raised. The species is highly abundant in eDNA detection but notably absent in actual catch results, presenting a significant issue. Our discussion concluded that this species was indeed captured. However, due to the high similarity among O spp. during the identification process, errors occurred in the identification work. Since no captured samples are currently available and photographs cannot provide definitive identification, we did not include supplementary notes on O. mossambicus. We fully recognize the significance of this issue and will strengthen our understanding of this aspect in future research.

 

27. Lines 280 – 288: Authors claimed to change this section but it was not done. This section is confusing with too much detail. Please simplify on how eDNA was compared.

Answer: We have received your valuable comments. We are very sorry and thank you for your valuable suggestions. Based on your suggestions (21), we have simplified the content of the eDNA analysis section.

 

28. Figure 3: Header is not clear, poor English. Rewrite.

Answer: Thank you for your valuable questions and comments. We have revised the title accordingly.

 

29. Figure 3: Were you able to pick up similar seasonal changes in capture methodology? This might provide some amount of biomass comparison for some species.

Answer: Thank you for your valuable questions and comments. Our discussions indicate that capture methods should indeed reflect seasonal variations. However, our research focus is not on documenting seasonal changes in these species, but rather on identifying dominant species. We have already conducted relevant analyses on dominant species in traditional fishing practices, so seasonal analysis is not required here. That said, we fully acknowledge your viewpoint and will consider incorporating this aspect into future studies to enrich our findings.

 

30.Table 4: Authors did not make change, still not sorted by dominance like table 3.

Answer: We have followed your comments. We apologize for the misunderstanding regarding your previous recommendations, which led to our failure to make the necessary adjustments. We have now reordered the data in descending numerical order, as shown in Table 3, to enhance clarity and readability.

 

31. Lines 381 – 382: This stocking information needs to be discussed in Introduction.

Answer: We have followed your comments, and corrected it.

 

32. Lines 390 – 391: Clarify what you mean by contribution rates – by restocking? Or survival from one year to the next?

Answer: We have followed your comments. The contribution rate we refer to is the survival rate from one year to the next. Based on the survival rates, we calculated the annual contribution rate to the H. molitrix to A. nobilis populations in the water body following each year's stocking.

 

33. Lines 391 – 392: Where did the average come from? Average of what?

Answer: We have followed your comments. The average value refers to the mean contribution rate since the release began in 2017, calculated by averaging the annual contribution rates.

 

34. Lines 393 – 397: Still no explanation of size and market price information for the value of the fish, what is the value you are calculating? Value to fishers? Value in markets? Artisanal value – how calculated?

Answer: We have followed your comments. We sincerely apologize for the lack of clarity in our previous explanations. After further discussion, we have refined our response.

2. molitrix and A. nobilis are common economic fish species in Fujian Province, where their market prices remain relatively low due to the abundance of marine fish species. According to our survey, the market prices of H. molitrix and A. nobilis are 2.5 CNY/kg and 4 CNY/kg, respectively—which are the values used in our calculations. In this study, the average body weight of H. molitrix was 464.01 g, and that of A. nobilis was 435.26 g.

The economic value was estimated by multiplying the predicted population size by the market price. The population size was derived based on mortality rates and stocking quantity, and then further multiplied by the average individual weight and market price. This calculation provided an estimated economic value of the populations.

 

35. Table 6: Confidence limits in wrong columns, mortality rates?

Answer: We have followed your comments and have revised this section accordingly. We acknowledge that the phrasing in the table was indeed inappropriate and have clarified it in the main text. The calculation refers to the confidence interval for the mortality rate.

 

36.Results section: Need to explain the results clearly and simply. You have plugged information into fisheries statistics – is that appropriate? Were your choices of parameters (e.g. M) correct and reflect that species in an altered environment? What do the results mean?

Answer: We have followed your comments. We fully agree with your recommendations. We have presented only the key numerical values in our results. The inclusion of information in fishery statistics serves to facilitate calculations, particularly within the FiSAT framework. The ELEFAN and length-based mortality estimation programs employed therein are effective and generally recommended methods for assessing fish stock status when complete age-at-catch data are unavailable. We acknowledge that calculations within these systems may be influenced by the balance between stocking activities and environmental variations. Nevertheless, they provide the best available quantitative framework for generating preliminary estimates of mortality and growth.

The parameters selected in this study were based on established methodologies from previous research, including official calculation formulas recommended by the FAO. Key parameters such as natural mortality, total mortality, and fishing mortality were estimated using empirical formulas from Pauly (1980) and Gayanilo et al. The adoption of these widely recognized classical formulas ensures the credibility of the parameter selections.

For instance, water temperature was used as a key variable for estimating natural mortality, as it is relatively stable and serves as a suitable predictive factor. Moreover, temperature significantly influences most physiological behaviors of fish, enabling it to reflect environmental variations during the study period.

While we acknowledge that these estimates may involve certain uncertainties, they represent a robust approach in the absence of direct estimation methods. In future research, we aim to explore and adopt methodologies that can more directly capture dynamic changes in relevant parameters.

 

37. Discussion: What does the contribution rate indicate about the stocking? These should be right at the beginning of the discussion.

Answer: We have followed your comments. We agree with your comments and have made corresponding adjustments to the content of the relevant sections in the manuscript. We wish to clarify the contribution rate of H. molitrix to A. nobilis.

 

38. Line 419: Strengths of eDNA were not clear in this study, results not directly comparable with capture, reliability not clear, cost factor not clear relative to capture methods, ....

Answer: We have followed your comments. We acknowledge that our explanation in this section was indeed inadequate. The comparison referenced here primarily stems from literature where authors contrasted eDNA technology with traditional survey methods. We fully agree with your points regarding capture methods and cost factors, which can vary significantly across different watersheds. Therefore, we primarily cited these references for a brief overview, noting that while eDNA methods may offer practical implementation advantages, we have not yet conducted comparative analyses of our specific research content.

 

39. Line 425 – 429: You have not proven that these results provide similar results to other studies. You are not able to provide any indication of biomass and your eDNA techniques did not pick up your stocked species. The best you can state is that your eDNA methodology for this river is developing, needs improvement for identification of species present, need to compare eDNA results with accurate biomass estimates to find a ratio for comparison and provide suggestions for improving the methodology.

Answer: Thank you for your valuable comments. We have made the revisions based on your recommendations. Integrating traditional methods with eDNA results reveals that Cypriniformes and certain species within Perciformes are the dominant fish groups in the Xixi River basin, particularly H. molitrix. However, the eDNA approach only detected the target species — H. molitrix, indicating unsatisfactory effectiveness. Thus, further refinement of eDNA methodologies tailored to the Xixi River basin is necessary to improve species detection, thereby enabling more accurate comparisons between eDNA results and biomass estimates, and providing recommendations for future methodological enhancements.

 

40. Line 432: “This demonstrates...”

Answer: We have followed your comments, and we have made the necessary revisions accordingly.

 

41. Lines 488-489: What do you mean by “differential stocking proportions”? Suggest delete this whole sentence.

Answer: We have followed your comments. And we have adopted and made changes to this section.

 

42. Lines 489 – 494: Authors don’t need to address why one species is caught more often than the other....and the logic and comments are incorrect in these sentences. A nobilis has higher survival rate, or is more likely to get caught. The key point is that one species (A nobilis) appears to have a higher than expected proportion in the population given the original stocking ratio. So, either A nobilis has a higher survival rate (which authors data suggest it does) or A nobilis is easier to catch in these studies (which the authors suggest is not the case). Perhaps, A nobilis is harder to catch by fishers?

Answer: Thank you for your valuable comments, which we have adopted and made changes to this section.

 

43. Lines 502 – 503: You need to provide proof that fishery resources have increased. You need a baseline before stocking, or at some time in the past, to compare with the current measurement of biomass.

Answer: Thank you for your valuable comments. Given the absence of prior biological surveys in the Xixi River basin and the lack of comparative data, our discussion focused on stocking and release efforts. We acknowledge that this approach may introduce bias, but indeed, no prior data exists for comparison. In future research, we will certainly prioritize conducting such surveys to enrich the biological data.

 

44. Lines 504 -505: I suggest deleting “optimising”. The use of the word “optimising” suggests that you are changing the population for some optimal goal? What is that? How do you know it is optimal? Provide the proof. I expect that the purpose of stocking those fish is presumably because people like to eat them. Maybe they are also good for cleaning up the river.

Answer: We have followed your comments and have adopted them. We have deleted and modified this section. "Optimization" is indeed not quite appropriate to be placed here. Because of the increase in the amount of resources, we adjusted it to play a positive role.

 

45. Lines 510 – 514: Need reference for the survey results.

Answer: Thank you for your valuable comments. We have partially revised this section and incorporated some references to the survey results. Through questionnaire surveys of local residents and fishermen, we analyzed public perceptions of the stock enhancement outcomes. The findings indicate that residents perceive improvements in water quality, heightened ecological conservation awareness, and increased social recognition of stock enhancement activities, yielding positive societal benefits.  Concurrently, these efforts have boosted fishermen's incomes, aligning with our findings that fishery resources have been partially replenished.

 

46. Line 511: Delete “riparian”, use “local”

Answer: Thank you for your valuable comments, which we have adopted and made changes to this section.

 

47. Lines 523 – 525：You discussed this already above

Answer: We have followed your comments, and we have adopted and made changes to this section.

 

48. Lines 525 – 527: I cannot understand what is meant in this sentence

Answer: We have followed your comments. This sentence does have ambiguity. After discussion, we thought it would be better to delete. What we originally intended to convey was to summarize the benefits brought by the release of H. molitrix and A. nobilis but it seemed unclear and confusing. Therefore, it was deleted in light of the previous text.

 

49. Lines 523-530: To me this should have been discussed earlier on why these species are stocked in the Xixi. Any benefits of the stocking program? You state there are but provide no proof. More fish for people? Cleaner water for consumption, swimming? Reduced algal blooms?

Answer: We have followed your comments. The reasons for releasing these species are explained in the Introduction. H. molitrix and A. nobilis are selected for release due to their fast growth rate, strong adaptability, high breeding yield and economic value, as well as their typical filter-feeding method. Releasing them has indeed increased fishermen's catch income and brought about certain economic benefits. The quantity of fishery resources in the river has been improved, and the water ecology has been enhanced. However, there are currently few systematic studies on this aspect, which cannot provide specific evidence. These improvements are only known from relevant official government documents and visits and investigations of residents along the river. There are indeed significant problems. Therefore, we have deleted this part of the content. In the subsequent investigation of fishery resources and water ecology, we will draw on the previous deficiencies and continuously optimize and improve, truly promoting the improvement of the local environment.

 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for providing the 3rd manuscript. Comments are below. I will not review it again.

Line 119  - “crucial ecological barrier” not used correctly. I’m really not sure what you are trying to say here. An ecological barrier is something that blocks the ecology from functioning properly, you are saying this ecological barrier is helpful?

• I think generally the authors are applying the term “ecological” in an incorrect manner.

Line 132-133 – “age-class fish” is not meaningful by itself because it refers generally to any size or age class. Need to add what age-class you are referring to.

Lines 131 – 133 – need a reference to support these overfished and fishery claims

Line 207 - Figure 1 – more information needed in header about what is in the figure, if the figure and header were viewed by themselves, could the reader understand what they were looking at?  Brief mention of location, analysis of fish population and years....

Figure 1 – the little box in the corner of the China map is confusing and out of place. The figures should flow from small box to large box, where the little box is the larger area showing the location of what is in the larger box.  The little box should show the location of the larger map of the Zhanzhou region. I suggest making the little red square a little smaller on the China map fit the region of the study and then delete the small box in the corner.

Figure 1 – I suggest showing the borders of the Xihu Ecological Park if it is large enough. The star suggests it is small and at a specific location

Figure 2 d – suggest leveling the picture so the horizon is horizontal

Section 2.3 – your methodology for water sampling is still confusing

• you use the word parallel incorrectly (simultaneous?),
• you then state you are getting 3 replicate samples but then state you are collecting 2 x 5L samples at each location?
• Step us through the water sampling clearly.

Lines 238-239 – for a total of how many 1 l samples? (18 x 1 l samples?)

Were 18 samples analysed for eDNA? Explain the replication clearly so we know how many eDNA samples were taken and how the replication was used. You used 2 membranes for each sample, were they both used (so 36 samples?)  Combined?

Section 2.4.1 - you apply Pauly methodology for M and Z, which is used for wild populations. And neither of your species is published in this paper. But is this really appropriate in such a modified artificial setting? Please clarify the appropriateness/justification of this in your methodology given the highly modified unnatural setting.  Or at least acknowledge that this is unknown.

Lines 331-333 – need to refer to table 3 here, not next sentence.

Lines 395-396 – provide date for market prices, provide reference for surveys

Lines 416 – 417 – survival rate is NOT higher for molitrix, it is higher for nobilis. Perhaps you mean population is higher for molitrix?

Author Response

Ms. Ref. No.: water-3810940

Title: Assessment of Stock Enhancement Efficacy for Hypophthalmichthys molitrix and Aristichthys nobilis in the Xixi of Jiulong River Basin

Reviewer:

1. Comments and Suggestions for Authors. Thank you for providing the 3rd manuscript. Comments are below. I will not review it again.

Answer: We sincerely thank you for reading our two revisions and providing valuable comments and suggestions that helped us improve the paper. We have made a third revision based on your comments.

 

2. Line 119: “crucial ecological barrier” not used correctly. I’m really not sure what you are trying to say here. An ecological barrier is something that blocks the ecology from functioning properly, you are saying this ecological barrier is helpful?

I think generally the authors are applying the term “ecological” in an incorrect manner.

Answer: Thank you for your comment. We agree with your point of view. This word can easily lead to various misunderstandings, so we have revised it. We modified it as follows:

“The Jiulong River is an important ecological waterway in southern Fujian Province. It has been designated by the state as a key mangrove biodiversity protection area and a Class III water conservation functional area on the southeast coast.”

 

3. Line 132-133: “age-class fish” is not meaningful by itself because it refers generally to any size or age class. Need to add what age-class you are referring to.

Answer: We appreciate your constructive feedback. The term "age-grade fish" is ambiguous and difficult to define accurately. We have revised it to "smaller fish."

We modified it as follows: “The catch is mainly smaller fish and juveniles, and the resource amount is declining.”

 

4. Lines 131 – 133: need a reference to support these overfished and fishery claims.

Answer: We have received your suggestion, and the evidence for these questions has been supplemented. Two references have also been added. This analysis is primarily based on relevant data and information published by the Fujian Provincial Oceanic and Fisheries Administration.

 

5. Line 207: Figure 1 – more information needed in header about what is in the figure, if the figure and header were viewed by themselves, could the reader understand what they were looking at?  Brief mention of location, analysis of fish population and years...

Answer: We have received your valuable comments. We have supplemented this section accordingly. We modified it as follows: “This map shows the sampling area and the Xixi River basin. Stations A, B, and C represent the three sampling sites. The star denotes the Xihu Ecological Park, which is the stocking site for H. molitrix and A. nobilis. The black shaded areas at the ends of the two rivers are the estuaries.”

 

6. Figure 1: the little box in the corner of the China map is confusing and out of place. The figures should flow from small box to large box, where the little box is the larger area showing the location of what is in the larger box.  The little box should show the location of the larger map of the Zhanzhou region. I suggest making the little red square a little smaller on the China map fit the region of the study and then delete the small box in the corner.

Answer: We have received your valuable suggestions. We have revised this section by reducing the size of the block area and removing the corner blocks.

 

7. Figure 1: I suggest showing the borders of the Xihu Ecological Park if it is large enough. The star suggests it is small and at a specific location.

Answer: We have received your comments. We fully agree with your perspective, but the park's area is not very large. Marking its entire boundary would appear inappropriate and create confusion regarding the boundaries. Therefore, we have only marked the location on the map.

 

8. Figure 2 d: suggest leveling the picture so the horizon is horizontal.

Answer: We have followed your comments, and revised it.

 

9. Section 2.3: your methodology for water sampling is still confusing.

• you use the word parallel incorrectly (simultaneous?),
• you then state you are getting 3 replicate samples but then state you are collecting 2 x 5L samples at each location?
• Step us through the water sampling clearly.

Answer: Thank you for your comments regarding Section 2.3. We apologize for the confusion caused by the earlier description. In our previous version, the mention of “5 L of surface water and 5 L of bottom water” was a writing error. The correct procedure was that, at each sampling point, 1 L of surface water and 1 L of bottom water were collected. At each station, three simultaneous sampling points were set approximately 1 m apart, resulting in a total of 6 L of water (including both surface and bottom layers). These were pooled, thoroughly mixed, and then 3 L of the composite sample was subsampled for eDNA analysis. We have revised the methodology section accordingly to clearly describe this step-by-step process.

 

10. Lines 238-239: for a total of how many 1 l samples? (18 x 1 l samples?).

Were 18 samples analysed for eDNA? Explain the replication clearly so we know how many eDNA samples were taken and how the replication was used. You used 2 membranes for each sample, were they both used (so 36 samples?) Combined?

Answer: Thank you for your comments. In the previous version, our description was not sufficiently clear and may have caused confusion regarding sample replication. Initially, we described the dataset as consisting of 18 composite water samples (three replicates per station × three stations × two months), but we acknowledge that this wording could be misleading. In the revised version, we have clarified the methodology by removing the reference to “18 samples” and instead specifying that at each station, 6 L of water (surface and bottom) was collected, 3 L was filtered, and three membranes were obtained and treated as biological replicates. We have revised the methodology section accordingly to ensure greater clarity.

 

11. Section 2.4.1: you apply Pauly methodology for M and Z, which is used for wild populations. And neither of your species is published in this paper. But is this really appropriate in such a modified artificial setting? Please clarify the appropriateness/justification of this in your methodology given the highly modified unnatural setting.  Or at least acknowledge that this is unknown.

Answer: We have received your comments. We have revised and adjusted this section once again, incorporating relevant additions into the article. Although the species studied were not published in the referenced papers, the Pauly methodology provided a basis for analyzing the natural mortality coefficient (M) and total mortality (A) of fish. The species have resided in this body of water since their release and have grown naturally without artificial intervention, making them suitable for analysis. Furthermore, the Pauly methodology is broadly applicable. It is suitable for the vast majority of temperate, subtropical, and tropical fisheries studies where water temperatures range from 5 to 30 °C. The Xixi waters are located in a subtropical region with an average measured temperature of 24.7°C, which makes this method appropriate. Environmental changes, such as the construction of hydroelectric power stations, have little impact on water temperature, which is primarily influenced by climatic and meteorological factors. It is important to note that this method provides indirect estimates and that these values may be uncertain.

 

12. Lines 331-333: need to refer to table 3 here, not next sentence.

Answer: We have followed your comments, and corrected it.

 

13. Lines 395-396: provide date for market prices, provide reference for surveys.

Answer: We have followed your comments, and corrected it. The date of our market price survey is October 2024.

 

14. Lines 416 – 417: survival rate is NOT higher for molitrix, it is higher for nobilis. Perhaps you mean population is higher for molitrix?

Answer: We have received your valuable comments. We intended to convey the same point as you mentioned—that the surviving population is higher. We have made the necessary revisions. The surviving population of H. molitrix is significantly higher than that of A. nobilis.