Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors should please check the attached for comments

Comments for author File: Comments.pdf

Comments on the Quality of English Language

English language is adequate 

Author Response

Dear reviewer,

We sincerely appreciate the time and effort you have dedicated to evaluating our manuscript. We found the comments to be extremely constructive and helpful in improving the quality of our work. We have carefully addressed each suggestion and revised the manuscript accordingly. We have carefully addressed each suggestion and revised the manuscript accordingly. All changes have been incorporated into the revised manuscript for your reference. Please find our point-by-point responses below. Please find our point-by-point responses below.

 

Comment 1

State the current status of the increase.

Response 1:

We appreciate the reviewer’s suggestion to clarify the background. We have revised the Introduction (Section 1, Lines 43-45) to explicitly state the current status of the rapid expansion in aquaculture and the resulting surge in aquafeed demand, citing the work of (Action, B, 2020).

 

Comment 2

such as ....mention some of the drawbacks.

Response 2:

We thank the reviewer for this constructive suggestion. In the revised manuscript, we have elaborated the limitations of plant protein sources, specifically mentioning drawbacks such as amino acid imbalances and the presence of anti-nutritional factors (Section 1, Lines 52-55).

 

Comment 3

Since the study is on replacement of FM, it is very crucial that the author examine the proximate composition of the QGM as well as anti-nutritional levels.

Response 3:

We sincerely thank the reviewer for this constructive suggestion regarding the characterization of the test ingredient. Regarding proximate composition: We would like to respectfully note that the detailed nutritional profile of the Quinoa Germ Meal (QGM) has been analyzed and is presented in Table 1. Regarding anti-nutritional levels: To provide a more comprehensive assessment as suggested, we have quantitatively analyzed the saponin content in the current study. The results indicated a saponin content of 0.19% in QGM, resulting in trace levels (0.02%–0.03%) in the formulated diets. These findings have been incorporated into Section 3.1, Lines 250-252 and are further discussed in Section 4.2, Lines 522-531 to substantiate the analysis of palatability and growth performance.

 

Comment 4

write the scientific name.

Response 4:

Thank the reviewer for this reminder. We have ensured that the full scientific name is correctly provided at the first mention in the text (Section 1, Line 98).

 

Comment 5

 You can use only common name it has earlier mention. Also scientific names must be italicize.

Response 5:

We appreciate the reviewer’s careful reading. We have corrected the formatting to ensure all scientific names are italicized (Scophthalmus maximus), and we have used the common name in subsequent mentions to maintain conciseness (Section 2.2, Line 139).

 

Comment 6

state the mean body weight and standard deviation.

Response 6:

We thank the reviewer for this constructive suggestion. We have revised the text to strictly include the mean body weight and standard deviation as suggested, providing a more precise description of the fish specifications (Section 2.2, Line 139).

 

Comment 7

Are water quality parameters herein within the recommended values for culturing turbot? Provide the reference.

Response 7:

We thank the reviewer for this constructive suggestion. We confirm that the water quality parameters were within the suitable range for turbot culture. As requested, we have added the relevant references (Boeuf, G. 1999) to support this statement in the Materials and Methods section (Section 2.2, Line 152).

 

Comment 8

what is does of MS222 used with reference?

Response 8:

We thank the reviewer for this constructive suggestion. We have clarified the specific dosage of MS-222 used for anesthesia in method and cited the corresponding reference (Topic Popovic, 2012) in the revised manuscript to ensure methodological reproducibility (Section 2.3, Line 158).

 

Comment 9

Subsection.

Response 9:

We thank the reviewer for this constructive suggestion. We have checked and corrected the formatting of the subsections to ensure they adhere to the journal's guidelines (Section 3.2, Line 254).

Reviewer 2 Report

Comments and Suggestions for Authors

Review Report
Manuscript Title: Effect of Quinoa Germ Meal as a Replacement for Fish Meal in Diets on Intestinal Health of Juvenile Turbot
Recommendation: Major Revision

General Assessment

The manuscript addresses a relevant and timely topic: the partial replacement of fish meal with quinoa germ meal (QGM) in juvenile turbot diets, integrating growth performance, intestinal histology, and transcriptomic analysis.

The experimental approach is comprehensive and technically appropriate. However, important methodological and interpretative limitations must be addressed before the manuscript can be considered for publication.

The main concerns focus on diet formulation design, significant reduction in growth performance from 10% inclusion onward, overinterpretation of transcriptomic results, and insufficient production-oriented discussion.

1.Materials and Methods (2.1) and Discussion – Diet Formulation

Although the diets are described as isonitrogenous and isolipidic, additional ingredients were simultaneously modified (wheat gluten, wheat flour, crystalline amino acids). This introduces multiple variables beyond QGM inclusion.

The authors should explicitly clarify in Materials and Methods (2.1) that the substitution required additional adjustments in the dietary matrix. In Discussion (4.2), they should include a specific paragraph acknowledging that the observed effects correspond to the reformulated dietary system and not exclusively to QGM. Direct causal attribution to QGM should be avoided unless this limitation is clearly stated.

2.Results (3.2), Discussion (4.2), and Abstract – Growth Reduction from 10% Inclusion

From 10% inclusion onward, FI, WGR, SGR, and CF significantly decreased. This is critical from a production perspective. The Abstract must be revised to explicitly state that no improvement in growth was observed, that productive performance decreased from 10% inclusion onward, and that practical applicability requires further optimization. The current statement describing QGM as a “feasible substitute” must be softened and aligned with the growth data.

3. Discussion (4.2) – Possible Palatability Effect

The reduction in FI from Q10 suggests a potential feed acceptance issue. The authors should include a specific paragraph discussing saponins, possible antinutritional factors, and changes in dietary matrix that may affect feed intake. They should also acknowledge that reduced feed intake may be the primary factor explaining the decline in growth performance.

4. Results (3.7) and Discussion (4.4) – Cellular Senescence: Moderation of Claims

The current conclusions state that cellular senescence was induced. The wording should be modified to indicate activation of senescence-related pathways or possible induction of related processes. Direct causal statements should be avoided, and the absence of functional validation must be acknowledged.

5. Discussion (4.4) – Interpretation of Ribosomal Genes

Ribosomal overexpression is interpreted as indicative of adequate nutritional status, which contradicts the observed reduction in growth. The authors should include alternative interpretations, such as a possible compensatory response or metabolic stress response, and avoid presenting ribosomal activation as evidence of improved nutritional status.

6. Materials and Methods (2.9) and Figures – Statistical Inconsistency

Tukey is mentioned in the methods, whereas Duncan is indicated in the figures 1. The authors should standardize the post hoc test used and correct either the figure legends or the methodological section accordingly.

The manuscript may be considered for publication if productive performance results and conclusions are clearly aligned, the limitation of diet formulation design is acknowledged, mechanistic interpretations are moderated, the statistical inconsistency is corrected, and the Abstract accurately reflects the productive results.

Author Response

Dear reviewer,

We sincerely appreciate the time and effort you have dedicated to evaluating our manuscript. We found the comments to be extremely constructive and helpful in improving the quality of our work. We have carefully addressed each suggestion and revised the manuscript accordingly. We have carefully addressed each suggestion and revised the manuscript accordingly. All changes have been incorporated into the revised manuscript for your reference. Please find our point-by-point responses below. Please find our point-by-point responses below.

 

Comment 1

Materials and Methods (2.1) and Discussion – Diet Formulation. Although the diets are described as isonitrogenous and isolipidic, additional ingredients were simultaneously modified (wheat gluten, wheat flour, crystalline amino acids). This introduces multiple variables beyond QGM inclusion. The authors should explicitly clarify in Materials and Methods (2.1) that the substitution required additional adjustments in the dietary matrix. In Discussion (4.2), they should include a specific paragraph acknowledging that the observed effects correspond to the reformulated dietary system and not exclusively to QGM.

Response 1:

We sincerely appreciate the reviewer’s rigorous attention to the formulation details. We strictly followed the standard practice in aquaculture nutrition to maintain isonitrogenous and isolipidic conditions across all groups, which necessitated the adjustment of "filler" ingredients (wheat gluten/flour) and the supplementation of crystalline amino acids to prevent nutritional imbalances. Following the reviewer's valuable suggestion to increase transparency, we have revised the manuscript: In Section 2.1 Lines 109-111, we explicitly clarified that wheat gluten, wheat flour, and oil were adjusted to maintain nutrient profiles. In Section 4.2, Lines 514-518, we added a paragraph discussing the "holistic diet effect," acknowledging that while the QGM substitution is the primary driver, the observed physiological changes occur within the context of the reformulated dietary matrix.

 

Comment 2

Results (3.2), Discussion (4.2), and Abstract – Growth Reduction from 10% Inclusion. From 10% inclusion onward, FI, WGR, SGR, and CF significantly decreased. The Abstract must be revised to explicitly state that no improvement in growth was observed, that productive performance decreased from 10% inclusion onward, and that practical applicability requires further optimization. The current statement describing QGM as a “feasible substitute” must be softened.

Response 2:

We thank the reviewer for this constructive suggestion. We acknowledge that the significant reduction in growth performance starting from the 10% inclusion level is a critical finding. We have revised the Abstract and Section 4.2 to reflect this accurately. We removed the unqualified term "feasible substitute." Instead, we now explicitly state that productive performance decreased from 10% inclusion onward and describe QGM as a "potential alternative source that requires optimization" for practical application at higher inclusion levels.

 

Comment 3

Discussion (4.2) – Possible Palatability Effect. The reduction in FI from Q10 suggests a potential feed acceptance issue. The authors should include a specific paragraph discussing saponins, possible antinutritional factors, and changes in dietary matrix that may affect feed intake. They should also acknowledge that reduced feed intake may be the primary factor explaining the decline in growth performance.

Response 3:

We appreciate the reviewer’s insight regarding the link between feed intake and growth performance. We have added a dedicated paragraph in Section 4.2, lines 521-530 to address the feed acceptance issue. Specifically, we elaborated on the negative impact of saponins and anti-nutritional factors on palatability, citing relevant literature (e.g., Suarez-Estrella et al.). Furthermore, we explicitly acknowledged that the reduced feed intake is likely the primary driver for the observed growth depression in the QGM-fed groups.

 

Comment 4

Results (3.7) and Discussion (4.4) – Cellular Senescence: Moderation of Claims. The wording should be modified to indicate activation of senescence-related pathways or possible induction of related processes. Direct causal statements should be avoided, and the absence of functional validation must be acknowledged.

Response 4:

We thank the reviewer for this constructive suggestion. We agree that our claims should be strictly aligned with the transcriptomic nature of the evidence. We have carefully modified the wording in Section 3.7, Lines 480-482 and Section 4.4, Lines651-654. We removed definitive terms such as "induced" or "accelerated." Instead, we adopted more precise descriptions indicating the "activation of senescence-related pathways" or "potential regulation of related processes." We also added a statement in the Discussion to explicitly acknowledge the study's limitations regarding functional validation.

 

Comment 5

Discussion (4.4) – Interpretation of Ribosomal Genes. Ribosomal overexpression is interpreted as indicative of adequate nutritional status, which contradicts the observed reduction in growth. The authors should include alternative interpretations, such as a possible compensatory response or metabolic stress response.

Response 5:

We thank the reviewer for this astute observation. We agree that our previous interpretation linking ribosomal upregulation to adequate nutrition was inconsistent with the phenotypic growth data. We have substantially revised the relevant discussion in Section 4.4 Lines 572-579. We removed the claim that upregulation indicates improved nutritional status and instead reinterpreted this phenomenon as a compensatory metabolic response. The revised text now clarifies that the organism likely upregulates protein synthesis machinery in an attempt to maintain homeostasis and counteract the nutritional stress induced by high QGM inclusion.

 

Comment 6

Materials and Methods (2.9) and Figures – Statistical Inconsistency. Tukey is mentioned in the methods, whereas Duncan is indicated in the figures 1. The authors should standardize the post hoc test used.

Response 6:

We apologize for the oversight regarding the description of the statistical method. We confirm that Duncan’s multiple range test was the method used for the analysis presented in the figures. Accordingly, we have corrected Section 2.9, Line 230 to replace "Tukey’s test" with "Duncan’s multiple range test," ensuring strict consistency between the methodology section and the figure legends throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The present manuscript is a well written manuscript. Presents the use of quinoa as an alternative to fish meal protein source in juvenile turbot nutrition. The methodology is clear with some minor amendments which are included below. The results are presented well but I would suggest replacing figures 1,2 and 4 with tables including the ± SD and the actual p-values. Some extra attention should be paid to figures quality.

More specifically:

Line 132 :”change to italics “Scophthalmus maximus”

In the methodology section the amino acid profile analysis is missing

Paragraph 3.2 change the title to “Growth performance” or something similar. Additionally, please include the p-values especially when referring to significant differences.

Figure 5 and figure 9: please improve the quality and the size of the fonts in the figures to readable

Line 493 why “in contrast”?? I would suggest replacing with “similarly” since it follows the same pattern as methionine concentration.

Section 4.1 is like a summary of the results. No critical thoughts or discussion.

Sections 4.2 and 4.3 need better interpretation and discussion of the results.

Author Response

Dear reviewer,

We sincerely appreciate the time and effort you have dedicated to evaluating our manuscript. We found the comments to be extremely constructive and helpful in improving the quality of our work. We have carefully addressed each suggestion and revised the manuscript accordingly. We have carefully addressed each suggestion and revised the manuscript accordingly. All changes have been incorporated into the revised manuscript for your reference. Please find our point-by-point responses below. Please find our point-by-point responses below.

 

Comment 1

 Line 132 :”change to italics “Scophthalmus maximus”

Response 1:

We sincerely appreciate the reviewer’s attention to detail. To avoid redundancy and ensure the manuscript is concise, we have removed the repetition of the scientific name (Scophthalmus maximus) in Line 113, as it was already fully defined earlier in the text.

 

Comment 2

In the methodology section the amino acid profile analysis is missing.

Response 2:

We apologize the description of the methodology was not sufficiently clear. We would like to respectfully note that the protocol for the amino acid profile analysis was included in the original manuscript and can be found in Section 2.5, Lines 176-178. We have re-examined this section to ensure it is accurate and easy to locate.

 

Comment 3

 Paragraph 3.2 change the title to “Growth performance” or something similar. Additionally, please include the p-values especially when referring to significant differences.

Response 3:

We agree with the reviewer’s constructive suggestion. We have revised the title of Section 3.2 to "Growth performance" to better reflect the content. Regarding the P-values, to present the statistical results more comprehensively and clearly, we have compiled the specific P-values and detailed comparisons into a new Supplementary Table 3-5. We believe this approach improves the readability of the main text while providing full statistical transparency.

 

Comment 4

 Figure 5 and figure 9: please improve the quality and the size of the fonts in the figures to readable.

Response 4:

We appreciate the reviewer’s suggestion to improve the figure quality. Accordingly, we have redrawn Figures 5 and 9 and converted them into vector graphics format to ensure maximum clarity and resolution to make all labels and data points easily readable in the revised manuscript.

 

Comment 5

 Line 493 why “in contrast”?? I would suggest replacing with “similarly” since it follows the same pattern as methionine concentration.

Response 5:

We thank the reviewer for this logical correction. We have replaced the phrase "in contrast" with "similarly" in Line 505 to accurately describe the results.

 

Comment 6

Section 4.1 is like a summary of the results. No critical thoughts or discussion.

Response 6:

We thank the reviewer for this constructive suggestion. We have revised Section 4.1 to include more critical discussion. While retaining the original data comparisons, we added interpretations regarding the nutritional implications of specific amino acids. Specifically, we analyzed how the deficiencies in lysine and threonine likely act as limiting factors for protein synthesis, thereby explaining why QGM serves better as a partial rather than a complete replacement (Section 4.1, Lines 507-510).

 

Comment 7

Sections 4.2 and 4.3 need better interpretation and discussion of the results.

Response 7:

We sincerely appreciate this constructive suggestion. We have extensively rewritten these sections to provide a more critical and mechanism-based discussion. Specifically, we expanded the analysis to link the observed growth retardation directly to the physiological effects of anti-nutritional factors (e.g., saponins) and amino acid imbalances (Section 4.2, Lines 521-529). Furthermore, we reinterpreted the physiological and morphological changes not merely as fluctuations in data, but as compensatory metabolic responses to nutritional stress (Section 4.3, Lines 558-561). These revisions ensure that the discussion now logically explains the biological mechanisms underlying the observed results.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for their careful and detailed responses to all the comments raised during the review process. The revisions have significantly improved the clarity, rigor, and balance of the manuscript.

The clarification regarding diet reformulation enhances methodological transparency. The abstract and discussion now more accurately reflect the growth performance results and appropriately moderate the claims about practical applicability. The additional discussion on palatability, antinutritional factors, and feed intake strengthens the biological interpretation of the observed growth reduction. I also appreciate the moderation of the transcriptomic interpretations and the acknowledgment of the limitations regarding functional validation. The reinterpretation of ribosomal gene expression as a potential compensatory response is more consistent with the phenotypic data.

Overall, the manuscript has improved substantially, and the authors have satisfactorily addressed the concerns raised.

Author Response

Dear reviewer, we sincerely appreciate the time and effort you have dedicated to evaluating our manuscript.