Influence of the Silkworm-Derived (Bombyx mori) Functional Substance (Silkrose-BM) on the Fish Meat Quality of Yellowtail (Seriola quinqueradiata)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This research evaluated the effects of silkrose, a silkworm-derived bioactive polysaccharide, on whole-body composition, collagen content and meat quality parameters of large yellowtail. This research presents innovative and relevant information regarding flesh quality intended for sushi and sashimi. The results and discussion sections are well-described and organized and need minor consideration. However, the abstract, introduction, material and methods section need further consideration. Furthermore, the abstract contemplates 33% of words to justify this research and should be more balanced, including data on experimental design, diet composition (energy, lipids and protein contents), and feed allowance. Additionally, it should be more precise in presenting details of genes and meat quality parameters affected by silkworm-derived compounds. The key role of a sustainable compound from silkworms regarding reduced wild fish caught to produce fish meal has not been considered in this manuscript's introduction. One of the biggest concerns of this manuscript regards the lack of details on experimental design, number of replicates and experimental unit used. Furthermore, this manuscript has not considered detailed information on sample collection and fish processing regarding trained personnel for filleting, equipments and methods. In the material and methods section, there is a need for more detailed information on water quality parameters, including dissolved oxygen and salinity parameters. Data on diet composition need to be presented, including main ingredients, energy, protein, crude lipids and ahs, which are the basic chemical composition of aquafeeds. Additionally, data on primers used and general methods presentations should be revisited, and the resumed methodology should be described once referenced. Although not considered for statistical analysis as only two cages were used for each experimental group, descriptive information on total feed intake and feed conversion ratio would be of great interest to the readers, considering the high importance of this fish species. Figure 2 should be presented as table to present more detailed information of data (mean, SP and P-value). 
Figure 2, which presents body composition data, is imprecise and would be more effectively presented as a table. The statistical analysis of total collagen content should be revisited, as the reported mean ± SD suggests a significant effect. These revisions should be carefully addressed in the second review round to improve the clarity and scientific rigor of the manuscript. Statistical analysis of gene expression should be detailed as normalized on non-normalized data. Authors should revisit the collagen content data (Figure 3), as mean ± SD implicates possible statistical effects. For this, data on homogeneity and normalized information in material and methods, mainly statistical analysis, will be described and used. 

 

Author Response

Thank you very much for taking the time to review this manuscript. We would like to express our appreciation to the reviewers for their constructive comments, which have helped us significantly improve the manuscript.

 

Comment 1. However, the abstract, introduction, material and methods section need further consideration. Furthermore, the abstract contemplates 33% of words to justify this research and should be more balanced, including data on experimental design, diet composition (energy, lipids and protein contents), and feed allowance. Additionally, it should be more precise in presenting details of genes and meat quality parameters affected by silkworm-derived compounds.

Answer 1: We greatly appreciate your suggestion regarding the abstract. We have revised the abstract section to give more clarity and comprehensive information for the reader.

Comment 2. The key role of a sustainable compound from silkworms regarding reduced wild fish caught to produce fish meal has not been considered in this manuscript's introduction.

Answer2: The utilization of insects as fish meal replacement to reduce wild fish caught is indeed a remarkable topic in recent years. Silkworm is one of the popular insects that can be used as fish meal replacement due to its rapid grow, easy to culture, and high nutritional profile. But, in the current study, we utilize a very small dose of bioactive substance from Silkworm (silkrose-BM; 0.1%) into feed, which is the nutritional influence of silkrose-BM in food composition was considered negligible. Thus, this is the reason why we did not include fish meal replacement in the manuscript`s introduction and focus on the beneficial compound from insects

 

Comment 3. One of the biggest concerns of this manuscript regards the lack of details on experimental design, number of replicates and experimental unit used. Furthermore, this manuscript has not considered detailed information on sample collection and fish processing regarding trained personnel for filleting, equipments and methods.

Answer3: The current experiment was carried out in the aquaculture fish cages that are actually used by fishermen for production to represent the real field condition in the aquaculture industry. In other words, the fish used in the experiments are farming products. Tank/cage replications are indeed an important factor to obtain the reliability of the data collection as pointed out by the reviewer, however, since the risk of failure is high when using novel substances, and large-scale trials require a budget of over $1 million per one trial, which is financially impossible as a practical matter. We believe that demonstration trials using actual fish pens are the most important for the widespread use of additives.

 

We now added the number of sampled fish and experimental unit in the Materials and Methods section as the reviewer mentioned. We also revised the Materials and Methods section to give more comprehensive information regarding experimental design, statistical analysis, feed composition, and meat quality analysis.

 

Comment 4. In the material and methods section, there is a need for more detailed information on water quality parameters, including dissolved oxygen and salinity parameters.

Answer4: We now include the overall water quality parameter in the manuscript that can be seen in Table 2.

 

Comment 5. Data on diet composition need to be presented, including main ingredients, energy, protein, crude lipids and ash, which are the basic chemical composition of aquafeeds.

Answer5: General feed composition and the proximate profile of diets are added to the Materials and Methods section in Table 1, as requested by the reviewer.

 

Comment 6. Additionally, data on primers used and general methods presentations should be revisited, and the resumed methodology should be described once referenced.

Answer6: Data for primer`s sequence are available in supplementary file Table S1. In addition, we have revised the Materials and Methods section to give more comprehensive information regarding experimental design, statistical analysis, feed composition, and meat quality analysis.

 

Comment 7. Although not considered for statistical analysis as only two cages were used for each experimental group, descriptive information on total feed intake and feed conversion ratio would be of great interest to the readers, considering the high importance of this fish species.

Answer7: Total feed intake is now presented in Table 1.

 

Comment 8. Figure 2 should be presented as table to present more detailed information of data (mean, SP and P-value). Figure 2, which presents body composition data, is imprecise and would be more effectively presented as a table.

Answer8: We have changed the format of Figure 2 into table format (Table 3) and provided detailed information including mean, SE, and P-value to give more clarity to the reader.

 

Comment 9. The statistical analysis of total collagen content should be revisited, as the reported mean ± SD suggests a significant effect.

Answe9r: We agreed with your comments about the collagen data, so we have re-checked all data and processed it again as we have attached in the supplementary additional data file (especially for collagen S10) using Mann-Whitney U Test to compare two different diet groups and we did not found a significant difference either.

 

Comment 10. Statistical analysis of gene expression should be detailed as normalized on non-normalized data.

Answer10: We have done so.

 

Comment 11. Authors should revisit the collagen content data (Figure 3), as mean ± SD implicates possible statistical effects. For this, data on homogeneity and normalized information in material and methods, mainly statistical analysis, will be described and used.

Answer11: We agreed with your comments about the collagen data, so we have re-checked all data and processed it again as we have attached in the supplementary additional data file (especially for collagen S10) using Mann-Whitney U Test to compare two different diet groups and we did not find a significant difference either.

 

Additional clarifications

We found a technical error in writing the number of samples per group where we previously wrote n =10 which means there are 10 fish analyzed in each group. We would like to clarify this. n = 10 is the total of both groups, so we revised it to n = 5 to avoid misunderstanding in the interpretation of the statistical analysis data we have obtained. I hope this oversight can be forgiven. We would also like to confirm regarding the analysis of the color change data using two-way ANOVA because this data meets the requirements where the two factors in question are storage time and different treatments, and in each individual fish, the color change data was taken for three replicates so this meets the requirements for statistical testing.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an interesting investigation into the effects of silkrose-BM, a bioactive polysaccharide derived from silkworms, on the quality of yellowtail (Seriola quinqueradiata) fish meat. The results, including improvements in color retention, collagen content, drip loss, and histological structure, are valuable contributions to the aquaculture field, particularly concerning the enhancement of fish meat quality through dietary additives. However, some areas in the study require further clarification and additional data to strengthen the conclusions.

 

Major Comments:

 

1. The manuscript would benefit from a more detailed explanation of the mechanisms by which silkrose-BM might influence fish meat quality at the molecular level. While the study focuses on antioxidant properties and collagen enhancement, a deeper discussion on the molecular pathways related to lipid oxidation, collagen formation, and protein denaturation in fish meat would be helpful.

 

2. The experimental setup is clear and well-organized. However, the justification for the selection of the silkrose-BM dosage (0.1%) should be clarified. More details on previous studies supporting this specific dosage or potential dose-response relationships would be beneficial.

 

3. While no significant differences were found in proximate composition between the treatment groups, further analysis on the fat content would be valuable, given its relevance to the texture and overall sensory characteristics of fish meat.

 

4. The study’s findings on slower discoloration in the silkrose-BM treatment group are compelling. However, the data would be more robust if the color measurement was also linked to sensory analysis, such as taste tests, to correlate color preservation with perceived quality.

 

5. The findings of increased collagen content and reduced drip loss in the silkrose-BM group are notable. However, statistical significance was not achieved for collagen content. It would be helpful to include more detailed statistical analysis or explore the potential biological mechanisms behind the observed trend. Additionally, a discussion of how collagen influences the meat texture and its potential impact on consumer preference would provide a more comprehensive understanding of the results.

 

6. The gene expression findings align with the hypothesis that silkrose-BM enhances muscle quality by reducing protein degradation and lipid metabolism. A clearer link between gene expression changes and the sensory attributes of fish meat (such as texture, juiciness, and flavor) would strengthen the interpretation of the results.

 

7. The discussion provides good context for the results, especially in terms of lipid oxidation and collagen formation. However, it would be useful to compare the findings with other studies on similar insect-derived bioactive substances in aquaculture to position the contribution of silkrose-BM in the broader field.

Author Response

Thank you very much for taking the time to review this manuscript. We would like to express our appreciation to the reviewers for their constructive comments, which have helped us significantly improve the manuscript.

 

Comment1: The manuscript would benefit from a more detailed explanation of the mechanisms by which silkrose-BM might influence fish meat quality at the molecular level. While the study focuses on antioxidant properties and collagen enhancement, a deeper discussion on the molecular pathways related to lipid oxidation, collagen formation, and protein denaturation in fish meat would be helpful.

Answer1 : We agree with your comments. We realized that this study still needed further analysis to clarify the molecular pathway of silkrose-BM effects on fish meat quality but in this research we have a limitation to talk about the comprehensive molecular pathway or underlying mechanism, remembering we did not conduct whole transcriptomics analysis (RNA-sequencing) before, so we considering to explain more detail about the molecular pathway after we do the RNA-seq in our further analysis.

 

Comment2: The experimental setup is clear and well-organized. However, the justification for the selection of the silkrose-BM dosage (0.1%) should be clarified. More details on previous studies supporting this specific dosage or potential dose-response relationships would be beneficial.

Answer2: The concentration of silkrose-BM (0.1%) was tested at the most effective concentration with reference to our previous papers (Ali et al., 2018[1]; Miura et al., 2022). Dietary silkrose-BM at concentration of 0.1% effectively prevents vibriosis in penaeid prawns (Litopenaeus vannamei and Marsupenaeus japonicus) and significantly reduced skin parasitism in yellowtail and white trevally (Pseudocaranx dentex). Beside that, silkrose-BM also significantly reduced cortisol level and altered expression of various genes, including those involved in immunity, stress response, wound healing, and heat responses, in yellowtail, white trevally and Japanese medaka (Oryzias latipes) (Ali et al., 2018; Ali et al., 2021; Miura et al., 2022). Based on our previous results, we decided to use concentration of silkrose-BM at 0.1% in the current study.

To give clarity for the reader, we added the above information to the manuscript.

 

Comment3: While no significant differences were found in proximate composition between the treatment groups, further analysis on the fat content would be valuable, given its relevance to the texture and overall sensory characteristics of fish meat.

Answer3: Further fat content analysis is indeed an important factor to consider when analyzing meat quality (texture, smell, taste, other sensory characteristics), especially for fish that mainly distributed as sushi and sashimi such yellowtail. This consideration will be our focus on our future study.

 

Comment4: The study’s findings on slower discoloration in the silkrose-BM treatment group are compelling. However, the data would be more robust if the color measurement was also linked to sensory analysis, such as taste tests, to correlate color preservation with perceived quality.

Answer4: Thank you for pointing this out. We agreed with your comments about sensory analysis, but given that this study was conducted, it will not possible to do it under exactly the same conditions, so we are considering this analysis in the future by looking at the correlation between visible sensory signs and whole gene expression (RNA-seq) in fish meat between treatment groups.

 

Comment5:  The findings of increased collagen content and reduced drip loss in the silkrose-BM group are notable. However, statistical significance was not achieved for collagen content. It would be helpful to include more detailed statistical analysis or explore the potential biological mechanisms behind the observed trend. Additionally, a discussion of how collagen influences the meat texture and its potential impact on consumer preference would provide a more comprehensive understanding of the results.

Answer5: We agreed with your comments about the collagen data, so we have re-checked all data and processed it again as we have attached in the supplementary additional data file (especially for collagen S10) using Mann-Whitney U Test to compare two different diet groups and we did not find a significant difference either. Further analysis on this regards will be our main concern in the future study.

In addition, we have revised the Materials and Methods section to give more comprehensive information regarding experimental design, statistical analysis, feed composition, and meat quality analysis.

 

Comment6:  The gene expression findings align with the hypothesis that silkrose-BM enhances muscle quality by reducing protein degradation and lipid metabolism. A clearer link between gene expression changes and the sensory attributes of fish meat (such as texture, juiciness, and flavor) would strengthen the interpretation of the results.

Answer6: In the current study, we used eight genes representing functional categories on the fish meat quality. However, only two genes (acadm and fbxo32) were significantly affected by dietary silkrose-BM. It is noteworthy to understand that silkrose-BM is a novel substances, and the information regarding its influence on the fish meat quality is still limited to date, especially in farmed fish. Therefore, we suggest to conduct more comprehensive gene expression analysis, such as RNA-sequencing to get a better understand and clearer link between gene expression changes and the sensory attributes of fish meat. Thus, in the future study, we will conduct whole transcriptomic analysis to answer the linkage.

 

Comment7: The discussion provides good context for the results, especially in terms of lipid oxidation and collagen formation. However, it would be useful to compare the findings with other studies on similar insect-derived bioactive substances in aquaculture to position the contribution of silkrose-BM in the broader field.

Answer7:  Thank you for the reviewer for pointing this out. Currently, we are conducting study on similar insect-derived bioactive substances from several insects that have a promising capability to alter various responses in fish and mammal, including lipid oxidation and collagen formation. We hope that in the future publication, we can share this promising results.