Effects of Capsaicin on the Growth, Development, and Nutritional Profiles of Black Soldier Flies Fed a High-Fat Diet

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

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

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Must be improved

Improved.

Are the results clearly presented?

Must be improved

Improved.

Are the conclusions supported by the results?

Must be improved

Improved.

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

Comments 1: Although statistical analysis and P-value calculations are mentioned, the manuscript does not include any P-values. P-values must be reported, even when results are not statistically significant. It is difficult to interpret results based solely on numerical values without indicating whether differences are statistically significant. Therefore, I strongly recommend including P-values in the Results section and revising the manuscript accordingly.

Response 1: Thank you very much for this comment. All P-values were added.

Comments 2: Ensure that table and figure titles are self-explanatory and understandable without referring back to the main text. In other words, the titles and captions should include sufficient information about the experiment and context of the study.

Include both scientific names and common names of the organism at first mention at the title.

Response 2: All titles corrected. Thank you.

Comments 3: If tables present mean values, please indicate the sample size (n = ?) and P-values in a footnote below each table. Also, mention have used SE or SD.

Response 3: All changed. Thank you for pointing this out.

Comments 4: Make sure that all figures and tables clearly describe the experimental conditions, treatments, and units of measurement where applicable.

Response 4: All changed. Thank you.

Comments 5: Any abbreviations used in figures and tables should be clearly explained in a footnote below the respective figure or table.

Response 5: Abbreviations (CK and HF) were explained in each figure and table.

Comments 6: Data should be presented only once (either in tables or figures), as duplication is considered redundant.

Response 6: Corrected. We deleted some figures. Thank you for pointing this out.

Comments 7: The discussion and explanation of tables and figures always appear before their presentation in the manuscript.

Response 7: All relevant contents have been changed. Thank you for your suggestion.

Comments 8: Figure 2, 3, &4: the information presented in these figures already shown once in table 1. So, these figures are redundant and should be removed. Only Figure 5. Show new data but missing P-value.

Response 8: Already changed according to previous comments. Thank you.

Comments 9: Line 40: Since the focus is on the tolerance of BSF to spicy food, please switch the order of the sentence to “spicy substrate and high-fat…”.

Response 9: Changed. Thank you.

Comments 10: Line 58: The word “however” is unnecessary here. Please consider using “meanwhile” or “in contrast” instead.

Response 10: Changed. Thank you.

Comments 11: Line 62: Scientific names should be italicised, e.g., Hermetia illucens.

Response 11: All reviewers mentioned this. This is indeed a negligence. Thank you.

Comments 12: Lines 62–63: A reference is required.

Response 12: We believe that the background knowledge about BSF can be provided in the reference at the end of the next sentence. Thank you for this comment any way.

Comments 13: Lines 65–67: Please include a reference for the origin and distribution of BSF.

Response 13: We have deleted this part according to another reviewer’s comment. Thanks.

Comments 14: Lines 73–75: A reference is needed.

Response 14: We have put 3 references of this part at the end of the sentence. Thanks.

Comments 15: Line 79: Up to this point, the authors have used the full name “black soldier fly,” but later switched to the abbreviation “BSFL,” and then back again to the full name. Please use consistent abbreviations throughout the manuscript (e.g., BSF and BSFL), after first introducing the full term.

Response 15: Thank you very much for pointing this out. We used BSFL, but we did’t use BSF. However, there are indeed two places in the following text where the abbreviation BSFL is not used (excluding the titles of tables and figures). Both corrected, thanks.

Comments 16: Lines 81–82: Please include studies addressing the effects of capsaicin in feed.

Response 16: According to another reviewer’s comment, we deleted some content in this part, and about the effects of capsaicin in feed on BSFL, such as the pupation rate, we have put 5 references at the end of the next sentence. Thank you for pointing this out.

Comments 17: Line 90: There are recent studies (2026) investigating BSF diets with high-fat content. It would be useful to include and discuss these studies.

Response 17: We found an article studying BSFL fat-rich substrates (Protein- or fat-rich substrates have divergent effects on waste reduction, biomass yield, and gas emissions in black soldier fly bioconversion), and the results are similar to ours. We added it in the discussion part, and also changed the expression of the relevant sentence in the introduction part. Thanks for your comment.  

Comments 18: Lines 98–103: This section is redundant and overly lengthy. Please remove it or incorporate it into the previous section.

Response 18: We deleted this part. Thanks.

Comments 19: Lines 105–110: This section can also be shortened, with a clear focus on the study aim in a single sentence.

Response 19: Some contents in this part were deleted. Thanks.

Comments 20: Line 130: Scientific names should be italicised: Capsicum annuum L.

Response 20: Corrected. Mentioned by others too. Thank you very much.

Comments 21: Line 131: Is “CK” referring to chicken feed? If so, please replace “standard diet” with “chicken feed.”

Response 21: No, CK doesn’t refer to chicken feed, but refer to control check. Thanks.

Comments 22: Line 163: Please provide a reference for the ECD equation.

Response 22: Added. Thanks a lot.

Comments 23: Line 174: Please include the appropriate AOAC reference for the crude protein method.

Response 23: Thank you for pointing this out. This is an omission in the manuscript, and we should add the references. Actually, the analytical methods for crude protein and crude fat are based on the Chinese national standards (GB/T 6432-2018 and GB/T 6433-2025). We have added them in the reference.

Comments 24: Line 181: Please include the appropriate AOAC reference for the crude fat method.

Response 24: Same as above.

Comments 25: Line 212: A few points for the table:

The title of the table would benefit from more information about the experiment. For example: Table 1. Growth performance of black soldier fly (BSF: Hermetia illucens) larvae under six dietary treatments including capsaicin (0, 0.01, 0.05, …%) and high-fat conditions across different treatment groups.

Response 25: All titles improved, as mentioned in previous responses. Thank you very much for your comments.

Comments 26: Please also add an explanation below the table for all abbreviated treatment names.

Response 26: Changed. See above please. Thanks.

Comments 27: The survival ratio is often presented as a percentage (%) to allow easier comparison, so please change it to %.

Response 27: Changed. Thanks.

Comments 28: P-values should be calculated and reported for all items in the table.

Response 28: All problems relating to P-values and significance were all corrected. Other reviewers also mentioned these. Thanks a lot.

Comments 29: Line 215: The table should be discussed and referred to before it appears in the paper. Please correct this.

Response 29: Corrected as mentioned in the response to comment 7.

Comments 30: Line 216: The significance between treatments can only be determined by P-values. Please include P-values in the tables to explain your results accurately.

Response 30: Same as above.

Comments 31: Lines 223–226: A similar point applies here; P-values are needed.

Response 31: Same as above.

Comments 32: Line 229: Figure and table titles should be independent and able to stand alone from the main text. Therefore, please improve the figure title and add P-values to the figure. If the P-value is not significant, please include a statement indicating this below the figure, along with an explanation of all abbreviations, as mentioned previously in the table comments.

Response 32: All revised as previous responses. Thank you very much for your help.

Comments 33: Line 258-269: This section does not belong under the topic of changes in nutritional value. It should be placed under a separate subheading on waste reduction efficiency.

Response 33: A separate subheading was added here according to your comment.

Comments 34: Line 273 & 301: Tables 2 & 3. Please refer to general comments about Tables.

Response 34: Changed as mentioned above.

Comments 35: Results & Discussion:

Please include P-values in the Results section and revise the discussion & conclusion accordingly.

Response 35: Changed as mentioned above.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

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

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Improved.

Are the results clearly presented?

Can be improved

Improved.

Are the conclusions supported by the results?

Can be improved

Improved.

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

Comments 1: Please specify the source, purity, and solubility method of the capsaicin used. How was homogeneous distribution in the high-fat diet ensured? Was capsaicin stability verified over the experimental period, given potential degradation under rearing conditions?

Response 1: Thank you for raising the questions regarding the stability verification of capsaicin. We fully understand the routine significance of this issue, but considering the unique in vitro digestion and feeding method of black soldier fly larvae, we believe that simply detecting the chemical stability of capsaicin in the substrate in this study may not truly reflect the actual exposure level of the larvae and is also difficult to explain the experimental results. Before feeding, the larvae of black soldier flies secrete oral secretions rich in digestive enzymes (such as protease, lipase, chitinase, etc.) into the substrate for pre-digestion, and then inhale the liquefied nutrients into their bodies. This means that once the larvae are introduced to the substrate, the degradation and metabolic transformation of capsaicin have already begun, and this process mainly depends on the secretory activity of the larvae and the participation of intestinal microbiota, rather than the chemical stability of the substrate itself. Therefore, even if we verified the short-term stability of capsaicin in the diet without inoculating the larvae (such as no significant change in content within 72 hours), this result still cannot represent the true lineage of capsaicin and its metabolites in the "dynamic substrate" faced by the larvae under actual rearing conditions.

Based on the above considerations, the chemical stability of capsaicin in the substrate was not separately verified in this study. We believe that the changes in the growth, development and nutritional composition of the larvae measured in the experiment have comprehensively reflected the biological effects of capsaicin on black soldier flies under real feeding conditions (including the in vitro digestion process). This is more ecologically valid and of practical significance than simply determining the residue of capsaicin in the substrate. Of course, we also agree with your concern about the metabolic destination of capsaicin. In the subsequent research, we plan to adopt more direct methods, such as determining the concentrations of capsaicin and its main metabolites in the intestinal contents of larvae or the homogenate of the larvae, and combine them with in vitro digestion simulation experiments to further reveal the metabolic transformation rules of capsaicin in the black soldier fly larvae. Thank you again for your valuable comments, which have pointed out the direction for our subsequent research.

Comments 2: Table 1: Some values lack standard deviations (e.g., "1584" for HF-0.1 total daily increment). Please ensure all mean values are reported with appropriate measures of variability.

Response 2: All added. Thank you.

Comments 3: While statistical methods are described, the Results section would benefit from explicit notation of significant differences (e.g., superscript letters or asterisks) in tables and figure legends to aid interpretation.

Response 3: All corrected. Others also mentioned this. Thanks a lot.

Comments 4: The observation that pupation age does not change monotonically with capsaicin concentration is intriguing. Please expand the discussion on potential physiological mechanisms (e.g., hormonal regulation, stress response pathways) that could explain this pattern.

Response 4: Thanks for your comments. We added the following discussion to the manuscript.

It is worth noting that the age of pupation did not show a single and monotonous change with the addition level of capsaicin (Table 1, Figure 2) : the age of pupation in the 0.1% treatment group was significantly shortened, while that in the 0.2% treatment group returned to the level of the high-fat control group. This dual-phase effect of "low-dose stimulation and high-dose inhibition" is precisely a typical feature of the excitatory effect of toxins. The systematic review by Rix and Cutler (2022) indicates that such biphase responses are widespread in insects and can manifest as phenotypes such as accelerated growth and development, enhanced reproductive capacity, prolonged lifespan, and improved tolerance to environmental stress. This review further reveals that the molecular pathways related to the excitatory effect of toxins mainly include alterations in antioxidant enzyme activity, upregulation of heat shock proteins, enhanced expression of detoxification genes, and changes in gene expression in the IIS/TOR signaling pathway that directly regulates growth, development, and reproduction. In addition, the changes in the levels of juvenile hormones and ovoproteinogen induced by stress are also associated with the toxic excitatory effect. Cutler et al. (2022) also pointed out that mild chemical, high temperature or nutritional stress can all induce excitatory effects in insects [24-25]. In addition to hormone regulatory pathways, energy redistribution is also an important dimension for understanding the mechanism of the excitatory effect of poisons. In this study, the weight gain rate of the high-dose capsaicin treatment group decreased significantly (the final weight of the HF-0.2 group was only 23% of that of the HF-0 group), suggesting that capsaicin stress may interfere with the feeding or digestive absorption efficiency of larvae. Under the state of "perceived nutritional deficiency", according to the discussion by Rix and Cutler (2022) on the core role of the IIS/TOR signaling pathway in the toxic excitation effect, larvae may, by down-regulating the activity of this pathway, prioritize the allocation of limited energy reserves to stress defense responses and homeostasis maintenance rather than for developmental processes and tissue growth [24]. Thereby delaying the onset of abnormal reactions. In Table 1, the average weight gain and weight gain rate of the HF-0.2 group were the lowest among all treatments, which was highly consistent with the above explanation of energy redistribution. Based on the above analysis, we believed that the possible reason for the non-monotonic developmental response observed in this study was that 0.1% capsaicin, as a mild source of chemical stress, may indirectly promote the synthesis and release of molting hormone by moderately activating the antioxidant defense system and IIS/TOR signaling pathway of the larvae, thereby enabling the larvae to enter the pre-pupa stage earlier. When the capsaicin dose rose to 0.2%, the oxidative stress and metabolic burden caused by excessive stress exceeded the compensatory capacity of the larvae, resulting in the inhibition of developmental initiation and the recovery of the pupation age to the level of the high-fat control group.

Comments 5: The sensitivity of C12:0, C18:1n-9, and C18:2n-6 to capsaicin treatment is well noted. However, the discussion would be strengthened by referencing known metabolic pathways in BSFL or related insects that could underlie these shifts.

Response 5: We also added a paragraph to discuss this point. Thanks for your comments.

Comments 6: The exclusion of tryptophan due to acid hydrolysis is standard practice but should be explicitly acknowledged as a limitation when interpreting protein quality.

Response 6: The following sentence added in the manuscript states this point: Tryptophan was easily destroyed by acid hydrolysis, so it was not reported separately in this study. Thanks.

Comments 7: The conclusion that capsaicin levels should be "controlled" for optimal BSFL rearing is valuable. Please consider adding quantitative guidance (e.g., recommended maximum capsaicin concentration in substrate) based on the observed thresholds for growth inhibition.

Response 7: We added our recommendation in the last paragraph in the discussion part. Thank you for your advice.

Comments 8: Ensure consistent use of units (e.g., "mg·d⁻¹" vs. "mg/d") and chemical notation (e.g., "C18:1n-9" vs. "C18:1n9") throughout.

Response 8: All changed, thank you.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

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

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Can be improved

Improved.

Are the conclusions supported by the results?

Yes

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

Comments 1: This research is relevant and interesting. Especially for the various cultures in the world that are used to consume spicy foods which are difficult to process. Moreover, these components are sometimes used as insecticides. The manuscript is written fluently in proper English. I only have a few remarks that could improve the manuscript:

Could you add the crude protein and fat contents to the amino acid and fatty acid tables respectively?

Response 1: Thank you for your constructive suggestions. Unfortunately, in the current study, we did not separately determine the crude protein and crude fat content of the larvae, as our main research focus was on the composition of amino acids and fatty acids. Therefore, we are unable to add these data to Tables 2 and 3. We fully recognize that this is a limitation of this work. In the discussion section of the revised draft, we have added a sentence in the discussion part to point out this deficiency and explain that the determination of crude protein and crude fat should be included in subsequent research to improve nutritional assessment. Thank you again for your attention to this important aspect.

Comments 2: Can you add a table with the composition of the substrates?

Response 2: We added a new table according to your advice.

Comments 3: What were the capsaicin levels in the reared larvae?

Response 3: Thank you for raising this question. Here we honestly state that the residual levels of capsaicin in black soldier fly larvae were not detected in this study. The main considerations are as follows:

Firstly, the research objective of this study focuses on the physiological effects of capsaicin on the growth performance, development process, feed conversion efficiency and nutritional composition (amino acids, fatty acids) of black soldier fly larvae, rather than the metabolic accumulation pattern of capsaicin in the larvae. We believe that the above indicators have been able to effectively answer the core scientific question of "whether capsaicin has an impact on black soldier flies" and "to what extent". Second, the larvae of black soldier flies have a strong ability to metabolize and detoxify exogenous substances. Previous studies have shown that insects can efficiently metabolize capsaicin through the cytochrome P450 enzyme system, and its metabolic products may be rapidly excreted from the body. Therefore, even if the daily diet contains a relatively high concentration of capsaicin, its residual level in the insects may be very low, and it can only be detected by using a highly sensitive LC-MS/MS method. Due to the resource conditions of this study, we were unable to carry out this test. Thirdly, we fully agree that the residual level of capsaicin in the larvae is of great significance for assessing the safety of the larvae as animal feed. We have supplemented this limitation in the discussion section of the revised draft and listed it as an important direction for subsequent research. Thank you again for your valuable comments.

Response to Reviewer 4 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

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

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Can be improved

Improved.

Are the conclusions supported by the results?

Yes

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

Comments 1: The title should be “Effects of capsaicin on the growth, development and nutritional profiles of black soldier flies fed a high-fat diet”.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have changed the the title.

Comments 2: Line 9, the indentation of paragraphs should be adjusted.

Response 2: Agree. We have, accordingly, changed the indentation to emphasize this point. Thank you.

Comments 3: Line 19, replace “have a relatively small effect” with “have relatively small effects”.

Response 3: Changed. Thank you for the comment.

Comments 4: Line 29, replace “BFSL” with “BSFL”.

Response 4: Changed. Thank you.

Comments 5: Line 52, changed "metropolis" with "metropolises".

Response 5: Changed. Thank you.

Comments 6: Line 55, delete “utilization”.

Response 6: Deleted. Thank you for pointing this out.

Comments 7: Line 62, Hermetia illucens should be italicized.

Response 7: Changed. Thank you.

Comments 8: Line 62-67, delete these sentences, as they are not highly relevant to this study.

Response 8: Deleted 3 sentences. Thank you.

Comments 9: Line 78-92, please condense these sentences into a single paragraph.

Response 9: We have condensed these sentences into one paragraph and deleted some sentences. Thank you for your comment.

Comments 10: Line 114, please supply references to the previous works.

Response 10: Thank you for pointing this out. Because the previous works were not published, we deleted this part.

Comments 11: Line 119, replace “include” with “included”.

Response 11: Changed. Thank you.

Comments 12: Line 125, replace “shall be” with “were”.

Response 12: Replaced. Thank you.

Comments 13: Line 130, Capsicum annuum should be italicized.

Response 13: Changed. Thank you.

Comments 14: Line 136, what are the specifications of the food cans? What is the material?

Response 14: Each aquaculture container is an open polypropylene (PP) plastic box with an open top. Its dimensions are 15 cm in length × 10 cm in width × 5 cm in height, with a volume of approximately 0.75 liters. This material is heat-resistant and moisture-resistant, and is suitable for aquaculture environments with a temperature of 28±2 ℃ and a relative humidity of around 80%. The specifications and material of the food cans were added to the manuscript. Thank you for pointing out this.

Comments 15: Line 153, what is the mesh size of the screen?

Response 15: Thank you for pointing this out. Actually, we didn't express clearly here. After the breeding is completed, the larvae and the residual substrates are separated by manual sorting. Sterile tweezers were used to pick out the larvae one by one to avoid mechanical damage. The net in the text is not a sieve but merely a tool to help remove the larvae.

Comments 16: In methods, verbs should be used in the past tense.

Response 16: A total of 7 places were corrected. Thank you very much.

Comments 17: Line 163, add “was” before “calculated”.

Response 17: Added. Thank you.

Comments 18: Remove the comma in the formula “ECD = m4/(m2-m5),”.

Response 18: Removed. Thank you.

Comments 19: Line 170, replace “stopped feeding” with “starved”.

Response 19: Changed. Thank you.

Comments 20: Line 170-171, the surface attachments were rinsed with distilled water, and the larvae were weighed after drying on filter paper.

Response 20: Changed. Thank you for your comment.

Comments 21: Line 172, replace “larva” with “larval”.

Response 21: Changed. Thank you.

Comments 22: Line 190, add a space between Excel and 2019. This problem also exists in IBMSPSS.

Response 22: Both changed. Thank you.

Comments 23: Line 191, data are presented as mean ± standard.

Response 23: . The symbol has been replaced. Thank you.

Comments 24: Page 5, please optimize the page layout and reduce blank spaces before final submission.

Response 24: . The format has been corrected. Thank you.

Comments 25: Line 210, add spaces after "A.", "B.", "C.", etc.

Response 25: All changed. Thank you.

Comments 26: Line 212, replace “under” with “in”.

Response 26: Changed. Thank you.

Comments 27: Please mark significant differences with letters or symbols in all figures and tables.

Response 27: All corrected. Thanks.

Comments 28: Line 406-413, these sentences do not support the conclusion in the first sentence.

Response 28: This reference is indeed not quite appropriate. We deleted it. Thank you for your suggestion.

Comments 29: In the references, the journal abbreviations, volume numbers, and citation formats for dissertations are incorrect. Please revise them carefully.

Response 29: All checked according to the guide file of the journal including the theses  (mdpi-acs-references-guide-2025.12-online.pdf). For some Chinese journals we can only find their full names, but all DOIs were added. Thanks.

Comments 30: The materials and methods used in this study is reliable, but the data analysis lacks sufficient rigor. The statistical results should be presented in greater detail. Please label significant differences with letters or symbols in all figures and tables. Besides, the English writing requires polishing by a native speaker.

Response 30: All corrected. Other reviewers also mentioned this. Thank you very much.

CK

34.8 ± 0.51ᵇ

185.3 ± 17ᵇ

< 0.000X

HF-0

49.0 ± 0.02ᵃ

259.7 ± 13ᵃ

< 0.00X

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript again. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

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

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Must be improved

Improved.

Are the conclusions supported by the results?

Must be improved

Improved.

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

Comments 1: The authors have made effort to revise the manuscript, particularly by including P-values. However, significant issues remain.

First, although statistical analyses (including post hoc tests) are mentioned in the Methods section, the results of these analyses are still not properly reported in the tables or text. Reporting only P-values without appropriate post hoc comparisons does not allow clear interpretation of differences among treatments. The authors are encouraged to follow standard practices in scientific reporting, particularly in table presentation. When significant differences are identified, they should be followed by appropriate post hoc tests, and differences among treatments should be clearly indicated (e.g. using superscript letters).

Values are mean ± SD. Different superscript letters (a,b,c, e) indicate significant differences within the same column (P < 0.05).

Response 1: Thank you very much for this comment. All appropriate post hoc tests were added, and all differences among treatments have been indicated with different superscript letters. All tables and figures were improved.

Comments 2: Second, the Results section remains unclear and repetitive. Statements such as “increased or decreased” are imprecise and not appropriate for scientific reporting.

Response 2: Thank you very much for this comment. All checked and revised. All statements including “increased or decreased” were added p values if necessary.

Comments 3: Third, the language throughout the manuscript requires substantial improvement. Many sentences remain ambiguous or poorly structured, which limits the clarity and scientific quality of the work.

Response 3: Thank you for your advice. We checked the whole manuscript and made some changes to improve the English quality.

Comments 4: In addition, the newly added sections need to be better integrated into the existing Discussion.

Response 4: We rearranged some paragraphs and also deleted some contents according to other reviewers’ comments. Thanks.

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1: The English has been improved to more clearly express the research.

Response to Reviewer 4 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

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

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Can be improved

Improved.

Are the conclusions supported by the results?

Yes

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

Comments 1: Line 455, replace “ovoproteinogen” with “vitellogenin”.

Response 1: Changed, thanks a lot for pointing this out.

Comments 2: Line 560, replace “reached” with “reaches”; replace “the weight accumulation of larvae” with “larval weight gain”.

Response 2: Agreed. All corrected. Thanks.

Comments 3: The discussion section is too long; it is recommended to condense it to 6–8 paragraphs.

Response 3: In the last round we added many new contents according to reviewers’ comments, so the discussion part is long. We deleted some contents. Thank you for the comment.

Comments 4: Additionally, some paragraphs are too long, for example, paragraphs 6, 7, and 8, which are also recommended to be shortened.

Response 4: We deleted some contents. Thank you.

Comments 5: In the references, you did not revise carefully; some journal names are given in full, some are abbreviated, and some have extra punctuation marks.

Response 5: We checked the reference part again and made some changes. Thank you. However, some Chinese journals are given in full because we can only find their full names.