Bioactive Substance Derived from Mealworm Larvae (Tenebrio molitor) Potentially Induces Immune Performance of Zebrafish (Danio rerio)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study makes a valuable contribution to the emerging field of insect-based immunonutrition in aquaculture. The investigation of specific bioactive compounds from Tenebrio molitor larvae represents an important step forward from previous studies that examined whole mealworm meal. The experimental design is appropriate, combining transcriptomic analysis with functional challenge tests to evaluate immunomodulatory effects.

1. Please clarify the purification methodology for the bioactive substance - what criteria were used to determine purity, and what is the proposed chemical nature of this compound?
2. Consider including an additional intermediate dose (e.g., 50 μg/g) to better characterize the dose-response relationship.

3. The 14-day feeding period seems appropriate, but was this duration determined based on preliminary studies?

4. For the challenge test, please provide more details about the metrics used to assess "pathogen resistance" (e.g., survival rates, bacterial loads, clinical signs).

5. The observation that effects were "not consistently significant" warrants discussion. Could this relate to individual variation, sample size, or the specific immune parameters measured?

6. How do your findings compare with previous studies using whole mealworm meal? Does the isolated bioactive reproduce all observed effects?

7. The tissue-specific responses (intestinal vs. liver) deserve more attention. Do the transcriptional changes suggest different mechanisms of action in these tissues?

8. Please discuss the practical implications of the 100 μg/g optimal dose - is this concentration feasible for commercial feed formulations?

9. There are some minor errors in the manuscript. For example, IL-1b should be "IL-1β",; Some abbreviations are not fully written or defined when they first appear; The format of references was not uniform, including the case of the first letter of the word in the title of the article and the name of the magazine; There should be space between some numbers and quantifiers, please check and modify.

Comments on the Quality of English Language

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

Author Response

1. Please clarify the purification methodology for the bioactive substance - what criteria were used to determine purity, and what is the proposed chemical nature of this compound?

In the first step, we performed water extraction followed by ethanol precipitation. The second step involved purification using anion exchange chromatography (AEC). Since our target was a bioactive compound, we assessed nitric oxide (NO) activity in each collected fraction. Additionally, we used the phenol-sulfuric acid (PSA) method to detect sugar content, as our previous study indicated that the bioactive substance may be classified as a polysaccharide. To confirm the purity of the active compound, we proceeded with further purification using size exclusion chromatography (SEC). Fractions were analyzed for NO activity, sugar content, protein content, and organic acids. The final purified fraction exhibited high NO activity but contained no detectable sugar, protein, or organic acids (see line 73-115).

Based on these findings, potential classifications for the mealworm-derived bioactive substance include polysaccharide derivatives, polyphenols, or possibly a novel biopolymer. However, its precise chemical identity remains undetermined. Further chemical and structural characterization will be conducted in subsequent experiments (lines 426-432).

2. Consider including an additional intermediate dose (e.g., 50 μg/g) to better characterize the dose-response relationship.

Thank you for your valuable suggestion. We acknowledge the importance of including an intermediate dose, such as 50 μg/g, to better characterize the dose-response relationship. We will take this into consideration in our future research.

3. The 14-day feeding period seems appropriate, but was this duration determined based on preliminary studies?

Yes, the 14-day feeding period was determined based on our previous studies using zebrafish as a model organism. These prior findings indicated that a 14-day duration is sufficient to elicit measurable physiological and immune responses, making it an appropriate timeframe for this experiment.

4. For the challenge test, please provide more details about the metrics used to assess "pathogen resistance" (e.g., survival rates, bacterial loads, clinical signs).

We sincerely appreciate your suggestion. We have now included survival rate as a metric for assessing pathogen resistance (see line 435). Additionally, details regarding the measurement of bacterial load have been provided in the methods section (lines 234–239).

5. The observation that effects were "not consistently significant" warrants discussion. Could this relate to individual variation, sample size, or the specific immune parameters measured?

We consider two possible explanations for the inconsistent significance observed in our results. First, the bioactive substance derived from mealworms may be less compatible with zebrafish compared to the bioactive compound we previously identified from silkworms. Second, the mealworm-derived bioactive substance may require synergistic interactions with other dietary components to fully enhance immune responses. This is supported by previous studies showing that while mealworm meal replacement did not impair growth, it also improved survival rates compared to fish meal-fed groups. These points are discussed in more detail in lines 442–453.

6. How do your findings compare with previous studies using whole mealworm meal? Does the isolated bioactive reproduce all observed effects?

Thank you for your insightful question. We regret to inform you that our previous study using whole mealworm meal focused primarily on fish growth performance and physiological parameters, without specific assessment of immune-related effects. Therefore, the current study represents, to the best of our knowledge, the first attempt to isolate and evaluate the immunomodulatory effects of a mealworm-derived bioactive substance in fish.

7. The tissue-specific responses (intestinal vs. liver) deserve more attention. Do the transcriptional changes suggest different mechanisms of action in these tissues?

Thank you for this insightful comment. As our current study relies primarily on RNA sequencing and qRT-PCR analysis of selected immune-related genes, we are unable to fully elucidate the underlying mechanisms of action in the intestine and liver. However, the observed transcriptional differences suggest the possibility of tissue-specific immune responses. In our future research, we plan to investigate the mechanisms further using mouse macrophage cells, with a focus on structural characterization of the bioactive compound and its interaction with toll-like receptor (TLR) signaling pathways.

8. Please discuss the practical implications of the 100 μg/g optimal dose – is this concentration feasible for commercial feed formulations?

We appreciate your thoughtful question. Given the high cost of producing purified insect-derived bioactive substances, the practical application of the 100 μg/g dose in commercial feed formulations remains a challenge. Our ongoing work is focused on improving the cost-effectiveness of this approach. For example, we have observed that the defatting process enhances the absorption of the bioactive compound in fish. However, the use of alcohol-based solvents in the extraction process significantly increases production costs, making it less feasible for widespread use by farmers. Therefore, we are currently evaluating alternative alcohol solvents to identify the most cost-effective option that still maintains high bioactivity.

9. There are some minor errors in the manuscript. For example, IL-1b should be "IL-1β",; Some abbreviations are not fully written or defined when they first appear; The format of references was not uniform, including the case of the first letter of the word in the title of the article and the name of the magazine; There should be space between some numbers and quantifiers, please check and modify.

Thank you for your thorough review and valuable observations. We have corrected the instances of "IL-1b" to the appropriate form "IL-1β" in all relevant figures and text. We have also reviewed and revised the manuscript to ensure that all abbreviations are defined at their first appearance. Additionally, we have standardized the reference formatting to ensure consistency, particularly regarding capitalization in article titles and journal names. For journal or publisher abbreviations, we have retained the formats provided on the respective journal websites to maintain accuracy and consistency with their official citation guidelines.

Reviewer 2 Report

Comments and Suggestions for Authors

REVIEW FISHES - 03 -3646546

Bioactive substance derived from mealworm larvae (Tenebrio molitor) potentially induces immune performance of zebrafish (Danio rerio)

Reviewer’s comments:

The present study investigating the effects of a bioactive substance isolated form Tenebrio molitor on the immune system and disease resistance of zebrafish is of scientific interest and could provide some insight on the wider interest of using dietary insects in aquaculutre.

The use of a 14-days immunostimulating period was chosen with 2 low doses of the isolated bioactive subtance. It would be interesting to prolong this period to question whether longer immunostimulation periods would overwhelm and suppress the immune system when stimulated for a long feeding period that would be more practical in fish farm settings.

The Materials and Methods section needs to be clearer especially concerning the pooling of fractions, protein determination, characterisation of the fractions but also the in vivo trial. You need to justify why you used NO production in mouse macrophages to choose the bioactive franctions?

The results need to be presented in a better manner with self-explanatory captions of the figures and with a logical order so that no confusion is introduced about when the gene expression was determined. Some parts of the result section need to be transferred to the discussion.

The discussion needs to explain better the isolation and characterisation of the bioactive compound.

The immunomodulation of the mealworm extract tended to show a suppression of the immune response and gene expression results are explained in a somehow confusing manner.

How do you explain the stimulating action of the mealworm-extracted bioactive compound towards NO production by mouse macrophages while gene expression showed an immunosuppression of zebrafish.

Overall, the extensive work described by the present manuscript is interesting but the results and discussion part are very confusing and do not describe the results acurately. 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English should also be revised thoroughly.  Extensive rewriting is needed to consider the manuscript for publication.

Author Response

Reviewer 2

The present study investigating the effects of a bioactive substance isolated form Tenebrio molitor on the immune system and disease resistance of zebrafish is of scientific interest and could provide some insight on the wider interest of using dietary insects in aquaculture.

We sincerely appreciate your kind and encouraging feedback on our research. Our current objective is indeed to explore the potential of insects as promising immunostimulants in aquaculture. We hope that in the future, insect-derived compounds may serve not only to enhance fish health but also as feasible alternatives or supplements to fish meal in aquafeeds, contributing to more sustainable and resilient aquaculture practices.

The use of a 14-days immunostimulating period was chosen with 2 low doses of the isolated bioactive subtance. It would be interesting to prolong this period to question whether longer immunostimulation periods would overwhelm and suppress the immune system when stimulated for a long feeding period that would be more practical in fish farm settings.

Thank you for your valuable insight. We agree that extending the feeding period while using lower doses could provide a deeper understanding of the long-term immunological effects of the bioactive substance. We will consider this approach in our future research to better evaluate its practicality and potential impacts within aquaculture settings.             

The Materials and Methods section needs to be clearer especially concerning the pooling of fractions, protein determination, characterisation of the fractions but also the in vivo trial. You need to justify why you used NO production in mouse macrophages to choose the bioactive franctions?

Thank you for your thorough review and insightful comments. We believe the methodology has been described in detail, particularly the pooling of fractions, protein determination, and fraction characterization (see lines 91–115). Additionally, we have now included a justification for the use of NO production in mouse macrophage cells (RAW264.7) as a screening tool to identify bioactive fractions (see lines 109–111 and 413–414).

The results need to be presented in a better manner with self-explanatory captions of the figures and with a logical order so that no confusion is introduced about when the gene expression was determined. Some parts of the result section need to be transferred to the discussion.

Thank you for your great insight. In response, we have revised the order of the results section to ensure a more logical and coherent presentation of the findings (see line 305). Additionally, we have transferred selected parts of the results section into the discussion (see lines 479–493 and 504–510) to enhance clarity.

The discussion needs to explain better the isolation and characterisation of the bioactive compound.

Thank you for your comment. The isolation and characterization of the bioactive compound have been described in detail in the methods and results sections.

The immunomodulation of the mealworm extract tended to show a suppression of the immune response and gene expression results are explained in a somehow confusing manner.

Thank you for your comment. We apologize for any lack of clarity in our explanation of the gene expression results. To help us improve the manuscript more effectively, we would greatly appreciate it if you could kindly clarify which specific aspects of the immunomodulatory findings or gene expression discussion were confusing or require further elaboration.

How do you explain the stimulating action of the mealworm-extracted bioactive compound towards NO production by mouse macrophages while gene expression showed an immunosuppression of zebrafish.

We presume that the observed immunosuppression in zebrafish may reflect a transition into the immune resolution phase, in which the organism seeks to restore homeostasis and prevent excessive or chronic inflammation (see line 482–484). In contrast, the stimulation of NO production in mouse macrophages reflects an early-stage immune activation, which is expected during in vitro screening of bioactive compounds. These differences likely reflect tissue- and species-specific immune regulation mechanisms, as well as differences between in vivo and in vitro responses.

Overall, the extensive work described by the present manuscript is interesting but the results and discussion part are very confusing and do not describe the results accurately.

Thank you very much for your comment. We acknowledge that the Results and Discussion section may have lacked clarity, and we revised it to more accurately present and interpret the findings. We have made every effort to improve the structure and flow to ensure clearer communication of the results and their implications.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all my concerns. I recommend accepting it in current form.

Author Response

Thank you for your peer review.

Reviewer 2 Report

Comments and Suggestions for Authors

REVIEW FISHES - 03 -3646546

Bioactive substance derived from mealworm larvae (Tenebrio molitor) potentially induces immune performance of zebrafish (Danio rerio)

Reviewer’s comments v2:

Authors have addressed most required revisions but some suggestions have been overlooked. Moreover, some of the reviewer’s comments should not only be answered in a letter but should be explained in the maunscript to make it clearer.

More particularly:

L45: insects have gained traction???

L53-54: the suggested 2021 review JIFF paper is in aquaculture and terrestrial farmed animals, not pets. (Gasco et al., 2021, JIFF, 7(5):715-741)

L55-57: you explained the link of PAMP with the present study very well to me ‘What we are implying in this paragraph is that our previous research demonstrated that insect-derived bioactive substances, such as dipterose-BC, dipterose-BSF, silkrose-AY, and silkrose-BM, can enhance innate immune responses. These effects are likely mediated through PAMP-like activity, as these substances may be recognized by pattern recognition receptors (PRRs) on immune cells, thereby triggering innate immune activation.’ but not in the text. L57: you added ‘through the use of macrophage’ please rephrase.

L66: I understand you focused on sugar content because of your previous experience in other insects but here you also looked at proteins and organic acid so don’t restrain your explanation to sugar which is not really pertinent in the present study.

L75: did you mean successively, continuously or successfully?

L98-100: ‘A linear gradient of NaCl at concentrations of 20%, 40%, and 100% was used at a flow rate of 2.0 mL/min to create the fractions.’ Are you sure this represents a linear gradient of NaCl when you used 3 different concentrations rather than a continuous increase of concentration?

L99&110: you need to add that fractions were diluted and using which solution, either here or in the NO assay section?

L100: you need to add a reference for the method of protein determination.

L103: Which fractions were further used in SEC? Only the ones showing NO production at dil x1000? Were they pooled? The ones in the oval? You need to explain this here in the Mat&Met and why you chose those fractions rather than the AMPs in fractions 9 and 30-32.

L113: add ‘as explained above’. 

L117 and/or L300: You assessed different dilutions of the mealworm-derived bioactive substances in fig 2. Which fractions were pooled, only fractions 5-7 (tested at dil 2000x on RAW cells)? As you pooled them, how can you know if there is only one bioactive substance? Please add a HPLC profile to check the purity of the bioactive substance.

L149: you state: ‘Since the bioactive substance was barely detected by RI, fractions were collected and subjected to NO assay’ Were they 1ml fractions? Did you use them neat or did you dilute or concentrate them?

L150: You need to add a reference for RI and organic acids in the Mat&Met which you mention only in the results.

L167: the bioactive substance (dry, liquid?) was mixed with other dry ingredients, oil or water?

L179: I’m still confused about the photoperiod. When was 16:8 L:D followed? Challenge was also done at 14:10?

L176-178: ‘A total of 120 zebrafish were used in this study. They were randomly distributed into six aquariums (45 × 25 × 25 cm), with 20 fish per aquarium. Each treatment was applied to two aquariums (i.e., in duplicate) and repeated three times’ You mean that you had 360 fish in total?.

L240-244: The challenge description is not in the right order. First describe E.tarda growth then concentration adjustement then challenge. Also state the final E.tarda concentration in the challenge immersion bath.

L244: three replicate per what? Diet, challenge? Be more clear (10 fish/diet, 3 replicates of the challenge experiment, 90 fish in total for the challenge experiments?).

L281:  Why do you present NO results in fig 1 for dil 20x and 80x when you state that ‘At 20- and 80-fold dilutions, the NO activity was so high that it caused cell death in the RAW cells, rendering them unreadable by the spectrophotometer.’

L274 & 294: In the figures 1&2 captions, you still need to mention the units of NO activity, protein and sugar on the y axis. How do you explain that the same fractions diluted at x1000 stimulate NO production by RAW cells while there was no activity when less diluted (20x or 80x)?

L283-285: you say fractions 9, 30-32 are AMP but fraction 9 was also rich in sugar.

L286: Why did you decide to focus on fractions 18-26? you need to explain it here or in the Mat & Met.

L287: Did you experience high cellular mortality with low dilution of the fractions of SEC?

L401: please, be precise. IL10 is not decreased in intestine of fish fed 10μg/g.

L403: why ‘in contrast’

L407: letters in IL6 of intestine need to be corrected.

L417: “Meanwhile, the 100 µg/g treatment showed expression levels nearly identical to the control group” No this is not true for IL1B (are you sure they are no statistical difference there?

L458-463: The fact that partial and total replacement of fishmeal with mealworm showed different effects on host resistance to disease cannot be linked with synergetic interaction because you compare different host species (shrimp & catfish) and pathogens were only tested in catfish not in shrimp?

L464-487: I still think that without showing first an immunostimulation/inflammation, you cannot speak about resolution phase to explain the downregulation. You should specify that you suspect a previous immunostimulation/inflammation but that you do not have evidence of this. You replied to my comment ‘How do you explain the stimulating action of the mealworm-extracted bioactive compound towards NO production by mouse macrophages while gene expression showed an immunosuppression of zebrafish.’:

‘We presume that the observed immunosuppression in zebrafish may reflect a transition to the immune resolution phase, through which the organism seeks to restore homeostasis and prevent excessive or chronic inflammation (see lines 467–469). In contrast, the stimulation of NO production in mouse macrophages reflects early-stage immune activation, which is expected during in vitro screening of bioactive compounds. These differences likely reflect tissue- and species-specific immune regulation mechanisms, as well as differences between in vivo and in vitro responses.’ You should explain this in the discussion rather than make assumptions you cannot explain from your results.

Please check that all the statements about your results correspond to the actual results and further check the corresponding discussion part.

* The use of a 14-days immunostimulating period was chosen with 2 low doses of the isolated bioactive subtance. It would be interesting to prolong this period to question whether longer immunostimulation periods would overwhelm and suppress the immune system when stimulated for a long feeding period that would be more practical in fish farm settings.

Authors could mention in the conclusion that longer immunostimulation periods could overwhelm and suppress the immune system and further study with low dose for longer periods, more practical in fish farm settings, should be studied in the future.

Author Response

Reviewer 2

Authors have addressed most required revisions but some suggestions have been overlooked. Moreover, some of the reviewer’s comments should not only be answered in a letter but should be explained in the maunscript to make it clearer.

More particularly:

1. L45: insects have gained traction???

We apologize for the confusion caused by our phrasing. The sentence has been revised to: “insects have gained attention.”

2. L53-54: the suggested 2021 review JIFF paper is in aquaculture and terrestrial farmed animals, not pets. (Gasco et al., 2021, JIFF, 7(5):715-741)

Thank you for your correction, and we apologize for the earlier misinterpretation. We have now cited the correct reference (Gasco et al., 2021) and incorporated it appropriately in the manuscript.

3. L55-57: you explained the link of PAMP with the present study very well to me ‘What we are implying in this paragraph is that our previous research demonstrated that insect-derived bioactive substances, such as dipterose-BC, dipterose-BSF, silkrose-AY, and silkrose-BM, can enhance innate immune responses. These effects are likely mediated through PAMP-like activity, as these substances may be recognized by pattern recognition receptors (PRRs) on immune cells, thereby triggering innate immune activation.’ but not in the text. L57: you added ‘through the use of macrophage’ please rephrase.

We appreciate your detailed observation. The paragraph has been revised for clarity as suggested.

4. L66: I understand you focused on sugar content because of your previous experience in other insects but here you also looked at proteins and organic acid so don’t restrain your explanation to sugar which is not really pertinent in the present study.

Thank you for this insightful comment. We have revised the sentence to reflect a broader perspective by including proteins and organic acids.

5. L75: did you mean successively, continuously or successfully?

Thank you. We intended to convey "continuously" and have updated the wording accordingly.

6. L98-100: ‘A linear gradient of NaCl at concentrations of 20%, 40%, and 100% was used at a flow rate of 2.0 mL/min to create the fractions.’ Are you sure this represents a linear gradient of NaCl when you used 3 different concentrations rather than a continuous increase of concentration?

We appreciate your attention to detail. The description has been corrected to “stepwise gradient” rather than “linear gradient,” which more accurately represents our methodology.

7. L99&110: you need to add that fractions were diluted and using which solution, either here or in the NO assay section?

Thank you for pointing this out. We have now added the relevant details in the NO assay section, specifying the dilution steps and the solution used for the assay.

8. L100: you need to add a reference for the method of protein determination.

Thank you. The reference for the protein determination method has been added.

9. L103: Which fractions were further used in SEC? Only the ones showing NO production at dil x1000? Were they pooled? The ones in the oval? You need to explain this here in the Mat&Met and why you chose those fractions rather than the AMPs in fractions 9 and 30-32.

Yes, we used the fractions within the oval section of Figure 1, which showed NO activity at high dilution. The manuscript has been updated to explain the rationale behind selecting these fractions rather than those potentially containing AMPs (fractions 9 and 30–32).

10. L113: add ‘as explained above’.

Thank you. We have added “as explained above”.

11. L117 and/or L300: You assessed different dilutions of the mealworm-derived bioactive substances in fig 2. Which fractions were pooled, only fractions 5-7 (tested at dil 2000x on RAW cells)? As you pooled them, how can you know if there is only one bioactive substance? Please add a HPLC profile to check the purity of the bioactive substance.

Thank you for the valuable suggestion. Yes, only fractions 5–7, which exhibited strong NO activity even at high dilution, were pooled. We acknowledge that pooling may not guarantee a single bioactive compound. However, based on parallel findings from our ongoing studies on similar insect-derived substances (currently being prepared for publication), these fractions are likely to contain a single bioactive entity. We are planning to further confirm the molecular identity and purity of the mealworm-derived substance in a follow-up study, which will include HPLC profiling.

12. L149: you state: ‘Since the bioactive substance was barely detected by RI, fractions were collected and subjected to NO assay’ Were they 1ml fractions? Did you use them neat or did you dilute or concentrate them?

Thank you for your observation. Yes, the collected fractions were 1 mL each, and for the NO assay, we used a dilution step and took only 20 µL from each fraction. We have clarified this information in the NO Activity Assay section as suggested.

13. L150: You need to add a reference for RI and organic acids in the Mat&Met which you mention only in the results.

Thank you for pointing this out. We have now added the appropriate references.

14. L167: the bioactive substance (dry, liquid?) was mixed with other dry ingredients, oil or water?

We appreciate the suggestion. The bioactive substance was dry (freeze-dried) before being mixed with the dry ingredients. This has now been clearly specified in the revised sentence.

15. L179: I’m still confused about the photoperiod. When was 16:8 L:D followed? Challenge was also done at 14:10?

Thank you for catching this inconsistency. The correct photoperiod used throughout the study—including acclimation, feeding, and challenge test—was 14 hours light and 10 hours dark (14:10). We have corrected all related statements for clarity.

16. L176-178: ‘A total of 120 zebrafish were used in this study. They were randomly distributed into six aquariums (45 × 25 × 25 cm), with 20 fish per aquarium. Each treatment was applied to two aquariums (i.e., in duplicate) and repeated three times’ You mean that you had 360 fish in total?

Thank you for identifying the ambiguity. You are correct—we used a total of 360 zebrafish. We have revised it.

17. L240-244: The challenge description is not in the right order. First describe E.tarda growth then concentration adjustement then challenge. Also state the final E.tarda concentration in the challenge immersion bath.

Thank you for the suggestion. We have reorganized the Challenge Test section accordingly.

18. L244: three replicate per what? Diet, challenge? Be more clear (10 fish/diet, 3 replicates of the challenge experiment, 90 fish in total for the challenge experiments?).

Thank you for your attention to detail. We have clarified that the three replicates were applied per dietary treatment group.

19. L281:  Why do you present NO results in fig 1 for dil 20x and 80x when you state that ‘At 20- and 80-fold dilutions, the NO activity was so high that it caused cell death in the RAW cells, rendering them unreadable by the spectrophotometer.’

We included the results from 20x and 80x dilutions to illustrate the exceptionally strong NO-stimulating capacity of the insect-derived bioactive substance, even at relatively high dilution factor.

20. L274 & 294: In the figures 1&2 captions, you still need to mention the units of NO activity, protein and sugar on the y axis. How do you explain that the same fractions diluted at x1000 stimulate NO production by RAW cells while there was no activity when less diluted (20x or 80x)?

Thank you for the suggestion. We have updated the figure captions to include the appropriate units: NO activity is expressed in µM (based on sodium nitrite standards), and protein detection is shown in AU (absorbance units), since protein data were obtained via UV-Vis spectrophotometry without absolute quantification. As for the observed phenomenon, we hypothesize that the lower dilutions (20x and 80x) were too cytotoxic, leading to cell death.

21. L283-285: you say fractions 9, 30-32 are AMP but fraction 9 was also rich in sugar.

Thank you for the correction. We have revised the statement for clarity.

22. L286: Why did you decide to focus on fractions 18-26? you need to explain it here or in the Mat & Met.

Thank you for your important question. Because our previous study show the insect-derived bioactive substance never contain protein. Thus we select fractions 16-26.

23. L287: Did you experience high cellular mortality with low dilution of the fractions of SEC?

Thank you for your question. No, we did not observe high cellular mortality at low dilutions of the SEC fractions. This is why we did not report it in the manuscript. In that particular sentence, our intention was to highlight that the bioactive substance retained NO-inducing activity even at a 2,000-fold dilution.

24. L401: please, be precise. IL10 is not decreased in intestine of fish fed 10μg/g.

Thank you for the correction. We have revised the statement to accurately.

25. L403: why ‘in contrast’

We agree that "in contrast" was not the appropriate transition word. It has been removed for clarity.

26. L407: letters in IL6 of intestine need to be corrected.

Thank you for your attention to detail. The labeling of IL-6 has been corrected accordingly.

27. L417: “Meanwhile, the 100 µg/g treatment showed expression levels nearly identical to the control group” No this is not true for IL1B (are you sure they are no statistical difference there?

We appreciate the concern. We have re-checked the statistical analysis and confirmed that there were no statistically significant differences.

28. L458-463: The fact that partial and total replacement of fishmeal with mealworm showed different effects on host resistance to disease cannot be linked with synergetic interaction because you compare different host species (shrimp & catfish) and pathogens were only tested in catfish not in shrimp?

Thank you for the important clarification. Although the challenge test was only conducted in catfish, the shrimp study measured immune parameters (e.g., hemocyte counts) after mealworm meal substitution. Both studies reported no negative impacts on growth and improved immune responses. Thus, we hypothesize that whole mealworm meal may exert broader immunological effects, potentially due to the presence of other synergistic components. We acknowledge the limitations due to different host species and pathogens and already few sentence before.

29. L464-487: I still think that without showing first an immunostimulation/inflammation, you cannot speak about resolution phase to explain the downregulation. You should specify that you suspect a previous immunostimulation/inflammation but that you do not have evidence of this. You replied to my comment ‘How do you explain the stimulating action of the mealworm-extracted bioactive compound towards NO production by mouse macrophages while gene expression showed an immunosuppression of zebrafish.’:

‘We presume that the observed immunosuppression in zebrafish may reflect a transition to the immune resolution phase, through which the organism seeks to restore homeostasis and prevent excessive or chronic inflammation (see lines 467–469). In contrast, the stimulation of NO production in mouse macrophages reflects early-stage immune activation, which is expected during in vitro screening of bioactive compounds. These differences likely reflect tissue- and species-specific immune regulation mechanisms, as well as differences between in vivo and in vitro responses.’ You should explain this in the discussion rather than make assumptions you cannot explain from your results.

We agree and thank you for the excellent suggestion. We have revised and add into discussion.

30. Please check that all the statements about your results correspond to the actual results and further check the corresponding discussion part.

Thank you. We have conducted a thorough cross-check. 

31. * The use of a 14-days immunostimulating period was chosen with 2 low doses of the isolated bioactive subtance. It would be interesting to prolong this period to question whether longer immunostimulation periods would overwhelm and suppress the immune system when stimulated for a long feeding period that would be more practical in fish farm settings.

Authors could mention in the conclusion that longer immunostimulation periods could overwhelm and suppress the immune system and further study with low dose for longer periods, more practical in fish farm settings, should be studied in the future.

Thank you for this valuable recommendation. We have added a statement in the conclusion as reviewer suggested.