Nutrient Attraction and Secondary Metabolites Induce Eogystia hippophaecola (Lepidoptera: Cossidae) Larvae Transfer from Sea Buckthorn Trunks to Roots

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 revisions/corrections highlighted/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?

Can be improved

Some changes were made to this thesis to add the following peer-to-peer responses to the letter The same as below

Are all the cited references relevant to the research?

Can be improved

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

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

Comments 1: The Conclusions section is too vague. Please, be more specific, in particular in the lines 524-528: 'notable differences' is too general.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the Conclusions section to provide specific details regarding the "notable differences". The Conclusions section has been systematically refined, with particular attention to Lines 524-528, to ensure methodological specificity and conceptual clarity.

Original:

“Notable differences in steroid biosynthesis, arachidonic acid metabolism, and glycer-ophospholipid metabolism suggested that the trunk's defense response was weaker than that of the root. This, in turn, likely encouraged the larvae to feed on the trunk.”

Revised: The Conclusions section has been thoroughly rewritten

“The feeding behavior of E. hippophaecola larvae on H. rhamnoides (sea buckthorn) trunks and roots induced significantly altered both the nutritional profiles and secondary metabolites. Comparative analysis showed that infested roots had significantly higher concentrations of sugars, proteins, lipids, and amino acids compared to damaged trunks, creating a nutritional gradient that led to larval migration from the trunks to the roots. Concurrently, the downregulation of lecithin - a pivotal metabolite in the secondary metabolic network - triggered subsequent decreases in arachidonate and betaine within the resistance metabolism of trunks compared to roots, indicating weakened defense responses. This metabolic shift enhanced the adaptive capacity of trunk-feeding larvae, ultimately driving partial recolonization of trunks. Consequently, elucidating the mechanistic basis of this host-shifting behavior in E. hippophaecola larvae holds significant potential for developing targeted biological control strategies.”

Comments 2: Please, provide Latin and common names (if they exist) and other taxonomical details (Authors, systematic assignement) for all organisms mentioned in the manuscript, especially in the Discussion. Line 66. it should be: Hippophae rhamnoides、Line 67. Hippophae belongs to Eleagnaceae. Please, correct.、Line 70: (Hua, Chou, Fang & Chen) - no brackets should be used, according to taxonomy rules, in this case. Line 268: The name of the species should be in italics.

Response 2: We sincerely appreciate your valuable suggestion. As requested, we have now provided the Latin names, common names (where applicable), and taxonomic details for all organisms mentioned in the manuscript, particularly in the Discussion section. The revisions include:

Line 66: Corrected to the accepted Latin name: "Hippophae rhamnoides L."

Line 67: Updated the family name spelling, change Hippophae to Elaeagnaceae

Line 70: Removed brackets around taxonomists' names as per ICN rules, Original: (Hua, Chou, Fang & Chen) → Revised: Hua, Chou, Fang & Chen.

Line 268: Italicized the species name: Hippophae rhamnoides (previously non-italicized).

Table 1: Included full taxonomic assignments for all reference species.

All modifications follow the International Code of Nomenclature (ICN) and are marked in red in the revised manuscript.

Comments 3: Lines 96-96: How were these plant samples collected? Did you collect samples of specific tissues or the whole organs? What tisseues the larvae feed on? Please explain. A photo of feeding damage would be helpful. Please, give more detail on the feeding habits of the larvae in the Introduction.

Response 3: We appreciate the reviewer’s constructive suggestions. Below are our detailed responses:

Plant Sample Collection: Six 5-8-year-old sea buckthorn trees, spaced at least 50 m apart, were randomly selected from the forest interior, avoiding forest edges and roads. Trunk and root borings were made in each tree. We collected 400 g of combined phloem and xylem tissues from both trunks and roots of unaffected sections of each sampled tree(line 102-106, marked in red).

Larval Feeding Tissues: The larvae primarily feed on the phloem and xylem of trunks and roots, causing extensive tunneling damage.

The photo of feeding damage: The image(left) displays the signs of damage at the trunk base and root base of the sea buckthorn. The image(right) shows larvae within insect gallery.

(Should it be considered necessary, this content may be included in the main body of the text.)

Expanded Discussion in Introduction: A detailed description of larval feeding behavior has been incorporated into the Introduction section. (line 79-84, marked in red)

“Previous studies have shown [33]: newly hatched larvae of E. hippophaecola initially infest the phloem of sea buckthorn trunks. By the onset of winter in the same year, approximately 70.5% of the larvae migrate through the phloem and shallow xylem to the base of the trunk and the subcortical region of the roots; fewer penetrate deeper into the trunk xylem. Larvae infesting roots often excavate them, ultimately killing the plant.”

Comments 4: Lines 252-254; 366-368: These statements, and others like that, belong to the Discussion section.

Response 4: We appreciate the reviewer’s constructive suggestion. As recommended, we have relocated the statements in Lines 252-254 and 366-368 (original manuscript) to the Discussion section.

Original: Lines 252-254

“This suggests that inorganic salt ions, which typically act as insect inhibitors in plant, accumulate in the insect's body and are excreted, potentially stimulating further feeding.”

Revised: lines 523-530,marked in red

In addition, metallic elements can influence insect feeding behavior to a certain extent. Studies on desert locusts (Schistocerca gregaria) have demonstrated that trace iron (Fe) modulates oxidative stress responses in these insects [73]. Conversely, elevated sodium (Na) levels have been shown to negatively impact grasshopper(Melanoplus differentialis) survival, morphological development, and locomotor performance[74].This suggests that inorganic salt ions, which typically act as insect inhibitors in plant, accumulate in the insect's body and are excreted, potentially stimulating further feeding.

Lines 366-368: “These findings suggest differences in the defense responses between the roots and trunks of sea buckthorn to E. hippophaecola larvae infestation, potentially influencing the larvae’s feeding site shift.”

Moving speculative interpretations from Results to Discussion(lines 615-617,mared in red), where they better fit the narrative. Ensuring the Results section remains focused on objective data presentation.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Dear Reviewer, Thank you very much for your evaluation of the English language quality in our manuscript. Your assessment is highly encouraging and gives us confidence in the clarity and readability of our writing. We understand that there is still room for improvement, and we will continue to strive for excellence in this regard. If you have any specific suggestions on how we can further enhance the English language quality, such as grammar, vocabulary, or sentence structure, we would greatly appreciate your input. Once again, thank you for you.

5. Additional clarifications

Thank you again for your guidance.

If you need any further information, please don't hesitate to let us know!

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 revisions/corrections highlighted/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?

Can be improved

Some changes were made to this thesis to add the following peer-to-peer responses to the letterThe same as below

Are all the cited references relevant to the research?

Can be improved

Is the research design appropriate?

Yes

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

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

Comments 1: Firstly, the introduction provides general background information but lacks a clear statement of research gaps. The authors should explicitly state why this study is necessary and how it builds on previous research. Also, some references are outdated, from 1193 and 1999. Including more recent studies on plant-insect interactions and chemical ecology would strengthen the manuscript.

Response 1: We sincerely appreciate the reviewers’ constructive comments, which have helped us significantly improve the manuscript. Below are our point-by-point responses to the concerns raised.

"The introduction lacks a clear statement of research gaps... explicitly state why this study is necessary."

We fully agree with this suggestion. In the revised manuscript, we have restructured the Introduction to highlight the research gaps more clearly (line 84-94, marked in red).

"Some references are outdated (e.g., 1993, 1999)... include recent studies."

We apologize for the oversight. The outdated citations (Wu et al., 1993; Huang et al., 1998; Ritsuo,1999) were replaced with recent advances(Line 40-41,44,47,53, marked in red).

We hope these revisions meet the reviewer’s expectations. Thank you again for the valuable feedback. Please let us know if any additional clarifications are needed.

Comments 2: In the materials and methods, the description of the chemical analysis methods needs more detail (how were the metabolites identified and quantified? were standards used for calibration?). The behavioral assay setup should also be clarified (how were the larvae exposed to the different chemical treatments, and what were the controls?), and the statistical methods used for data analysis should be better explained (were normality tests conducted before applying parametric tests?).

Response 2: We sincerely appreciate the reviewer's constructive comments. In our original submission, certain methodological descriptions in the Materials and Methods section were intentionally concise due to considerations of manuscript length and potential redundancy. In response to the reviewer's valuable suggestions, we have now comprehensively revised this section to provide more detailed explanations of our experimental procedures.

We have comprehensively refined the Materials and Methods section with the following enhancements:

1. Section 2.1 now includes expanded descriptions of Plant specimen selection criteria, Larval handling protocols, Control group establishment(line 101-106, line108-110, line111-113, line115-117, line121-124; all marked in red)
2. Section 2.2 now provides an extended description of the analytical protocols for nutrient and mineral element quantification, supported by additional references (Lines 127-129, line 134-141, line 142-184; marked in red).

We have added Section 2.2.2 to provide a detailed description of the data analysis process for this part (190-196).

3.      Section 2.3 has been augmented with Midgut enzyme activity assay procedures, Midgut enzyme activity assay procedures (line 201-203, line 208-209, line 231-238; marked in red)

4.      Section 2.4.1 now incorporates supplementary information regarding LC-MS sampling procedures (line242-244, marked in red)

5.      Section 2.4.3 has been augmented with refined metabolite identification criteria (Lines 277-280, marked in red)

Comments 3：The results are generally well presented, but additional statistical validation is needed. The significance levels for all findings should be explicitly stated in the text and tables. The authors should discuss the variability observed in the data (were there any outliers or unexpected trends?).

Response 3: We sincerely appreciate the reviewer's constructive suggestions to strengthen the statistical rigor of our study. It was an oversight on our part that the statistical analyses of both nutrient quantification and enzymatic activity measurements were not adequately presented in the Results section.

We have addressed each point as follows:

First, we have supplemented the methodological descriptions for data analysis pertaining to both nutrient measurements and enzyme activity assays, as detailed in Sections 2.2.2(line189-195, marked in red) and 2.3.4(line 230-237, marked in red), respectively.

In the Results section, we have now included additional statistical analyses as presented in Sections 3.1 (line 296-342, marked in red) and 3.2 (line360-381, marked in red). Should the reviewers deem it necessary, we would be pleased to submit the complete statistical test results tables as supplementary material.

Comments 4: The discussion section should better integrate findings with broader ecological and pest management contexts. The role of secondary metabolites in pest behavior should be further expanded, with references to comparable (recent) studies in other plant-insect systems, and the practical applications of the findings should be discussed in more detail (how can this knowledge be used for developing pest control strategies?).

Response 4: We sincerely appreciate the reviewer’s constructive suggestions to strengthen the ecological and applied dimensions of our discussion. Below we outline the modifications made to address these points:

We have incorporated additional relevant literature on effective pest control strategies within plant-insect interaction systems and provided concise interpretations.(line 577-579, marked in red) Reference:Tong, Xiwen, Yundan Wang, Pengcheng Yang, Chengshu Wang, and Le Kang. "Tryptamine Accumulation Caused by Deletion of Mrmao-1 in Metarhizium Genome Significantly Enhances Insecticidal Virulence." PLOS Genetics 16, no. 4 (2020): e1008675

Building upon recent studies, we provide preliminary perspectives on translating these research findings into practical applications.

“Recent advancements in synthetic biology and biotechnology have led to the in-creasing application of RNA interference (RNAi) and gene-editing technologies in pest control research[94]. A variety of plant-derived metabolites have already been commercialized for agricultural applications. For example, azadirachtin, derived from Azadirachta indica (neem), has shown exceptional efficacy in pest management [95]. Furthermore, breakthroughs like the de novo synthesis of cembra-trien-ol—an insect repellent derived from natural products—through the mevalonate (MVA) pathway in engineered yeast (Saccharomyces cerevisiae) have established a foundation for the development of next-generation plant-based insecticidal agents [96]. These advancements offer a strategic framework for the future develop-ment of environmentally friendly control methods targeting E. hippophaecola larvae, utilizing natural compounds derived from sea buckthorn.” (line 526-635, marked in red)

Comments 5: The conclusion is somewhat repetitive. The authors should succinctly summarize the key findings and their significance rather than reiterating previous sections.Also, future research directions should be briefly suggested.

Response 5: We thank the reviewer for the constructive suggestion to improve the conciseness and forward-looking perspective of our conclusion.

We have thoroughly revised the Conclusion section to provide a more concise and focused presentation of our key findings and their significance. The modified content is as follows:

“The feeding behavior of E. hippophaecola larvae on H. rhamnoides (sea buckthorn) trunks and roots induced significantly altered both the nutritional profiles and secondary metabolites. Comparative analysis showed that infested roots had significantly higher concentrations of sugars, proteins, lipids, and amino acids compared to damaged trunks, creating a nutritional gradient that led to larval migration from the trunks to the roots. Concurrently, the downregulation of lecithin - a pivotal metabolite in the secondary metabolic network - triggered subsequent decreases in arachidonate and betaine within the resistance metabolism of trunks compared to roots, indicating weakened defense responses. This metabolic shift enhanced the adaptive capacity of trunk-feeding larvae, ultimately driving partial recolonization of trunks. Consequently, elucidating the mechanistic basis of this host-shifting behavior in E. hippophaecola larvae holds significant potential for developing targeted biological control strategies.” （line 637-648，marked in red）

Comments 6: Last but not least, you should double check the whole manuscript to fix minor issues. For example, in the abstract when you mention for the first time Eogystia hippophaecola you should cite the nominators of the species. Something you do on line 70 but wrongly: it is Eogystia hippophaecola Hua, Chou, Fang & Chen, 1990 without parenthesis (you also forgot to mention the year).

Response 6: We sincerely thank the reviewer for their meticulous attention to taxonomic nomenclature details.

The original description (basionym) was published as:
[Original genus] hippophaecola Hua, Chou, Fang & Chen, 1990

The current accepted combination is:
Eogystia hippophaecola (Hua, Chou, Fang & Chen, 1990)

And we have had changed it (line 11, marked in red).

“Yakovlev, R. V. "A New Species and New Records of the Genus Meharia Chretien 1915 (Lepidoptera, Cossidae) from the Middle East and Central Asia." Zoologichesky Zhurnal 97, no. 9 (2018): 1127-31.”

Comments 7: Also, check the whole reference list; it does not follow the MDPI standards, and you must use italics in the scientific names e.g. Lines546, 551, 553 etc.

Response 7: We sincerely appreciate the reviewer's careful reading of our manuscript and their valuable feedback regarding the reference list and scientific nomenclature. We have thoroughly addressed all the points raised.

Following the reviewer’s comments, we have revised the entire reference list to strictly adhere to MDPI’s formatting guidelines and ensured italics for all scientific names throughout the manuscript. Also, we have Double-checked consistency in formatting using reference management software EndNote and manual verification.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Dear Reviewer, Thank you very much for your evaluation of the English language quality in our manuscript. Your assessment is highly encouraging and gives us confidence in the clarity and readability of our writing. We understand that there is still room for improvement, and we will continue to strive for excellence in this regard. If you have any specific suggestions on how we can further enhance the English language quality, such as grammar, vocabulary, or sentence structure, we would greatly appreciate your input. Once again, thank you for you.

5. Additional clarifications

We appreciate the opportunity to improve our manuscript and hope these revisions meet the journal’s standards. Should any additional adjustments be needed, we are happy to comply.

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 revisions/corrections highlighted/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

Some changes were made to this thesis to add the following peer-to-peer responses to the letterThe same as below

Are all the cited references relevant to the research?

Must be improved

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

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

Comments 1: First of all, at the very beginning of the methodological part of the MS in section 2.1. Test Plants and Insects reports: “Six sea buckthorn plants, aged 5 to 8 years and infested with larvae, were selected for the study.” Accordingly, the presentation of the analysis results in the MS raises a legitimate question: how and in what way was this point taken into account, why is the effect of the plant factor in the sample nowhere characterized? And in this regard, there is a legitimate suspicion that by describing the variation of certain analysis results, the authors of the MS violated the methodological requirements that ensure the omission of the formation of pseudoreplications. Let me remind you that the problem of pseudoreplications is very important and requires constant attention (see Hurlbert, S. H. (1984). Pseudoreplication and the design of ecological field experiments. Ecological monographs, 54(2), 187-211.).

Response 1: We sincerely appreciate the reviewer's insightful comments regarding potential pseudoreplication issues in our experimental design. We fully acknowledge the critical importance of proper replication in ecological studies, and have now implemented the following revisions to address these concerns.

To clarify our original methodology:

The six sea buckthorn plants represent independent biological replicates, with each selected individual spaced at least 50 meters apart and situated away from forest edges and interior roads. All measurements were performed separately for each plant, with no cross-pooling of tissues across individuals. The selected sea buckthorn plants exhibited similar infestation levels by larvae of Eogystia hippophaecola, with both trunks and roots being damaged, each individual plant harboring more than 10 observable larvae.

We have substantially improved the methodological descriptions in Section 2.1(line101-108, marked in red).

Statistical control:

First, we have supplemented the methodological descriptions for data analysis pertaining to both nutrient measurements and enzyme activity assays, as detailed in Sections 2.2.2(line190-196, marked in red) and 2.3.4(line 231-238, marked in red), respectively.

In the Results section, we have now included additional statistical analyses as presented in Sections 3.1 (line 296-343, marked in red) and 3.2 (line360-381, marked in red). Should the reviewers deem it necessary, we would be pleased to submit the complete statistical test results tables as supplementary material.

Comments 2: Further, the presentation of the methodological part in the MS often looks very unsatisfactory, for example, in sections 2.2 and 2.3.1. there is not a single reference to the methods used in the work.

Response 2: We sincerely appreciate this insightful comment. We sincerely appreciate the reviewer's constructive comments. In our original submission, certain methodological descriptions in Sections 2.2 and 2.3.1 were intentionally concise due to considerations of manuscript length and potential redundancy. In response to the reviewer's valuable suggestions, we have now comprehensively revised this section to provide more detailed explanations of our experimental procedures with the following improvements:

For Section 2.2, we have systematically documented the analytical protocols for nutrient and mineral element quantification, with detailed methodological descriptions (Lines 127-184,190-196, marked in red) and 12 additional supporting references (References 34-45) corresponding to each analytical procedure.

In Section 2.3.1, all operations for digestive enzyme assays were strictly performed according to the manufacturer's protocols, for which we have specified the commercial kit providers (including company names and locations) and all relevant catalog numbers, with additional citations (References 46-48) incorporated to elucidate the underlying biochemical principles, as detailed in the revised text (Lines 201-203, marked in red).

Concurrently, we have conducted a comprehensive review of the Materials and Methods section and implemented necessary methodological refinements to address identified limitations(section2.4.1, line242-244; section 2.4.3, line 277-280. All the modifications have been marked in red).

Comments 3: Further, the discrepancy between the text and the referenced links indicates negligence in the preparation of the MS. So, the authors write: “Essential amino acids from host plants are crucial for insect protein synthesis and growth. Pieris rapae and Spodoptera exigua larvae prefer host plants with higher levels of essential amino acids [49, 57]”. However, the article under #49 mentions the diamondback moth, but Pieris rapae and Spodoptera exigua are not mentioned at all! In the article under #57, the work was carried out only with Spodoptera exigua and Pieris rapae is mentioned only in the discussion when the article by Huang and Renwick (1992) is cited. Or here we read: “Both butterflies and Spodoptera littoralis preferred sugar solutions containing glutamic acid over the control solution [61, 62],” however, verification indicates that the article at link # 61 deals with a completely different species of Lepidoptera, namely Bicyclus anynana! And here it should be noted that the review did not cover all the articles from the list given in the article, just a few… The spelling of species names in the article indicates the lack of biological education of the authors: they capitalize the species name everywhere: Spodoptera Litura, Chorthippus Parallelus, Plutella Xylostella, Carposina Sasakii, Pinus Sylvestris, Chrysomela Vigintipunctata, etc.! The list of references, by the way, is completely ugly, for example, we read: Wu, K. J., and M. H. Li. "Nutritional Ecology of the Cotton Bollworm, Helicoverpa Annigera (Hubener): Life Table of the Population on the Artificial Diets with Different Protein Levels." (1993), the number of errors here is simply off the scale.

Response 3: We sincerely apologize for these citation inaccuracies and taxonomic formatting errors, which resulted from insufficient verification during literature compilation. We have now conducted a systematic review of all citations and species nomenclature throughout the manuscript, implementing the following corrections:

“Essential amino acids from host plants are crucial for insect protein synthesis and growth. Pieris rapae and Spodoptera exigua larvae prefer host plants with higher levels of essential amino acids [49, 57]”

We have changed into: Plutella xylostella [67]and Spodoptera exigua [75]larvae prefer host plants with higher levels of essential amino acids.

“Both butterflies and Spodoptera littoralis preferred sugar solutions containing glutamic acid over the control solution [61, 62],”

We have changed into: Under starvation stress, Bicyclus anynana larvae tend to feed on sugar solutions containing glutamate[79]. Results from the "Styropor method" assay on Spodoptera littoralis larvae demonstrated that a mixture of 0.125 M L-glutamic acid and 0.125 M sucrose exhibited a phagostimulatory effect[80].

“The spelling of species names in the article indicates the lack of biological education of the authors: they capitalize the species name everywhere: Spodoptera Litura, Chorthippus Parallelus, Plutella Xylostella, Carposina Sasakii, Pinus Sylvestris, Chrysomela Vigintipunctata, etc.!”

We sincerely appreciate your meticulous review of our manuscript. Regarding the issue of the capitalization of Latin names in the references, we apologize for not conducting a thorough proofreading after using EndNote. Therefore, we have examined and reorganized all the Latin names mentioned throughout the full text to ensure, to the greatest extent possible, that there will be no further errors.

“Wu, K. J., and M. H. Li. "Nutritional Ecology of the Cotton Bollworm, Helicoverpa Annigera (Hubener): Life Table of the Population on the Artificial Diets with Different Protein Levels." (1993), the number of errors here is simply off the scale.”

We are extremely grateful to you for pointing out the errors in the references of this article. We have carefully reviewed all the references cited in new latest article, replaced those with problems, and added more rigorous references(line 40,44,47,53).

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: Dear Reviewer, Thank you for your feedback regarding the English language in our manuscript. We understand your concern about the need for clearer expression of our research. Before submission, we had our paper reviewed and revised by a native English speaker to enhance clarity and fluency. However, we acknowledge that there might still be room for improvement. In response to your comment, we have conducted a thorough in - house review of the manuscript.  We have focused on simplifying complex language, ensuring logical flow, and making the content more accessible to a wide range of readers. We hope that the revised version will meet your expectations in terms of clear and effective communication of our research.

5. Additional clarifications

Once again, we sincerely appreciate the suggestions you provided regarding our experimental design, materials and methods, reference standardization and etc. We have made corresponding revisions and improvements to these parts. If you require any further information, please feel free to let us know.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/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?

Must be improved/

Some changes were made to this thesis to add the following peer-to-peer responses to the letter. The same as below

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?

Yes

Are the conclusions supported by the results?

Yes

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

Comments 1: At the same time, when re-reading the MS, a phrase was found in the text (unfortunately it was omitted during the previous review), which attracted very close attention: “In Liaoning, China, E. hippophaecola completes a four-year life cycle, with larvae going through 16 instars.” The question is that the above-mentioned life cycle length of a moth and the number of molts of larvae are more than extremely long. Indeed, in some species of moths, 8 and even 9 molts are known in larvae, as well as the duration of development sometimes lasts 2 years, but the development characteristics given in the MS are simply amazing. In the MS, this phrase is given in the Introduction and is not supported by any reference to a literary source. Obviously, if the above phrase is saved in the MS, it cannot be given without a supporting link.

Response 1: Thank you for pointing this out. We agree with this comment. We apologize for the omission of relevant literature citations in our previous manuscript and hereby provide supplemental references. Regarding the life history of Eogystia hippophaecola, the statement "In Liaoning, China, E. hippophaecola completes a four-year life cycle, with larvae going through 16 instars" is neither erroneous nor unsubstantiated but is supported by documented research. As explicitly stated in the doctoral dissertation by Zong Shixiang (2006): "Studies on the Bio-Ecological Characteristics of Seabuckthorn Carpenter Moth: Holcocerus hippophaecolus (Lepidoptera: Cossidae)", this species exhibits a quadrennial developmental cycle accompanied by 16 larval instars under Liaoning’s climatic conditions. The original text is as follows:

“The research results of biological characteristics indoor and outdoor showed that a generation of H. hippophaecolus lasted four years in Liaoning province, the mature larvae begin to pupate in the first of May under the ground and the moths emerge from the end of May to the early of September with two peaks of eclosion, the middle of June and the last of July respectively.”

“The larvae were identified to be sixteen instars through measuring the width of head, the length and width of body and mandible. The length of prothorax lateral seta and the size of abdominal prolegs can be used to identify the different instars of larvae.”

The Latin name Holcocerus hippophaecolus was revised to Eogystia hippophaecola (Hua, Chou, Fang & Chen, 1990) in the 2018 publication: Yakovlev, R. V. "A New Species and New Records of the Genus Meharia Chrétien, 1915 (Lepidoptera, Cossidae) from the Middle East and Central Asia." Zoologichesky Zhurnal 97, no. 9 (2018): 1127–31.

Therefore, we have supplemented the original sentence with the corresponding reference, and the modification can be found in lines 74–79and line723-724 of the original manuscript.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Dear Reviewer, Thank you very much for your evaluation of the English language quality in our manuscript. Your assessment is highly encouraging and gives us confidence in the clarity and readability of our writing. We understand that there is still room for improvement, and we will continue to strive for excellence in this regard. If you have any specific suggestions on how we can further enhance the English language quality, such as grammar, vocabulary, or sentence structure, we would greatly appreciate your input. Once again, thank you for you.

5. Additional clarifications

Thank you again for your guidance.

If you need any further information, please don't hesitate to let us know!