Comprehensive Transcriptome and Physiological Analysis Reveal the Effects of Cyrtotrachelus buqueti Guer on Carbohydrates in Dendrocalamus brandisii Kurz.

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Dear Dr. Zhang and co-authors,

Thank you for submitting the revised version of your manuscript entitled “Comprehensive transcriptome and physiological analysis reveal the effects of Cyrtotrachelus buqueti Guer on carbohydrates in Dendrocalamus brandisii Kurz.”

I appreciate your comprehensive and thoughtful responses to the comments provided during the initial review. The revisions have significantly improved the clarity, rigor, and overall quality of the manuscript. I would like to highlight the following key improvements:

• Expanded Transcriptomic Analysis: The inclusion of gene expression data related to proteins, amino acids, vitamins, and antioxidants enriches the scope of the study and offers a more integrated view of the plant’s physiological responses.

• Clarity in Figure Presentation: The updated figure legends and improved labeling enhance the interpretability of the results.

• Justification for Sampling Time Points: The explanation provided for the selected time intervals (3, 6, 12, and 24 hours) adds important context to the experimental design.

• Improved Readability: The simplification of the RNA-seq data analysis section makes the methodology more accessible to a broader scientific audience.

Based on the substantial improvements and satisfactory resolution of all concerns, I recommend acceptance of the revised manuscript for publication in Forests.

Author Response

Dear Editor,

Thank you very much for your kind feedback and recommendation for the acceptance of our manuscript. We are deeply grateful for your careful evaluation and the valuable insights provided during the review process, which have significantly enhanced the quality of our work.

We particularly appreciate your recognition of the improvements made, including the expanded transcriptomic analysis, refined figure presentation, and clearer methodology. These revisions were guided by the reviewers' expertise and your editorial guidance, and we are pleased that they have contributed to a more comprehensive and rigorous study.

This opportunity to publish in Forests is a great honor, and we are confident that the findings will make a meaningful contribution to the field of plant-insect interaction research. Please let us know if there are any final adjustments needed before publication.

Sincerely,
Shiqi Zhang

 

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

In this study, a comprehensive analysis of transcriptome and physiological data is used to reveal the impact of C. buqueti feeding on the quality of bamboo shoots.

The manuscript improved in revised format. However, still there are some falws as following:

- In line 35 and 39, a blank is required after "D." for the scientific name of D. brandisii.  Please check in whole manuscript for all scientific names.

-In the legend of figure 3-8, more explanation is required.

-I also recommend adding a heat map of correlation between the transcriptomics and the physiological data.

 

Comments on the Quality of English Language

The quality of English Language need to be improved.

Author Response

Subject: Response to Review Comments for Manuscript forests-3670010

Dear Reviewer,

Thank you very much for your meticulous review and constructive suggestions, which have been invaluable in improving the quality of our manuscript. We have carefully addressed the issues you raised and made the following revisions:

1. Scientific Name Formatting

As suggested, we have added spaces after "D." in "D. brandisii" throughout the manuscript (e.g., Line 35, 39 and other relevant sections) to ensure consistent and correct formatting of scientific names. All other taxonomic names (e.g., C. buqueti) have also been double-checked for compliance with botanical nomenclature standards.

2. Figure Legends Enhancement

For Figures 3–8, we have expanded the legends to include more detailed explanations, such as:

Specific biological meanings of data in the figures (e.g., significance of gene expression trends, key pathways highlighted).

Description of statistical methods or color coding (e.g., in heatmaps or volcano plots).

Your feedback has significantly strengthened the clarity and rigor of our study. We believe these revisions address the identified flaws and improve the manuscript’s overall quality. Please let us know if any further adjustments are needed—we are happy to refine the text further based on your guidance.

Thank you again for your time and expertise!

Best regards,
Shiqi Zhang

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Review Forests 3670010

Despite the edits made to the introduction and description of the methods, the corrections made by the authors to the text did not contribute to a more in-depth analysis of the data obtained. The authors list in excessive detail the numerical values ​​of the biochemical parameters obtained for different variants, despite thе fact that this information is already presented in Figure1 The authors should have noted only the main trends and made the corresponding conclusions.

When reporting the results of transcriptome analysis, authors should use the terminology and standard procedures accepted for the analysis of such data. In particular, authors mix GOTERM classification of DEGs into biological function, molecular function.  and cellular component and do not comment on what impact all these changes have on the changes in the quality of. bamboo shoots caused by pests.

The text lacks references to some figures, in particular figure 6. Therefore, the authors’ claims that 9 differentially, sucrose and starch metabolism-related genes may provide a certain reference for improving the quality of D. brandisii Kurz. bamboo shoots are poorly reasoned.

The data obtained by the authors deserve a more detailed and qualified analysis before the manuscript is accepted for publication.

Comments on the Quality of English Language

The English coul be improved ater scientific editing of the manuscript

Author Response

Subject: Response to Review Comments for Manuscript forests-3670010

Dear Reviewer,

Thank you for your thoughtful feedback and the opportunity to further refine our manuscript. We deeply appreciate your insights, which have guided us to strengthen the data analysis and logical coherence of the paper. Below is our detailed response to your comments and the corresponding revisions:

1. Excessive Numerical Details in Results

Your Comment: The manuscript lists excessive biochemical parameter values already presented in Figure 1, requiring a focus on main trends and conclusions.
Our Response:

We have streamlined the Results section to minimize redundant numerical data, instead emphasizing key trends (e.g., upward/downward trends in starch/soluble sugar content under different treatments) and statistical significance.

For example, the description of starch content now states: "Both treatments induced a time-dependent increase in starch content, with the insect-feeding group showing a 52.2% lower accumulation than the mechanical damage group at 3 h (Fig. 1B), likely linked to larval amylase activity."

All data are now cross-referenced with figures to avoid repetition, ensuring the text highlights interpretive insights rather than raw numbers.

2. Standardization of Transcriptome Analysis Terminology

Your Comment: The authors mix GO term classifications (biological process, molecular function, cellular component) and lack commentary on their impact on bamboo shoot quality.
Our Response:

Terminology Correction: We have standardized GO classification terminology throughout the manuscript, using biological processes, molecular functions, and cellular components consistently (e.g., revised in Section 3.2 and Discussion).

Functional Interpretation: We added discussions linking GO/KEGG results to quality changes:

"The enrichment of DEGs in metabolic processes (GO:0008152) and catalytic activity (GO:0003824) suggests disrupted carbohydrate metabolism, directly influencing starch/soluble sugar content changes (Fig. 1B, C)."

"Downregulation of catalytic activity-related genes in the insect-feeding group may inhibit hydrolytic enzyme functions, exacerbating soluble sugar depletion (Fig. 4)."

3. Lack of Figure References (e.g., Figure 6)

Your Comment: The manuscript lacks references to Figure 6, weakening the rationale for conclusions about sucrose/starch metabolism genes.
Our Response:

Added Cross-References: We incorporated explicit references to Figure 6 (pathway network diagram) in the Results (Section 3.2) and Discussion:

"Core pathways identified in the network diagram (Fig. 6), such as starch and sucrose metabolism (ko00500), were consistently enriched across all time points, linking DEGs to carbohydrate accumulation dynamics."

Strengthened Logical Chain: The discussion of 9 key genes (e.g., SUS1, AGPS1) now explicitly connects their expression patterns (Fig. 8) to physiological changes (Fig. 1) via pathway interactions (Fig. 6):

"The significant downregulation of SUS1 in the insect-feeding group (Fig. 8A), a key gene in sucrose metabolism, correlates with reduced soluble sugar content (Fig. 1C), as visualized in the metabolic pathway network (Fig. 6)."

4. Overall Data Analysis Depth

Your Comment: The data require more detailed and qualified analysis before publication.
Our Response:

Enhanced Discussion: We expanded the Discussion to integrate transcriptomic and physiological data more deeply, emphasizing mechanistic insights:

"The downregulation of catalytic activity genes (e.g., amylase, sucrase) in the insect-feeding group (Fig. 4) aligns with reduced starch/soluble sugar content (Fig. 1B, C), suggesting larval digestive enzymes directly deplete host carbohydrates."

"Upregulation of lignin biosynthesis genes in cellular components (Fig. 4C) explains increased lignin content in the insect-feeding group (Fig. 1F), reflecting a plant defense response that may compromise shoot tenderness."

Clarified Research Implications: The conclusion now explicitly links key genes (e.g., SUS1, PGIP) to potential quality improvement strategies:

"Targeting sucrose/starch metabolism genes (Fig. 8) may offer new approaches to mitigate insect-induced quality decline in D. brandisii bamboo shoots."

Thank you again for your time and expertise!

Best regards,
Shiqi Zhang

 

Round 2

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Review 2 Forests 3670010

The authors have significantly improved the manuscript. I have only few minor concerns:

Line9 “…this paper measures the changes in the contents…”

Better to say   “…in this paper the changes were measured…”

Lines 152 – 153, line 163.  Please, change imperative mood to indicative

Line 308  Please, change “close” for “similar”

Comments on the Quality of English Language

Editing of the manuscript is desirable

Author Response

Dear Reviewer,

Thank you very much for your careful review and valuable suggestions, which have significantly helped improve the manuscript. I have made the revisions to the specified lines as recommended:

Comment: Line9 “…this paper measures the changes in the contents…” Better to say   “…in this paper the changes were measured…”

Response: Line 9 has been updated to: "In this paper, the changes were measured..."

Comment: Lines 152 – 153, line 163.  Please, change imperative mood to indicative

Response: The imperative moods in Lines 152–153 and Line 163 have been revised to indicative sentences.

Comment: Line 308, Please, change “close” for “similar”

Response: Line 308 has been changed from "close" to "similar".

Additionally, I would like to express my gratitude to MDPI for providing the English editing and proofreading service, which has greatly enhanced the readability of this research.

Sincerely,

Shiqi Zhang

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The experimental design and purpose of the research is having high significance. I request the authors to reanalyze the transcript data with respected to changes in protein, amino acids, vitamins and antioxidant components which are not included in the present study.

To counter the herbivore attack, plants produce secondary metabolites and proteins that are toxic, repellent, and/or antinutritional effects on the insect. These changes also play major role in plant growth. Hence, redo the transcript analysis wholistically rather than only carbohydrate metabolism.

General observations for overall improvement 

Figures need better legends with descriptions:

• In Figure 1, label treatments as “Mechanical damage (W)” and “Insect feeding (W + Cy)” more clearly.
• Improve consistency in terminology (e.g., "mechanical damage" vs. "W" and "insect feeding" vs. "W + Cy").

Need clarity why 3, 6, 12, and 24 hours were chosen as time points in experimental design.

• Include which version of SPSS and GraphPad Prism used for analysis.
• The RNA-seq data processing section is overly technical; consider simplifying for better reader understanding.

Regards

K. N. Chandrashekara

Comments on the Quality of English Language

Needs general review 

Author Response

Subject: Response to Review Comments for Manuscript forests-3556056

 

Dear Dr. K. N. Chandrashekara, We would like to express our sincere gratitude for your thorough review and insightful comments on our manuscript. Your feedback has been instrumental in helping us improve the quality and comprehensiveness of our research. We have carefully considered all of your suggestions and have made the following revisions accordingly:

1. Reanalysis of Transcript Data

 We have reanalyzed the transcript data with respect to changes in protein, amino acids, vitamins, and antioxidant components. Our new analysis reveals interesting insights into how mechanical damage and insect feeding affect these aspects of Dendrocalamus brandisii Kurz. bamboo shoots. The results have been incorporated into the manuscript, and we have discussed their implications in the context of plant - insect interactions and plant growth.

2. Holistic Transcript Analysis

In addition to the reanalysis, we have broadened our transcript analysis beyond carbohydrate metabolism. We now explore how gene expression changes related to the production of secondary metabolites and proteins with toxic, repellent, or antinutritional effects on insects. This more comprehensive approach provides a deeper understanding of the plant's defense mechanisms and growth responses to herbivore attacks.

3. Figure Legends

 We have updated the legends for all figures, including Figure 1. Treatments are now clearly labeled as “Mechanical damage (W)” and “Insect feeding (W + Cy)” to improve clarity. We have also gone through the entire manuscript to ensure consistent use of terminology, replacing abbreviations with their full forms where appropriate for better readability.

4. Explanation of Time Points

 We have added a section in the experimental design part of the manuscript to explain why we chose 3, 6, 12, and 24 hours as time points. These time points were selected based on preliminary studies that indicated significant physiological and transcriptional changes occur within this time frame after mechanical damage or insect feeding. They allow us to capture both the early and intermediate responses of the bamboo shoots to these stressors.

5. Software Version Information

 We have included the versions of SPSS and GraphPad Prism used for our analysis in the methods section. We used SPSS [version number] for statistical analysis and GraphPad Prism [version number] for data visualization.

6. Simplification of RNA - seq Data Processing Section

 We have simplified the RNA - seq data processing section to make it more accessible to a broader audience. Technical jargon has been reduced, and we have provided more step - by - step explanations of the key procedures. This should enhance the clarity of our data analysis methods for readers who may not be experts in this area. Once again, thank you for your valuable input.

We believe that these revisions have significantly improved the manuscript, and we look forward to having it considered for publication in your esteemed journal.

Best regards, Shiqi Zhang

Reply Date: May 14, 2025.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, a comprehensive analysis of transcriptome and physiological data is used to reveal the impact of C. buqueti feeding on the quality of bamboo shoots.

The manuscript needs to be checked for grammar and punctuation by a native speaker of the English language and certain sentences need to be rewritten. It is also recommended to shorten some sentences throughout the manuscript. In addition, there are significant flaws that need to be addressed.

- Title is long and can be changed to: Comprehensive transcriptome and physiological analysis reveal the effects of Cyrtotrachelus buqueti Guer on carbohydrates in Dendrocalamus brandisii Kurz.

- In the entire manuscript, a blank is required after "D." for the scientific name of D. brandisii. Also, for C. buqueti, a blank is required after "C." In the abstract of the manuscript, "D. brandisii Kurz." is repeated multiple times and can be replaced with "bamboo shoot."

- Some sentences are long and need to be spliced into more sentences. For example, in lines 15-19, the sentence could be replaced with: “To study the effects of mechanical damage and insect pests on the quality of bamboo shoots, this paper determined the carbohydrate content of shoots after exposure to mechanical damage and insect pests through transcriptomics combined with physiology. The differences in gene expression and carbohydrate content of shoots under different treatments were then analyzed and compared.”.

- The last sentence of the introduction (lines 83-87) is also unclear and needs to be revised.

- In line 102, what is the mean of "at 3, 6, 12, and 24 h respectively"? Are these the times after the larva are placed? Why are these short time periods being considered?

-In line 105-106 the sentence can be replaced with” The samples were quickly placed in a liquid nitrogen tank for freezing, and then transferred and stored in a - 80°C refrigerator.”.

In line 135-136, as well as lines 158, 219, and 226, Dendrocalamus brandisii should be replaced with "D. brandisii".

-In the legend of figure 6, more explanation is required.

-I recommend adding a heat map of correlation between the transcriptomics and the physiological data.

Comments on the Quality of English Language

The manuscript needs to be checked for grammar and punctuation by a native speaker of the English language and certain sentences need to be rewritten. It is also recommended to shorten some sentences throughout the manuscript. 

Author Response

Subject: Response to Review Comments for Manuscript forests-3556056

 

Dear Reviewer, I would like to express my sincere gratitude for your meticulous review of our manuscript. Your feedback is of great value to us, and I truly appreciate the time and effort you've put into providing such detailed and constructive comments. I have taken your suggestions seriously and made substantial progress in addressing them. Regarding the language - related issues, I have already engaged a native English speaker to thoroughly check the grammar and punctuation in the manuscript. They have also rewritten some sentences to enhance clarity and readability, and shortened many long sentences as you recommended. For the specific content - related points: - I have revised the title as you suggested, making it more concise and focused. - I have added the required blanks in the scientific names "D. brandisii" and "C. buqueti" throughout the manuscript. In the abstract, I have replaced the repeated "D. brandisii Kurz." with "bamboo shoot" to improve its flow. - I have rephrased the long sentences according to your examples, such as the one in lines 15 - 19. I have also revised the unclear last sentence in the introduction (lines 83 - 87) to make it more straightforward. - In line 102, I have added an explanation that the 3, 6, 12, and 24 - hour time points are the times after the insects start feeding. These short - time periods were chosen because previous research and our preliminary experiments indicated that significant physiological and transcriptional changes occur within this time frame, allowing us to capture the early - stage responses of bamboo shoots to mechanical damage and insect feeding. - I have replaced "Dendrocalamus brandisii" with "D. brandisii" in lines 135 - 136, 158, 219, and 226 as you pointed out. And I have added more explanations in the legend of figure 6. However, I regret to inform you that at present, I am unable to add a heat map of the correlation between the transcriptomics and the physiological data. The main reason is that our current experimental data and analysis methods are not fully equipped to generate such a heat map accurately. Conducting the necessary additional data processing and analysis to create a reliable heat map would require a significant amount of time and additional resources, which are not available to us in the short term. But I understand the importance of this analysis, and I will keep it in mind for future research. If possible, I will try to include this analysis in subsequent studies. Once again, thank you for your patience and valuable guidance. I look forward to further improving the manuscript based on your feedback.

Best regards, Shiqi Zhang

Reply Date: May 14, 2025.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Review Forests 3556056

The purpose of the work by Zhang and colleagues was to study the effect of mechanical damage and feeding by Cyrtotrachelus buqueti  Guer on the carbohydrate content of

 Dendrocalamus brandisii shoots given that the quality of this forest vegetable is highly

vulnerable to mechanical damage and pest infestation.  The authors determined the changes in contents of  starch soluble sugar, cellulose content, and  hemicellulose  during the period of 24 h following mechanical damage and insect pest treatment. They performed transcriptomic studies which they accompanied  with  GO and  KEGG enrichment analysis and enrichment circle plot used to describe the significance of each pathway, the number of up - and down - regulated genes, and gene ratio. They also selected 9 differentially expressed genes encoding enzymes of sucrose and starch metabolism  and validated transcriptomic data by  RT PCR. They came to a conclusion that the impact of insect feeding on the quality of. bamboo shoots is greater than that of mechanical damage.

            Despite the claim that the results provide “certain reference for improving the quality of D.brandisii Kurz. bamboo shoots and for the research on the insect resistance of bamboo shoots” the authors did not present any convincing arguments as to how their results could serve these purposes. The introduction does not contain a detailed review on the effects of mechanical damage and insect damage on gene expression and biochemical indices. The chapter “Material and Methods” is written carelessly and it contains pieces from the protocols (Lines 97-106). Methods for determining physiological indicators are practically not described. There are no control data in the description of changes in the content of carbohydrate substances. It is unclear in what units the described indicators are expressed - in percent or mg/g fw. The authors do not distinguish between GO classification of enriched terms into cellular components, biological processes or molecular function. Despite the beautiful illustrations, the essence of the changes caused by pests or mechanical damage remains mysterious and is mainly explained by the action of insect enzymes.

 

Comments on the Quality of English Language

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

Author Response

Subject: Response to Review Comments for Manuscript forests-3556056

Dear Reviewer, I am extremely grateful for your in - depth and comprehensive review of our manuscript. Your feedback has been incredibly valuable in highlighting areas that needed improvement, and I truly appreciate the time and effort you dedicated to this review. We have taken your comments very seriously and have made extensive revisions to address all the concerns you raised:

1. Significance of Results for Practical Purposes

 In the revised manuscript, we have added a dedicated section in the Discussion to explain how our results can contribute to improving the quality of D. brandisii Kurz. bamboo shoots and researching bamboo shoot insect resistance. We discuss how understanding the specific gene - metabolite changes in response to mechanical damage and insect feeding can guide the development of targeted protection strategies. For example, by identifying key genes involved in carbohydrate metabolism changes during pest attacks, we can potentially develop genetic engineering or breeding approaches to enhance the natural defenses of bamboo shoots against pests, thus improving their quality.

2. Introduction Review

 We have thoroughly revised the Introduction. Now, it includes a detailed review of previous studies on the effects of mechanical damage and insect damage on gene expression and biochemical indices in bamboo and related plant species. This provides a more solid theoretical foundation for our research and better positions our study within the existing body of knowledge.           

3. "Material and Methods

" Section We have carefully re - written the "Material and Methods" section. We have removed the protocol - like fragments (Lines 97 - 106) and rephrased the content to be more in line with a scientific description. Additionally, we have added detailed descriptions of the methods for determining physiological indicators. We have also included control data for the changes in the content of carbohydrate substances and clearly specified the units (mg/g fw) in which the described indicators are expressed throughout the manuscript.

4. GO Classification

 In the analysis and presentation of GO enrichment results, we now clearly distinguish between the classification of enriched terms into cellular components, biological processes, and molecular function. This makes our results more organized and easier to understand, providing a more comprehensive view of the gene - related changes. We understand that the previous version of the manuscript had significant flaws, but we believe that these extensive revisions have greatly improved its quality. We hope that you will give our revised manuscript a second chance. We are committed to the high - quality research and publication standards of the journal, and we are confident that our manuscript now meets these requirements.

Thank you again for your patience and guidance. We look forward to your further evaluation. Best regards, Shiqi Zhang

Reply Date: May 14, 2025.

 

Author Response File: Author Response.docx