Specific Function and Assembly of Crucial Microbes for Dendroctonus armandi Tsai et Li

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Corresponding Author
I checked your paper. In fact, the comparison of gut vs. cuticular microbiome in D. armandi is a new aspect. But, parts of the discussion part of the paper overstate the uniqueness of the research. Some research on Dendroctonus and Ips beetles (for example, Hu et al. 2014, 2015; Chakraborty et al. 2020) already evaluated similar things. The paper should more clearly focus what is truly novel about this project.
1) Guts and cuticles from 70 beetles were pooled per replicate. Pooling is understandable for DNA yield, but it masks intra-individual variation and may inflate statistical independence. The limitations of this design should be explicitly acknowledged.
2) Only three replicates per tissue type were sequenced. While typical in metagenomic studies, this reduces statistical robustness. More emphasis on biological vs. technical replication is needed.
3) Reads were mapped to Dendroctonus valens genome. This may bias assembly and annotation. Justification is needed for not using D. armandi genomic resources, or at least the implications of using a related species should be discussed.
4) The claim that gut microbes are more enriched in pathways degrading terpenoids, phenolics, and polysaccharides is interesting. However, statistical evidence is limited. Most comparisons rely on unigene counts; formal statistical testing (e.g., DESeq2 for functional gene abundance) should be applied.
5) The βNTI approach is appropriate, but the thresholding and interpretation are sometimes oversimplified. For example, the distinction between stochasticity and determinism is not binary; the combined effects should be better emphasized.
6) The authors note higher fungal diversity on cuticles, but the lack of statistical significance (p > 0.05) contradicts the narrative. The text should more accurately reflect the results.
Regards

Author Response

I checked your paper. In fact, the comparison of gut vs. cuticular microbiome in D. armandi is a new aspect. But, parts of the discussion part of the paper overstate the uniqueness of the research. Some research on Dendroctonus and Ips beetles (for example, Hu et al. 2014, 2015; Chakraborty et al. 2020) already evaluated similar things. The paper should more clearly focus what is truly novel about this project.

Response: Thank you for pointing this out. We have revised the whole manuscript, particularly, introduction (see in lines 35-63 and 70-72) and discussion (see in lines 311-314, 316-319, 330-335 and 356-359). And we supplemented the novel sentence in the revised manuscript (see in lines 56-63).

Comments 1: Guts and cuticles from 70 beetles were pooled per replicate. Pooling is understandable for DNA yield, but it masks intra-individual variation and may inflate statistical independence. The limitations of this design should be explicitly acknowledged.

Response 1: Thank you for pointing this out. A total of 70 bark beetles were pooled for every sample to minimize the differences among individuals. We supplemented this sentence in revised manuscript (see in lines 87-88).

Comments 2: Only three replicates per tissue type were sequenced. While typical in metagenomic studies, this reduces statistical robustness. More emphasis on biological vs. technical replication is needed.

Response 2: Thank you for the reviewers' suggestions. We agree that three replicates may limit the statistical robustness, but it may be conventional practice for the metagenomic research, for example, only one metagenome for a sample in the article by Adams (2013), and two metagenomes for every sample in the article by Ceriani-Nakamurakare (2018). And it had sufficient sequencing depth for species and genes in the gut and cuticular samples to confirm the feasibility analysis of the data (see Figure S1). Future research will increase the biological or technological replication in order to further verify findings.

Adams, A.S.; Aylward, F.O.; Adams, S.M.; Erbilgin, N.; Aukema, B.H.; Aukema, C.R.; Suen, G.; Raffa, K.F. Mountain pine beetles colonizing historical and naive host trees are associated with a bacterial community highly enriched in genes contributing to terpene metabolism. Applied and environmental microbiology, 2013, 79, 3468-3475.

Ceriani-Nakamurakare, E.; Ramos, S.; Robles, C.A.; Novas, M.V.; D´Jonsiles, M.F.; Gonzalez-Audino, P.; Carmarán, C. Metagenomic approach of associated fungi with Megaplatypus mutatus (Coleoptera: Platypodinae). Silva Fennica, 2018, 52.

Comments 3: Reads were mapped to Dendroctonus valens genome. This may bias assembly and annotation. Justification is needed for not using D. armandi genomic resources, or at least the implications of using a related species should be discussed.

Response 3: We agree with the reviewer's point of view. using the D. valens genome instead of the reference sequence of the closely related species D. armandi might bias assembly and annotation. But D. valens is currently the only closely related species that has completed the assembly at the chromosomal level in China. And ecological habits of D. valens are more compatible with those of D. armandi.

Liu, Z.; Xing, L.; Huang, W.; Liu, B.; Wan, F.; Raffa, K.F.; Hofstetter, R.W.; Qian, W.; Sun, J. Chromosome-level genome assembly and population genomic analyses provide insights into adaptive evolution of the red turpentine beetle, Dendroctonus valens. BMC Biology, 2022.

Comments 4: The claim that gut microbes are more enriched in pathways degrading terpenoids, phenolics, and polysaccharides is interesting. However, statistical evidence is limited. Most comparisons rely on unigene counts; formal statistical testing (e.g., DESeq2 for functional gene abundance) should be applied.

Response 4: We thank you for reviewer's comments. The statistical evidence is limited. We conducted formal statistical testing, such as Figure 3a (see in line 200), it showed that it had a slight difference in the galactose metabolism pathway. There was no difference for other pathways. However, we found that the number of unigenes for degrading complex compounds was distinct in the gut and cuticular samples. therefore, we studied the enzymes and unigenes for degradation of complex compounds.

Comments 5: The βNTI approach is appropriate, but the thresholding and interpretation are sometimes oversimplified. For example, the distinction between stochasticity and determinism is not binary; the combined effects should be better emphasized.

Response 5: Thank you for your suggestions. We have made revisions to the results (see in lines 271-272) and discussion (see in lines 386-388) sections accordingly.

Comments 6: The authors note higher fungal diversity on cuticles, but the lack of statistical significance (p > 0.05) contradicts the narrative. The text should more accurately reflect the results.

Response 6: Thank you for pointing out this key issue. We have adjusted this sentence in the revised manuscript of lines 173-175.

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction

 

Line 38 It would be beneficial to talk a bit about taxonomy and classification of bark beetles

 

Materials and Methods

Line 84 How many logs were used for the study and was there proper authorization to collect both the logs and the beetles

Line 87 I am not sure how many samples were pooled for each of the two groups (cuticle and gut). The authors say that 70 samples were pooled, but the total number of samples is 210. It would be better to provide clarification on the actual number of samples. Do the authors mean that each three groups for gut and three groups for cuticles were obtained from the total samples and the statistical analyses conducted aimed to assess differences among a total of 6 samples (3 guts and 3 cuticles)?

If this is the case, could the author provide the rational of pooling data into three groups?

Was a normality test conducted on the data?

Line 88 Could you provide some information on how the different samples (cuticle and guts) were kept separated during dissection without causing contamination?

 

Table 3 the text in the first column should be fixed

 

I think that some clarifications on the points listed above should be provided to improve understanding of the paper; having said that, this is a valuable paper that discusses important results obtained through well-established techniques. Great job

Author Response

Comments 1: Introduction

Line 38 It would be beneficial to talk a bit about taxonomy and classification of bark beetles.

Response 1: We have added the meaning of study bark beetles (see in lines 35-36).

Comments 2: Materials and Methods

Line 84 How many logs were used for the study and was there proper authorization to collect both the logs and the beetles

Response 2: We adopted the bark beetles from different tunnels of the same log with the help of local forestry workers. And the data has been uploaded to NCBI publicly.

Comments 3: Line 87 I am not sure how many samples were pooled for each of the two groups (cuticle and gut). The authors say that 70 samples were pooled, but the total number of samples is 210. It would be better to provide clarification on the actual number of samples. Do the authors mean that each three groups for gut and three groups for cuticles were obtained from the total samples and the statistical analyses conducted aimed to assess differences among a total of 6 samples (3 guts and 3 cuticles)?

If this is the case, could the author provide the rational of pooling data into three groups?

Response 3: We randomly selected 70 individuals for each sample, and placed their gut and remanent tissues into two sterile tubes respectively. All the operations were carried out on ice.

Comments 4: Was a normality test conducted on the data?

Response 4: We used SPSS to analyze the microbial diversity. During this process, we conducted a normality test (see in line 109).

Comments 5: Line 88 Could you provide some information on how the different samples (cuticle and guts) were kept separated during dissection without causing contamination?

Response 5: A handful of samples were taken from the -80°C freezer and placed on ice (to prevent DNA degradation). Firstly, the midgut and hindgut were removed, then the head and prothorax were separated using sterile forceps and pipettes, and the anterior gut was extracted. All the guts were placed in one sterile tube, and the remaining tissues were placed in another sterile tube, which was taken as a gut sample and a cuticular sample. The other samples are operated in the same way as the above ones. All the processes were conducted on the ice. After the operation, the samples were rapidly frozen in liquid nitrogen, placed in the -80°C freezer, and sent for analysis within one week.

Comments 6: Table 3 the text in the first column should be fixed

Response 6: We have revised it (see in line 273).

Comments 7: I think that some clarifications on the points listed above should be provided to improve understanding of the paper; having said that, this is a valuable paper that discusses important results obtained through well-established techniques. Great job

Response 7: Thank the reviewer for the positive feedback on our manuscript. We have revised the whole manuscript carefully and hope to meet the standard.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript provides useful insights into the gut and cuticular microbiomes of Dendroctonus armandi using metagenomics. The topic is important for both basic science and potential applications in pest management. The study is well-designed overall, but some areas need clarification and improvement before the paper can be accepted.

Major Comments

1. Abstract:

• The practical importance of the study needs to be highlighted
• Strong claims are made based on bioinformatics inference rather than experimental validation. So soften the claims.

2. Introduction:

• Should cite more recent studies (after 2020) on metagenomics in bark beetles and other forest insects
• Novelty is not clearly stated. Please explain how this study differs from prior work on valens, D. rhizophagus, and Ips species.

3. Materials and Methods:

• Since 70 guts were pooled together, it becomes hard to see how much variation exists between individual insects. Please explain why this approach was chosen.
• More detail is needed on sequencing depth, data filtering, and how contamination was avoided.
• The choice of valens as a reference genome for annotation should be justified, as it may influence functional predictions.
• Mention which software and exact parameters were used for statistical analysis?

4. Result:

• Bacterial dominance linked to diet is speculative. These should be moved to the Discussion.
• Ecological meaning of “deterministic vs stochastic” processes should be explained more cautiously
• Figure 2, 3: Overcrowded. Legends need to more clearer

5. Discussion:

• Functional inferences on terpenoid degradation, phenolic detoxification are based on prediction not by experimental validation
• Discussion repeats some results. Provide more interpretation than repetitive data
• Expand applied perspective like pest management, microbial manipulation.
• Many sentences are too long.

Comments on the Quality of English Language

Some sentences are too long need to be improved

Author Response

The manuscript provides useful insights into the gut and cuticular microbiomes of Dendroctonus armandi using metagenomics. The topic is important for both basic science and potential applications in pest management. The study is well-designed overall, but some areas need clarification and improvement before the paper can be accepted.

Major Comments

Comments 1: Abstract: The practical importance of the study needs to be highlighted Strong claims are made based on bioinformatics inference rather than experimental validation. So soften the claims.

Response 1: Thank you to the reviewers for suggestions. We have revised the Abstract with softened words (see in lines 18-19, 21-23 and 26-27).

Comments 2: Introduction: Should cite more recent studies (after 2020) on metagenomics in bark beetles and other forest insects Novelty is not clearly stated. Please explain how this study differs from prior work on valens, D. rhizophagus, and Ips species.

Response 2: We thank the reviewers for the valuable comments, and we have revised the paper accordingly. Firstly, we supplemented references (after 2020) (see in lines 57-58, 466-470, 477-478 and 483-488); secondly, we supplemented the reason for studying the microbes associated with Dendroctonus armandi (see in lines 56-62 and 70-72).

It was found that using 16S rRNA genes to analyze the gut microbial (bacteria or fungi) community structure and metabolism for D. valens and D. Rhizophagus. But the differences in community structure and function between intestinal and non-intestinal microbes have not yet been compared. Chakraborty (2020) used 16S rRNA genes for extensive research on studies of gut bacteria and fungi associated with Ips bark beetles. And our team studied the difference between different habitat environments intestinal and non-intestinal microbes associated with Ips bark beetles, including microbial community structures, functional characteristics, and community assembly. However, During the process of data analysis, we found that the microbial diversity, particularly, dominant microbial groups of D. armandi were distinct with Ips bark beetles', they may play important roles in degrading complex compounds.

Comments 3: Materials and Methods: Since 70 guts were pooled together, it becomes hard to see how much variation exists between individual insects. Please explain why this approach was chosen.

Response 3: Thank you for pointing this out. A total of 70 guts were pooled together may reduce the differences between individual bark beetle. And it may promote the detection of rare microbes.

Comments 4: More detail is needed on sequencing depth, data filtering, and how contamination was avoided. The choice of valens as a reference genome for annotation should be justified, as it may influence functional predictions.

Response 4: We have supplemented the related expression of sequencing depth (see in lines 145-148) in Results, data filtering (see in lines 148-152), and avoiding contamination (see in lines 90-92).

Comments 5: Mention which software and exact parameters were used for statistical analysis?

Response 5: SPSS (t-test) and STAMP (Wilcox test) were used for statistical analysis (see in lines 109 and 118-119, Figure 2a and 2b, and Figure 3a).

Comments 6: Result: Ecological meaning of “deterministic vs stochastic” processes should be explained more cautiously

Response 6: We have supplemented the sentence in the results (see in lines 271-272). And relevant description was also provided in the Discussion (see in lines 386-388).

Comments 7: Figure 2, 3: Overcrowded. Legends need to more clearer

Response 7: Thank you to the reviewers for suggestions. We have revised Figure 2 (see in line 183) and Figure 3 (see in lines 200).

Comments 8: Discussion: Functional inferences on terpenoid degradation, phenolic detoxification are based on prediction not by experimental validation

Response 8: We agree with the reviewer's point. We revised some sentences in lines 331-334 and 341.

Comments 9: Discussion repeats some results. Provide more interpretation than repetitive data

Response 9: Thank you to the reviewers for comments. We have revised carefully the discussion of whole article (see in lines 309-312, 317-319, 357-359 and 386-388).

Comments 10:  Expand applied perspective like pest management, microbial manipulation. Many sentences are too long.

Response 10: Thank you to the reviewers for suggestions. We have revised the long sentences (see in lines 371-373, 379, 385), and supplemented the perspective of control for pests and diseases (see in lines 397-398).

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for sharing your interesting work on bark beetle microbiomes. It's a compelling study with a valuable dataset at its core. My main feedback is that the manuscript's current presentation doesn't yet do justice to the solid science within. My suggestions are aimed at sharpening the narrative and improving clarity, which will significantly elevate the paper's impact.

The first thing a reader encounters—the title and abstract—needs to be direct and engaging. The current title is a bit long and could be streamlined to signal your key finding immediately. Similarly, the abstract would be stronger if it led with your most significant result rather than a detailed list of methods. Tell us the answer first, then how you found it. This creates a much more powerful hook.

Looking at the introduction, it's clear you have a strong command of the literature. The challenge here is to weave those facts into a smoother story that logically leads the reader to the specific gap your research fills. End that section with a crystal-clear statement of your study's goals and its novelty. Why was this work needed, and what does it uniquely address?

For the methods, the priority is ensuring reproducibility. A few key details would help immensely. Please clarify how the beetles were collected and how the biological replicates were handled. Also, briefly mention the steps taken to control contamination during dissection. When discussing statistics, consistently naming the tests used (like the Wilcoxon test) and the reason for choosing them will make your analysis more transparent.

The results section is rich with data, but it can feel like a list of numbers. I would encourage you to focus more on interpreting the patterns you see. What do these differences mean? The figures, especially 2 and 4, are quite dense and could be simplified to make the main takeaways more accessible to the reader. Sometimes, moving detailed data to supplementary materials can make the main story much clearer.

The discussion is your opportunity to explain the "so what?" of your findings. Right now, it tends to restate the results. Instead, delve into their ecological significance. What does it mean that stochastic processes are more important on the cuticle? How could this knowledge potentially influence forest management practices? Making these broader connections will greatly strengthen the paper's significance.

Finally, a thorough revision of the language throughout will make everything flow better. Some sentences are lengthy and grammatically awkward, making the text harder to follow. Shortening and simplifying these sentences will make your excellent science much easier to appreciate.

I am genuinely enthusiastic about the potential of this research. With these revisions focused on narrative and clarity, I am confident this will become a strong and impactful publication.

Author Response

Comments 1: Thank you for sharing your interesting work on bark beetle microbiomes. It's a compelling study with a valuable dataset at its core. My main feedback is that the manuscript's current presentation doesn't yet do justice to the solid science within. My suggestions are aimed at sharpening the narrative and improving clarity, which will significantly elevate the paper's impact.

Response 1: Thank you for pointing this out. We agree with fully. And we have reorganized the first (see in lines 35-42) and second (see in lines 56-63) paragraphs of the introduction, and we deleted some sentences of introduction to make the expression more concise.

Comments 2: The first thing a reader encounters—the title and abstract—needs to be direct and engaging. The current title is a bit long and could be streamlined to signal your key finding immediately. Similarly, the abstract would be stronger if it led with your most significant result rather than a detailed list of methods. Tell us the answer first, then how you found it. This creates a much more powerful hook.

Response 2: Thank you for your valuable suggestions on the title and abstract of our manuscript. We have changed the title to “Specific function and assembly of crucial microbes for Dendroctonus armandi”. For “Abstract”, we deleted the methods to highlight the important results (see in lines 18-26). 

Comments 3: Looking at the introduction, it's clear you have a strong command of the literature. The challenge here is to weave those facts into a smoother story that logically leads the reader to the specific gap your research fills. End that section with a crystal-clear statement of your study's goals and its novelty. Why was this work needed, and what does it uniquely address?

Response 3: We thank the reviewer' comments for introduction, we have revised this part to improve logicality, for example, we reviewed the latest literature and supplemented the meaning of study (see in lines 59-62 and 70-72).

Comments 4: For the methods, the priority is ensuring reproducibility. A few key details would help immensely. Please clarify how the beetles were statement and how the biological replicates were handled. Also, briefly mention the steps taken to control contamination during dissection. When discussing statistics, consistently naming the tests used (like the Wilcoxon test) and the reason for choosing them will make your analysis more transparent.

Response 4: We thank the reviewer' comments for methods. We have added the significance of collecting mixed samples (see in line 89), t-test for gut and cuticular microbial communities using SPSS (see in line 109), and the reason for choose the Wilcoxon test (see in line 119). And the statement for the method of sample collection and biological replicates were descripted in lines 84-92. For test of statistics, particularly, STAMP analysis, firstly, it was used to analyze the differences of all pathway (level 3) between gut and cuticular samples with T-test. it showed no difference. Then we used Wilcoxon test to conduct STAMP analysis, it showed a slight difference at galactose metabolism (Figure 3a). 

Comments 5: The results section is rich with data, but it can feel like a list of numbers. I would encourage you to focus more on interpreting the patterns you see. What do these differences mean? The figures, especially 2 and 4, are quite dense and could be simplified to make the main takeaways more accessible to the reader. Sometimes, moving detailed data to supplementary materials can make the main story much clearer.

Response 5: Thank you so much for constructive suggestions. Just as reviewer has pointed out that the results feel like a list of number. So we added some sentences to make sure a complete story (see in lines 311-312, 320-321, and 347). About “Figure 2 and Figure 3”, they are the two most important diagrams to compare the difference with gut and cuticular microbes. So we would like to temporarily retain them. Additionally, we supplemented a Figure and a table for metagenomic sequencing (see in lines 145-152).

Comments 6: The discussion is your opportunity to explain the "so what?" of your findings. Right now, it tends to restate the results. Instead, delve into their ecological significance. What does it mean that stochastic processes are more important on the cuticle? How could this knowledge potentially influence forest management practices? Making these broader connections will greatly strengthen the paper's significance.

Response 6: Thank you for this critical and helpful feedback on the Discussion section. We agree that the current version does not sufficiently articulate the broader significance of our findings. We appreciate the specific guidance to explore the ecological meaning of stochastic processes on the cuticle and the potential implications for forest management. In revision, we have thoroughly revised the Discussion (see in lines 385-389) to show the effect on forest management practices.

Comments 7: Finally, a thorough revision of the language throughout will make everything flow better. Some sentences are lengthy and grammatically awkward, making the text harder to follow. Shortening and simplifying these sentences will make your excellent science much easier to appreciate.

Response 7: We have revised the sentences to show concise results (see in lines 371-373, 379, 385).

Comments 8: I am genuinely enthusiastic about the potential of this research. With these revisions focused on narrative and clarity, I am confident this will become a strong and impactful publication.

Response 8: Thank you so much for encouragement to us and enthusiasm about our research. It is incredibly motivating to receive such positive feedback on its potential. We have revised the manuscript carefully under the guidance of the reviewer. We will look forward to submitting a much-improved version.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Corresponding Aythor

I think for the moment your paper is OK for acceptance.

Regards

Reviewer 3 Report

Comments and Suggestions for Authors

MS can be accepted