Developmental Dynamics of Bacterial Microbiota in Aphis gossypii Revealed Using Full-Length 16S rRNA Sequencing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is clearly written and presents valuable results for the research community, particularly in the field of insect-microbiota interactions. The use of full-length 16S rRNA sequencing across multiple developmental stages of Aphis gossypii provides novel insights into the structure and potential function of the microbiota throughout the aphids life cycle. Some comments have been added directly to the PDF, which I believe would help improve clarity, strengthen the discussion, and enhance the overall scientific quality of the paper

Comments for author File: Comments.pdf

Author Response

A: Thanks for your kind comments, all revisions were in manuscript with revision mode, the details described as follows:

1. Deleting “and” in authors names.
2. Adding reference in line 51.
3. Italic “defensa” in line 74, italic “symbiotica” in line 75.
4. Remade previous Fig. 3.
5. Rewriting section 3.3.
6. Moving previous Fig. 6 to present Fig. S3.
7. Corrections in Discussion section based on reviewer’s comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the reviewed MS a group of scientists from China provides results of their study on metagenomics changes in aphid species, Aphis gossypii. They established a laboratory culture of this insect and investigated three groups of samples: eggs, nymphs and adults, each subdivided by age: 1day, 3day, 5 day, 7 day. The number of sample size is very low in this study (3-4). Because of this the results may be severely biased and interpretation of the results could be doubtful. The MS is well-written (in terms of English) but it contains a lot of inaccuracies and typos. Some work (functional analysis) is not explained in the Material and Methods section. The section Results contains contradictory data on the abundance of bacteria, which itself is presented in suboptimal way. The Discussion is too long and contains doubtful interpretations of the Results. The figures and captions for the figures need revision. The MS needs serious revision. Additionally, the authors are requested to state that the same MS was not submitted to other journals and that AI was not used for writing (some passages from Intro and Discussion provide this impression). Some more remarks are given below.

Why the MS pdf lacks numeration of lines?

Abstract:

underscore the stability of core symbionts – this not clear in the context of what was said abobe. Please, explain or revise.

insights into aphid-microbe coevolution – it is not clear why do you consider the results of this study to reflect the aphid-microbe coevolution

subsection 2.1. please explain in detail how aphids of different age were determined? E.g. how did you separate 3 days nymph from 5 days nymph and 3 days adults from 5 days adults? Please connect this with the content of the coding from the first column of the Table 1.

subsection 2.1. How many eggs, nymphs and adults in total were used in the experiments? for DNA extraction? What is a sample in this study? Please connect this with the content of the coding from the first column of the Table 1.

subsection 2.3. please, indicate the software used for phylogenetic analyses. Also follow the journal format when specifying software

Fig 1. Please, indicate relative abundance and degree of Blast similarity near every bacterium. Or specify that you consider exactly these prokaryote taxa to present in your samples (unrealistic).

Fig.2 caption Aphis – italic. Check entire text. Additionally, italicize all bacterial names in all figures and remove “_”

Fig3. Do you give averaged values in this figure? Please, add explanation in the caption. Please, specify the sample size for each category: n=…

Fig3 vs Fig1 – please explain why you give 20 bacteria in Fig 1 and only 15 in Fig3?

Fig 4. Please, specify in the figure the sample size for each category (eggs, nymph, adult): n=…

Fig3. Please, explain the differences of Enterobacter in different nympal stages? Zhang et al. 10.1186/s13071-021-05053-1. How could you proof that presence of this bacteria is not a result of contamination?

Fig.4. According to Fig.3 Enterobacter hormaecheci is absent in nymph 5d, however according to Fig. 4 it is generally present in nymphs. This is confusing. Please, find another way for presenting your data.

Fig. 5,6,7 please explain a, b and ab abbreviations in the caption

3.3. proportions of the Buchnera aphidicola – please, explain the methodology for estimation this proportion in detail. Why do you think that this methodology is relevant? According to your figures the sample sizes are very low and some of specimens lack this Bacteria at all. Please (specify) how many specimens lack this bacteria, (b) in the light of this absence data, please reconsider your conclusion that in adults… the proportion of Buchnera was significantly higher than in nymphs…

3.4. KEGG database, Functional analysis - this work is not described in the section Material and methods. Please, explain the methodology and the material that was analyzed using Kegg database.

3. Discussion. First paragraph duplicates the information given in the Introduction. Please, revise this or completely remove

“Buchnera aphidicola is a primary endosymbiotic bacterium that has co-evolved with aphids for over 160 million years” – but only 100 million years in Introduction. Please, revise and give references. Check the entire text and remove all inaccuracies.

“In this study, Buchnera aphidicola is abundant in all growth stages of A. gossypii” – but according to some of your figures this bacetria is absent in some specimens in each group (egg, nymph, adult). Please, revise to avoid self-contradictions.

Delftia and Enterobacter – please, discuss the possibility that presence of these bacteria, often assotiated with human deseases in hospitals, could be a result of contamination

The Discussion is unfocused and contains speculative statements. It needs major revision to provide stronger connectivity with the results of this study.

Author Response

1. Q: Why the MS pdf lacks numeration of lines?

A: Thanks for your kind comments, and the numerations added in manuscript.

Abstract:

2. Q: underscore the stability of core symbionts – this not clear in the context of what was said abobe. Please, explain or revise.

A: Thanks for your comments, and this contents revised in Abstract: “These findings highlight the persistent dominance of the obligate symbiont Buchnera aphidicola across all developmental stages, despite quantitative fluctuations in its abundance, alongside stage-specific shifts in facultative bacterial communities."

3. Q: insights into aphid-microbe coevolution – it is not clear why do you consider the results of this study to reflect the aphid-microbe coevolution.

A: Thanks for your comments, and related contents about the aphid-microbe coevolution removed in manuscript.

4. Q: subsection 2.1. please explain in detail how aphids of different age were determined? E.g. how did you separate 3 days nymph from 5 days nymph and 3 days adults from 5 days adults? Please connect this with the content of the coding from the first column of the Table 1. Subsection 2.1. How many eggs, nymphs and adults in total were used in the experiments? for DNA extraction? What is a sample in this study? Please connect this with the content of the coding from the first column of the Table 1.

A: Thanks for your comments, the sample names corrected in Table 1 and related contents added in section 2.1 with marked mode.

5. Q: subsection 2.3. please, indicate the software used for phylogenetic analyses. Also follow the journal format when specifying software.

A: Detailed information added in section 2.3.

6. Q: Fig 1. Please, indicate relative abundance and degree of Blast similarity near every bacterium. Or specify that you consider exactly these prokaryote taxa to present in your samples (unrealistic).

A: Thanks for your comments, and related information added in supplemental materials, and all raw data of this experiment were submitted in NCBI database with number SUB15302330.

7. Q: Fig.2 caption Aphis – italic. Check entire text. Additionally, italicize all bacterial names in all figures and remove “_”

A: Thanks for your comments, and contents in all figures captions corrected.

8. Do you give averaged values in this figure? Please, add explanation in the caption. Please, specify the sample size for each category: n=…

A: Thanks for your comments, explanations in caption of Fig. 3 corrected in manuscript.

9. Fig3 vs Fig1 – please explain why you give 20 bacteria in Fig 1 and only 15 in Fig3?

A: Figure 1 displays the top 20 most abundant bacterial taxa at the genus or broader taxonomic level across developmental stages, providing an overview of the dominant bacteria within the microbiota. Including more taxa enhances the reliability of phylogenetic analysis. In contrast, Figure 3 focuses on the top 15 bacterial species; most of these species have relative abundances below 1%, making it difficult to effectively display their abundances in the figure. Based on these observations, we believe that the difference in the number of species shown between Figures 1 and 3 is justified and convincing.

10. Q: Fig 4. Please, specify in the figure the sample size for each category (eggs, nymph, adult): n=…

A: Thanks for your comments, and contents in all figures captions corrected.

11. Q: Fig3. Please, explain the differences of Enterobacter in different nympal stages? Zhang et al. 10.1186/s13071-021-05053-1. How could you proof that presence of this bacteria is not a result of contamination?

A: Related contents added in Discussion section 5^th paragraph.

12. Q: Fig.4. According to Fig.3 Enterobacter hormaecheci is absent in nymph 5d, however according to Fig. 4 it is generally present in nymphs. This is confusing. Please, find another way for presenting your data.

A: Thanks for your comments, and Figure 4 was intended to summarize presence across 3 growth stages including egg, nymph and adult, while nymph stages including 1d-7d, which may have obscured variation among specific time points.

13. Q: Fig. 5,6,7 please explain a, b and ab abbreviations in the caption.

A: Contents added in figure captions.

14. Q: 3.3. proportions of the Buchnera aphidicola – please, explain the methodology for estimation this proportion in detail. Why do you think that this methodology is relevant? According to your figures the sample sizes are very low and some of specimens lack this Bacteria at all. Please (specify) how many specimens lack this bacteria, (b) in the light of this absence data, please reconsider your conclusion that in adults… the proportion of Buchnera was significantly higher than in nymphs….

A: As manuscript described, 16S rRNA amplicon sequencing is a standard, widely used approach for estimating the relative abundance of bacterial taxa in insect microbiota studies. While this method accurately reflects the proportional representation of Buchnera compared to other bacteria, we acknowledge that technical limitations can influence detection, particularly with a symbiont present at very low abundance or in samples with low sequencing depth. Buchnera aphidicola is a primary symbiont for Aphids, which means all aphid individulas harbor this symbiont, as well as in all aphid samples used in this experiment.

15. 4. KEGG database, Functional analysis - this work is not described in the section Material and methods. Please, explain the methodology and the material that was analyzed using Kegg database.

A: Detailed information about function predict added in section 2.3.

16. Q: Discussion. First paragraph duplicates the information given in the Introduction. Please, revise this or completely remove.

A: Removed 1^st paragraph of Discussion.

17. “Buchnera aphidicola is a primary endosymbiotic bacterium that has co-evolved with aphids for over 160 million years” – but only 100 million years in Introduction. Please, revise and give references. Check the entire text and remove all inaccuracies.

A: Corrected.

18. Q: “In this study, Buchnera aphidicola is abundant in all growth stages of A. gossypii” – but according to some of your figures this bacetria is absent in some specimens in each group (egg, nymph, adult). Please, revise to avoid self-contradictions.

A: Based on results of Fig. 3, all microbiota of different developmental stages of A. gossypii include Buchnera aphidicola.

19. Q: Delftia and Enterobacter – please, discuss the possibility that presence of these bacteria, often assotiated with human deseases in hospitals, could be a result of contamination.

A: Detailed information added in Discussion section in 5^th and 6^th paragraphs.

20. Q: The Discussion is unfocused and contains speculative statements. It needs major revision to provide stronger connectivity with the results of this study.

A: Corrections revised in Discussion section.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Please find my recommendation for "Developmental Dynamics of Bacterial Microbiota in Aphis gossypii Revealed by Full-Length 16S rRNA Sequencing" manuscript.

General comment: Please use line numbered template, this help the reviewers to provide targeted comments, and will help authors to better identify the weaknesses and perform improvements

1. Introduction:

• The introduction section should present clearly the proposed research hypothesis or objectives statement
• In my opinion the manuscript should be explicit when presenting the research gaps that this study aims to address
• In my opinion the introduction doesn't adequately explain why developmental stages are important for microbiota studies
• The manuscript should provide more background on how microbiota might influence aphid biology across different life stages
• The rationale for using full-length 16S rRNA gene sequencing is mentioned but not adequately justified
• In this version of the manuscript are limited information about the ecological and agricultural significance of the research
• This section should include also a clear statement of the study's significance or potential applications

2. Materials and methods 

For experimental design and sampling

• The authors should mention how individuals were selected for sampling and clearly mention and justify the samples number and replicate
• Describe how eggs were collected and handled
• Provide information about cotton leaf conditions and how this was assessed if, and ensure adequate details about potential environmental contamination control
• Provide information if aphid health was verified
• Provide details about how consistent conditions across stages were maintained

For analytics

• Provide details about DNA quality assessment and quantification
• Please detail the measures taken to prevent cross-contamination

For statistics

• Provide detail about normality testing procedures
• Please specify the significance thresholds and provide information about multiple testing corrections
• Please clarify the criteria for choosing between parametric and non-parametric tests

Results

• The section presents a table (Table 1) summarizing sequencing data for different developmental stages of Aphis gossypii. However, the table is very large and not all columns are clearly explained - please see “Effective CCS”...
• The section claims that “all samples from different growth stages all clustered together,” suggesting no significant influence of developmental stage on microbiota structure. However, no statistical test results (e.g., p-values, confidence intervals, or test statistics) are reported to support this claim. Fig. S1 - is not available 
• The description of “insignificant influence” is vague; explicit statistical results and the methods used should be provided for scientific rigor
• The basis for reported percentages (e.g., “Buchnera aphidicola is the most dominant bacteria (>23.83%)”) is not clearly explained—whether this is relative abundance or another metric.

Discussion - this is section 4 not 3

• There is a tendency to over-interpret correlative data without experimental validation, as claims regarding bacterial functions are made in the absence of direct evidence.
• While statistical significance (e.g., P < 0.05) is reported, the biological relevance of these findings is not thoroughly addressed, and conclusions about the “vital influence” of Buchnera are not robustly substantiated by the presented data.
• Critical analysis of the results is limited, with insufficient exploration of alternative explanations for the observed patterns.
• The discussion does not adequately connect microbiota composition to aphid biology, nor does it explain the variation in bacterial proportions across developmental stages
• The integration of literature specific to A. gossypii microbiota is insufficient. There is an overemphasis on general microbiome studies rather than aphid-focused literature
• Comparisons with other aphid species are minimal, reducing the broader relevance of the findings
• The manuscript omits a critical evaluation of its own methodological constraints
• In my opinion, introduction of study limitations, future research directions, and practical applications are important to increase the value and relevance of this study

Author Response

1. Q: General comment: Please use line numbered template, this help the reviewers to provide targeted comments, and will help authors to better identify the weaknesses and perform improvements.

A: Thanks for your comments, and contents in all figures captions corrected.

Introduction:

2. Q: The introduction section should present clearly the proposed research hypothesis or objectives statement. In my opinion the manuscript should be explicit when presenting the research gaps that this study aims to address.

A: Related contents added in last paragraph of Introduction section.

3. In my opinion the introduction doesn't adequately explain why developmental stages are important for microbiota studies. The manuscript should provide more background on how microbiota might influence aphid biology across different life stages.

A: Related contents added in 4^th paragraph of Introduction section.

4. The rationale for using full-length 16S rRNA gene sequencing is mentioned but not adequately justified.

A: Related contents added in the 5^th paragraph of Introduction section.

5. In this version of the manuscript are limited information about the ecological and agricultural significance of the research. This section should include also a clear statement of the study's significance or potential applications.

A: Related contents added in the last paragraph of Introduction section.

Materials and methods 

6. For experimental design and sampling

• The authors should mention how individuals were selected for sampling and clearly mention and justify the samples number and replicate
• Describe how eggs were collected and handled
• Provide information about cotton leaf conditions and how this was assessed if, and ensure adequate details about potential environmental contamination control
• Provide information if aphid health was verified
• Provide details about how consistent conditions across stages were maintained

A: All related information added in section 2.1 in manuscript.

7. For analytics

• Provide details about DNA quality assessment and quantification
• Please detail the measures taken to prevent cross-contamination

A: Detailed information added in section 2.2.

8. For statistics

• Provide detail about normality testing procedures
• Please specify the significance thresholds and provide information about multiple testing corrections
• Please clarify the criteria for choosing between parametric and non-parametric tests

A: Detailed information added in section 2.3.

Results

• The section presents a table (Table 1) summarizing sequencing data for different developmental stages of Aphis gossypii. However, the table is very large and not all columns are clearly explained - please see “Effective CCS”...

A: Revised in notes of Table 1.

• The section claims that “all samples from different growth stages all clustered together,” suggesting no significant influence of developmental stage on microbiota structure. However, no statistical test results (e.g., p-values, confidence intervals, or test statistics) are reported to support this claim. Fig. S1 - is not available 
• The description of “insignificant influence” is vague; explicit statistical results and the methods used should be provided for scientific rigor

A: Results of (PERMANOVA: R² = 0.045, p = 0.18) added in section 3.2.

• The basis for reported percentages (e.g., “Buchnera aphidicola is the most dominant bacteria (>23.83%)”) is not clearly explained—whether this is relative abundance or another metric.

A: Thanks for your comments, “23.83% mean relative abundance” added in this sentence.

Discussion - this is section 4 not 3

A: Corrected.

• There is a tendency to over-interpret correlative data without experimental validation, as claims regarding bacterial functions are made in the absence of direct evidence.

A: We appreciate the reviewer's valuable feedback and agree that caution is necessary when interpreting correlative data. While our study provides comprehensive insights into the microbial community structure and potential functional capabilities based on bioinformatic predictions, we acknowledge that these findings are inherently correlative and do not establish direct causal relationships. We explicitly state that functional inferences drawn from 16S rRNA data require further validation through targeted experimental approaches, such as gene expression analyses, functional assays, and microbiome manipulation studies. Future research will focus on directly testing these microbial functions to substantiate their roles in aphid physiology and development, thereby strengthening the biological relevance of our findings.

Q: While statistical significance (e.g., P < 0.05) is reported, the biological relevance of these findings is not thoroughly addressed, and conclusions about the “vital influence” of Buchnera are not robustly substantiated by the presented data.

• Critical analysis of the results is limited, with insufficient exploration of alternative explanations for the observed patterns.
• The discussion does not adequately connect microbiota composition to aphid biology, nor does it explain the variation in bacterial proportions across developmental stages
• The integration of literature specific to A. gossypii microbiota is insufficient. There is an overemphasis on general microbiome studies rather than aphid-focused literature
• Comparisons with other aphid species are minimal, reducing the broader relevance of the findings

A: Thank you for your kind comments, and revised part added in Discussion 4^th paragraph.

• The manuscript omits a critical evaluation of its own methodological constraints. In my opinion, introduction of study limitations, future research directions, and practical applications are important to increase the value and relevance of this study.

A: Thank you for your valuable feedback. We agree that addressing the study limitations, future research directions, and practical applications can significantly enhance the impact and relevance of our work. Contents added in the last of Discussion section.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors carefully addressed all reviewers' criticisms and revised the manuscript accordingly. While the manuscript has improved, some issues remain: it is still too long, with redundant figures/tables and an overly lengthy discussion. Specifically: (1) Table 1 is highly technical and would be better placed in the Supplement. (2) Some figures (e.g., Fig. 1) and diagrams could also be moved to the Supplement. (3) The Discussion should be shortened and refocused to highlight the main conclusions more clearly. The authors are requested to revise the manuscript to address these points.

Author Response

1. Q: Table 1 is highly technical and would be better placed in the Supplement.

A: Thanks for your comments, and the Table 1 moved to supplemental materials as Table S1.

2. Q: Some figures (e.g., Fig. 1) and diagrams could also be moved to the Supplement.

A: Fig. 1 moved to supplemental materials as present Fig. S1.

3. Q: The Discussion should be shortened and refocused to highlight the main conclusions more clearly. The authors are requested to revise the manuscript to address these points.

A: Thanks for your comments, and Discussion section revised in manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for considering my recommendations in "Developmental Dynamics of Bacterial Microbiota in Aphis gossypii Revealed by Full-Length 16S rRNA Sequencing". Reading carefully the new version of the manuscript, I found some parts that needs to be clarified and improved. Please see below:

• L91-103: Please better highlight gaps in previous research and justify why full-length 16S rRNA sequencing is uniquely valuable for this study
• L104-106: Please check for clarity; Similar for L406-408
• In the method section please better clarify how contamination was controlled or how negative controls were handled
• In the supplementary material - Table - the authors should reduce the number of decimals, the current version of table is crowded and difficult to follow
• Please reconsider Figure 1 from supplementary material
• L546-556: In my opinion the authors should also consider the potential biases in 16S rRNA sequencing, lack of functional validation, or the absence of environmental/contextual data as potential study limitation. The authors should also better present how the findings could be affected by technical artifacts (e.g., PCR bias, sequencing errors)
• In my opinion, the implications for pest management are mentioned but not enough developed, with specific recommendations or experimental follow-up. Please extend a bit this

 

 

 

Author Response

1. Q: L91-103: Please better highlight gaps in previous research and justify why full-length 16S rRNA sequencing is uniquely valuable for this study.

A: Related contents added in lines 98-102.

2. Q: L104-106: Please check for clarity; Similar for L406-408

A: Deleting similar contents in Discussion section.

3. Q: In the method section please better clarify how contamination was controlled or how negative controls were handled

A: Contents added in lines 149-152.

4. Q: In the supplementary material - Table - the authors should reduce the number of decimals, the current version of table is crowded and difficult to follow

A: Previous Table S1 corrected.

5. Q: Please reconsider Figure 1 from supplementary material

A: Thanks for your comments, and previous Fig. 1 moved as Fig. S1.

6. Q: L546-556: In my opinion the authors should also consider the potential biases in 16S rRNA sequencing, lack of functional validation, or the absence of environmental/contextual data as potential study limitation. The authors should also better present how the findings could be affected by technical artifacts (e.g., PCR bias, sequencing errors)

A: Thanks for your comments, and detailed information added in the second last paragraph of Discussion.

7. Q: In my opinion, the implications for pest management are mentioned but not enough developed, with specific recommendations or experimental follow-up. Please extend a bit this

A: Thanks for your comments, and related contents added in the last paragraph of Discussion.

Author Response File: Author Response.pdf