Differences in the Structure and Function of the Gut Microbiota of the Earthworm (Amynthas aspergillum) Under Different Habitat Conditions

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors have adequately addressed all the comments and queries raised in my previous review. I have no further comments or concerns.

Author Response

We sincerely thank Reviewer 1 for the positive evaluation and for confirming that all previous comments have been adequately addressed. We have no further modifications needed for this round.

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have addressed my previous comments and made the necessary revisions to the manuscript. However, in my opinion, the Discussion section still requires some further improvement. In particular, the repeated presentation of results should be removed, as the Discussion should focus more on the interpretation and significance of the findings rather than reiterating the results.

 

Author Response

We sincerely thank Reviewer 2 for the constructive feedback and valuable suggestions. The Discussion section has been thoroughly expanded and refined in the revised manuscript to further improve clarity, ecological depth, and articulation of our findings. Detailed point-by-point explanations and tracked changes are provided in our comprehensive Response Letter and the revised manuscript.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The article explains the microbial community in the gut of the earthworm Amynthas aspergillum sampled at various niches viz. fertile farmland, barren farmland, plantation forest, primary forest, artificial grassland, wasteland slope, and a breeding farm. The authors have employed metagenome sequencing to identify the OTUs. If somebody aims to culture those microbes and then study their identity, they may miss some hard to culture bacteria. So their choice of studying partial 16S rRNA gene segments is worthy. 
They have listed the abundant microbial genera in each type of location. Then they compare and inform that anthropogenic factors could be the reason for change in the gut microflora. 

In other earthworms bacteria were found to exist in symbiotic relationship with earthworm. For example, bacterial symbiont (gut) of Eudrilus eugeniae supply riboflavin to the worm which is used for successful regeneration. In case of Eisenia fetida, Verminephrobacter eiseniae (nephridial symbiont) supports the worm with reproductive health. A similar relationship could have been established for A. aspergillum and then the importance of gut microflora would be an acceptable argument. If no such relationship had been established yet, then the type of microflora inhabiting the earthworm gut does not matter, in scientific context.

1. The NCBI SRA identifier provided is not working. Kindly provide the correct identifier. 
2. Line 48, current breeding models largely rely on what?
3. In Table 1, convert 'Sem' to 'SEM'; what does a,b,c,d,e,f superscripts indicate?
4. Were the labels a,ab,ac,bc,... provided in Figure 5 discussed anywhere in the paper?
5. The abbreviations FW, RGL, HP, etc., donot apply well with their full forms. And it is confusing for a reader.
6. Figure 6, what is the basis for selecting the colour coding for individual species? It does not corroborate with other figures, say figure 5.
7. In several places the earthworm name is misspelt, example Figure 10 caption. 
8. A list of all microbial genus observed could be provided as a supplementary material.
9. Earthworms like Dendrobaena veneta are freeze tolerant. Other worms are not. Line 124, the authors have sampled adult earthworms and kept in liquid nitrogen. The worms would have died instantly. How come they anesthetize the worm again using ice in lab? (line 150)
10. In Figure 2, 5. Multiomics: Where does multiomics techniques adapted in the paper. It is just 16SrRNA sequencing and bioinformatics analysis. Consider changing the term.

Author Response

We sincerely thank Reviewer 3 for the thorough and constructive comments. All requested experimental details and technical clarifications—including earthworm morphometrics, DNA extraction amounts, standard soil physicochemical measurement protocols, and OTU clustering parameters—have been fully addressed and incorporated into the revised manuscript. Please refer to our comprehensive Response Letter and the revised manuscript for complete point-by-point details.

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

The study is generally well designed and presented. However, before making a final decision on the manuscript, I would like the authors to address a few important points.

First, the manuscript should include a Data Availability Statement. The authors should clearly indicate whether the sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) or another public repository and provide the corresponding accession number(s). This information is essential to ensure transparency and to allow independent verification of the analyses.

In addition, a few methodological details require clarification:

• How much gut content was used for DNA extraction?
• Which sequencing platform was used for sequencing?
• What sequencing chemistry was employed?
• Were the libraries sequenced using paired-end or single-end reads?

Addressing these points will improve the clarity and reproducibility of the study.

Author Response

We are very grateful for the valuable comments and constructive suggestions regarding our manuscript. We have carefully studied all comments and made corresponding revisions in the revised manuscript (marked in RED font).

RESPONSES TO REVIEWER 2's COMMENTS

Comment 1 (Methodological Details for Transparency): In addition, a few methodological details require clarification: How much gut content was used for DNA extraction? Which sequencing platform was used for sequencing? What sequencing chemistry was employed? Were the libraries sequenced using paired-end or single-end reads? Addressing these points will improve the clarity and reproducibility of the study.

Response: We highly appreciate the reviewer's constructive suggestions to enhance transparency and reproducibility. We have added these exact parameters to Section 2.4 and Section 2.5 of the revised manuscript:

1.For each biological replicate, approximately 0.25 g of pooled gut content was used for total genomic DNA extraction.

2.The high-throughput sequencing was conducted on the Illumina NovaSeq 6000 platform.

3.The sequencing chemistry utilized the NovaSeq Reagent Kit.

4.The libraries were sequenced using a paired-end (2 x 300 bp) sequencing strategy.

We hope these comprehensive revisions fully address all queries and meet the high standards of Life. Thank you again for your time and guidance.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This study addresses an important topic: how habitat diversity (a gradient ranging from natural to highly artificial environments) affects the gut microbiota of the species Amynthas aspergillum. This is a significant issue from the perspective of species conservation, and also has potential practical implications.

The manuscript presents valuable data on the links between soil properties and the gut microbiota of this species. The results provide strong support for the main hypothesis. The authors observed very strong habitat specificity (only 25 shared taxonomic units (OTUs) across all habitats) and a simplification of the microbiota’s function under artificial breeding.

However, the paper has certain shortcomings, particularly in the ‘Results’ and ‘Discussion’ sections. In my opinion, the ‘Results’ section contains an excessive amount of numerical data. There were also some repetitions between the text and the tables. The authors should remove all interpretations contained in this section. The ‘Discussion’ section also requires improvement, as it contains some repetitions of the results.

The following missing information should also be provided:

• the date of sampling (season),
• at the breeding farm (YZ) – how much time elapsed between the preparation of the substrate (a mixture of fermented cattle manure and straw) and the sampling? a surprisingly low C:N ratio, given the presence of straw
• Data on soil moisture and the microclimate would be useful?
• How do the authors define the intensity of anthropogenic disturbances?

Author Response

We are very grateful for the valuable comments and constructive suggestions regarding our manuscript. We have carefully studied all comments and made corresponding revisions in the revised manuscript (marked in RED font).

Below is our point-by-point response to the reviewers' comments.

RESPONSES TO REVIEWER 3's COMMENTS

Comment 1 (Missing Information - Sampling Season): Provide the date of sampling (season).

Response: Thank you for pointing this out. The field sampling was conducted in mid-October 2025, which corresponds to the autumn season in the subtropical area. This detail has been added to Section 2.2.

Comment 2 (Missing Information - Breeding Farm Details): At the breeding farm (YZ) - how much time elapsed between the preparation of the substrate (a mixture of fermented cattle manure and straw) and the sampling? A surprisingly low C/N ratio, given the presence of straw.

Response: We appreciate this astute question. At the breeding farm (YZ) site, the mixed substrate of fermented cattle manure and straw had been fully matured and stabilized for approximately 45 days prior to our sampling to ensure a stable colonizing microenvironment for the earthworms. The surprisingly low C/N ratio (9.38) is attributed to the intense pre-fermentation process where labile carbon was rapidly respired by hyperactive microbes, leaving a nitrogen-dense consolidated matrix. This clarification has been incorporated into Section 2.2 and Section 4.1.

Comment 3 (Missing Information - Macro-environment): Data on soil moisture and the microclimate would be useful?

Response: We agree that microclimate data add value. While transient moisture and microclimate fluctuations were not individually monitored for all sites, all field operations were synchronized within the same stable meteorological window to ensure background homogeneity. We have noted this limitation and contextual framework in Section 2.3.

Comment 4 (Missing Information - Anthropogenic Disturbances): How do the authors define the intensity of anthropogenic disturbances?

Response: We have provided a clear operational definition in Section 2.1. The gradient of anthropogenic disturbance intensity was classified into three tiers based on land-use types, management frequency, and input levels: natural control undisturbed (YSL), semi-natural low-intensity disturbed (RGL, HP), and high-intensity artificial continuous managed intervention (FW, PJ, RGCD, YZ).

We hope these comprehensive revisions fully address all queries and meet the high standards of Life. Thank you again for your time and guidance.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript focuses on Amynthas aspergillum, a key soil invertebrate and traditional Chinese medicine. By collecting earthworms across seven representative habitats, the authors intend to systematically explore the driving mechanisms of habitat heterogeneity on the structure and functional succession of the gut microecology. While this study has potential value for the conservation of wild germplasm resources and the optimization of ecological breeding techniques, the manuscript in its current form suffers from severe conceptual errors and methodological flaws that must be thoroughly addressed. 

1.  The normal body weight of a sexually mature adult of A. aspergillum typically ranges from 2.0 to 5.0 g，or even heavier, and its body length regularly exceeds 10 cm. However, the collected mature adult in the manuscript weighing only 0.50 g with the body length of 5 cm - 7 cm cannot possibly be a "mature adult" of this species. 
2. Since soil physicochemical factors represent the absolute core environmental filter used to interpret gut microbial assembly in this study, their measurements must be highly accurate. Commercial rapid testers are designed for quick, crude agronomic diagnostic estimations in the field, not for rigorous, peer-reviewed scientific publications. The authors are required to validate these soil properties using internationally accepted standard laboratory protocols.
3. In Figure 5, the YZ (breeding farm) group's Simpson index boxplot sits at a medium-to-low level compared to the others, not the highest Simpson dominance index in 4.1. Additionally, the y-axis scale of the Simpson boxplot in Figure 5 only spans from 0.000 to 0.020, making the text claim of "~0.92" in 4.1 mathematically impossible. 
4. OTU and ASV represent entirely different bioinformatic denoising and clustering philosophies. The authors must be precise about which pipeline was actually executed and correct the figures (e.g. Figure 3) or text accordingly to maintain methodological integrity.  
5. The species name should be in italic.
6. It is ecologically impossible to harvest the exact same number of earthworms (25 individuals) from a single quadrat across habitats with drastically different anthropogenic disturbance levels and soil properties. Furthermore, a yield of 25 large earthworms in a tiny 0.09 m² area was unormal.
7. What is your meaning of Table 1 " Table 1. Soil nutrient and physicochemical properties under different habitats.earthworm gut samples from different habitats. "?

Author Response

We are very grateful for the valuable comments and constructive suggestions regarding our manuscript. We have carefully studied all comments and made corresponding revisions in the revised manuscript (marked in RED font).

Below is our point-by-point response to the reviewers' comments.

RESPONSES TO REVIEWER 1's COMMENTS

Comment1 (General Quality): The English could be improved to more clearly express the research. Figures and tables must be improved.

Response: Thank you very much for this critical guidance. We have thoroughly polished the English throughout the manuscript with the help of professional editors to ensure scientific accuracy and clarity. All figures and tables have also been updated to top-tier journal presentation quality.

Comment2 (Methodological Clarification): OTU and ASV represent entirely different bioinformatic denoising and clustering philosophies. The authors must be precise about which pipeline was actually executed and correct the figures (e.g. Figure 3) or text accordingly to maintain methodological integrity.

Response: We sincerely apologize for the nomenclature confusion between OTUs and ASVs in our preliminary draft. We wish to clarify that the pipeline executed was an Operational Taxonomic Unit (OTU) clustering strategy based on a 97% similarity threshold using the UPARSE algorithm. We have thoroughly checked and revised the text to exclusively use "OTUs" and have updated the axis label of Figure 3 from "Number of ASVs" to "Number of OTUs" to maintain strict methodological integrity.

Comment3 (Nomenclature): The species name should be in italic.

Response: We appreciate this reminder. We have meticulously checked the entire manuscript and italicized all instances of the species name (Amynthas aspergillum).

Comment4 (Ecological Sampling Rigor): It is ecologically impossible to harvest the exact same number of earthworms (25 individuals) from a single quadrat across habitats with drastically different anthropogenic disturbance levels and soil properties. Furthermore, a yield of 25 large earthworms in a tiny 0.09 m2 area was unormal.

Response: We completely agree with the reviewer's expert ecological insight and realize our previous description was misleading. In actual field operations, we employed a multi-point random sampling strategy rather than harvesting all 25 individuals from one single quadrat. For each habitat, 25 healthy, adult earthworms were collected from across five separate quadrats (30 cm x 30 cm each) and pooled as a representative composite sample for that habitat group. We have revised Section 2.2 to clarify this multi-point sampling strategy.

Comment 5 (Table Title Clarification): What is your meaning of Table 1 "Table 1. Soil nutrient and physicochemical properties under different habitats.earthworm gut samples from different habitats. "?

Response: We apologize for the clerical error in the title of Table 1, which resulted from a copy-paste overlay. The redundant phrase "earthworm gut samples from different habitats." has been removed. The title has been corrected to "Table 1. Soil nutrient and physicochemical properties under different habitats."

We hope these comprehensive revisions fully address all queries and meet the high standards of Life. Thank you again for your time and guidance.

Author Response File: Author Response.docx