Enhanced Cultivation of Actinomycetota Strains from Millipedes (Diplopoda) Using a Helper Strain-Assisted Method

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an innovative method, however, the way it is done not convincing.

Photos of the plates are required with the results not hand drawn figures.

When the helper strain is plated out and agar is poured onto it how the authors prove that metabolites are produced especially for the growth promoting ones. 

Authors have to work more on the project and resubmit.

Comments on the Quality of English Language

can be improved

Author Response

Comments 1: This manuscript presents an innovative method, however, the way it is done not convincing. Photos of the plates are required with the results not hand drawn figures.

Response 1: Thank you very much for your positive assessment of the innovation in our method and for your constructive feedback regarding the presentation of the results. We fully agree with your comment that photographic evidence would provide more objective and convincing support for our findings. In the original submission, we used schematic diagrams to illustrate the principle of interaction tests between bacterial strains. However, as you rightly pointed out, actual photographs are essential to demonstrate the raw experimental outcomes. Therefore, we have now added Figure 3 with original photographs of the assay plates. The manuscript has been updated accordingly with expiations of new figure like bellows.

P5, L234-237

The specific growth conditions are shown in Figure 3a and Figure 3b. The selected representative helper strains significantly promoted the growth of type strains. In the agar medium, the closer the type strain is to the helper bacteria, the denser the number of strains. And it forms a distinct halo around the candidate helper colony.

P6, L248-251

The specific growth conditions are shown in Figure 3c and 3d. The selected representative helper strains significantly promoted the growth of type strains. In the agar medium, the number of type strains in the experimental group was notice-ably higher than in the control group.

P8, L328-334

Figure 3. Growth promotion of type strains by helper strains. (a, b) Double-layer plate assay. Dotted circles in (a) and (b) highlight the colonies of the type strains (a) Growth of type strain K3 with helper N6. (b) Growth promotion of type strain TB5 by helper strain N8. (c, d) Conditioned medium growth assay. Growth of the helper-dependent strain was tested using supernatant of helper strain. (c) Control (K1 on unconditioned medium). (d) K1 forms colonies on medium supplemented with supernatant from helper strain N4.

 

Comments 2: When the helper strain is plated out and agar is poured onto it how the authors prove that metabolites are produced especially for the growth promoting ones.
Response 1:Thank you for raising this important methodological point. We appreciate the opportunity to clarify how our evidence supports the production of growth-promoting metabolites in the double-layer plate method.Our primary and direct proof comes from the conditioned medium experiment (Figure 3). The logic is as follows:

1. The double-layer plate method showed a growth zone of the indicator (type) strain around the helper strain colony, suggesting the diffusion of a growth-promoting factor(s).
2. The confirmation experiment using conditioned medium was designed to definitively test this hypothesis. By culturing the helper strain in liquid medium, removing all cells via sterile filtration, and then applying this cell-free supernatant to the dependent strain, we demonstrated that the growth-promoting effect is entirely reproducible without any direct cell-to-cell contact.
3. Therefore, the positive result from the confirmation experiment serves as mechanistic validation for the agar overlay assay. It confirms that the observed growth zone in the overlay is indeed caused by diffusible, secreted metabolites produced by the helper strain, and not by other indirect factors like nutrient depletion or pH alteration caused by the living helper cells themselves.

While we agree that identifying the specific chemical nature of these metabolites is a crucial next step (and we have added a discussion on this future direction in the revised manuscript), the combination of the overlay assay (double-layer plate method) and the confirmation test using conditioned medium provide solid, multi-layered evidence for our core conclusion regarding metabolite-based interaction.

P14, L440-444

While our findings demonstrate that the eight helper strains promote the growth of the type strain through secreted metabolites, the chemical identities and biosynthetic origins of these compounds remain to be elucidated. Future work will therefore focus on isolating the active components via HPLC-MS and validating their biosynthetic pathways through genetic approaches such as targeted gene knockout.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments:

1. The authors should specify which millipede species were used in this study. Additionally, please clarify how many individual millipedes were used for the isolation process—was a single specimen used, or were multiple millipedes combined after surface sterilization and homogenization?
2. In my opinion, Prediction of BCGs using 16S rRNA gene is too rough.
3. Typically, BLAST results obtained from EzBioCloud report sequence similarity values with two decimal places (e.g., 98.68%). I am unsure why the authors reported the similarity values only as whole numbers.
4. If the authors intend to propose these strains as new species, they should clearly provide the criteria used for species delineation along with appropriate citations supporting those criteria.
5. Line 168-168: The authors state that the isolates were randomly picked. It would be helpful to clarify how selection bias was minimized during this process and what criteria or procedures were used to ensure that the isolates were truly selected at random.

Other comments:

Line 105: What is the meaning of “Among the 74 operational taxonomic units (OTUs) identified,” were author isolated 74 strains from the millipede

Line 106-108: If author have published growth promoting test for the strain selected from culture collection. Please provide the citation.

Line 110-118: Please provide the full name of the “NIMM” culture collection. If these strains are deposited as type strains in a valid culture collection, the authors should present the strain designation in the appropriate format, for example: Kitasatospora cheerisanensis NIMM 60144ᵀ (with “ᵀ” in superscript).

Line 175: “Taq” italic

Line 177: EzBioCloud 

Line 200: I think this is antimicrobial activity screening not susceptibility testing. Please check

 

Author Response

Comment 1: The authors should specify which millipede species were used in this study. Additionally, please clarify how many individual millipedes were used for the isolation process-was a single specimen used, or were multiple millipedes combined after surface sterilization and homogenization?

Response 1: Thank you for this important clarification. In the revised manuscript, we have addressed both points in the Materials and Methods section as follows:

1. Species Identification: We have now specified that the millipede host used in this study was Prospirobolus joannsi.
2. Sample processing: To clarify the isolation procedure, we have added that two individual millipedes were processed separately. Each specimen was surface-sterilized, homogenized individually, and its homogenate was inoculated onto cultivation media as an independent sample.

P3, L95-97

Candidate helper strains consisted of 19 strains isolated from millipede samples (Prospirobolus joannsi) and 10 strains from a strain bank (Figure 1a).

P3, L97-99

We collected two millipedes from Xiala Mountain (Ningbo, Zhejiang, China) in October 2023 and transported them alive to the laboratory for bacterial cultivation.

P3, L101-102

Under sterile conditions, each sample was individually homogenized in 1 mL of PBS using a vortex mixer for 15 minutes.

 

Comment 2: In my opinion, Prediction of BCGs using 16S rRNA gene is too rough.

Response 2: Thank you for raising this critical point. We fully agree that predicting biosynthetic gene clusters (BGCs) based solely on 16S rRNA gene phylogeny is an approximate method, as it cannot accurately infer the presence or function of specific secondary metabolite pathways due to its limited resolution at the species level and the loose coupling between phylogeny and metabolic traits.We would like to clarify that this approach was employed strictly as a practical and efficient preliminary screening tool, in this study. Faced with a large library of environmental isolates, our goal was to rationally select a phylogenetically diverse subset of candidate strains for downstream, labor-intensive empirical antibacterial assays. This strategy was intended to increase the probability of encountering unique bioactivities by ensuring phylogenetic coverage, rather than to predict specific activities.To prevent any misunderstanding, we have explicitly clarified the purpose and limitation of this screening step in the revised results section, stating that it was used for strain selection based on diversity, not for activity prediction.

P6, L277-282

We emphasize that the 16S rRNA-based phylogenetic analysis was used not to predict antibacterial activity, but as a preliminary diversity-based screening strategy. Its purpose was to select a phylogenetically representative subset of strains from our large isolate collection for subsequent empirical antibacterial testing, thereby improving overall screening efficiency.

 

Comment 3: Typically, BLAST results obtained from EzBioCloud report sequence similarity values with two decimal places (e.g..98.68%). I am unsure why the authors reported the similarity values only as whole numbers.

Response 3: We used sequence similarity values with two decimal places per your comment.

 

Comment 4: If the authors intend to propose these strains as new species, they should clearly provide the criteria used for species delineation along with appropriate citations supporting those criteria.

Response 4: Thank you for this suggestion. In the revised manuscript, we have now clearly stated the species delineation criteria applied in this study, which are in accordance with one reference.

P6, L259-261

A total of 540 bacterial isolates were identified, representing 244 operational taxonomic units (OTUs), 78 of which were novel species (< 98.5% 16S rRNA similarity to the closest known relative among the isolates in EzBioCloud) ^[20].

P15, L517-518

Kim, M.; Oh, H. S.; Park, S. C.; Chun, J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 2014, 64(Pt 2), 346–351.

 

Comment 5: Page 4, line 171. The authors state that the isolates were randomly picked. It would be helpful to clarify how selection bias was minimized during this process and what criteria or procedures were used to ensure that the isolates were truly selected at random.

Response 5: We thank the reviewer for this important methodological question. We agree that the term "random selection" requires precise definition to avoid ambiguity. In this study, our aim was to implement a structured and unbiased sampling method to capture the broadest possible phenotypic diversity of culturable bacteria from the primary plates, thereby minimizing human selection bias.To achieve this, sterile disposal needles were used to pick colonies at predetermined, evenly spaced intervals across the entire surface of the agar plate, regardless of colony size, morphology, or pigmentation. Particular attention was paid to include microcolonies. This method ensures that every colony on the plate had a roughly equal probability of being selected, as the selection was based on location within a fixed grid rather than researcher preference for specific phenotypes. We have revised the relevant section in the Materials and Methods to clarify this procedure in detail.

Line 172-174

To construct an unbiased isolate library, bacterial colonies were picked using a sterile disposal needle at evenly spaced intervals across the entire agar plate, without selection based on colony morphology.

 

Comment 6: Page 3, line 105: What is the meaning of "Among the 74 operational taxonomic units (OTUs) identified, were author isolated 74 strains from the millipede

Response 6: Thank you for your comment. We agree that our original wording was unclear. Following your suggestion, we have revised the sentence to improve its clarity.

Line 105-107

From the 74 bacterial strains ultimately isolated and identified from the millipedes, 19 actinobacterial strains were randomly selected as candidate helper strains.

 

Comment 7: Page 3, line 107. If author have published growth promoting test for the strain selected from culture collection. please provide the citation.

Response 7: This paper has not been published yet, so it cannot be cited at this time.

 

Comment 8: Page 3, line 114-120. Please provide the full name of the “NlMM" culture collection. If these strains are deposited as type strains in a valid culture collection, the authors should present the strain designation in the appropriate format, for example: Kitasatospora cheerisanensis NlMM 60144^T (with “^T” in superscript).

Response 8: We edited per your comments.

 

Comment 9: Page 4, line 178. “Tag” italic. “27F and 1492R” should be “27F and 1492R”

Response 9: We edited per your comment.

 

Comment 10: Page 4, line 181-182. EzBioCloud. “EZBIOCLOUD” should be “EzBioCloud”

Response 10: We edited per your comment.

 

Comment 11: Page 5, line 203. I think this is antimicrobial activity screening not susceptibility testing. Please check.

Response 11: We edited per your comment.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors should use the current systematics, Actinobacteria does not exist any longer, it is Actinomycetota (Phylum) and Actinomycetes (Class).

Although growth promoting compounds can be applied in pure culture situation, it is still not clear how this is demonstrated in the isolation stage as there are many different competitive microorganisms. Antagonism should be demonstrated against over competing bacteria as well. 

Although the concept is excellent more evidence is needed.

Comments on the Quality of English Language

can be improved

Author Response

Comments 1:Authors should use the current systematics, Actinobacteria does not exist any longer, it is Actinomycetota (Phylum) and Actinomycetes (Class).

Response 1:Thank you very much for your careful review and valuable comments regarding the taxonomic nomenclature.

1. As suggested, we have replaced all mentions of the phylum name 'Actinobacteria' with the currently accepted term 'Actinomycetota' throughout the tables and the text.
2. Regarding the term "actinobacterial strains": We acknowledge that "Actinomycetota" is the correct phylum name. However, "actinobacterial" remains a widely recognized and commonly used adjective to describe strains within this phylum. To ensure clarity, we have introduced the phrase "actinobacterial strains (phylum Actinomycetota)" upon its first use in both the Abstract and the Introduction. Subsequently, we use "actinobacterial strains" as a general descriptor for strains belonging to this phylum, consistent with prevailing usage in the literature.

We believe these revisions fully address your concern while maintaining readability for our audience. Thank you again for this important correction.

Comments 2:Although growth promoting compounds can be applied in pure culture situation, it is still not clear how this is demonstrated in the isolation stage as there are many different competitive microorganisms. Antagonism should be demonstrated against over competing bacteria as well. Although the concept is excellent more evidence is needed.

Response 1:We fully acknowledge the limitations of our initial findings as pointed out in the comment and have revised the manuscript accordingly to address them as comprehensively as possible.

1. We acknowledge that our original discussion on the mechanisms within environmentally complex cultivation experiments was limited. To strengthen this, we have added new experimental data (Figure S2) to show grow-promoting effects from helper strains, and have significantly expanded the accompanying explanation and discussion in the main text.
2. As suggested, we have now explicitly discussed the potential role of antagonistic interactions in suppressing fast-growing competitors. This discussion has been integrated into the manuscript and is supported by additional relevant references.

The core of these revisions is reflected in the following newly added paragraph in the Discussion section. We hope these additions directly respond to your comment by providing more nuanced evidence and discussion, thereby strengthening the manuscript's conclusions and contextualizing its scope. We are grateful for the feedback, which has undoubtedly improved the quality of our work.

P6, L261-263

In media supplemented with the supernatants from helper strains, we consistently observed a greater diversity and abundance of colonies compared to control (Figure S2a-d).

P13, L400-411

Although the growth-promoting activity of cell-free supernatants from helper strains has been demonstrated in pure culture, its efficacy during the initial isolation stage from a sample remains unclear due to the presence of complex competitive mi-crobiota. A successful isolation strategy should therefore incorporate not only positive growth factors (Figure S2a-d) but also antagonistic factors that can suppress fast-growing dominants ^[10]. In laboratory co-culture, faster-growing species often out-compete others for resources, even if these slower growers are dominant in their natu-ral habitat ^[8,29]. Consequently, applying negative pressure (e.g., via antagonism) on these fast growers can shift the cultivated community's composition, potentially en-riching it with species that would otherwise be missed. The underlying mechanisms of these key growth factors (both positive and negative) require further experimentation, which should be a priority for follow-up studies.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

1] Actinobacteria name is not used any longer, authors please use the correct definitions and refer to the publication by Oren, Aharon, and George M. Garrity. "Valid publication of the names of forty-two phyla of prokaryotes." International journal of systematic and evolutionary microbiology 71, no. 10 (2021): 005056.

2] Place photos not diagrams with treatment and control plates to demonstrate your hypothesis please.

Comments on the Quality of English Language

can be improved

Author Response

Comments 1: Actinobacteria name is not used any longer, authors please use the correct definitions and refer to the publication by Oren, Aharon, and George M. Garrity. "Valid publication of the names of forty-two phyla of prokaryotes." International journal of systematic and evolutionary microbiology. 71, no. 10 (2021): 005056.

Respone 1: Thank you for the comment. We have revised the terminology throughout the manuscript to reflect the updated phylum name 'Actinomycetota' as suggested. We also added reference ‘Oren et al 2021’ as reviewer suggested. Specifically, in the title and key instances, we have used the phrase 'actinobacterial (phylum Actinomycetota) strains' to maintain clarity and align with the current taxonomy. We have used the term 'actinobacterial strains' sparingly and only where contextually necessary as same as other recent references.

Examples)

[1] Chavarría-Pizarro, L.; Núñez-Montero, K.; Gutiérrez-Araya, M.; Watson-Guido, W.; Rivera-Méndez, W.; Pizarro-Cerdá, J. Novel strains of Actinobacteria associated with neotropical social wasps (Vespidae; Polistinae, Epiponini) with antimicrobial potential for natural product discovery. FEMS Microbes. 2024, 5, xtae005.

[2] Jarmusch, S. A.; Schostag, M. D.; Yang, Z.; Wang, J.; Andersen, A. J. C.; Weber, T.; Ding, L. Lydicamycins Induce Morphological Differentiation in Actinobacterial Interactions. Environ. Microbiol. 2025, 91, e00295-25.

[3] Maud, L.; Barakat, N.; Bornot, J.; Snini, S.P.; Mathieu, F. Biocontrol of Mycotoxigenic Fungi by Actinobacteria. Fungi. 2025, 11, 4.

[4] M. L. Saez, J.; E. Raimondo, E.; B. Costa-Gutierrez, S.; D. Aparicio, J.; Mosca Angelucci, D.; Donati, E.; A. Polti, M.; C. Tomei, M.; S. Benimeli, C. Enhancing Environmental Decontamination and Sustainable Production through Synergistic and Complementary Interactions of Actinobacteria and Fungi. Heliyon. 2025, e42135.

Comments 2: Place photos not diagrams with treatment and control plates to demonstrate your hypothesis please.

Respone 1: Thank you for your constructive comment. As suggested, we have replaced the diagrams in Figure 1 with actual photographs of the treatment and control plates to visually demonstrate our experimental hypothesis more clearly.

The revised figure is now included in the updated manuscript.

Author Response File: Author Response.pdf