The Metabolism of Streptomyces avermitilis Isolated from Agricultural Soils During the Four Seasons of the Year

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript titled “Metabolic Profiling of Streptomyces avermitilis from Agricultural Soils Across the Four Seasons” is informative, well-organised, and presents valuable findings. However, certain aspects require refinement to enhance clarity and scientific rigour.

1. The introduction should be better connected, as it currently consists of several short and loosely linked paragraphs.
2. Several sentences are long and complex; they should be simplified or split into shorter, more concise statements to improve readability.
3. Some phrases could be clarified, e.g., “morphological characteristics of the metabolism” could be rephrased as “morphological and metabolic characteristics” for better understanding.
4. Address grammatical inconsistencies and awkward phrasings that currently reduce the manuscript’s clarity.
5. Provide the rationale for selecting the four soil types, indicate whether they differ in nutrient composition, pH, or texture to strengthen the ecological context.
6. Although the study associates pigment and growth variations with seasonal changes, specific environmental variables (e.g., temperature, pH, moisture, nutrient content, or organic matter in soil) were not measured or correlated quantitatively.
7. Conducting repeated seasonal sampling over a few years is recommended to improve reproducibility and ecological relevance.

Author Response

"Ver archivo adjunto"

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The revised manuscript (previously submitted under applmicrobiol-3891928) demonstrates a notable effort by the authors to improve clarity and add additional data.

• The methodological section is more detailed.
• Growth curve results are now included.
• The preliminary HPLC analysis provides useful information on secondary metabolites.
• The rewritten abstract clearly describes seasonal variations and pigment profiles, improving readability.
• Taxonomic identification still relies on morphological characteristics. Molecular analyses, such as 16S rRNA sequencing, would allow for a more robust identification. The authors must provide a justification for not using this approach.
• Functional analyses and molecular ecological data are not yet included. The authors must explain their absence.

Overall, the manuscript has gained in clarity and presentation. With additional methodological improvements, it could make a significant contribution to understanding the biology and ecology of Streptomyces avermitilis.

Author Response

"Ver archivo adjunto"

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors clarify the logistical and administrative constraints encountered during the study. However, to ensure adequate scientific transparency, I request that these limitations be explicitly mentioned and discussed in the manuscript, particularly in the Discussion section. This includes the lack of molecular confirmation of the species, the preliminary nature of the metabolite characterization, and the planned steps to complete the study later. Clearly presenting these limitations will contribute to strengthening the scientific rigor and readability of the manuscript.

Author Response

"Por favor, consulte el archivo adjunto."

Author Response File: Author Response.pdf

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 authors discussed Metabolic Evaluation of Streptomyces avermitilis from Agricultural Soils Across Four Seasons. The manuscript is informative and well-structured; however, I have a few comments and suggestions for improvement:

1. Figure 1 is unclear and does not add significant value. Consider removing it.
2. Include error bars in all figures to reflect variability and improve clarity.
3. Some sentences are long and dense. Breaking them into shorter, focused sentences will enhance readability.
4. Replace vague phrases such as “any drastic environmental change” with more precise terms like “environmental fluctuations” or “abiotic stressors”.
5. The term “dipsticks” should be clarified (type and purpose) as it may be ambiguous in a microbiological context.
6. Doubling time is reported in both hours and days; standardising to a single unit (e.g., hours) will make comparisons clearer.
7. Other environmental factors, such as temperature fluctuations, moisture, pH, and nutrient availability, were not explored but may also influence metabolism. Including or discussing these factors would strengthen the study.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the isolation and morphological characterization of Streptomyces avermitilis from soils in Mexico. While the topic is potentially interesting, the study suffers from several critical methodological and interpretational shortcomings.

For Taxonomic Identification: The species assignment relies solely on morphological characteristics (Bergey’s Manual). Recent studies consistently use 16S rRNA sequencing, phylogenetic analysis, and accession numbers to confirm S. avermitilis identity. Perform 16S rRNA gene sequencing for all isolates, construct a phylogenetic tree, and deposit sequences in a public database.

For metabolite Verification: Claims of pigment and ivermectin production are unsupported by chemical analysis. Visual observation alone is insufficient. Recent studies employ HPLC, LC-MS, NMR,

The manuscript does not test the biological activity of the reported metabolites.

Microbiome Analysis: Soil microbial communities can influence the growth and metabolite production of S. avermitilis. Recent study Perform 16S rRNA amplicon sequencing of soil samples to characterize microbial communities and evaluate interactions with S. avermitilis. Correlate community composition with metabolite production or growth patterns.

Statements regarding the non-endemic status of S. avermitilis in Japan are unsupported by the presented data.

The manuscript lacks molecular verification, analytical confirmation of metabolites, functional assays, and ecological context through microbiome and metabolomic analyses. The conclusions are overstated relative to the data presented. Given these substantial deficiencies, the manuscript cannot be recommended for publication in its current form.