Acaricidal Activity of Biosurfactants Produced by Serratia ureilytica on Tetranychus urticae and Their Compatibility with the Predatory Mite Amblyseius swirskii

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review report

Journal: Microbiology research

Manuscript number:  microbiolres-3691120

Title: Acaricidal Activity of Biosurfactants Produced by Serratia ureilytica on Tetranychus urticae and Their Compatibility with the Predatory Mite Ambliseus swirski

Comprehensive Review of Manuscript

Typos: Thes fatty acids change to-These fatty acids.

-Progresses logically: genus background - biocontrol potential - specific pest problem - study aim.

-Paragraphs transition smoothly from general to specific.

-Sufficient background is given on the problem (T. urticae) and the potential solution (Serratia ureilytica).

-Consider integrating a sentence comparing biosurfactants to existing commercial biopesticides.

The manuscript is well-organized, following a standard scientific paper format

 Introduction, Materials and Methods, Results, Discussion, and Conclusions. However, some sections could be refined for better flow: 

- Introduction: The transition from general Serratia species to S. ureilytica is abrupt. Consider adding a sentence bridging the two topics. 

- Materials and Methods: Subsection 2.10 ("Biosurfactants Characterization by GC-MS") could be merged with 2.3 (Characterization of Biosurfactants) to avoid redundancy. 

Line 86–87: - to reach a final concentration of 10% (v/v) in the culture medium.

Suggested: resulting in a final inoculum concentration of 10% (v/v).

Line 91: the ethanolic solution was stirred.

Suggested: “The solution was stirred with ethanol.

Line 106–111: EI24 (%) = (HE/HT) 100 (29; 30).

 Suggested calculated as: EI₄₂ (%) = (HE/HT) × 100, following standard protocols [29, 30].

- Results: The subsections are clear, but Figure/Tables 1–4 are referenced before their legends appear (e.g., Figure 1 is discussed on page 5 but appears on page 6). Ensure figures/tables are placed close to their first mention. 

Line 199: By 72 h, mortality induced by S. ureilytica NOD-3 and UTS reached 99%, mirroring the efficacy of abamectin (Fig. 1).

Suggested: At 72 h post-treatment, S. ureilytica NOD-3 and UTS achieved 99% mortality, comparable to abamectin.

Line 204–212: Very informative, but sentence structure is repetitive with (caused) and (induced). Varying the verbs helps maintain interest.

Line 239–246: Suggested Revision:

GC-MS analysis revealed fatty acids including hexadecanoic, pentanoic, octadecanoic, decanoic, and tetradecanoic acids. Among these, hexadecanoic and octadecanoic acids were present in all three strains. Notably, metabolites such as hexadecanamide and octadecanamide were also detected. The amino acid γ-guanidinobutyric acid was exclusively present in the UTS strain.

The writing is generally clear, but some sentences are overly complex or ambiguous: 

- Example (Page 2, Lines 50–52): The use of bacteria or their derivatives... has emerged as a promising alternative due to their safety for human health, lower risk of resistance compared to conventional pesticides, and absence of residual environmental impact.

- Suggestion: "Bacterial derivatives offer a promising alternative to chemical pesticides because they are safer for humans, reduce resistance risks, and minimize environmental residue." 

Minor grammatical errors and stylistic improvements are needed: 

- Mix of past and present tense (e.g., This study evaluated vs. These results show). Stick to past tense for methods/results. 

-Replace informal phrases like lined up with (Page 10, Line 322) with (aligned with). 

-Overuse of passive voice (e.g., were detected). Active voice is preferable where possible (e.g. We detected). 

-The study provides robust experimental data, but the Discussion could better contextualize findings. For example: 

- Compare biosurfactant efficacy with other Serratia species (e.g., S. marcescens). 

- Discuss practical implications for field applications (e.g., scalability, cost). 

- Bioinformatics: The NRPS gene clusters (Table 2) are intriguing but need more explanation. Link these genes directly to the observed acaricidal activity. 

The work is good, but some sections closely mirror prior studies (e.g., GC-MS methods resemble Zamorano-González et al., 2025). Differentiate by emphasizing novel findings (e.g., specific fatty acids' roles). 

- General Clarity: Define acronyms (e.g., NRPS at first use) and avoid jargon like "biorational" without explanation. 

7. Figures and Tables,

Legends: 

Figure 1: Specify statistical test used (Tukey’s test) in the caption. 

  - Table 1: Clarify "RT (min)" as "Retention Time (minutes).

- Page 1, Abstract: 

These findings highlight the potential of S. ureilytica-derived biosurfactants 

  - Revised: These findings demonstrate that S. ureilytica biosurfactants are promising eco-friendly acaricides, reducing T. urticae populations by >95% while partially sparing A. swirskii. 

Discussion: 

  -The high mortality in A. swirskii (75–85%) suggests selectivity issues, warranting formulation optimizations to enhance predator compatibility. 

Line 285:

adding a novel property for this bacterial species.

Suggested revealing a novel acaricidal property of this bacterial species.

Line 294–297:

Action of lytic enzymes that have been found in the biosurfactant fraction of the crude extract from the liquid cultures.

Suggested likely due to lytic enzymes and specific fatty acids detected in the biosurfactant-rich crude extracts.

Line 305–308:

Consider combining:

These fatty acids may likely be implicated in the observed acaricidal effect. The identified fatty acids included.

Suggested: These fatty acids are likely responsible for the acaricidal activity, particularly hexadecanoic, octadecanoic, and tetradecanoic acids, which have been reported to have insecticidal effects [36].

Line 316–321:

The comparison with known NRPS products is excellent.

- Address minor grammatical errors (e.g., "lytic enzymes that have been found" - lytic enzymes found). 

- Figures: Ensure high resolution and consistent formatting. 

The manuscript is well-written and impactful but would benefit from these refinements to enhance clarity and engagement.

- Line 322: Replace lined up with - aligned with.

- Table 1: Add Retention Time (minutes) footnote. 

- Figure 3: Fix y-axis label truncation. 

Author Response

Response to reviewers

Reviewer #1: 

Title: Acaricidal Activity of Biosurfactants Produced by Serratia ureilytica on Tetranychus urticae and Their Compatibility with the Predatory Mite Ambliseus swirski

Comment. Comprehensive Review of Manuscript

• Typos: Thes fatty acids change to-These fatty acids.
• Progresses logically: genus background - biocontrol potential - specific pest problem - study aim.
• Paragraphs transition smoothly from general to specific.
• Sufficient background is given on the problem (T. urticae) and the potential solution (Serratia ureilytica).
• Consider integrating a sentence comparing biosurfactants to existing commercial biopesticides.

Response. Thank you for your thorough and constructive feedback.

• The typo “Thes fatty acids” has been corrected to “These fatty acids” (Line 28)
• We appreciate your positive comments regarding the logical structure and flow of the manuscript.
• Following your suggestion, we have added a sentence comparing biosurfactants to existing commercial biopesticides in the discussion section (lines 285-286), to better contextualize the potential of S. ureilytica.

The manuscript is well-organized, following a standard scientific paper format

Introduction, Materials and Methods, Results, Discussion, and Conclusions. However, some sections could be refined for better flow:

Comment. Introduction: The transition from general Serratia species to S. ureilytica is abrupt. Consider adding a sentence bridging the two topics.

Response.

To improve the flow and clarity of the paragraph, we added a bridging sentence to smoothly transition from the general discussion of Serratia species to the specific focus on S. ureilytica (lines 43-46). 

Comment. Materials and Methods: Subsection 2.10 ("Biosurfactants Characterization by GC-MS") could be merged with 2.3 (Characterization of Biosurfactants) to avoid redundancy.

Response.

We have merged subsection 2.10 into 2.3 under a unified heading “Characterization of Biosurfactants” to avoid redundancy and improve section cohesion. The description of GC-MS analysis now appears as part of this comprehensive characterization (Lines 116-128).

Comment. Line 86–87: - to reach a final concentration of 10% (v/v) in the culture medium. Suggested: resulting in a final inoculum concentration of 10% (v/v).

Response. Corrected (Line 88).

Comment. Line 91: the ethanolic solution was stirred. Suggested: “The solution was stirred with ethanol.

Response. Corrected (Line 93).

-Line 106–111: EI24 (%) = (HE/HT) 100 (29; 30). Suggested calculated as: EI₄₂ (%) = (HE/HT) × 100, following standard protocols [29, 30].

Response. Corrected (Lines 114-115).

Comment. Results: The subsections are clear, but Figure/Tables 1–4 are referenced before their legends appear (e.g., Figure 1 is discussed on page 5 but appears on page 6). Ensure figures/tables are placed close to their first mention.

Response. Corrected.

Figures 1-4 (Lines 209, 214, 224, and 238, respectively) and Tables 1 and 2 (Lines 263 and 277). Figures and Tables appear immediately after their first mention in the text, improving the flow and readability of the Results section.

Comment. Line 199: By 72 h, mortality induced by S. ureilytica NOD-3 and UTS reached 99%, mirroring the efficacy of abamectin (Fig. 1). Suggested: At 72 h post-treatment, S. ureilytica NOD-3 and UTS achieved 99% mortality, comparable to abamectin.

Response. Corrected (208-209).

Comment. Line 204–212: Very informative, but sentence structure is repetitive with (caused) and (induced). Varying the verbs helps maintain interest. Line 239–246: Suggested Revision: GC-MS analysis revealed fatty acids including hexadecanoic, pentanoic, octadecanoic, decanoic, and tetradecanoic acids. Among these, hexadecanoic and octadecanoic acids were present in all three strains. Notably, metabolites such as hexadecanamide and octadecanamide were also detected. The amino acid γ-guanidinobutyric acid was exclusively present in the UTS strain.

Response.Corrected (Lines 256-260).

Comment. The writing is generally clear, but some sentences are overly complex or ambiguous:

- Example (Page 2, Lines 50–52): The use of bacteria or their derivatives... has emerged as a promising alternative due to their safety for human health, lower risk of resistance compared to conventional pesticides, and absence of residual environmental impact. Suggestion: "Bacterial derivatives offer a promising alternative to chemical pesticides because they are safer for humans, reduce resistance risks, and minimize environmental residue."

Response. Corrected (Lines 66-68).

Minor grammatical errors and stylistic improvements are needed:

Comment. Mix of past and present tense (e.g., This study evaluated vs. These results show). Stick to past tense for methods/results.

Response.

We revised the entire manuscript to ensure consistent use of the past tense throughout the Methods and Results sections. Phrases such as “These results show” and “This study evaluates” were changed to “These results showed” and “This study evaluated” to align with conventional scientific reporting in past tense.

Comment. Replace informal phrases like lined up with (Page 10, Line 322) with (aligned with).

Response

Informal expressions were replaced with more appropriate academic language. For example, “lined up with” (Page 10, Line 322) was revised to “aligned with (Line 340).

Comment. Overuse of passive voice (e.g., were detected). Active voice is preferable where possible (e.g. We detected).

Response.

Where possible and appropriate, we revised passive constructions to active voice to improve clarity and reader engagement. For example, in Line 269.

Comment. The study provides robust experimental data, but the Discussion could better contextualize findings. For example:

- Compare biosurfactant efficacy with other Serratia species (e.g., S. marcescens). 

Response.

We strengthened the Discussion section to better contextualize the findings. We compared the efficacy of the biosurfactants with that of the chemical acaricide abamectin, emphasizing the comparable mortality rates observed for S. ureilytica strains (e.g., UTS and NOD-3) (Lines 309-311). 

- Discuss practical implications for field applications (e.g., scalability, cost).

Response.

The reviewer's observation is very accurate; however, at this stage of the research development, we are evaluating biosurfactants produced by Serratia ureilytica in various biological models to assess their effect. We are also evaluating optimal yield, for which we are defining the culture conditions by assessing factors such as carbon and nitrogen sources, lipophilic substrate concentration, micronutrient availability, inoculum size, temperature, pH, aeration, and agitation speed, with the goal of producing it economically.

- Bioinformatics: The NRPS gene clusters (Table 2) are intriguing but need more explanation. Link these genes directly to the observed acaricidal activity.

Response.

One of the detected NRPSs showed similarity to 5-dimethylallylindole-3-acetonitrile, an antibacterial compound (Long et al. 2024). Another NRPS exhibited structural homology to xenotetrapeptide, a metabolite with insecticidal properties (Mahar et al., 2005; Kegler et al., 2014). Additionally, a third NRPS cluster displayed homology to kolossin, a compound reported with bioactivity against unicellular protists (Bode et al., 2015; Tobias et al., 2018). (lines 333-337)

The work is good, but some sections closely mirror prior studies (e.g., GC-MS methods resemble Zamorano-González et al., 2025). Differentiate by emphasizing novel findings (e.g., specific fatty acids' roles).

Response.

The methodology is the same because it is the same technique and equipment used to analyze the biosurfactants in the work of Zamorano-González et al., 2025; however, the biological activity was evaluated in nematodes and fungi, and the presence of fatty acids and amino acids is related to the structure of the lipopeptide.

Comment. General Clarity: Define acronyms (e.g., NRPS at first use) and avoid jargon like "biorational" without explanation.

Response.

All acronyms were carefully reviewed and defined at their first mention in the manuscript (Lines 30-31, 272-273, 332).

Comment. Figures and Tables,

Legends:

Figure 1: Specify statistical test used (Tukey’s test) in the caption.

  - Table 1: Clarify "RT (min)" as "Retention Time (minutes).

Response.

We have revised the legend of Figure 1 to include the statistical test used. The abbreviation “RT (min)” has been clarified for better understanding

Comment. Page 1, Abstract: 

These findings highlight the potential of S. ureilytica-derived biosurfactants 

  - Revised: These findings demonstrate that S. ureilytica biosurfactants are promising eco-friendly acaricides, reducing T. urticae populations by >95% while partially sparing A. swirskii.

Response. Corrected (Lines 32-33).

Comment. Discussion:

  -The high mortality in A. swirskii (75–85%) suggests selectivity issues, warranting formulation optimizations to enhance predator compatibility

Response. Corrected (295-297).

adding a novel property for this bacterial species.

Suggested revealing a novel acaricidal property of this bacterial species.

Response. Corrected (Lines 300-302).

Line 294–297: Action of lytic enzymes that have been found in the biosurfactant fraction of the crude extract from the liquid cultures. Suggested: likely due to lytic enzymes and specific fatty acids detected in the biosurfactant-rich crude extracts.

Response. Corrected (Lines 312-314).

Comment. Line 305–308: Consider combining: These fatty acids may likely be implicated in the observed acaricidal effect. The identified fatty acids included. Suggested: These fatty acids are likely responsible for the acaricidal activity, particularly hexadecanoic, octadecanoic, and tetradecanoic acids, which have been reported to have insecticidal effects [36].

Response. Corrected (Lines 322-324).

Comment. Line 316–321: The comparison with known NRPS products is excellent

- Address minor grammatical errors (e.g., "lytic enzymes that have been found" – lytic enzymes found).

Response. Corrected (Line 312-314).

Comment. Figures: Ensure high resolution and consistent formatting.

Response.

We have carefully reviewed all figures to ensure they meet the journal’s requirements for resolution and formatting

The manuscript is well-written and impactful but would benefit from these refinements to enhance clarity and engagement.

Comment. Line 322: Replace lined up with - aligned with.

Response. Corrected (Line 340).

Comment. Table 1: Add Retention Time (minutes) footnote.

Response. Corrected

Comment. Figure 3: Fix y-axis label truncation. 

Response. Corrected

1. Zamorano-González CA, Ramírez-Trujillo JA, Pilotzi-Xahuentitla H, Yáñez-Ocampo G, Hernández-Núñez E, Suárez Rodríguez R, Orea-Flores MLA, Gómez-Rodríguez O, Espinosa-Zaragoza S, Rangel-Zaragoza JL, Aguilar-Marcelino L, Aguilar-Fuentes J, Wong-Villarreal A (2025) In Vitro Evaluation of the Biosurfactant Produced by Serratia ureilyticaUTS with Antifungal and Nematicidal Activity Against Nacobbus aberrans. Curr Microbiol 82(2):63. https://doi.org/10.1007/s00284-024-04042-8

Reviewer #2

This manuscript presents relevant and original work on the use of biosurfactants from Serratia ureilytica as a potential control agent against Tetranychus urticae. The integration of laboratory bioassays, greenhouse validation, chemical profiling, and genomic analysis gives the study both scientific depth and applied value. The topic is timely and addresses a concrete need for sustainable alternatives to chemical acaricides.

At the same time, several important aspects require clarification and improvement before the manuscript can be considered for publication. The following points are intended to help strengthen the scientific quality, readability, and practical relevance of the work.

Comment. Non-Target Toxicity: The high mortality observed in Amblyseius swirskii (up to 82%) raises concerns for compatibility with integrated pest management systems. This issue should be addressed more critically in the discussion, including possible explanations and future directions to reduce non-target effects.

Response.

We acknowledge the importance of evaluating compatibility with IPM systems. The discussion has been expanded to include possible explanations for the high sensitivity observed in Amblyseius swirskii. We also emphasize the need to optimize application methods to mitigate adverse effects on beneficial mites (Lines 295-300).

Comment. Mechanism of Action: The mode of action of the biosurfactants is not explored or discussed. Even a brief explanation of possible mechanisms such as contact toxicity, membrane disruption, or enzyme inhibition, would strengthen the interpretation of results and align the biological findings with the chemical and genomic data.

Response.

A detailed explanation of the potential mechanisms of action of biosurfactants has been added, including contact toxicity through membrane disruption and cuticular dehydration. These mechanisms are supported by previous studies and align with the observed biological effects (Lines 287-295)

Methodological Details: Please clarify the following:

On what basis was the 0.5% (w/v) biosurfactant concentration selected?

Response.

This concentration was selected taking as a reference the article, In Vitro Evaluation of the Biosurfactant Produced by Serratia ureilytica UTS with Antifungal and Nematicidal Activity Against Nacobbus aberrans, where they evaluate different concentrations of biosurfactants against phytopathogenic fungi and nematodes, this is the concentration where a significant effect was observed, based on this background it was proposed to evaluate this concentration against Tetranychus urticae (Zamorano- González et al., 2025)

Were the biosurfactants applied immediately after extraction, or stored? If stored, specify how.

Response.

They have been used in both forms after 24 and 48 hours of extracting the biosurfactants and also after storing at room temperature for a period. They have been evaluated on the same pathogens and the same effect on mortality has not been observed, which is why mentioning this condition has not been considered.

What was the pest density at the start of the greenhouse trial?

Response.

The pest density was measured prior to the treatment application (day 0) and at 7-, 14-, and 21-days post-treatment. At the start of the trial (day 0) the pest density was in average 10-17 adults per cm2, 10-15 nymphs per cm2, and 20-35 eggs cm2. This information has been added to the manuscript

Which database or criteria were used for GC-MS compound identification?

Response

The NIST Mass Spectroscopy library was used, considered the most reliable due to its quality, wide coverage and accessibility.

Comment. Terminology and Consistency: Terms such as "biosurfactant," "extract," and "compound" should be used more precisely and consistently. Additionally, units (e.g. °C, mg/L), strain names, and abbreviations should be standardized throughout the manuscript.

Response.

We have thoroughly revised the manuscript to ensure consistency and precision in terminology.

Comment. Figures and Tables: Figures are informative and statistically supported. To improve readability: Consider using shading or visual markers to distinguish treatments more clearly.

Response.

 As recommended, we applied shading to Figures 1 and 3 to enhance the visual distinction between treatments and facilitate interpretation.

Comment. Summarize key quantitative results in the figure legends or main text, especially in the greenhouse section.

Response.

To improve clarity and readability, we revised the greenhouse results section to include a concise summary of the key quantitative findings and highlight the most effective biosurfactant treatments at the beginning of the paragraph (Lines 227-230).

Comment. Provide a key for the +/– symbols used in Table 1.

Response.

We have added a key to Table 1 explaining the meaning of the +/– symbols to clarify the presence or absence of each compound.

Comment. Broader Impact and Limitations: Some claims regarding environmental sustainability and potential application may be overstated, given the non-target effects and the lack of field testing. These points should be presented more cautiously, acknowledging that further work is needed to support practical use.

Response.

We have revised the relevant sections of the Discussion and Conclusion to present the environmental implications and potential applications of the biosurfactants more cautiously (Lines 296-300, 349-353, respectively).

Comment. Comments on the Quality of English Language.

Language and Clarity: The manuscript contains numerous grammatical errors, awkward sentence structures, and imprecise wording. While the main ideas are understandable, the overall clarity would improve significantly with careful editing. A professional English language revision is strongly recommended.

Response.

We acknowledge the reviewer’s comment regarding the language quality. To address this, the manuscript has been thoroughly revised for grammar, syntax, and clarity.

Ben Khedher S, Boukedi H, Laarif A, Tounsi S (2020). Biosurfactant produced by Bacillus subtilis V26: A potential biological control approach for sustainable agriculture development. Organic Agriculture, 10, 117124. https://doi.org/10.1007/s13165-020-00316-0

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Mahar AN, Munir M, Elawad S, Gowen SR, Hague NG (2005) Pathogenicity of bacterium, Xenorhabdus nematophila isolated from entomopathogenic nematode (Steinernema carpocapsae) and its secretion against Galleria mellonella larvae. J Zhejiang Univ Sci B. 6(6):457-63. doi: 10.1631/jzus.2005.B0457.

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Zamorano-González CA, Ramírez-Trujillo JA, Pilotzi-Xahuentitla H, Yáñez-Ocampo G, Hernández-Núñez E, Suárez Rodríguez R, Orea-Flores MLA, Gómez-Rodríguez O, Espinosa-Zaragoza S, Rangel-Zaragoza JL, Aguilar-Marcelino L, Aguilar-Fuentes J, Wong-Villarreal A (2025) In Vitro Evaluation of the Biosurfactant Produced by Serratia ureilytica UTS with Antifungal and Nematicidal Activity Against Nacobbus aberrans. Curr Microbiol 82(2):63. https://doi.org/10.1007/s00284-024-04042-8

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This manuscript presents relevant and original work on the use of biosurfactants from Serratia ureilytica as a potential control agent against Tetranychus urticae. The integration of laboratory bioassays, greenhouse validation, chemical profiling, and genomic analysis gives the study both scientific depth and applied value. The topic is timely and addresses a concrete need for sustainable alternatives to chemical acaricides.

At the same time, several important aspects require clarification and improvement before the manuscript can be considered for publication. The following points are intended to help strengthen the scientific quality, readability, and practical relevance of the work.

1. Non-Target Toxicity: The high mortality observed in Amblyseius swirskii (up to 82%) raises concerns for compatibility with integrated pest management systems. This issue should be addressed more critically in the discussion, including possible explanations and future directions to reduce non-target effects.
2. Mechanism of Action: The mode of action of the biosurfactants is not explored or discussed. Even a brief explanation of possible mechanisms such as contact toxicity, membrane disruption, or enzyme inhibition, would strengthen the interpretation of results and align the biological findings with the chemical and genomic data.
3. Methodological Details: Please clarify the following:

• On what basis was the 0.5% (w/v) biosurfactant concentration selected?
• Were the biosurfactants applied immediately after extraction, or stored? If stored, specify how.
• What was the pest density at the start of the greenhouse trial?
• Which database or criteria were used for GC-MS compound identification?

4.Terminology and Consistency: Terms such as "biosurfactant," "extract," and "compound" should be used more precisely and consistently. Additionally, units (e.g. °C, mg/L), strain names, and abbreviations should be standardized throughout the manuscript.

5. Figures and Tables: Figures are informative and statistically supported. To improve readability:

• Consider using shading or visual markers to distinguish treatments more clearly.
• Summarize key quantitative results in the figure legends or main text, especially in the greenhouse section.
• Provide a key for the +/– symbols used in Table 1.

6. Broader Impact and Limitations: Some claims regarding environmental sustainability and potential application may be overstated, given the non-target effects and the lack of field testing. These points should be presented more cautiously, acknowledging that further work is needed to support practical use.

Comments on the Quality of English Language

Language and Clarity: The manuscript contains numerous grammatical errors, awkward sentence structures, and imprecise wording. While the main ideas are understandable, the overall clarity would improve significantly with careful editing. A professional English language revision is strongly recommended.

Author Response

Reviewer #2

This manuscript presents relevant and original work on the use of biosurfactants from Serratia ureilytica as a potential control agent against Tetranychus urticae. The integration of laboratory bioassays, greenhouse validation, chemical profiling, and genomic analysis gives the study both scientific depth and applied value. The topic is timely and addresses a concrete need for sustainable alternatives to chemical acaricides.

At the same time, several important aspects require clarification and improvement before the manuscript can be considered for publication. The following points are intended to help strengthen the scientific quality, readability, and practical relevance of the work.

Comment. Non-Target Toxicity: The high mortality observed in Amblyseius swirskii (up to 82%) raises concerns for compatibility with integrated pest management systems. This issue should be addressed more critically in the discussion, including possible explanations and future directions to reduce non-target effects.

Response.

We acknowledge the importance of evaluating compatibility with IPM systems. The discussion has been expanded to include possible explanations for the high sensitivity observed in Amblyseius swirskii. We also emphasize the need to optimize application methods to mitigate adverse effects on beneficial mites (Lines 295-300).

Comment. Mechanism of Action: The mode of action of the biosurfactants is not explored or discussed. Even a brief explanation of possible mechanisms such as contact toxicity, membrane disruption, or enzyme inhibition, would strengthen the interpretation of results and align the biological findings with the chemical and genomic data.

Response.

A detailed explanation of the potential mechanisms of action of biosurfactants has been added, including contact toxicity through membrane disruption and cuticular dehydration. These mechanisms are supported by previous studies and align with the observed biological effects (Lines 287-295)

Methodological Details: Please clarify the following:

On what basis was the 0.5% (w/v) biosurfactant concentration selected?

Response.

This concentration was selected taking as a reference the article, In Vitro Evaluation of the Biosurfactant Produced by Serratia ureilytica UTS with Antifungal and Nematicidal Activity Against Nacobbus aberrans, where they evaluate different concentrations of biosurfactants against phytopathogenic fungi and nematodes, this is the concentration where a significant effect was observed, based on this background it was proposed to evaluate this concentration against Tetranychus urticae (Zamorano- González et al., 2025)

Were the biosurfactants applied immediately after extraction, or stored? If stored, specify how.

Response.

They have been used in both forms after 24 and 48 hours of extracting the biosurfactants and also after storing at room temperature for a period. They have been evaluated on the same pathogens and the same effect on mortality has not been observed, which is why mentioning this condition has not been considered.

What was the pest density at the start of the greenhouse trial?

Response.

The pest density was measured prior to the treatment application (day 0) and at 7-, 14-, and 21-days post-treatment. At the start of the trial (day 0) the pest density was in average 10-17 adults per cm2, 10-15 nymphs per cm2, and 20-35 eggs cm2. This information has been added to the manuscript

Which database or criteria were used for GC-MS compound identification?

Response

The NIST Mass Spectroscopy library was used, considered the most reliable due to its quality, wide coverage and accessibility.

Comment. Terminology and Consistency: Terms such as "biosurfactant," "extract," and "compound" should be used more precisely and consistently. Additionally, units (e.g. °C, mg/L), strain names, and abbreviations should be standardized throughout the manuscript.

Response.

We have thoroughly revised the manuscript to ensure consistency and precision in terminology.

Comment. Figures and Tables: Figures are informative and statistically supported. To improve readability: Consider using shading or visual markers to distinguish treatments more clearly.

Response.

 As recommended, we applied shading to Figures 1 and 3 to enhance the visual distinction between treatments and facilitate interpretation.

Comment. Summarize key quantitative results in the figure legends or main text, especially in the greenhouse section.

Response.

To improve clarity and readability, we revised the greenhouse results section to include a concise summary of the key quantitative findings and highlight the most effective biosurfactant treatments at the beginning of the paragraph (Lines 227-230).

Comment. Provide a key for the +/– symbols used in Table 1.

Response.

We have added a key to Table 1 explaining the meaning of the +/– symbols to clarify the presence or absence of each compound.

Comment. Broader Impact and Limitations: Some claims regarding environmental sustainability and potential application may be overstated, given the non-target effects and the lack of field testing. These points should be presented more cautiously, acknowledging that further work is needed to support practical use.

Response.

We have revised the relevant sections of the Discussion and Conclusion to present the environmental implications and potential applications of the biosurfactants more cautiously (Lines 296-300, 349-353, respectively).

Comment. Comments on the Quality of English Language.

Language and Clarity: The manuscript contains numerous grammatical errors, awkward sentence structures, and imprecise wording. While the main ideas are understandable, the overall clarity would improve significantly with careful editing. A professional English language revision is strongly recommended.

Response.

We acknowledge the reviewer’s comment regarding the language quality. To address this, the manuscript has been thoroughly revised for grammar, syntax, and clarity.

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Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript demonstrates evident and meritorious advancement. The authors have addressed the main points raised during the initial review, including the high non-target toxicity, the lack of discussion on the mode of action, and the need for more precise terminology and clearer presentation of the results.

The present discussion provides a more considered interpretation of the risks for beneficial mites and introduces reasonable strategies to mitigate these effects in future applications. The potential mechanisms of action of the biosurfactants are also well integrated and supported by the findings of chemical and genomic research. Furthermore, the figures have been enhanced to improve their clarity and legibility, thereby facilitating the comprehension of the key quantitative outcomes.

The language and structure of the manuscript have been significantly improved, rendering it more comprehensible and professional.

It should be noted that a minor detail remains to be addressed: the section on methods does not provide a comprehensive explanation as to whether the biosurfactants were used in a fresh state or stored, despite the authors mentioning it in their reply. Incorporating this directly into the manuscript would enhance transparency and reproducibility.

In conclusion, this is a strong and original contribution to the field of biological pest control. It is recommended that the manuscript be accepted following minor revision, with the addition of a brief clarification on biosurfactant storage conditions.

Author Response

Were the biosurfactants applied immediately after extraction, or stored? If stored, specify how.

Response.

They have been used after 24 to 48 hours of extraction of the biosurfactants, storage was at room temperature (30-32 °C).