Green Solutions for Agriculture: Topical and Oral Effect of Botanical Extracts in the Sustainable Management of Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

The manuscript deals with an important pest, responsible for significant economic losses. It´s an up to date subject, significant from the scientific and economic point of view. Overall is well written, methodology and results are described with sufficient detail; presenting good perspectives for the development of novel bionematicides. The employed methodology sounds solid and well defined. Method schematics are a great support to text, even understandable without it.

However, there´s the need for some very small improvements.

My notes and recommendations are the following ones:

The introduction includes several angles (mode of action, economic importance, etc.), but in my opinion author’s could write a paragraph on the state of the art of such type of research (is everything new, other extracts with activity in diamondback moth, similar research, others).

Authors could also explain (or in the discussion) why the 4 plant species were chosen, based in their economic/agronomic importance and prevalence, in a Brazilian or worldwide perceptive. 

Line 42: please check “certain plant species”. Did you intend to mean “insects”?

Line 51: perhaps “into” instead of “on the”. “insecticides that are ingested by the insect after translocation into the plant are called systemic” – it´s a unnecessary information.

Line 59: please check “of this group”. Which group are you referring to?

Please change the symbol for temperatures ( ̊ C / ̊ F instead of the “circle with the line underneath it") – lines 84, 85 and 105. Please check throughout the text.

Line 101: please change from Portuguese to English.

Line 102: Itahum, MS – MS means?

Please check if “AECh” is “each” throughout the text – lines 103, 106, 119, 137, 154, 166, 179, 207, 209, 210, 215 and 218.

Line 105: for accuracy 40 ºC are 104 ºF. Please add “º” before F.

Line 107: DDMS-UFGD Herbarium – DDMS-UFGD means?

Table 1 header: Please change “Especies” to “Species”.

Line 110: Please change “coordenadas” to “coordinates”.

Lines 111 and 114: As you referred to the 4 extracts, please change “extract was prepared” to “extracts were prepared”.

Line 120, Fig 1 and Fig 2: please try to use the same terminology – collard versus cabbage.

Line 121: please add “paper” before disks – not to be confused with the collard disks.

Line 178: Please leave a space between “24” and “h”.

Lines 146, 150 and 151: Please change “5 cm de Ø” to “5 cm in Ø”.

Line 147: Please replace “Micropipeta Biopet 8 Canais BSN023A” to English.

Line 151: Brassica oleraceae in italic.

Line 177: Please change “diameter” to “Ø”.

Line 178: Please leave a space between “24” and “h”.

Lines 298 and 299: reduce font size.

Table 9 header: “tannins” to “Tannins”.

Line 312: place a “*” after Treatment in header. Please replace “Hydrangeifolia” by hydrangeifolia” and “Furfuraceae” by “furfuraceae”.

Table 10 description from Portuguese to English. Change “Treatments” to “Treatment”. Place a “*” after Treatment in header. Please replace “Hydrangeifolia” by hydrangeifolia” and “Furfuraceae” by “furfuraceae”.

Reference 15: please include accession date.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. The rationale behind the selection of the four plant species is not clearly explained. Were these species chosen based on ethnobotanical relevance or previous evidence of insecticidal properties?

2. While an average mortality rate of 45% is promising, please clarify whether this level of efficacy is considered sufficient for practical pest control applications. A comparison with a positive control (a synthetic insecticide) would be valuable.

3. The manuscript lacks details on the statistical tests applied to determine the significance of the observed effects. Please specify the tests used, sample sizes, and p-values or confidence intervals.

4. The discussion would benefit from a deeper exploration of the possible modes of action of the major phytochemical groups, especially in relation to the observed oral toxicity.

5. The reported reductions in pupal duration and fecundity are relevant. Were these sublethal effects quantified statistically? A more detailed analysis would strengthen the conclusions.

6. The abstract would benefit from clearer segmentation between the two types of bioassays (oral vs. topical). Highlighting the novelty and practical relevance of the study in the first sentence would enhance impact.

7. Expressions such as "significant effect" should be used with caution. Indicate whether statistical significance is meant, and if so, at what threshold.

8. There appears to be a typographical error in Duguetia furfura-cea; it should be Duguetia furfuracea. Please double-check the spelling of all species names and ensure consistency.

9. Clearly define all plant extract abbreviations (AEAa, AECh, etc.) upon first mention and use them consistently throughout the text.

10. The topic is timely and relevant, addressing sustainable pest management and targeting an important agricultural pest (Plutella xylostella).

11. Evaluating both oral and topical toxicity provides a holistic assessment of the extracts’ potential.

12. Including a phytochemical profile adds mechanistic depth to the biological findings.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comment on the Title:
The current title, “Green Solutions for Agriculture,” is quite broad and may not fully capture the scientific focus of your study. To help readers quickly understand the core of your work, we suggest using more specific and descriptive terms such as botanical insecticides, plant-based pest control, or bioinsecticides.

Also, including the type of study (e.g., toxicity assessment, laboratory bioassays) or the scientific name of the target pest (Plutella xylostella) could make the title clearer and more informative.

Q1. Would you consider revising the title to better reflect the content and purpose of your study?

Comments on the Abstract and Keywords section:

Q2. Why do you mention inhalation toxicity in the abstract, although this route was not evaluated in your study? Please clarify or consider removing this reference to avoid confusion.

Q3. What method(s) did you use for the phytochemical screening to identify the major compound classes ? Please include this information in the abstract for greater clarity.

Q4. Could you refine your keywords to make them more specific to your study? For instance, consider including the names of the plant species investigated or the types of toxicity assessed (oral toxicity, topical toxicity).

Comment on the Introduction :
We strongly recommend a thorough revision of the Introduction section. While the general context is well presented, the text contains several redundancies, unclear transitions, and a few imprecise or overly broad statements.

In particular, the sentence "Plutella xylostella is currently the world’s biggest pest" appears exaggerated and should be reformulated for accuracy. We suggest replacing it with:
"one of the most destructive pests of cruciferous crops globally" — which better reflects the scientific consensus.

Q5. Overall, we encourage the authors to revise this section to improve clarity, precision, and scientific tone, and to reduce repetition.

Line 83 : Comments on Section 2.1 – Stock Rearing of Plutella xylostella :

Q6. Could you clarify whether the insects used in the experiments were taken directly from the field (F0 generation), or whether they were reared for one or more generations in the lab (e.g., F1, F2) before use ? This information is important for understanding the consistency and adaptation of the test population.

Q7. Line 88-90 : The sanitation protocol for collard greens is well described. However, could you briefly explain why both sodium hypochlorite and UV light were used? Was this based on a specific protocol or previous studies?

Q8. line 90 : Please put a space between the values ​​and the units used “l55 μW/cm²”.

Q9. Line 94 : You mention “collard green discs” and “filter paper discs” with a surface area of 4 cm². Did you mean 4 cm in diameter rather than area? Please consider clarifying this point, as it may affect the interpretation of the oviposition setup.

Q10. Line 94-95 : Please, Regarding the 10 mg/mL honey solution used to feed adults, was this concentration chosen based on existing literature or preliminary observations? A brief justification would help readers understand the reasons for this choice.

Line 101 : Comment on Section 2.2 – Preparation of Aqueous Botanical Extracts:

Q11. Line 102-107 : The method for preparing the extract is clearly described. However, most of the process initially focuses on Coussarea hydrangeifolia (AECh), and only later are the other plant species mentioned. Could you consider restructuring the paragraph to clarify from the outset that the same protocol was applied to all four plant species?

Q12. Line 113 : Could you specify the exact temperature used for the cold maceration?

Q13. Line 101 : Please, the subtitle must be in English.

Q14. Line 108 : You selected four plant species from different botanical families (Bignoniaceae, Rubiaceae, Anacardiaceae, and Annonaceae), which is scientifically interesting. However, the rationale behind this taxonomic diversity is not explained in the manuscript. Could you clarify whether there was a specific objective or hypothesis guiding the selection of plants from such diverse families?

Line 117 : Comment on Section 2.3 – 2.3. Plutella xylostella oral toxicity

Q15. Lines 118-121 : Please reword and clarify this paragraph.

Q16. Although the protocol is described in detail for the AECh treatment, we assume that the same procedure was applied to the other botanical extracts (AETg, AEDf, AEAa). Could you confirm this? If there are any differences, please specify them.

Line 144 : Comment on Section 2.4. Topical toxicity on P. xylostella larvae :

Q17. In the sentence "The AECh treatment consisted of 30 replicates...", it is unclear whether this applies only to AECh or to all four treatments. Could you specify the number of replicates per treatment and whether all treatments were tested under identical conditions ?

Line 158: Check language

Line 160. Comment on Section 2.5. Topical toxicity on Plutella xylostella pupae :

Q18. Still the same comment: "You mention that 'the AECh treatment consisted of 30 replicates.' Could you clarify whether each plant extract treatment (AETg, AEDf, AEAa, AECh) was tested with the same number of replicates? If not, please provide the exact number of replicates for each."

Line 172. 2.6. Topical toxicity on Plutella xylostella eggs

Q19. Could you clarify whether all botanical extracts (not just AECh) were tested on eggs using the same protocol and number of replicates?

 

Q20. Line 175-176 : The protocol mentions that the eggs were immersed for 5 seconds. Did you consider monitoring the amount of solution absorbed by the filter paper or the eggs themselves?

Q21. Line 179.  The sentence “AECh treatment consisted of 10 replicates, AECh with 10 eggs” could be reworded for greater clarity. Would you like to simplify it to avoid confusion?

Comment on Section 2.7 – Phytochemical Analysis:

The methods used for determining phenolics, flavonoids, tannins, and alkaloids are standard and widely accepted for preliminary screening. However, as they rely on general colorimetric assays, they may not fully capture the complexity and specificity of the active compounds involved in the observed insecticidal effects.

Would you consider complementing this section with a more detailed chemical characterization, such as HPLC-MS analysis, in future work or as part of the discussion? This could provide deeper insight into the bioactive molecules responsible for the effects observed in Plutella xylostella, and would significantly enhance the scientific strength of your study.

Comment on Oral Toxicity Results:

Q22. Line 240-241-243-24244-252 : Could you please provide the full definitions of abbreviations such as GL, MSI, and RANK KW at their first mention in the text or in a footnote?

Q23. Could you specify how many larvae were tested per treatment and their exact stage?

Also, if possible, could you distinguish between males and females among the emerged adults? This could enrich the interpretation of the results.

Q24. Could you add a positive control (a local insecticide used against Plutella xylostella)? This would allow readers to better compare the effectiveness of plant extracts with standard treatments. If this control is not included, a brief explanation of the methods would be appreciated.

Comment on the discussion section

Q25. Thank you for this detailed discussion. Could you consider simplifying or reorganizing some sections for greater clarity? For example: Try to group ideas (effects on larvae, effects on adults, current results) into more focused paragraphs.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction section
- Briefly introduce the scientific background to the choice of plants: their origin, traditional use or insecticidal potential,
- Explain why Plutella xylostella was chosen as the biological model,
- Justify the relevance of testing both oral and topical toxicity, explaining their ecological and practical implications.
Materials and Methods section
- Specify the concentrations or doses used in the bioassays (e.g. mg/mL or µg/insect).
- Indicate the number of replicates and the number of individuals per treatment to assess statistical robustness.
- Clarify the duration of exposure for each test (oral and topical), and the rearing conditions (temperature, humidity, photoperiod).
- Mention the statistical criteria used to assess significant effects 
Section results
- Present the results separately for each plant and each test: oral toxicity results and topical toxicity results.
- Highlight differences in efficacy between extracts.
- Include a link between the biological results and the presence of certain secondary metabolites (mechanism of action)

Conclusion Section
- Emphasise the insecticidal potential by ingestion of certain extracts,
- Insist on the need to assess the selectivity of these extracts to preserve beneficial insects.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to thank the authors for the thorough revision and clear responses to my comments. The manuscript has significantly improved, and the topic is highly relevant for the sustainable management of Plutella xylostella using botanical extracts.

I recommend acceptance of this article pending minor revision.

The main points to address are:

• Refining the structure of certain paragraphs to improve the overall flow.

• Adding relevant references not only in the introduction and discussion but also in the Materials and Methods section to support the experimental choices and protocols used.

Congratulations to the authors for this interesting and valuable contribution to the field.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf