Laboratory Evaluation of Contact and Feeding Deterrent Effects of Selected Essential Oils Against Different Life Stages of Cylas formicarius (Coleoptera: Brentidae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for the Authors:

1. In section 2.2 Essential oils and solution preparation, please provide the specific producers of each EO that was purchased from amazon. At the current information level, researchers would not be able to reproduce the studies of your experiments because they could not test the same Eos
2. Please explain how you have an analytical grade essential oil? were you using analytical grades of the active ingredient from the oil? Were the oils synthetic blends or natural sources?
3. In table 2, please list reference where you got the main active compounds and relevant biological activity. I suggest adding a column and listing the supporting references there
4. Please list the provider of the analytical-grade acetone
5. Please provide a reference for why discs were immersed for 30 sec in acetone. That seems like a very long time, did this immersion process result in damage to the disc?
6. Line 162 it states that 200ul of the corresponding treatment solution was used to moisten the filter paper. Please verify that it was acetone /EO used to moisten the filter paper. Why was this used and not water?
7. Across the various experiments, it states that each treatment was replicated five times, totaling 50 insects per treatment. Please provide some justification on why the experiment was only replicated 5 times. Were the experiments replicated across a minimum of two time points? Were discs from different roots or the same root? Was each individual insect considered a replicate for statistical analysis?
8. In section 2.4 evaluation of larval survival please verify that treatments were applied prior to the drilling of cavities
9. In section 2.7 Adult survival assay, line 204-205 states that if deterioration of the disc was observed it was replaced. Please explain how the integrity of the experiment was maintained if some treatments were being replaced while other treatments were not.
10. The statistical analysis presented in section 2.8 is likely not appropriate nor sufficient for this research. I suggest the researchers consult a statistician for guidance. An anova is an inappropriate test for the discrete data presented in this manuscript, especially with the sample size only being 5. I suggest a logistic regressions or an exact logistic regression with a binomial distribution.
11. The results section of this article is insufficient and needs to be redone. It presents no summary statistics (means, standard deviation, standard error), nor does it provide any inferential statistics. Please update this section to report the means, standard error, P-values, degrees of freedom, and F stats for the experiments conducted. Move sections discussing the results of the experiments to the discussion section.
12. Line 346, the authors state that the EO treatments revealed gender specific responses but gender specific studies were not outlined in the methods or results section of the article
13. Remove references toward repellency effects from the treatments as the experiments did not evaluate repellency
14. Lines 375-377 do not make sense and go against the results presented. please revise
15. Conclusion section, based on the methodologies outlined in this study, especially for adult mortality, comparisons across EO is not appropriate as they treatment of the discs was not the same across treatments. 

Author Response

      1.    In section 2.2 Essential oils and solution preparation, please provide the specific producers of each EO that was  purchased from Amazon. At the current information level, researchers would not be able to reproduce the studies of your experiments because they could not test the same Eos.

Response:

Essential oil	Specific Producers	Botanical species	Main active compounds	Relevant biological activity
Eucalyptus	Kukka	Eucalyptus globulus	1,8-cineole (eucalyptol), α-pinene, limonene	Repellent, neurotoxic, feeding deterrent
Garlic	Sheer Essence	Allium sativum	Allicin, diallyl disulfide, diallyl trisulfide	Fumigant, insecticidal, feeding deterrent
Marigold	Deve Herbes	Calendula officinalis	Flavonoids, triterpenoids, sesquiterpenes	Moderate deterrent effect, growth inhibitor
White mustard	Talya	Sinapis alba	Allyl isothiocyanate, glucosinolates	Insecticidal, feeding deterrent, fumigant
Peppermint	Gya Labs	Mentha piperita	Menthol, menthone, pulegone	Strong repellent, feeding deterrent, neurotoxic
Rosemary	Kukka	Rosmarinus officinalis	1,8-cineole, camphor, α-pinene, borneol	Repellent, growth inhibitor, insect toxicant
Thyme	Hiqili	Thymus gobicus	Thymol, carvacrol, p-cymene	Strong feeding deterrent, repellent, neurotoxic

 

2. Please explain how you have an analytical grade essential oil? were you using analytical grades of the active ingredient from the oil? Were the oils synthetic blends or natural sources?

Response: The essential oils used in this study were obtained from commercial suppliers which were extracted from natural botanical sources rather than synthetic blends or purified analytical-grade active ingredients. The products consisted of whole essential oils derived from the respective plant species, including Kukka (Eucalyptus globulus and Rosmarinus officinalis), Sheer Essence (Allium sativum), Deve Herbes (Calendula officinalis), Talya (Sinapis alba), Gya Labs (Mentha piperita), and Hiqili (Thymus gobicus). The study did not employ analytical-grade isolated compounds or synthetic formulations of the active ingredients. Instead, naturally derived essential oils containing the complex mixture of volatile bioactive constituent’s characteristic of each plant species were used. The oils were utilized as commercially available natural extracts according to the manufacturers’ specifications.

3. In Table 2, please list the reference where you got the main active compounds and relevant biological activity. I suggest adding a column and listing the supporting references there.

Response: Thank you for the valuable suggestion. We revised Table 2 by adding a new column listing supporting references for the major active compounds and their reported biological activities for each essential oil.

4. Please list the provider of the analytical-grade acetone.

Response: Analytical-grade acetone used for dilution of the essential oils and as the control treatment was obtained from Fisher Chemical.

5. Please provide a reference for why discs were immersed for 30 sec in acetone. That seems like a very long time, did this immersion process result in damage to the disc?

Response: Thank you for this observation. We apologize for the mistake in the original manuscript. The previously reported immersion time of 30 s was incorrect; the discs were actually immersed in acetone for only 10 s. This short immersion period was sufficient to ensure uniform coverage of the discs without causing any visible physical damage or structural alteration. The discs remained intact throughout the bioassays after solvent had completely evaporated before insect exposure.

6. Line 162 it states that 200ul of the corresponding treatment solution was used to moisten the filter paper. Please verify that it was acetone /EO used to moisten the filter paper. Why was this used and not water?

Response: Thank you for this observation. Water was used to moisten the filter paper, not the acetone/essential oil solution. The filter paper was moistened with water to maintain humidity within the experimental container and to prevent desiccation of the insects during the bioassay period. The essential oil treatments were applied separately, while water was used only to provide a suitable microenvironment for insect survival throughout the experiment.

7. Across the various experiments, it states that each treatment was replicated five times, totaling 50 insects per treatment. Please provide some justification on why the experiment was only replicated 5 times. Were the experiments replicated across a minimum of two time points? Were discs from different roots or the same root? Was each individual insect considered a replicate for statistical analysis?

Response: Thank you for these important observations. The experiments were conducted using six replicates per treatment, with each replicate consisting of 10 insects, totaling 50 insects per treatment. This level of replication was selected based on methodologies commonly adopted in laboratory bioassays involving essential oils and insect mortality/behavioral evaluations, in which 3-6 replicates are frequently considered sufficient to detect treatment effects under controlled conditions. The experiments were conducted under standardized laboratory conditions and repeated over multiple experimental periods to ensure consistent results. In addition, the discs within each replicate were prepared from the same root; however, different sweet potato roots were used in the treatments to minimize possible bias associated with individual root variability. For the statistical analyses, each experimental unit (Petri dish/container containing 10 insects) was considered one replicate rather than each individual insect. Therefore, the mean response obtained from each experimental unit was used in the analyses to avoid pseudoreplication and maintain replicate independence.

8. In section 2.4 evaluation of larval survival please verify that treatments were applied prior to the drilling of cavities.

Response: Thank you for this observation. We confirm that the treatments were applied before cavity preparation and the introduction of the larvae. Specifically, sweet potato cubes were first immersed in the essential oil solutions and control treatments and subsequently air-dried at room temperature. Only after complete drying of the treatments were the cavities prepared, followed by the introduction of second- and third-instar larvae of Cylas formicarius. We revised the text to improve the methodological clarity of this procedure.

9. In section 2.7 Adult survival assay, line 204-205 states that if deterioration of the disc was observed it was replaced. Please explain how the integrity of the experiment was maintained if some treatments were being replaced while other treatments were not.

Response: Thank you for this important observation. All sweet potato discs were replaced simultaneously across all treatments and controls every 48 hours to prevent deterioration. This standardized replacement procedure was adopted to ensure uniform exposure conditions across treatments and to minimize potential bias associated with differences in disc quality, moisture loss, or microbial deterioration over time. Therefore, the integrity and comparability of the experiment were maintained throughout the bioassay period.

10. The statistical analysis presented in section 2.8 is likely not appropriate nor sufficient for this research. I suggest the researchers consult a statistician for guidance. An anova is an inappropriate test for the discrete data presented in this manuscript, especially with the sample size only being 5. I suggest a logistic regressions or an exact logistic regression with a binomial distribution.

Response: Mortality and survival data were analyzed using logistic regression models with a binomial distribution and a logit link function, as recommended for discrete response variables and for experiments with relatively small sample sizes. The number of dead or surviving insects relative to the total number of insects per experimental unit was used in the analyses. Essential oil type, concentration, and their interaction were included as fixed effects in the models. The significance of treatment effects was evaluated using likelihood ratio chi-square tests. When significant differences were detected, comparisons among treatments were performed using estimated marginal means at P < 0.05. Each Petri dish containing 10 insects was considered one experimental replicate, with six replicates per treatment. Individual insects were not treated as independent replicates to avoid pseudoreplication. Oviposition and feeding data were summarized as mean ± standard error (SE). All statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC, USA).

11. The results section of this article is insufficient and needs to be redone. It presents no summary statistics (means, standard deviation, standard error), nor does it provide any inferential statistics. Please update this section to report the means, standard error, P-values, degrees of freedom, and F stats for the experiments conducted. Move sections discussing the results of the experiments to the discussion section.

Response: Thank you for this important observation. We agree that the original Results section needed additional statistical detail and clearer separation between results and discussion. Accordingly, the Results section was thoroughly revised to include descriptive and inferential statistics for all experiments conducted. Mean values ± standard error (SE), P-values, degrees of freedom, likelihood ratio chi-square statistics, and treatment comparisons were added throughout the manuscript where appropriate.

12. Line 346, the authors state that the EO treatments revealed gender specific responses but gender specific studies were not outlined in the methods or results section of the article.

Response to Reviewer 1: Thank you for this observation. We agree that the statement regarding gender-specific responses was not adequately supported by the methodology and results presented in the manuscript. This statement has been corrected and removed from the revised version of the manuscript to ensure consistency between the Methods, Results, and Discussion sections.

13. Remove references toward repellency effects from the treatments as the experiments did not evaluate repellency.

Response: Thank you for this important observation. We agree that the experiments in this study did not directly assess repellency effects. Accordingly, all references and interpretations related to repellency were removed from the revised manuscript to ensure that the discussion and conclusions accurately reflect the experimental data obtained.

14. Lines 375-377 do not make sense and go against the results presented. please revise.

Response: Thank you for this observation. We agree that the statements in lines 375–377 were inconsistent with the results presented. Therefore, these sentences were removed from the revised manuscript to improve clarity and ensure consistency between the Results and Discussion sections.

15. Conclusion section, based on the methodologies outlined in this study, especially for adult mortality, comparisons across EO is not appropriate as they treatment of the discs was not the same across treatments. 

Response: Thank you for this important observation. We agree that direct comparisons among essential oils in the adult survival assay should be interpreted cautiously. Accordingly, the Conclusion section was revised to avoid overgeneralized comparisons among essential oils and to ensure that the conclusions remain strictly supported by the methodologies and results presented in the study. The revised conclusion now focuses on the overall effects of the essential oils and their potential for incorporation into integrated pest management programs without overstating comparative efficacy among treatments.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses a relevant topic and provides useful preliminary information on the biological activity of essential oils against Cylas formicarius. However, several significant issues limit its scientific credibility. The following recommendations are provided to improve the manuscript:

Title:

The title is too broad and does not specify the type of bioassay or the life stage of the test organism. It also does not clearly reflect that the study was conducted under laboratory conditions. It is recommended to revise the title as “Laboratory Evaluation of Contact and Feeding Deterrent Effects of Selected Essential Oils Against Different Life Stages of Cylas formicarius”

Simple Summary:

The phrase “could be used as an alternative”’ is overly conclusive. It is recommended to revise this to a more cautious statement, such as “These findings suggest potential for further investigation under semi-field and field conditions”.

Abstract:

The abstract lacks quantitative data (e.g., %mortality, mean values, and p-values). It is recommended to include key quantitative results, along with statistical significance (p-values) and the number of replicates (n), to strengthen the scientific rigor and clarity of the abstract.

Introduction:

The Introduction contains a high level of redundancy, including some overlapping or repetitive paragraphs. It is recommended to streamline the text and reduce its length by approximately 20–30% to improve clarity and focus.

In addition, the research gap is not clearly articulated. A concise closing statement should be added to the Introduction to highlight this gap, for example “However, limited studies have systematically compared the effects of multiple essential oils across different developmental stages of Cylas formicarius under controlled conditions.”

Finally, the study objective is not sufficiently specific. It is recommended to revise it as “This study aims to quantify stage-specific susceptibility, evaluate dose-dependent responses, and identify the most effective essential oil for each life stage.”

Materials and Methods:

- Source of essential oils (critical issue):
The use of essential oils purchased from Amazon raises concerns regarding quality and reproducibility. It is recommended to specify reliable and traceable suppliers (e.g., Sigma-Aldrich), including batch numbers. Alternatively, the authors should perform chemical characterization (e.g., GC-MS analysis) to ensure consistency.

- Lack of GC–MS analysis (very important):
The manuscript does not include chemical composition analysis of the essential oils. It is strongly recommended to add a subsection titled “Chemical Composition Analysis”, including GC-MS conditions, retention indices (RI), and relative percentage composition of identified compounds.

- Use of Table 2 (literature-based):
Table 2 appears to present literature, derived data rather than experimental results. It is recommended to either replace it with experimentally obtained data or clearly rename it as “Reported compounds (literature)” to avoid confusion.

- Inappropriate concentration design:
The concentration range used is not sufficient to establish a clear dose-response relationship. It is recommended to include a broader concentration series (e.g., 0.5%, 1%, 2.5%, 5%, 10%) and adopt a dose-response framework.

- Absence of LC₅₀/LD₅₀ values:
The study does not report toxicity parameters. It is recommended to conduct probit analysis to estimate LC₅₀ or LD₅₀ values, including 95% confidence intervals and slope parameters.

- Non-standardized feeding assay:
The feeding assay lacks standardization. It is recommended to quantify feeding using objective methods, such as weight loss (mg consumed) or image-based analysis (area consumed).

- No positive control:
The experimental design lacks a reference insecticide. It is recommended to include a positive control (e.g., deltamethrin or malathion) for comparative evaluation.

- Low replication:
The number of replicates appears insufficient. It is recommended to increase replication to at least n ≥ 8-10 to improve statistical strength.

- Incomplete statistical analysis:
The statistical methods are not adequately described. It is recommended to include a factorial ANOVA model (essential oil × concentration × life stage), along with tests for normality and homogeneity of variance, to ensure the validity of the analysis.

Results:

- Use of qualitative language:
The Results section relies on qualitative descriptions. It is recommended to replace terms such as “strong effect” with quantitative, statistically supported statements (e.g., “significantly higher, p<0.05”).

- Lack of statistical reporting:
Key statistical values are not presented. It is recommended to include mean ± standard error (SE), p-values, and, where appropriate, F-values to support the results.

- Absence of dose–response analysis:
The manuscript does not demonstrate a clear dose–response relationship. It is recommended to include regression analysis (e.g., dose-response plots) and a table reporting LC₅₀ values.

- Data inconsistency:
There appear to be inconsistencies between the text and figures. The authors should carefully cross-check all reported values and avoid overstated terms such as “complete inhibition” unless the effect is truly 100%.

Discussion:

- Overly generic discussion:
The Discussion is largely descriptive and lacks direct linkage to the study’s findings. It is recommended to explicitly relate the discussion to the results (e.g., highlighting comparative efficacy such as peppermint > thyme).

- Overstated mechanism:
The statement “essential oils inhibit AChE” is not supported by the presented data. It is recommended to revise this to a more cautious interpretation, such as: “Essential oils may interfere with neural signaling pathways, although this was not directly tested in the present study.”

- Lack of comparative context:
The Discussion does not adequately compare the findings with existing literature. It is recommended to include comparisons of LC₅₀ values or other relevant parameters with those reported in previous studies.

- Absence of study limitations:
The manuscript does not acknowledge its limitations. It is recommended to include a statement such as: “This study was conducted under laboratory conditions; therefore, further validation under semi-field and field conditions is required.”

Conclusion:

- Overstated claims:
The phrase “can be used” is overly conclusive. It is recommended to revise this to a more cautious statement, such as “show promise for further development.”

- Lack of future research directions:
The manuscript does not outline potential future work. It is recommended to include perspectives on further development, such as formulation optimization, nanoencapsulation approaches, and validation through field trials.

Tables:

- Table 1 should be streamlined for conciseness or moved to the Supplementary Materials.

- Table 2 should be replaced with GC-MS analytical data; alternatively, it should be clearly labeled as a “review table” if it presents literature-based information.

- Table 3 contains redundancy and should be simplified, for example, by presenting the data in a concise essential oil × concentration matrix format.

Figures:

- Complex color scheme and legend:
The figures use a complex and potentially confusing color scheme. It is recommended to adopt a consistent and simplified color scheme across all figures.

- Lack of error bar details:
Error bars are not clearly defined. It is recommended to include a statement such as “Mean ± SE (n=5)” in the figure captions.

- Inappropriate Y-axis labeling:
The Y-axis labels are not clearly defined. It is recommended to use standardized metrics, such as percentage mortality (%) or percentage feeding reduction (%), where appropriate.

- Absence of dose-response visualization:
The manuscript lacks graphical representation of dose-response relationships. It is recommended to include line graphs (e.g., concentration vs. response).

- Unclear statistical annotations:
The meaning of statistical letters is not explained. It is recommended to include a note such as: “Different letters indicate significant differences (Tukey’s HSD, p<0.05).”

Comments on the Quality of English Language

In general, the manuscript is understandable and uses appropriate scientific terminology; however, the English language requires moderate revision to improve clarity and accuracy. There is some redundancy, particularly in the Introduction and Discussion sections, which should be reduced to enhance conciseness. In addition, the text relies heavily on qualitative or promotional terms (e.g., “most effective,” “potent”), which should be replaced with quantitative, evidence-based descriptions. Some sentences are overly long or not well structured, affecting readability and logical flow, and there are minor grammatical errors and inconsistencies in word choice throughout the manuscript. A careful language revision is therefore recommended to improve clarity, consistency, and the overall scientific tone.

Author Response

This manuscript addresses a relevant topic and provides useful preliminary information on the biological activity of essential oils against Cylas formicarius. However, several significant issues limit its scientific credibility. The following recommendations are provided to improve the manuscript:

Title:

The title is too broad and does not specify the type of bioassay or the life stage of the test organism. It also does not clearly reflect that the study was conducted under laboratory conditions. It is recommended to revise the title as “Laboratory Evaluation of Contact and Feeding Deterrent Effects of Selected Essential Oils Against Different Life Stages of Cylas formicarius”.

Response: Thank you for this valuable suggestion. We agree that the original title was too broad and did not adequately reflect the experimental conditions, bioassay approach, or developmental stages evaluated in the study. Accordingly, the title was revised to more clearly describe the research's scope, including the laboratory conditions and the evaluation of different life stages of Cylas formicarius.

Simple Summary:

The phrase “could be used as an alternative”’ is overly conclusive. It is recommended to revise this to a more cautious statement, such as “These findings suggest potential for further investigation under semi-field and field conditions”.

Response: Thank you for this valuable suggestion. The change was made to the text in red.

Abstract:

The abstract lacks quantitative data (e.g., %mortality, mean values, and p-values). It is recommended to include key quantitative results, along with statistical significance (p-values) and the number of replicates (n), to strengthen the scientific rigor and clarity of the abstract.

Response: Thank you for this valuable suggestion. We agree that the original abstract lacked sufficient quantitative and statistical information. These additions were incorporated to improve the scientific rigor, clarity, and overall interpretation of the study findings.

Introduction:

The Introduction contains a high level of redundancy, including some overlapping or repetitive paragraphs. It is recommended to streamline the text and reduce its length by approximately 20–30% to improve clarity and focus.

In addition, the research gap is not clearly articulated. A concise closing statement should be added to the Introduction to highlight this gap, for example “However, limited studies have systematically compared the effects of multiple essential oils across different developmental stages of Cylas formicarius under controlled conditions.”

Finally, the study objective is not sufficiently specific. It is recommended to revise it as “This study aims to quantify stage-specific susceptibility, evaluate dose-dependent responses, and identify the most effective essential oil for each life stage.”

Response: Thank you for these valuable suggestions. We carefully revised the Introduction to improve clarity, reduce redundancy, and enhance the manuscript's overall focus. The text was streamlined and shortened substantially to avoid overlapping information and improve readability.

In addition, the research gap was clarified by adding a concise statement emphasizing the limited number of studies systematically comparing multiple essential oils across different developmental stages of Cylas formicarius under controlled conditions.

Finally, the study objective was revised to make it more specific and aligned with the experimental design. The updated objective now clearly states that the study aimed to evaluate stage-specific susceptibility, dose-dependent responses, and identify the most effective essential oils against different life stages of C. formicarius.

Materials and Methods:

- Source of essential oils (critical issue):
The use of essential oils purchased from Amazon raises concerns regarding quality and reproducibility. It is recommended to specify reliable and traceable suppliers (e.g., Sigma-Aldrich).

Response: Thank you for this important observation. We agree that the use of commercial essential oils requires clear supplier information to improve reproducibility and traceability. Accordingly, the manuscript was revised to include the specific commercial providers and botanical sources for all essential oils used in the study, replacing the previous generalized description. In addition, detailed supplier information was added to the Materials and Methods section to ensure transparency and facilitate reproducibility of the experiments.

- Use of Table 2 (literature-based):
Table 2 appears to present literature, derived data rather than experimental results. It is recommended to either replace it with experimentally obtained data or clearly rename it as “Reported compounds (literature)” to avoid confusion.

Response: Thank you for this important observation. We agree that Table 2 could lead to confusion about whether the information was experimentally obtained or literature-derived. Accordingly, the table was revised and clearly identified as containing literature-based information on previously reported compounds and biological activities associated with the evaluated essential oils. The title and description of the table were modified to improve clarity and avoid misinterpretation of the data presented.

- Inappropriate concentration design:
The concentration range used is not sufficient to establish a clear dose-response relationship. It is recommended to include a broader concentration series (e.g., 0.5%, 1%, 2.5%, 5%, 10%) and adopt a dose-response framework.

Response: Thank you for this valuable suggestion. We agree that a broader concentration range would provide a more detailed dose–response evaluation and improve the interpretation of concentration-dependent effects. The concentrations used in the present study were selected as an initial screening approach to evaluate the bioactivity of the essential oils against different developmental stages of Cylas formicarius.

Following the reviewer’s recommendation, we acknowledge that future studies should incorporate a wider concentration series, including intermediate and lower concentrations, to establish dose-response relationships better and determine more precise toxicity thresholds. This consideration has been acknowledged in the revised manuscript as an important direction for future research.

- No positive control:
The experimental design lacks a reference insecticide. It is recommended to include a positive control (e.g., deltamethrin or malathion) for comparative evaluation.

Response: Thank you for this valuable suggestion. The inclusion of a positive control insecticide would strengthen the comparative assessment of the evaluated essential oils. However, the main objective of this study was to investigate the bioactivity of plant-derived essential oils as natural alternatives to synthetic insecticides, and therefore no conventional insecticide was included in the experimental design. Nevertheless, following the reviewer’s recommendation, we have highlighted in the revised manuscript that future studies should include standard insecticides, such as deltamethrin or malathion, to enable direct comparisons with chemical controls.

- Low replication:    
The number of replicates appears insufficient. It is recommended to increase replication to at least n ≥ 8-10 to improve statistical strength.

Response: Thank you for this important observation. We acknowledge that increasing the number of replicates could further improve statistical power and experimental precision. However, the number of replicates used in this study was based on methodologies commonly adopted in laboratory bioassays involving essential oils and insect mortality evaluations, where a minimum of three to five replicates is frequently considered statistically acceptable under controlled experimental conditions.

In the present study, each treatment consisted of six independent replicates, with 10 insects per experimental unit, totaling 60 insects per treatment. In addition, we reanalyzed the data using logistic regression models with binomial distribution, which are appropriate for discrete mortality data and relatively small sample sizes.

- Incomplete statistical analysis:
The statistical methods are not adequately described. It is recommended to include a factorial ANOVA model (essential oil × concentration × life stage), along with tests for normality and homogeneity of variance, to ensure the validity of the analysis.

Response: Thank you for this important observation. We agree that the original statistical analysis section required clarification and improvement. Accordingly, the statistical analyses were revised and updated in the manuscript. Instead of factorial ANOVA, the data were reanalyzed using logistic regression models with binomial distribution and logit link function, which are more appropriate for the discrete mortality and survival data generated in this study.

The revised statistical analysis section now clearly describes the effects evaluated, including essential oil type, concentration, and their interaction. In addition, likelihood ratio chi-square tests were used to evaluate treatment effects. Because the response variables were discrete and proportional rather than continuous, logistic regression was considered more appropriate than factorial ANOVA, and assumptions of normality and homogeneity of variance were therefore not required for these analyses. The revised methodology and statistical approach were fully updated throughout the manuscript to improve rigor and clarity.

Results:

- Use of qualitative language:
The Results section relies on qualitative descriptions. It is recommended to replace terms such as “strong effect” with quantitative, statistically supported statements (e.g., “significantly higher, p<0.05”).

- Lack of statistical reporting:
Key statistical values are not presented. It is recommended to include mean ± standard error (SE), p-values, and, where appropriate, F-values to support the results.

- Absence of dose–response analysis:
The manuscript does not demonstrate a clear dose–response relationship. It is recommended to include regression analysis (e.g., dose-response plots) and a table reporting LC₅₀ values.

- Data inconsistency:
There appear to be inconsistencies between the text and figures. The authors should carefully cross-check all reported values and avoid overstated terms such as “complete inhibition” unless the effect is truly 100%.

Response: Thank you for these important observations. The Results section was thoroughly revised to address all of these concerns and improve the scientific rigor and clarity of the manuscript.

Qualitative expressions such as “strong effect” were replaced with quantitative and statistically supported statements throughout the manuscript. The revised Results section now consistently reports mean values ± standard error (SE), P-values, degrees of freedom, and likelihood ratio chi-square statistics obtained from the logistic regression analyses.

In addition, the statistical analyses were updated using logistic regression models with binomial distribution and logit link function, which were considered more appropriate for the discrete mortality and survival data evaluated in this study. The figures and corresponding text were revised to ensure consistency between reported values and graphical presentation. Overstated expressions such as “complete inhibition” were carefully reviewed and corrected to accurately reflect the observed results.

Furthermore, concentration-dependent effects were clarified throughout the Results and Discussion sections, emphasizing the observed increase in biological activity with increasing essential oil concentration. All numerical values and interpretations were carefully cross-checked and corrected where necessary to ensure accuracy and consistency throughout the manuscript.

Discussion:

- Overly generic discussion:
The Discussion is largely descriptive and lacks direct linkage to the study’s findings. It is recommended to explicitly relate the discussion to the results (e.g., highlighting comparative efficacy such as peppermint > thyme).

- Overstated mechanism:
The statement “essential oils inhibit AChE” is not supported by the presented data. It is recommended to revise this to a more cautious interpretation, such as: “Essential oils may interfere with neural signaling pathways, although this was not directly tested in the present study.”

- Lack of comparative context:
The Discussion does not adequately compare the findings with existing literature. It is recommended to include comparisons of LC₅₀ values or other relevant parameters with those reported in previous studies.

- Absence of study limitations:
The manuscript does not acknowledge its limitations. It is recommended to include a statement such as: “This study was conducted under laboratory conditions; therefore, further validation under semi-field and field conditions is required.”

Response: Thank you for these valuable comments. The Discussion section was extensively revised to address all concerns raised by the reviewer. The revised version now directly relates the discussion to the study findings by explicitly comparing the efficacy of the evaluated essential oils across developmental stages of Cylas formicarius, including comparative interpretations such as the higher efficacy of peppermint oil relative to other treatments.

In addition, the mechanistic interpretation was revised to avoid unsupported conclusions. Statements suggesting direct inhibition of acetylcholinesterase (AChE) were removed or rewritten in a more cautious manner, indicating that essential oils may interfere with neural signaling pathways and other physiological processes, although these mechanisms were not directly evaluated in the present study.

The Discussion was also expanded to include broader comparisons with previous literature, including studies involving essential oils, botanical insecticides, repellency, oviposition inhibition, and nanoemulsion-based formulations against C. formicarius and other insect pests. Comparative context involving LC₅₀ values, mortality responses, and behavioral effects reported in previous studies was incorporated to strengthen the scientific interpretation of the results.

Finally, a specific paragraph acknowledging the limitations of the study was added. The revised manuscript now clearly states that the experiments were conducted under laboratory conditions and that further validation under semi-field and field conditions is necessary before practical implementation in integrated pest management programs.

Conclusion:

- Overstated claims:
The phrase “can be used” is overly conclusive. It is recommended to revise this to a more cautious statement, such as “show promise for further development.”

Response: Thank you for this important observation. The conclusion section was revised accordingly, and the overly conclusive phrase “can be used” was replaced with more cautious and scientifically appropriate language, such as “with promise for further development,” to better reflect the scope and limitations of the present study.

- Lack of future research directions:
The manuscript does not outline potential future work. It is recommended to include perspectives on further development, such as formulation optimization, nanoencapsulation approaches, and validation through field trials.

Response: Thank you for these important observations. The Conclusion section was revised to avoid overstated claims and to present the findings more cautiously and accurately. Statements implying definitive applicability were modified to indicate that the evaluated essential oils show strong potential for future development and possible incorporation into integrated pest management programs.

In addition, future research perspectives were incorporated into the revised conclusion. The manuscript now highlights the need for further studies involving formulation optimization, evaluation of residual stability and phytotoxicity, investigation of nanoencapsulation approaches, and validation through greenhouse and field trials to better assess the practical applicability of these essential oils under commercial production conditions.

Tables:

- Table 1 should be streamlined for conciseness or moved to the Supplementary Materials.

Response: Thank you for this suggestion. We agree that Table 1 contained information that was already sufficiently described in the main text and could be simplified to improve manuscript conciseness. Accordingly, Table 1 was removed from the revised manuscript, and the relevant information was incorporated directly into the text to improve readability and avoid redundancy.

- Table 2 should be replaced with GC-MS analytical data; alternatively, it should be clearly labeled as a “review table” if it presents literature-based information.

Response: Thank you for this important observation. We agree that the table could lead to confusion about whether the information was experimentally obtained or literature-derived. Accordingly, the table title and description were revised to clearly identify it as a literature-based review table presenting previously reported compounds and associated biological activities from published studies, rather than GC-MS analytical data generated in the present research.

- Table 3 contains redundancy and should be simplified, for example, by presenting the data in a concise essential oil × concentration matrix format.

Response: Thank you for this valuable suggestion. We agree that the original Table 2 contained unnecessary redundancy and could be presented in a more concise format. Accordingly, the table was revised and simplified into a matrix-style format organized by essential oil and concentration, which improved clarity, reduced repetition, and enhanced the overall readability of the manuscript.

Figures:

- Complex color scheme and legend:
The figures use a complex and potentially confusing color scheme. It is recommended to adopt a consistent and simplified color scheme across all figures.

- Lack of error bar details:
Error bars are not clearly defined. It is recommended to include a statement such as “Mean ± SE (n=5)” in the figure captions.

- Inappropriate Y-axis labeling:
The Y-axis labels are not clearly defined. It is recommended to use standardized metrics, such as percentage mortality (%) or percentage feeding reduction (%), where appropriate.

- Absence of dose-response visualization:
The manuscript lacks a graphical representation of dose-response relationships. It is recommended to include line graphs (e.g., concentration vs. response).

- Unclear statistical annotations:
The meaning of statistical letters is not explained. It is recommended to include a note such as: “Different letters indicate significant differences (Tukey’s HSD, p<0.05).”

Response: Thank you for these valuable suggestions. The figures and figure captions were thoroughly revised to improve clarity, consistency, and scientific presentation throughout the manuscript. A consistent and simplified color scheme was adopted across all figures to improve readability and reduce visual complexity.

In addition, all figure captions were updated to clearly define the error bars as mean ± standard error (SE) with the corresponding number of replicates (n = 6). Y-axis labels were standardized and revised to more accurately reflect the evaluated biological responses, including feeding activity, larval mortality, pupal mortality, oviposition, and adult survival.

The statistical annotations were also clarified in all figure captions by explicitly explaining that different letters indicate significant differences among treatments based on logistic regression analyses followed by estimated marginal mean comparisons at P < 0.05. Furthermore, the figures and corresponding text were revised to better emphasize concentration-dependent responses and improve consistency between graphical and statistical presentation throughout the manuscript.

Comments on the Quality of the English Language

In general, the manuscript is understandable and uses appropriate scientific terminology; however, the English language requires moderate revision to improve clarity and accuracy. There is some redundancy, particularly in the Introduction and Discussion sections, which should be reduced to enhance conciseness. In addition, the text relies heavily on qualitative or promotional terms (e.g., “most effective,” “potent”), which should be replaced with quantitative, evidence-based descriptions. Some sentences are overly long or not well structured, affecting readability and logical flow, and there are minor grammatical errors and inconsistencies in word choice throughout the manuscript. A careful language revision is therefore recommended to improve clarity, consistency, and the overall scientific tone.

Response: Thank you for these valuable observations. The manuscript was carefully revised throughout to improve the clarity, consistency, and overall scientific quality of the English language. Redundant text, particularly in the Introduction and Discussion sections, was reduced to improve conciseness and readability. In addition, qualitative and overly promotional expressions such as “most effective” and “potent” were revised and replaced, where appropriate, with more objective and quantitatively supported descriptions based on the statistical results obtained in the study. Several long or unclear sentences were restructured to improve logical flow and readability, and grammatical inconsistencies and word choice issues were corrected throughout the manuscript. The manuscript underwent extensive language editing to improve clarity, scientific tone, and consistency across all sections.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a timely and relevant study on the potential of plant-derived essential oils (EOs) as eco-friendly alternatives to synthetic insecticides for managing Cylas formicarius. The research is well-structured, and the findings are significant for integrated pest management (IPM) strategies. The introduction provides adequate background, and the results clearly demonstrate the bioactivity of peppermint, rosemary, and thyme oils.

However, before the manuscript can be accepted for publication, several minor issues regarding chemical terminology, data interpretation consistency, and formatting require attention.

Specific Comments:

1. Chemical Nomenclature and Terminology:

Abstract & Keywords: The manuscript uses the term "Essential Oils (EOs)" throughout. However, in the Keywords section, the term "pest management" is listed. While acceptable, standardizing keywords to match the specific terminology used in the text (e.g., "botanical pesticides" or "alternative control") might improve indexing. Furthermore, in Section 2.2, please ensure the chemical names (e.g., 1,8-cineole, α-pinene) are consistently formatted in italics or regular font as per journal style.

2. Statistical Reporting:

Section 2.8 (Statistical Analysis): that a two-way ANOVA was performed. Please clarify the specific factors used in this model. Based on the text, it is implied these are "Essential Oil Type" and "Concentration," but explicitly stating this in the methods section will improve clarity.

Section 3 (Results):In the "Adult mortality" section, "Mortality was consistently lowest at 1%, intermediate at 5%, and highest at 10%." While this is likely true, the text should also briefly mention the statistical significance (p-values) or the results of the ANOVA to reinforce that these differences are statistically valid, not just observational.

3. Methodological Details:

Section 2.2 (Source of Oils):the oils were purchased from "Amazon." For scientific rigor and reproducibility, it is highly recommended to provide the specific brand names and lot numbers (if available) or at least the specific product identifiers/ASINs in the supplementary materials. The quality and composition of EOs can vary drastically between brands.

Section 2.3 (Feeding Assay):using "commercial sweet potato roots (var. Beauregard)." Please specify the source (e.g., local grocery store, specific farm) and whether the roots were washed or peeled before cutting discs, as surface residues could theoretically interfere with the EO application.

4. Discussion and Conclusion:

Section 4 (Discussion):The discussion mentions "thyme oil" consistently showing high efficacy. However, the specific oil used in the study is listed as Thymus gobicus (Mongolian Thyme). Please ensure you refer to the specific species used (T. gobicus) in the discussion to avoid generalizing the results to all thyme species (like Thymus vulgaris), as their chemical profiles differ.

Conclusion:The conclusion is strong but slightly repetitive of the results. Consider shortening the specific mortality percentages and focusing more on the practical implications for farmers or future formulation studies.

Author Response

The manuscript presents a timely and relevant study on the potential of plant-derived essential oils (EOs) as eco-friendly alternatives to synthetic insecticides for managing Cylas formicarius. The research is well-structured, and the findings are significant for integrated pest management (IPM) strategies. The introduction provides adequate background, and the results clearly demonstrate the bioactivity of peppermint, rosemary, and thyme oils.

However, before the manuscript can be accepted for publication, several minor issues regarding chemical terminology, data interpretation consistency, and formatting require attention.

Specific Comments:

1. Chemical Nomenclature and Terminology:

Abstract & Keywords: The manuscript uses the term "Essential Oils (EOs)" throughout. However, in the Keywords section, the term "pest management" is listed. While acceptable, standardizing keywords to match the specific terminology used in the text (e.g., "botanical pesticides" or "alternative control") might improve indexing.

Response: Thank you for this valuable suggestion. We agree that standardizing the keywords to better reflect the terminology used throughout the manuscript can improve indexing and searchability. Accordingly, the Keywords section was revised, and more specific terms related to the study scope, such as “botanical pesticides” and “alternative control,” were added to improve consistency with the manuscript's terminology.

1. Statistical Reporting:

Section 2.8 (Statistical Analysis): that a two-way ANOVA was performed. Please clarify the specific factors used in this model. Based on the text, it is implied these are "Essential Oil Type" and "Concentration," but explicitly stating this in the methods section will improve clarity.

Section 3 (Results): In the "Adult mortality" section, "Mortality was consistently lowest at 1%, intermediate at 5%, and highest at 10%." While this is likely true, the text should also briefly mention the statistical significance (p-values) or the results of the ANOVA to reinforce that these differences are statistically valid, not just observational.

Response: Thank you for these important observations. The manuscript was revised accordingly to improve clarity and consistency in both the Materials and Methods and Results sections.

In Section 2.8 (Statistical Analysis), the factors included in the statistical models were clarified explicitly. In addition, following recommendations from previous reviewers, the original two-way ANOVA approach was revised and replaced with logistic regression models using binomial distribution and logit link function, which were considered more appropriate for the discrete mortality and survival data evaluated in this study. The revised statistical section now clearly identifies essential oil type, concentration, and their interaction as the evaluated factors.

Furthermore, the Results section was revised to include statistical support for the observed treatment effects. Statements regarding concentration-dependent mortality were updated to include the corresponding statistical significance, including P-values and likelihood ratio chi-square statistics, to ensure that the reported differences are statistically supported rather than purely observational.

3. Methodological Details:

Section 2.2 (Source of Oils): The oils were purchased from "Amazon." For scientific rigor and reproducibility, it is highly recommended to provide the specific brand names and lot numbers (if available) or at least the specific product identifiers/ASINs in the supplementary materials. The quality and composition of EOs can vary drastically between brands.

Response: Thank you for this important observation. We agree that detailed supplier information is essential to improve scientific rigor and reproducibility. Accordingly, the manuscript was revised to include the specific commercial brands and suppliers for all essential oils used in the study, and this information was incorporated into the corresponding table in the Materials and Methods section. These additions were included to improve traceability and reproducibility of the experiments.

Section 2.3 (Feeding Assay): Using "commercial sweet potato roots (var. Beauregard)." Please specify the source (e.g., local grocery store, specific farm) and whether the roots were washed or peeled before cutting discs, as surface residues could theoretically interfere with the EO application.

Response: Thank you for this important observation. The manuscript was revised to provide additional detail regarding the source and preparation of the sweet potato roots used in the feeding bioassays. Commercial sweet potato roots (cv. Beauregard) were purchased from Walmart in Tallahassee, Florida, USA. In addition, the roots were thoroughly washed with water prior to the preparation of the discs to minimize potential interference from surface residues before the beginning of the experiments.

4. Discussion and Conclusion:

Section 4 (Discussion): The discussion mentions "thyme oil" consistently showing high efficacy. However, the specific oil used in the study is listed as Thymus gobicus (Mongolian Thyme). Please ensure you refer to the specific species used (T. gobicus) in the discussion to avoid generalizing the results to all thyme species (like Thymus vulgaris), as their chemical profiles differ.

Response: Thank you for this important observation. We agree that referring generally to “thyme oil” could lead to overgeneralization, since different Thymus species may present distinct chemical compositions and biological activities. Accordingly, the Discussion section was revised to consistently refer specifically to Thymus gobicus oil throughout the manuscript in order to accurately reflect the species evaluated in this study and avoid confusion with other thyme species such as Thymus vulgaris.

Conclusion: The conclusion is strong but slightly repetitive of the results. Consider shortening the specific mortality percentages and focusing more on the practical implications for farmers or future formulation studies.

Response: Thank you for this valuable suggestion. We agree that the original Conclusion section contained some repetition of the Results section and included excessive numerical detail. Accordingly, the conclusion was revised and streamlined to improve conciseness and readability. Specific mortality percentages were reduced, and greater emphasis was placed on the practical implications of the findings, including the potential application of essential oils in integrated pest management programs and the need for future studies involving formulation optimization and field validation.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

The manuscript deals with an important pest (Cylas formicarius), responsible for significant economic losses. It's an up to date subject, significant from the scientific and economic point of view.

Overall it is well written, reviewed data covers several aspects on nematode economic importance, damage, mode of action and, of course, control. The chosen structure seems logical, solid and well conducted. Discussion seems exhaustive, supported by several and recent bibliography.

I´ve performed an exhaustive review, even checking a few formatting glitches (manly in figures).

My main concerns are if “the moistened filter paper” does not affect the overall concentrations / the EOs degradation overtime and the procedure / time for solvent (acetone) evaporation. Information on the number of time repetitions in the different bioassays isn´t stated – although I´ve no control if you just state that more repetitions were performed...

Tables need some minor adjustments in order to facilitate readers' work and to standardize legends. Table 3, in my opinion, could be erased, since no additional information is provided. 

My notes and recommendations are the following ones:

Line 17: Please replace “strengths” by “concentrations”.

Same idea in lines 67-69 and lines 79-81. Please condensate in just one.

Same idea in lines 71-73 and lines 83-85. Please condensate in just one.

In introduction, authors could include some previous research evolving EOs (or their compounds) in C. formicarius. As an example:

- Screening of five Lamiaceae essential oils as repellents for sweet potato weevil, Cylas formicarius (F.) (Coleoptera: Brentidae). Asian Journal of Agriculture and Rural Development, 11(4), 327–333.

- Chemical composition and biological activity of the essential oil from Jamaican Cleome serrata. Nat Prod Commun. 2012 Sep;7(9):1231-2.

Figure 1: Please, if possible, include a scale bar for each image.

Line 118 – 119: As written seems that EOs and active compounds were evaluated. Please divide in 2 sentences: the seven EOs evaluated + additionally active compounds of each EOs (and respective biological activity) were presented.

Table 2: In my opinion, authors could support the "Relevant biological activity" with some references.

Line 130: Please elaborate on "controlled environment" (chamber? room temperature?).

Table 3: Except the treatment codes, all other information is already presented before table 3. The table just summarizes them. Concentration is reflected in the code designation. Oil volume is explained in line 133 to 135. Solvent (except control) & volume tested were always the same. After all, it seems that Codes aren't employed throughout the text.

Line 155: “complete evaporation of the solvent” - For how long (approximately)? How was the solvent evaporation confirmed? Does the solvent evaporation time could affect EO evaporation also? In the acetone control bioassay - if you allow solvent to evaporate, nothing remains in the disc!

Line 162: Why the 200 µL does not affect the final EO concentration in the sweet potato disc? The solvent on filter paper was also allowed to evaporate?

Line 173: “air-dried at room temperature”

Line 182: “air-dried at room temperature”

Line 191: “air-dried at room temperature” - Please check similar comments for line 155.

Line 167; 179; 187, 198 & 211: Please state how many time repetitions?

Line 204: “replaced every 48 h,” - The replacement discs were always moistened with the EO solution? - does this affect the EO solution degradation in the sweet potato discs?

Line 240: Perhaps 10 perforations? (at least Control 2 in Marigold, Mustard & Peppermint is under 15 perforations). Or describe the result for Control 1 and for Control 2 in 2 sentences.

Figure 2: Number feeding or number or perforations (axis label)? Mean is referred to how many repetitions (n=?)? The square before "Conc" is unnecessary.

If space allows, please change "Cont1" and "Cont2" to "Control 1" and "Control 2" (as in Figure 3).

Figure 3: Please remove the header in A and B. Are you able to change 2 and 3 to 2nd and 3rd in the axis label? Mean is referred to how many repetitions (n=?)? The square before "Conc" is unnecessary.

Figure 4: The square before "Conc" is unnecessary. If space allows, please change "Cont1" and "Cont2" to "Control 1" and "Control 2" (as in Figure 3).

Figure 5: If the space allows, please change "Cont1" and "Cont2" to "Control 1" and "Control 2" (as in Figure 3).

Concerning just the 2 controls:

Why in Cylas formicarius feeding / Number of Cylas formicarius eggs, Control 1 was higher than Control 2? In mortality of 2nd and 3rd instar / Mortality of Cylas formicarius pupae / Mortality of Cylas formicarius adults, Control 2 was higher than Control 1? Please elaborate.

Line 359 to 363: Please include species names for Oregano (Origanum vulgare?); lemon (Citrus × limon?) and basil (Ocimum basilicum?). Common names embrace some species.

Line 377: Plant extract-based nanoemulsion for controlling sweet potato pests and weeds. Sci Rep 16, 2565. Also deals with this issue. Authors may consider it as a reference.

Line 381: In line 375, authors stated that Thyme showed the highest efficacy. One is not accurate.

Author Response

Dear authors,

The manuscript deals with an important pest (Cylas formicarius), responsible for significant economic losses. It's an up to date subject, significant from the scientific and economic point of view.

Overall it is well written, reviewed data covers several aspects on nematode economic importance, damage, mode of action and, of course, control. The chosen structure seems logical, solid and well conducted. Discussion seems exhaustive, supported by several and recent bibliography.

I´ve performed an exhaustive review, even checking a few formatting glitches (manly in figures).

My main concerns are if “the moistened filter paper” does not affect the overall concentrations / the EOs degradation overtime and the procedure / time for solvent (acetone) evaporation.

Response to Reviewer 4: Thank you for this important observation. We would like to clarify that only a very small volume of water (microliter scale) was added to the filter paper, exclusively to maintain the sweet potato discs hydrated and prevent desiccation during the bioassays. The water was applied only to the lower surface of the sweet potato disc, which remained in direct contact with the filter paper. The upper surface of the disc, which was exposed to the insects for feeding, did not remain in contact with the moistened filter paper.

In addition, the essential oil solutions were applied to the sweet potato discs and allowed to air-dry completely before the insects were introduced. Therefore, the acetone solvent had already evaporated and the essential oil residues were dry on the root surface prior to insect exposure. Since the insects fed only on the upper exposed surface of the sweet potato disc, contact with the moistened lower surface was minimal or absent. Consequently, we do not believe that the small amount of water used for moisture maintenance significantly altered the effective concentrations of the essential oils or promoted substantial degradation of the treatments during the experimental period.

Tables need some minor adjustments in order to facilitate readers' work and to standardize legends. Table 3, in my opinion, could be erased, since no additional information is provided. 

Response: Thank you for this suggestion. We agree that Table 3 was redundant and did not provide substantial additional information beyond what was already described in the manuscript text. Accordingly, Table 3 was removed from the revised version of the manuscript, and the relevant information was maintained directly in the text to improve conciseness and readability.

My notes and recommendations are the following ones:

Line 17: Please replace “strengths” by “concentrations”.

Same idea in lines 67-69 and lines 79-81. Please condensate in just one.

Same idea in lines 71-73 and lines 83-85. Please condensate in just one.

Response: Thank you for these observations. The suggested corrections were implemented throughout the revised manuscript. Specifically, the term “strengths” was replaced with “concentrations” in line 17, and the repetitive information presented in lines 67–69 and 79–81, as well as lines 71–73 and 83–85, was condensed and reorganized to improve conciseness, readability, and overall manuscript flow.

In introduction, authors could include some previous research evolving EOs (or their compounds) in C. formicarius. As an example:

- Screening of five Lamiaceae essential oils as repellents for sweet potato weevil, Cylas formicarius (F.) (Coleoptera: Brentidae). Asian Journal of Agriculture and Rural Development, 11(4), 327–333.

- Chemical composition and biological activity of the essential oil from Jamaican Cleome serrata. Nat Prod Commun. 2012 Sep;7(9):1231-2.

Response: Thank you for this valuable suggestion. The recommended studies were added to the Introduction section to strengthen the background information regarding the use of essential oils against Cylas formicarius. The manuscript now includes previous research addressing both insecticidal and repellent activities of essential oils and their compounds against this pest, providing better contextualization and supporting the present study.

Line 118 – 119: As written seems that EOs and active compounds were evaluated. Please divide in 2 sentences: the seven EOs evaluated + additionally active compounds of each EOs (and respective biological activity) were presented.

Response: Thank you for this observation. The text was revised accordingly to improve clarity and avoid ambiguity. The description of the seven evaluated essential oils was separated from the description of their major active compounds and associated biological activities, and this information was reorganized into distinct sentences in the revised manuscript.

Table 2: In my opinion, authors could support the "Relevant biological activity" with some references.

Response: Thank you for this valuable suggestion. References supporting the reported biological activities of the major compounds were added to Table 2 in the revised manuscript to improve scientific support, clarity, and traceability of the information presented.

Table 3: Except the treatment codes, all other information is already presented before table 3. The table just summarizes them. Concentration is reflected in the code designation. Oil volume is explained in line 133 to 135. Solvent (except control) & volume tested were always the same. After all, it seems that Codes aren't employed throughout the text.

Response: Thank you for this important observation. We agree that Table 3 was largely redundant because most of the information presented was already sufficiently described in the text and Materials and Methods section. In addition, the treatment codes were not consistently used throughout the manuscript. Accordingly, Table 3 was removed from the revised version of the manuscript to improve conciseness and avoid unnecessary repetition.

Line 155: “complete evaporation of the solvent” - For how long (approximately)? How was the solvent evaporation confirmed? Does the solvent evaporation time could affect EO evaporation also? In the acetone control bioassay - if you allow solvent to evaporate, nothing remains in the disc!

Response: Thank you for this important observation. The manuscript was revised to clarify the solvent evaporation procedure. The treated discs were allowed to air-dry for approximately 5 minutes at room temperature prior to insect exposure, which was considered sufficient for acetone evaporation due to its high volatility and the small volume applied to the discs. Solvent evaporation was confirmed visually by the absence of residual liquid on the disc surface before the beginning of the bioassays.

We acknowledge that prolonged drying periods could potentially contribute to partial loss of volatile essential oil compounds; therefore, a short drying period was intentionally adopted to minimize essential oil volatilization while still allowing acetone evaporation. In the acetone control treatment, the discs were not subjected to the same evaporation period, since the objective was to evaluate the direct effect of acetone exposure under the experimental conditions used in the study.

Figure 2: Number feeding or number or perforations (axis label)? Mean is referred to how many repetitions (n=?)? The square before "Conc" is unnecessary.

Response: Thank you for this observation. The requested modifications were implemented in the revised figure. The Y-axis label was corrected and standardized, the number of replicates used to calculate the means (n = 6) was clarified in the figure caption, and the unnecessary square symbol before “Conc” was removed to improve figure clarity and presentation.

If space allows, please change "Cont1" and "Cont2" to "Control 1" and "Control 2" (as in Figure 3).

Response: Thank you for this suggestion. The figure was revised accordingly, and the labels “Cont1” and “Cont2” were replaced with “Control 1” and “Control 2” to improve consistency and clarity across all figures in the manuscript.

Figure 3: Please remove the header in A and B. Are you able to change 2 and 3 to 2nd and 3rd in the axis label? Mean is referred to how many repetitions (n=?)? The square before "Conc" is unnecessary.

Response: Thank you for these observations. The requested modifications were implemented in the revised figure. The headers in panels A and B were removed, the axis labels were corrected from “2” and “3” to “2nd” and “3rd” instar, the number of replicates used to calculate the means (n = 6) was clarified in the figure caption, and the unnecessary square symbol before “Conc” was removed to improve clarity and consistency of the figure presentation.

Figure 4: The square before "Conc" is unnecessary. If space allows, please change "Cont1" and "Cont2" to "Control 1" and "Control 2" (as in Figure 3).

Response: Thank you for these suggestions. The requested modifications were implemented in the revised figure. The unnecessary square symbol before “Conc” was removed, and the labels “Cont1” and “Cont2” were replaced with “Control 1” and “Control 2” to improve clarity, consistency, and readability across all figures in the manuscript.

Figure 5: If the space allows, please change "Cont1" and "Cont2" to "Control 1" and "Control 2" (as in Figure 3).

Response: Thank you for this suggestion. The requested modification was implemented in the revised figure, and the labels “Cont1” and “Cont2” were replaced with “Control 1” and “Control 2” to improve consistency and readability across the manuscript figures.

Line 377: Plant extract-based nanoemulsion for controlling sweet potato pests and weeds. Sci Rep 16, 2565. Also deals with this issue. Authors may consider it as a reference.

Response to Reviewer 4: Thank you for this valuable suggestion. The recommended reference regarding plant extract-based nanoemulsions for sweet potato pest management was added to the revised manuscript to strengthen the discussion and support the perspectives related to formulation development and sustainable pest control strategies.

Line 381: In line 375, authors stated that Thyme showed the highest efficacy. One is not accurate.

Response to Reviewer 4: Thank you for this observation. We agree that the previous statement was inconsistent and could lead to an inaccurate interpretation of the results. Therefore, the sentence was revised to avoid stating that Thymus gobicus oil showed the highest efficacy. The revised text now presents the results more cautiously and consistently with the data, indicating that T. gobicus showed high activity at certain concentrations but was not identified as the single most effective treatment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form

Author Response

We sincerely thank the reviewer for the constructive comments and valuable suggestions. We agree that understanding the chemical composition of the tested essential oils, their modes of action, and the relationship between concentration and efficacy is important for improving the scientific rigor and practical applicability of this study. The revised file of the manuscript is attached with highlighted areas where improvements were made. 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses an important topic concerning the potential use of plant-derived essential oils for the management of the sweet potato weevil, Cylas formicarius. The study provides useful preliminary laboratory evidence regarding feeding deterrence, oviposition inhibition, and insecticidal activity against different developmental stages of the insect. The manuscript has improved in overall organization and presentation; however, several important scientific and methodological issues still require substantial revision. In particular, the lack of chemical characterization of the tested essential oils, the absence of a positive insecticide control, inconsistencies in replication and statistical descriptions, and the absence of LC₅₀ or dose–response analyses reduce the scientific rigor of the study. In addition, some interpretations and conclusions remain stronger than supported by the laboratory-based data presented. The Results and Discussion sections would also benefit from reduced repetition and more concise scientific interpretation. Overall, the manuscript has potential value, but additional methodological clarification, improved statistical consistency, and more cautious interpretation of the findings are necessary before the study can be considered for publication.

Comments on the Quality of English Language

The English language is generally clear and understandable. However, the manuscript still contains repetitive wording, some overly strong statements, and minor grammatical inconsistencies. Several sentences could be revised for greater clarity and conciseness. Additional language editing is recommended to improve readability and maintain a consistent scientific tone.

Author Response

This manuscript addresses an important topic concerning the potential use of plant-derived essential oils for the management of the sweet potato weevil, Cylas formicarius. The study provides useful preliminary laboratory evidence regarding feeding deterrence, oviposition inhibition, and insecticidal activity against different developmental stages of the insect. The manuscript has improved in overall organization and presentation; however, several important scientific and methodological issues still require substantial revision.

Response: We sincerely thank the reviewer for the constructive comments and valuable suggestions. We agree that understanding the chemical composition of the tested essential oils, their modes of action, and the relationship between concentration and efficacy is important for improving the scientific rigor and practical applicability of this study. We have revised the manuscript based on the following suggestion:

In particular, the lack of chemical characterization of the tested essential oils, the absence of a positive insecticide control, inconsistencies in replication and statistical descriptions, and the absence of LC₅₀ or dose–response analyses reduce the scientific rigor of the study. In addition, some interpretations and conclusions remain stronger than supported by the laboratory-based data presented. The Results and Discussion sections would also benefit from reduced repetition and more concise scientific interpretation.

Response: We have expanded the Discussion section to include additional information on the major bioactive constituents reported in the tested essential oils and their potential roles in insect mortality, feeding deterrence, and oviposition inhibition. We have also added a discussion on the positive implications of botanical insecticides as environmentally friendly alternatives to conventional synthetic pesticides. Furthermore, we elaborated on the observed concentration-dependent responses and discussed how increasing EO concentrations may influenced weevil mortality, feeding activity, and reproductive performance. However, we have not used the LC₅₀ values for this preliminary study. For our future work, we would like to add this information.

Overall, the manuscript has potential value, but additional methodological clarification, improved statistical consistency, and more cautious interpretation of the findings are necessary before the study can be considered for publication.

Response: We have carefully revised the manuscript to provide greater detail in the Materials and Methods section, including additional information on experimental procedures, data collection, and statistical analyses. We have also expanded the Results and Discussion sections to provide a more comprehensive interpretation of the findings, emphasizing their biological significance and practical implications for the management of Cylas formicarius. Furthermore, we improved the clarity of several sections throughout the manuscript to enhance readability and facilitate understanding by researchers, extension personnel, and other end-users.

The English language is generally clear and understandable. However, the manuscript still contains repetitive wording, some overly strong statements, and minor grammatical inconsistencies. Several sentences could be revised for greater clarity and conciseness. Additional language editing is recommended to improve readability and maintain a consistent scientific tone.

Response: We have carefully revised the manuscript to improve the overall quality of the English language, sentence structure, and readability. Several sections were edited to eliminate grammatical inconsistencies, improve clarity, and ensure a more concise and professional scientific tone. We also revised lengthy and complex sentences to enhance comprehension and improve the logical flow of information throughout the manuscript. In addition, terminology and phrasing were standardized to maintain consistency across all sections of the paper.

We believe that these revisions have substantially improved the manuscript and addressed the reviewer's concerns. We greatly appreciate the thoughtful comments, which have helped us strengthen the scientific rigor, clarity, and overall impact of this study.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

This revised manuscript provides valuable laboratory data on the biological activity of selected essential oils against the sweet potato beetle (Cylas formicarius) and contributes to the growing research in plant-based pest management. This revised edition has been significantly improved; however, several key issues remain to be addressed before publication. In particular, the lack of chemical characterization of the tested essential oils limits the interpretation of observed biological activity, while the absence of positive insecticide control effects diminishes the practical significance of efficacy comparisons. Furthermore, inconsistencies in duplicate reporting and statistical analysis should be rectified throughout the manuscript. The results and discussions should also be further improved to reduce duplication and ensure that conclusions are fully supported by the presented laboratory evidence. Addressing these issues will greatly enhance the scientific quality and reliability of the study.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English language is generally clear and understandable. However, minor grammatical errors, repetitive wording, and a few awkward sentence constructions remain. Additional language editing is recommended to improve clarity, conciseness, and consistency.

Author Response

The states "five replicates (n = 6)." These values are contradictory.

Response: Thank you this has been update to six replicated.

The conclusion that EOs have potential for IPM is premature because all experiments were conducted under laboratory conditions only.

Response: Thank you. This has been removed from the manuscript.

The most important weakness of the manuscript remains unresolved. The EOs were purchased commercially from Amazon and used according to manufacturer specifications, but no chemical characterization was performed. Why this is problematic, EO composition varies among manufacturers and production batches. Biological activity cannot be linked confidently to specific compounds. Subsequent discussion attributes efficacy to compounds that were never analytically verified. It is recommended to request the certificates of analysis from the suppliers. However, GC-MS analysis should still be performed.

Response: Thank you for this important comment. We agree that chemical characterization of the essential oils would strengthen the study. However, the essential oils were commercially purchased and no certificates of analysis were available from the suppliers at the time of the study. Furthermore, GC–MS analysis was beyond the scope and resources of the present investigation. To address this limitation, we have revised the manuscript to avoid attributing biological activity to specific compounds and instead discuss the results in terms of the overall essential oil formulations tested.

The reporting of the number of replicates is inconsistent as follows:

Five replicates (n = 6)

Five replicates in feeding assay

Six replicates in larval assay

Six replicates in pupal assay

Statistical section refers again to five replicates
This inconsistency needs to be corrected to avoid confusion.

Response: Thank you. This has been modified.

Acetone controls exhibited measurable mortality in several assays, yet no Abbott correction was reported.

Response: Thank you for this observation. We acknowledge that acetone controls exhibited measurable mortality in several assays. Abbott’s correction was not applied because control mortality remained below the threshold generally considered to require correction and did not substantially affect the interpretation of treatment effects. To improve clarity and transparency, we have now included a statement in the revised manuscript reporting the observed control mortality and explaining the rationale for not applying Abbott’s correction. We agree that documenting this information is important for readers to accurately interpret the bioassay results.

Many sections repeatedly state that mortality increased with concentration without providing additional biological interpretation.

Response: Thank you for this valuable comment. We agree that simply stating mortality increased with concentration provides limited biological insight. In the revised manuscript, we have expanded the discussion to better interpret the observed dose-dependent responses. Specifically, we explain that the increasing mortality with higher concentrations suggests a concentration-dependent toxic effect of the tested essential oils on Cylas formicarius, likely resulting from greater exposure to bioactive compounds and enhanced physiological disruption at higher concentrations. These additions provide greater biological context and improve the overall interpretation of the results.

The discussion attributes efficacy to menthol, thymol, carvacrol, cineole, and other compounds. However, these compounds were not verified in the tested oils.

Response: Thank you for this important observation. We agree that the chemical composition of the tested essential oils was not analyzed in the present study; therefore, attributing the observed biological activity to specific compounds such as menthol, thymol, carvacrol, cineole, or other constituents would be speculative. In the revised manuscript, we have modified the discussion to avoid direct attribution of efficacy to individual compounds. Instead, we now state that the observed insecticidal effects are likely associated with the complex mixture of bioactive constituents present in the essential oils, as reported in the literature, while acknowledging that the specific compounds responsible for the activity were not verified in this study. We have also identified chemical characterization of the essential oils as an important area for future research.

The manuscript repeatedly suggests practical applicability in IPM programs. The evidence remains limited to laboratory bioassays.

Response: Thank you for this insightful comment. We agree that the current study was conducted entirely under laboratory conditions and that direct recommendations for implementation in Integrated Pest Management (IPM) programs should be made cautiously. In the revised manuscript, we have tempered our conclusions and clarified that the findings demonstrate the potential of these essential oils as candidate botanical insecticides rather than confirming their immediate applicability in operational IPM programs. We now emphasize that additional research, including greenhouse and field evaluations, assessment of persistence under environmental conditions, non-target effects, compatibility with other management tactics, and economic feasibility, is needed before practical adoption can be recommended. These revisions ensure that the discussion more accurately reflects the scope and limitations of the study.

Author Response File: Author Response.pdf