Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes a novel physical method, PEF, for the disinfestation of a major secondary pest Tribolium castaneum. The study is novel and follows a scientific methodology. However, the following points may be incorporated into the manuscript.

In the introduction, give a few sentences on the novel physical disinfestation methods for stored grain protection, such as RF, microwave, cold plasma, etc. Also provide the advantages of PEF over these methods.

The discussion section is devoted entirely to the proposed mechanism of lethality of PEF. The discussion section should also address the depth of penetration of PEF in the grain bulk, its uniformity in the grain bulk, its effects on eggs, grubs, and pupae, and its impact on the quality of the treated produce.

Author Response

Response to Reviewer 1

Manuscript ID: agriculture-4020338

Title: Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Reviewer: Reviewer 1

Review Date: 02 Dec 2025

We sincerely thank Reviewer 1 for the professional and constructive comments. These suggestions, particularly regarding the comparison with other physical methods and the discussion on application potential, have been extremely valuable in improving the scientific depth and practical relevance of our manuscript. Below is our detailed point-by-point response.

Detailed Responses：

1. Reviewer Comment：

In the introduction, give a few sentences on the novel physical disinfestation methods for stored grain protection, such as RF, microwave, cold plasma, etc. Also provide the advantages of PEF over these methods.

Responses：

We have incorporated an overview of emerging physical disinfestation methods for stored grain (e.g., Radio Frequency (RF), Microwave (MW), and Cold Plasma (CP)) into the Introduction. We then clarified the distinct advantages of PEF compared to these alternatives, which better highlights the context and innovation of our study. Specifically, PEF is fundamentally a non-thermal technique, avoiding quality degradation caused by the high temperatures typical of thermal methods (RF/MW), and it demonstrates superior macroscopic penetration capability compared to CP, making it suitable for bulk grain processing.

Location of Change in Manuscript:

Introduction: Lines [60-68].

2. Reviewer Comment

The discussion section is devoted entirely to the proposed mechanism of lethality of PEF. The discussion section should also address the depth of penetration of PEF in the grain bulk, its uniformity in the grain bulk, its effects on eggs, grubs, and pupae, and its impact on the quality of the treated produce.

Responses：

To enhance the practical relevance and completeness of the Discussion, we restructured the chapter and added a new section, 4.2 Application Potential: Penetration, Uniformity, and Quality Preservation, addressing all the applied issues raised by the reviewer. The specific additions include:

1. Penetration and Uniformity: Detailed discussion combining results from Section 3.2 simulation (Figure 9a, 9b) and Section 3.1.4 "in-rice" validation, focusing on how PEF overcomes dielectric limitations to achieve uniform treatment in bulk grain.
2. Impact on Quality: Discussion on how the non-thermal nature of PEF (confirmed by Section 3.3 results)ensures the preservation of nutritional and functional grain quality.
3. Different Life Stages: Added a section, 4.2.3 Limitations and Future Perspectives, discussing potential efficacy variance across developmental stages (eggs, larvae, pupae) and justifying the focus on the more resistant adult stage in this optimization study.
4. Conclusion Support: We refined and strengthened Section 5 (Conclusion). The revised conclusion ensures that every stated scientific finding (including optimal parameters, FEM mechanism, and AChE inhibition) is directly and clearly supported by the expanded discussion and the experimental data presented in Section 3 (Results). This improves the overall robustness of the manuscript as required.

Location of Change in Manuscript:

New Section 4.2 Application Potential: Lines [520-559].

Conclusion (Summary of advantages): Lines [594-601].

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity” has valuable data that are of interest to readers of the Journal. The MS needs minor revision in order to be published in the journal.

Below there are some comments that could help the improvement of the MS. 

Abstract:

• Lines 13-14: I suggest the authors to replace the part “. It…investigated” with “and to investigate its electro-neurotoxicity mechanism.”

Introduction:

• Lines 40, 43, 45: I think that refs 3-7 don’t support sufficient the statements. So please replace the refs 3,4 and use refs the deal with the insect biology and the refs 5,6,7 with others more relevant with the statements.

Material and Methods:

• Line 101: What do you mean “standard strain”? if you want to say that it was not phosphine resistance strain, then you have to describe the examination you have done for the resistance. Also as it is written, it seems that the farm family have Tribolium stock culture which is unusual. Probably collected from the farm and cultured in the lab. Please calcify.    
• Line 105: Please define the term “healthy” and how the unhealthy but active insects was putted aside.
• Line 122: Please provide additionally a picture of the real experimental scheme with the instruments reported at chap 2.1.2
• Line 140: Please report the range of control mortality
• Line 141: The “specific protocols were as follows” is after your preliminary tests or what you follow to do them? Please clarify.
• Line 151-152: Please report which was the three factors, the three levels and the 17 experimental run
• Line 155: Please report the combinations
• Line 185: Please report the condition of the “Surviving insects” if it was active or immobile or their difference with the control because of the drop of AChE activity mentioned in the results. Also report the treatment used.
• Line 195: Please report the calculation made

Results:

• Line 236: Did the authors conclude to the “cost-effective” combination if so please report it
• Line 240: At table 3 line 4 the combination results 100% mortality but the same combination at fig 3c resulted to 95% mortality. Are these experiments done separately? How you explain the difference.
• Line 285-286: Please omit “As shown in Figure 7, the”
• Line 287: After full stop add “Figure 7 shows the insect condition before and after PEF”
• Line 377: add in parenthesis the fixed conditions ex a (20 kHz, 300 s). Do this also for b and c

Author Response

Response to Reviewer 2

Manuscript ID: agriculture-4020338

Title: Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Reviewer: Reviewer 2

Review Date: 06 Dec 2025

We are grateful to Reviewer 2 for the detailed review and insightful comments. Your specific suggestions regarding the experimental design descriptions and method clarifications have significantly enhanced the rigor and clarity of our methodology. Below is our detailed point-by-point response.

Detailed Responses：

Abstract

1. Reviewer Comment:

Lines 13-14: I suggest the authors to replace the part “. It…investigated” with “and to investigate its electro-neurotoxicity mechanism.”

Responses:

We have adopted the suggested phrasing to improve the clarity and conciseness of the abstract's aim statement.

Location of Change in Manuscript:

Abstract: Lines [12-14].

Introduction

2. Reviewer Comment:

Lines 40, 43, 45: I think that refs 3-7 don’t support sufficient the statements. So please replace the refs 3, 4 and use refs the deal with the insect biology and the refs 5, 6, 7 with others more relevant with the statements.

Responses:

We have critically reviewed the references cited in these lines. We replaced the existing citations (Refs 3, 4, 5, 6, 7) with more recent and more relevant literature that specifically supports the biological statements regarding T. castaneum biology, economic losses, and the accumulation of carcinogenic quinone secretions.

Location of Change in Manuscript:

Introduction:Lines [40-47].

Material and Methods

3. Reviewer Comment:

Line 101: What do you mean “standard strain”? if you want to say that it was not phosphine resistance strain, then you have to describe the examination you have done for the resistance. Also as it is written, it seems that the farm family have Tribolium stock culture which is unusual. Probably collected from the farm and cultured in the lab. Please clarify.

Responses:

We have clarified the insect source description in Section 2.1.1. We replaced "standard strain" with "a stable colony established in our laboratory for multiple generations from stock originally obtained from Wotian Breeding Family Farm." This clarifies that the farm was the original source, not the stock culture location. Furthermore, we clarify that the colony was a "laboratory stock culture" and was not specifically tested for phosphine resistance, as phosphine resistance testing was outside the scope of this PEF physical control study.

Location of Change in Manuscript:

Section 2.1.1: Lines [112-123].

4. Reviewer Comment:

Line 105: Please define the term “healthy” and how the unhealthy but active insects was putted aside.

Responses:

We have explicitly defined the inclusion criteria for the adults selected for the experiment. An active adult was considered "healthy" if it exhibited intact appendages and normal motility. Individuals showing physical defects, sluggish movement, or any visible damage were excluded via visual inspection prior to randomization.

Location of Change in Manuscript:

 Section 2.1.1:Lines [116-119].

5. Reviewer Comment:

Line 122: Please provide additionally a picture of the real experimental scheme with the instruments reported at chap 2.1.2

Responses:

We acknowledge the need for clearer visualization of the experimental setup. We have added a schematic diagram (New Figure 1) that illustrates the complete experimental platform in detail. This diagram shows the core high-voltage Pulsed Electric Field (PEF) system, including the pulse power supply and the constant temperature and humidity chamber housing the electrode assembly. Additionally, we have included photographic references to the key analytical instruments used in the study, specifically the UV-visible spectrophotometer (UV-1800) for AChE activity and the digital microscope (AD3800) for insect observation.

Location of Change in Manuscript:

Section 2.1.2 : Line [136]. 

6. Reviewer Comment:

Line 140: Please report the range of control mortality

Responses:

We have explicitly reported the mortality range for the control group. Throughout the preliminary tests, the natural mortality remained consistently low, ranging from 0% to 5%. This information has been added to the Materials and Methods section to confirm the validity of the Abbott's formula correction.

Location of Change in Manuscript:

Section 2.3.1 : Line [166-171].

7. Reviewer Comment:

Line 141: The “specific protocols were as follows” is after your preliminary tests or what you follow to do them? Please clarify.

Responses:

We have clarified that the "specific protocols" refer to the two sets of single-factor preliminary experiments carried out to systematically evaluate the parameter effects and to establish the appropriate range for the subsequent RSM optimization.

Location of Change in Manuscript:

Section 2.3.1:Line [166-173].

8. Reviewer Comment:

Line 151-152: Please report which was the three factors, the three levels and the 17 experimental run

Responses:

We have clarified the experimental design details in Section 2.3.2. The three independent factors were Electric Field Strength (A), Pulse Frequency (B), and Treatment Time (C), each set at three levels, resulting in 17 experimental runs (12 factorial points and 5 center points), as detailed in Table 1.

Location of Change in Manuscript:

Section 2.3.2: Lines [180-188].

9. Reviewer Comment:

Line 155: Please report the combinations

Responses:

The 17 experimental combinations are fully detailed in Table 1, which is cited immediately after the design description. We ensure the citation of Table 1 is prominent and clear.

Location of Change in Manuscript:

Section 2.3.2: Line [181-188] [295](Referring to Table 1).

10. Reviewer Comment:

Line 185: Please report the condition of the “Surviving insects” if it was active or immobile or their difference with the control because of the drop of AChE activity mentioned in the results. Also report the treatment used.

Responses:

We have revised the method description in Section 2.3.5 to clarify the sampling conditions. We used insects collected 24 h post-treatment from groups representing the minimum and maximum levels of key parameters. We also added a descriptive statement in Section 3.4 (Neurotoxic Mechanism) noting that individuals in the high-intensity treatment groups exhibited distinct neurotoxic behaviors such as sluggishness, tremors, and immobility prior to sampling, which supports the observed AChE inactivation.

Location of Change in Manuscript:

Section 2.3.5: Lines [222-227];

Section 3.4: Lines [4234-427].

11. Reviewer Comment:

Line 195: Please report the calculation made

Responses:

We have included the specific formula (Equation 3) used for calculating AChE activity, along with the definition of each variable (∆ A, W, and the coefficient 2250), directly in Section 2.3.5.

Location of Change in Manuscript:

Section 2.3.5: Lines [236-253] (Equation 3 and definitions).

Results

12. Reviewer Comment:

Line 236: Did the authors conclude to the “cost-effective” combination if so please report it

Responses:

We have clarified that the optimization criteria prioritized selecting the minimum electric field strength necessary to achieve 100% mortality, thus balancing maximum efficacy with minimum energy input (minimized field strength and time). The determined optimal parameters (26 kV/cm, 20 kHz, 140 s) represent this cost-effective combination. This rationale is explicitly stated in Section 3.1.4.

Location of Change in Manuscript:

Section 3.1.4:Lines [337-344].

13. Reviewer Comment:

Line 240: At table 3 line 4 the combination results 100% mortality but the same combination at fig 3c resulted to 95% mortality. Are these experiments done separately? How you explain the difference.

Responses:

We appreciate the reviewer’s keen observation regarding the apparent discrepancy between the results in Table 1 and the data in Figure 3c.

We confirm that these experiments were conducted separately. The difference in mortality rates (100% vs. 95%) is due to an unintentional error in the parameter labeling in Figure 3c caption, where the condition was intended to be 20 kV/cm but was mistakenly marked as 28 kV/cm, rather than an experimental inconsistency.

We confirm that both sets of actual experimental data are accurate for their respective distinct conditions and fall within the acceptable measurement error range. We have rectified the labeling error in the revised Figure 3c caption.

Location of Change in Manuscript:

Section 3.1.1: Lines [286].

14. Reviewer Comment:

Line 285-286: Please omit “As shown in Figure 7, the”

Responses:

We have deleted the phrase to improve the flow of the sentence structure.

Location of Change in Manuscript:

Section 3.1.4: Lines [349]

15. Reviewer Comment:

Line 287: After full stop add “Figure 7 shows the insect condition before and after PEF”

Responses:

We have ensured that the image showing the insect condition before and after PEF treatment (New Figure 8) is properly introduced and captioned.

Location of Change in Manuscript:

Figure 8 Caption: Line [350].

16. Reviewer Comment:

Line 377: add in parenthesis the fixed conditions ex a (20 kHz, 300 s). Do this also for b and c

Responses:

To enhance the clarity and independence of the figures, we have added the fixed parameters (Electric Field Strength, Pulse Frequency, and Treatment Time) in parenthesis to the captions for Figure 10a, 10b, and 10c.

Location of Change in Manuscript:

Figure 10 Caption: Lines [433-449].

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The main issue addressed in this work is reducing the use of chemical fumigants in the preservation and protection of agricultural crops from pests.
I believe this work is aimed at ensuring food security and addresses a relevant topic.
This work utilizes computer modeling and experimental research methods.
Although the use of electric fields of varying frequencies and intensities in pest control has been studied for some time, the authors present several relatively new approaches to modeling the process under study.
The mechanism of field action and the reversible and irreversible reactions in the body are adequately described. Despite the importance of the work, a number of criticisms exist:
1. The harm caused by the use of chemical reagents is not assessed.
2. The effectiveness of chemical and alternative pest control methods is not assessed.
3. Regulatory requirements for the implementation of the disinfestation process under consideration are not provided. 4. Estimated losses in stored crop volume and quality due to the impact of cutting agents are not provided.
5. Existing developments in the application of electric fields in the process under consideration are not presented.
6. Theoretical assumptions and justification for the applied field frequency, for example, based on the dielectric properties of insects, are not provided.
7. The properties of grain at the selected frequency, and therefore the depth of field penetration for processing, are not considered.
8. It is unclear whether the computer model of the processing chamber assumed the presence of a dense layer of processed material.
9. The choice of electric field strength for processing at a level close to the air breakdown strength is unclear.
10. The conclusions do not contain a comparative analysis of the developed method with existing ones.

Author Response

Response to Reviewer 3

Manuscript ID: agriculture-4020338

Title: Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Reviewer: Reviewer 3

Review Date: 25 Nov 2025

We deeply appreciate Reviewer 3 for the constructive feedback regarding the background assessment and theoretical foundations. Your comments have helped us to better contextualize our study within the existing literature and to strengthen the theoretical basis for our parameter selection. Below is our detailed point-by-point response.

Detailed Responses：

1. Reviewer Comment:

The harm caused by the use of chemical reagents is not assessed.

Responses:

We agree that an explicit assessment of the harm caused by chemical fumigants will strengthen the background of our study. We have expanded the Introduction to quantify and describe the negative environmental and health impacts of traditional chemical reagents. 

Location of Change in Manuscript:

Introduction: Lines [46-59].

2. Reviewer Comment:

The effectiveness of chemical and alternative pest control methods is not assessed.

Responses:

We have expanded the Introduction to include a concise assessment of the current efficacy limitations of both traditional chemical methods (e.g., fumigant resistance) and emerging physical alternatives (e.g., RF and CP, focusing on their respective drawbacks concerning thermal damage and penetration depth). This establishes a clearer niche for the Pulsed Electric Field (PEF) method.

Location of Change in Manuscript:

Introduction: Lines [60-68].

3. Reviewer Comment:

Regulatory requirements for the implementation of the disinfestation process under consideration are not provided.

Responses:

Accepted and Clarified. We acknowledge the importance of explicitly stating the regulatory context. We have addressed this requirement in two locations:

Introduction: We retain and highlight the discussion regarding the trend toward stringent Maximum Residue Limits (MRLs) for chemical pesticides (Lines [76-80]) to establish the regulatory pressure driving the need for alternatives.

Discussion (New Content): We have added a statement to Section 4.2.3 (Limitations and Future Perspectives) to clarify that the residue-free nature of PEF intrinsically aligns with stringent global regulatory trends favoring the phase-out of chemical fumigants (like phosphine and methyl bromide), thus providing crucial support for its application as a sustainable technology.

Location of Change in Manuscript:

Introduction: Lines [55-57];

Section 4.2: Lines [557-559].

4. Reviewer Comment:

Estimated losses in stored crop volume and quality due to the impact of cutting agents are not provided.

Responses:

We have revised the Introduction to provide estimated quantitative data on the global economic losses and quality degradation of stored grain caused by T. castaneum infestation and the resulting fungal growth and mycotoxin contamination. This establishes a clearer justification for the urgency of effective non-chemical control.

Location of Change in Manuscript:

Introduction: Lines [37-45].

5. Reviewer Comment:

Existing developments in the application of electric fields in the process under consideration are not presented.

Responses:

We have significantly expanded the literature review in the Introduction to specifically discuss existing developments and prior research using electric fields (EF), including pulsed and static fields, in stored product pest control. We delineate our study's novelty by emphasizing its focus on the optimization of key PEF parameters and the mechanistic insight into electro-neurotoxicity—aspects which remain sparsely covered in the current EF literature.

Location of Change in Manuscript:

Introduction: Lines [74-90].

6. Reviewer Comment:

Theoretical assumptions and justification for the applied field frequency, for example, based on the dielectric properties of insects, are not provided.

Responses:

We acknowledge the omission of the theoretical basis for parameter selection. We have added a new subsection in the Materials and Methods (Section 2.3) to provide the theoretical justification for the selected kilohertz frequency range. This explanation is grounded in the Maxwell-Wagner polarization theory, which leverages the distinct dielectric properties of the rice bulk (low-loss dielectric) and the insect body (lossy dielectric) to ensure the electric field effectively bypasses the grain and concentrates selectively within the pest, guaranteeing targeted energy delivery at 20 kHz.

Location of Change in Manuscript:

Section 2.3: Lines [154-165].

7. Reviewer Comment:

The properties of grain at the selected frequency, and therefore the depth of field penetration for processing, are not considered.

Responses:

This point is crucial for industrial application. We have added a discussion in the expanded Section 4.2 Application Potential focusing on the dielectric properties of dry stored grain at the selected kHz frequency range. We explain that, unlike high-frequency microwaves, the relatively low frequency of PEF results in minimal absorption of energy by the dry grain bulk. This leads to superior penetration depth and uniform energy distribution within the bulk material, which is critical for effective disinfestation, confirming the viability shown in our FEM simulation.

Location of Change in Manuscript:

Discussion (Section 4.2.1 Penetration and Uniformity): Lines [524-539].

8. Reviewer Comment:

It is unclear whether the computer model of the processing chamber assumed the presence of a dense layer of processed material.

Responses:

We have revised the methodology description for the FEM simulation (Section 2.3.4) to explicitly state the geometric assumptions. We confirm that the space between the parallel electrode plates was not modeled as a completely dense layer. Instead, it was modeled strictly according to the actual physical distribution, consisting of a heterogeneous mixture of rice grains, insects, and the air filling the gaps (voids) between them. This detailed setup was essential to accurately estimate the effective electric field distribution within the granular processing environment, as verified by our in-rice mortality experiments.

Location of Change in Manuscript:

Section 2.3.4: Lines [208-220].

9. Reviewer Comment:

The choice of electric field strength for processing at a level close to the air breakdown strength is unclear.

Responses:

We acknowledge the need for clearer justification regarding the high electric field strength selected. This has been addressed in two locations:

Results (Section 3.1.4): We explicitly state the experimental criteria prioritizing the minimum electric field strength necessary for 100% mortality while maintaining a safe margin below the theoretical air breakdown strength (Lines [348-351]).

Discussion (Section 4.1.1): We provide the theoretical justification for why this high macroscopic strength (up to 28 kV/cm) is essential. We explain that this high level is necessary to overcome the high impedance and dielectric shielding effects posed by the stored grain material. Critically, only this high macroscopic field ensures that the effective localized field at the insect's anatomical extremities exceeds the critical threshold for irreversible electroporation, as confirmed by our FEM simulation ("focusing effect").

Location of Change in Manuscript:

Section 3.1.4: Lines [337-350];

Discussion : Lines [462-470].

10. Reviewer Comment:

The conclusions do not contain a comparative analysis of the developed method with existing ones.

Responses:

We have revised the Conclusion section to explicitly include a comparative summary. This comparative analysis highlights that the developed PEF method offers a highly effective, non-thermal, residue-free solution for bulk grain treatment, distinguishing it from conventional fumigants and emerging methods like RF/MW (due to thermal damage) and Cold Plasma (due to poor penetration depth).

Location of Change in Manuscript:

Conclusion: Lines [594-601].

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This study evaluates the effect of pulsed electric fields as a control tactic for Tribolium castaneum in stored rice, and provides a model of the lethal physiological 
mechanism of this treatment. The results demonstrate the high efficacy of the proposed methodology, making it an important alternative for controlling this pest. The reviewed literature is relevant. The experimental methodology is appropriate 
and described in detail, although the statistical analyses should be described more precisely. The results are presented appropriately and the conclusions are clear.

The following are some specific recommendations:

Section 2.4: Explain the statistical analysis methodology used for each experiment.
Lines 198-199: Since multiple regression and ANOVA were used, which assume normal distribution of residuals and homogeneity of variances, 
the authors should clarify whether they performed validation tests of these assumptions.

Figures: Explain that averages of the variables are shown and that SD stands for standard deviation, when the abbreviation is used for the first time.

 

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 4

Manuscript ID: agriculture-4020338

Title: Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Reviewer: Reviewer 3

Review Date: 05 Dec 2025

We thank Reviewer 4 for the specific and highly professional suggestions regarding the statistical analysis and data presentation. These comments have been crucial in refining our data processing descriptions and ensuring the accuracy of our statistical reporting. Below is our detailed point-by-point response.

Detailed Responses：

1. Reviewer Comment:

Section 2.4: Explain the statistical analysis methodology used for each experiment.

Responses:

We have expanded Section 2.4 (Statistical Analysis) to clearly specify the statistical methodology used for each distinct experiment presented in the manuscript, ensuring precision and clarity regarding data processing.

Specifically, we clarified:

The use of Response Surface Methodology (RSM) and ANOVA for optimizing PEF parameters and generating the regression model (Section 3.1).

The use of One-way ANOVA followed by Duncan's multiple range test for comparative analyses (e.g., in the single-factor experiments and AChE activity results).

Location of Change in Manuscript:

Section 2.4: Lines [244-253].

2. Reviewer Comment:

Lines 198-199: Since multiple regression and ANOVA were used, which assume normal distribution of residuals and homogeneity of variances, the authors should clarify whether they performed validation tests of these assumptions.

Responses:

We acknowledge the importance of validating these assumptions. We have revised Section 2.4 (Statistical Analysis) to explicitly state that prior to performing the multiple regression analysis (RSM) and ANOVA, validation tests were performed. These tests confirmed both the normal distribution of residuals (using the Shapiro-Wilk test) and the homogeneity of variances (using Levene's test), thereby ensuring the validity of the statistical model used.

Location of Change in Manuscript:

Section 2.4: Lines [244-253].

Section 3.1.2: Lines [304-307].

3. Reviewer Comment:

Figures: Explain that averages of the variables are shown and that SD stands for standard deviation, when the abbreviation is used for the first time.

Responses:

We have implemented the following changes to enhance the clarity of the figures and tables:

We have updated the captions for Figure 4 (previously Figure 3), Figure 5, Figure 8, and Figure 10 to explicitly state that the data represents the mean values of the variables.

We have ensured that the abbreviation SD (Standard Deviation) is clearly defined in the first figure caption where it appears, specifically in Figure 4. 

Location of Change in Manuscript:

Section 3: Lines [268][287][450]

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

he main issue addressed in this work is reducing the use of chemical fumigants in the preservation and protection of agricultural crops from pests.
I believe this work is aimed at ensuring food security and addresses a relevant topic.
This work utilizes computer modeling and experimental research methods.
Although the use of electric fields of varying frequencies and intensities in pest control has been studied for some time, the authors present several relatively new approaches to modeling the process under study.
The mechanism of field action and the reversible and irreversible reactions in the body are adequately described. 

The authors have answered the questions raised during the initial review quite thoroughly. I believe the article, in its current form, can be recommended for publication. The data obtained by the authors can serve as a basis for further research.

The conclusions should be supplemented with the obtained data and a numerical comparison with existing methods.

Author Response

Response to Reviewer 3

Manuscript ID: agriculture-4020338

Title: Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Reviewer: Reviewer 3

Review Date: 15 Dec 2025

We sincerely thank Reviewer 3 for the positive evaluation and the recommendation for publication. We greatly appreciate this final suggestion to strengthen the conclusion with specific data. This addition has significantly improved the precision and comparative value of our findings, allowing for a clearer demonstration of the technical advantages of the PEF method. Below is our detailed response.

Detailed Responses：

Reviewer Comment:

The conclusions should be supplemented with the obtained data and a numerical comparison with existing methods.

Response:

We have revised the Conclusion section to explicitly include specific numerical comparisons with existing methods, as suggested. We highlighted that:

Time: PEF significantly shortens the treatment time to 140 s (compared to 3–14 days for chemical fumigation).

Temperature: PEF maintains a negligible temperature rise of < 1.5 ℃, effectively avoiding thermal damage (compared to > 50 °C in thermal treatments like RF/MW).

Penetration: PEF achieves superior macroscopic penetration for bulk grain (compared to surface-limited treatment in cold plasma). These additions provide a clear quantitative assessment of the advantages of the developed PEF method.

Location of Change in Manuscript:

Conclusion: Lines [594-602].

Author Response File: Author Response.pdf