Toxicity of Nanoemulsified Eugenia uniflora (Myrtaceae) Essential Oil to Spodoptera frugiperda (Lepidoptera: Noctuidae) and Selectivity to Trichogramma pretiosum (Hymenoptera: Trichogrammatidae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This draft describes several experiments used to evaluate the toxicity of Eugenia uniflora nanoemulsions against fall armyworm (Spodoptera frugiperda) and selectivity to one of its parasitoids. I have a major question regarding the concentration used for these experiments (LC50 vs LC90). Besides that, the study was well conducted, but the discussion is not fully supported by the results provided. Major revisions are needed.

MAJOR COMMENTS

The introduction is short as it can be. Could the authors elaborate more on the use of nanoemulsions for essential oils. Is it something emerging, is there any product in the market with this type of technology? Can we have some background information on T. pretiosum (native or introduced? established in the landscape? commercially available? Natural rate of parasitism? Etc.). Same thing for fall armyworm. It is one of the most damaging pests worldwide, so the authors can certainly told us more about its host range, and impact in Brazil for instance.

The chemical analysis (L72) is not used at all in the rest of the manuscript. It is completely disconnected from the toxicology analysis. The logic path would have been to test the toxicity of some isolated compounds. Without this I do not really understand why it is there.

L101: I do not understand why the authors used the LC50 value instead of the LC90 for all their experiments. As mentioned later in the draft (L223), below 80% toxicity there is no way this would be used commercially.

L213-214: this seems like cherry picking. The main effect on the parasitoid is the drastic drop in parasitism rate up to 48h after application. Combined with a decrease from 85% to 61% emergence rate, there is a risk that NEO may disrupt the biological control program.

L249-250: I don’t believe this will compensate for a reduction in parasitism by 90% up to two days (maybe more) after application.

L253: Is it really evident? Toxicity didn’t increase as compared to EO and declined way below EO as conservation time increase. After 60 days of conservation, NEO is basically inefficient (> 80% survival).

L368: why immerse the eggs for this experiment and spraying for the other ones?

MINOR COMMENTS

L23. Please include the common name of Spodoptera frugiperda (fall armyworm) same thing for Eugenia uniflora (L26)

L38: indicate the level (%) of toxicity.

Figure 1: I would flip the y-axis so that 1 is close to zero, not 30. °

L120: a bracket is missing.

Table 3: Are these separations correct? How 25°C/1 day (26.06 nm) can be significantly different from 4°C/1 day (27.36 nm), but not from 40C/1 day (23.86 nm)?

L133: I don’t understand how the mean diameter of nanoparticles is around 28 nm in table 3, and size of the same nanoparticles is 283 nm in Figure 2. Is size different from diameter? How?

L133: spaces before and after ±

L140: The experiment tested NEO stability at 1, 15, and 30 days storage time.

L159-160: Conflicting results, table 4 indicates no significant difference between the treatments.

Table 4 and 5: what lowercase and uppercase letters refer to, lines or columns?

 

 

Author Response

Reviewer #1:

“The introduction is short as it can be. Could the authors elaborate more on the use of nanoemulsions for essential oils. Is it something emerging, is there any product in the market with this type of technology? Can we have some background information on T. pretiosum (native or introduced? established in the landscape? commercially available? Natural rate of parasitism? Etc.). Same thing for fall armyworm. It is one of the most damaging pests worldwide, so the authors can certainly told us more about its host range, and impact in Brazil for instance.”

Response: The Introduction section has been expanded to provide a more comprehensive background addressing all the points raised. Specifically, we added: (i) an expanded discussion on nanoemulsion technology for essential oils, highlighting its emergence as a promising strategy to overcome physicochemical limitations such as low solubility, volatility, and instability, as well as mentioning its increasing investigation for agricultural applications;
(ii) background information on Trichogramma pretiosum, including its native occurrence in Brazil, wide distribution, commercial availability, ecological role, and relevance as one of the most widely used egg parasitoids in biological control programs;
(iii) additional contextualization of Spodoptera frugiperda, emphasizing its polyphagous behavior, wide host range, global economic impact, and particular relevance as one of the most damaging pests in Brazilian agriculture. These additions improve the scientific context and reinforce the relevance of evaluating both insecticidal efficacy and selectivity within an integrated pest management (IPM) framework.

 

Reviewer #1:

“The chemical analysis (L72) is not used at all in the rest of the manuscript. It is completely disconnected from the toxicology analysis. The logic path would have been to test the toxicity of some isolated compounds. Without this I do not really understand why it is there.”

Response: The chemical characterization of E. uniflora essential oil was included to establish the chemical profile of the material tested, ensuring reproducibility and comparability with previous and future studies using this species. Moreover, earlier studies cited in the manuscript, including those based on molecular docking approaches, indicate that the insecticidal activity of this essential oil is associated with synergistic interactions among its constituents rather than the action of a single isolated compound. For this reason, the present study focused on evaluating the biological activity of the whole essential oil and its nanoemulsion, instead of testing isolated constituents. We acknowledge that assessing the toxicity of individual compounds could provide complementary information; however, the major constituent of this essential oil was not commercially available at the time the experiments were conducted. Currently, our research group is working on the isolation of this compound to support future studies addressing its individual and combined biological effects.

Reviewer #1:

“L101: I do not understand why the authors used the LC50 value instead of the LC90 for all their experiments. As mentioned later in the draft (L223), below 80% toxicity there is no way this would be used commercially.”

Response: The choice of using the LC₅₀ instead of the LC₉₀ was intentional and aligned with the specific objectives of this study. Our primary aim was not to simulate a commercial field application, but rather to assess the physiological selectivity and potential sublethal effects of the essential oil nanoemulsion (NEO) on the non-target organism Trichogramma pretiosum. The use of LC₅₀ concentrations is widely adopted in ecotoxicological and selectivity studies, as it allows the detection of subtle effects on development, parasitism, emergence, longevity, and transgenerational parameters that could be masked under higher mortality levels. Applying LC₉₀ concentrations would likely result in excessive mortality or severe impairment of biological traits, preventing a meaningful evaluation of sublethal and transgenerational responses. We agree that LC₉₀ values are more relevant from a regulatory and commercial perspective, as highlighted later in the manuscript. For this reason, LC₉₀ was estimated and reported, and its relevance for pest management applications is discussed. However, LC₅₀ was considered more appropriate for addressing selectivity and compatibility with biological control agents, which is a key requirement for integration into IPM programs.

Reviewer #1:

“L213-214: this seems like cherry picking. The main effect on the parasitoid is the drastic drop in parasitism rate up to 48h after application. Combined with a decrease from 85% to 61% emergence rate, there is a risk that NEO may disrupt the biological control program.”

Response: We thank the reviewer for this important observation and for highlighting the need to clearly distinguish between lethal, sublethal, and residual effects of the nanoemulsion on T. pretiosum. The statement cited by the reviewer refers specifically to the assays conducted on the immature stages of the parasitoid (egg–larva and pupa), in which the NEO caused only minimal or no effects on emergence. In the egg–larval stage, emergence of the F₁ generation was reduced by only 3.40%, with no negative effect on F₂ emergence, while no reduction in emergence was observed when the parasitoid was treated at the pupal stage. These results support the conclusion of low direct toxicity to immature stages, which has now been clarified in the revised discussion. However, we fully agree that the residual exposure assays, in which treated host eggs were offered to adult females (F₀), revealed a marked reduction in parasitism capacity, with decreases of 83.80%, 91.40%, and 81.40% at 12, 24, and 48 h after treatment, respectively. In addition, daughters (F₁) originating from these treatments showed a reduced parasitism capacity even when offered untreated eggs, indicating sublethal and transgenerational effects. To avoid any overinterpretation, we revised the discussion to explicitly separate these two experimental contexts and removed the term “harmless.” The manuscript now states that the NEO presents low direct toxicity to immature stages of T. pretiosum but can induce significant sublethal and residual effects on parasitism behavior, which may compromise biological control under certain exposure scenarios. We also acknowledge that these effects represent a potential risk for integration into biological control programs if application timing and exposure are not properly managed. We believe these revisions resolve the perceived contradiction and provide a more balanced and accurate interpretation of the results.

Reviewer #1:

“L249-250: I don’t believe this will compensate for a reduction in parasitism by 90% up to two days (maybe more) after application.”

Response: We agree that the transgenerational parameters alone do not compensate for the marked reduction in parasitism observed shortly after application in the residual exposure experiment. We would like to clarify that the statement referring to minimal transgenerational effects (Lines 249–250) specifically concerns the assays in which NEO was applied to host eggs containing T. pretiosum in the immature stages (egg–larva and pupa), where no biologically relevant reductions were observed in emergence, sex ratio, or parasitism in the F₁ and F₂ generations. In contrast, the residual effects experiment (Experiment 2) clearly demonstrated a substantial and temporary reduction in parasitism by F₀ females exposed to treated eggs within 12–48 h after application, as well as a reduced parasitism capacity in the F₁ generation. We have revised the Discussion to clearly distinguish between lethal, sublethal, and residual effects, and to avoid any implication that transgenerational outcomes offset the short-term reduction in parasitism. These results reinforce that the timing of NEO application relative to parasitoid release is critical for its compatibility within IPM programs.

Reviewer #1:

“L253: Is it really evident? Toxicity didn’t increase as compared to EO and declined way below EO as conservation time increase. After 60 days of conservation, NEO is basically inefficient (> 80% survival).”

Response: We agree that the insecticidal activity of NEO decreases with prolonged storage time. To address this point and avoid overinterpretation, we have revised the text to clarify that NEO efficacy is most pronounced at shorter storage periods, while longer storage results in reduced bioactivity.

The revised discussion now frames this reduction as a common and well-documented challenge associated with essential oil-based nanoemulsions, related to gradual physicochemical changes that may affect the availability of active compounds. Importantly, we emphasize that the high efficacy observed within the initial storage period supports the practical potential of NEO when applied within an appropriate shelf-life window. Thus, the decrease in activity over time is presented as a formulation optimization challenge rather than a limitation of the biological effectiveness of the nanoemulsion. The manuscript has been modified accordingly to reflect this interpretation more accurately.

Reviewer #1:

“L368: why immerse the eggs for this experiment and spraying for the other ones?”

Response: There was a misunderstanding regarding the experimental procedures. In both experiments involving Trichogramma pretiosum, host eggs were treated by immersion in NW or NEO solutions for 5 s, following a methodology widely adopted in studies evaluating the selectivity of insecticides to egg parasitoids. This approach ensures uniform exposure of the egg surface and allows direct assessment of effects on parasitoid development and parasitism behavior. In contrast, spraying using a Potter spray tower was employed exclusively in the experiments with Spodoptera frugiperda, as the standard approach for evaluating insecticidal efficacy against larval pests.

 

Reviewer #1:

“L23. Please include the common name of Spodoptera frugiperda (fall armyworm) same thing for Eugenia uniflora (L26)”

Response: Done.

 

Reviewer #1:

“L38: indicate the level (%) of toxicity.”

 

Response: Done.

 

Reviewer #1:

“Figure 1: I would flip the y-axis so that 1 is close to zero, not 30°”

 

Response: Done.

 

Reviewer #1:

“L120: a bracket is missing.”

Response: We add the bracket.

 

Reviewer #1:

“Table 3: Are these separations correct? How 25°C/1 day (26.06 nm) can be significantly different from 4°C/1 day (27.36 nm), but not from 40C/1 day (23.86 nm)?”

Response: We thank the reviewer for carefully examining Table 3. We identified a typographical error in the lettering used to indicate statistical groupings, in which an extra letter was inadvertently included. This error has been corrected, and the statistical separations are now consistent with the Tukey test results. The table and corresponding text have been revised accordingly to avoid any misinterpretation.

 

Reviewer #1:

“L133: I don’t understand how the mean diameter of nanoparticles is around 28 nm in table 3, and size of the same nanoparticles is 283 nm in Figure 2. Is size different from diameter? How?”

Response: We thank the reviewer for raising this important point, which allowed us to clarify the distinction between the two measurements. Table 3 reports the individual nanosphere diameters obtained from direct measurements of discrete particles using image analysis (ImageJ) based on microscopy images. In contrast, the value presented in Figure 2 corresponds to the mean hydrodynamic size obtained by Dynamic Light Scattering (DLS), which reflects the average circumferential diameter of all nanospheres present in the analyzed aliquot, including the hydration layer and possible particle–particle interactions in suspension. Therefore, the apparent discrepancy arises from the fundamentally different principles and scales of the two techniques, rather than from an inconsistency in the data. This clarification has now been explicitly added to the revised manuscript to avoid confusion.

 

Reviewer #1:

“L133: spaces before and after ±”

Response: We add the spaces.

 

Reviewer #1:

“L140: The experiment tested NEO stability at 1, 15, and 30 days storage time.”

Response: We acknowledge that the mention of “60 days” in line 140 was a textual error. The nanoemulsion stability assay was conducted at 1, 15, and 30 days of storage, as correctly described in the Methods section. The reference to 60 days applies exclusively to the bioassay evaluating the insecticidal activity of NEO over extended storage time, and not to the physicochemical stability analysis. The text has been corrected to clearly distinguish between these two experimental approaches and to avoid any misunderstanding.

 

Reviewer #1:

“L159-160: Conflicting results, table 4 indicates no significant difference between the treatments.”

Response: We thank the reviewer for this comment, which helped us identify an important issue related to the interpretation of Table 4. The apparent inconsistency was due to the absence of a clear explanation of the statistical notation used in the table. We have now clarified in the table caption that lowercase letters indicate comparisons within columns (between treatments), whereas uppercase letters indicate comparisons within rows (between developmental stages), according to Tukey’s test (p < 0.05). With this information made explicit, it becomes clear that the statistical comparison discussed in the text refers to differences between developmental stages (egg–larva vs. pupa) within the NEO treatment, and not between NW and NEO treatments. Accordingly, Table 4 supports the statement that parasitism by F1 females was significantly higher when the parasitoid was treated during the egg–larval stage compared to the pupal stage under NEO exposure, as indicated by different uppercase letters. The table caption and the Results text have been revised to prevent further misinterpretation.

 

Reviewer #1:

“Table 4 and 5: what lowercase and uppercase letters refer to, lines or columns?”

Response: We add in legend of table 4 this sentence: “…followed by the lowercase letter in the column (between treatments) and uppercase in the row (between development stages) do not differ …” and add in the legend of the table 5: “… followed by the lowercase letter in the column (between treatments) and uppercase in the row (between contact hours) do not differ …”.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Toxicity of nanoemulsified Eugenia uniflora (Myrtaceae) essential oil to Spodoptera frugiperda (Lepidoptera: Noctuidae) and selectivity to Trichogramma pretiosum (Hymenoptera: Trichogrammatidae)“ investigates the toxicity of Eugenia uniflora essential oil (EO) and its nanoemulsion (NEO) against Spodoptera frugiperda, while also assessing the selectivity of the NEO toward the beneficial parasitoid Trichogramma pretiosum. The topic is relevant to sustainable pest management and aligns well with the scope of Plants, especially given the growing interest in botanical insecticides and nanoformulations.

One of the main strengths of the study is the integrated approach, combining chemical characterization (GC–MS), nanoemulsion physicochemical properties, toxicity bioassays, and selectivity/transgenerational effects on a non-target organism. The chemical profile of the EO is clearly presented, and the dominance of curzerene and sesquiterpenes is consistent with previous reports, supporting the reliability of the material studied. The nanoemulsion characterization is also comprehensive, including SEM, DLS, PDI, and zeta potential measurements, which strengthens the formulation aspect of the work.

However, several points require improvement:

1. Lines 118-123, Table 3: The particle size data appear inconsistent between SEM (mean sizes in the range of approximatelly18–31 nm) and DLS results (283.2 nm; Line 132). This discrepancy should be discussed more clearly, with an explanation of the methodological differences and their implications for biological activity.
2. Lines 159-176, Tables 4 and 5: Although the NEO is described as “selective” to T. pretiosum, the results show significant reductions in parasitism rates under certain conditions. The discussion (Lines 242-246) partially addresses this issue, but the conclusions (Lines 424-430) somewhat overstate selectivity. A more cautious interpretation is recommended, clearly distinguishing between effects on emergence, parasitism, and transgenerational responses.
3. Lines 221-223: The experimental design justification could be strengthened. The rationale for selecting only the LC₅₀ concentration for most NEO bioassays should be better supported, and the absence of dose response testing for the nanoemulsion should be acknowledged as a limitation.
4. Minor issues related to English clarity and consistency are present (e.g., “a harmless to the parasitoid” in line 156; inconsistent use of NOE/NEO in some tables). Careful language editing is recommended to improve readability and precision.

Overall, the manuscript presents valuable data but would benefit from clearer interpretation of selectivity, a more thorough discussion of particle size data, and more cautious conclusions.

Comments on the Quality of English Language

Minor issues related to English clarity and consistency are present (e.g., “a harmless to the parasitoid” in line 156; inconsistent use of NOE/NEO in some tables). Careful language editing is recommended to improve readability and precision.

Author Response

Reviewer #2:

“Lines 118-123, Table 3: The particle size data appear inconsistent between SEM (mean sizes in the range of approximatelly18–31 nm) and DLS results (283.2 nm; Line 132). This discrepancy should be discussed more clearly, with an explanation of the methodological differences and their implications for biological activity.”

 

Response: We note that a similar concern regarding the apparent discrepancy between SEM and DLS particle size measurements was also raised by Reviewer #1. In response to both comments, we have revised the manuscript to clarify the methodological differences between these techniques and their implications. Specifically, we now explicitly state that SEM provides the physical diameter of individual nanoemulsion droplets based on direct imaging, whereas DLS measures the hydrodynamic diameter of particle populations in suspension, which includes the solvation layer and possible aggregation effects. As a result, larger values obtained by DLS compared to SEM are expected and widely reported in the literature for nanoemulsion systems. These clarifications have been incorporated into the Results section (Lines 149-178) and further discussed in the Discussion section (Lines 267–275) to improve transparency and ensure consistency in data interpretation. We believe these revisions resolve the apparent inconsistency and strengthen the understanding of the nanoemulsion characterization and its relevance to biological activity.

 

Reviewer #2:

“Lines 159-176, Tables 4 and 5: Although the NEO is described as “selective” to T. pretiosum, the results show significant reductions in parasitism rates under certain conditions. The discussion (Lines 242-246) partially addresses this issue, but the conclusions (Lines 424-430) somewhat overstate selectivity. A more cautious interpretation is recommended, clearly distinguishing between effects on emergence, parasitism, and transgenerational responses.”

 

Response: We acknowledge that, in the previous version of the manuscript, the use of the term “selective” could be interpreted as overstated, given the observed reductions in parasitism under specific exposure scenarios. This concern was also raised by Reviewer #1, and in response, we have carefully revised both the Discussion and Conclusion sections to provide a more cautious, nuanced, and biologically accurate interpretation of the results.

The revised discussion explicitly states that NEO exhibits low impact on T. pretiosum during protected immature stages (egg–larva and pupa), with minimal effects on emergence and no biologically relevant transgenerational impairment. However, we now clearly acknowledge that residual exposure of adult females to treated host eggs caused pronounced short-term reductions in parasitism, representing sublethal effects that may affect biological control performance depending on application timing. Accordingly, the term “selective” is now used conditionally, emphasizing that compatibility with T. pretiosum is dependent on developmental stage and timing of application, rather than implying an absence of adverse effects. The conclusion was also revised to avoid overgeneralization and to explicitly mention the residual sublethal effects on adult parasitism. These revisions can be found in: Discussion: Lines 267-275 and 323-381 and Conclusion: Lines 543-554. We believe these changes fully address the reviewer’s concern and provide a balanced interpretation consistent with the experimental evidence and IPM principles.

 

Reviewer #2:

“Lines 221-223: The experimental design justification could be strengthened. The rationale for selecting only the LC₅₀ concentration for most NEO bioassays should be better supported, and the absence of dose response testing for the nanoemulsion should be acknowledged as a limitation.”

 

Response: The rationale for selecting the LC₅₀ concentration was clarified in the revised manuscript. This concentration was chosen because it represents a biologically relevant and standardized reference point, commonly used in ecotoxicological studies to evaluate both efficacy against target pests and potential sublethal effects on non-target organisms. Moreover, preliminary screening assays indicated that the LC₅₀ value (36.05 mg·mL⁻¹) achieved biologically meaningful mortality in S. frugiperda. We also acknowledge that the absence of a full dose–response evaluation for the nanoemulsion constitutes a limitation of the present study. This point has now been explicitly recognized in the Discussion section (lines 285-294), where we emphasize that future studies should investigate multiple NEO concentrations to better characterize dose-dependent effects, optimize selectivity, and refine risk assessment for non-target organisms. This clarification strengthens the experimental justification while transparently addressing the study’s scope and limitations.

 

 

Reviewer #2:

“Minor issues related to English clarity and consistency are present (e.g., “a harmless to the parasitoid” in line 156; inconsistent use of NOE/NEO in some tables). Careful language editing is recommended to improve readability and precision. Overall, the manuscript presents valuable data but would benefit from clearer interpretation of selectivity, a more thorough discussion of particle size data, and more cautious conclusions.”

Response: The manuscript has been carefully revised to address issues related to English clarity, grammatical accuracy, and terminology consistency. Specifically, inappropriate expressions such as “a harmless to the parasitoid” were corrected, and the nomenclature was standardized throughout the text, figures, and tables to consistently use “NEO.” In addition, the interpretation of selectivity was refined to more clearly distinguish between effects on emergence, parasitism, and transgenerational responses, avoiding overgeneralization. The discussion regarding particle size data was expanded to explicitly explain the methodological differences between SEM and DLS measurements and their respective implications for nanoemulsion characterization and biological activity. Finally, the conclusions were revised to adopt a more cautious and balanced tone, emphasizing the conditional nature of selectivity and the importance of application timing, formulation stability, and exposure route. These changes were made to improve readability, precision, and alignment with the experimental evidence, as recommended by the reviewer.

 

 

Reviewer #2:

“Comments on the Quality of English Language Minor issues related to English clarity and consistency are present (e.g., “a harmless to the parasitoid” in line 156; inconsistent use of NOE/NEO in some tables). Careful language editing is recommended to improve readability and precision.”

 

Response: The manuscript has undergone careful language revision to improve readability and precision. Specifically, ambiguous expressions such as “a harmless to the parasitoid” have been corrected to more accurate formulations, avoiding absolute terms and clearly distinguishing lethal from sublethal effects. Additionally, inconsistencies in the use of abbreviations (NOE/NEO) across the text and tables have been standardized. These revisions aim to enhance clarity while ensuring that interpretations remain scientifically cautious and consistent with the presented data.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have carefully revised the manuscript and have substantially addressed the main remarks raised in the first review round. Overall, the revised version represents a clear improvement in scientific rigour, balance of interpretation, and clarity. The manuscript now presents a balanced and well-supported evaluation of nanoemulsified Eugenia uniflora essential oil within an IPM context.

Comments on the Quality of English Language

Several language issues and inconsistencies have been corrected. The English is now clear and precise.