Review Reports - Piper fuligineum (Piperaceae) Essential Oil on Euschistus heros: Lethal and Sublethal Toxicities and Selectivity to an Egg Parasitoid Used in Applied Biological Control

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the Author

Reviewer report

Manuscript title: Piper fuligineum (Piperaceae) essential oil on Euschistus heros: lethal and sublethal toxicities and selectivity to an egg parasitoid used in applied biological control

General assessment

The present manuscript investigates the chemical composition of the essential oil of Piper fuligineum and evaluates its bioactivity against different developmental stages of Euschistus heros, a major soybean pest. In addition, it assesses its selectivity toward the egg parasitoid Telenomus podisi.

The topic is timely and well aligned with the scope of the Journal. The manuscript has the potential to make a valuable contribution to the literature on pest management by advancing our understanding of the use of plant-derived essential oils as promising alternatives to conventional insecticides, owing to their broad spectrum of biological activity and their potential to support more sustainable pest management strategies.

Minor Comments

Overall, the manuscript demonstrates good potential but requires minor revision before it is suitable for publication. I believe that satisfactorily addressing these comments will strengthen the manuscript and make it suitable for consideration.

- Materials and Methods should be Section 2. Please reorder the manuscript so that this section appears immediately after the Introduction.

Line 381: The experimental unit is the blue paper strip containing the 10 attached eggs; therefore, the sample size is n = 10 for each treatment. According to the experimental design, there are nine treatments: six EOPF concentrations, two negative controls, and one positive control.
Line 99: "nymph hatching" is not an appropriate expression. The standard term used in entomology to refer to this process is "egg hatching" rather than "nymph hatching."
Please organize the Results section according to the subheadings used in the Materials and Methods section.

Additional comments and corrections have been incorporated directly into the manuscript file.

Comments for author File: Comments.pdf

Author Response

LIST OF CHANGES AND ANSWERS TO REVIEWERS

Ms. Ref. No.: molecules-4498453

Title: Piper fuligineum (Piperaceae) essential oil on Euschistus heros: lethal and sublethal toxicities and selectivity to an egg parasitoid used in applied biological control

Dear Editor,

Thank you for your careful review of our manuscript and for your comments. The manuscript has been revised according to your suggestions, as detailed below. The corrections have been highlighted in red text.

Responses to reviewers

Reviewer 1:

Reviewer 1: Materials and Methods should be Section 2. Please reorder the manuscript so that this section appears immediately after the Introduction.

Response: We thank the Reviewer for this suggestion. However, according to the current Molecules manuscript template, Section 2 corresponds to the Results, whereas the Materials and Methods section is present later in the manuscript. We have also verified that recently published articles in Molecules follow this same structure. Therefore, we have retained the current organization of the manuscript to ensure consistency with the journal’s formatting guidelines.

Reviewer 1: Line 381: The experimental unit is the blue paper strip containing the 10 attached eggs; therefore, the sample size is n = 10 for each treatment. According to the experimental design, there are nine treatments: six EOPF concentrations, two negative controls, and one positive control.

Response: We thank the Reviewer for this important clarification. We agree that the experimental unit corresponds to each blue paper strip containing 10 attached eggs; therefore, the correct sample size is n = 10 experimental units per treatment, rather than n = 100 eggs. Please confirm the change to the lines 400-403 and 419-420.

Reviewer 1: Line 99: "nymph hatching" is not an appropriate expression. The standard term used in entomology to refer to this process is "egg hatching" rather than "nymph hatching."

Response: The expression “nymph hatching" has been replaced with the standard entomological term “egg hatching” in line 99.

Reviewer 1: Line 432: Could the authors describe how the parasitism rate was calculated?

Response: We thank the Reviewer for this suggestion. The request information describing how the parasitism rate was calculated has been added to the Materials and Methods section (lines 443-446).

Reviewer 1: Line 466: Could the authors clarify what is meant by the term "behavioral deterrence"?

Response: We thank the Reviewer for this comment. To improve clarity and avoid ambiguity regarding the term “behavioral deterrence”, we have rewritten the sentence in lines 454-457.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General Comments

This manuscript evaluated the insecticidal activity of Piper fuligineum essential oil (EOPF) against Euschistus heros and its effects on the egg parasitoid Telenomus podisi. The study includes chemical composition analysis, toxicity tests against different insect stages, and selectivity evaluation toward a natural enemy. The topic is interesting and the experimental data are valuable for the development of botanical insecticides. However, some problems regarding data accuracy, chemical identification, and interpretation of IPM application need to be addressed before publication.

Major Comments

The LC50 and LC90 values in Table 2 should be carefully checked.

There is an obvious inconsistency between the text and Table 2. For example, the LC50 value of 1-day-old eggs is reported as about 1,100 mg L⁻¹ in the text, but Table 2 shows 0.11. This may be a calculation or decimal error. The authors should carefully check all LC values based on the original data and confirm that the units are correct in the text, tables, and figures.

The identification of 1-naphthalenol needs further confirmation.

The authors reported that 1-naphthalenol was one of the major compounds (9.88%) in EOPF. However, this compound is unusual as a major component in Piper essential oils. The authors should clarify whether this compound was confirmed by retention index and/or standard compounds, and discuss whether it could be related to analytical artifacts during extraction or GC-MS analysis.

The chemical standardization of EOPF should be discussed.

Essential oils are natural products with high variation depending on plant origin, extraction method, and environmental conditions. Although the authors identified several major compounds, the manuscript does not discuss how EOPF can be standardized as a botanical pesticide. I suggest the authors discuss possible chemical markers and the relationship between chemical composition and insecticidal activity.

The practical application value of EOPF needs further discussion.

The insecticidal activity was mainly evaluated under laboratory conditions using relatively high concentrations. Whether these concentrations are realistic for field application should discuss. Some information about field efficacy, persistence, formulation, and possible phytotoxicity to soybean plants would improve the manuscript.

The conclusion about IPM compatibility should be reconsidered.

The evaluation of EOPF effects on T. podisi is an important part of this study. However, the results showed that EOPF negatively affected parasitism and adult emergence, especially at higher concentrations.

In addition to toxicity, the authors should discuss the strong repellency observed in free-choice tests. Avoidance behavior of parasitoids may reduce the efficiency of biological control in IPM programs.

Therefore, EOPF should be considered as a potential botanical insecticide candidate, but its compatibility with biological control requires further evaluation.

Minor Comments

Table 1 in the submitted manuscript appears incomplete. Please provide the complete table.
Chemical names should be consistent throughout the manuscript. For example, "caryophyllene" and "Cariofilene" should be corrected.
The typo in the abstract "40,00 mg L⁻¹" should be corrected to "40,000 mg L⁻¹".
The description of parasitoid bioassay in Section 4.3.3.2 needs language correction for clarity.
Please check Figure 3. The positive control (Engeo Pleno™ S) appears missing in some panels.
"Egg sensibility" (Line 256-7) should be changed to "egg susceptibility".
The reference format should be carefully checked according to the requirements of Molecules.

Author Response

LIST OF CHANGES AND ANSWERS TO REVIEWERS

Ms. Ref. No.: molecules-4498453

Title: Piper fuligineum (Piperaceae) essential oil on Euschistus heros: lethal and sublethal toxicities and selectivity to an egg parasitoid used in applied biological control

Dear Editor,

Responses to reviewers

Reviewer 2

Reviewer 2: The LC50 and LC90 values in Table 2 should be carefully checked.

Response: We thank the Reviewer for this correction in the table. The values in the table were originally in percentage and were changed to mg L⁻¹ to align with the results presented in the text. Please confirm the lines 155-159.

Reviewer 2: The identification of 1-naphthalenol needs further confirmation.

Response: We thank the reviewer for this important observation. In this study, the chemical constituents of EOPF were identified by comparing their mass spectra with those available in commercial spectral libraries, as described in the manuscript. Using this approach, 1-naphthalenol was identified and represented 9.88% of the total identified constituents. Therefore, it was considered one of the main constituents detected in the essential oil.

Importantly, our results do not indicate that 1-naphthalenol originated from contamination during the extraction procedure or from analytical artifacts related to the GC-MS analysis.

The presence of different chemical constituents, including compounds that are not commonly reported as major components of Piper essential oils, may be related to the marked chemical variability observed in this genus. This variability can be influenced by several factors, such as geographic location, soil and climate conditions, seasonality, plant developmental stage, genetic background, cultivation practices, and post-harvest conditions. Therefore, the chemical profile found in this study may reflect the specific environmental and biological conditions under which the plant material was grown.

This point has been clarified in the revised manuscript.

Reviewer 2: The chemical standardization of EOPF should be discussed.

The practical application value of EOPF needs further discussion.

Response: We appreciate the assistance in improving the discussion. These suggestions were added in lines 339–362, as were the additional references in lines 612–627.

Reviewer 2: The conclusion about IPM compatibility should be reconsidered.

The evaluation of EOPF effects on T. podisi is an important part of this study. However, the results showed that EOPF negatively affected parasitism and adult emergence, especially at higher concentrations. In addition to toxicity, the authors should discuss the strong repellency observed in free-choice tests. Avoidance behavior of parasitoids may reduce the efficiency of biological control in IPM programs. Therefore, EOPF should be considered as a potential botanical insecticide candidate, but its compatibility with biological control requires further evaluation.

Response: We have rewritten the conclusion. Please check lines 494–498.

Reviewer 2: Table 1 in the submitted manuscript appears incomplete. Please provide the complete table.

Response: The current table presents 19 constituents and their retention indices, as well as the percentage of each constituent found in the essential oil. Please check lines 97–99.

Reviewer 2: Chemical names should be consistent throughout the manuscript. For example, "caryophyllene" and "Cariofilene" should be corrected.

Response: We thank the reviewer for the correction. We made the change to Table 1 (lines 97–99).

Reviewer 2: The description of parasitoid bioassay in Section 4.3.3.2 needs language correction for clarity.

Response: We have rewritten section 4.3.3.2 in lines 446–463.

Reviewer 2: The typo in the abstract "40.00 mg L⁻¹" should be corrected to "40.000 mg L⁻¹".

Response: We thank the reviewer for the correction. We made the change to the line 31.

Reviewer 2: Please check Figure 3. The positive control (Engeo Pleno™ S) appears missing in some panels.

Response: We re-examined Figure 3 and observed that the Engeo Pleno™ S control is present in all four panels (lines 172-178).

Reviewer 2: "Egg sensitivity" (Line 256-257) should be changed to "egg susceptibility".

Response: The expression “egg sensitivity" has been replaced with the standard entomological term “egg susceptibility” in line 256-257.

Reviewer 2: The reference format should be checked carefully according to the requirements of Molecules.

Response: We verified the references to comply with Molecules' standards (lines 517-643).