From Attraction to Repellency: The Olfactory Response Pattern of Papilio polytes to Shared Volatiles from Frass and Host Plants Driven by Chemical Composition

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Wu et al. reports electrophysiological and behavioral responses of the Mormon butterfly Papilio polytes to odors emitted from both host plants and caterpillar frass. The findings highlight the role of the shared plant-frass VOCs in host selection of oligophagous insects and advances theories on manipulating P. polytes’ behavior. The topic aligns with the journal’s scope, and the research is well-designed with appropriate methods. Here are some issues need to be addressed:

 

Major comments:

1. Lines 138−139, “This data was then compared against published profiles". Please cite the relevant literatures. Also, the vocabulary “published profiles” is confused, please modify.
2. Section 3.2.1 & 3.3.1 (electrophysiological and behavioral tests): When first introducing the three key compounds (linalool, citronellal, and geraniol), the authors should briefly explain the rationale for selecting them specifically for the blend/combination assays.
3. Behavioral results: Please report proportion of the 'no choice' individuals. Moreover, were the insects that made no choice included in the statistical analysis? To avoid confusions, please add an explanation in the M&M section.
4. To deepen the interpretation, the authors could add a discussion of the potential neural and cognitive mechanisms underlying blend composition detection.
5. Please add an explanation of the potential ecological or evolutionary implications of the phenomenon: the ternary blend repels both sexes, while binary blends are sex-specific.

Minor comments:
     1. Table 1: Please standardize the number of decimal places in the "Relative abundance (%)" column. Also, check row 25 ("Tau-Cadinol"), the value "0.290±3.99" appears to have a typographical error in the standard error.

2. The subsection numbering is duplicated. There are two subsections labeled 2.1.2 ("Frass" and "Volatiles"). Please perform a full consistency check on all numbering (sections, equations, figures) in the manuscript.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "From Attraction to Repellency: The Olfactory Response Pattern 2 of Papilio polytes to Shared Volatiles from Frass and Host 3 Plants Driven by Chemical Composition"  explores, using EAG and a Y-shaped olfactometer, how P. polytes butterflies respond to odors released by both their host plants and their caterpillar excrement. This manuscript appears to be of interest to potential readers of this journal, and I have some questions about it.

Lines 62-63, 68: Given that the authors studied a diurnal lepidopteran, do they believe that vision is a relatively unimportant sense in the search for optimal host plants for oviposition?

Lines 123-128: Please provide a complete description of this section, remembering that it must be reproducible. How were the insects obtained? How were they identified? If collected from the field, were the insects quarantined? How many insect generations were present before the experiments? On what basis and with what criteria did I select the healthy butterflies? Was the interval 1 to 3 days? Three days without food can affect an insect's response; if I used 4-day-old adult butterflies, some were 5 or 7 days old.

Line 131: You need to explain how you standardized the method in that section.

Lines 130-133: The method is not precise enough to replicate. What is the justification for using 3-5 instar larvae? Was the same amount of waste used for extraction? How much time elapsed between waste storage and extraction (VOC release varies rapidly over time)? Were the residues standardized by mass or density?

Line 136: You did not include a section on host plant conditions.

Line 154: By "fresh excrement," do you mean it came from the freezer?

Line 158: What type of fiber? What are its characteristics? How was it conditioned before use?

Lines 352-355: The graphs in Figures 1 and 2 are illegible; tables would be more appropriate for explaining the results.

Line 425: "To our knowledge." Please ensure your work is pioneering in a specific area of study; if you are unsure, do not mention it.

Line 461: "Host-plant volatiles can mimic signaling profiles of healthy or suitable hosts." You make this comparison, but you did not evaluate it. Healthy or damaged hosts? This is not supported by your data.

Lines 483-486: Can you further justify what you mention? If the concentration increases, the behavioral response will not always increase; there should be a response threshold.

Lines 487-504: Can you be more precise in your conclusion and limit yourself to your results?

Comments on the Quality of English Language

The style of the English language could be improved

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Congratulations, there is a lot of work in this study, and there are dose-response curves in EAG and behavior, which is very nice. I did not read the manuscript, but went directly to the statistical analysis and data presentation, where I have some comments that follow. The paper needs resubmission after these changes are introduced, and then I will review the manuscript if it is the case.

1-Comment 1: EAG dose-response curves. Please perform non-linear dose-response curve analysis and calculate the ED50 (that is, the mid-point of the sigmoidal curve) and the slope of the curves. Then compare this mid-point and slope among compounds for each sex separately, and between males and females within each compound. Consider not subtracting hexane from each puff but using hexane as a zero concentration in the curve. I recommend using R "drc" package https://cran.r-project.org/web/packages/drc/drc.pdf and following non-linear fitting advice such as "The R Book", Non-linear Regression, by Michael Crawley. (example with supplementary R code: https://www.nature.com/articles/s41598-022-10954-x).

2-EAG graphs: please show the ACTUAL mass of compound loaded (instead of the volume concentration) in the x-axis

3-Y-tube assay. Since you have done doses, instead of PLOTTING each dose in a separate plot, I would put the 4 doses of each compound side by side and then do a STATISTICAL comparison among them (R GLM family binomial, e.g., supplementary R code https://www.nature.com/articles/s41598-019-44581-w

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

To Authors:

The manuscript needs complete rehaul of statistical analysis and results display. In the link below there are 4 files. One of them contains the reviewer report, which explains everything. It is based on the statistical analysisi (another .docx file) and 2 excel files with the data iused for the statistical analysis.

https://saco.csic.es/s/FbgtH8ywt6LRC5W

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript needs a few more changes. Everything is indicated in the attached file which is the main text PDF converted to WORD and with comments in track changes, and at the beginning of the document there are further comments in blue color font.

 Thanks

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf