Review Reports - Integrating Molecular Docking and Electrophysiology Reveals Sesquiterpenes as Candidate Attractants for <i>Ceratitis capitata</i> Wiedemann (Diptera: Tephritidae)

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

In this manuscript the authors have improved significance while updating molecular docking analysis.

Author Response

Dear Reviewer,

We sincerely appreciate the time and effort you devoted to reviewing our manuscript. Your insightful comments and constructive suggestions have been invaluable in improving the clarity, accuracy, and overall quality of our work. We carefully considered each of your remarks and incorporated the necessary revisions.

Thank you again for your thoughtful contribution, which has significantly enhanced the manuscript.

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Most of the issues have been appropriately addressed, leading to a substantial improvement of the manuscript. The revisions made have significantly strengthened the description of the experimental design and have improved the clarity of the results and their discussion. Overall, the manuscript is now much clearer and more robust than the original submission.

I have included a small number of minor comments in the revised version of the manuscript, which are intended to further improve the manuscript, should the authors consider them useful. These comments are minor in nature and do not affect the main conclusions of the study.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you again for your thoughtful contribution, which has significantly enhanced the manuscript.

Here are detailed responses to your suggestions

This sentence appears to repeat the first line of the paragraph and does not provide any clarification of the experimental design. In particular, the rationale for analyzing only males should be explicitly stated, so that readers can better understand and contextualize the study.

Our reply: The sentence was modified, and the reason for the use of males was incorporated. Please see lines 101-106

“Because C. capitata is a quarantined pest eradicated from Mexico, experimental work is restricted to irradiated sterile males. Accordingly, all electrophysiological assays were conducted on males. This design allowed us to evaluate whether computational screening can prioritize structurally diverse ligands with potential behavioral relevance, providing a basis for subsequent biochemical validation and behavioral testing.”

This line does not specify the database from which the molecular models were retrieved. To address this, I suggest adding: “Molecular models of C. capitata ORs were retrieved from the UniProt database (EMBL-EBI).”

Our reply: Thank you for the suggestion. It was added, please see lines 109-110

Similarly to the previous comment, the source database should be specified. I suggest adding: “were obtained from the UniProt database.”

Our reply: Done, please, see lines 112-113, and 117

Please provide and/or cite the list of chemical compounds included in this study and their main characteristics.

Our reply: The list was included as supplementary material, and it was cited in the main text

I consider that Figure 1 should present the results of binding affinities of chemical compounds across OBPs, rather than a description of trimedlure isomers. Please revise accordingly and move the current figure to the Supplementary Material.

Our reply: Thank you for this comment. Now, Figure 1 represents the results of binding affinities, and the previous figure was removed to supplementary information as Supplementary Figure 1

I therefore suggest relocating this descriptive paragraph to the Materials and Methods section (Section 2.2).

Our reply: Done

Which trimedlure isomer was used (1R,2R,4S or 1S,2S,4R)? Please clarify this point and ensure that it is consistently corrected throughout the text and figure legends.

Our reply: We apologize for this misunderstanding, the isomer is 1R, 2R, 4S. It was clarified and is now consistent throughout the text

The results shown in Figure 3 are not described in the text. Please add a sentence describing these results.

Our reply: The results for figure 3 (Now, figure 4) are described in lines 253-255, as follows: “We calculated the binding energy values for the 14 OBPs with compounds reported as attractants for medfly, focusing on essential oil-based terpenoids and fermented-origin compounds (F27, 362 = 34.16, P < 0.001, Figure 4 and Supplementary Table 4),”

Please add "(Figure 4A)"

Our reply: Done

Please add "(Figure 4B)"

Our reply: Done

As I understand the statistical analysis described, where “different letters denote significant differences,” Z-E-α-, Z-β-, E-Z-α- and E-β-farnesene showed lower binding energies than β- and γ-cadinene. No significant differences were observed between these molecules and trimedlure C.

Our reply: Reviewer is right. It was corrected in the text

Please add "observed in our results"

Our reply: Done

Please add "across the 14 OBPs and 4 ORs"

Our reply: Done

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have carefully addressed all my comments and suggestions. The revised manuscript is clearer, more precise, and better organized. The authors corrected problems with redundancy, scientific names, and anatomical descriptions. They also clarified methods, improved figure references, and fixed statistical reporting. Some overstatements were reduced to better match the real scope of the study. The previous discrepancies in statistical results and figure annotations were corrected. The discussion and conclusions were also revised according to the corrected analyses and comments about multiple comparisons.

The authors admitted the limitations of their study. They clearly stated that behavioral experiments were not performed and explained that future studies are needed to confirm the computational and electrophysiological findings. This makes the conclusions more accurate and transparent.

The claims about molecular insights and behavioral relevance were also changed. Now the authors clearly show the difference between computational docking, electrophysiology, and future behavioral studies. These changes improve the scientific quality and clarity of the manuscript.

Overall, the study shows a well-done combination of computational screening and electrophysiology. It gives useful information about medfly olfaction and compound prioritization for integrated pest management. In its current form, the manuscript is suitable for publication after minor editorial checks.

Author Response

Dear Reviewer,

Thank you again for your thoughtful contribution, which has significantly enhanced the manuscript.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript review report

Journal: MDPI- insects

Manuscript number: insects-4064549

Title MS: Integrating molecular docking and electrophysiology reveals sesquiterpenes as candidate attractants for Ceratitis capitata (Diptera: Tephritidae).

The manuscript integrates in silico molecular docking with electroantennography (EAG) to prioritize sesquiterpenes as candidate attractants for C. capitata. The approach is timely and relevant to reverse chemical ecology and IPM. Overall, the study is well motivated and clearly written, but several methodological clarifications, statistical assumptions, and interpretative overextensions need to be addressed to strengthen reproducibility and biological inference.

Below is a structured focused specifically on M&M, results, and discussion. I focus on scientific rigor, reproducibility, interpretation, and alignment between methods, results, and conclusions. The study is scientifically sound and publishable, but methodological transparency, interpretative limitation and corrections are required before acceptance.

Materials and Methods

The workflow (protein modeling, docking, EAG validation) is logically structured and appropriate and uses of multiple OBPs and ORs increases robustness relative to single-target docking. As I think the limitations of docking are acknowledged in the manuscript, which is commendable.

1.1 Protein modeling and validation

The manuscript states that models were “energy-minimized and validated, but no validation metrics are reported (e.g., Ramachandran statistics, QMEAN, GMQE, MolProbity scores). AlphaFold models and homology models are treated equivalently, but confidence scores (pLDDT) for AlphaFold structures are not discussed.

I would like to recommend some points. Add a short subsection or table in the Supplementary Material reporting, template coverage and identity, model quality metrics (Ramachandran %, QMEAN/GMQE or equivalent) and pLDDT ranges for AlphaFold-derived models this is important given that docking accuracy strongly depends on model quality.

1.2 Docking strategy and averaging approach

The averaging of docking energies across 14 OBPs and 4 ORs is a central methodological choice. While the authors justify this as a prioritization tool, averaging implicitly treats all proteins as independent biological replicates, which they are not.

OBPs and ORs differ fundamentally in structure, binding mode, and biological role. Treating them as replicates inflates degrees of freedom in ANOVA (e.g., F15,208), potentially violating independence assumptions. Openly state that docking values are pseudo-replicates used for ranking, not statistical inference. Consider rephrasing statistical tests on docking data as descriptive ranking analyses, or alternatively use mixed-effects models with protein identity as a random factor or present docking results without inferential statistics, emphasizing effect sizes and rankings.

1.3 Docking parameters

The grid box size (80 × 80 × 80) covering “most of the protein surface, is unusually large. Blind docking across entire OR models (membrane proteins) raises concerns about biological plausibility of binding poses, especially given transmembrane helices. Clarify whether docking to ORs targeted extracellular/lumenal regions or was fully blind. Discuss limitations of docking to ORs without membrane context or lipid embedding.

1.4 Electroantennography (EAG)

The EAG protocol is generally sound and well described. However, only irradiated males were tested, which may differ physiologically from wild males’ solvent control subtraction is mentioned but not shown statistically. Briefly justify the exclusive use of irradiated males. Indicate whether solvent controls differed significantly from zero after correction. Clarify whether antennae from different flies were treated as independent biological replicates (they appear to be).

Results

Results are clearly organized by OBPs, ORs, and EAG. Figures are informative and well referenced. The authors avoid overclaiming receptor specificity based on docking alone.

2.1 Statistical treatment of docking data repeated use of ANOVA on docking energies (e.g., F31,416) implies statistical independence of binding energies derived from different proteins. This is statistically problematic and should be interpreted cautiously. Reframe these analyses as comparative trends, not definitive statistical tests. Explicitly state in results that p-values are used for ranking consistency, not hypothesis testing.

2.2 OR docking inconsistencies

Figure 7 reports no significant differences, yet the caption includes an ANOVA result with P < 0.001, which contradicts the text. As I think this appears to be an error. as title says docking analysis should be draw docking figures. Ensure consistency between figure captions, main text, and supplementary tables. Carefully recheck all F and P values for OR docking figures.

2.3 EAG results interpretation the compound effect is reported as non-significant (P = 0.08), yet the discussion refers to comparable responses to trimedlure. Dose dependence is clear, but compound-specific differences are statistically weak. Tone down compound-specific claims and emphasize that EAG confirms antennal detectability, not differential potency.

The Discussion is comprehensive, well referenced, and demonstrates strong command of medfly chemical ecology. The limitations of docking and EAG are overtly acknowledged. The link between receptor plasticity and ecological strength is conceptually strong.

Major comments in the discussion section

`3.1 Interpretation of stereoselectivity. The conclusion that ORs show minimal stereoselectivity is based entirely on docking. Docking alone cannot reliably predict stereochemical discrimination at ORNs.

I would like to give some recommendation to improve discussion rephrase more cautiously Replace do not discriminate with did not show predicted stereoselectivity under the docking framework used. Emphasize that SSR or heterologous expression assays are required to validate this claim.

3.2 Linking docking, EAG, and behavior. The manuscript correctly states that antennal activation does not imply attraction. However, later sections implicitly suggest that sesquiterpenes may substitute trimedlure please clear this statement. Clearly separate detectability, neural activation, and behavioral attraction throughout the Discussion. Avoid language suggesting equivalence to trimedlure without behavioral assays.

3.3 Cost and application arguments. The argument that mixed sesquiterpenes could reduce costs is compelling. However, release rate, oxidation stability, and field longevity are not discussed. Add a short paragraph acknowledging formulation and release kinetics as key future constraints.

Explain and justify statistical treatment of docking data, add protein model validation metrics, correct inconsistencies in OR docking statistics, moderate claims regarding stereoselectivity, functional equivalence to trimedlure, clarify irradiated male use, improve language thoroughly in the MS and ensure all figure captions match reported statistics.

Author Response

Reviewer 1

Title MS: Integrating molecular docking and electrophysiology reveals sesquiterpenes as candidate attractants for Ceratitis capitata (Diptera: Tephritidae).

Our reply: Thank you for your valuable comments and suggestions

Materials and Methods

1.1 Protein modeling and validation

Our reply: We agree that molecular docking accuracy largely depends on the quality of the structural models. Therefore, following the reviewer’s suggestion, we have added a supplementary table reporting the quality parameters of the models used in this study.

The quality of homology models generated using SWISS-MODEL was evaluated based on template coverage, sequence identity, GMQE values, and QMEAN Z-scores. In contrast, models obtained from AlphaFold were assessed using previously unreported pLDDT confidence scores.

In addition, all models were validated using the MolProbity server, from which Ramachandran statistics, MolProbity scores, and clashscores were obtained. These parameters are presented in a new supplementary table, showing that all models used in this study exhibit desirable quality metrics and are suitable for reliable docking analyses.

1.2 Docking strategy and averaging approach

Our reply: We thank the reviewer for raising this important point. To clarify, OBPs and ORs were not combined into a single dataset. Docking values were analyzed separately for the 14 OBPs and for the 4 ORs, and comparisons were made within each protein group. Our intention was to use the average binding energy across proteins of the same class (OBPs or ORs) as a descriptive ranking tool, not as independent biological replicates. We have revised the Methods and Results sections to explicitly state this distinction and to emphasize that docking values represent pseudo-replicates used for prioritization rather than statistical inference.

Please see lines 182-190, and 304-310

1.3 Docking parameters

Our reply: We thank the reviewer for this important observation. Docking analyses were performed using a blind docking approach with a large grid box (80 × 80 × 80) to cover the entire protein surface and enable unbiased exploration of potential ligand-binding regions. This approach was adopted because the binding sites of insect OBPs and ORs have not been experimentally characterized.

We acknowledge that insect ORs are membrane proteins with multiple transmembrane helices. We also recognize that the docking analyses were performed on full-length receptor models without explicitly accounting for the membrane environment or lipid embedding, thereby lacking full biological context. In light of these limitations, we interpret the docking results as comparative and exploratory, as the primary aim of this study was to identify relative binding affinities rather than to define definitive binding sites.

1.4 Electroantennography (EAG)

Our reply: We thank the reviewer for the constructive comments regarding the EAG protocol. The exclusive use of irradiated males was due to the availability of flies provided by the Moscafrut program, which routinely supplies sterile males for experimental assays. These individuals are physiologically comparable to wild-type males in terms of antennal olfactory responses, as confirmed in previous studies, and their use ensured consistency and biosafety during experimentation. We have now added a brief justification in the Methods section to clarify this choice.

Regarding solvent controls, we confirm that hexane responses did not differ significantly from zero after baseline correction. All data from EAG has been added to the supplementary information.

Finally, antennae from different flies were indeed treated as independent biological replicates, with each preparation corresponding to a distinct individual. We have clarified this point in the Methods section to avoid ambiguity. Please, see lines 174-176.

Results

Results are clearly organized by OBPs, ORs, and EAG. Figures are informative and well referenced. The authors avoid overclaiming receptor specificity based on docking alone.

Our reply: We appreciate the reviewer’s careful observation regarding the statistical treatment of docking data. We agree that repeatedly using ANOVA on binding energies may imply statistical independence among proteins, which is not strictly valid. Our intention was not to perform hypothesis testing on receptor-specific selectivity, but rather to use p-values as a descriptive tool to highlight comparative trends and ranking consistency among ligands.

In the revised manuscript, we have reframed these analyses as exploratory comparisons rather than definitive statistical tests. Specifically, we now state in the Results section that p-values are used only to support ranking consistency and should not be interpreted as evidence of statistical independence. Please, see lines 184-195

2.2 OR docking inconsistencies

Our reply: We acknowledge the reviewer's careful revision. Yes, it was our mistake, and we corrected it. Additionally, all the figures were rechecked to avoid inconsistencies

Our reply: We thank the reviewer for this valuable clarification. We agree that compound-specific differences in EAG responses were statistically weak (P = 0.08) and should not be interpreted as evidence of differential potency. In the revised manuscript, we have toned down compound-specific claims and now emphasize that the EAG results confirm the antennal detectability of the tested sesquiterpenes, rather than demonstrating stronger or weaker responses than trimedlure.

We have modified the Discussion accordingly to highlight dose-dependent activation as the main finding, while clarifying that compound-specific differences remain tentative and require further behavioral assays for confirmation. Please see lines 401-404

The Discussion is comprehensive, well referenced, and demonstrates strong command of medfly chemical ecology. The limitations of docking and EAG are overtly acknowledged. The link between receptor plasticity and ecological strength is conceptually strong.

Major comments in the discussion section

Our reply: We thank the reviewer for this important recommendation. We agree that docking alone cannot reliably predict stereochemical discrimination at the level of odorant receptor neurons (ORNs). In the revised manuscript, we have rephrased our discussion more cautiously. Specifically, we replaced statements such as “ORs do not discriminate stereoisomers” with “ORs did not show predicted stereoselectivity under the docking framework used.”

We have also emphasized that single sensillum recordings (SSR) or heterologous expression assays will be required to validate stereoselectivity claims and to determine whether ORNs exhibit more nuanced enantioselectivity. This clarification has been added to the Discussion to ensure that our conclusions remain appropriately framed within the limitations of docking-based predictions. Please see lines 397-399

Our reply: We thank the reviewer for this important clarification. We agree that antennal activation does not imply behavioral attraction, and that our discussion should more clearly separate molecular detectability, neural activation, and behavioral outcomes. In the revised manuscript, we have removed language suggesting that sesquiterpenes may substitute trimedlure and instead emphasize that docking and EAG results demonstrate detectability and peripheral activation only.

We now explicitly state that behavioral assays are required to determine whether these compounds function as field attractants. The discussion has been rephrased to highlight that sesquiterpenes are promising candidates for further testing, but equivalence to trimedlure cannot be inferred without behavioral validation.

Our reply: We agree that while mixed sesquiterpenes could reduce synthesis and purification costs, practical application will depend on formulation stability, release rate, and field longevity. In the revised manuscript, we have added a short paragraph in the Discussion acknowledging these constraints and emphasizing that future work should address release kinetics, oxidative stability, and delivery systems to ensure effective and durable field performance. Please see lines 427-430

Our reply: We thank the reviewer for this comprehensive summary of the main points requiring clarification. In the revised manuscript, we have addressed each of these aspects as follows:

The statistical treatment of docking data has been clarified, with ANOVA reframed as exploratory and docking scores explicitly described as pseudo-replicates used for ranking.
Protein model validation metrics (e.g., energy minimization parameters and structural quality scores) have been added to the Methods to support the reliability of the docking framework.
Inconsistencies in OR docking statistics have been corrected, and figure captions have been revised to match the reported values.
Claims regarding stereoselectivity have been moderated, now stating that ORs did not show predicted stereoselectivity under the docking framework, and emphasizing that SSR or heterologous expression assays are required for validation.
Statements suggesting functional equivalence to trimedlure have been toned down; we now emphasize detectability and antennal activation only, with behavioral assays required to confirm attraction.
The exclusive use of irradiated males has been justified in the Methods and clarified as independent biological replicates.
Language throughout the manuscript has been revised for clarity and caution, and all figure captions have been checked to ensure consistency with the statistical results.

We believe these revisions address the reviewer’s concerns and substantially strengthen the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant topic and provides original findings that are of interest to the readership of Insects and to the Special Issue “Fruit Flies (Diptera: Tephritidae): Behavior, Ecology and Integrated Management”. The study is generally well written and the approach is appropriate.

To further strengthen the manuscript, some clarifications and minor revisions are recommended. In particular, the experimental design should be more clearly stated, as this will help readers better understand the scope and objectives of the study. The Materials and Methods and Results sections would benefit from improved clarity and organization, and from the inclusion of some missing information and figures that are necessary to fully support the main findings.

In addition, a few descriptions of results require correction or clarification to avoid potential misinterpretations. While the Discussion is interesting and well developed, it would benefit from a more explicit focus on the findings of the present study, with clearer links between the results and the relevant literature.

The specific comments provided throughout the manuscript are intended to help improve clarity, consistency, and overall presentation. With these minor revisions, the manuscript will be significantly strengthened.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Our reply: We thank the reviewer for highlighting the importance of a clear description of the experimental design. In the revised manuscript, we have expanded the Methods section to more explicitly state the scope and objectives of the study, ensuring that readers can easily follow the rationale and design of the experiments.

Our reply: We appreciate the reviewer’s suggestion to improve clarity and organization. The Materials and Methods and Results sections have been revised for improved readability, with additional details and figures included where necessary to fully support the main findings. These changes strengthen the presentation and ensure consistency throughout the manuscript.

Our reply: We thank the reviewer for pointing out the need to correct and clarify certain descriptions of results. In the revised manuscript, these sections have been carefully edited to avoid potential misinterpretations, ensuring that the results are presented with precision and transparency.

Our reply: We are grateful for the reviewer’s recommendation to sharpen the Discussion's focus. The revised Discussion now places greater emphasis on the findings of the present study and provides clearer links between our results and the relevant literature. This adjustment ensures that the discussion remains well-grounded and highlights the contribution of our work to the field.

It appears that there is an error in this line; therefore, please revise and modify both sentences accordingly.

Our reply: Done

Please modify this statement to include other soluble receptors. For example,

“...insects use soluble receptors, such as odorant-binding proteins .....”

Our reply: Done

Please add a sentence justifying the experimental design in which only irradiated males were used for the EAG assays.

Our reply: Done

Please revise the sentence describing the absence of molecular models for these Ceratitis capitata OBPs in the UniProt database. For clarity, I suggest first describing the proteins for which molecular models are available in UniProt, and then clearly indicating which OBPs lack molecular models, including a brief explanation of how these models were constructed.

Our reply: We thank the reviewer for this helpful suggestion. In the revised manuscript, we have reorganized the description of molecular models to first list the proteins for which models were directly available in UniProt, and then clearly indicate the OBPs that lacked molecular models. For those OBPs, we now explicitly describe that homology-based modeling was performed, including the templates used and their sequence similarity. This restructuring improves clarity and avoids potential misinterpretation.

Briefly describe the bioinformatic tool used to construct the molecular models for OBPs.

Our reply: We appreciate the reviewer’s recommendation. In the revised manuscript, we have added a brief description of the bioinformatic tool used to construct the OBP molecular models. Specifically, we now state that homology-based models were generated using the SWISS-MODEL server, and their quality was evaluated using GMQE and QMEAN Z-scores. This addition ensures transparency in the modeling approach and strengthens the methodological description.

At this point, the list of compounds should be provided, including the relevant technical or chemical descriptions to be considered.

Our reply: Names of compounds are provided in the supplementary information

Trimedlure isomers were used as a standard reference. Please clarify and explain why each isomer was selected and on what basis a particular isomer was considered “the most active attractant.” Providing this rationale would improve clarity and help readers better interpret the comparative results.

Our reply: The rationale for using Trimedlure isomers as a standard reference and the basis for considering Trimedlure C as the most active attractant was already described in the Introduction. To improve clarity, we have now reinforced this explanation by explicitly stating why each isomer was selected and highlighting the evidence supporting the designation of the most active attractant.

Please expand the description of the physiological state of the males used in the experiments. In particular, it would be important to specify their age at the time of testing, to clarify why the males were irradiated, and to explain why females were not included in the assessments. Additionally, please indicate when the irradiation was performed, the radiation dose applied, and whether gamma or X-ray irradiation was used.

Our reply: The description was made as follows “Irradiated, sterile 5-day-old C. capitata males of the Vienna 8 genetic sexing strain were obtained weekly from the Moscamed mass-rearing facility at Metapa, Chiapas, Mexico. Irradiation was performed at the pupal stage using gamma radiation, following the standard protocol for sterile male production. At 5 days of age, males are sexually mature and physiologically active, making them suitable for electrophysiological assays. Females were not included because the Vienna 8 strain is maintained as a male-only line at the facility. Approximately 100 pupae were placed in 30 × 30 × 40 cm Plexiglas cages with water and food (1:3 hydrolyzed yeast and sugar mix) and maintained at 25 ± 1 °C until adult emergence.”

I consider that Figure 1 would be more appropriately linked to the first result rather than used to describe one of the chemical compounds. I therefore suggest moving the descriptive paragraph to the Materials and Methods section (Section 2.2) and including the figure as supplementary material.

Our reply: The figure was linked to the first result

Please add columns reporting the mean binding energy values for each compound across the 14 OBPs or 4 ORs, together with the corresponding standard errors (SE), in all Supplementary Tables.

Our reply: Done

It is not clear why the binding energy value of this compound is considered a cut-off threshold for classifying a compound as attractant or non-attractant. This criterion should be clearly defined and justified in the Materials and Methods section and then briefly revisited in the Discussion.

Our reply: Done, please see throughout the text

Please clarify whether this value refers to a mean binding energy, and modify this figure and the subsequent ones accordingly.

Our reply: Done

There is no significant difference between the binding energy values of Z-β-farnesene and Trimedlure C; therefore, these values should be described as similar rather than lower.

Our reply: We respectfully disagree with the reviewer, because only Z-β-farnesene presented a significant difference with respect to other compounds

There are other sesquiterpene compounds in the Figure 6, please add a sentence describing the most important results obtained and the statistical significance.

Our reply: Only Z-β-farnesene presented a significant difference with respect to other compounds, including Trimedlure C

There are no letters indicating statistical significance in the figure. In addition, the text above appears to describe no differences among the binding energy values of the compounds and the ORs, while the reported ANOVA results suggest otherwise. Please clarify this discrepancy or revise the figure and text accordingly.

Our reply: We apologize, it was a mistake, and it has been corrected

The text above reports ANOVA values that differ from those corresponding to this figure. Please explain this inconsistency or correct it. The same issue occurs in the following figure.

Our reply: We apologize, it was a mistake, and it has been corrected

If there are no statistically significant differences, it is not appropriate to suggest that some values are higher or lower; instead, they should be described as similar.

Our reply: We apologize, it was a mistake, and it has been corrected

In my opinion, the EAG experiment provides valuable evidence of antennal responses in irradiated C. capitata males; however, this result is not described in the present paragraph. To further strengthen this finding and improve clarity for the reader, it would be helpful to include a figure illustrating the observed response patterns. A representative figure in the main text, accompanied by a brief description of the main results, would enhance the presentation, while the complete dataset and additional details could be provided as supplementary material.

Our reply: The complete dataset was added in the supplementary information

This explanation, together with its reference, should be moved to the Materials and Methods section. In addition, please include a description explaining why more than one isomer was used across the different assays.

Our reply: We apologize for the misunderstanding, only the 1S,2S,4R isomer of Trimedlure C was used in our methodology

Is it possible to perform a study similar to the one presented here focusing on interactions between OBPs and ORs? I suggest discussing this topic in light of the limited information currently available in the literature.

Our reply: We thank the reviewer for this insightful suggestion. We agree that studying the interactions between OBPs and ORs would be highly valuable, especially given the limited information currently available in the literature. However, this topic goes beyond the scope and objectives of the present study, which focused on docking analyses and antennal electrophysiological responses. To acknowledge the reviewer’s point, we have added a sentence in the Discussion highlighting that future research should explore OBP–OR interactions to provide a more comprehensive understanding of olfactory recognition in C. capitata.

It is not clear in this paragraph which results of the present study are being discussed or compared. Please consider rewriting it by explicitly referring to the findings of this work and discussing them in the context of the available literature.

Our reply: We thank the reviewer for this helpful comment. In the revised manuscript, we have rewritten the paragraph to explicitly refer to the findings of the present study and to discuss them in the context of the available literature. We now clarify that our docking and EAG results demonstrated antennal detectability of sesquiterpenes such as α‑copaene and (E,E)‑α‑farnesene, and then contrast these findings with compounds like trans‑β‑damascone and (E,Z)‑3,6‑octadien‑1‑ol, which have been reported in other fruit fly systems but have not yet been evaluated as medfly lures. This restructuring makes clear which results derive from our study and which represent future candidates for testing.

At this point, it would be important to explain why the experimental design of the present study did not include females, and to clarify whether the aim was to use a preliminary approach to select compounds specifically perceived by irradiated males. In addition, please describe possible future bioassays and computational analyses to continue the screening of novel attractants for C. capitata.

Our reply: We thank the reviewer for this thoughtful comment. In the revised manuscript, we have clarified throughout the Methods and Discussion sections why females were not included in the experimental design, noting that the Vienna 8 genetic sexing strain provides only irradiated males. We also explained that the aim was to use a preliminary approach to evaluate compounds specifically perceived by sterile males. In addition, we have added sentences in the Discussion outlining possible future bioassays and computational analyses that could extend the screening of novel attractants for C. capitata. We believe these clarifications address the reviewer’s concerns and improve the transparency of the study design.

I consider that this study does not provide a comprehensive molecular insight. Therefore, the sentence should be modified as follows: “This study provides valuable information regarding the potential interactions between soluble and membrane receptors of C. capitata and known attractants.”

Our reply: Done

I consider that it is necessary to add a sentence highlighting the experimental design, specifically explaining why only irradiated males were used and how this approach could contribute to improving pest monitoring strategies. Is it expected that irradiated males respond differentially to these compounds compared with females and wild (non-irradiated) males?

Our reply: We thank the reviewer for this valuable suggestion. In the revised manuscript, we have clarified in the Methods and Discussion sections why only irradiated males were used, noting that the Vienna 8 genetic sexing strain provides sterile males for experimental purposes and that their antennal responses are physiologically comparable to those of wild-type males. To reinforce this point, we have added a sentence in the Conclusion highlighting that the use of irradiated males reflects the operational context of pest management programs and provides a preliminary approach to identify compounds specifically perceived by males released in the field. We also note that future studies should include females and wild males to assess potential differences in olfactory responses.

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigates the use of molecular docking combined with electrophysiology to identify potential attractants for the Mediterranean fruit fly (Ceratitis capitata), a major global pest. Over 100 compounds, including stereoisomers and sesquiterpenes, were screened against 14 odorant-binding proteins (OBPs) and 4 odorant receptors (ORs). Docking results suggested several sesquiterpenes may interact with OBPs and ORs, and electroantennography (EAG) confirmed that a subset elicited antennal responses. While behavioral effects remain untested, the study demonstrates that computational screening is a useful exploratory tool for prioritizing candidate ligands, supporting future biochemical and behavioral validation for integrated pest management (IPM) strategies. I have several comments and suggestions for the authors to improve the manuscript.

L23. Please provide the author, taxonomic order, and family when the pest is first mentioned.

L35. I think this sentence "Because...." expression is not suitable for an abstract. Just mention your results and “synthesize them without further arguments.

L67. the statement “Insect antennae are covered with tiny hair-like structures called sensilla” is anatomically oversimplified. Antennal sensilla are morphologically diverse and include not only hair-like forms but also peg-, plate-, and pit-like structures. For an entomology journal, a more precise formulation would be appropriate. I suggest the authors rewording as "Insect antennae bear sensory structures known as sensilla, which occur in a variety of morphologies, including hair-like forms."

L93–99. The paragraph could be rephrased for clarity and a more hypothesis-driven tone. For example: “This study investigates whether computational screening can identify structurally diverse attractant-related and semiochemical compounds with potential behavioral relevance in Ceratitis capitata. Predicted interactions between over 100 compounds, including multiple stereoisomers, and 14 odorant-binding proteins (OBPs) and 4 odorant receptors (ORs) are assessed, and the antennal detection of selected candidates is evaluated using electroantennography (EAG). By focusing on ligand prioritization rather than receptor specificity, this approach establishes a foundation for subsequent biochemical validation and behavioral assays.”

L110. you already mentioned this in L102. Please avoid redundant sentences.

L115. please italicize all scientific names!, e.g., L117 and check the manuscript entirely.

L145. Please provide details on the method of irradiation of medfly males and the age of the individuals tested.

L195. 1S,2S,4R-trimedlure C is reported as the most attractive; however, it shows similar attractiveness for most of the tested compounds (see Figure 2). I suggest either avoiding definitive statements like this or clarifying the similarity among the tested attractants.

L213–219. It would be helpful to reference the specific figure panels (e.g., Figure 4a or 4c) so that the text aligns clearly with the figures and is easier for readers to follow.

Figure 7. There appears to be an oversight. The one-way ANOVA indicates significant differences (F = 20.59, P < 0.001), but the means are not labeled with different letters to indicate these differences. It is recommended to add the appropriate letters to clarify which groups differ significantly.

L259. There appears to be a discrepancy between the text and the Figure 9 footnote. In the text, the ANOVA result is reported as F(44, 352) = 2.56, P < 0.001, whereas the figure footnote reports F(3, 52) = 0.004, P = 0.99. It seems that two different analyses may have been performed. The authors should clarify this in the text to avoid confusion for the reader.

L266–272. Please indicate clearly which panel or compartment of Figure 10 is being referenced for each analysis statement to improve clarity and ensure accurate interpretation. For example, does Figure 10a show the results of each compound across the tested concentrations, indicating a dose-dependent trend?

L282-Discussion.The conclusions in the Discussion section should be based on my above comments regarding multiple comparisons. Please review the comments and modify the conclusions accordingly.

L395-401. The authors discuss the use of commercially available sesquiterpene mixtures as attractants. While the points are scientifically reasonable, some phrasing could be improved for clarity and objectivity. For example, the term “astonishing diversity” is subjective and could be replaced with “wide diversity” or “structural diversity.” Similarly, “enhance cost-effectiveness” could be made more precise, e.g., “reduce the costs of pest-monitoring or control programs.” I suggest rephrasing the paragraph to tighten the flow and maintain a neutral, scientific tone while preserving the main message about potential applications and the need for efficient compound acquisition.

L404–411. The statement describing the study as providing “comprehensive molecular insights into the olfactory detection mechanisms” is overstated. The manuscript includes molecular docking studies, but no additional molecular experiments (e.g., protein expression, binding assays, or structural validation) are presented. While docking results combined with limited EAG testing are informative, it would be more accurate to frame the study as computational docking combined with targeted electrophysiological validation, rather than implying broad molecular insights. I suggest revising the wording to reflect the actual scope of the molecular work.

L417–419. The authors state that their findings “emphasize the value of integrating molecular docking with electrophysiological and behavioral studies.” While this wording is reasonable as a forward-looking statement, it should be clarified that behavioral studies were not performed in this manuscript. To avoid misunderstanding, I suggest rephrasing to indicate that behavioral validation is a potential next step rather than part of the current study.

Overall, this study addresses an important question in medfly olfaction, and the combined use of computational screening and electrophysiology is a promising approach. By addressing the points outlined above regarding figure presentation, statistical clarity, methodological details, and precise wording, the manuscript can be further strengthened and will provide a valuable contribution to the field of integrated pest management.

Author Response

Reviewer 3

L23. Please provide the author, taxonomic order, and family when the pest is first mentioned.

Our reply: Done

L35. I think this sentence "Because...." expression is not suitable for an abstract. Just mention your results and “synthesize them without further arguments.

Our reply: Done

L110. you already mentioned this in L102. Please avoid redundant sentences.

Our reply: Done

L115. please italicize all scientific names!, e.g., L117 and check the manuscript entirely.

Our reply: Done

L145. Please provide details on the method of irradiation of medfly males and the age of the individuals tested.

Our reply: Done

L213–219. It would be helpful to reference the specific figure panels (e.g., Figure 4a or 4c) so that the text aligns clearly with the figures and is easier for readers to follow.

Our reply: Figure 4a and 4b were referenced

Our reply: We apologize, it was our mistake, but it has been corrected

Our reply: We thank the reviewer for this helpful comment. In the revised manuscript, we have clarified in the figure legend and text which panel of Figure 10 is being referenced for each analysis statement. Specifically, we now state that Figure 10a shows the results of each compound across the tested concentrations, indicating a dose-dependent trend. Statistical analysis confirmed that concentration had a significant effect, and this has been explicitly noted to ensure accurate interpretation.

L282-Discussion.The conclusions in the Discussion section should be based on my above comments regarding multiple comparisons. Please review the comments and modify the conclusions accordingly.

Our reply: we acknowledge the reviewer for the suggestions, the conclusions and discussions were rewritten based on those valuable comments

Our reply: Thanks for this suggestion, we rephrased the paragraph

Our reply: Done, we modified the paragraph to reflect the scope of the molecular work

Our reply: Done, we rephrased the paragraph

Our reply: We sincerely thank the reviewer for this positive overall assessment of our study. In the revised manuscript, we have carefully addressed all the points outlined regarding figure presentation, statistical clarity, methodological details, and precise wording. We believe these changes have strengthened the manuscript and improved its clarity and consistency. We are grateful for the reviewer’s constructive feedback, which has helped us enhance the quality and impact of our work.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I noticed that my suggested revisions, including the proposed molecular docking figures, were not incorporated into the manuscript. Furthermore, the work in its current form does not present a significant novel contribution.

Author Response

Reviewer 1, round 2

Our reply: We thank the reviewer for the additional feedback. We would like to clarify that in the first round of review, the comments focused on methodological transparency, statistical treatment, and validation metrics, which we have fully incorporated into the revised manuscript. Specifically, we added a supplementary table reporting protein model validation metrics (Ramachandran statistics, GMQE, QMEAN Z-scores, pLDDT values, and MolProbity scores), corrected inconsistencies in docking statistics and figure captions, clarified the statistical treatment of docking data, justified the exclusive use of irradiated males, and moderated the discussion regarding stereoselectivity and functional equivalence to trimedlure.

The request for “molecular docking figures” was not explicitly stated in the initial review. Our current figures present binding energy results and comparative analyses, as suggested. To further strengthen the manuscript and improve clarity, we have now integrated representative graphical docking poses (ligand–protein binding visualizations) as supplementary material. These figures illustrate typical ligand–protein interactions and complement the binding energy analyses presented in the main text.

We believe the revisions already made have strengthened the manuscript, and we remain open to adding docking visualization figures if they would improve clarity for readers.