Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript examines how Calabrian pine responds to prolonged feeding by the pine processionary moth. By tracking terpene concentrations and terpene synthase gene expression over 195 days, it explores whether the tree’s chemical response changes as infestation progresses.

Abstract

The abstract summarizes the 195-day study clearly, but changes attributed to infestation should be described more cautiously in light of the experimental design.

Introduction
2. The introduction provides a thorough account of terpene-based defence in pines and clearly explains the value of examining gene expression alongside metabolite accumulation.

Materials and Methods
3. The main concern is that infested and control plants were kept in one separate mesh-covered enclosure per treatment, making it impossible to distinguish an infestation effect from an enclosure effect (lines 151–155).
4. Individual saplings are biological replicates for sampling, but they do not provide independent replication of the infestation treatment.
5. The authors should clarify whether data from additional, independent control and infested enclosures are available; otherwise, the study must be presented as exploratory.
6. Larval numbers and needle consumption were not quantified, so exposure time cannot be separated from larval development and the intensity of feeding damage.

Results
7. The contrasting temporal patterns of terpenoids and TPS transcripts are interesting, particularly the decline in δ-3-carene and the later accumulation of abietane-type resin acids.
8. However, without independent enclosure replication, the reported treatment-related p-values and PERMANOVA results cannot establish an effect caused by infestation.
9. Given the baseline difference in α-thujene, baseline differences for all measured variables should be presented consistently.

Discussion
10. The shared treatment and time trends may contribute to the transcript–metabolite correlations, which therefore cannot establish direct gene–product relationships.
11. The authors appropriately acknowledge that TPS products and volatile emissions were not measured directly, and this limitation should also be maintained throughout the ecological interpretation.

Conclusions
12. The manuscript reports potentially valuable patterns, but the present design does not demonstrate that the observed changes were specifically caused by the moth.

Author Response

We sincerely thank the Reviewer for the careful and constructive assessment of our work. We fully recognize that the absence of independent enclosure-level replication prevents a formal separation of the PPM condition from a possible enclosure-specific contribution and therefore limits enclosure-level causal inference. At the same time, we respectfully consider that this limitation does not invalidate the biological information provided by the experiment. Several features of the design support the biological relevance of the observations: saplings were randomly assigned to the two groups; the two enclosures were identical, located approximately 10 m apart under the same open-field environmental conditions, covered with the same mesh and managed using the same watering regime; and each infestation-assigned sapling received a separate larval nest collected from a different host tree, so the biological challenge was applied separately at the plant level.

Moreover, most variables did not differ between groups before nest attachment, whereas progressive and compound-specific divergence emerged after larval feeding had begun. Although these features cannot substitute for independent enclosure-level replication or exclude a possible enclosure contribution, they provide temporal and biological coherence consistent with PPM infestation and support the value of the resulting time-course as biologically informative.

We have therefore adopted a balanced interpretation throughout the revised manuscript: we distinguish plant-level biological sampling replication from enclosure-level treatment replication, present the statistical analyses as exploratory, and interpret the findings as associations observed within this experiment rather than as conclusive evidence of effects caused exclusively by PPM infestation. In our opinion, this approach directly addresses the Reviewer’s principal concern regarding the experimental design while preserving the biological relevance of the plant-level evidence generated under semi-natural conditions. Nevertheless, to address the Reviewer’s concerns as fully as possible, we have carefully revised the manuscript and Supplementary Materials in response to all the observations raised, as detailed point by point below. All changes made in response to Reviewer 1 are highlighted in yellow in the revised manuscript and Supplementary Materials.

General assessment

Reviewer comment: This manuscript examines how Calabrian pine responds to prolonged feeding by the pine processionary moth. By tracking terpene concentrations and terpene synthase gene expression over 195 days, it explores whether the tree’s chemical response changes as infestation progresses.

Response: We thank the Reviewer for this accurate summary of the study.

Comment 1 — Abstract

Reviewer comment: The abstract summarizes the 195-day study clearly, but changes attributed to infestation should be described more cautiously in light of the experimental design.

Response: We thank the Reviewer for this careful and constructive observation. We agree that the wording in the Abstract required greater caution in light of the experimental design. Accordingly, we revised the Abstract to describe time-dependent differences between saplings in the control enclosure and those exposed to PPM in the separate infested enclosure. We removed direct causal wording and added an explicit concluding statement that, because experimental condition and enclosure were not independently replicated, the observed differences cannot be attributed exclusively to PPM infestation.

Location of changes: Revised manuscript: p. 1, lines 24–26, 31–35, and 39–40.

Comment 2 — Introduction

Reviewer comment: The introduction provides a thorough account of terpene-based defence in pines and clearly explains the value of examining gene expression alongside metabolite accumulation.

Response: We thank the Reviewer for this positive assessment. We retained the conceptual framework while revising the final aim statement to define the study as exploratory and to refer to differences between experimental groups rather than a definitive PPM-triggered response.

Location of changes: Revised manuscript: p. 3, lines 134–135 and 138–139.

 

Materials and Methods

Comment 3

Reviewer comment: The main concern is that infested and control plants were kept in one separate mesh-covered enclosure per treatment, making it impossible to distinguish an infestation effect from an enclosure effect (lines 151-155).

Response: We thank the Reviewer for this careful and constructive comment. We recognize that the use of one enclosure per experimental condition prevents formal statistical separation of the PPM condition from a possible enclosure-specific contribution, and we have revised the manuscript to state this limitation explicitly. We respectfully note, however, that the experiment incorporated several safeguards against arbitrary pre-existing group differences: saplings were randomly assigned; the two enclosures were identical, approximately 10 m apart, covered with the same mesh and maintained under the same semi-natural conditions and watering regime; and each infestation-assigned sapling received a separate nest collected from a different host tree.

In addition, most variables measured at T0, after the two-week acclimation period and before nest attachment, did not differ significantly between groups (Tables S4–S7), providing limited evidence of widespread pre-existing group differences; progressive and compound-specific divergence emerged after larval feeding had begun.

We acknowledge that baseline similarity cannot substitute for independent enclosure-level replication. Nevertheless, it provides a sound starting point for interpreting the subsequent time-course patterns as biologically informative associations consistent with PPM exposure. To address the Reviewer’s concern fully, we have explicitly stated both the inferential limitation and the relevant biological features of the design in the Materials and Methods, Statistical Analysis, Results, and Discussion, as well as in Figure S2 and the note to Table S9.

Location of changes: Revised manuscript: p. 4, lines 155–159 and 175–190; p. 8, lines 347–353 and 365–369; p. 9, lines 383–393; p. 25, lines 911–917, continuing on p. 26, lines 918–919. Supplementary Materials: p. 4, Figure S2 legend; and p. 18, Table S9 note.

Comment 4

Reviewer comment: Individual saplings are biological replicates for sampling, but they do not provide independent replication of the infestation treatment.

Response: We thank the Reviewer for this important and constructive clarification. In the revised manuscript, we have corrected the terminology throughout and now describe individual saplings as plant-level biological sampling units used to estimate within-enclosure variability, while explicitly stating that they are not independent treatment-level or enclosure-level replicates. At the same time, we respectfully emphasize that these saplings are genuine biological, rather than technical, replicates: each was a distinct plant, and each infestation-assigned sapling received a separate nest originating from a different host tree. This plant-level replication therefore provides relevant information about the variability and consistency of biological responses among saplings within each enclosure, even though it cannot by itself establish an enclosure-independent causal PPM effect. We believe that this distinction appropriately acknowledges the statistical limitation identified by the Reviewer without discounting the biological value of the sampling design.

Location of changes: Revised manuscript: p. 4, lines 182–190; p. 5, lines 201–206, 210–212, and 227–229; p. 7, lines 299–302; p. 8, lines 347–353 and line 359; p. 9, line 377; p. 10, line 420; p. 11, line 452; p. 13, lines 514–515; p. 14, lines 521–522; p. 15, line 534; p. 17, lines 590–591; p. 18, line 603 and lines 617–618; and p. 20, line 651. Supplementary Materials: p. 4, Figure S2 legend; p. 6, Figure S4 legend; p. 7, Figure S5 legend; p. 8, Figure S6 legend; p. 11, Table S2 note; p. 12, Table S3 note; and p. 18, Table S9 note.

Comment 5

Reviewer comment: The authors should clarify whether data from additional, independent control and infested enclosures are available; otherwise, the study must be presented as exploratory.

Response: We thank the Reviewer for raising this important point. No additional independent control or infested enclosures were used, and no data from other enclosures are available. We have now stated this explicitly in Section 2.1 and present the study as exploratory throughout the Abstract, Introduction, Statistical Analysis, Results, and Discussion. The results are therefore interpreted as descriptive temporal and group-related patterns observed within this experiment, rather than as enclosure-independent causal evidence of a PPM infestation effect.

Location of changes: Revised manuscript: p. 1, lines 24–26, 31–35, and 39–40; p. 3, lines 134–135 and 138–139; p. 4, lines 155–159; p. 8, lines 347–353 and 365–369; p. 9, lines 383–393; p. 21, lines 696–708; p. 22, lines 739–753; p. 25, lines 911–917, continuing on p. 26, lines 918–919. Supplementary Materials: p. 4, Figure S2 legend; and p. 18, Table S9 note.

Comment 6

Reviewer comment: Larval numbers and needle consumption were not quantified, so exposure time cannot be separated from larval development and the intensity of feeding damage.

Response: We thank the Reviewer for highlighting this important limitation. We now state in Section 2.1 that larval abundance, larval biomass, needle consumption, and defoliation severity were not quantified. Direct quantification of larval biomass and needle consumption would have required repeated handling or destructive sampling of larvae, nests, or plant material, potentially altering larval development, feeding activity, and the longitudinal sampling protocol. We have also clarified in the Discussion that sampling time integrates exposure duration, larval development, and feeding intensity; therefore, the individual contributions of these factors cannot be separated. These measurements should be incorporated into future experiments specifically designed to separate these factors, as now stated in the Discussion.

 

Location of changes: Revised manuscript: p. 4, lines 167–169; p. 25, lines 866–869; and p. 26, lines 930–935.

Results

Comment 7

Reviewer comment: The contrasting temporal patterns of terpenoids and TPS transcripts are interesting, particularly the decline in δ-3-carene and the later accumulation of abietane-type resin acids.

Response: We thank the Reviewer for highlighting these findings. We agree that the contrasting temporal patterns of terpenoids and TPS transcripts are important. These patterns remain central to the Results, but we now describe them as temporal differences between experimental groups rather than as changes proven to have been caused by PPM.

Location of changes: Revised manuscript: p. 12, lines 485–486 and 497–498; p. 14, lines 527–529; p. 15, lines 537–540, 553–558, and 563–564; p. 16, line 575 and lines 581–583; p. 19, lines 620–625 and 627–628; p. 20, line 655 and lines 664–666; and p. 22, lines 739–747.

 

Comment 8

Reviewer comment: However, without independent enclosure replication, the reported treatment-related p-values and PERMANOVA results cannot establish an effect caused by infestation.

Response: We thank the Reviewer for this important and constructive clarification. We recognize the inferential limitation identified and have revised Section 2.6 to state explicitly that experimental condition and enclosure were not independently replicated, preventing their respective contributions from being statistically separated. The statistical analyses are now explicitly described as exploratory, and the main text and Table S9 note clarify that the reported p-values and PERMANOVA results describe patterns observed within this experiment but cannot establish an enclosure-independent causal effect of PPM infestation. In the Results and Discussion, wording such as ‘confirmed’ and ‘caused’ has been replaced with descriptive terms such as ‘indicated’, ‘showed’, or ‘was higher/lower in the PPM-infested group’.

 

Location of changes: Revised manuscript: p. 8, lines 347–353 and 365–369; p. 9, lines 383–393; p. 21, lines 696–708; and p. 25, lines 870–875. Supplementary Materials: p. 18, Table S9 note.

 

Comment 9

Reviewer comment: Given the baseline difference in α-thujene, baseline differences for all measured variables should be presented consistently.

Response: We thank the Reviewer for this careful observation. We agree that baseline differences should be presented consistently for all measured variables. Tables S4–S7 already contain the 0-DPI contrast for every measured metabolite and transcript. We now summarize these contrasts explicitly in Section 3.1. Four variables differed between groups before nest attachment: α-thujene and bornyl acetate concentrations, and Pnl MTPS5 and Pnl MTPS6 transcript abundances, were lower in the group assigned to subsequent infestation. All DRA and Pnl DTPS contrasts and all remaining monoterpenoid and Pnl MBOS/MTPS contrasts were not significant at baseline.

 

Location of changes: Revised manuscript: p. 11, lines 474–478, continuing on p. 12, lines 479–480. Supplementary Materials: Tables S4–S7, pp. 13–16; the 0-DPI contrast columns were already present and remain unchanged.

 

Discussion

Comment 10

Reviewer comment: The shared treatment and time trends may contribute to the transcript-metabolite correlations, which therefore cannot establish direct gene-product relationships.

Response: We thank the Reviewer for raising this important interpretive point. We agree that shared experimental-condition and time trends may contribute to the transcript–metabolite correlations. Sections 2.6, 3.4, and 4.3 now identify these correlations as exploratory. Because the analyses pool both experimental groups and all sampling times, shared condition and time trends may contribute to the coefficients. In the revised manuscript, we explicitly state that these correlations describe covariation, cannot distinguish shared trends from within-group coupling, and do not establish direct gene–product relationships.

Location of changes: Revised manuscript: p. 9, lines 401–403; p. 22, lines 724–726 and 734–738; and p. 24, lines 829–831 and 855–856.

Comment 11

Reviewer comment: The authors appropriately acknowledge that TPS products and volatile emissions were not measured directly, and this limitation should also be maintained throughout the ecological interpretation.

Response: We thank the Reviewer for this important and constructive comment. We agree that the limitations arising from the lack of direct measurements of TPS products and volatile emissions should be maintained throughout the ecological interpretation. In the revised manuscript, we retained the distinction between internal solvent-extractable pools and airborne emissions throughout the Discussion and removed wording that overstated ecological implications. The overlap with previously reported volatile compounds is now used only to identify candidates for future headspace and behavioral studies. TPS–terpenoid relationships remain explicitly described as putative for monoterpenoids and at the pathway level for DRAs.

Location of changes: Revised manuscript: p. 23, lines 787–788; p. 24, lines 855–856; and p. 26, lines 930–935.

 

Conclusions

Comment 12

Reviewer comment: The manuscript reports potentially valuable patterns, but the present design does not demonstrate that the observed changes were specifically caused by the moth.

Response: We thank the Reviewer for this careful and constructive assessment. We agree that the present design does not support attributing the observed differences exclusively to PPM infestation. To avoid repeating material already presented in the Discussion, the separate Conclusions section has been removed. The principal design limitation is now stated explicitly in the Discussion, which presents the work as an exploratory description of a semi-natural PPM-exposure time-course and interprets the observed patterns as biologically informative associations rather than causal effects. A concise final sentence identifies the relevant terpene branches and compounds as candidates for validation in future independently replicated PPM experiments.

 

Location of changes: Revised manuscript: p. 25, lines 911–917, continuing on p. 26, lines 918–919; the former standalone Conclusions section was removed in full, and the retained concluding sentence now appears on p. 26, lines 940–943.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors demonstrate that Calabrian pine exhibits selective and temporally structured reprogramming of mono- and diterpenoid pathways in response to PPM infestation, rather than a generalized induction of terpene metabolism. The study also identifies specific terpene compounds and associated pathways that warrant further investigation, particularly with respect to volatile emissions and their potential roles in PPM behavior and interactions with natural enemies. Overall, the manuscript is scientifically sound and provides valuable insights into the chemical and molecular responses of Calabrian pine to PPM infestation. However, some minor revisions and clarifications are needed to improve the clarity and presentation of the manuscript, as outlined below:

1. How did the authors determine the sampling time points used in this study? A clear rationale for the selection of these time points would improve the methodological clarity of the manuscript and facilitate reproducibility by other research groups.
2. Figure 1: The relative abundance of individual Pnl MTPS transcripts does not appear to directly correspond to the abundance of their putatively associated monoterpenoid products. For example, Pnl MTPS4 shows lower transcript abundance than Pnl MTPS2, whereas β-pinene, putatively associated with Pnl MTPS4, is more abundant than a-terpineol, putatively associated with Pnl MTPS2. Although the authors appropriately acknowledge that the MTPS-product assignments remain putative, it may be helpful to briefly clarify that differences in transcript abundance among MTPS genes are not necessarily expected to correspond directly to the relative abundance of their associated metabolites, given potential differences in enzyme activity, substrate availability, metabolic flux, product turnover, and post-transcriptional or post-translational regulation. This clarification would help readers interpret the relationship between the transcript and metabolite profiles presented in Figure 1.
3. The Discussion is comprehensive and appropriately acknowledges the limitations of the transcript-metabolite associations. However, Sections 4.3~4.6 contain some overlap in the discussion of selective metabolic reprogramming, temporal responses, ecological interpretation, and the limitations of functional TPS assignments. The authors may consider combining or streamlining overlapping points across these sections and reducing repetition, while retaining the key interpretations, to improve conciseness and strengthen the overall flow of the Discussion.
4. The Conclusion provides a comprehensive summary of the main findings. However, it is relatively detailed and repeats several points already discussed in the Discussion section, particularly the responses of individual terpenes and TPS transcripts, the limitations of TPS-product assignments, and detailed future research directions. The authors may consider shortening this section by focusing on the principal findings, their broader biological significance, and the most important limitations and future directions, thereby providing a more concise and focused conclusion.

 

Author Response

We thank the Reviewer for the positive and constructive assessment. We have revised the manuscript and Supplementary Materials to clarify the biological rationale for the sampling schedule and to explain why transcript and metabolite abundances are not expected to be proportional. In addition, we streamlined the Discussion and removed the standalone Conclusions section because it repeated information already presented in the Discussion. All changes made in response to Reviewer 2 are highlighted in green in the revised manuscript and Supplementary Materials.

 

General assessment

Reviewer comment: In this manuscript, the authors demonstrate that Calabrian pine exhibits selective and temporally structured reprogramming of mono- and diterpenoid pathways in response to PPM infestation, rather than a generalized induction of terpene metabolism. The study also identifies specific terpene compounds and associated pathways that warrant further investigation, particularly with respect to volatile emissions and their potential roles in PPM behavior and interactions with natural enemies. Overall, the manuscript is scientifically sound and provides valuable insights into the chemical and molecular responses of Calabrian pine to PPM infestation. However, some minor revisions and clarifications are needed to improve the clarity and presentation of the manuscript, as outlined below:

Response: We thank the Reviewer for this favourable evaluation and for the focused suggestions, which helped us to improve the methodological clarity and the conciseness of the manuscript.

 

Comment 1

Reviewer comment: How did the authors determine the sampling time points used in this study? A clear rationale for the selection of these time points would improve the methodological clarity of the manuscript and facilitate reproducibility by other research groups.

Response: We thank the Reviewer for raising this important point. We agree that the rationale for the sampling schedule should be stated explicitly. As illustrated in Figure S4, the four sampling times were selected to represent broad phases of the natural developmental and feeding sequence under the conditions of the experimental site: T0 was the pre-feeding baseline immediately before nest attachment; T21 represented early feeding by larvae approximately at the L2–L3 stages; T84 represented winter trophic activity with progressive defoliation; and T195 represented late feeding by L5 larvae before procession. We added this rationale to Section 2.2 and the Figure S4 legend. These stages explain the biological basis of the schedule without implying that exposure duration, larval development, and feeding intensity were quantified independently or can be separated in the present experiment.

 

Location of changes: Revised manuscript: p. 5, lines 196–201. Supplementary Materials: p. 6, Figure S4 legend.

 

Comment 2

Reviewer comment: Figure 1: The relative abundance of individual Pnl MTPS transcripts does not appear to directly correspond to the abundance of their putatively associated monoterpenoid products. For example, Pnl MTPS4 shows lower transcript abundance than Pnl MTPS2, whereas β-pinene, putatively associated with Pnl MTPS4, is more abundant than α-terpineol, putatively associated with Pnl MTPS2. Although the authors appropriately acknowledge that the MTPS-product assignments remain putative, it may be helpful to briefly clarify that differences in transcript abundance among MTPS genes are not necessarily expected to correspond directly to the relative abundance of their associated metabolites, given potential differences in enzyme activity, substrate availability, metabolic flux, product turnover, and post-transcriptional or post-translational regulation. This clarification would help readers interpret the relationship between the transcript and metabolite profiles presented in Figure 1.

Response: We thank the Reviewer for this careful and constructive comment. We agree that this distinction should be made explicit. The Results now characterize the correspondence between basal MTPS transcript and monoterpenoid profiles as qualitative. Section 4.3 further clarifies that transcript and metabolite abundances should not be interpreted as proportional. In addition to multiproduct or functionally overlapping enzymes and incomplete gene–product assignments, gene-specific translation and enzyme activity, substrate availability and metabolic flux, product turnover and storage, and post-transcriptional or post-translational regulation can decouple transcript abundance from steady-state metabolite levels.

 

 

Location of changes: Revised manuscript: p. 10, lines 435–436; and p. 24, lines 831–837.

Comment 3

Reviewer comment: The Discussion is comprehensive and appropriately acknowledges the limitations of the transcript-metabolite associations. However, Sections 4.3–4.6 contain some overlap in the discussion of selective metabolic reprogramming, temporal responses, ecological interpretation, and the limitations of functional TPS assignments. The authors may consider combining or streamlining overlapping points across these sections and reducing repetition, while retaining the key interpretations, to improve conciseness and strengthen the overall flow of the Discussion.

Response: We thank the Reviewer for this careful and constructive suggestion. We agree and have substantially streamlined this part of the Discussion. Former Sections 4.3 and 4.4 were merged into a single revised Section 4.3, ‘Transcript-metabolite coordination and temporal organization’, and the subsequent sections were renumbered. We also removed a repetitive cross-system comparison, condensed the discussion of functional TPS validation, and shortened the future and applied perspectives. The main interpretations and key limitations, including the lack of independent enclosure-level replication, the distinction between internal pools and volatile emissions, the putative nature of TPS assignments, and the need for quantitative entomological measurements, were retained.

 

Location of changes: Revised manuscript: p. 24, line 821, line 839, lines 856–857, and lines 860–863; and p. 26, lines 928–930.

Comment 4

Reviewer comment: The Conclusion provides a comprehensive summary of the main findings. However, it is relatively detailed and repeats several points already discussed in the Discussion section, particularly the responses of individual terpenes and TPS transcripts, the limitations of TPS-product assignments, and detailed future research directions. The authors may consider shortening this section by focusing on the principal findings, their broader biological significance, and the most important limitations and future directions, thereby providing a more concise and focused conclusion.

Response: We thank the Reviewer for this important and constructive suggestion. We agree that the former Conclusions section was relatively detailed and repeated information already presented in the Discussion. We therefore removed the standalone Conclusions section in full because, in our view, it did not add substantive information beyond the Discussion. A single concise sentence was retained at the end of Section 4.5 to conclude the Discussion by identifying the relevant terpene branches and candidate compounds for validation in independently replicated PPM experiments.

Location of changes: Revised manuscript: the former standalone Conclusions section was removed in full; the retained concluding sentence appears on p. 26, lines 940–943.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This present form is suitable for publishing. Thank you!