A Multi-Year Study on the Presence and Infestation Levels of Cameraria ohridella and Guignardia aesculi on Horse Chestnut in North-Eastern Italy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper presents the results of high-quality and significant research. However, there are some misprints in the manuscript. Some illustrations are difficult to understan. See the attached file. 

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Comments

The paper presents the results of high-quality and significant research. However, there are some misprints in the manuscript. Some illustrations are difficult to understan. See the attached file. 

 

Authors' answer

The answers are reported in the attached pdf file where the reviewer inserted his comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction section

This section presents a comprehensive overview of the biological invasion of the horse-chestnut leaf miner, Cameraria ohridella, yet several aspects require elaboration and clarity to support a robust scientific foundation for the study. Here are a few comments for the authors to consider:

1. Clearly state hypotheses and objectives. This section sets the stage by detailing the spread and impact of C. ohidellaon Aesculus hippocastanum, originating from the Balkans and expanding across Europe, including Italy. However, it still lacks clearly stated hypothesis and explicitly outlined research objectives, which are crucial in framing the study. 

2. Explain the long-term approach. While some publications spend 7 years to investigate the influence of leaf damage by C. ohridella on Aesculus hippocastanum*, this study spend multi-decade time frame for an extensive data collection, which is a major strength. If the authors can thoroughly explain why they conducted a multi-year study in the Introduction section, that can help readers understand the importance of this study (to increase research impact).

*reference: Tyburska-Woś J, Nowak K, Kieliszewska-Rokicka B. Influence of leaf damage by the horse chestnut leafminer (Cameraria ohridella Deschka & Dimić) on mycorrhiza of Aesculus hippocastanum L. Mycorrhiza. 2019 Jan;29:61-7.

3. Address gaps in recent research. While this section references historical spread and impact assessments, it overlooks recent studies concerning the genetic variability and adaptive mechanism of C. ohidella in new environments. Highlighting recent insights into its genetic makeup or dispersal mechanisms would bolster the paper’s relevance and connection to current scientific dialogues. Here’s one paper as an example, Łaszczyca P, Nakonieczny M, Kędziorski A, Babczyńska A, Wiesner M. Towards understanding Cameraria ohridella (Lepidoptera: Gracillariidae) development: effects of microhabitat variability in naturally growing horse-chestnut tree canopy. International Journal of Biometeorology. 2021 Oct;65:1647-58.

4. Address logical gap in rationale. This section notes that native predators and parasitoids don’t keep C. ohidella below damaging levels (Lines 61-65), but it doesn’t fully explain why the study’s focus on (i) multi-year damage progression, (ii) leaf removal, altitude, climate, G. aesculin, and (iii) abamectin injections follows from that fact. Show how investigating these factors directly addresses the shortcomings of natural enemy control and leads to more efficient management strategies. 

 

Materials and Methods section

1. Sampling protocol consistency. The leaf damage classification scheme (classes 1 to 10) is adequately explained, and it was good to know that “samplings were always conducted by the same two members of the research team” (Line 106). But it would be helpful to note whether the same trees (or same subset of trees) were monitored repeatedly, or whether new trees were selected in some years within this multi-decade time frame. It is commonly understandable that long-term data are susceptible to changes in tree selection if not carefully standardized.

2. Replication details. Please indicate how many replicates (tree per site) were used each sampling year. For instance, sampling “5 branches from 5 trees” is mentioned in some places; clarify whether this was consistent across all sites and years.

3. Statistical analyses. 1) Multiple regression rationale: Please justify why rainfall, altitude, temperature, etc. were included together. For instance, the authors note that altitude and temp are strongly correlated. The final approach (excluding one variable in favor of the other) is logical but should be emphasized up front. 2) Model diagnostics: Please consider providing brief information on checking assumptions (normality of residuals, homoscedasticity) for the multiple regressions.

 

Discussion and Conclusion sections

The finding that abamectin can remain lethal for ~19 years is striking. However, the manuscript also states that residues were not chemically detected. If the authors can discuss the mechanisms of the extremely prolonged effect of abamectin, that could better highlight their novel findings- since that sets this study apart from routine “altitude/temperature effect” findings. 

The European Union has implemented bans and restrictions on outdoor use of certain neonicotinoids due to their impact on pollinators. While abamectin was previously approved for use as a biocide in the European Economic Area and Switzerland, this approval has now expired*. There is ongoing legal and regulatory review regarding abamectin. This research finding could strongly evidence the long-term environment impact due to the abamectin applications and be informative for the European Food Safety Authority or the EU to better assess scientific arguments related to its approval. Therefore, the reviewer highly suggests incorporating a discussion on the current insecticide regulations to further strengthen the authors’ findings and recommendations with a broader impact.

*https://echa.europa.eu/substance-information/-/substanceinfo/100.113.437

Author Response

Reviewer 2

 

Introduction section

This section presents a comprehensive overview of the biological invasion of the horse-chestnut leaf miner, Cameraria ohridella, yet several aspects require elaboration and clarity to support a robust scientific foundation for the study. Here are a few comments for the authors to consider:

1. Clearly state hypotheses and objectives. This section sets the stage by detailing the spread and impact of C. ohidellaon Aesculus hippocastanum, originating from the Balkans and expanding across Europe, including Italy. However, it still lacks clearly stated hypothesis and explicitly outlined research objectives, which are crucial in framing the study.

Answer: see the new version of the study objectives, including the hypotheses.

 

2. Explain the long-term approach. While some publications spend 7 years to investigate the influence of leaf damage by C. ohridellaon Aesculus hippocastanum*, this study spend multi-decade time frame for an extensive data collection, which is a major strength. If the authors can thoroughly explain why they conducted a multi-year study in the Introduction section, that can help readers understand the importance of this study (to increase research impact).

*reference: Tyburska-Woś J, Nowak K, Kieliszewska-Rokicka B. Influence of leaf damage by the horse chestnut leafminer (Cameraria ohridella Deschka & Dimić) on mycorrhiza of Aesculus hippocastanum L. Mycorrhiza. 2019 Jan;29:61-7.

Answer

The study was conducted over many years because the trees treated with abamectin remained uninfested or only slightly infested many years after treatment and we want to see whether after many years the infestation decreased in relation to the hypothesis of an increase in biological control. In this context, we also wanted to understand whether the long-term effect was on egg-laying or newly-hatched larvae. We added a new sentence in the introduction as suggested by the reviewer.

We added also the suggested reference in the introduction.

3. Address gaps in recent research. While this section references historical spread and impact assessments, it overlooks recent studies concerning the genetic variability and adaptive mechanism of C. ohidella in new environments. Highlighting recent insights into its genetic makeup or dispersal mechanisms would bolster the paper’s relevance and connection to current scientific dialogues. Here’s one paper as an example, Łaszczyca P, Nakonieczny M, Kędziorski A, Babczyńska A, Wiesner M. Towards understanding Cameraria ohridella (Lepidoptera: Gracillariidae) development: effects of microhabitat variability in naturally growing horse-chestnut tree canopy. International Journal of Biometeorology. 2021 Oct;65:1647-58.

Answer

We added a study on genetic variability of Cameraria ohridella in the Introduction (Kirichenko et al., 2023). About the suggested paper ( Łaszczyca et al., 2021), we don't know how to fit it into the context of our study.

 

4. Address logical gap in rationale. This section notes that native predators and parasitoids don’t keep C. ohidella below damaging levels (Lines 61-65), but it doesn’t fully explain why the study’s focus on (i) multi-year damage progression, (ii) leaf removal, altitude, climate, G. aesculin, and (iii) abamectin injections follows from that fact. Show how investigating these factors directly addresses the shortcomings of natural enemy control and leads to more efficient management strategies.

Answer

The part on the aims was rewritten.

 

Materials and Methods section

1. Sampling protocol consistency. The leaf damage classification scheme (classes 1 to 10) is adequately explained, and it was good to know that “samplings were always conducted by the same two members of the research team” (Line 106). But it would be helpful to note whether the same trees (or same subset of trees) were monitored repeatedly, or whether new trees were selected in some years within this multi-decade time frame. It is commonly understandable that long-term data are susceptible to changes in tree selection if not carefully standardized.

Answer: always the same plants were sampled. We added this information in the manuscript.

 

2. Replication details. Please indicate how many replicates (tree per site) were used each sampling year. For instance, sampling “5 branches from 5 trees” is mentioned in some places; clarify whether this was consistent across all sites and years.

Answer: The five branches were collected from 5 different trees, when the number of plants in the site was equal to or greater than five. If the number of plants was less than 5, multiple branches per tree were considered. We added this information in the manuscript.

 

3. Statistical analyses. 1) Multiple regression rationale: Please justify why rainfall, altitude, temperature, etc. were included together. For instance, the authors note that altitude and temp are strongly correlated. The final approach (excluding one variable in favor of the other) is logical but should be emphasized up front. 2) Model diagnostics: Please consider providing brief information on checking assumptions (normality of residuals, homoscedasticity) for the multiple regressions.

Answer: In the regression analyses, altitude and temperature were not considered together (see Tables 1 and 2), precisely because they are strictly correlated, as indicated in the Materials and Methods at lines 136-137 of the version read by the reviewer. In Materials and Methods we wrote that, before performing multiple regression analyses, assumptions were checked.

 

 

Discussion and Conclusion sections

The finding that abamectin can remain lethal for ~19 years is striking. However, the manuscript also states that residues were not chemically detected. If the authors can discuss the mechanisms of the extremely prolonged effect of abamectin, that could better highlight their novel findings- since that sets this study apart from routine “altitude/temperature effect” findings.

Answer: In the conclusions we reiterated the novelty of this data.

 

The European Union has implemented bans and restrictions on outdoor use of certain neonicotinoids due to their impact on pollinators. While abamectin was previously approved for use as a biocide in the European Economic Area and Switzerland, this approval has now expired*. There is ongoing legal and regulatory review regarding abamectin. This research finding could strongly evidence the long-term environment impact due to the abamectin applications and be informative for the European Food Safety Authority or the EU to better assess scientific arguments related to its approval. Therefore, the reviewer highly suggests incorporating a discussion on the current insecticide regulations to further strengthen the authors’ findings and recommendations with a broader impact.

Answer: We added a sentence on this aspect in Discussion.

*https://echa.europa.eu/substance-information/-/substanceinfo/100.113.437

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “A Multi-Year Study on the Factors Influencing Cameraria ohridella Infestation Levels on Horse Chestnut” is an important study that explores the monitoring of Aesculus hippocastanum in a multi-year study (1997–2020). The research focuses on Cameraria ohridella and Guignardia aesculi, conducted at altitudes ranging from 200 to 1300 m a.s.l. in north-eastern Italy. However, the manuscript requires major revisions before it can be considered for publication.The title does not fully reflect the scope of your work. Consider revising it to: “A Multi-Year Study on the Presence and Infestation Levels of Cameraria ohridella and Guignardia aesculi on Horse Chestnut in North-Eastern Italy.”

·      In the abstract, include the family and order names for Cameraria ohridella.

·      Specify whether data were recorded during the study; if so, ensure the title reflects this.

·      Mention the economic significance of both Cameraria ohridella and Guignardia aesculi.

·      Add a brief statement on how temperature and rainfall data were recorded.

·      Provide values or ranges for the abiotic factors analyzed.

·      Discuss whether parameters for inter-specific competition were assessed.

·      Replace Google Maps with GIS-based maps to highlight the study areas, particularly where both C. ohridella and G. aesculi coexist.

·      Address why data recording was not consecutive and cite references for sampling techniques.

·      Ensure all scientific terms, such as "eggs," are precisely written.

·      Provide more details about the statistical analysis methods used.

·      Make the figures self-explanatory and include standard errors (SE) and statistical notes where applicable.

·      In the introduction, include economic loss values (in percentages) caused by C. ohridella.

·      Add detailed information on management practices for C. ohridella and the economic impact of the fungus G. aesculi.

·      Discuss the damage patterns of the fungus, current management practices in the study area, and its competition with C. ohridella based on previous studies.

·      Propose the best solution for managing both pests, supported by your hypothesis.

·      Clearly explain why abamectin was chosen over other insect growth regulators (IGRs).

·      Include ranges for abiotic factors such as temperature and rainfall.

·      While the discussion is thorough, avoid concluding that host plants should not be planted due to abiotic factors. Instead, propose alternative solutions based on your findings.

 

·      Summarize the main conclusions of your study and provide actionable recommendations for future research or management strategies.

Please check annotated pdf file

Comments for author File: Comments.pdf

Author Response

Reviewer 3

 

The manuscript entitled “A Multi-Year Study on the Factors Influencing Cameraria ohridella Infestation Levels on Horse Chestnut” is an important study that explores the monitoring of Aesculus hippocastanum in a multi-year study (1997–2020). The research focuses on Cameraria ohridella and Guignardia aesculi, conducted at altitudes ranging from 200 to 1300 m a.s.l. in north-eastern Italy. However, the manuscript requires major revisions before it can be considered for publication.The title does not fully reflect the scope of your work. Consider revising it to: “A Multi-Year Study on the Presence and Infestation Levels of Cameraria ohridella and Guignardia aesculi on Horse Chestnut in North-Eastern Italy.”

 

Authors' answers to the reviewer's comments.

1. Unfortunately, it is not possible to expand the text of the abstract as there is a maximum limit of 200 words. However, some of the requests have been included in the new version of the abstract.
2. Since the comments below are also listed in the PDF file, we respond to the comments in the PDF file.

In the abstract, include the family and order names for Cameraria ohridella. Answer: see PDF file.

Specify whether data were recorded during the study; if so, ensure the title reflects this. Answer: see PDF file.

Mention the economic significance of both Cameraria ohridella and Guignardia aesculi. Add a brief statement on how temperature and rainfall data were recorded. Answer: see PDF file.

Provide values or ranges for the abiotic factors analyzed. Answer: see PDF file.

Discuss whether parameters for inter-specific competition were assessed. Answer: see PDF file.

Replace Google Maps with GIS-based maps to highlight the study areas, particularly where both C. ohridella and G. aesculi coexist. Answer: see PDF file.

Address why data recording was not consecutive and cite references for sampling techniques. Answer: see PDF file.

Ensure all scientific terms, such as "eggs," are precisely written. Answer: see PDF file.

Provide more details about the statistical analysis methods used. Answer: see PDF file.

Make the figures self-explanatory and include standard errors (SE) and statistical notes where applicable. Answer: see PDF file.

In the introduction, include economic loss values (in percentages) caused by C. ohridella. Answer: see PDF file.

Add detailed information on management practices for C. ohridella and the economic impact of the fungus G. aesculi. Answer: see PDF file. Answer: see PDF file.

Discuss the damage patterns of the fungus, current management practices in the study area, and its competition with C. ohridella based on previous studies. Answer: see PDF file.

Propose the best solution for managing both pests, supported by your hypothesis. Answer: see PDF file.

Clearly explain why abamectin was chosen over other insect growth regulators (IGRs). Answer: see PDF file.

Include ranges for abiotic factors such as temperature and rainfall. Answer: see PDF file.

While the discussion is thorough, avoid concluding that host plants should not be planted due to abiotic factors. Instead, propose alternative solutions based on your findings. Answer: see PDF file.

Summarize the main conclusions of your study and provide actionable recommendations for future research or management strategies. Answer: see PDF file.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I am writing to express concerns regarding the manuscript titled a Multi-year Study on the Factors Influencing Cameraria ohridella Infestation Levels on Horse.. Despite previous comments, several critical issues remain unaddressed, particularly the absence of standard error bars in the figures, which is essential for accurately interpreting the data presented. Additionally, other comments provided were not adequately addressed, raising concerns about the overall rigor and transparency of the study. Given these unresolved issues, it is recommended that the manuscript should be rejected.

Author Response

I am writing to express concerns regarding the manuscript titled a Multi-year Study on the Factors Influencing Cameraria ohridella Infestation Levels on Horse.. Despite previous comments, several critical issues remain unaddressed, particularly the absence of standard error bars in the figures, which is essential for accurately interpreting the data presented. Additionally, other comments provided were not adequately addressed, raising concerns about the overall rigor and transparency of the study. Given these unresolved issues, it is recommended that the manuscript should be rejected.

 

Answer

Although the indication is to reject the manuscript, we respond to the only explicit comment regarding the lack of standard error in the figures.

Figure 1 does not present a comparison between treatments but simply the trend over time of the percentage of locations with the presence of Cameraria ohridella and therefore cannot be a standard error.

In figure 3, a comparison among three percentages is shown (Ryan's test is a test based on chi square for more than three treatments) and not among three means and therefore there cannot be a standard error.

We are unable to answer any further questions as it is not specified which other comments we have not answered satisfactorily.