Sensitivity of Yponomeuta padella and Yponomeuta cagnagella (Lepidoptera: Yponomeutidae) to a Native Strain of Steinernema feltiae (Filipjev, 1934)
, Dorota Tumialis 1,*, Lidia Florczak 1, Barbara Zajdel 2 and Iwona Skrzecz 3Round 1
Reviewer 1 Report (New Reviewer)
These are my main comments on the manuscript (Agriculture-2517508) entitled “Sensitivity of Yponomeuta padella and Yponomeuta cagnagella (Lepidoptera: Yponomeutidae) to a Native Strain of Steinernema feltiae (Filipjev, 1934)”. This work investigates the effects in Yponomeuta species to Steinernema feltiae under laboratory and field conditions. Following substantial revisions should be incorporated in the manuscript prior to acceptance.
1. I have concerns about the manuscript sections that I believe need to be addressed in order to improve its clarity.
2. A hypothesis for this study is needed.
3. In results section, data should be analyzed with Student test, without this the manuscript cannot published.
4. Other revisions could be checked in PDF attached.
Comments for author File: 
Comments.pdf
Moderate editing of English language required
Author Response
Dear Reviewer,
We would like to thank for remarks and suggestions that improve our paper. All changes in the manuscript are marked with using the "Track Changes" function. The manuscript has been edited by native speaker and all editorial errors were corrected. Below we provide the detailed answers to the comments:
1: „A hypothesis for this study is needed”.
.Answer: Suggestion taken into account.
2: „In results section, data should be analyzed with Student test, without this the manuscript cannot published”.
Answer: Suggestion taken into account.
3: „More information about methods in this study is needed”.
Answer: added
4: „Keywords should be in alphabetic order. Also, keywords serve to widen the opportunity to be retrieved from a database. To put words that already are into title and abstracts makes KW not useful. Please choose terms that are neither in the title nor in abstract.”
Answer: Suggestion taken into account.
5: „Summarize and combine these sentences”
Answer: Suggestion taken into account.
6: „Combine these sentences”
Answer: Suggestion taken into account.
7: „Rephrase”: The meteorological data for the study area for the dates of application and the following seven-day period after application are given in Table 1.
Answer: corrected
Reviewer 2 Report (New Reviewer)
Abstract: The abstract provides a clear and concise summary of the study, outlining the objectives, methodology, key findings, and conclusions.
I-Introduction section: The introduction section presents a comprehensive overview of the study's objectives and background information. It effectively introduces the problem of ermine moths (Yponomeuta spp.) as pests in fruit and ornamental trees, emphasizing the need for alternative pest control methods. The introduction discusses the limitations of chemical insecticides and the potential of biological control agents, specifically entomopathogenic nematodes, as a promising alternative. It also highlights the challenges of controlling Yponomeuta moths and the lack of practical applications for natural enemies identified thus far. The section concludes by stating the specific objectives of the study, which focuses on assessing the effectiveness of Steinernema feltiae against Y. padella and Y. cagnagella larvae and pupae through laboratory and field experiments.
Suggestions for improvement:
1. Clearly state the study's novelty: While the introduction provides background information and reviews previous studies, it could benefit from a clearer statement about the novelty or unique contribution of the present study.
2. Clarify the study's scope: The introduction mentions specific locations in Poland, but it would be helpful to specify the geographic scope more explicitly to provide context for readers outside of Poland.
II-Materials and Methods: The section provides a clear description of the entomopathogenic nematode used (Steinernema feltiae ZAG15) and its isolation process. The methodology for conducting laboratory tests on Y. padella and Y. cagnagella larvae and pupae is well-defined, including the use of Petri dish tests and container tests. The dosage of nematodes and control groups are clearly stated. The statistical analysis is mentioned, indicating the use of one-way ANOVA tests.
Suggestion for improvement:
1. Provide more details on the sample size: While the number of replicates is mentioned, it would be helpful to provide more information on the sample size for each experimental group to ensure statistical validity.
III-Results: The results section presents the findings of the laboratory tests and field trials. The mortality rates of Y. padella and Y. cagnagella larvae and pupae are reported for both Petri dish and container tests. The results indicate higher mortality rates for Y. padella compared to Y. cagnagella in the laboratory tests. However, the effectiveness of the nematode application in the field trials is not demonstrated, as the survival rate of larvae and pupae remains at 100%.
Suggestion for improvement:
1. Provide more information on the field trial results: The section briefly mentions the field trials but does not provide specific results or observations. Adding details on the outcomes of the field trials would enhance the comprehensiveness of the study.
IV-Discussion: The discussion section interprets the findings of the study, comparing them with previous research and addressing the limitations and implications of the results. It discusses the effectiveness of entomopathogenic nematodes against Yponomeuta moths, the challenges encountered in field trials, and the potential for further research on biological control methods.
Suggestions for improvement:
1. Elaborate on the implications: While the discussion addresses the limitations and potential future research directions, it could further elaborate on the broader implications of the findings and their practical significance for pest control strategies.
2. Address the lack of field trial results: Since the field trial results were not provided in detail, it would be beneficial to acknowledge this limitation explicitly and discuss the impact it has on drawing conclusive findings.
V-Conclusion: The conclusion section summarizes the study's objectives, methodology, and key findings. It restates the higher susceptibility of Y. padella to entomopathogenic nematodes in laboratory tests but acknowledges the limitations in demonstrating their effectiveness in field trials. The conclusion also reiterates the need for further research on biological control methods for Yponomeuta moths.
Suggestion for improvement:
1. Connect the conclusion to the broader context: While the conclusion highlights the need for further research, it could benefit from further connecting the study's findings to the broader context of pest control strategies and the potential impact on agricultural or ecological systems.
Author Response
Dear Reviewer,
We would like to thank for remarks and suggestions that improve our paper. All changes in the manuscript are marked with using the "Track Changes" function. Below we provide the detailed answers to the comments:
Introduction section, 1: „Clearly state the study's novelty: While the introduction provides background information and reviews previous studies, it could benefit from a clearer statement about the novelty or unique contribution of the present study.”
Answer: Suggestion taken into account.
2: „Clarify the study's scope: The introduction mentions specific locations in Poland, but it would be helpful to specify the geographic scope more explicitly to provide context for readers outside of Poland.”
Answer: We do not understand this suggestion, since we have indicated that the investigation was carried out in Warsaw and have given the geographical coordinates. We added: “Warsaw - Central Poland”.
Materials and Methods, 1: „Provide more details on the sample size: While the number of replicates is mentioned, it would be helpful to provide more information on the sample size for each experimental group to ensure statistical validity.”
Answer: Sample size n=60 is given in manuscript (line121).
Results, 1: „Provide more information on the field trial results: The section briefly mentions the field trials but does not provide specific results or observations. Adding details on the outcomes of the field trials would enhance the comprehensiveness of the study.”
Answer: Suggestion taken into account.
Discussion, 1: „Elaborate on the implications: While the discussion addresses the limitations and potential future research directions, it could further elaborate on the broader implications of the findings and their practical significance for pest control strategies.”
Answer: Suggestion taken into account.
2: „Address the lack of field trial results: Since the field trial results were not provided in detail, it would be beneficial to acknowledge this limitation explicitly and discuss the impact it has on drawing conclusive findings.”
Answer: Suggestion taken into account.
Conclusion, 1: „Connect the conclusion to the broader context: While the conclusion highlights the need for further research, it could benefit from further connecting the study's findings to the broader context of pest control strategies and the potential impact on agricultural or ecological systems.”
Answer: Suggestion taken into account.
Round 2
Reviewer 2 Report (New Reviewer)
The authors have addressed all the requests. In my opinion, the manuscript could be published in the present form.
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
Review report
Manuscript ID: agriculture-2426029
Sensitivity of Yponomeuta padella and Yponomeuta cagnagella (Lepidoptera: Yponomeutidae) to native strains of Steinernema feltiae (Filipjev, 1934)
This study focused on the larvae and pupae of Yponomeutidae, a moth family that feeds on fruit and ornamental trees. Certain species within this family pose a significant threat to the environment due to defoliation and the use of insecticides for their control. The researchers aimed to assess the sensitivity of Yponomeuta padella and Y. cagnagella larvae and pupae to the Steinernema feltiae Zag 15 native strain nematode in laboratory conditions. They also conducted field studies to test the effectiveness of these nematodes against ermine moth larvae and pupae. The results indicated that Y. padella showed higher susceptibility to the nematodes (81.7% larvae, 88.3% pupae) compared to Y. cagnagella (50% larvae, 33.3% pupae). However, despite promising laboratory outcomes, the application of nematodes in the field trials proved to be ineffective.
Comments:
Introduction
P1: Imagines fly out in summer [5, 6, 7, 8, 9.
Please correct to: Imagines fly out in summer [5-9].
P2: …under laboratory conditions [11, 16, 17, 18]
Please correct to: …under laboratory conditions [11, 16-18]
P2: Entomophilous nematodes of the genera Stei[1]nernema and Heterorhabditis were extensively used as biological means against pest insects [20, 21, 22].
Please correct to: Entomophilous nematodes of the genera Stei[1]nernema and Heterorhabditis were extensively used as biological means against pest insects [20-22].
Materials and methods
Please explain how did you prepair the dose of nematodes. Dilution method? If so, please refer to Jagodič et al. 2017.
Jagodič, A., Ipavec, N., Trdan, S., Laznik, Ž., 2017. Attraction behaviours: are synthetic volatiles, typically emitted by insect-damaged Brassica nigra roots, navigation signals for entomopathogenic nematodes (Steinernema and Heterorhabditis)? BioControl 62, 515-524.
P3:…applied on filter paper at a dose of 50 IJs/1 insect. Please correct 50 IJs/insect.
Overall Recommendation: Accept after minor revision (corrections to minor methodological errors and text editing)
Author Response
We have considered all suggestions given by the Reviewers. All changes in the manuscript are marked with using the "Track Changes" function.
Below we provide the detailed answers to the comments:
P1: Imagines fly out in summer [5, 6, 7, 8, 9.
„Please correct to: Imagines fly out in summer [5-9].”
Answer: corrected
P2: …under laboratory conditions [11, 16, 17, 18]
„Please correct to: …under laboratory conditions [11, 16-18]”
Answer: corrected
P2: Entomophilous nematodes of the genera Steinernema and Heterorhabditis were extensively used as biological means against pest insects [20, 21, 22.
„Please correct to: Entomophilous nematodes of the genera Steinernema and Heterorhabditis were extensively used as biological means against pest insects [20-22].”
Answer: corrected
Materials and methods
„Please explain how did you prepair the dose of nematodes. Dilution method? If so, please refer to Jagodič et al. 2017.”
Answer: Suggestion taken into account.
P3:…applied on filter paper at a dose of 50 IJs/1 insect.
„Please correct 50 IJs/insect.”
Answer: corrected
Reviewer 2 Report
Comments to the Authors
The introduction part of the manuscript is effectively structured, providing a thorough explanation of the reasons behind conducting this study. The introduction section provides a general overview of entomopathogenic nematodes (EPN). However, the study focuses on a specific strain of these pathogens, namely S. feltiae Zag 15. Therefore, additional specific information regarding this particular strain is required.
In paragraph 2, lines 10-17, it is my opinion that providing the commercial names of the pesticides is unnecessary. Instead, the argument could be generalized by only mentioning the active ingredients of the pesticides.
The first two sentences in this section should be rephrased for better understanding of where the EPN was isolated from.
It would be appropriate to provide brief information about the identification of the moth species used in the experiments. Regarding the bioassay trials, it should be explained how the dosage of EPN used in the experiments was determined and based on what criteria. In the experiments, only the measurements of larval size have been provided, without specific information about their stages. If there is any information regarding the correlation between larval size and stages, it should be referenced. It should also be explained why the studies were conducted at a temperature of 20°C instead of 25°C. Although information has been provided about the obtaining of larvae, there is no explanation about how the pupae used in the experiments were obtained. This topic has not been addressed. Providing answers to these questions will help improve the understanding of the Materials and Methods section.
Page 3, (2.3.2 Container Test, line 4): It was previously stated that a dose of 50 IJs was used for larvae and pupae in the experiments. However, in this section, the nematode suspension is reported as 100 IJs/insect. This issue should be clarified. Furthermore, it would be appropriate to provide an explanation for why there was a need for such an experiment (container experiments)?"
Do the authors have any information about the stages of the larvae in the pods in field trials? Additionally, were there webs in these pods due to species' web-building behavior, and if so, how did EPN (Entomopathogenic nematodes) reach the larvae within the web? Furthermore, a more detailed explanation of the methodology used in studies related to the pupal stage is needed. Since pupae are generally found in protected places, what approach was followed to deliver EPN to them?
My other comments regarding the manuscript are provided in the text.
Comments for author File: Comments.pdf
Minor editing of English language required.
Author Response
We have considered all suggestions given by the Reviewers. All changes in the manuscript are marked with using the "Track Changes" function.
Below we provide the detailed answers to the comments:
- „….However, the study focuses on a specific strain of these pathogens, namely S. feltiae Zag 15. Therefore, additional specific information regarding this particular strain is required.”
Answer: We belive we covered the most important information about the S. feltiae Zag 15 isolate relevant to this manuscript in section 2. Materials and methods, 2. 1. Entompathogenic nematodes. Can you please clarify what other information the Reviewer would like to see included?
- „In paragraph 2, lines 10-17, it is my opinion that providing the commercial names of the pesticides is unnecessary. Instead, the argument could be generalized by only mentioning the active ingredients of the pesticides.”
Answer: Suggestion taken into account.
- „The first two sentences in this section should be rephrased for better understanding of where the EPN was isolated from.”
Answer: Suggestion taken into account.
- „It would be appropriate to provide brief information about the identification of the moth species used in the experiments.”
Answer: Suggestion taken into account.
- „Regarding the bioassay trials, it should be explained how the dosage of EPN used in the experiments was determined and based on what criteria.”
Answer: Suggestion taken into account.
- 6. „In the experiments, only the measurements of larval size have been provided, without specific information about their stages. If there is any information regarding the correlation between larval size and stages, it should be referenced.”
Answer: Since the larvae used in the study were collected from the field and were not reared from the egg stage, it was difficult to accurately determine the larval stage, so we reported only the size of the larvae.
- „It should also be explained why the studies were conducted at a temperature of 20°C instead of 25°C.”
Answer: The studies were conducted at a temperature of 20°C because the field application was carried out in the late afternoon when temperatures usually do not exceed 20°C.
- „Although information has been provided about the obtaining of larvae, there is no explanation about how the pupae used in the experiments were obtained.”
Answer: Section 2.2. „Test insects” provides the following detail: „Larvae and pupae of Y. padella and Y.cagnagella were used in laboratory tests. They were collected in June and July 2019, respectively, during their mass appearance on the wild plum (Prunus domestica L. subsp. syriaca (Borkh.) Janch. var. cerea) and on the common spindle tree (Euonymus europaeus L.) in Warsaw (Ursynów district) (520 9’52.198”N, 210 2’38.409”E and 520 8’35.043”N, 210 4’7.631”E).”
- „Page 3, (2.3.2 Container Test, line 4): It was previously stated that a dose of 50 IJs was used for larvae and pupae in the experiments. However, in this section, the nematode suspension is reported as 100 IJs/insect. This issue should be clarified.”
Anawer: In Petri dish tests, a dose of 50 IJs was used, for both larvae and pupae, and in container tests 100 IJs (also for larvae and pupae). It is described in the methodology section and the information on why these doses were used has also been added.
- „Furthermore, it would be appropriate to provide an explanation for why there was a need for such an experiment (container experiments)?"
Answer: The tests were conducted to assess the extent to which webs surrounding larvae and pupae might be a barrier to penetration of EPN IJs.
- „Do the authors have any information about the stages of the larvae in the pods in field trials? Additionally, were there webs in these pods due to species' web-building behavior, and if so, how did EPN (Entomopathogenic nematodes) reach the larvae within the web?
Furthermore, a more detailed explanation of the methodology used in studies related to the pupal stage is needed. Since pupae are generally found in protected places, what approach was followed to deliver EPN to them?”
Answer: Nematode application in the field studies was performed on larvae that were similar in size to the larvae used in the Peteri dish and container tests. In the manuscript, by „pod” we meant „web”. We came to the conclusion that the term „web” is more accurate and updated the manuscript accordingly. In tests on pupae, we used the standard method of EPNs application by hand sprinkler. We have added this clarification in the „Container tests” section.
- „Why were two different species within the same genus included in the experiment for two different applications?”
Answer: For two reasons: firstly, they feed on two different plant species; secondly, the authors' previous research has shown that even closely related species have different susceptibility to nematodes (this was mentioned in the discussion).
- „By what criteria were the dosages used in this case determined?”
Answer: The dose used was twice as high as in the container ones due to the low survival of IJs in the field application.
14. „As a general rule, before analyzing the data, the homogeneity of variances and normality should be checked. Based on the analyses conducted here, it is assumed that the data follows a normal distribution from the beginning, which is an incorrect assumption.”
Answer: Suggestion taken into account.
Reviewer 3 Report
This is a short manuscript about the use of EPN in controlling some important pests. It is very interesting because they do not just test the effectiveness of the EPN in laboratory essays but also in field essays, and considering both larvae and pupae stages. I think it could be published in Agriculture, but after making some changes, especially in the results, discussion and conclusions sections. Here I give some comments that I hope will help to improve the manuscript.
Do not use an acronym in the abstract, or if you are going to use it you have to say what it means.
Introduction
Page 1, line 7 in second paragraph: there is a missing bracket.
I think you need to indicate the source of information about the pesticides used for the moth. Also, some of the pesticides you mentioned are considered environmentally friendly (i.e., the Bacillus-based products), so you need to explain a little more why to you need other environmentally safe methods (and what is the relationship between these sentences: “All these means mainly act gastrically and through the surface contact” and “The ermine moths are, however, resistant to insecticides used on crops”), in order to state clearly the knowledge gap and the problem you are trying to solve.
Materials and methods
Page 2, section 2.2., please put “syriaca” into italics.
Did you treat every Petri dish and every container as a replicate for analyses?
Which was the humidity in the Sanyo incubation chambers?
Please indicate how/where you obtained the meteorological data.
There is a missing bracket in the Table 1’s legend.
Page 3, line 3 after Table 1: How were you able to count the mean number of caterpillars in the pods from the laboratory?
Why do you have a different number of replicates for the nematodes application and for the control samples in the field trials (10 and 6 pods, respectively)? Or it is that you “artifitially” put the pods in the field for the trial?
Results
You are showing many data from the statistical test, but almost none from the actual results! I would like to see the mean survival values in every test, and also what you obtained in the control samples.
Discussion
There is a little mess in the format, with different font types and sizes.
The last two sentences of the first paragraph are contradictory: you first say that “there is no example in the literature showing that two closely related species have so different susceptibility to EPNs”, but then you show an example where that happened.
In the next two paragraphs, you are only comparing your data with others’, but without giving any explanation about differences. For instance, why did you get a higher mortality in Y. padella than other authors? Might it be because of the EPN species used or might be any other reason? It might also be because you used a native species, and you could discuss a little further about the benefits of using native EPN instead of commercial ones. Also, you are not discussing about differences in mortality of larvae and pupae, or differences in mortality regarding the type of test (Petri dishes and containers).
You mention the need of adiuvants, but you should mention some examples of the type of adiuvants that are commonly used when applying EPN.
Conclusions
Conclusion section should not be a summary of the main results, it should present the take-home message for readers.
Author Response
We have considered all suggestions given by the Reviewers. All changes in the manuscript are marked with using the "Track Changes" function.
Below we provide the detailed answers to the comments:
- „Do not use an acronym in the abstract, or if you are going to use it you have to say what it means.”
Answer: We have explained the acronym in the abstract
- “Page 1, line 7 in second paragraph: there is a missing bracket.”
Answer: added
- „I think you need to indicate the source of information about the pesticides used for the moth. Also, some of the pesticides you mentioned are considered environmentally friendly (i.e., the Bacillus-based products), so you need to explain a little more why to you need other environmentally safe methods (and what is the relationship between these sentences: “All these means mainly act gastrically and through the surface contact” and “The ermine moths are, however, resistant to insecticides used on crops”), in order to state clearly the knowledge gap and the problem you are trying to solve.”
Answer: The part of text was amended and edited (also taking into account the comments of the Reviewer 2).
- „Page 2, section 2.2., please put “syriaca” into italics.”
Answer: corrected
- „Did you treat every Petri dish and every container as a replicate for analyses?”
Answer: In petri dish tests, one repetition included 6 dishes, 5 insects in each. In container tests, one repetition included 5 containers, one web in each.
- „Which was the humidity in the Sanyo incubation chambers?”
Answer: Average humidity in the incubation chambers was approximately 60%
- „Please indicate how/where you obtained the meteorological data.”
Answer: added
- „There is a missing bracket in the Table 1’s legend.”
Answer: corrected
- „Page 3, line 3 after Table 1: How were you able to count the mean number of caterpillars in the pods from the laboratory?”
Answer: During the assessment of insect mortality in container tests, the caterpillars were precisely counted in each web. From this data, the average number of insects in the web was calculated.
- „Why do you have a different number of replicates for the nematodes application and for the control samples in the field trials (10 and 6 pods, respectively)? Or it is that you “artifitially” put the pods in the field for the trial?”
Answer: The different number of replications for the nematodes application and for the control samples results from the fact that the number of webs with similar exposure was limited. In field trials, to our knowledge, a smaller control sample is often used.
- „You are showing many data from the statistical test, but almost none from the actual results! I would like to see the mean survival values in every test, and also what you obtained in the control samples.”
Answer: Suggestion taken into account and added table with results.
12.”There is a little mess in the format, with different font types and sizes.”
Answer: corrected
- „Discussion: The last two sentences of the first paragraph are contradictory: you first say that “there is no example in the literature showing that two closely related species have so different susceptibility to EPNs”, but then you show an example where that happened.In the next two paragraphs, you are only comparing your data with others’, but without giving any explanation about differences. For instance, why did you get a higher mortality in Y. padella than other authors? Might it be because of the EPN species used or might be any other reason? It might also be because you used a native species, and you could discuss a little further about the benefits of using native EPN instead of commercial ones. Also, you are not discussing about differences in mortality of larvae and pupae, or differences in mortality regarding the type of test (Petri dishes and containers).You mention the need of adiuvants, but you should mention some examples of the type of adiuvants that are commonly used when applying EPN.”
Answer: Discussion was amended and edited according to the Reviewer’s suggestion.
- „Conclusion section should not be a summary of the main results, it should present the take-home message for readers.”
Answer: Suggestion taken into account.
Round 2
Reviewer 3 Report
After reviewing the new version of the manuscript, I am not entirely happy with the corrections made.
There is some new text in the introduction that is intended to amend the problem about the need of new control methods for the Yponomeuta species, but now it looks somewhat forced, now it seems like there are many different methods (we do not know if they have proved to be useful or not because the authors do not say it) and that there is no need for new ones. Also, there are different font sizes. Now authors have to justify the need for the EPN if there are already so many control options.
I am still not sure about the experimental design, it is not clear in the text. When authors say repetition do they mean replicate? If it is so, only two repetitions (=two replicates) in every essay is too little for any statistical test, so results in that case would not be valid. Authors should reconsider the experimental design, or be more precise.
Some of the answers made to the report 1 are not reflected in the manuscript.
I feel that the discussion can still be improved. In second paragraph authors only compare data, with no explanation or further comments. Why those differences? Is it because of the EPN species or because of the dose? And the same concerns to the first part of the next paragraph (lines 211-220). Also, there are some statements that are partially true, such this one: “Higher mortality of larvae and pupae in the Petri dish tests than in the container tests proves the protective role of these structures, on the other hand, higher mortality of larvae than pupae in container tests indicates that pupal cocoons provide an additional protective barrier against nematodes” (lines 227-229). It only happened for one of the species, and not even the same in both cases, so those can not be the only reasons. Differences in mortality can not be completely explained by the protection of the webs nor by that from the cocoon. I suggest authors should take some quality time to rewrite the discussion properly.
There are also some minor mistakes:
Line 26: “or polyphagous”.
Line 47: there is an extra bracket.
Lines 87-88: the sentence is repeated.
Line 116: there is a missing period.
Line 204: “two species”.
Line 236: “adjuvants”.
I suggest a revision of the English grammar and spelling, some minor mistakes are present.
Author Response
Dear Reviewer,
Below we provide the detailed answers to the comments:
- “There is some new text in the introduction that is intended to amend the problem about the need of new control methods for the Yponomeuta species, but now it looks somewhat forced, now it seems like there are many different methods (we do not know if they have proved to be useful or not because the authors do not say it) and that there is no need for new ones. Also, there are different font sizes. Now authors have to justify the need for the EPN if there are already so many control options.”
Answer: We reworded the Introduction and standardized the font.
- “I am still not sure about the experimental design, it is not clear in the text. When authors say repetition do they mean replicate? If it is so, only two repetitions (=two replicates) in every essay is too little for any statistical test, so results in that case would not be valid. Authors should reconsider the experimental design, or be more precise.”
Answer: The experiment involving larvae was conducted using n=30 individuals. The results were validated in a separate experiment (maintaining the same conditions) also including n=30 larvae. In total n=60 larvae were used in the studies. The same approach was used for pupae. The 60 individuals in each group is a sufficient number of records for statistical analysis. The relevant part of the methodology has been reworded.
- “Some of the answers made to the report 1 are not reflected in the manuscript.”
Answer: We added a sentence referring to humidity in the chamber.
- “I feel that the discussion can still be improved. In second paragraph authors only compare data, with no explanation or further comments. Why those differences? Is it because of the EPN species or because of the dose? And the same concerns to the first part of the next paragraph (lines 211-220).”
Answer: A relevant comment was added to the part where data are compared.
- “Also, there are some statements that are partially true, such this one: “Higher mortality of larvae and pupae in the Petri dish tests than in the container tests proves the protective role of these structures, on the other hand, higher mortality of larvae than pupae in container tests indicates that pupal cocoons provide an additional protective barrier against nematodes” (lines 227-229). It only happened for one of the species, and not even the same in both cases, so those can’t be the only reasons. Differences in mortality can’t be completely explained by the protection of the webs nor by that from the cocoon. I suggest authors should take some quality time to rewrite the discussion properly.”
Answer: We believe that we have grounds for making such a statement. Differences in mortality between larvae in Petri dishes and bucket tests are evident in both species of Yponomeuta (Y. padella: 81.7% - Petri dish vs. 47.7% - container test; Y. cagnagella: 50.0% - Petri dish vs. 44.2% - container test). The same pattern was observed in the case of pupae (Y. padella: 88.3% - Petri dish vs. 35.6% - container test; Y. cagnagella: 33.3% - Petri dish vs. 15.4% - container test). In container tests, comparing the mortality of larvae and pupae in both species, there were differences (Y. padella: 47.7% - larvae vs. 35.6% - pupa; Y. cagnagella: 44.2% - larvae vs. 15.4% - pupae).
- “There are also some minor mistakes:”
Answer: We corrected any minor mistakes.
The manuscript was checked by a native English speaker.
