European Spruce Bark Beetle, Ips typographus (L.) Males Are Attracted to Bark Cores of Drought-Stressed Norway Spruce Trees with Impaired Defenses in Petri Dish Choice Experiments

Round 1

Reviewer 1 Report

The paper is original and well written, clear and easy to read. In contributes to the understanding of how males spruce bark beetle selects a host trees. This is an interesting research, and their results can be related to a widely spread and specific forest areas – spruce forests. The results can be relevant for management of the spruce forests.

The article requires only a few corrections. Those little editorial corrections I put directly onto the article.

• Each first mentioning of species name within the text and in table and figure titles should be provided with at least an abbreviation author(s)' name(s). Apply on lines: 90, 119, 120, 177, 192, 269, 306, 341, 372, 451 and 457.
• Shorten the name of the insect on lines: 15 and 170.
• Add space on line 140.
• Correct name on line 227.
• Use plural on lines: 255, 298 and 319.
• Use the full name (not the abbreviation) and plural on lines: 287 and 439.
• Use the full name (not the abbreviation) on line 435.
• Prepare the references section as required by the Insects Editorial Board (example on line 513).

Comments for author File: Comments.pdf

Author Response

Reviewer 1

The paper is original and well written, clear and easy to read. In contributes to the understanding of how males spruce bark beetle selects a host trees. This is an interesting research, and their results can be related to a widely spread and specific forest areas – spruce forests. The results can be relevant for management of the spruce forests.

The article requires only a few corrections. Those little editorial corrections I put directly onto the article.

We are glad about this positive feedback and carefully considered the requested editorial corrections.

• Each first mentioning of species name within the text and in table and figure titles should be provided with at least an abbreviation author(s)' name(s). Apply on lines: 90, 119, 120, 177, 192, 269, 306, 341, 372, 451 and 457.

Done.

• Shorten the name of the insect on lines: 15 and 170.

Done.

• Add space on line 140.

Done.

• Correct name on line 227.

Done.

• Use plural on lines: 255, 298 and 319.

Done.

• Use the full name (not the abbreviation) and plural on lines: 287 and 439.

Done.

• Use the full name (not the abbreviation) on line 435.

Done.

• Prepare the references section as required by the Insects Editorial Board (example on line 513).

Done according to Forests requirements.

Reviewer 2

1. General Comments

The present paper offers a good review about Ips typographus damages and ecological performance.  The work done includes an innovative way to test bark beetles host choice and acceptance behaviour and a video to illustrate this situation.  The result of such approach is very interesting.  I really appreciate all the information provided and the originality existing in the work. As a scientific reader, I found the paper very interesting, appealing, motivating and very understandable.

We are happy about the reviewer’s appreciative and valuable comments on our manuscript and addressed all issues.

1. Section by section

2.1. - Introduction

This section is very comprehensible, interesting and has a lot of recent bibliography to consolidate the affirmations made. The authors present a very good review about the subject presented and about the interaction established between climatic events - insect – fungi – tree. The problem is very well posed.

2.2. - Material and Methods

Material and Methods are very easy to understand and allow to follow data collection and bioassays conducted. The description of methodology allow to replicate the assay which is encouraged by the authors for other similar biological models.

However, from my point of view, some improvements should be done.

1. It is not clear if there is some difference between trees C1 and C2 or if both are in the same situation to control the experiments.

Thank you for this comment, which refers to a very important point. Yes, the control trees C1 and C2 were in the same situation to control the experiments. To make this clear, we added the sentence: “These control trees were of similar age and diameter, faced the same site and water supply conditions, and showed similar vigorous crown conditions” (lines 131-133).

2. Table 1 presents some results and for this reason should be modified (i) explaining only methodology used or (ii) moved for results.

We moved table 1 to results.

3. I don’t understand the criteria to combine the choice experiments performed and presented in Table 1. For instance, why R1 is tested with C1; R2 tested with C2 and R3 tested with C1 and C2? Please clarify this aspect.

This publication is based on the experiments performed in a Bachelor thesis (Rentsch Verena) and a Master thesis (Morgante Giuseppe). One student tested against C1, the other against C2; the groups of tested trees were partly overlapping. We chose the test trees according to the main questions of the theses (BA: comparison of most/least water stressed trees; MA: focus on wound reaction to blue-stain fungi). We could not sample and test all 20 trees of the experimental site against both C1 and C2 due to limitations in personnel and time resources.

2.3. - Results

Results are well presented; graphic component is interesting and gives a lot of information easy to understand. The authors presented a small video, interesting, very friendly and appealing.

 2.4. Discussion:

Discussion is well conducted, well supported in bibliographic references, explains clearly the results observed and present new aspects of interactions established. In this section, the authors point out the effect of drought-stressed trees in the resistant to biotic attack and highlights the role of Grosmannia species on Ips typographus mass aggregation.

Reviewer 3

Overall evaluation and major comments

This is an interesting description of a series of simple bioassays done to test if male Ips typographus distinguish between bark cores from water-deprived Norway spruce trees and trees receiving regular rainfall. My main criticism of the study is that it is a bit thin on data. The authors discuss their results in the context of primary attraction of beetles to the bark cores, yet they did not make any attempt to measure and characterize the headspace volatiles in their test arenas. This would have been easy to do and would have added a mechanistic dimension to their study.

We are fully aware of this point and agree that a characterisation of volatile substances in the test arena or of bark compounds would have delivered interesting information to interpret beetle choice, as pointed out in our conclusions: ”To deepen our understanding of host tree-mediated primary attack, we recommend a future combination of bark beetle behavioural studies and electroantennography with tree physiological and biochemical analyses” (lines 502-504).

In the present study, however, we did not include the measurement of volatiles or bark compounds for several reasons: First, the laboratory bioassays were intended as first attempts to examine the feasibility and significance of such Petri dish choice experiments, which we nicely show indeed in our study. Furthermore, the lab studies were performed as part of a Bachelor and a Master thesis, which also explains why the choice of test trees seems to be a bit coincidental (see comment and answer below). Data of the BA and MA theses were merged after the experiments for joint interpretation.

Another aspect regarding possible chemical analyses: as the choice tests were developed as extra study in addition to the very comprehensive Rosalia Roof field study (including measurements of volatiles and bark compounds), there were simply no financial resources available to support further chemical analyses. Now that we know about the relevance of such choice experiments, we plan to conduct more comprehensive studies combining behavioural tests and chemical analyses.

While reading the description of the sampling of bark plugs and setup of the Petri dish arenas I thought about an alternative interpretation of the test results. The beetles might not simply be selecting bark plugs based on whether they come from water-deprived or regular trees. Since each bark plug exposes a large area of wounded bark it is possible that the beetles were reacting to wound-induced chemicals emitted from the wounded bark. Furthermore, it is possible that bark from water-stressed trees react differently to wounding than bark from normal trees. This possibility should be discussed briefly as an alternative explanation for beetle choice.

This aspect can indeed not be fully excluded. However, the bark cores were immediately (within seconds) shock frozen with liquid nitrogen after sampling in the field and then stored at -80°C (and at -20°C prior to the experiments) to stop any chemical processes in the tissues. The samples were taken out of the freezer a couple of minutes before the experiments started and used for a maximum of 60 minutes, as described in the Materials and Methods part (lines 184-186). To make this clear, we added to the discussion: “We consider it unlikely that beetles were mainly reacting to wound-induced volatiles emitted from bark cores, as samples were immediately shock-frozen in the field to stop degradation processes in the tissues.” in lines 411-413.

Abbreviations: in general, I think abbreviations should be kept to a minimum since they make the text harder to read. Abbreviations that are used only a couple of times can definitely be scrapped, such as ‘RRS II’ for the Rosalia Roof Study II. I am not too fond of ‘PDAC’ for ‘Petri dish arena choice’ either. It is much better to simply refer to it as ‘arena’ or similar. There is only one kind of arena used in your study, so there is no risk of confusion. ‘WRZ’ for wound reaction zones is also unnecessary – why not simply refer to this as ‘lesion size’?

We removed these abbreviations and instead used the terms “Rosalia study”, “arena” experiments, and “lesion size”.

Experimental design: this is not optimal or balanced and looks a bit coincidental. See more about this under my comment to Figure 1 and line 158-160 below.

As mentioned before, the experimental design results from merging the experiments conducted in the course of a Bachelor thesis and a Master thesis study. The groups of test trees were chosen regarding the specific questions posed in both theses (BA: comparison between most/least water stressed trees; MA: focus on wound reaction to blue-stain fungus inoculation), which also explains why some trees were tested solely against C1 or C2 and some against both control trees. We are also fully aware that the number of replicates in future arena experiments needs to be higher in order to achieve significant statistical results, for instance from Chi-square tests (see our replies below). However, we are convinced that our study gives valuable insights in the feasibility of arena experiments for such kind of research questions.

Specific comments:

Line 13: Add “During outbreaks, the beetles can colonize…”

Done.

Line 45: perhaps you should explain what ‘thigmotactic’ means or use another word

We complemented “thigmotactic (haptic) cues.”

Line 49: “beetle’s olfactory system is sensitive to …”

Done.

Line 78-81: I think this information is a bit out-of-place here – what is the logical link to the previous sentence?

We changed the wording to “While empirical evidence for increased attractiveness of drought-stressed trees for pioneer beetles is still scarce” – the logical link to the previous sentence is that the behaviour of pioneer spruce bark beetles has rarely been studied with regard to drought stress of trees and therefore, we miss empirical data on beetle/tree level proving this relationship.

Line 102: “2) how long the beetles remained in Petri…”

We changed the phrase to “2) the time beetles remained in Petri dish sections was related to their final choice”

Line 107: “and the beetles’ potential to…” Done.

Line 111: the mentioning of ‘resistant’ here is a little confusing. You probably mean to say that the trees were both differentially drought-stressed and differed in resistance, but this was not immediately clear to me.

We changed the phrase to: “To understand the host selection behaviour of male I. typographus towards Norway spruce trees differing in drought-stressed status and resistance”.

Line 144-146: you should mention that the inoculations were evenly spread around the trunk (I assume they were?). Also, why were they fungus inoculated at different heights in R and nR trees? Inoculation height is probably not very important, but it looks a bit curious with the different heights and the reader will want to know why.

Inoculations were performed at different tree heights because R trees were surrounded by roofs and we had to climb an 1.4 m high platform to reach the trunks. To leave enough space between inoculations and the bark area where field behavioural bioassays were conducted, we had to inoculate the fungi at a lower height at R trees compared to the control trees. The reason is that we could reach up higher for installing the attack boxes at the control trees without a roof, because we were able to place a ladder to the stem. We also think that inoculation heights are not so relevant, therefore we did not add this quite complicated explanation for the differences.

Line 150: there were six lesions per tree, not four, right?

We deleted “four”.

Figure 1: I suggest you highlight the trees that were used by e.g. using grey (or black) background on the tree labels. How did you select which trees to include? There doesn’t seem to be pairs of neighboring R and nR trees.

Thank you for this good idea – we highlighted roofed, non-roofed and control trees in different colours and changed the legend accordingly:

Figure 1. Rosalia Roof Study II – the drought manipulation site involves ten roofed (R1-10) and ten non-roofed (nR1-10) study trees. We sampled 7 roofed (R1, R2, R3, R4, R5, R6, R9, in yellow), 8 non-roofed (nR1, nR3, nR4, nR5, nR7, nR8, nR9, nR10, in blue) and two additional, non-manipulated control trees (C1, C2, in grey) for Petri dish arena choice (arena) experiments.

Selection of trees: The original study design of the Rosalia Roof field experiment was not based on pairs of roofed and non-roofed trees located next to each other, but R and nR trees were randomly distributed (as illustrated by Figure 1). As already mentioned, the choice experiments were conducted in addition to the comprehensive field studies at the drought manipulation site. For the arena experiments, we chose test trees according to their water stress status (the most and the least stressed ones regarding pre-dawn twig water potential) measured in the field. A further aspect in the decision for specific study trees were the attack rates observed in the field bioassays (“attack box” experiments, which we do not report and discuss in this manuscript (but see Netherer et al. 2015, New Phytologist, for a description of this type of experiment/bioassay), as we want to focus on the results of the laboratory choice tests and not mix them up with observational data from the field. While the optimal approach would have been to include all 20 test trees, this was simply not possible due to time and personnel resource limitations.

Line 158-160: you should explain more clearly that you collected multiple bark cores from each tree. Perhaps you could provide a range of numbers? You should also give the number of R and nR trees in the text. And why is n not equal, but n = 7 for R and 8 for nR? Your experimental design is not optimal, since not all R/nR trees were paired up with both control trees. What was the rationale for the specific pairing of trees? The design looks a bit coincidental. You succinctly sum up the unbalanced design in line 242-245 and I think the design requires an explanation.

We sampled between 20 and 40 bark cores per tree (numbers were added to the text in the Materials and Methods part, line 163). We also mention now that we sampled 7 R and 8 nR trees (which are almost equal numbers). Concerning our study design we refer to the replies to previous questions.

Figure 2: you should define R and nR in the figure caption (or, better, spell it out and skip the abbreviations).

We added: “cores of test (roofed trees, R or non-roofed trees, nR)”.

Line 195: “The beetles could move freely in the Petri dish and enter the…”

Done.

Line 262-266: descriptions like this are very hard to follow for a regular reader. There are simply too many comparisons going on here at the same time. What exactly do you want to get across here?

We think that this description of further tests is very important. You raised the question why we did not compare R against nR trees (see comment on lines 393-396 below), but in fact we performed exactly such tests with selected study trees: for instance, R3 (a highly preferred tree against C samples) was preferred over R2 and also over nR10 (both less attractive to beetles than C samples) and R2 was preferred over nR8. We complemented the text:” Additional “T and T” tests to directly compare study trees…” (lines 271-272).

Figure 3: here and in subchapter 3.1 there are no statistical comparisons of C vs. R, C vs. nR, etc. I think these contrasts could be tested using e.g. a Chi-square test.

We performed Fisher’s exact test, which is a Chi-square test for small sample sizes for all experiments, and checked whether the expected number of choices (e.g. 5 choices for T, 5 choices for C and 0 “no choice” outcome in case of 10 test runs) significantly differed from the actually observed number of choices. This was the case in only two experiments (R2 against C2 and R9 against C1). Not even a relationship of 2 to 8 resulted in a significant difference from the expected 5 T / 5 C / 0 “no” choices. For this reason, we did not document Fisher’s exact tests for the experiments in the manuscript, but we now state in the conclusions: “The number of replicates needs to be increased in future experiments to improve statistical significance of results.” (lines 497-498).
To include statistical comparisons of R/nR and C1/C2, we conducted Fisher’s exact tests for R/C1, R/C2, nR/C1, and nR/C2 experiments in total (statistical results added to lines 266-270). In the sub-chapter data analysis, we included: “We used Fisher’s exact test to examine whether the number of T and C choices significantly deviated from an expected equal distribution.” (lines 228-229).

Line 277-278: “the test run ended after 60 minutes.”

Done.

Figure 4: perhaps you need to explain in more detail how you calculated relative time. Isn’t relative time just a simple function of time, i.e. isn’t it just a different way of presenting the same data?

We calculated relative time, i.e. the proportion of time a beetle spent in each section (added in line 218) because absolute time was highly variable (between some minutes and a maximum of 60 minutes) and therefore not very well suited for comparisons. In line 218, we now explain: “relative time, i.e. the proportion of time a beetle spent in each section”.

Figure 5: I suggest that you include R-square values in the plots + perhaps the function for the lines.

We included Pearson correlation (r) values in figures 5a and b and also in the text part, but not the function of the regression line. We rather intended to illustrate the linear relationship in these figures (which is very nicely shown).

Figure 6: same comment as for Figure 5. Also, you should explain what T is. The figure caption is incredibly difficult to follow – I think it is written as one very long sentence! The text needs a thorough clean-up.

We included Pearson correlation (r) values in figures 6a-d. To break up the very long sentence, we exchanged semicolon by full stop, but did not delete any information because we think it is needed to understand the plots. We don’t think it is necessary to explain which samples are represented by T, this can be seen in Table 1. Moreover, an explanation would make the legend even longer and more complicated.

Line 383-388: your discussion of primary attraction would have been much more relevant if you had made an attempt to characterize the headspace volatiles around the bark plugs from R and nR trees.

Please consider our replies regarding this issue above.

Line 393-396: this is perhaps to overplay the clarity of your results, since you only showed clearly that the bark beetles are attracted to spruce bark. The contrast between R and nR trees is less clear. And you don’t compare R and nR directly – only indirectly through the comparison with C1 or C2.

There are conceptual reasons for not comparing R and nR trees, which we thoroughly thought about when designing the study: We did not work with Norway spruce clones, which means that each tree has individual characteristics regarding tissue quality, stress state etc. When comparing pairs of trees, the different choice experiments have nothing in common except for the fact of testing R and nR trees against each other. We therefore chose the approach of testing always the same two, very similar control trees (similar regarding site and water supply conditions, tree age and dimension, crown vigour conditions) against our study trees. To make this clear, we added the following sentence in the Material and Methods part: “These control trees were of similar age and diameter, faced the same site and water supply conditions, and showed similar vigorous crown conditions” (lines 131-133). The control trees could be any vital, well water supplied trees in the forest stand. Our question was whether these trees would be preferred over stressed (R) or not water stressed (nR) trees. In addition, we tested particular R and nR trees against each other (see lines 272-277).

Author Response File: Author Response.docx

Reviewer 2 Report

Overall evaluation and major comments

This is an interesting description of a series of simple bioassays done to test if male Ips typographus distinguish between bark cores from water-deprived Norway spruce trees and trees receiving regular rainfall. My main criticism of the study is that it is a bit thin on data. The authors discuss their results in the context of primary attraction of beetles to the bark cores, yet they did not make any attempt to measure and characterize the headspace volatiles in their test arenas. This would have been easy to do and would have added a mechanistic dimension to their study.

While reading the description of the sampling of bark plugs and setup of the Petri dish arenas I thought about an alternative interpretation of the test results. The beetles might not simply be selecting bark plugs based on whether they come from water-deprived or regular trees. Since each bark plug exposes a large area of wounded bark it is possible that the beetles were reacting to wound-induced chemicals emitted from the wounded bark. Furthermore, it is possible that bark from water-stressed trees react differently to wounding than bark from normal trees. This possibility should be discussed briefly as an alternative explanation for beetle choice.

Abbreviations: in general, I think abbreviations should be kept to a minimum since they make the text harder to read. Abbreviations that are used only a couple of times can definitely be scrapped, such as ‘RRS II’ for the Rosalia Roof Study II. I am not too fond of ‘PDAC’ for ‘Petri dish arena choice’ either. It is much better to simply refer to it as ‘arena’ or similar. There is only one kind of arena used in your study, so there is no risk of confusion. ‘WRZ’ for wound reaction zones is also unnecessary – why not simply refer to this as ‘lesion size’?

Experimental design: this is not optimal or balanced and looks a bit coincidental. See more about this under my comment to Figure 1 and line 158-160 below.

Specific comments:

Line 13: Add “During outbreaks, the beetles can colonize…”

Line 45: perhaps you should explain what ‘thigmotactic’ means or use another word

Line 49: “beetle’s olfactory system is sensitive to …”

Line 78-81: I think this information is a bit out-of-place here – what is the logical link to the previous sentence?

Line 102: “2) how long the beetles remained in Petri…”

Line 107: “and the beetles’ potential to…”

Line 111: the mentioning of ‘resistant’ here is a little confusing. You probably mean to say that the trees were both differentially drought-stressed and differed in resistance, but this was not immediately clear to me.

Line 144-146: you should mention that the inoculations were evenly spread around the trunk (I assume they were?). Also, why were they fungus inoculated at different heights in R and nR trees? Inoculation height is probably not very important, but it looks a bit curious with the different heights and the reader will want to know why.

Line 150: there were six lesions per tree, not four, right?

Figure 1: I suggest you highlight the trees that were used by e.g. using grey (or black) background on the tree labels. How did you select which trees to include? There doesn’t seem to be pairs of neighboring R and nR trees.

Line 158-160: you should explain more clearly that you collected multiple bark cores from each tree. Perhaps you could provide a range of numbers? You should also give the number of R and nR trees in the text. And why is n not equal, but n = 7 for R and 8 for nR? Your experimental design is not optimal, since not all R/nR trees were paired up with both control trees. What was the rationale for the specific pairing of trees? The design looks a bit coincidental. You succinctly sum up the unbalanced design in line 242-245 and I think the design requires an explanation.

Figure 2: you should define R and nR in the figure caption (or, better, spell it out and skip the abbreviations).

Line 195: “The beetles could move freely in the Petri dish and enter the…”

Line 262-266: descriptions like this are very hard to follow for a regular reader. There are simply too many comparisons going on here at the same time. What exactly do you want to get across here?

Figure 3: here and in subchapter 3.1 there are no statistical comparisons of C vs. R, C vs. nR, etc. I think these contrasts could be tested using e.g. a Chi-square test.

Line 277-278: “the test run ended after 60 minutes.”

Figure 4: perhaps you need to explain in more detail how you calculated relative time. Isn’t relative time just a simple function of time, i.e. isn’t it just a different way of presenting the same data?

Figure 5: I suggest that you include R-square values in the plots + perhaps the function for the lines.

Figure 6: same comment as for Figure 5. Also, you should explain what T is. The figure caption is incredibly difficult to follow – I think it is written as one very long sentence! The text needs a thorough clean-up.

Line 383-388: your discussion of primary attraction would have been much more relevant if you had made an attempt to characterize the headspace volatiles around the bark plugs from R and nR trees.

Line 393-396: this is perhaps to overplay the clarity of your results, since you only showed clearly that the bark beetles are attracted to spruce bark. The contrast between R and nR trees is less clear. And you don’t compare R and nR directly – only indirectly through the comparison with C1 or C2.

Author Response

Please see attachment

Author Response File: Author Response.docx

Reviewer 3 Report

The authors present a study about  bark beetle attracted to bark cores of drougth stressed. From my understanding, the technical parts of this work were done carefully and correctly. Moreover, I appreciate the effort the authors collecting the data. The research question is a very interesting and original topic. However, this article has some weaknesses that I list below:

• The title is long and must to be reduced
• An international context about climate change and outbreaks should be provided.
• Hypothesis are lacking could be improved.
• Future research and recommendations are not indicated.
• There are important elements that must be more strongly highlighted in this paper:

-          What are the original elements of this research?

-          Need to study this subject?

-         Implications for forest management?

At this stage, I propose the authors to consider these suggestions in a moderate revision (not major- not minor), and request the editor not to accept the manuscript until and unless the authors make the changes. Congratulations to the authors, they have realized a good job in this original research. I have also provided the following specific comments that would help to improve the quality of this manuscript.

Title: remve the latin name of the study species. Remove From “Norway spruce to Choice experiments”

Keywords must to be in alphabetic order

L32. Start the paper with a context about the importance to study natural disturbances under climate change in boreal forests:

Exemple: Natural disturbances will increase in terms od severity and frequency in the future due to the impact of climate change (Seidl). Insect outbreaks Insect outbreaks are showing changes in the distribution area, and the severity has increased around the last century in the boreal forests (Navarro et al. 2018). Insect outbreaks are major drivers in forest ecoystems modifieng the structure and species composition in time (Girona et al. 2018…),

Seidl, R., Thom, D., Kautz, M., Martin-Benito, D., Peltoniemi, M., Vacchiano, G., ... & Reyer, C. P. (2017). Forest disturbances under climate change. Nature climate change, 7(6), 395-402.

 Navarro, L., Morin, H., Bergeron, Y., & Girona, M. M. (2018). Changes in spatiotemporal patterns of 20th century spruce budworm outbreaks in eastern Canadian boreal forests. Frontiers in Plant Science, 9, 1905.

Montoro Girona, M., Navarro, L., & Morin, H. (2018). A secret hidden in the sediments: Lepidoptera scales. Frontiers in Ecology and Evolution, 6, 2.

L79-80: Explain more the link between drought and outbreaks. Provide examples of other outbreaks.

L99-108: add hypothesis

Figure 1: add a map of the study country

L376. More general context with other outbreaks

DL473: Add section with forest management implications.

Author Response

Please see attachment

Author Response File: Author Response.docx

Reviewer 4 Report

1. General Comments

The present paper offers a good review about Ips typographus damages and ecological performance.  The work done includes an innovative way to test bark beetles host choice and acceptance behaviour and a video to illustrate this situation.  The result of such approach is very interesting.  I really appreciate all the information provided and the originality existing in the work. As a scientific reader, I found the paper very interesting, appealing, motivating and very understandable.

2. Section by section

2.1. - Introduction

This section is very comprehensible, interesting and has a lot of recent bibliography to consolidate the affirmations made.

The authors present a very good review about the subject presented and about the interaction established between climatic events - insect – fungi – tree. The problem is very well posed.

2.2. - Material and Methods

 Material and Methods are very easy to understand and allow to follow data collection and bioassays conducted. The description of methodology allow to replicate the assay which is encouraged by the authors for other similar biological models.

However, from my point of view, some improvements should be done.

1. It is not clear if there is some difference between trees C1 and C2 or if both are in the same situation to control the experiments.

2. Table 1 presents some results and for this reason should be modified (i) explaining only methodology used or (ii) moved for results.

3.  I don’t understand the criteria to combine the choice experiments performed and presented in Table 1. For instance, why R1 is tested with C1; R2 tested with C2 and R3 tested with C1 and C2? Please clarify this aspect.

2.3. - Results

Results are well presented; graphic component is interesting and gives a lot of information easy to understand. The authors presented a small video, interesting, very friendly and appealing.

 2.4. Discussion:

Discussion is well conducted, well supported in bibliographic references, explains clearly the results observed and present new aspects of interactions established. In this section, the authors point out the effect of drought-stressed trees in the resistant to biotic attack and highlights the role of Grosmannia species on Ips typographus mass aggregation.

Author Response

Please see attachment

Author Response File: Author Response.docx