Initial Location Preference Together with Aggregation Pheromones Regulate the Attack Pattern of Tomicus brevipilosus (Coleoptera: Curculionidae) on Pinus kesiya

Round  1

Reviewer 1 Report

See attached suggestions and comments for improving the clarity and rigor of the manuscript, especially the Introduction. These are mostly minor editorial points and need for clarification.

The science is fine.

Comments for author File: Comments.pdf

Author Response

Thank you very much for your advice regarding to our manuscript. We have tried best to revise our manuscript according to the comments. Most of the attached suggestions and comments, especially the Introduction, have been modified in the manuscript. And some questions have been clarified as follows:

Point 1: Page 1, line 14: what is "special" attack. I don't know what this means.

Response 1: Thanks for your question. The "special" attack was a description of the attack pattern of T. brevipilosus on P. kesiya. We have changed the "special attack" to "top-down attack" in the manuscript.

Point 2: Page 1, line 39: "and then" implies that one follows the other. Is this always the case?

Response 2: Thanks for your reminding. The life cycle of Tomics contains two phases: a feeding maturation and a reproduction stage. Newly emerged callow adults fly toward healthy trees and tunnel into vigorous shoots to become sexually maturmature. Subsequently adults seek recently cut or dying pines for reproduction.

Point 3: Page 2, line 44: where? In China?

Response 3: Thanks for your question. We have added the location in our manuscript. Page 2, line 45.

Point 4: Page 2, line 47: technically "reproduction" (i.e. mating) does not cause tree mortality, but feeding during reproduction does. Perhaps re-word this sentence.

Response 4: Thanks for your advice. We have reworded this sentence as “Gallery construction and larval feeding are regarded as the direct causes of P. yunnanensis tree mortality” in our manuscript. Page 2, line 48.

Point 5: Page 2, line 49: when you mean "on its own", do you mean with no other bark beetle species, or with no other stress-causing agents??

Response 5: Thanks for your question. The colonization pattern of T. brevipilosus varied dramatically on P. yunnanensis, depending on the other two Tomicus species were already present on the tree trunks or not. On trees that were already infested by the other two Tomicus species, T. brevipilosus colonized areas of the trunk that were not already occupied. When there were no P. yunnanensis newly infested by the other two Tomicus species, T. brevipilosus attacked P. yunnanensis by itself, infesting the lower parts of the trunk first and then infesting progressively upward along the trunk into the crown, exhibiting a down-up attack pattern. it can infest vigorous P. yunnanensis to death only by itself.

Point 6: Page 2, line 57: just nutrient flow, or water also?

Response 6: Thanks for your suggestion. The galleries of bark beetles were mainly distributed within the phloem tissue of the host. The nutrients of trees are mainly transported by sieve tubes in phloem, while water is mainly transported by ducts in xylem. So, the longitudinal galleries mainly disrupt the nutrient flow within the phloem tissue.

Point 7: Page 2, line 80: the host trees species, or the bark beetle species?

Response 7: Thanks for your reminding. “This species” was indicated the bark beetle species. We have modified in our manuscript. Page 2, line 86.

Point 8: Page 3, line 95-97: how long are the galleries generally? Perhaps indicate this, as puts your classes (I, II, III etc.) into context.

Response 8: Thanks for your question. We have descripted the length of the galleries of this bark beetle in the manuscript. Page 3; Line 101.

Point 9: Page 4, line 172 do you mean a plantation?perhaps just say "...a plantation of P. kesiya..."

Response 9: Thanks for your advice. We have modified it as “a plantation of P. kesiya...” in our manuscript.

Point 10: Page 10, line 288: re-word this to clarify. Do you mean that your traps were too low, because attack began in the upper crown?

Response 10: Thanks for your question. In our field trapping, fewer numbers of beetles were caught than expected. For the optimization of lures in follow-up study, we descripted this in our manuscript.

Point 11: Page 11, line 327: when the other species are present??

Response 11: Thanks for your question. In our study area, T. brevipilosus is the dominant species, we did not find other species bark beetles on P. kesiya. However, in previous study (Chen et al. J Insect Sci. 2015, 15(1).), T. brevipilosus coexisted with T. minor and T. yunnanensis. Its attack pattern was affected by the other two Tomicus species on P. yunnanensis. In order to explanation the to-down attack pattern of T. brevipilosus on P. kesiya, we descripted the other two beetles (T. yunnanensis and T. minor) in our manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper reports on the chemical ecology and attack pattern of Tomicus brevipilosus on Pinus kesiya. This work is novel because T. brevipilosus has been a relatively understudied species until recently. Moreover, attacks on Pinus kesiya have not yet been studied, although the attack pattern of this beetle on Pinus yunnanensis has been published. Overall, the methodology is sound, and the results are easy to interpret for the most part.

The most important thing to be addressed is the chemical analysis by GC-MS. The authors have listed several compounds which were purchased as synthetic standards. However, there are compounds identified in Table 1 for which synthetic standards were not acquired, such as styrene, α-pinene, S-(­–)-β-pinene, β-phellandrene, linalool, and terpinen-4-ol. These compounds may not be commercially available, but I think it is important to indicate which compounds were confirmed with standards, and which compounds were identified with NIST library reference alone. Please make that change.

It appears that the attack pattern of T. brevipilosus on P. yunnanensis had previously been published (Chen et al. 2015). What is particularly interesting to me is that the attack pattern presented in the present study on P. kesiya is opposite of what was described for their attacks on P. yunnanensis: attacking from the top down rather than from the middle/bottom up. This means that T. brevipilosus may alter its attack pattern depending on its host. The implications of this shift could be discussed in light of competitor beetles species or other factors. This phenomenon should be addressed in the introduction to lay the foundation for what is known about this species.

The English in this paper is mostly adequate, with a few areas that need to be addressed. These are described in greater detail below. There are also numerous spelling or grammatical errors, so I recommend a native English speaker proof read the manuscript before resubmission.

Lines 20, 161, and 259: Replace the words “Walking bioassays” with “Olfactometer bioassays”. This makes it more clear what kind of bioassay it is, and it sounds more natural in English.

Lines 65-68: This sentence is confusing and should be reworded.

Line 73: The word “semiochemical” is misspelled.

Section 2.2 The stages are described here, but I suggest adding the word “Stage” before each roman numeral for clarity. For example, “Stage I (<10 mm)” on line 95. This is especially helpful because “Stage I,” “Stage II,” etc., are referred to in the results section.

Lines 97-99: Please clarify the differences between Stage IV and Stage V galleries. It sounds like the presence of males was used as the determining factor, but Stage IV is described as “mostly females in a spawning state (males had gradually left the mating chamber). How does one tell if the males have “gradually left” for Stage IV, rather than “left entirely” in Stage V?

Lines 179-185: The list of lure combinations is difficult to read in paragraph form. Consider listing them in a table.

Line 187: This paragraph should have a “Statistical analysis” header. Right now, it reads like a part of field trapping, but you discuss statistical analyses for many different experiments in the study.

Lines 230-231: Is it odd that hind gut extracts of males and females did not differ no matter which stage of galleries they were collected from?

Figures 4 and 5: please indicate whether the error bars are standard error or standard deviation.

The references are neat and orderly, with only a few minor typographical inconsistancies throughout, including commas instead of colons between the issue number and the page numbers; n-dashes instead of m-dashes between page numbers; and the absence of commas after some last names.

Author Response

Point 1: The most important thing to be addressed is the chemical analysis by GC-MS. The authors have listed several compounds which were purchased as synthetic standards. However, there are compounds identified in Table 1 for which synthetic standards were not acquired, such as styrene, α-pinene, S-(­)-β-pinene, β-phellandrene, linalool, and terpinen-4-ol. These compounds may not be commercially available, but I think it is important to indicate which compounds were confirmed with standards, and which compounds were identified with NIST library reference alone. Please make that change.

Response 1: Thank you for your suggestion. We are really sorry that we didn’t describe it clear. In the experiment of chemicals analysis, all the compounds were confirmed with standards. These synthetic standards were commercially available, which listed in Table 2. Finally, the purity and merchants of these compounds were added to our manuscript. in Page 3; lines 112-116.

Point 2: It appears that the attack pattern of T. brevipilosus on P. yunnanensis had previously been published (Chen et al. 2015). What is particularly interesting to me is that the attack pattern presented in the present study on P. kesiya is opposite of what was described for their attacks on P. yunnanensis: attacking from the top down rather than from the middle/bottom up. This means that T. brevipilosus may alter its attack pattern depending on its host. The implications of this shift could be discussed in light of competitor beetles species or other factors. This phenomenon should be addressed in the introduction to lay the foundation for what is known about this species.

Response 2: Thanks for your positive advice. We have addressed this phenomenon in the introduction. Page 2; Lines 50-57.

Point 3: The English in this paper is mostly adequate, with a few areas that need to be addressed. These are described in greater detail below. There are also numerous spelling or grammatical errors, so I recommend a native English speaker proof read the manuscript before resubmission.

Response 3: Thanks for your suggestion. The language of this manuscript has been edited by a native English speaker.

Point 4: Lines 20, 161, and 259: Replace the words “Walking bioassays” with “Olfactometer bioassays”. This makes it more clear what kind of bioassay it is, and it sounds more natural in English.

Response 4: Thanks for your reminding. We have replaced the words “Walking bioassays” as “Olfactometer bioassays” in our manuscript. Lines 20, 169, and 262.

Point 5: Lines 65-68: This sentence is confusing and should be reworded.

Response 5: Thanks for your advice. We have reworded it as “T. piniperda used host volatiles to locate trees suitable for brood production, the host monoterpenes were effective lures for this bark beetle” in our manuscript. Lines 67-69.

Point 6: Line 73: The word “semiochemical” is misspelled.

Response 6: We are really sorry that we didn’t spell it correctly. We have revised it in our manuscript. Line 79.

Point 7: Section 2.2 The stages are described here, but I suggest adding the word “Stage” before each roman numeral for clarity. For example, “Stage I (<10 mm)” on line 95. This is especially helpful because “Stage I,” “Stage II,” etc., are referred to in the results section.

Response 7: Thanks for your advice. We have modified it in our manuscript. Page 3; line 101-105.

Point 8: Lines 97-99: Please clarify the differences between Stage IV and Stage V galleries. It sounds like the presence of males was used as the determining factor, but Stage IV is described as “mostly females in a spawning state (males had gradually left the mating chamber). How does one tell if the males have “gradually left” for Stage IV, rather than “left entirely” in Stage V?

Response 8: Thanks for your question. In our research, the colonization stages were then sampled by the length of the galleries, the presence or absence of males is not the decisive factor in judging the fourth and fifth stages. It is a phenomenon that mostly females in a spawning state (males had gradually left the mating chamber) in the fourth stage (30-50 mm); and only females and eggs that were inside a completed gallery system (males had left entirely) in the fifth stage (> 50 mm).

Point 9: Lines 179-185: The list of lure combinations is difficult to read in paragraph form. Consider listing them in a table.

Response 9: Thanks for your suggestion. We have listed the lures in table 1 in our manuscript. Page 5 Table1.

Point 10: Line 187: This paragraph should have a “Statistical analysis” header. Right now, it reads like a part of field trapping, but you discuss statistical analyses for many different experiments in the study.

Response 10: Thanks for your advice. The “Statistical analysis” header was added in our manuscript. Page 5; line 190.

Point 11: Lines 230-231: Is it odd that hind gut extracts of males and females did not differ no matter which stage of galleries they were collected from?

Response 11: Thanks for your question. We have modified as “Similarly, there were no significant differences for oxygenated monoterpenes (cis-verbenol, trans-verbenol, myrtenol, and verbenone) between the females and males of T. brevipilosus collected from galleries of the same length (P > 0.05).” in our manuscript. Page 6; lines 232-234.

Point 12: Figures 4 and 5: please indicate whether the error bars are standard error or standard deviation.

Response 12: Thanks for your advice. The error bars are standard error in Figures 4 and 5. Figures 4 and 5 have adjusted according to the comments in our manuscript.

Point 13: The references are neat and orderly, with only a few minor typographical inconsistancies throughout, including commas instead of colons between the issue number and the page numbers; n-dashes instead of m-dashes between page numbers; and the absence of commas after some last names.

Response 13: Thanks for your reminding. We have revised these typographical inconsistancies of references in our manuscript.

 Author Response File: Author Response.docx

Reviewer 3 Report

The work examined factors governing host colonization by the bark beetle Tomicus brevipilosus in Pinus kesiya. While not unique, the project did combine multiple aspects of the beetle’s colonization (pattern and chemistry) to provide an in-depth examination of the attack process. It also examined the patterns in a single tree species which does add to the knowledge we have on this species.

 In the discussion, (if possible), the authors should put their new data into the context of predation (were any predators found associated with the beetles in the trees or in the baited traps), competition (which they do mention but they also state that it is the only scolytid in the trees – also, were any potential competitors captured in the traps) and phloem thickness (most scolytids are limited by thickness and attacking in the crown where phloem thickness is smaller and then moving to attack the thicker phloem seems counter-intuitive to me).

 The authors took a few liberties with the description of the mating status and I feel the study would have benefited from using newly emerged adults. That way, the actual (and physiological) age of the organisms would not be in question. That said, I still feel that the manuscript should be published following minor revisions.

 The title represents bark beetles as a stand-alone family. The group is currently classified as a subfamily (Scolytinae) within the family Curculionidae. The title should represent the proper taxonomic listing for the group.

 The ‘mating status’ as presented on page 3; lines 95-99 is understandable but (as mentioned above) I feel the beetles could have been selected so that a more uniform group of beetles were being tested.

 The ‘age’ of the beetles is also a bit confusing on page 3; lines 100-103. How long were they stored prior to being used in the tests? I am assuming that they were only stored for 24 hours (as is stated on page 4; line 143) but this should be clarified.

 I would like to see some description of the ‘modified’ open arena olfactometer that was used in the project (page 4; lines 161-163). The reference is needed, but just a sentence or two would help the reader.

Author Response

Point 1: In the discussion, (if possible), the authors should put their new data into the context of predation (were any predators found associated with the beetles in the trees or in the baited traps), competition (which they do mention but they also state that it is the only scolytid in the trees – also, were any potential competitors captured in the traps) and phloem thickness (most scolytids are limited by thickness and attacking in the crown where phloem thickness is smaller and then moving to attack the thicker phloem seems counter-intuitive to me).

Response 1: Thanks for your advice. In previous study, T. brevipilosus coexisted with T. minor and T. yunnanensis, caused extensive mortality of P. yunnanensis. Interestingly, we found T. brevipilosus was the only bark beetle on P. kesiya. Meanwhile, we did not find other species on the host. And, when attacked alone, this beetle showed a top-down attack pattern on P. kesiya (manuscript), while exhibited a bottom-up attack pattern on P. yunnanensis [Reference 17. 17. Chen, P., Lu, J., Haack, R.A., Ye, H. Attack pattern and reproductive ecology of Tomicus brevipilosus (Coleoptera: Curculionidae) on Pinus yunnanensis in southwestern china. J Insect Sci. 2015, 15(1)]. The reason was still unclear that T. brevipilosus showed an opposite attack pattern on two hosts. The volatiles and phloem thickness of the host might affect the attack pattern, which needed an intensive study.

Point 2: The authors took a few liberties with the description of the mating status and I feel the study would have benefited from using newly emerged adults. That way, the actual (and physiological) age of the organisms would not be in question. That said, I still feel that the manuscript should be published following minor revisions.

Response 2: Thanks for your advice very much. In our experiment, the colonization stages of beetles were just sampled by the length of the galleries (page 3; lines 101). The chemical constituents would have some differences between the mated and non-mated beetles. The different states of hindgut volatiles should be analyzed in the following study.

Point 3: The title represents bark beetles as a stand-alone family. The group is currently classified as a subfamily (Scolytinae) within the family Curculionidae. The title should represent the proper taxonomic listing for the group.

Response 3: Thanks for your advice. The title has been modified as “Initial location preference together with aggregation pheromones regulate the attack pattern of Tomicus brevipilosus (Coleoptera: Curculionidae) on Pinus kesiya”.

Point 4: The ‘mating status’ as presented on page 3; lines 95-99 is understandable but (as mentioned above) I feel the beetles could have been selected so that a more uniform group of beetles were being tested.

Response 4: Thanks for your advice. In our experiment, the colonization stages of beetles were just sampled by the length of the galleries, but the mated and non-mated beetles were not separated. The beetles should be selected by different states, and tested separately in following study.

Point 5: The ‘age’ of the beetles is also a bit confusing on page 3; lines 100-103. How long were they stored prior to being used in the tests? I am assuming that they were only stored for 24 hours (as is stated on page 4; line 143) but this should be clarified.

Response 5: Thanks for your advice. The beetles were stored at 4°C for 24 hours before testing, we have descripted it in the manuscript, page 3; line 108-109.

Point 6: I would like to see some description of the ‘modified’ open arena olfactometer that was used in the project (page 4; lines 161-163). The reference is needed, but just a sentence or two would help the reader.

Response 6: Thanks for your suggestion. Some description of the ‘modified’ open arena olfactometer that was used in the project have been added in the manuscript. Page 4; line 171-174.

  Author Response File: Author Response.docx