Cold Tolerance and Cold-Resistant Substances in Two Tomicus Species during Critical Transferring Periods

Round 1

Reviewer 1 Report

The document is well written, it contributes to the knowledge about phytosanitary treatments against two species of insects with economic importance, Tomicus minor and Tomicus yunnanensis. In addition, they determine the importance of threalose, sorbitol, protein, water content in development. The introduction is clear and precise. The materials and methods are well written. However, the experimental design is not clear, two species are included (the results are clear in the presentation, as well as the discussion), the supercooling point (SCP), freezing point (FP) and antifreeze protective substances of two Tomicus are determined. species adults in two periods. But the results of Table 1 and figure 1 include the results of the transfer-ringperiods, two species by sex for the SCP and FP. It is understood that these are the temperatures at which SCP and FP are achieved, is that correct?

But, Figure 2. Contents of substances in female and male adults of two Tomicus during the shoot to trunk and trunk to shoot periods, and Figure 3. Contents of substances in adults of two Tomicus species at two periods, with these titles not describing the differences between the substances in the supercooling and freezing insects. But the title of the manuscript implies that the substances were determined in chilled insects in two transferring periods "Cold tolerance and cold-resistant substances of two Tomicus species during two transferring periods". About this, the authors should describe more clearly the results and the corresponding experimental design, and I am willing to review it again. Considering that the document has an important and valuable contribution that should be published, above all because it includes some substances such as water content, trehalose, sorbitol and proteins that may be involved in tolerance to cold, and not only is this knowledge important for the application of cooling as a phytosanitary treatment, this knowledge is also important to improve the cooling process of released insects for the application of the sterile insect technique; as well as parasitoids that are cooled to 0-3°C. something like FP and that the published documents indicate that they are susceptible to chilling.

I have included some remarks in the pdf.

 

 

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

 

Point 1: However, the experimental design is not clear, two species are included (the results are clear in the presentation, as well as the discussion), the supercooling point (SCP), freezing point (FP) and antifreeze protective substances of two Tomicus are determined.

 

Response 1: Thank you for your valuable advice. The corresponding changes have been made. To study cold tolerance of Tomicus, we designed three experimental treatments, (1) different sex of two Tomicus species, (2) different species (T. minor and T. yunnanensis) and (3) different development stages, namely the shoot to trunk period and trunk to shoot period of two Tomicus species.

 

Point 2: “But the results of Table 1 and figure 1 include the results of the transfer-ringperiods, two species by sex for the SCP and FP. It is understood that these are the temperatures at which SCP and FP are achieved, is that correct”?

 

Response 2: Thank you so much for your comments, that these are the temperatures at which SCP and FP are achieved of two Tomicus species.

 

Point 3: “But, Figure 2. Contents of substances in female and male adults of two Tomicus during the shoot to trunk and trunk to shoot periods, and Figure 3. Contents of substances in adults of two Tomicus species at two periods, with these titles not describing the differences between the substances in the supercooling and freezing insects”.

 

Response 3: Thank you for your innovative ideas. The substances corresponding to the SCP and FP is a very important index. Describing the differences between the substances in the supercooling and freezing two species of Tomicus was not studied in this paper, we are also trying to solve the shortage of low temperature incubators so that we could conduct related experiments in the next step, which could guide the physical prevention and control of quarantine pests, and that's what we're going to do next.

Point 4:I have included some remarks in the pdf.

4.1 Explain the experimental design and the cooling curves

 

Response 4.1: Thank you for your valuable advice. The corresponding changes have been made. To study cold tolerance of Tomicus, we designed three experimental treatments, (1) different sex of two Tomicus species, (2) different species (T. minor and T. yunnanensis) and (3) different development stages, namely the shoot to trunk period and trunk to shoot period of two Tomicus species.

The temperature at which the insects started to freeze was taken as the SCP. After insects freeze, they release heat, which can be seen shown as a jump in the temperature curve, and this peak was considered the FP.

 

4.2 Adjust the format

Response 4.2: Thank you for your valuable advice.Table 1 has been adjusted accordingly ，Please see Table 1 .

 

4.3 Comple the phrase

 

Response 4.3: I’m sorry, these are all errors caused by the author’s carelessness and have been 3.2 conte changed 3.2 Content of cold-resistant substances .

 

4.4 This paragraph is in results format, is not discussion

 

Response 4.3: Thank you so much for your patient and thoughtful comments, the corresponding changes have been made. Please see Line 291-300.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This study examined the cold tolerance mechanisms of Tomicus minor and T. yunnanensis, the important stem borer of the pine. Overall, the results are interesting and will be very useful for further investigation. However, I think the paper needed significant English improvement. In addition, it will be useful to add information or provide a discussion on the behavioral adaption for cold tolerance of these species, if any.

Following are my specific comments and suggestions:

- Line 41 – 44, Suggest changing to “Two species, T. minor Hartig and T. yunnanesis Kirkendall & Faccoli are well-known pests infested Pinus yunnanensis in southwest China. Tomicus yunnanensis occur in southwest China previously recognized as a member of T. piniperda but molecular examination revealed that it was genetically very different from other populations thus, formally described as new species. This species is an unusually highly aggressive pine shoot beetle. [1,2]”

- Line 69, suggests changing to “For example, a pine beetle, Dendroctonus armandi Tsai and Li …..”

- Line 100, replace “experiment” with “further study.”

- Line 178 – 201, I suggest changing to “Sex difference: During the shoot to trunk period, there were no differences in the SCP and FP between female and male adults of the two Tomicus specie. However, the SCP and FP were significantly lower in females during the trunk to shoot period (Table 1).”

Species difference: There were no significant differences in the SCP and FP between T. minor and T. yunnanensis in the same period (Table 1).

Period difference: There were significant differences in the SCP and FP between T. minor and T. yunnanensis in the different period. The SCP and FP of both Tomicus species during the trunk to shoot period were significantly lower than those of the shoot to trunk period. T. minor (Figure 1).

 

The df and P values can be added as an additional columns in Table 1.

 

- Table 1 and Figure 1, what is the difference between the information report in Table 1 and Figure 1?

 

- Line 189 – 190, I am confused about the values reported in the text e.g. “the median SCP and FP of the two Tomicus species were (T. minor, SCP: -20.68 ℃, FP: -16.77 ℃, T. yunnanensis, SCP: -20.56 ℃ FP: -

16.00 ℃) respectively,…” Values of SCP and FP reported here are different from those reported in Table 1. Why? Please clarify this.

 

- Line 225 – 231, suggests changing to “…. species during the shoot to trunk period. The protein concentration, water, and glycogen contents of females were significantly higher in T. minor. However, the trehalose contents (Figure 2-C) in the male of T. minor was significantly higher than that of T. yunnanensis. At the same period, there was no significant difference in the contents of the other substances between the two Tomicus species in both sexes.”

 

- Line 336-337, “The water content of the female of T. minor was higher than that of the female of T. yunnanensis during the shoot to trunk period.”

It will be useful to explain or provide a discussion here why females of these species are different in water contents.

 

-Line 358 – 364, “Glycogen is also an important energy substance, and the total sugar content can also indicate the cold tolerance of insects [29]. The results showed that the content of glycogen in T. minor during the shoot to trunk period was higher than that of T. yunnanensis, this content of the adults of the two Tomicus species between the two periods was significantly different. This may be because the metabolism of the two Tomicus species needs more energy during the shoot to trunk period, at the same time, Tomicus in this phase faced overwintering and breeding offspring [36].”

It will be useful to explain why glycogen content in T. minor is higher than T. yunnanensis.

 

 

Author Response

Response to Reviewer 2 Comments

 

Point 1 - Line 41 – 44, Suggest changing to “Two species, T. minor Hartig and T. yunnanesis Kirkendall & Faccoli are well-known pests infested Pinus yunnanensis in southwest China. Tomicus yunnanensis occur in southwest China previously recognized as a member of T. piniperda but molecular examination revealed that it was genetically very different from other populations thus, formally described as new species. This species is an unusually highly aggressive pine shoot beetle. [1,2]”

Response 1: Thank you so much for your patient and thoughtful comments, the corresponding changes have been made. Please see Line 38-43.

Point 2 - Line 69, suggests changing to “For example, a pine beetle, Dendroctonus armandi Tsai and Li …..”

Response 2: Thank you so much for your patient and thoughtful comments, the corresponding changes have been made. Please see Lines 67.

Point 3 “- Line 100, replace “experiment” with “further study.”

Response 3: Thank you so much for your patient and thoughtful comments, the corresponding changes have been made. Please see Lines 100.

 

Point 4 - Line 178 – 201, I suggest changing to “Sex difference: During the shoot to trunk period, there were no differences in the SCP and FP between female and male adults of the two Tomicus specie. However, the SCP and FP were significantly lower in females during the trunk to shoot period (Table 1).”Species difference: There were no significant differences in the SCP and FP between T. minor and T. yunnanensis in the same period (Table 1).

Period difference: There were significant differences in the SCP and FP between T. minor and T. yunnanensis in the different period. The SCP and FP of both Tomicus species during the trunk to shoot period were significantly lower than those of the shoot to trunk period T. minor (Figure 1).

Response 4: Thank you so much for your patient and thoughtful comments, the corresponding changes have been made. Please see Lines 189-200.

Point 5 The df and P values can be added as an additional columns in Table

Response 5: Thank you so much for your patient and thoughtful comments, and we have changed Table 1. Please see Table 1 and description.

Point 6 - Table 1 and Figure 1, what is the difference between the information report in Table 1 and Figure 1?

Response 6:

Table 1. SCP and FP (the difference between male and female) of two species Tomicus in different periods.

Figure 1, SCP and FP (difference between two species) of two species Tomicus adults at different period.

Point 7 - Line 189 – 190, I am confused about the values reported in the text e.g. “the median SCP and FP of the two Tomicus species were (T. minor, SCP: -20.68℃, FP: -16.77℃, T. yunnanensis, SCP: -20.56℃ FP: -16.00℃) respectively,…” Values of SCP and FP reported here are different from those reported in Table 1. Why? Please clarify this.

Response 7: Reported here SCP and FP are (difference between two species )of two species Tomicus adults at different period.Table 1. SCP and FP (the difference between male and female) of two species Tomicus in different periods.

Point 8 - Line 225 – 231, suggests changing to “…. species during the shoot to trunk period. The protein concentration, water, and glycogen contents of females were significantly higher in T. minor. However, the trehalose contents (Figure 2-C) in the male of T. minor was significantly higher than that of T. yunnanensis. At the same period, there was no significant difference in the contents of the other substances between the two Tomicus species in both sexes.”

Response 8: Thank you so much for your patient and thoughtful comments, the corresponding changes have been made. Please see Lines 215-219.

Point 9 - Line 336-337, “The water content of the female of T. minor was higher than that of the female of T. yunnanensis during the shoot to trunk period.”It will be useful to explain or provide a discussion here why females of these species are different in water contents.

Response 9: Thank you so much for your patient and thoughtful comments, “why females of these species are different in water contents”. The corresponding discussion have been made. Please see Lines 326-333.

Point 10 -Line 358 – 364, “Glycogen is also an important energy substance, and the total sugar content can also indicate the cold tolerance of insects [29]. The results showed that the content of glycogen in T. minor during the shoot to trunk period was higher than that of T. yunnanensis, this content of the adults of the two Tomicus species between the two periods was significantly different. This may be because the metabolism of the two Tomicus species needs more energy during the shoot to trunk period, at the same time, Tomicus in this phase faced overwintering and breeding offspring [36].”It will be useful to explain why glycogen content in T. minor is higher than T. yunnanensis.

Response 10: Thank you so much for your patient and thoughtful comments, “why glycogen content in T. minor is higher than T. yunnanensis”. The corresponding explain have been made. Please see Lines 349-359.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I have reviewed the new version and checked the changes and the authors have made an effort to improve the document, so I have no problem recommending it for publication.