PONE-D-22-20345The title is

Semiochemicals produced by fungal bark beetle symbiont Endoconidiophora rufipennis and the discovery of an anti-attractant for Ips typographusPLOS ONE

Dear Dr. Unelius,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers found your study to be of considerable interest and worthy of publication subject to minor textual changes. Reviewer 2 has provided quite a long list of these and I draw your particular attention to their detailed comments running between "Lines 204..." to "Lines 477...". Their comments regarding the possible use of generalised linear models and calibration of your SPME data I regard as optional. 

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PLOS ONE

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“This work was supported by two grants from The Carl Trygger Foundation, https://www.carltryggersstiftelse.se/this-is-the-carl-trygger-foundation/ and Linnaeus University, Kalmar, Sweden https://lnu.se/en/ (CRU and SG). This research was also financially supported by the Regional Development Fund for Västernorrland and Jämtland/Härjedalen county and the Swedish Agency for Economic and Regional Growth (EAW and ML). FS was supported by project EXTEMIT-K CZ.02.1.01/0.0/0.0/15_003/0000433 financed by OP RDE at the Czech University of Life Sciences, Prague. We thank Dr J-E Englund at SLU, Alnarp for statistical advice and SCA Forest AB for allowing the field trial to be conducted on their land.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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“This work was supported by two grants from The Carl Trygger Foundation, (CRU and SG), and Linnaeus University, Kalmar, Sweden. This research was also financially supported by the Regional Development Fund for Västernorrland and Jämtland/Härjedalen county and the Swedish Agency for Economic and Regional Growth (EAW and ML). FS was supported by project EXTEMIT-K CZ.02.1.01/0.0/0.0/15_003/0000433 financed by OP RDE at the Czech University of Life Sciences, Prague. We thank Dr J-E Englund at SLU, Alnarp for statistical advice and SCA Forest AB for allowing the field trial to be conducted on their land.”

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“This work was supported by two grants from The Carl Trygger Foundation, https://www.carltryggersstiftelse.se/this-is-the-carl-trygger-foundation/  and Linnaeus University, Kalmar, Sweden https://lnu.se/en/ (CRU and SG). This research was also financially supported by the Regional Development Fund for Västernorrland and Jämtland/Härjedalen county and the Swedish Agency for Economic and Regional Growth (EAW and ML). FS was supported by project EXTEMIT-K CZ.02.1.01/0.0/0.0/15_003/0000433 financed by OP RDE at the Czech University of Life Sciences, Prague. We thank Dr J-E Englund at SLU, Alnarp for statistical advice and SCA Forest AB for allowing the field trial to be conducted on their land.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an excellent manuscript that describes a very well designed and thoroughly conducted investigation that combines chemical analyses of volatiles from bark beetle fungal symbionts and behavioral responses of those beetles to identified volatiles. A new 'anti-attractant' was identified from a blue stain fungus that may have practical utility against I. typographus beetles. The manuscript is clear and concise. The conclusions are based on the results. The methods can be fully replicated based on the description. The results are clear cut. The data are well placed within the context of the literature.

Couple minor edits:

Line 269: Write out ANOVA when first used.

Line 379: Missing full stop (period).

Reviewer #2: This paper analyzed volatile emissions from laboratory cultures of the blue stain fungus Endoconidiophora rufipennis associated with the North American spruce bark beetle Dendroctonus rufipennis which is closely related to the fungus associated with the Eurasian spruce bark beetle Ips typographus. Several volatile compounds were identified of which 3 were selected and field tested in Europe for attraction or inhibition of Ips typographus. Volatiles of the North American fungus were analyzed as a potential source of new attractants or anti-attractant compounds and because Ips typographus is often intercepted in North America and could eventually be introduced and expand its geographic range there where it may encounter or become associated with this fungal species. One of the fungal volatiles tested significantly inhibited attraction of Ips typographus to its pheromone and thus could have potential as an inexpensive anti-attractant. Overall, the paper is well-written and the methods and results are clearly described and presented. It is the first study to test fungal volatiles’ long range attraction or inhibition in field trapping experiments. Ips typographus is a major pest of spruce trees in Europe and one of the most commonly intercepted bark beetles or woodborers in solid wood packing material and is of great concern as a potential invasive threat to North America. Therefore this study is of interest as it contributes new knowledge about Ips typographus responses to a North American fungus and identifies a compound which may be useful for anti-aggregation. I believe it is of interest and fits the scope of PLOSone and would be suitable for publication with minor revisions. I have no major concerns with the paper, and only a few minor suggestions.

For the statistical analysis of trap catch data, it sounds like the authors tried various transformations and models (raw data, log transformed, relative or proportional catch) and models (all factorial combinations of treatment and block effects) to find a model that fit best. It would seem more appropriate to look at the distribution of the data and use a generalized linear mixed model with the appropriate distribution and link function that fit the observed distribution.

Overall the number of replicates seems somewhat low (only 3 flasks of fungal culture used for volatile collections, and only 3 field sites with one replicate per site and 4 collection periods with treatment positions re-randomized during each collection period for a total of 12 replicates). That being said, a very high number of beetles were captured, there were no time or block by treatment interactions and the sterile media and pure fungal cultures would have little variation, so replication and experimental power appeared to be adequate.

Lines 204-214 - indicate which compounds were provided by which chemical supplier.

Line 222. I suggest adding a brief explanation of how the 3 compounds were selected for field testing in the methods – and not just in the discussion. After “Three chemicals were” insert “selected for testing in the field based on published reports of their activity in bark beetles (see Discussion for detailed rationale). The chemicals were”….

Line 232. Change “For each dispenser, 1 mL of fungal compound was distributed onto the felt in the dispenser bag” to “For each of the test fungal compounds, 1 mL of synthetic pure compound was distributed onto the felt in a dispenser bag”

Line 243. For the Lindgren funnel traps, how many funnel units did they have? What killing agent was used in the collection cups – dry cups with insecticide strip, or wet cups with liquid preservative?

Line 389. What species were odour receptor neurons found in for 2-Phenethyl acetate? Is Ips typographus known to have receptors for this compound?

Line 396. Add a reference for the statement that geranyl acetone has been reported as a common green leaf volatile

Line 398. Add a reference for the statement that geranyl acetate has been demonstrated as an attractant or antiattractant in several taxa.

Line 404. Add a reference for the statement that geranyl acetate as EAD activity in Ips subelongatus.

Line 410-411. Reword sentence – it seems awkward. “but electophsioloical activity alone does not inform of valence of an odour for behavior” Maybe something like “but electophsyiology activity alone does not infer behavioral activity (attraction, inhibition, or none)”

Line 420. Summarize and cite any literature of known physiological or behavioral reponses to geranyl acetate.

Line 440. “have been reported as electophysiologically active” in what species?

Line 467. Add a reference for the statement that Geranyl acetone is relatively low cost compound compared to other anti attractants, and for the anti-attraction activity of 1-octen-3-ol, trans-conophthorin, and thujanol.

Line 468. Add a reference for the lower volatility of geranyl acetone compared to 1,8-cineole

Line 477-478. Add a reference for the limited availabilityia nd high host of other anti-attractants trans-conophthorin and verbenone.

Table 1. In addition to the relative amounts, it might be useful to give the total daily quantity of volatiles emitted. Although there is no quantitative internal standard when using SPME fibers for volatile collection, since the volume of the airspace was the same each day, and the length of time that the SPME fiber was exposed in the airspace, it seems that there should be a way to standardize the total amount of volatiles captured on each day and report them as ng or pg per mL of headspace or something like that? How do the volatile rates compare to the release rates of lures?

Table 2. List the chemical supplier for each compound – perhaps as a footnote

Figure 5. I suggest adding “+ Synth Pher” to each of the treatment labels on the X-axis t clarify and emphasize that each compound was tested in combination with the synthetic pheromone. I also suggest using the letter “a” on the three bars with the highest values and the letter “b” on the bar for Geranyl acetone with the lowest value. The insert with the schematic for the block layout is not necessary and kind of confusing.

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Reviewer #1: Yes: Lukasz Stelinski

Reviewer #2: No

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Semiochemicals produced by fungal bark beetle symbiont Endoconidiophora rufipennis and the discovery of an anti-attractant for Ips typographus

PONE-D-22-20345R1

Dear Dr. Unelius,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Christopher Walton, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Therese Poland

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