PONE-D-23-00043Aphid facultative symbionts confer no protection against the fungal entomopathogen Batkoa apiculataPLOS ONE

Dear Dr. Parker,

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.

Please submit your revised manuscript by Mar 25 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Nafiu Bala Sanda, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"RI was part of the NSF Research Experience for Undergraduates Program “Microbial community interactions and functions” DBI-2050743. This work was also funded by National Science Foundation (NSF) Grant IOS-2152954 to BJP. "

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"This work was funded by US National Science Foundation (https://www.nsf.gov) Grant IOS-2152954 to BJP. RI was part of the NSF Research Experience for Undergraduates Program “Microbial community interactions and functions” DBI-2050743. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

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: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: No

**********

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: It has been well known that some facultative symbiotic bacteria protect aphids from infection by certain fungal strains. However it is not clear whether this protection is universal in term of lines of aphids, strains of symbiotic bacteria and pathogenic fungi. In this manuscript, the authors present solid evidences showing such symbiont-mediated protection does not exist to infection against Batkoa apiculate. These results will lead further study on ecological and molecular mechanisms of symbiont-mediated protection in aphids.

Reviewer #2: Review of PONE-D-23-00043 - Aphid facultative symbionts confer no protection against the fungal entomopathogen Batkoa apiculata by Inchauregui, Tallapragada, and Parker

Comments for authors

I like the idea of the MS. Pea aphids have a number of microbial endosymbionts, some of which have protective functions. Here the authors focus on antifungal activities, and attempt to show that the mechanisms that protect the aphids against one common fungal pathogen may not protect against a closely related fungus. This is a limited objective, but potentially, an informative and consequential one.

I have two sets of reservations about the MS. In each case, I suspect that the authors may have the required information, but simply haven’t yet provided it in the MS. Please note, my expertise does not extend to the more molecular methods used to identify pathogens. I cannot speak to that work.

Methodology

- Pea aphids have a variety of (mostly) vertically transmitted, microbial endosymbionts including some not mentioned here (e.g., Buchnera aphidicola, Hamiltonella defensa - Buchnera is unlikely to be defensive, but is an obligate symbiont, Hamiltonella has several roles.) The bacteria mediate a number of services to the aphids, and occur in various combinations which may interact and may be functionally and mechanistically important (see refs below). This makes me ask the following methodological questions:

1) How was the aphid stock ‘cured’ of R. insecticola (lines 108-110)? - in particular, which other endosymbionts were (likely) cured in the same process, and what endosymbionts (likely) remained in the stock?

2) Did you assess the effectiveness of the endosymbiont infection injections in establishing infections with the desired endosymbionts (lines 115-118)? Similarly, did you attempt to assess which other endosymbionts (if any) were harbored by the various lines produced?

3) Were there any procedural controls that assessed the effectiveness and uniformity of success of the methods used to infect the aphids with the fungi? I ask particularly because the strongly significant block effect (line 167), when the blocks were treated as fixed variables, suggests not just that repeated blocks differed, but that block 1 differed systematically from block 2. Unless, blocks 1 & 2 differed systematically as part of the experimental design, the fungal infection rates should not have differed strongly and significantly. If that effect is real, it should be presented and explained.

Without those clarifications, it is difficult to assess the meaning of Fig. 2.

Statistics

I agree that the fungal infection/protection data should be analyzed via a generalized liner model, but:

4) We need a better description of the model used. What type of GLM was used? The methods suggest that a logistic/binomial-family GLM was most appropriate, but the symmetric error bars on Fig. 2 and the use of the ‘multcomp’ package made me suspect that a normal/gaussian GLM was used. With infection rates running as low as 10%, I suspect that the logistic GLM is much more defensible.

5) I was surprised that you chose to model the block effect as a fixed effect. Unless Block 1 really did differ systematically from Block 2 in the design of the experiment (e.g., Block 1 was done first and there was a time/duration/extinction effect), it seems more defensible to treat the blocks as random (blocking) effects in a mixed model (e.g., via the ‘glmer’ function in the lme4 package). This is particularly important when comparing the honest-to-goodness fixed effects (here the lines/symbionts) because the blocking effects, in classical theory, become part of the error variance for testing the fixed effects. The ‘glmer’ function handles this properly. - - I think we can ignore the ‘shrinkage’ effects commonly discussed in mixed model random effects because you don’t get much shrinkage with only two blocks with nearly balanced sample sizes.

6) I have always been nervous about using the ‘multcomp’ package for logistic regressions when some of the effects are close to 0 or 1 (e.g., 0.1 Fig. 2). For GLM’s, multcomp uses a likelihood ratio test that assumes the likelihood is symmetrically distributed around the MLE, which may well not be true in logistic regressions (with some extreme effects) because of the non-linear transformation of the linear result (the logit) to the probability value, and the hard bound on probabilities of infection at 0 and 1. One way to get around this is to use a Bayesian regression and directly test the ‘Bayesian p values’ comparing each line to the ‘None’ control.

Smaller points - Fig. 2

- I was confused by the ‘Percent sporulated’ label on the dependent axis. Isn’t this just percent cadavers (line 137-138)?

- The confidence intervals for each line will be asymmetric in a logistic regression, so the ‘+/- 1 SE’ error bars can be misleading. I’d suggest converting to confidence intervals.

References:

Oliver, K. M., Moran, N. A., & Hunter, M. S. (2005). Variation in resistance to parasitism in aphids is due to symbionts not host genotype. PNAS 102(36), 12795-12800.

Rock, D. I., et al.. (2018). Context-dependent vertical transmission shapes strong endosymbiont community structure in the pea aphid, Acyrthosiphon pisum. Molecular Ecology, 27(8), 2039-2056.

Smith, A. H., et al. (2015). Patterns, causes and consequences of defensive microbiome dynamics across multiple scales. Molecular Ecology, 24(5), 1135-1149.

Reviewer #3: I read the manuscript by Inchauregui et al. with interest. The paper aims to investigate the impact of the aphid facultative symbiont against the the fungal entomopathogen Batkoa apiculata. In general the topic is relevant however there is some comments:

I suggest authors to motivate the choice of facultative bacteria tested in this study. On the basis of what they were chosen? Please improve this aspect because it is the purpose of the work.

Section Material and Methods:

Line 109: Please explain how you can verify the infection status of both subclones cured and infected aphid with Regiella insecticola. The infection of the aphid was verified using diagnostic PCR? If yes, please give a full description of the method.

Line 110: please add your laboratory conditions used in this experiment. Aphids were reared on V. faba seedlings at which temperature? Photoperiod?

Line 113: you used species-specific primers? Give details please? And what about other facultative symbionts such as Hamiltonella defensa? You are sure that your aphid without this bacteria? Have you used specific primers?

Line 128: are you sure for the age of your aphids used in this experiment? 11 days old?!!! I think it’s too old.

Section Discussion:

The discussion section is very short and should be supported with previous work. For example:

1.The aphid facultative symbiont Serratia symbiotica influences the foraging behaviors and the life-history traits of the parasitoid Aphidius ervi (Entomologia Generalis)

2. Effects of pea aphid secondary endosymbionts on aphid resistance and development of the aphid parasitoid Aphidius ervi: A correlative study. (Entomologia Experimentalis et Applicata)

**********

6. 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 #1: No

Reviewer #2: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Review of PONE-D-23-00043 - Aphid facultative symbionts.pdf

PONE-D-23-00043R1Aphid facultative symbionts confer no protection against the fungal entomopathogen Batkoa apiculataPLOS ONE

Dear Dr. Parker Benjamin,

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.

Please submit your revised manuscript by Jun 03 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Nafiu Bala Sanda, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The manuscript can be accepted pending minor revision suggested by the reviewers.

[Note: HTML markup is below. Please do not edit.]

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 #1: (No Response)

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

Reviewer #6: (No Response)

**********

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 #1: (No Response)

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

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 #1: (No Response)

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

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 #1: (No Response)

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

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 #1: (No Response)

Reviewer #4: In their paper, Inchauregui et al. studied the influence of facultative symbionts on the ability of aphids to resist a generalist entomopathogenic fungus. They showed that the facultative symbionts tested did not confer any protection against the fungus Batkoa apiculata. Although this is more of a case study (only one species of fungus tested) with negative results that are difficult to generalize, the study is very well done and addresses an area of research that remains understudied. Despite this and its concise form, the study is well written, to the point, and sets the stage for studying the mechanisms behind these protective effects. I have a few minor comments below.

L48: Perhaps also cite Hansen et al. 2012 (1).

L77: Where do these uninfected aphids exposed to spores come from? From the field? Are these aphids infected by facultative symbionts?

L113-114: Was the screening done before or after the 10 generations were obtained? It is not clear to me if it was done after (which is necessary to ensure that the relationship between host and symbiont has stabilized).

L120: Do you know more about the Serratia symbiotica 509 strain? Are there any studies that have characterized the associated induced phenotypic effects?

L128: The aphids are 11 days old. They are young adults. But is this the same developmental stage as in previous studies that showed protective effects against entomopathogenic fungi?

L186-187: The subject behind "strains" is not clear. I assume that the authors are referring to Regiella. But since only one strain of Regiella has been tested, the subject should be expressed in the singular.

I have reread the article several times and have no further comments. Perhaps the discussion could have been more in depth (2), but perhaps it is best to keep it concise here due to the brevity of the experience. Perhaps raise the idea that fungal pathogens can switch from parasitism to mutualism as in cicadas with an interest in studying these interactions in other Hemiptera (3–6). I would have many more thoughts to share, but I would stray from the basic article. I hope the authors will teach us more about insect-symbiont-fungus interactions.

1. Hansen AK, Vorburger C, Moran NA. 2012. Genomic basis of endosymbiont-conferred protection against an insect parasitoid. Genome Res 22:106–114.

2. Gibson CM, Hunter MS. 2010. Extraordinarily widespread and fantastically complex: comparative biology of endosymbiotic bacterial and fungal mutualists of insects. Ecol Lett 13:223–234.

3. Huang Z, Zhou J, Zhang Z, He H, Wei C. 2023. A Study on Symbiotic Systems of Cicadas Provides New Insights into Distribution of Microbial Symbionts and Improves Fluorescence In Situ Hybridization Technique. 3. Int J Mol Sci 24:2434.

4. Matsuura Y, Moriyama M, Łukasik P, Vanderpool D, Tanahashi M, Meng X-Y, McCutcheon JP, Fukatsu T. 2018. Recurrent symbiont recruitment from fungal parasites in cicadas. Proc Natl Acad Sci 115:E5970–E5979.

5. Michalik A, Franco DC, Deng J, Szklarzewicz T, Stroiński A, Kobiałka M, Łukasik P. 2023. Variable organization of symbiont-containing tissue across planthoppers hosting different heritable endosymbionts. Front Physiol 14.

6. Kobiałka M, Michalik A, Walczak M, Szklarzewicz T. 2018. Dual “Bacterial-Fungal” Symbiosis in Deltocephalinae Leafhoppers (Insecta, Hemiptera, Cicadomorpha: Cicadellidae). Microb Ecol 75:771–782.

Reviewer #5: (No Response)

Reviewer #6: (No Response)

**********

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 #1: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: Yes: Chen Luo

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: answer to authors.docx

Aphid facultative symbionts confer no protection against the fungal entomopathogen Batkoa apiculata

PONE-D-23-00043R2

Dear Dr. Benjamin Parker,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nafiu Bala Sanda, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Having addressed the concerned raised by the reviewers, the manuscript can be accepted for publication in PLOS ONE, congratulations!

Reviewers' comments: