A novel mitovirus associated with the fungal entomopathogen Zoophthora radicans

Meaghan J. Adler; McKayla M. Martin; Paula Rozo-Lopez; Benjamin J. Parker

doi:10.1371/journal.pone.0331239

Peer Review History

Original SubmissionMay 14, 2025
1 Jul 2025 Decision Letter - Humberto Julio Debat, Editor PONE-D-25-25639A novel mitovirus associated with the fungal entomopathogen Zoophthora radicansPLOS ONE Dear Dr. Adler, 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 revise and improve as mentioned by reviewer #2 data availability, methods descriptions, and phylogenetic relationships, as their suggestions are relevant for reproducibility and transparency. I do not expect a virus isolation experiment, just keep in mind the comments to revise in the discussion the limitations for host assignment. ============================== Please submit your revised manuscript by Aug 15 2025 11:59PM. 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MJA was supported by the SMART scholarship funded by OUSD/R&E (The Under Secretary of Defense-Research and Engineering), National Defense Education Program (NDEP) / BA-1, Basic Research. MMM was supported by a Microbiology Summer Research Award from the Department of Microbiology at UTK. A portion of this project was carried out by MMM in association with a course-based undergraduate research experience (CURE), which was funded by the College of Arts and Sciences at the University of Tennessee Knoxville. BJP is a Pew Scholar in the Biomedical Sciences, funded by the Pew Charitable Trusts.]. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. 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MMM was supported by a Microbiology Summer Research Award from the Department of Microbiology at UTK. A portion of this project was carried out by MMM in association with a course-based undergraduate research experience (CURE), which was funded by the College of Arts and Sciences at the University of Tennessee Knoxville. BJP is a Pew Scholar in the Biomedical Sciences, funded by the Pew Charitable Trusts.]. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. 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: Please revise and improve as mentioned by reviewer #2 data availability, methods descriptions, and phylogenetic relationships, as their suggestions are relevant for reproducibility and transparency. I do not expect a virus isolation experiment, just keep in mind the comments to revise in the discussion the limitations for host assignment. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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: No ****** 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 ****** 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: The manuscript entitled “A novel mitovirus associated with the fungal entomopathogen Zoophthora radicans” by Adler et al. is a straightforward manuscript that reports the molecular characterization of a novel mitovirus associated to a pea aphid´s fungal entomopathogen. It is great that authors conducted the 18S amplicon sequencing and further PCR screening to unequivocally assign the host to the identified mitovirus. Reviewer #2: The study by Adler et al. identifies several viruses present in metatranscriptomic sequencing data for field-collected aphids. Of these viruses, the researchers recovered a contig encoding a novel mitovirus, and its presence in aphids was associated with the co-presence of reads attributed to the fungal entomopathogen, Zoophthora radicans. The data provided is original and provides novel insights into aphid virome diversity, which also includes viruses from plants and fungi. However, there are several minor revisions regarding data availability and methods descriptions, as well major revisions regarding the phylogenetic relationships and the true host of “Zoophthora radicans mitovirus”: - Individual aphid sample metadata should be provided either in main figures or supplemental table: o Which samples were from winged (alate) versus unwinged (aptera) aphids? o Which samples were aphids from T. repens versus T. pratense? o Which individual aphid samples were put into pool 1 and pool 2 used for metatranscriptomic sequencing? - For data reproducibility purposes, the method of cDNA synthesis used for PCR screening individual aphid samples for the novel mitovirus should be stated; it is unclear what “constructed cDNA as above” (line 127) is referring to. - The data provided for RT-PCR confirmation of the mitovirus across the 10 aphid samples is insufficient in Figure 2. PCR gel images should be shown including expected/observed molecular weight of mitovirus amplicons as well as appropriate control groups, such as reference gene controls, cDNA synthesis reactions plus and minus reverse transcriptase, and/or mitovirus RT-PCR for the corresponding gDNA samples. - A statement of what criteria was chosen to call this a “novel” species of Mitoviridae should be included. For example, refer to the International Committee on Taxonomy of Viruses (ICTV) Mitoviridae proposal for this criteria (https://ictv.global/taxonomy). A phylogenetic tree comparing the novel mitovirus RdRp to several other known mitovirus RdRps would also provide a Mitoviridae genus-level assignment and provide a measure of novelty of Zoophthora radicans mitovirus compared to other mitoviruses - For data reproducibility purposes, statements for the parameters used with following tools should be included, even if simply “default parameters were used”: o STAR o Trimmomatic o PriceSeqFilter o Mini-map2 o Diamond o SPADES o bowtie2 o Geneious Prime o NCBI ORFfinder o InterPro - The “true” host of the novel mitovirus could be established by in vitro isolation and culture of Z. radicans from a mitovirus-positive aphid, then showing that Z. radicans alone is RT-PCR positive for the mitovirus. This experiment should be performed if it’s trivial, as this would confirm the host of the novel mitovirus (otherwise it should not be called “Zoophthora radicans mitovirus”). ****** 6. 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Revision 1
7 Aug 2025 Author Response Reponses to reviewers for PLOS One Additional Editor Comments: Please revise and improve as mentioned by reviewer #2 data availability, methods descriptions, and phylogenetic relationships, as their suggestions are relevant for reproducibility and transparency. I do not expect a virus isolation experiment, just keep in mind the comments to revise in the discussion the limitations for host assignment. Thanks to the editor for handling this manuscript. Reviewer #1: The manuscript entitled “A novel mitovirus associated with the fungal entomopathogen Zoophthora radicans” by Adler et al. is a straightforward manuscript that reports the molecular characterization of a novel mitovirus associated to a pea aphid´s fungal entomopathogen. It is great that authors conducted the 18S amplicon sequencing and further PCR screening to unequivocally assign the host to the identified mitovirus. Thanks to Reviewer #1 for their review of this manuscript. Reviewer #2: The study by Adler et al. identifies several viruses present in metatranscriptomic sequencing data for field-collected aphids. Of these viruses, the researchers recovered a contig encoding a novel mitovirus, and its presence in aphids was associated with the co-presence of reads attributed to the fungal entomopathogen, Zoophthora radicans. The data provided is original and provides novel insights into aphid virome diversity, which also includes viruses from plants and fungi. However, there are several minor revisions regarding data availability and methods descriptions, as well major revisions regarding the phylogenetic relationships and the true host of “Zoophthora radicans mitovirus”: Thanks to Reviewer #2 for their comments on the manuscript. Comment #1: - Individual aphid sample metadata should be provided either in main figures or supplemental table: • Which samples were from winged (alate) versus unwinged (aptera) aphids? • Which samples were aphids from T. repens versus T. pratense? • Which individual aphid samples were put into pool 1 and pool 2 used for metatranscriptomic sequencing? As suggested, we now include a table in the Supporting Information (S1 Table) that describes this metadata. Readers are directed to S1 Table in the following methods section sentence: “Samples were a mix of winged (alate) and unwinged (aptera) adults, metadata in S1 Table.” Host plants were identified at a genus level and to a species level, when confident. Comment #2 - For data reproducibility purposes, the method of cDNA synthesis used for PCR screening individual aphid samples for the novel mitovirus should be stated; it is unclear what “constructed cDNA as above” (line 127) is referring to. We added the specific cDNA synthesis kit to the referenced sentence. It now reads, “We extracted RNA from field-collected aphids and constructed cDNA via iScript cDNA Synthesis Kit by BIO-RAD, as directed by the manufacturer.” Comment #3 - The data provided for RT-PCR confirmation of the mitovirus across the 10 aphid samples is insufficient in Figure 2. PCR gel images should be shown including expected/observed molecular weight of mitovirus amplicons as well as appropriate control groups, such as reference gene controls, cDNA synthesis reactions plus and minus reverse transcriptase, and/or mitovirus RT-PCR for the corresponding gDNA samples. We have added a supporting figure, S2 Figure, which is gel images of the Mitovirus PCR screening of aphid samples used in amplicon sequencing in Figure 2. Comment #4 - A statement of what criteria was chosen to call this a “novel” species of Mitoviridae should be included. For example, refer to the International Committee on Taxonomy of Viruses (ICTV) Mitoviridae proposal for this criteria (https://ictv.global/taxonomy). A phylogenetic tree comparing the novel mitovirus RdRp to several other known mitovirus RdRps would also provide a Mitoviridae genus-level assignment and provide a measure of novelty of Zoophthora radicans mitovirus compared to other mitoviruses In the revised results we now clarify: “Species demarcation in this study was based on ICTV Mitoviridae Study Group (SG) criteria of less than 70% amino acid sequence identity to other viral genome sequences (Walker, et al. 2022). Zoopthora radicans mitovirus 1 exhibits less than 70% amino acid sequence identity to the best match in the nr database (see above). We, therefore, concluded that this sequence is a newly described species of Mitovirus that is likely associated with Z. radicans, and in the NCBI submission we refer to this virus as Zoophthora radicans mitovirus 1.” Comment #5 - For data reproducibility purposes, statements for the parameters used with following tools should be included, even if simply “default parameters were used”: o STAR o Trimmomatic o PriceSeqFilter o Mini-map2 o Diamond o SPADES o bowtie2 o Geneious Prime o NCBI ORFfinder o InterPro We have added specific parameters to the bioinformatic processing methods section. The CZ ID Illumina mNGS metagenomic pipeline is publicly available on their github so we have clarified when the pipeline starts and ends in our analysis. The remaining programs (Geneious Prime, NCBI ORFfinder, InterPro) are now described with the version used and the description “with default parameters” as suggested by Reviewer #2 version. It now reads, “With completion of the CZ ID pipeline, we de novo assembled reads with homology to each putative virus using Geneious Prime version 2025.0 using default parameters for the De Novo Assemble tool. With the assembled genomes, we used the NCBI Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/, last modified 2021-05-04) to identify protein-coding regions of the viral genome (using genetic code 1 for putative viruses, and genetic code 3 Yeast Mitochondrial for the putative mitovirus). We used interpro (https://www.ebi.ac.uk/interpro/ release 103.0) to look for conserved genes to annotate viral genomes.” Comment #6 - The “true” host of the novel mitovirus could be established by in vitro isolation and culture of Z. radicans from a mitovirus-positive aphid, then showing that Z. radicans alone is RT-PCR positive for the mitovirus. This experiment should be performed if it’s trivial, as this would confirm the host of the novel mitovirus (otherwise it should not be called “Zoophthora radicans mitovirus”). This would be a definitive way to associate the mitovirus to Z.radicans, but unfortunately, neither in vitro isolation nor culture of a Z.radicans from a known mitovirus-positive field collected aphid is possible. We have used the following sentence to describe the inference made from our data, “We used these data sets to infer that our newly described Mitovirus is likely associated with an entomopathogenic fungus called Zoophthora radicans”. Further, we add an additional caveat in the discussion: “We note, however, that the true host of this novel mitovirus cannot be definitely established without in vitro isolation and culturing, and caution is warranted in the use of the name Zoophthora radicans mitovirus 1 without further investigation.” Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0331239.r002
13 Aug 2025 Decision Letter - Humberto Julio Debat, Editor A novel mitovirus associated with the fungal entomopathogen Zoophthora radicans PONE-D-25-25639R1 Dear Dr. Adler, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . 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, Humberto Julio Debat Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0331239.r003
Formally Accepted
Acceptance Letter - Humberto Julio Debat, Editor PONE-D-25-25639R1 PLOS ONE Dear Dr. Adler, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Humberto Julio Debat Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0331239.r004

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