Virus–prokaryote infection pairs associated with prokaryotic production in a freshwater lake

ABSTRACT Viruses infect and kill prokaryotic populations in a density- or frequency-dependent manner and affect carbon cycling. However, the effects of the stratification transition, including the stratified and de-stratified periods, on the changes in prokaryotic and viral communities and their interactions remain unclear. We conducted a monthly survey of the surface and deep layers of a large and deep freshwater lake (Lake Biwa, Japan) for a year and analyzed the prokaryotic production and prokaryotic and viral community composition. Our analysis revealed that, in the surface layer, 19 prokaryotic species, accounting for approximately 40% of the total prokaryotic abundance, could potentially contribute to the majority of prokaryotic production, which is the highest during the summer and is suppressed by viruses. This suggests that a small fraction of prokaryotes and phages were the key infection pairs during the peak period of prokaryotic activity in the freshwater lake. We also found that approximately 50% of the dominant prokaryotic and viral species in the deep layer were present throughout the study period. This suggests that the “kill the winner” model could explain the viral impact on prokaryotes in the surface layer, but other dynamics may be at play in the deep layer. Furthermore, we found that annual vertical mixing could result in a similar rate of community change between the surface and deep layers. These findings may be valuable in understanding how communities and the interaction among them change when freshwater lake stratification is affected by global warming in the future. IMPORTANCE Viral infection associated with prokaryotic production occurs in a density- or frequency-dependent manner and regulates the prokaryotic community. Stratification transition and annual vertical mixing in freshwater lakes are known to affect the prokaryotic community and the interaction between prokaryotes and viruses. By pairing measurements of virome analysis and prokaryotic production of a 1-year survey of the depths of surface and deep layers, we revealed (i) the prokaryotic infection pairs associated with prokaryotic production and (ii) the reset in prokaryotic and viral communities through annual vertical mixing in a freshwater lake. Our results provide a basis for future work into changes in stratification that may impact the biogeochemical cycling in freshwater lakes.


Overview:
The authors have responded to most of my suggestions, and I am mostly satisfied with their responses.I think that the manuscript reads well and their effort in revision is appreciated.I recommend further clarification of the statistical methods, including specific tests used, which should be mentioned throughout the manuscript and in associated figure legends.I also note that one of my more major comments was not addressed (viral identification reanalysis/comparison; see below), which I leave up to the editor to adjudicate.

Minor comments:
Lines 367-369: "Although…" I don't fully agree with this sentiment as the reason that we think most viruses are dsDNA viruses is because of a historical attention focused on dsDNA viruses (partially due to technological and methodological limitations).I would recommend phrasing that reflects this, such as "Although ssDNA and RNA viruses infect prokaryotes, most of those described prokaryote-infecting viruses are dsDNA viruses, and as such we have focused our attention on this group."I would recommend a similar approach in your Discussion paragraph about this (lines 247-255), but I do appreciate the efforts made by the authors to address this throughout the manuscript, as well as associated limitations.
Lines 367-369: "raw reads were assembled using SPAdes …" Thank you for clarifying that you used default SPAdes.I will say that I find it curious that you chose to use SPAdes in its default format as opposed to metaspades or single cell spades, which is preferred for metagenomic data.I suspect that this would likely result in more misassemblies and merging of contigs from unrelated taxa (as default spades assumes the input is from an isolate genome), but given that the rest of your methods are fairly conservative, my guess/hope is that the effect of any associated misassembly would be mostly mitigated?Just a guess.I would recommend using single cell SPAdes in your future work, as Simon Roux put out a good paper a few years back (https://peerj.com/articles/6902/)examining the effect of different assembly approaches and found scSPAdes to generate the best assemblies and this has been my experience as well.

Major comments:
Response to "As suggested, these tools can detect more viral contigs than Virsorter1, which we used in this study…" This isn't really a sufficient response.Yes, you found viruses associated with prokaryotic production, but there's no ground truth that says you found all/most of them.In fact, it is almost certain that most other methods would more accurately detect viral contigs, and, importantly help with accurate annotation in light of potential misassembly.Virsorter1 is an antiquated tool that is outperformed by all other methods, which may be of particular importance given your usage of default SPAdes.I will leave it up to the editor to decide how to respond here, but I do not find this to be a satisfactory response.I think a comparison with another tool should be made or a contemporary tool like geNomad should be used if a comparison is not desired.
Please expand on the statistical analyses used.This aspect of the study is far too scant.There is a brief section under the data processing, but the authors should specify the specific tests used for each set of analyses, (eg.for ecological analysespermanova?) and how multiple hypothesis correction was performed.This is still very underdeveloped and needs expanded upon pretty significantly throughout the manuscript.Please also list the units and tests used for each analysis in the associated figure legends (the supp figs would be difficult for readers to interpret if they were not also looking at the manuscript text).I found myself often looking at figures/reading text and wondering what tests were used and if the results were significantly different or just passed the eyeball test.

Reviewer 1
Overview: The authors have responded to most of my suggestions, and I am mostly satisfied with their responses.I think that the manuscript reads well and their effort in revision is appreciated.I recommend further clarification of the statistical methods, including specific tests used, which should be mentioned throughout the manuscript and in associated figure legends.I also note that one of my more major comments was not addressed (viral identification reanalysis/comparison; see below), which I leave up to the editor to adjudicate.

Minor comments:
Lines 367-369: "Although..." I don't fully agree with this sentiment as the reason that we think most viruses are dsDNA viruses is because of a historical attention focused on dsDNA viruses (partially due to technological and methodological limitations).I would recommend phrasing that reflects this, such as "Although ssDNA and RNA viruses infect prokaryotes, most of those described prokaryote-infecting viruses are dsDNA viruses, and as such we have focused our attention on this group."I would recommend a similar approach in your Discussion paragraph about this (lines 247-255), but I do appreciate the efforts made by the authors to address this throughout the manuscript, as well as associated limitations.

Response:
Thank you for this suggestion.We have revised the relevant parts accordingly (Lines 250, 367-369).
Lines 367-369: "raw reads were assembled using SPAdes ..." Thank you for clarifying that you used default SPAdes.I will say that I find it curious that you chose to use SPAdes in its default format as opposed to metaspades or single cell spades, which is preferred for metagenomic data.I suspect that this would likely result in more misassemblies and merging of contigs from unrelated taxa (as default spades assumes the input is from an isolate genome), but given that the rest of your methods are fairly conservative, my guess/hope is that the effect of any associated misassembly would be mostly mitigated?Just a guess.I would recommend using single cell SPAdes in your future work, as Simon Roux put out a good paper a few years back (https://peerj.com/articles/6902/)examining the effect of different assembly approaches and found scSPAdes to generate the best assemblies and this has been my experience as well.

Response:
Thank you for your comment and for recommending a good paper for reference.
Misassembly was carefully addressed through bioinformatics and PCR amplification experiments when establishing this method in the below papar.Finally we chose SPAdes with "careful" flags based on a previous work in which several assemble tools were compared and SPAdes was identified as the best assembler (Table 1 in Nishimura et al., 2017).Collinearity with the isolated viruses was observed in the assembled genomes.And although there were surprisingly few SNPs, the sequences were confirmed by PCR amplification of some parts with relatively low coverage from environmental samples.Thus, we conclude that the genomes assembled using the method is not derived from a single virus particle, but is a consensus genome among closely related viruses as long as they exist in that environment (Nishimura et al., 2017).
Per your suggestion, we aim to use scSPAdes (or metaSPAdes) as an assembler in the subsequent viral metagenome investigations.

Major comments:
Response to "As suggested, these tools can detect more viral contigs than Virsorter1, which we used in this study..." This isn't really a sufficient response.Yes, you found viruses associated with prokaryotic production, but there's no ground truth that says you found all/most of them.
In fact, it is almost certain that most other methods would more accurately detect viral contigs, and, importantly help with accurate annotation in light of potential misassembly.
Virsorter1 is an antiquated tool that is outperformed by all other methods, which may be of particular importance given your usage of default SPAdes.I will leave it up to the editor to decide how to respond here, but I do not find this to be a satisfactory response.
I think a comparison with another tool should be made or a contemporary tool like geNomad should be used if a comparison is not desired.

Response:
Thank you for this comment.We agree that most of the other tools are more accurate than VirSorter 1.However, we wanted to emphasize that we purified the viral particles using CsCl step-gradient ultracentrifugation and DNase I steps, by which the remaining prokaryotic cells and external DNA were removed before bioinformatic analysis.This method was originally used for the purification of lambda phages (dsDNA) and has occasionally been used to purify viruses before virome analysis.Indeed, in our study, 86% of the assembled contigs (>10 kb) were detected to be of viral origin by VirSorter1 (Table S4).In contrast, a previous study that did not use a CsCl step-gradient ultracentrifugation step detected only half of the contigs to be of viral origin using VirSorter1 (EX. 2 in Luo et al., 2020).These observations indicate that our method (CsCl step-gradient ultracentrifugation + SPAdes + VirSorter1) worked well and could sufficiently capture a viral population.We believe that our method is sufficient to achieve our goal, such that we need not perform a reanalysis or comparison with other methods.In the method text, virus concentration and purification steps were described separately(Lines 366-369).The GeNomad paper was published on September 21, 2023, and our manuscript has been under review during this period.We will consider this tool for our subsequent virome analyses.Please expand on the statistical analyses used.This aspect of the study is far too scant.
There is a brief section under the data processing, but the authors should specify the specific tests used for each set of analyses, (eg.for ecological analysespermanova?) and how multiple hypothesis correction was performed.This is still very underdeveloped and needs expanded upon pretty significantly throughout the manuscript.Please also list the units and tests used for each analysis in the associated figure legends (the supp figs would be difficult for readers to interpret if they were not also looking at the manuscript text).I found myself often looking at figures/reading text and wondering what tests were used and if the results were significantly different or just passed the eyeball test.

Response:
Thank you for your comment.We have revised the Data Processing and Visualization section to include more details (Lines 451-460).We have also added units and tests used for each analysis in the figure captions (Lines 734-736, 746-748, caption of Figure S5).We have also added the results of the test in the Result section (Lines 116,121,139,160).

Reviewer 2
As far as my expertise goes, I do not have any more issues to discuss.I would like to thank the authors for taking the time to address the issues raised in the previous review round in such depth.
Just one final minor comment from my side: In Table S1 you

Response:
Thank you for your comment.We have revised the caption (added "between the surface and deep layer") and the figure number (Figure 1C-E) (Table S1) accordingly.
1st Revision -Editorial Decision Re: mSystems00906-23R1 (Virus-prokaryote infection pairs associated with prokaryotic production in a freshwater lake) Dear Dr. Shang Shen: Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the mSystems editorial office, and the reviewer comments.In principle I believe we can accept the paper but there are a couple minor points from one of the reviewers below that need to be addressed before we can do that.
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Reviewer #2 (Comments for the Author): I have nothing really substantial to add to the current point of debate in the review process.I could, however, try to disentangle the issue beyond discussions about using one method or the other, in which I lack the expertise to intervene.
According to reviewer#1, the authors "found viruses associated with prokaryotic production, but there's no ground truth that says [they] found all/most of them".Now if I am understanding correctly, this is the key issue at stake.I may even agree that it cannot be claimed that most or all viral diversity was detected.Assuming this, then my questions are: (1) is it implied by the authors in the manuscript that they found most/all viral diversity?If so, it may be worth it to nuance the text a bit.
(2) does the fact that not all viral diversity was captured substantially affect the authors claims?
Reviewer #2 (Comments for the Author): I have nothing really substantial to add to the current point of debate in the review process.I could, however, try to disentangle the issue beyond discussions about using one method or the other, in which I lack the expertise to intervene.
According to reviewer#1, the authors "found viruses associated with prokaryotic production, but there's no ground truth that says [they] found all/most of them".Now if I am understanding correctly, this is the key issue at stake.I may even agree that it cannot be claimed that most or all viral diversity was detected.Assuming this, then my questions are: (1) Is it implied by the authors in the manuscript that they found most/all viral diversity?If so, it may be worth it to nuance the text a bit.Response: No, we did not imply in the manuscript that we found all or most viral diversity.As you and Reviewer 1 have commented, not all viruses were captured in this study.The 13,761 viral contigs detected in this study were combined with approximately 4,000 viral contigs recovered from other 12 metagenomic samples collected from the same lake (Okazaki et al., 2019: https://doi.org/10.1111/1462-2920.14816),and all contigs were clustered.In total, 14,952 viral contigs were obtained (Figure below).It seems to be becoming saturated, but some viruses could still be detected.
Thus we want to correct our response in the previous round.We have responsed that "our method (CsCl step-gradient ultracentrifugation + SPAdes + VirSorter1) worked well and could sufficiently capture a viral population" and want to replace with "our method worked well and VirSorter 1 could sufficiently detect viruses from assembled contigs".Thank you for your comment.
(2) Does the fact that not all viral diversity was captured substantially affect authors' claims?
Response: No, we believe that this will not significantly affect our claims.Our goal was to extract virus-prokaryotic infection pairs associated with prokaryotic production.Using 13,761 viral contigs, we successfully extracted co-occurring viruses for every prokaryote associated with prokaryotic production.This indicates that our goal had already been achieved.If we could capture all viruses, more co-occurring viruses could be extracted, but this would not affect our results.Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the mSystems editorial office, and the reviewer comments.After reviewing the comments from Reviewer 2 it seems as though you have addressed everything except for one final issue.Reviewer 2 had a question about your choice of assembly protocol and viral identification and annotation methods.It seems that there have been other programs published recently.Can you add a few lines in the Results or Methods as to why you chose these particular methods?I believe if you can justify the choice then we will be able to publish the paper.Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration by mSystems.

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After reviewing the comments from Reviewer 2 it seems as though you have addressed everything except for one final issue.Reviewer 2 had a question about your choice of assembly protocol and viral identification and annotation methods.It seems that there have been other programs published recently.Can you add a few lines in the Results or Methods as to why you chose these particular methods?I believe if you can justify the choice then we will be able to publish the paper.
Thank you for your comments.We have added some sentences/phrases to explain why we have chose our methods (385)(386)(391)(392).
December 6, 2023 3rd Revision -Editorial Decision Re: mSystems00906-23R3 (Virus-prokaryote infection pairs associated with prokaryotic production in a freshwater lake) Dear Dr. Shang Shen: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.Data Availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact ASM production staff immediately with the expected release date.
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Editor mSystems Luo, E., Eppley, J.M., Romano, A.E. et al.Double-stranded DNA virioplankton dynamics and reproductive strategies in the oligotrophic open ocean water column.ISME J 14,1304-1315 (2020).https://doi.org/10.1038/s41396-020-0604-8 say original data are shown in Figure S6 which, as far as I can tell does not exist.It rather refers to Figure 1CDE if I am understanding it correctly.Authors should also explicitly clarify that what's being compared in the statistical analysis is surface vs deep layer (again, if I am getting it right).