Dear Phil,

Thank you for submitting your manuscript entitled "A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host" for consideration as a Short Reports by PLOS Biology.

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Roland G Roberts, PhD,

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PLOS Biology

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Please treat the checklist and elaboration as confidential as public release is planned for September 2019.

We would be grateful for any feedback you may have.

Dear Dr Newmark,

I am writing on behalf of my colleague Senior Editor, Roland Roberts, who is the Editor Handling your manuscript as he is out on vacation this week.

Thank you very much for submitting your manuscript "A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host" for consideration as a Short Reports at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by four independent reviewers. One of the reviewers chose to reveal his identity and his name appears with his review below. Please note that the fourth reviewer provided only brief comments as we wanted to move to a decision as expediently as was possible.

In light of the reviews (below), we are pleased to offer you the opportunity to address the comments from the reviewers in a revised version that we anticipate should not take you very long. Indeed, most of the comments can be addressed textually. However, the Academic Editor noted that addressing the comments from Reviewer #4 should not be too difficult by providing 16S amplification to check for the existence of a bacterial symbiont in the cultures, which we agree would strengthen your manuscript. As this is a Short Report, we do need you to keep the number of figures at no more than 4. Once you have revised, we will then assess your revision and your response to the reviewers' comments, and we may then consult the reviewers again.

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Chief Editor, PLOS Biology

eganley@plos.org

On behalf of:

Roland G Roberts, PhD,

Senior Editor

PLOS Biology

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Reviewer remarks:

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Reviewer #1

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This paper takes an observation made almost 40 years ago that rotifers produce a substance that interferes with schistosome cercariae ability to infect experimental animals. The authors identify the reactive component from the rotifer Rotaria rotatoria as Schistosome Paralysis Factor, demonstrate that it paralyzes the cercariae, and can prevent cercariae from infecting mice. They also perform a number of biophysical experiments to work out a potential structure of the molecule as a tetracyclic alkaloid.

The research is well done and the paper well written.

A few minor points:

1. Lines 109-111; Please clarify. An epimer differs at one carbon but R,S suggest enantiomers. Are you just trying to say that ht-13-A and thus maybe SPF are racemic? Alternatively are you trying to say that there are R & S forms and one is responsible for the paralysis.

2. Lines 114-115; Please clarify. 30 min exposure to their tails? Did you show or just suggest that SPF works on the tails of cercariae? Did you count tails that had dislodged from cercariae? Were the heads moving on paralyzed cercariae?

3. Line 147; produced by the rotifer

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Reviewer #2

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Review of manuscript PBIOLOGY-D-19-01881R1 entitled “A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host” by Gao, Sweedler, Newmark and others.

This is a very well-written paper that builds upon Stirewalt and Lewis’s 1981 finding that association of intermediate host snails with rotifers impeded output, motility and infectivity of schistosome cercariae emerging from them. This study has many significant strengths, including, identification of Rotaria rotatoria as the source rotifer species for the biological activity, logical and definitive schema for isolating and characterizing a tricyclic alkaloid that the authors dubbed Schistosome Paralysis Factor (SPF), careful structure activity analyses using SPF and two related alkaloids naturally occurring in Streptomyces bacteria along with structural analogs of these, and a careful analysis of dose-dependent decrements in infectivity in cercariae exposed to SPF and the Streptomyces alkaloids. The latter analysis supports that exposure to SPF in a fresh water environment could block the ability of schistosome cercariae to infect hosts and bolsters the authors conclusion that SPF could form the basis of an environmental or other treatment to prevent transmission of schistosome infection. At least two significant and testable hypotheses are framed in this paper to stimulate future studies. One is that the microbiomes of rotifers associated with intermediate host snails, rather than the rotifers themselves could be the source of the paralytic alkaloids. This hypothesis is plausible given the paralytic activities of structurally related alkaloids of prokaryotic origin. Similarly, the authors provide a strong basis for a hypothesis that SPF acts on serotonin signaling pathways necessary for normal motility in schistosome cercariae.

I noted only one minor substantive organizational shortcoming in the paper that related to the appropriate point to highlight the presence of a serotonin backbone in SPF and the bacterial alkaloids. Also, there were only a few glitches in copy editing and clarity in this very well-written manuscript.

SUBSTANTIVE ISSUES

The observation that SPF contains a serotonin backbone comes rather “out of the blue” in the Conclusion. For this reviewer, this is one of the most important findings in the paper, being consistent with previous reports of the importance of serotonin signaling in schistosome motility and providing a jumping off point for investigations of the mode of action of SPF against schistosome cercariae. Given this, shouldn’t this point be introduced in Results and Discussion? At or around line 90, where the possible structures of SPF are introduced would seem to be a logical point for this introduction. It might also be helpful to shade the serotonin backbone in the structures of SPF, ht-13-A, ht-13-A pr and ht-13-B in Fig. 3.

MINOR ISSUES OF COPY EDITING AND CLARITY

Line 147: Insert “by” between “produced” and “the rotifer”.

Line 162: Insert “a” before “100 um cell strainer”.

Line 205: Suggest substituting “using a” for “through”.

Lines 254-255: I assume the Tricaine incubation was to unpair the adult worms for counting, correct? If so, I suggest “…recovered by hepatic portal vein perfusion, and males and females were unpaired by a brief incubation in 2.5% Tricaine to facilitate counting.”

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Reviewer #3: Jon Clardy

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This in an interesting manuscript with findings of interest to fields as varied as chemical ecology and infectious disease. In brief, the authors have characterized a compound produced by a rotifer that paralyzes the snail stage of the schistosome responsible for schistosomiasis, a neglected disease of growing importance. The general outline of the story: the ability of some but not all rotifers to produce a schistosome paralysis factor (SPF) that render the cercariae life-cycle stage of the parasite ‘paralyzed’ – unable to infect human hosts.

The manuscript fleshes out this bare bones picture in significant ways. Most importantly it uses bioassay-guided fractionation to establish that SPF is a small molecule and uses chemical analysis – primarily mass spectrometry (MS) and nuclear magnetic resonance (NMR) to characterize SPF. All of the work described has been done at an acceptable technical level, and the procedures are described so that they could be readily reproduced. The findings reported in this manuscript form a solid basis for pursuing further studies both on small molecule signaling and therapeutic agents. I recommend publication with no changes.

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Reviewer #4

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I thought the paper was interesting, though the biosynthetic origin of the tetracyclic alkaloid (called Schistosome Paralysis Factor, SPF) should really be discussed in the manuscript. Other than SPF, the Streptomyces-derived alkaloids ht-13-A and ht-13-B are the only two examples of naturally occurring 3,4-oxepino-fused indoles, and thus it seems likely that the SPF is also bacterial origin, rather than “produced by the rotifer Rotaria rotatoria”, as the abstract suggests.

Some experiments to explore a potential bacterial origin of SPF would be in order. Did the authors check for the presence of any Streptomyces spp in Rotaria rotatoria? Sequencing would provide a quick means to get an overview of associated microbiota.

Dear Phil,

Thank you for submitting your revised Short Report entitled "A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host" for publication in PLOS Biology. The Academic Editor and I have now assessed your revisions and responses to reviewers, and we're delighted to let you know that we're now editorially satisfied with your manuscript.

However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication.

IMPORTANT: Please could you also address my Data Policy requests below, namely, provide underlying numerical data for some of the Figure panels, and cite the location of the data in respective legends?

Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

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To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript.

Please do not hesitate to contact me should you have any questions.

Best wishes,

Roli

Roland G Roberts, PhD,

Senior Editor

PLOS Biology

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ETHICS STATEMENT:

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DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

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Dear Dr Newmark,

On behalf of my colleagues and the Academic Editor, Chaitan Khosla, I am pleased to inform you that we will be delighted to publish your Short Reports in PLOS Biology.

The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.

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Kind regards,

Alice Musson

Publication Assistant,

PLOS Biology

on behalf of

Roland Roberts,

Senior Editor

PLOS Biology