No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: From mating interactions to differential gene expression

Julien Kincaid-Smith; Eglantine Mathieu-Bégné; Cristian Chaparro; Marta Reguera-Gomez; Stephen Mulero; Jean-Francois Allienne; Eve Toulza; Jérôme Boissier

doi:10.1371/journal.pntd.0009363

Peer Review History

Original SubmissionSeptember 4, 2020
13 Oct 2020 Decision Letter - Poppy H L Lamberton, Editor Dear Mrs Mathieu-Bégné, Thank you very much for submitting your manuscript "Pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. All three reviewers have commented on the level of English and grammar within this paper, that makes it hard to properly review this paper. I would recommend that in addition to all that scientific comments and specific language comments included that all authors really help rewrite this manuscript and if needed that you ask a native English speaker to help with the next version. I will send this out to reviewers again upon resubmission if the level of English has improved, but that really does need to be addressed. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts. Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Poppy Lamberton Deputy Editor PLOS Neglected Tropical Diseases ********************* All three reviewers have commented on the level of English and grammar within this paper, that makes it hard to properly review this paper. I would recommend that in addition to all that scientific comments and specific language comments included that all authors really help rewrite this manuscript and if needed that you ask a native English speaker to help with the next version. I will send this out to reviewers again upon resubmission if the level of English has improved, but that really does need to be addressed. Reviewer's Responses to Questions Key Review Criteria Required for Acceptance? As you describe the new analyses required for acceptance, please consider the following: Methods -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? -Is the study design appropriate to address the stated objectives? -Is the population clearly described and appropriate for the hypothesis being tested? -Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? -Were correct statistical analysis used to support conclusions? -Are there concerns about ethical or regulatory requirements being met? Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes -Is the study design appropriate to address the stated objectives? Yes but more discussion is needed on the limitations of the study design -Is the population clearly described and appropriate for the hypothesis being tested? More information is needed on the strains used and the details of the experiments -Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? Yes -Were correct statistical analysis used to support conclusions? I would like this checked by a statistician -Are there concerns about ethical or regulatory requirements being met? No Reviewer #2: - The overall objective is clear but the hypotheses could be more clearly formulated in the introduction. For example, somewhere in the paper it was stated that the authors expected the opposite outcome (more transcriptomic changes in females than in males), so this hypothesis could have been included at the end of the introduction, instead of the vague kind of conclusion in line 194-197. Additional testable hypotheses can be formulated. - The study design is appropriate although some issues arise regarding statistical power. The results discussed in line 362 (premature death of two hamsters) does raise the question whether 5 replica’s (5 hamsters) is enough to make robust conclusions as in this particular case no reliable statistics can be done for S. bovis males’ choice. - No concerns about ethical issues. For more comments on the Methods section see General Comments. Reviewer #3: Methods seem appropriate -------------------- Results -Does the analysis presented match the analysis plan? -Are the results clearly and completely presented? -Are the figures (Tables, Images) of sufficient quality for clarity? Reviewer #1: -Does the analysis presented match the analysis plan? Yes -Are the results clearly and completely presented? Improvement is needed for clarity of the results to allow interpretation -Are the figures (Tables, Images) of sufficient quality for clarity? More information and clarity is needed as detailed in the attached document Reviewer #2: The analysis is sound and the results are well presented. Table 2 is redundant I would say. For more comments on the Results section see General Comments. Reviewer #3: Analysis matches described goals of the work. REsults are clearly presented. Figures are sufficient quality -------------------- Conclusions -Are the conclusions supported by the data presented? -Are the limitations of analysis clearly described? -Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? -Is public health relevance addressed? Reviewer #1: -Are the conclusions supported by the data presented? Yes -Are the limitations of analysis clearly described? No -Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? Ye -Is public health relevance addressed? Yes Further comments on the discussion are available in the attached Reviewer #2: As put in my General comments, the Discussion needs reworking and should be more substantiated. The conclusions are not always clear or strong, I miss more references to similar studies, but also a proper discussion on the limitations and recommendations for future research are missing. The public health relevance is not really thoroughly discussed. Reviewer #3: Conclusions are supported by the data. They speculate quite a bit in the discussion about the potential importance of various DE genes. But this type of speculation is rampant in gene expression papers and they don't make any hard conclusions. -------------------- Editorial and Data Presentation Modifications? Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: The main modidications are needed in the clarity of the data and how it is presented. Comments are in the attached document. Reviewer #2: (No Response) Reviewer #3: (No Response) -------------------- Summary and General Comments Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed. Reviewer #1: This is an important and interesting study that shows the lack of pre zygotic isolation between S. haematobium and S. bovis supporting inter-species hyrbidisation an important scenario for human and animal health in Africa. Although, I consider the study of vlaue and there is a substantial body of high quality of work, which is not easy to do (particularly the generation of the isolates and the mating experiments) the paper needed considerable improvements before it can be reviewed further. The authors should consider that the readers will not be familiar with these types of experiments and there is a need to add the detail that allows the readers to understand the experimental procedures and the data produced. There are also english and gramma errors many of which can be improved with careful reading. In the attached these are highlighted in the text showing the errors and where changes are needed. I have tried to cover the whole document but due to time some places may have been missed and careful reading and revision is needed. There are also comments on the attached to highlight where more clarity is needed and also where further information or discuss is warranted. The discussion is also very long and could be condensed by not repeating what is in the results. Reviewer #2: This is a very interesting study, with interesting results. Experimental infections in hamsters show that there are no pre-zygotic barriers to mating between the human Schistosoma haematobium and the animal S. bovis parasite, neither are there any major transcriptomic responses following hetero-specific pairings. Even though a previous study by Webster and colleagues already showed that S. bovis and S. haematobium readily paired in laboratory hamsters (to which the present authors not refer, which I think is an omission), this is the first time that any transcriptomic study is done on hybrid crosses. I was frustrated by the sloppy grammar and spelling throughout the entire manuscript, which gave the impression that the authors were in a hurry to submit this manuscript or they didn’t really care about this. I also lack a discussion on the asymmetry in the direction of hybridisation and introgression between schistosomes, which is sometimes even unidirectional, but nothing is mentioned on this. Also, it is repeatedly said that isolation ‘by the host’ is apparently the only barrier to hybridisation between these two schistosome species, but it is never specified which host they mean with that, the final or the intermediate one. Since mate choice and reproduction takes place in the final host, this one is of particular interest of course, but if the different intermediate hosts are not sympatric, then hybridisation will also be less frequent (if they final host does not move around too much). For S. bovis and S. haematobium this is more complicated, as intermediate host specificity of the latter varies with geography, but still this should be properly discussed as it could also explain why we see such regional differences in the distribution of hybrids. Then finally, I also have some problems with parts of the discussion: some questions remain unanswered (e.g. why would only S. haematobium males in heterospecific pairing have this transcriptomic response?), the statistical power, and with the fact that I miss the broader picture, the reference to all the previous work on schistosome mating experiments. So therefore I think that these concerns should be addressed first before it can be accepted for publication. Abstract & author summary - line 44: make two sentences out of this long sentence - line 47: delete ‘allowing them to maintain…’ this is repetition from above - line 48: what do you mean with misunderstood? Not understood? Or are there really mistakes and / or misconceptions out there in the literature? - line 57: ‘by the host’: replace with ‘final host choice’ or something like that, because at the snail host level there is not always spatial isolation - line 67-68: evolutionary biology? - Line 68: replace ‘If’ by ‘While’ - Line 73: S. haematobium (species names always in full when first mentioning) and parasitize - Line 70-74: sentence too long, split in two and rephrase ‘including out of endemic areas’ - Line 75: ‘…rather than having a homo-specific mate preference’ - Line 78: mechanisms - Line 78: ‘but the one imposed by host specificity’? what do you mean? Please rephrase ‘except the one imposed by final host specificity’ - Line 81: ‘encounter each other’ Especially the author summary does not read very fluently, and there are quite some grammar mistakes. Introduction - line 88= mechanism - line 90: this sounds more like mate choice rather than behavioural isolation - line 91: individuals - line 91: I would replace copulation by reproduction - line 93: encounter - line 96: less fertile - line 96: hybrid lines - line 98: making it difficult to predict - line 104: terms - line 105 and others: free-living - line 108: compared to those of free-living… - line 109: hostile rather than inimical? - Line 112-113: rephrase this sentence, it is not really shaped through the host alone, it is shaped through the host – parasite interactions, and you first call this a strong isolation mechanism and a few words later you say ‘potentially’ preventing hybridization… the sounds less convinced. Also, you write ‘its hosts’, so plural, I would make the distinction already here between intermediate and final hosts, because in case of schistosomes sexual reproduction only takes place in the final host, so host choice at this level is more important than at the other level. You should discuss it at least somewhere, because in the abstract you only talk about ‘host’ choice. - Line 113: you mean ‘closely related’ species? Because all species are related somehow… - Line 116: plasmodium species (or you provide the genus names for the other two parasites you add here) - Line 123: rephrase ‘schistosomiasis debilitating diseases’, this is not an official term (also, the disease is of great concern, rather than the parasites themselves I would say) - Line 128: rephrase ‘among other trematodes’, you want to say here that they are an exception within the Trematoda - Line 133: ‘this can lead to hybridisation’ - Line 136: influence rather than interest - Line 136: First, - Line 138: same host individual and schistosome species - Line 144: female’s - Line 145: of a sexually … - Line 145: what does not depend upon species-specific pairing? Discuss this in a separate sentence in order to avoid too long sentences - Line 147: stimulate - Line 149: male worms’ physiology or the physiology of male worms - Line 153: groups instead of clades? - Line 155: schistosome - Line 157: others, you mean other combinations? - Line 158: S. mattheei - Line 156-159: in all these cases it should be mentioned that the viability of these crosses depend on the type of crosses, which parental species provides the male and which the female in the hybrid cross. Also, the way you write this you suggest that the first group of species can readily pair because there is less divergence between them (because this is what you write in the preceding sentence), while others are more selective because they are more divergent… but the divergence between S. haematobium and S. intercalatum is similar to the divergence between S. haematobium and S. mattheei. Also how can a combination be more selective or readily pair? It is the species that forms this combination that can be selective I would say. The grammar is quite sloppy in many cases, please take care of this. - Line 174: non-human; also Cetartiodactyla is a superorder, and a superorder or a genus cannot be infected by parasites, but their members can - Line 175: ruminants - Line 177: rodents - Line 191: outperforms the fitness of parental species - Line 194: the sentence starting with ‘Relying on such an integrative…’ is redundant as it is mainly repetition Material and methods - Line 214: in or with Bulinus - Line 219: B. truncatus - Line 223: were performed - Line 242: versus - Line 244: each worm and its - Figure 1: I am a bit confused why you use the same color red for S. haematobium and S. bovis females? - Table 1: line 248: ‘and’ should not be in italic. Line 250: the number of male and female S. haematobium and S. bovis worms - Line 254: rephrase this sentence, grammatically incorrect - Table 2: I think this table can be left out as everything is already explained in the text - Line 278-279: parasite species names not in italic since the title is in italic - Line 289: rephrase: as the volume of each reagent was halved - Line 296: library construction - Line 302: vs in full and not italic - Line 307: all sample reads - Line 318: each newly assembled gene or all newly assembled genes - Line 319: were extracted from the S. haematobium - Line 321: transcript - Line 324: hetero-specific paired worms (or elsewhere you write hetero-specifically paired worms) - Line 327: gene expression - Line 338: sets of genes Results - line 349: experiment - line 353: partners - line 362: these are the risks of experimental research of course, and this cannot be avoided, but it does raise the question whether 5 replica’s (5 hamsters) is enough to make robust conclusions as in this particular case no reliable statistics can be done for S. bovis males’ choice - line 367: find instead of found - Table 3, line 376: remaining single or that remained single - Line 379: elsewhere you write P-value - Line 380: experiments - line 385: male and female S. haematobium - line 412: providing from? - Line 426: does this 7% means 7% of all schistosome genes? Please specify this - line 427: vs. in full as elsewhere - line 462: GO terms - line 463: male S. haematobium - line 468: biological - line 489: and not in italic - line 500: correspond (in present tense) - line 503: involved in ion - line 506: functions Discussion - line 513: reproductive isolation mechanisms? - Line 521: male and female - Line 523: definitive host. This sentence is actually a final conclusion before starting the Discussion itself - Line 532: S. mattheei - Line 534: so this calls for more replica’s in future experiments to verify this possibility! - Line 539: there are no barriers or there is no barrier - Line 557: previous instead of precedent - Line 558: there are no - Line 560: what do you mean with ‘male and female S. haematobium and S. bovis’? Between male S. haematobium and female S. bovis? - Line 561: male S. haematobium - Line 571: display a more - Line 576: ‘females species sexual maturation’? - Line 587: female S. bovis - Line 602: gonad-specific - Line 579 – 612: this lengthy discussion is confusing and less convincing because right before this discussion you conclude that only few transcriptomic adjustments are associated with hetero-specific pairing and that the log2-Fold change in males were low, and that it seems difficult to conclude that one or the other sex is preferentially impacted, suggesting that your results are not so convincing. This is not so motivating for the reader then to follow this subsequent discussion - Line 627-628: these observations could have indeed important consequences with respect to drug treatment, but again, how serious do we have to take this, and why would only S. haematobium males in heterospecific pairing have this response, and not S. bovis males? - Line 632: avenues - Line 634: but these report unidirectional introgression of a few bovis genes into the haematobium genome, so a dominance of haematobium genomic DNA, how can you reconcile or link this with your results? Wouldn’t you expect more differences between the different crosses? It has been proven in co-infection experiments that crosses are not always reciprocal, i.e. one cross performing better than the reverse cross. This could also be expected in these crosses as in Senegal many hybrids that were found appeared to have arisen of a female S. bovis x male S. haematobium pairing (although backcrossing in nature obscures these patterns, and in other areas, like Niger, the reverse crosses are more frequently found). I do not see any reference to this or to the topic of unidirectional hybridisation and unidirectional introgression, which I think is missing. - line 648: I don’t completely agree, because the different ‘compartments’ (strange term) do not prevent them from meeting each other in the liver, where mating takes place, only after that stage the couple moves through the hepatic portal vein to the egg-laying site. So there is plenty of opportunity in the liver, irrespective of the difference in tropism - line 653: how can these parasites be co-occurring (I would use the word sympatric) of their hosts are not? Please adapt - line 657: rephrase ‘parental species individuals’ - line 655-659: what do the authors want to say here exactly? Does ‘such hybrids’ refer to those hybrids resulting from an ancient hybridisation event? And do only ‘those hybrids’ present heterosis, in contrast to ‘other, more recent hybrids’? Please rephrase. Also, how do your results explain the fact that previous studies show that mainly ‘ancient hybrids’ are found in nature (Platt et al, 2019), while your experiments show that hybridisation is so ‘easy’? In line 668 you write that your result ‘echoes recent evidence of introgression’. I am not sure what you mean with this. Do you mean ‘evidence of recent introgression’ or ‘recent evidence of (ancient) introgression’? I would say the opposite, the high prevalence of hybrids is an echo or a reflection of the weak pre-zygotic barrier that you observed. But still, I don’t see how your results can be matched to the outcome of Platt et all, suggestion ancient hybridisation. - Line 669: a higher - Line 670: if the hybrids always have higher fitness, why do you still see ‘pure S. haematobium’ and ‘pure’ S. bovis in places where they overlap? Wouldn’t the hybrids take over? - Line 672: ‘new issues in the disease control’ and ‘alteration in the efficacy’ sound rather vague as a closing sentence, please be more specific. Reviewer #3: Summary The goal of this work appeared to be to test for evidence of species-specific mate choice between S. haemotobium and S. bovis, and also to see whether there are differentially expressed genes in adults engaged in homo vs. hetero-specific pairings. The found minimal evidence for mate choice and few strongly DE genes. In the discussion they speculate on possible roles of the few DE genes, but make no strong conclusions about any of them. Comments The authors did a mate choice experiment using cercariae of known sex in hamsters. They found no strong evidence that species-specific mate choice occurs by males or females of either species. The statistical analysis of the mate choice experiments seemed appropriate to me. If they have the data, it would be interesting if the authors could comment on the physical location of the hetero vs homo-specific pairings within the hamster (urogenital vs. mesenteric). I wondered whether different behavior might be observed if the definitive host was a larger mammal such as a bovine or human. Perhaps the authors would care to speculate or at least mention the possibility. The authors also looked for evidence of differential gene expression in individuals of each sex and species when engaged in hetero vs. homo-specific pairings. They observed only a few dozen DE genes in females of either species. Oddly, they found zero DE genes in male S. bovis, but over 1000 in male S. haemotobium (although I wonder if figure 2a suggests the difference in S. haemotobium might be driven by one individual). The almost complete lack of even minimally DE genes in S. bovis males shown in figure 3d is puzzling. I have never seen a volcano plot like this one. I would have expected more by chance alone with only n = 3 per treatment. Analysis of gene expression data is not my expertise, so I defer to other reviewers to comment on this. Or perhaps the authors could head off puzzled readers by explaining why this pattern obtains. Minor comments: The manuscript could use some editing to fix various small grammatical errors and instances of odd English usage. Perhaps asking a native English speaker to read it through once for them would be helpful. It would help Figure 3 if the authors would label, on the figure, which combination of sex and species is represented by each panel. Going back and forth between the legend and the figure is tedious for the reader. Figure 4. Is there some measure of statistical significance associated with the difference between blue and red bars that could be indicated on the figure? Supplementary table S1 would be helped by species identifications. -------------------- 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 Figure 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. 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 us at figures@plos.org. Data Requirements: Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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29 Dec 2020 Author Response Attachments Attachment Submitted filename: 1_Letter_responses to reviewer comments_def.docx https://doi.org/10.1371/journal.pntd.0009363.r002
2 Feb 2021 Decision Letter - Poppy H L Lamberton, Editor Dear Mrs Mathieu-Bégné, Thank you very much for submitting your manuscript "No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic and we all agree that the manuscript is now greatly improved. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. I look forward to seeing an updated paper resubmitted very soon. Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Poppy H L Lamberton Deputy Editor PLOS Neglected Tropical Diseases Poppy Lamberton Deputy Editor PLOS Neglected Tropical Diseases ********************* The manuscript is now greatly improved and I look forward to seeing these minor changes suggested by two of the reviewers amended and an updated paper resubmitted very soon. Reviewer's Responses to Questions Key Review Criteria Required for Acceptance? As you describe the new analyses required for acceptance, please consider the following: Methods -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? -Is the study design appropriate to address the stated objectives? -Is the population clearly described and appropriate for the hypothesis being tested? -Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? -Were correct statistical analysis used to support conclusions? -Are there concerns about ethical or regulatory requirements being met? Reviewer #1: The methods are now clear and easy to follow Reviewer #2: yes Reviewer #3: Acceptable -------------------- Results -Does the analysis presented match the analysis plan? -Are the results clearly and completely presented? -Are the figures (Tables, Images) of sufficient quality for clarity? Reviewer #1: The results are clearly presented. Reviewer #2: yes Reviewer #3: Acceptable -------------------- Conclusions -Are the conclusions supported by the data presented? -Are the limitations of analysis clearly described? -Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? -Is public health relevance addressed? Reviewer #1: The discussion supported the data presented and is informative. The limitations are clearly presented and there relevance to public health is addressed. Reviewer #2: yes Reviewer #3: Acceptable -------------------- Editorial and Data Presentation Modifications? Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: See general comments Reviewer #2: The authors did a good job in addressing all the comments of the referees, they adapted the text and figures/tables where needed or suggested, and this resulted in a much-improved version of the manuscript. I think this version can be accepted for publication. A few minor comments: Line 43-44: before reproduction is also ‘during the life cycle’, so why not simply saying ‘These preventive barriers can act before reproduction, “pre-zygotic barriers”, or after reproduction/fertilisation, “post-zygotic barriers”? Line 561; draw Line 569: male transcriptomes Line 640; to overcome their divergence only I would say, not to overcome their relatedness Line 654: a lower sensitivity to PZQ of the hybrid being at the origin of the spread was not at all proposed by Huyse et al., 2009 so please adapt. Line 708: S. mansoni [28]). Line 711; I think the biased patterns in the field might indeed by due to post-zygotic isolation but not the rare encounter of first generation hybrids (or not only because of that). The latter is probably (also) due geographical variation in the level of sympatry between parental species, no? Reviewer #3: They adequately addressed the issues of language usage. -------------------- Summary and General Comments Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed. Reviewer #1: The paper is much improved, and the authors have taken the time and effort to amend the manuscript according to the reviewer’s comments. This is a highly informative paper that adds to our understand on the interactions between S. haematobium and S. bovis. It will also open several research avenues for future work. I have the following minor comments. Gramma punctuation is needed in several places so a good go over will help Some further modifications of the English is needed. Areas that I managed to pick up are highlighted. Line 120 – there are 23 species not 25 Line 121 – to be precise 20 infect animals if you include S. mansoni that is found in rodents and monkeys. Line 188-189 – make it clear that the heterosis has been observed in experimental infections not in the natural setting. Line 220-222- the animal license information should be checked with the editors regarding how it should be written. Line 229 – what do you mean by sympatric B. truncatus – do you mean local natural hosts? Line 250 – add “below” to the end of the sentence. Figure 2 – S. h = S. bovis needs correcting. Also, the symbols are both in the figure and legend. Only one is needed. Table 1: in Exp. 2 and 4 you say that this is a female choice experiment. Is this not more competition of the two male species? Line 290 – make it clear that this section is moving onto the molecular work and not the analysis of pairings. Line 296 – make it clear how the worms were separated. It is important here that there is not contamination between males and females, so the detail is needed. Line 327 – “effect” not needed Line 328 – reference the Galaxy instance. E.g., is it a software or a machine? Table 2 column 6 and 7 remove the words or change homo specific or hetero specific as they are not paired. Thy can just be called unpaired worms. It would also be better to have the exp. row above the data rather and below. For figure 4 is it important to distinguish between hetero and homo paired worms ? Line 481 – show what GO means, Line 553 – is there any evidence for female selecting their partners. They are very underdeveloped until they are paired? Line 555 – this S. intercalatum strain is actually now named S. guineensis – maybe add a note on this and also where it is referenced in the intro. Line 565 – reference the study Webster et al., 2012 that also suggested that there was no mate choice Line 599 - suggest you add here that the majority of the hybrids found in the field appear to be a result of a cross between S. h male and S. b female based on cox1 and ITS sequencing. It is better to say this than state that they are as more work is needed to clarify that. Line 655 – I think it is important to point out that any changes in PZQ response by hybrids is very theoretical and there is not current evidence that there is any difference in drug response in natural infections. Line 707- check if the S. intercalatum should be called S. guineensis. S. intercalatum is the Zaire/DRC strain. Cameroon and other areas is S. guineensis. Reviewer #2: (No Response) Reviewer #3: I believe the authors have adequately addressed my comments on their original submission. The main points of the paper, that there is random mating and few differentially expressed genes in interspecies pairings, are sufficiently supported and worth putting into the literature -------------------- 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: Yes: Dr Bonnie Webster Reviewer #2: No Reviewer #3: No Figure 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. 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 us at figures@plos.org. Data Requirements: Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5. Reproducibility: To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods Attachments Attachment Submitted filename: PNTD-D-20-01594_R1.pdf https://doi.org/10.1371/journal.pntd.0009363.r003
Revision 2
30 Mar 2021 Author Response Attachments Attachment Submitted filename: response reviewers.docx https://doi.org/10.1371/journal.pntd.0009363.r004
6 Apr 2021 Decision Letter - Poppy H L Lamberton, Editor Dear Mrs Mathieu-Bégné, We are pleased to inform you that your manuscript 'No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases. Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated. IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS. Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases. Best regards, Poppy H L Lamberton Deputy Editor PLOS Neglected Tropical Diseases Poppy Lamberton Deputy Editor PLOS Neglected Tropical Diseases *********************************************************** There is some minor type editing required, such as: Line 160: remove full stop and close bracket. Line 231: remove space at start Line 233: I think you can revert to sympatric here, I think it is clear, but maybe if clarification is needed write 'and sympatric B. truncatus molluscs, bred from snails collected from the same location as the parasites, were individually .....' and please calrify here also, or if these were collected straight from the field and exposed, then remove the 'bred from' Line 376: Please add 'worms of the limiting sex (i.e., choosing partners, such as female choice or male competition) and please include this in the table legend as well to explain it when it is first used. Several references need the Latin names in italics and the titles having the full capitalisation removed https://doi.org/10.1371/journal.pntd.0009363.r005
Formally Accepted
29 Apr 2021 Acceptance Letter - Poppy H L Lamberton, Editor Dear Mrs Mathieu-Bégné, We are delighted to inform you that your manuscript, "No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: from mating interactions to differential gene expression," has been formally accepted for publication in PLOS Neglected Tropical Diseases. We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication. The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly. Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers. Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases. Best regards, Shaden Kamhawi co-Editor-in-Chief PLOS Neglected Tropical Diseases Paul Brindley co-Editor-in-Chief PLOS Neglected Tropical Diseases https://doi.org/10.1371/journal.pntd.0009363.r006

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