Social flocculation in plant–animal worms

Individual animals can often move more safely or more efficiently as members of a group. This can be as simple as safety in numbers or as sophisticated as aerodynamic or hydrodynamic cooperation. Here, we show that individual plant–animal worms (Symsagittifera roscoffensis) can move to safety more quickly through flocculation. Flocs form in response to turbulence that might otherwise carry these beach-dwelling worms out to sea. They allow the worms to descend much more quickly to the safety of the substrate than single worms could swim. Descent speed increases with floc size such that larger flocs can catch up with smaller ones and engulf them to become even larger and faster. To our knowledge, this is the first demonstration of social flocculation in a wild, multicellular organism. It is also remarkable that such effective flocculation occurs where the components are comparatively large multicellular organisms organized as entangled ensembles.

comments. I have no major comments, but offer a few minor suggestions for improvement of the manuscript, and my thoughts regarding their responses to reviewer comments.

MINOR COMMENTS
Numbers and their units should always be separated by a space (e.g. 15 mm, not 15mm). l.129 -A fuller description should be provided by what is meant by "filtered seawater" (e.g. how it was filtered, what mesh size was used etc.). Also, words like "substantial" are open to interpretation, so I would suggest a more specific description of the size of this air bubble.
l.183-184 -Does this mean that the worms aggregated in less than 50 % of the runs (i.e. 17 out of 36)? If so, then I think this is an important result that should be elaborated upon in the discussion, given the important role this behaviour potentially plays in survival the authors are discussing in relation to the evolutionary advantage of this behaviour. l.214: Delete the comma after "above it". l.247: Why do the authors refer to "Our flocs"? I suggest that this use of language is too personal.

RESPONSES TO REFEREE 2 COMMENTS:
1) I agree with the authors that there is no need to increase the size of the experimental data set. If it was straightforward to carry out repeat experiments perhaps it would be of interest to do this. However, the patterns observed already and statistical analysis seems sufficiently robust as it stands, also given the nature of the fieldwork, it seasonality, and the need for self-funding, it would seem unnecessary to insist on further experiments.

RESPONSES TO REFEREE 1 COMMENTS:
2) The referee says that the "evidence for social behavior is preliminary. The rules of interaction are not fully addressed.". This may be true, but I don't see why this should prevent the current description of this flocculation behaviour being published in its current state.
3) I agree with the authors, that there is sufficient indication of social behaviour to leave this phrase within the current manuscript, and rely on future studies to more thoroughly dissect the nature and controls of this behaviour in these worms. 4) I agree with the authors, and do not find any inconsistencies regarding annotation for velocity.

04-Jan-2019
Dear Dr Franks, The editors assigned to your paper ("Social Flocculation in Plant-Animal Worms") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 27-Jan-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
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• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Thank you for submitting this extension to your earlier work in Royal Society Open Science. The reviewers broadly favour publication, but have queried your response to the absence of ethical approval being sought before conducting the research. While the work is carried out on invertebrates, it is nevertheless common to seek approval from your institution's ethical committee prior to conducting interventions such as this research (see, for instance, https://royalsociety.org/~/media/journals/ethics/abguidelines2017.pdf?la=en-GB).
The Editors would like the authors to provide an explanation of why they did not seek approvals from their ethics committee (even if none were subsequently required, it is best practice to ask the question)? We will look forward to receiving your response to this -and the the other reviewers' -query.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript by Worley et al demonstrated that the plant-animal worm Symsagittifera roscoffensis could contract and curve under disturbance which facilitates flocculation. Groups of worms flocculate so that they could descend faster than single worms to reach to bottom of shallow water to avoid being carried away by tide. The research is interesting and would complement the development of the S. roscoffensis in other subjects.
1. Line 34 and line 292: 'cooperative behavior' and 'social behavior' should be changed to 'collective behavior'. The horizontal circular behavior demonstrated in an earlier publication has not been shown to link to this flocculation behavior. 2. Line 23 and line 288: the aspect of energy saving was not demonstrated in the manuscript. 3. Table 1: The image number could be moved into supplementary data, keeping only Δt(i3-i1). 4. Legend of Figure 4, the color of medium and large was the same. Different marker shape could be used for better separation.

Reviewer: 2
Comments to the Author(s) OVERVIEW This study covers a very interesting topic and the authors appear to have addressed the previous referee comments satisfactorily in revising the manuscript and responding to individual comments. I have no major comments, but offer a few minor suggestions for improvement of the manuscript, and my thoughts regarding their responses to reviewer comments.

MINOR COMMENTS
Numbers and their units should always be separated by a space (e.g. 15 mm, not 15mm).
l.129 -A fuller description should be provided by what is meant by "filtered seawater" (e.g. how it was filtered, what mesh size was used etc.). Also, words like "substantial" are open to interpretation, so I would suggest a more specific description of the size of this air bubble.
l.183-184 -Does this mean that the worms aggregated in less than 50 % of the runs (i.e. 17 out of 36)? If so, then I think this is an important result that should be elaborated upon in the discussion, given the important role this behaviour potentially plays in survival the authors are discussing in relation to the evolutionary advantage of this behaviour. l.214: Delete the comma after "above it". l.247: Why do the authors refer to "Our flocs"? I suggest that this use of language is too personal.

RESPONSES TO REFEREE 2 COMMENTS:
1) I agree with the authors that there is no need to increase the size of the experimental data set. If it was straightforward to carry out repeat experiments perhaps it would be of interest to do this. However, the patterns observed already and statistical analysis seems sufficiently robust as it stands, also given the nature of the fieldwork, it seasonality, and the need for self-funding, it would seem unnecessary to insist on further experiments.

RESPONSES TO REFEREE 1 COMMENTS:
2) The referee says that the "evidence for social behavior is preliminary. The rules of interaction are not fully addressed.". This may be true, but I don't see why this should prevent the current description of this flocculation behaviour being published in its current state.
3) I agree with the authors, that there is sufficient indication of social behaviour to leave this phrase within the current manuscript, and rely on future studies to more thoroughly dissect the nature and controls of this behaviour in these worms. 4) I agree with the authors, and do not find any inconsistencies regarding annotation for velocity.

Author's Response to Decision Letter for (RSOS-181626.R0)
See Appendix A.

Comments to the Author(s)
The authors have addressed my comments.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Comments to the Author(s)
The authors have made a thorough job of this manuscript revision and I am happy with all the revisions made and responses to reviewer comments.

25-Feb-2019
Dear Dr Franks, I am pleased to inform you that your manuscript entitled "Social Flocculation in Plant-Animal Worms" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published.

Responses to Editor's and Referees' Comments
Associate Editor's comments: (1) Thank you for submitting this extension to your earlier work in Royal Society Open Science. The reviewers broadly favour publication, but have queried your response to the absence of ethical approval being sought before conducting the research. While the work is carried out on invertebrates, it is nevertheless common to seek approval from your institution's ethical committee prior to conducting interventions such as this research (see, for instance, https://royalsociety.org/~/media/journals/ethics/abguidelines2017.pdf?la=en-GB).
Thank you for the positive comments about our paper. Please see under (2) below our response to the query regarding ethics in research involving animals.
(2) The Editors would like the authors to provide an explanation of why they did not seek approvals from their ethics committee (even if none were subsequently required, it is best practice to ask the question)? We will look forward to receiving your response to this -and the the other reviewers' -query. We have updated our Animal ethics statement accordingly (ll. 300-304).
Reviewer: 1 (3) The manuscript by Worley et al demonstrated that the plant-animal worm Symsagittifera roscoffensis could contract and curve under disturbance which facilitates flocculation. Groups of worms flocculate so that they could descend faster than single worms to reach to bottom of shallow water to avoid being carried away by tide. The research is interesting and would complement the development of the S. roscoffensis in other subjects.
Thank you for these encouraging words.
(4) 1. line 34 and line 292: 'cooperative behavior' and 'social behavior' should be changed to 'collective behavior'. The horizontal circular behavior demonstrated in an earlier publication has not been shown to link to this flocculation behavior.
(5) 2. Line 23 and line 288: the aspect of energy saving was not demonstrated in the manuscript.
We have considered this suggestion carefully but with all due respect we disagree because we are concerned this would sever the link between Fig. 5 and Table 1. In addition, we do not think it would save any space.
(7) 4. Legend of Figure 4, the color of medium and large was the same. Different marker shape could be used for better separation.
We have used a blue open square for the large flocs so that both the shape and colour of the symbol are different for each floc size (Fig. 4, l. 230 This has been implemented throughout the manuscript and ESM. (10) l.129 -A fuller description should be provided by what is meant by "filtered seawater" (e.g. how it was filtered, what mesh size was used etc.). Also, words like "substantial" are open to interpretation, so I would suggest a more specific description of the size of this air bubble.
We had stated the air bubble size in the ESM but now we have added it to the main text as "height of 17.2 ± 8.0 mm (mean ± sd), n=16" (ll. 129-130).
(11) l.183-184 -Does this mean that the worms aggregated in less than 50 % of the runs (i.e. 17 out of 36)? If so, then I think this is an important result that should be elaborated upon in the discussion, given the important role this behaviour potentially plays in survival the authors are discussing in relation to the evolutionary advantage of this behaviour.
No, while it is true that 17 out of 36 runs yielded filmable flocs, the runs that did not yield filmable flocs may have been associated with smaller bubbles that did not cause sufficient disturbance. We have added a sentence to this effect in the Results (ll. 186-187). We have also added two sentences to the Discussion on the implications for the evolutionary advantage of floc behaviour: "Some replicates did not yield filmable flocs and we think this might be associated with a lack of sufficient perturbation. This suggests that the tendency to floc in individual worms is tuneable by natural selection." (ll. 257-259).
We edited the initial sentence "The floc appears to have a toroidal structure (see also figure S8), is descending into clear water and above it, are many single worms it is shedding." Now it is replaced by "The floc appears to have a toroidal structure (see also figure S8). It is descending into clear water and shedding many single worms that follow above it." (ll. 214-215).