Turning defence into offence? Intrusion of cladoceran brood chambers by a green alga leads to reproductive failure

Microalgae are the foundation of aquatic food webs. Their ability to defend against grazers is paramount to their survival, and modulates their ecological functions. Here, we report a novel anti-grazer strategy in the common green alga Chlorella vulgaris against two grazers, Daphnia magna and Simocephalus sp. The algal cells entered the brood chamber of both grazers, presumably using the brood current generated by the grazer's abdominal appendages. Once inside, the alga densely colonized the eggs, significantly reducing reproductive success. The effect was apparent under continuous light or higher light intensity. The algal cells remained viable following removal from the brood chamber, continuing to grow when inoculated in fresh medium. No brood chamber colonization was found when the grazers were fed the reference diet Raphidocelis subcapitata under the same experimental conditions, despite the fact that both algal species were readily ingested by the grazers and were small enough to enter their brood chambers. These observations suggest that C. vulgaris can directly inflict harm on the grazers' reproductive structure. There is no known prior example of brood chamber colonization by a microalgal prey; our results point to a new type of grazer–algae interaction in the plankton that fundamentally differs from other antagonistic ecological interactions.

conditions. Firstly, the photoperiod throughout their experiments were 18L:6D or even 24 L: 0D, which was not in accordance with the natural condition. This is particularly true when the longterm light intensity (130 µmol photons m-2 s-1) was also considered.
(2) Although with similar cell size, the morphology of the two used algal species was different, is this the reason for their difference in colonization ability? This possibility should be discussed. (3) The third experiment lasted for 10 days. Is it long enough for individuals of Simocephalus to reach their maximum body size? If not, your conclusion that Simocephalus individuals cannot be colonization by Chlorella vulgaris is not rigorous. Please explain. Discussion section (4) You suggest that the success of the C. vulgaris cells to remain inside the brood chambers was associated with its ability in high production of EPS under higher light availability? How do you know R. subcapitata do not have this ability? In other words, why R. subcapitata cannot invade grazers brood chamber? (5) In addition, the underlying mechanisms for the failure of C. vulgaris cells to invade brood chamber of Simocephalus should also be discussed. (6) Any microbial process facilitate the intrusion? Minor questions. 1.
Line 4-7, Page 3, the experimental procedure is not clear. For example, the 20 ml tube contain 17 ml Evian spring water, is oxygen availability of cladoceran grazers limited by this treatments? Was the tube capped with breathable polyethylene? 2.
Line 13-14, page 4, What kind of magnifying lens was used? Is the magnification large enough to do this work? 3.
Line 36-37, page 4, The culture condition for algae should be added here. 4.
Line 20-38, page 6, It would be better if you could give some pictures to prove your hypothesis about the specific process how C. vulgaris cells entrance the brood chamber of D. magna. 6.
Line 20-38, page 6, Is it possible for C. vulgaris cells entrance the brood chamber of D. magna from the postabdomen of D. magna, such as during the process of discharging neonates. 7.
For figure 2 and 3, it is not clear what data you used for these two figures. Is it at the end of the experiment on a specific day? Review of Manuscript RSOS-200249 General comments The results presented in this paper show the ability of one common species of microalga (Chlorella vulgaris) to colonise the brood chambers and induce reproductive failure in two cladoceran species. One of them, of great significance in the ecosystems and the scientific literature (Daphnia magna). Colonization of the brood chamber is positively related with longer light-photoperiod and stronger light intensity. There is some issue with the experimental design, that authors should solve, and its detailed in the specific comments below. I miss some discussion about an ecological interpretation (beyond that high light conditions increase EPS production by C. vulgaris) of the potential significance of this mechanism of stronger exposure to light in the real ecosystems. The authors claim that this is the first time that an interaction of this nature was found. However, something similar was found between two unicellular planktonic organisms: the haptophyte Prymnesium parvum and the dinoflagellate Oxhyrris marina (Tillmann et al. 2003, Aquatic Microbial Ecology 34: 73-84). This dinoflagellate is a heterotrophic organism that usually phagocytes phytoplankton cells, like P. parvum. P. parvum was observed to release cytolytic toxins that lyse O. marina cells, to phagocyte the small remains of the cells. This interaction is different that the one reported here because the predator actually becomes the prey, however, I think it needs to be referenced. I would recommend this paper for publication after the authors solve the few questions presented here.
Specific comments -Methods: In experiment 1, page 3, lines 60, it is said that "the experiment consisted of different combinations… ", referring to the experimental treatments. I would expect all the possible combinations to be shown, in a full crossed design. However, later, in the results (Fig. 2) I see that the combinations of the control diet at high food concentrations is missing for both photoperiods. This will affect the interpretation of the GLM performed, because the factor 'concentration' is not fully crossed. I don't see the reason for this. The authors should provide an explanation, since if R. subcapitata could colonize the brood chamber also at high concentrations, that would change the interpretation of results.

31-Mar-2020
Dear Dr Albini, The editors assigned to your paper ("Turning defence into offence? Intrusion of cladoceran brood chambers by a green alga leads to reproductive failure") 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.
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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. Comments to the Author(s) This is an interesting work. Adaptations of algae and other phytoplankton groups against grazers such as cladocerans are long known. These include formation of colonies, development of spines, elevated levels of toxin production, thickened cell wall formation, etc. Even without apparent morphological adaptations, simple and non-colonial alga such Chlorella can still develop resistance against grazers (Yoshida et al. Since the conditions in the brood pouch are not hostile to alga or bacteria, fungi, etc., such organisms can proliferate there.

Reviewer: 2
Comments to the Author(s) The authors did an interesting job. They analyzed the anti-grazer strategy in the green alga Chlorella vulgaris against two cladoceran grazers, Daphnia magna and Simocephalus sp. They found that C. vulgaris could directly inflict harm on the reproductive structure of D. magna, and the colonization process depended on light conditions. To our knowledge, this is indeed the first study demonstrating the harmful intrusion of cladoceran brood chambers by a freshwater microalga, although the underlying mechanism and the specific process has still not been well clarified in the present study. Therefore, I recommend publishing this novel discovery in the journal after a major revision. The specific opinions are as follows.
Major questions Method section (1) It is unclear whether this phenomenon could occur in naturally aquatic ecosystems given that the colonization of brood chamber by Chlorella vulgaris mainly depended on extremely light conditions. Firstly, the photoperiod throughout their experiments were 18L:6D or even 24 L: 0D, which was not in accordance with the natural condition. This is particularly true when the longterm light intensity (130 µmol photons m-2 s-1) was also considered.
(2) Although with similar cell size, the morphology of the two used algal species was different, is this the reason for their difference in colonization ability? This possibility should be discussed.
(3) The third experiment lasted for 10 days. Is it long enough for individuals of Simocephalus to reach their maximum body size? If not, your conclusion that Simocephalus individuals cannot be colonization by Chlorella vulgaris is not rigorous. Please explain. Discussion section (4) You suggest that the success of the C. vulgaris cells to remain inside the brood chambers was associated with its ability in high production of EPS under higher light availability? How do you know R. subcapitata do not have this ability? In other words, why R. subcapitata cannot invade grazers brood chamber? (5) In addition, the underlying mechanisms for the failure of C. vulgaris cells to invade brood chamber of Simocephalus should also be discussed. (6) Any microbial process facilitate the intrusion? Minor questions. 1.
Line 4-7, Page 3, the experimental procedure is not clear. For example, the 20 ml tube contain 17 ml Evian spring water, is oxygen availability of cladoceran grazers limited by this treatments? Was the tube capped with breathable polyethylene? 2.
Line 13-14, page 4, What kind of magnifying lens was used? Is the magnification large enough to do this work? 3.
Line 36-37, page 4, The culture condition for algae should be added here. 4.
Line 20-38, page 6, It would be better if you could give some pictures to prove your hypothesis about the specific process how C. vulgaris cells entrance the brood chamber of D. magna. 6.
Line 20-38, page 6, Is it possible for C. vulgaris cells entrance the brood chamber of D. magna from the postabdomen of D. magna, such as during the process of discharging neonates. 7.
For The results presented in this paper show the ability of one common species of microalga (Chlorella vulgaris) to colonise the brood chambers and induce reproductive failure in two cladoceran species. One of them, of great significance in the ecosystems and the scientific literature (Daphnia magna). Colonization of the brood chamber is positively related with longer light-photoperiod and stronger light intensity. There is some issue with the experimental design, that authors should solve, and its detailed in the specific comments below.
I miss some discussion about an ecological interpretation (beyond that high light conditions increase EPS production by C. vulgaris) of the potential significance of this mechanism of stronger exposure to light in the real ecosystems. The authors claim that this is the first time that an interaction of this nature was found. However, something similar was found between two unicellular planktonic organisms: the haptophyte Prymnesium parvum and the dinoflagellate Oxhyrris marina (Tillmann et al. 2003, Aquatic Microbial Ecology 34: 73-84). This dinoflagellate is a heterotrophic organism that usually phagocytes phytoplankton cells, like P. parvum. P. parvum was observed to release cytolytic toxins that lyse O. marina cells, to phagocyte the small remains of the cells. This interaction is different that the one reported here because the predator actually becomes the prey, however, I think it needs to be referenced. I would recommend this paper for publication after the authors solve the few questions presented here.
Specific comments -Methods: In experiment 1, page 3, lines 60, it is said that "the experiment consisted of different combinations… ", referring to the experimental treatments. I would expect all the possible combinations to be shown, in a full crossed design. However, later, in the results (Fig. 2) I see that the combinations of the control diet at high food concentrations is missing for both photoperiods. This will affect the interpretation of the GLM performed, because the factor 'concentration' is not fully crossed. I don't see the reason for this. The authors should provide an explanation, since if R. subcapitata could colonize the brood chamber also at high concentrations, that would change the interpretation of results.

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
Can the figures 2 & B be tested statistically using multiple comparisons?

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

Recommendation? Accept as is
Comments to the Author(s) I am satisfied with the revision that the authors made according to my previos comments. My main issue with this work was about the lack of a fully crossed experimental design, but now I understand that this was due to practical reasons, and also see that the "high food" level was tested in the second experiment. Although this is not exactly equivalent to a fully crossed design, it is acceptable, and authors are including this aspect in the discussion.

Decision letter (RSOS-200249.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Albini, On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-200249.R1 entitled "Turning defence into offence? Intrusion of cladoceran brood chambers by a green alga leads to reproductive failure" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
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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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Decision letter (RSOS-200249.R2)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Albini,
It is a pleasure to accept your manuscript entitled "Turning defence into offence? Intrusion of cladoceran brood chambers by a green alga leads to reproductive failure" in its current form 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 the production office (openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a co-author (if available) to manage the proofing process, and ensure they are copied into your email to the journal.
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Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. We are submitting a revised version of our manuscript "Turning defence into offence? Intrusion of cladoceran brood chambers by a green alga leads to reproductive failure." for consideration in Royal Society Open Science.
We have made every effort to fully address all of the reviewer comments. In particular, we have added new information to clarify the relevance of our experimental conditions to the light conditions experienced by Chlorella vulgaris within its natural range, how morphological differences between the algae species may have contributed to our results, further details about the Methodology, and how robust our interpretations are to the experimental design. Detailed point-by-point responses are given below, with our responses formatted in blue.
We believe the revisions we have made have improved the manuscript and are grateful to the reviewers and Editorial team for their feedback. We look forward to hearing back from you. Since the conditions in the brood pouch are not hostile to alga or bacteria, fungi, etc., such organisms can proliferate there. REPLY: We thank the reviewer for acknowledging that the article is interesting. We would like to reiterate that the novelty of our study lies not in the fact that Daphnia brood chamber is open to the external environment (as it has been elegantly demonstrated by Seidl et al. (2002) in their study of the brood current), or that pathogens and parasites could exploit the opportunity to inflict harm on Daphnia; rather, there is no precedent where a microalgal food species enters the brood chamber, colonises the eggs and causes reproductive failure. While some may expect Appendix A such a phenomenon to occur, we now provide the first scientific evidence to support such an expectation. Nevertheless, we thank the reviewer for the very interesting comments and suggestions, which we have now incorporated into the Introduction and Discussion. (P2 L18-19; P6 L2-5) Reviewer 2: The authors did an interesting job. They analyzed the anti-grazer strategy in the green alga Chlorella vulgaris against two cladoceran grazers, Daphnia magna and Simocephalus sp. They found that C. vulgaris could directly inflict harm on the reproductive structure of D. magna, and the colonization process depended on light conditions. To our knowledge, this is indeed the first study demonstrating the harmful intrusion of cladoceran brood chambers by a freshwater microalga, although the underlying mechanism and the specific process has still not been well clarified in the present study. Therefore, I recommend publishing this novel discovery in the journal after a major revision. The specific opinions are as follows. REPLY: We thank the reviewer for acknowledging that the findings are novel and the article is interesting, and for suggestions to improve the manuscript.

Major question
Method section (1) It is unclear whether this phenomenon could occur in naturally aquatic ecosystems given that the colonization of brood chamber by Chlorella vulgaris mainly depended on extremely light conditions. Firstly, the photoperiod throughout their experiments were 18L:6D or even 24 L: 0D, which was not in accordance with the natural condition. This is particularly true when the long-term light intensity (130 µmol photons m-2 s-1) was also considered. REPLY: C. vulgaris has a global distribution, stretching from the arctic (Matula et al 2007) to the tropics (Pham et al 2011), which suggests that the photoperiods and light intensities used in our experiments will be found within their natural range. This point has now been added to the Introduction (P2 L27 -30). Further, the light intensity we used is lower than the incipient light intensity and the mean surface mixed-layer light intensity of many lakes (e.g. Pérez et al. 2002, Staehr et al. 2016). We first used 24:0 L:D photoperiod, admittedly a rather extreme condition, to test for the occurrence of the new trait in Chlorella vulgaris, but we did not stop there. We repeated the experiment using the more common photoperiod of 18:6 L:D (which is a common photoperiod in summer across Chlorella's distribution) with a higher light intensity (~130 µmol photons m -2 s -1 ) and we showed the phenomenon also occurred in this case. These experiments, together, allow us to discover a novel predator-prey interaction in the plankton that warrants further investigation. Going beyond natural ecosystems, given the fact that Chlorella is widely used to feed cladocerans in experimental and aquaculture systems, our findings should prompt reconsideration of the practices as well. (P5 L54 -P6 L5) (2) Although with similar cell size, the morphology of the two used algal species was different, is this the reason for their difference in colonization ability? This possibility should be discussed. REPLY: We have discussed this possibility in the Discussion section (P5 L10 -14): "Both C. vulgaris and R. subcapitata cells were less than 12 μm in the longest dimension, whereas the gap when D. magna and Simocephalus sp. opened their abdominal processes was 577 ± 7.05 µm (mean ± s.d., n = 50; Figure S3) and 500 ± 33 µm (mean ± s.d., n = 50), respectively. Hence, the gap was clearly large enough for both algal species to enter the brood chamber via the brood current." Therefore, size alone cannot explain the different outcomes. If by "morphology" the reviewer was referring to cell shape, neither species have spiny structures or protrusions that may suggest entanglement inside the brood chamber. We were indeed surprised by our findings which suggest other interspecific differences leading to different outcomes between the two species. We have now also added the following . Therefore, C. vulgaris has previously been shown to have a competitive advantage over R. subcapitata." We hope that publication of these results will encourage others to build on this study to investigate the question further.
(3) The third experiment lasted for 10 days. Is it long enough for individuals of Simocephalus to reach their maximum body size? If not, your conclusion that Simocephalus individuals cannot be colonization by Chlorella vulgaris is not rigorous. Please explain. REPLY: Perhaps the reviewer may have confused the species we tested here. Between the two algal species R. subcapitata and C. vulgaris, only C. vulgaris showed brood chamber colonisation. However, both of the cladoceran species D. magna and Simocephalus sp. were colonised by C. vulgaris. Please refer to the third experiment where we showed brood chamber colonization of Simocephalus by Chlorella vulgaris, starting on day 7, where 33 out of 40 females were colonised ( Figure 1C). The fact that we demonstrated brood chamber colonisation with a second cladoceran species allows us to conclude that the phenomenon was not unique to D. magna.
Discussion section (4) You suggest that the success of the C. vulgaris cells to remain inside the brood chambers was associated with its ability in high production of EPS under higher light availability? How do you know R. subcapitata do not have this ability? In other words, why R. subcapitata cannot invade grazers brood chamber? REPLY: We can only speculate that the production of EPS was the reason based on the literature (P5 L30-40). Further experiments with both algae species would aim to discover the nature of these substances and to characterise it, to better understand this phenomenon. Please also see our response to question (2) above.
(5) In addition, the underlying mechanisms for the failure of C. vulgaris cells to invade brood chamber of Simocephalus should also be discussed. REPLY: As mentioned above (and in the original submission), we confirmed that C. vulgaris did invade and colonise the brood chamber of Simocephalus. Please refer to the third experiment, Figure 1C and our response to question (3) above.

7.
For figure 2 and 3, it is not clear what data you used for these two figures. Is it at the end of the experiment on a specific day? REPLY: Both figures show the data for the overall experimental time. We have updated the Figure captions to clarify this point.

Reviewer: 3
The results presented in this paper show the ability of one common species of microalga (Chlorella vulgaris) to colonise the brood chambers and induce reproductive failure in two cladoceran species. One of them, of great significance in the ecosystems and the scientific literature (Daphnia magna). Colonization of the brood chamber is positively related with longer light-photoperiod and stronger light intensity. REPLY: We thank the reviewer for the comment and for acknowledging the importance of our study.
There is some issue with the experimental design, that authors should solve, and its detailed in the specific comments below. I miss some discussion about an ecological interpretation (beyond that high light conditions increase EPS production by C. vulgaris) of the potential significance of this mechanism of stronger exposure to light in the real ecosystems. REPLY: We thank the reviewer for the suggestion, which we have incorporated into the Discussion. (P5 L61 -P6 L7).
subcapitata could colonize the brood chamber also at high concentrations, that would change the interpretation of results. REPLY: We have added the following point to the Discussion to clarify (P5 L18 -22): "While we did not fully cross all possible concentration combinations in Experiment 1 (due to practical and resource limitations), we did explicitly test the higher R. subcapitata concentrations (6×10 6 cells ml -1 ) in Experiment 2 and still did not record any brood chamber colonisation with this algal species. For this reason, we believe our general interpretations to be robust." Literature cited:  Table 1 (and other tables) provide sufficient information to interpret these figures without post-hoc analysis: each factor in the additive GLMs consists of only 2-levels (indicated by 1 d.f. in each row of Table 1). Therefore, any significant difference in that factor represents a difference between the two levels of that factor. Given the lack of interaction terms included in our GLMs, it is not appropriate to perform any further pairwise comparisons, as they would not correspond to these statistical models.
We did, however, perform further analyses including the 2-way interaction between Photoperiod x Algal Species where appropriate, but this did not show qualitative differences from the additive GLMs. Further this more complex statistical model did not describe the data more parsimoniously than the simpler additive model we originally considered. As our data are publicly available on Dryad, interested readers will be able to carry out any further analyses as desired.
Therefore, we have modified the Methods (Section 3.5) to clarify this issue: "Statistical analysis was performed with R-studio version 1.1.419. An additive, factorial generalized linear model (GLM) with a binomial error family tested for the effect of experimental treatments (algal species, initial concentration, photoperiod and/or light intensity) on the probability of colonisation by the algae. The effect of C. vulgaris colonisation on the reproductive output of the grazer was assessed using an additive factorial Negative Binomial GLM [24] examining the effects of the same factors listed above on the number of newborns at the end of the experiment. Given the lack of interaction terms in our analyses (in some cases due to lacking a fully crossed experimental design), we did not perform further pairwise comparisons on these statistical models. Each factor in the additive GLMs consists of only two levels (two photoperiods, two light intensities, two algal species, two initial algal concentrations), therefore any significant