Photosynthesis from stolen chloroplasts can support sea slug reproductive fitness

Some sea slugs are able to steal functional chloroplasts (kleptoplasts) from their algal food sources, but the role and relevance of photosynthesis to the animal host remain controversial. While some researchers claim that kleptoplasts are slowly digestible ‘snacks’, others advocate that they enhance the overall fitness of sea slugs much more profoundly. Our analysis shows light-dependent incorporation of 13C and 15N in the albumen gland and gonadal follicles of the sea slug Elysia timida, representing translocation of photosynthates to kleptoplast-free reproductive organs. Long-chain polyunsaturated fatty acids with reported roles in reproduction were produced in the sea slug cells using labelled precursors translocated from the kleptoplasts. Finally, we report reduced fecundity of E. timida by limiting kleptoplast photosynthesis. The present study indicates that photosynthesis enhances the reproductive fitness of kleptoplast-bearing sea slugs, confirming the biological relevance of this remarkable association between a metazoan and an algal-derived organelle.

Figures: In general, I think more detailed descriptions of the figures would be helpful. For example: Fig 1: Those less adept at microscopy or slug anatomy may not be able to determine where the 13C and 15N are localized. It would be helpful to clarify in the figure text what is going on in the pictures. Fig 2: The figures have the albumen glands labelled, but why not labels for the digestive tubule? Fig 3: What is the tissue on the left hand side of the 3H chase micrograph? Are those follicles? Again, why not label more regions for clarity?
Review form: Reviewer 2

Recommendation
Major revision is needed (please make suggestions in comments)

General interest: Is the paper of sufficient general interest? Excellent
Quality of the paper: Is the overall quality of the paper suitable? Good

Is the length of the paper justified? Yes
Should the paper be seen by a specialist statistical reviewer? No

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author Several papers have demonstrated that kleptoplasts in the sea slug provide photosynthate and support growth, but recent study has denied it. Therefore, the role of kleptoplasts in the sea slug is still controversial. This study demonstrated that photosynthate from kleptoplasts are translocated to kleptoplasts-free reproductive organs and it might enhance the reproductive fitness. Authors showed the translocation of photosynthate by tracking short-term lightdependent incorporation of inorganic carbon (13C) and nitrogen (15N) into animal tissues using compound specific isotope analysis (CSIA) of fatty acid methyl esters (FAME) and highresolution secondary ion mass spectrometry (NanoSIMS). This reviewer feels the experimental design is very reasonable and results are clear for this portion of the paper. However, authors also showed the physiological significance of the photosynthate from kleptoplasts by testing the effect of light on fecundity of sea slug. This reviewer feels the conclusion that the photosynthate from kleptoplasts is important for the reproductive fitness of the sea slug is not supported by the current data set. This reviewer feels the data reported in this paper is important to understanding the role of the kleptoplasts in sea slugs, however, in order to recommend publication either additional data needs to be added or the discussion/conclusion needs to be modified.

Major comment
In order to conclude that photosynthesis enhances the reproductive fitness of the sea slug, photosynthesis needed to be directly and specifically suppressed using an inhibitor such as DCMU, rather than reduced light intensity. The authors cannot exclude that reduced light intensity may directly reduce the fecundity of the sea slugs. Author need to add new data to support their conclusion. Alternatively, authors need to change this conclusion to discussion. Furthermore, throughout the text (and the title), it should be changed to say reduced light intensity and not inhibited/suppressed photosynthesis. I am writing to inform you that your manuscript RSPB-2021-1331 entitled "Photosynthesis from stolen chloroplasts increases sea slug reproductive fitness" has, in its current form, been rejected for publication in Proceedings B.
This action has been taken on the advice of referees, who have recommended that substantial revisions are necessary. With this in mind we would be happy to consider a resubmission, provided the comments of the referees are fully addressed. However please note that this is not a provisional acceptance.
The resubmission will be treated as a new manuscript. However, we will approach the same reviewers if they are available and it is deemed appropriate to do so by the Editor. Please note that resubmissions must be submitted within six months of the date of this email. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office. Manuscripts submitted after this date will be automatically rejected.
Please find below the comments made by the referees, not including confidential reports to the Editor, which I hope you will find useful. If you do choose to resubmit your manuscript, please upload the following: 1) A 'response to referees' document including details of how you have responded to the comments, and the adjustments you have made.
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Sincerely, Dr Daniel Costa mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: Thank you for submitting your manuscript entitled "Photosynthesis from stolen chloroplasts increases sea slug reproductive fitness" to Proceedings B. We have gotten back the reviews and both are generally positive. Both reviewers agree that the authors have strongly demonstrated that the sea slug makes use of and obtains nutritional benefits from kleptoplasts, and this contributes to the controversy around this topic. The demonstration of translocation of the photosynthates to reproductive cells also indicate potential enhancement of reproductive fitness. However, Reviewer 2 brings up an important concern which can weaken the conclusion that kleptoplast photosynthesis does enhance reproductive fitness. Reviewer 2 notes that the methodology used for this aspect of the study, in particular the different light treatments, can also have a direct impact on the fecundity of the sea slugs and cannot be said to necessarily inhibit or suppress photosynthesis. Reviewer 2 provides suggestions to address the comments.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) Review of Manuscript ID: RSPB-2021-1331 Photosynthesis from stolen chloroplasts increases sea slug reproductive fitness Summary: This is a strong paper which details careful experiments to demonstrate that the sea slug Elysia timida derives significant nutritional benefit from its kleptoplastic association with chloroplasts stolen from Acetabularia acetabulum. The authors use 13C and 15N labelling to show that organic molecules produced by photosynthesis are incorporated into the sea slug tissues, and translocated to the albumen gland and gonadal follicles (which are NOT located near chloroplasts). The use of NanoSIMS isotopic imaging was particularly useful in demonstrating this transfer. They also measured the amount and types of fatty acids, included those reported to be used in reproduction, in slugs using 13C labelling under light and dark conditions. In addition, they demonstrate that eggs produced by sea slugs under actinic light lay statistically significantly more eggs than those kept in non-actinic light. Taken together, these three lines of analysis strongly support the hypothesis that kleptoplasty provides nutrition for sea slugs, and improves their overall evolutionary fitness. The author's major conclusions are: 1. Sequestered chloroplasts in E. timida actively photosynthesize fixing inorganic carbon to organic molecules which are then translocated to non-photosynthetic tissues in the animal involved with reproduction 2. Radiolabelled carbon was shown to be incorporated in a range of fatty acids, including those associated with reproduction, in animals exposed to light only. This experiment also provided an opportunity to trace the biochemistry of fatty acid synthesis in the animal. 3. Egg fecundity was statistically higher in slugs reared under actinic conditions vs non-actinic. Comments: This is a very well written manuscript. The experiments are well designed, well executed, and conclusively support the hypothesis that E. timida rely on photosynthesis from their stolen algal chloroplasts. While there is significant data in the literature supporting this position, recent papers have cast doubt on the role of photosynthesis. In my opinion, this study is of particular importance because it is the first to prove the link between photosynthesis and actual reproductive fitness in the animals. I recommend this manuscript for publication and only have a few minor comments. Minor comments: Line 121: You state that the 13C level "generally leveled out". Could you clarify what this means? As in the rate of incorporation leveled out, or the total amount leveled out indicating no new incorporation of 13C? Figures Referee: 2 Comments to the Author(s) Several papers have demonstrated that kleptoplasts in the sea slug provide photosynthate and support growth, but recent study has denied it. Therefore, the role of kleptoplasts in the sea slug is still controversial. This study demonstrated that photosynthate from kleptoplasts are translocated to kleptoplasts-free reproductive organs and it might enhance the reproductive fitness. Authors showed the translocation of photosynthate by tracking short-term lightdependent incorporation of inorganic carbon (13C) and nitrogen (15N) into animal tissues using compound specific isotope analysis (CSIA) of fatty acid methyl esters (FAME) and highresolution secondary ion mass spectrometry (NanoSIMS). This reviewer feels the experimental design is very reasonable and results are clear for this portion of the paper. However, authors also showed the physiological significance of the photosynthate from kleptoplasts by testing the effect of light on fecundity of sea slug. This reviewer feels the conclusion that the photosynthate from kleptoplasts is important for the reproductive fitness of the sea slug is not supported by the current data set. This reviewer feels the data reported in this paper is important to understanding the role of the kleptoplasts in sea slugs, however, in order to recommend publication either additional data needs to be added or the discussion/conclusion needs to be modified.

Major comment
In order to conclude that photosynthesis enhances the reproductive fitness of the sea slug, photosynthesis needed to be directly and specifically suppressed using an inhibitor such as DCMU, rather than reduced light intensity. The authors cannot exclude that reduced light intensity may directly reduce the fecundity of the sea slugs. Author need to add new data to support their conclusion. Alternatively, authors need to change this conclusion to discussion. Furthermore, throughout the text (and the title), it should be changed to say reduced light intensity and not inhibited/suppressed photosynthesis. It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author My original review had only minor concerns which were adequately addressed in this revision. The majority of changes appear to be in response to the other reviewer. I feel that the author's adequately addressed these concerns as well.

Recommendation
Accept with minor revision (please list in comments) Scientific importance: Is the manuscript an original and important contribution to its field? Good General interest: Is the paper of sufficient general interest? Acceptable Quality of the paper: Is the overall quality of the paper suitable? Acceptable Is the length of the paper justified? Yes

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author
The authors conclude that photosynthesis of kleptoplasts enhances the reproductive fitness of kleptoplasts-bearing sea slugs. However, this conclusion is not supported in current data set provided, as this reviewer previously commented. The authors chose to revise the manuscript according to this reviewer's comment. However, the revised manuscript still states that photosynthesis enhances the reproductive fitness of kleptoplasts-bearing sea slugs. Therefore, this reviewer feels the revisions are insufficient. Authors need to understand that reporting this kind of unsupported conclusion discourages further research.
Specific comments: Title should be changed to something like "Photosynthesis of stolen chloroplast might support sea slug reproductive fitness".
In the abstract, "Finally, we report reduced fecundity of E. timida by limiting kleptoplasts photosynthesis" should be "Finally, we report reduced fecundity of E. timida in conditions where the kleptoplasts photosynthesis is limited". Also in the abstract, "The present study provides the first thorough experimental evidence that photosynthesis enhances the reproductive fitness of kleptoplasts-bearing sea slugs, …" is not appropriate. It should be "The present study suggests that photosynthesis enhances the reproductive fitness of kleptoplasts-bearing sea slugs, …".
Lines 90-91, sentence should be "We report strong experimental data supporting a role of photosynthesis in the reproductive investment and fitness of a kleptoplast-bearing sea slug.".
Lines 265-273, authors can suggest, but not conclude, that photosynthesis of kleptoplasts enhances the reproductive fitness of kleptoplasts-bearing sea slugs.

03-Sep-2021
Dear Dr Cruz I am pleased to inform you that your manuscript RSPB-2021-1779 entitled "Photosynthesis from stolen chloroplasts increases sea slug reproductive fitness" has been accepted for publication in Proceedings B.
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Sincerely, Dr Daniel Costa mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: Thank you for revising and resubmitting your manuscript. Reviewer 1 is fully satisfied with your revisions, while Reviewer 2 suggests minor revisions. Reviewer 2's comments still revolve around the first comment made regarding strong statements on the direct link between the kleptoplast photosynthesis and reproductive fitness. In your revision, you toned down some of these statements as a response to Reviewer 2, particularly in your conclusion section. You could possibly similarly fine tune at least the title and the last sentence of the abstract for consistency. The other sentences pointed out though seem to be already fine as is.
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Comments to Reviewers
Reviewer 1: Review of Manuscript ID: RSPB-2021-1331 Photosynthesis from stolen chloroplasts increases sea slug reproductive fitness Summary: This is a strong paper which details careful experiments to demonstrate that the sea slug Elysia timida derives significant nutritional benefit from its kleptoplastic association with chloroplasts stolen from Acetabularia acetabulum. The authors use 13C and 15N labelling to show that organic molecules produced by photosynthesis are incorporated into the sea slug tissues, and translocated to the albumen gland and gonadal follicles (which are NOT located near chloroplasts). The use of NanoSIMS isotopic imaging was particularly useful in demonstrating this transfer. They also measured the amount and types of fatty acids, included those reported to be used in reproduction, in slugs using 13C labelling under light and dark conditions. In addition, they demonstrate that eggs produced by sea slugs under actinic light lay statistically significantly more eggs than those kept in non-actinic light. Taken together, these three lines of analysis strongly support the hypothesis that kleptoplasty provides nutrition for sea slugs, and improves their overall evolutionary fitness.
The author's major conclusions are: 1. Sequestered chloroplasts in E. timida actively photosynthesize fixing inorganic carbon to organic molecules which are then translocated to non-photosynthetic tissues in the animal involved with reproduction 2. Radiolabelled carbon was shown to be incorporated in a range of fatty acids, including those associated with reproduction, in animals exposed to light only. This experiment also provided an opportunity to trace the biochemistry of fatty acid synthesis in the animal.
3. Egg fecundity was statistically higher in slugs reared under actinic conditions vs non-actinic.
Comments: This is a very well written manuscript. The experiments are well designed, well executed, and conclusively support the hypothesis that E. timida rely on photosynthesis from their stolen algal chloroplasts. While there is significant data in the literature supporting this position, recent papers have cast doubt on the role of photosynthesis. In my opinion, this study is of particular importance because it is the first to prove the link between photosynthesis and actual reproductive fitness in the animals. I recommend this manuscript for publication and only have a few minor comments.

Reply:
The authors sincerely thank the positive feedback on our work by Reviewer 1.

Minor comments:
Line 121: You state that the 13C level "generally leveled out". Could you clarify what this means? As in the rate of incorporation leveled out, or the total amount leveled out indicating no new incorporation of 13C?