Brightly coloured tissues in limid bivalves chemically deter predators

Members of the marine bivalve family Limidae are known for their bright appearance. In this study, their colourful tissues were examined as a defence mechanism towards predators. We showed that when attacked by the peacock mantis shrimp (Odontodactylus scyllarus), the ‘disco’ clam, Ctenoides ales, opened wide to expose brightly coloured tissues to the predator. The predator also significantly preferred to consume the internal, non-colourful clam tissues than the external, colourful tissues. Mass spectrometry-based metabolomic analysis confirmed that colourful tissues had significantly different chemical compositions than the non-colourful ones. The internal, non-colourful tissues had metabolite profiles more similar to an outgroup bivalve than to the species' own colourful external tissues. A number of the compounds that differentiated the colourful tissues from the non-colourful tissues appeared to be peptide-like, which potentially serve as the underlying defensive compounds. This is the first study demonstrating that colourful bivalve tissues are used for chemical defence.

the internal tissues were never touched, and that (like the disco clam) it was also not consumed, which does not really provide an adequate control. If the clams were attacked, this control is suitable for demonstrating that the disco clam opened more frequently than another bivalve species. However, for comparing predator behavior, I would have liked to see a comparison between successful predation attempts and predation attempts that were unsuccessful due to chemical defense. If V. philippinarum was not eaten, I would suggest clarifying the purpose of this control in the text. If you have videos of the control experiment with the mantis shrimp attacking and eating V. philippinarum it might be useful to add this to the ESM along with the predation attempt with the disco clam for the purpose of comparison. This way the reader can better understand what is meant when you say that the mantis shrimp did not react abnormally.
Line 118: Typo. I suggest replacing "Same patterns were" with "A similar pattern was" Referee: 3 Comments to the Author(s) This study investigates the defensive function of the red mantle tissue of the mussel Ctenoides ales. The authors conducted a set of behavioural experiments to investigate predators', peacock mantis shrimps, response to the tissue. First, the predators were given a choice between the mantle and adductor tissues of Ctenoides ales, and between the mantle and adductor tissues of another, palatable mussel species. This was repeated in the dark to conceal visual differences between the tissues. Finally, the predators were given a choice between the mantle tissues of Ctenoides ales and another, non-noxious mussel species, after the tissues had been dyed red to mask any differences in tissue conspicuousness. These choice experiments suggest that the mantle tissue of C. ales was distasteful to the predators. The presence or absence of the visual cues did not influence this outcome, suggesting that the avoidance response was induced by the chemical properties and not by the visual properties of the tissue. Hence, the results do not lend support to the hypothesis that the red mantle tissue would have a protective function based on aposematism (but it is important to bear in mind that the tissue may provide aposematic protection against other predators with different vision, such as fishes). A PCA based on metabolomics analysis showed differences in the chemical composition of different parts of C. ales as well as other mussel species. However, to me it seems to emphasise the difference of the abductor muscle from other tissue types and tells little about which chemical properties are distinct to the mantle tissue of C. ales and contribute to its distastefulness. I appreciate the use of two different ways to test the signalling function of the red mantle tissue in the experimental part of the study, but I also have some concerns regarding the manuscript. My main concern is that I think that the findings are interesting mainly from a taxon-specific point of view, but they contribute little to the broader theoretical framework of prey defences.

Detailed comments
The title: the emphasis on bright colour is not in agreement with your results showing no effect of colour L. 46 and elsewhere: Bright coloration/appearance and colourfulness are words that have been used a lot in the text. Yet, the concept is very subjective and fuzzy. What is experienced as bright or colourful depends on the visual system of the viewer and on the visual environment.
Methods: I would like to see some sentences about the biology of the study species, such as its distribution area and its natural predators. Also, please explain why the peacock mantis shrimp was chosen as the predator for the experiments.

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

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

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

Comments to the Author(s)
To the Authors, I appreciate the changes that the authors have made to the manuscript since it was last in review. In particular I believe the new behavioural trials have filled some important gaps in the experimental framework.
I have a few comments outlined line by line below: Line 46: Replace 'colorations' with 'colors' Line 49: Again, replace 'colorations' with 'colors' Lines 74-85: Were these three experiments conducted on the same seven mantis shrimp? Either way I think this should be stated here Lines 76-77: Replace ',' with ':' Lines 77-78: Was the left-right presentation order randomised or controlled on the choice stick? Lines 80-82: I think these two control experiments are a very nice touch Lines 115-121: When reporting percentages, perhaps it's worth reminding the reader of the sample size Lines 151: You could also mention that the red may act as an aposematic signal to other species of predator, some fish have been shown to be able to learn aposematic signals. On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-191298 entitled "Brightly-Colored Tissues in Limid Bivalves Deter Predators" 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.
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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. It seems the authors have not addressed any of mine or reviewer 3's comments from the last review, not even the correction of typos that were pointed out. So, my previous comments remain. I would also like for the authors respond to reviewer 3's specific comments as they point out several things that need clarification that I missed.
In addition to my previous comments: Reviewer 3 asks whether the same predators were used across trials. This information is of particular interest for the second experiment where mantis shrimp are first offered a choice of the two tissue types unmanipulated, then offered the same tissues but in the dark (without visual cues), and finally offered the two tissues dyed the same red colour. If the same mantis shrimp are used across each of these trials, this would show that they did not learn to avoid the red colour (lending no support for aposematism). But I am thinking that the first part of this experiment (n=7) was run at the same time as the previous experiment, whereas parts 2 and 3 (n=11, n=12) were run later and possibly with different mantis shrimp individuals? If so, the authors did not test whether the mantis shrimp learned to avoid the red colour and the role of the red colour is still potentially aposematic for the mantis shrimp (and other predators) but simply not tested yet. You would not expect the red colouration to deter naïve predators on its own (unless it is an innate aversion) until they had learned that it is associated with unpalatable tissues. If the experiments were run at different times of year and with different individuals this should be clearly stated. And if parts 2 & 3 of the experiment were run with different individuals, it would also be helpful to be very clear for the reader that you are testing whether the red tissues are unpalatable compared to other tissues, but that your experiments are not designed to test whether the red colouration is aposematic.

Previous comments from myself and reviewer 3:
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) This paper is greatly strengthened with the addition of the two new experiments, and I am now satisfied with the author's conclusion. I believe they have sufficiently disentangled colouration and chemical defense. I would still like some clarification regarding the first experiment with the live disco clam and the new palatable bivalve control. Otherwise, I have only a few suggestions for minor changes below.
Lines 32 -34: I think the abstract would read better with consistent terminology for the tissues. For instance saying "colourful tissues" and "non-colourful tissues" as in the previous sentence rather than "tissues pre-identified as noxious" and "tissues pre-identified as non-noxious." It seems odd to use these terms in the abstract since they are not used elsewhere in the text (except line 125 referring to the ESM file).
Methods: Please mention here that there is an ESM file with the collection information for the mantis shrimp and clams used in the experiments.
Line 113: If V. philippinarum was attacked and eaten by the mantis shrimp please say so in the text. The way it is presented it seems V. philippinarum was ignored by the mantis shrimp, that the internal tissues were never touched, and that (like the disco clam) it was also not consumed, which does not really provide an adequate control. If the clams were attacked, this control is suitable for demonstrating that the disco clam opened more frequently than another bivalve species. However, for comparing predator behavior, I would have liked to see a comparison between successful predation attempts and predation attempts that were unsuccessful due to chemical defense. If V. philippinarum was not eaten, I would suggest clarifying the purpose of this control in the text. If you have videos of the control experiment with the mantis shrimp attacking and eating V. philippinarum it might be useful to add this to the ESM along with the predation attempt with the disco clam for the purpose of comparison. This way the reader can better understand what is meant when you say that the mantis shrimp did not react abnormally.
Line 118: Typo. I suggest replacing "Same patterns were" with "A similar pattern was" Referee: 3 Comments to the Author(s) This study investigates the defensive function of the red mantle tissue of the mussel Ctenoides ales. The authors conducted a set of behavioural experiments to investigate predators', peacock mantis shrimps, response to the tissue. First, the predators were given a choice between the mantle and adductor tissues of Ctenoides ales, and between the mantle and adductor tissues of another, palatable mussel species. This was repeated in the dark to conceal visual differences between the tissues. Finally, the predators were given a choice between the mantle tissues of Ctenoides ales and another, non-noxious mussel species, after the tissues had been dyed red to mask any differences in tissue conspicuousness. These choice experiments suggest that the mantle tissue of C. ales was distasteful to the predators. The presence or absence of the visual cues did not influence this outcome, suggesting that the avoidance response was induced by the chemical properties and not by the visual properties of the tissue. Hence, the results do not lend support to the hypothesis that the red mantle tissue would have a protective function based on aposematism (but it is important to bear in mind that the tissue may provide aposematic protection against other predators with different vision, such as fishes). A PCA based on metabolomics analysis showed differences in the chemical composition of different parts of C. ales as well as other mussel species. However, to me it seems to emphasise the difference of the abductor muscle from other tissue types and tells little about which chemical properties are distinct to the mantle tissue of C. ales and contribute to its distastefulness. I appreciate the use of two different ways to test the signalling function of the red mantle tissue in the experimental part of the study, but I also have some concerns regarding the manuscript. My main concern is that I think that the findings are interesting mainly from a taxon-specific point of view, but they contribute little to the broader theoretical framework of prey defences.

Detailed comments
The title: the emphasis on bright colour is not in agreement with your results showing no effect of colour L. 46 and elsewhere: Bright coloration/appearance and colourfulness are words that have been used a lot in the text. Yet, the concept is very subjective and fuzzy. What is experienced as bright or colourful depends on the visual system of the viewer and on the visual environment.
Methods: I would like to see some sentences about the biology of the study species, such as its distribution area and its natural predators. Also, please explain why the peacock mantis shrimp was chosen as the predator for the experiments.

05-Sep-2019
Dear Dr Li, I am pleased to inform you that your manuscript entitled "Brightly-Colored Tissues in Limid Bivalves Chemically Deter Predators" is now accepted for publication in Royal Society Open Science.
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Editor
The reviewers and handling editors 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.
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Funding statement Please list the source of funding for each author.

Response:
We have updated the Ethic statement and funding statement: Ethics Animals used in the study were purchased from aquarium stores. Use of invertebrate animals does not require protocol approval by the Institutional Animal Care and Use Committee (IACUC), University of Colorado Boulder.
Funding Statement This study is supported by internal funds to JL from the Museum of Natural History, University of Colorado Boulder Reviewer: 1 Reviewer 3 asks whether the same predators were used across trials. This information is of particular interest for the second experiment where mantis shrimp are first offered a choice of the two tissue types unmanipulated, then offered the same tissues but in the dark (without visual cues), and finally offered the two tissues dyed the same red colour. If the same mantis shrimp are used across each of these trials, this would show that they did not learn to avoid the red colour (lending no support for aposematism). But I am thinking that the first part of this experiment (n=7) was run at the same time as the previous experiment, whereas parts 2 and 3 (n=11, n=12) were run later and possibly with different mantis shrimp individuals? If so, the authors did not test whether the mantis shrimp learned to avoid the red colour and the role of the red colour is still potentially aposematic for the mantis