Proactive avoidance behaviour and pace-of-life syndrome in Atlantic salmon

Individuals in a fish population differ in key life-history traits such as growth rate and body size. This raises the question of whether such traits cluster along a fast-slow growth continuum according to a pace-of-life syndrome (POLS). Fish species like salmonids may develop a bimodal size distribution, providing an opportunity to study the relationships between individual growth and behavioural responsiveness. Here we test whether proactive characteristics (bold behaviour coupled with low post-stress cortisol production) are related to fast growth and developmental rate in Atlantic salmon, Salmo salar. Boldness was tested in a highly controlled two-tank hypoxia test were oxygen levels were gradually decreased in one of the tanks. All fish became inactive close to the bottom at 70% oxygen saturation. At 40% oxygen saturation level a fraction of the fish actively sought to avoid hypoxia. A proactive stress coping style was verified by lower cortisol response to a standardized stressor. Two distinct clusters of bimodal growth trajectories were identified, with fast growth and early smoltification in 80% of the total population. There was a higher frequency of proactive than reactive individuals in this fast-developing fraction of fish. The smolts were associated with higher post-stress plasma cortisol than parr, and the proactive smolts leaving hypoxia had significant lower post-stress cortisol than the stayers. The study demonstrated a link between a proactive coping and fast growth and developmental ratio and suggests that selection for domestic production traits promotes this trait cluster.

I think the results of the study do address the hypothesis at hand, although it seems to be spread across several figures. Thus, I have comments/suggestions regarding data presentation and several major questions regarding the experiments. I also have more minor editorial comments for the authors. Major questions and comments: -Seeing that the goal was to investigate a possible relationship between growth and boldness in behaviour, and also that for both are continuous measurements, it should be possible to relate fish weight to frequency of Stayers and Leavers (Fig.4A measurement) and/or to plasma cortisol concentration (Fig.4B measurement) and to look at the correlation between the two variables? This way of data presentation would presumably directly address the hypothesis instead of having weight distribution in one set of figures (Fig.2) and the response variable in 2-3 other figures (Fig.3-4). - Figure 3C: It's not clear which of data points in here are from fish that were from UM and LM groups. Was there a difference in swimming depth between the two groups? Could the fish classified in the two different groups (if there was a spread) be indicated in different colors to indicate whether there was a difference in swimming depth? - Figure 4: Due to the distribution of fish weights, is there a size-dependency of plasma cortisol concentration at all? - Figure 4: Can the authors discuss whether because the frequency of LM fish was lower (Fig.2B) whether that created a bias in selecting fish with certain personalities for the hypoxia avoidance experiment? -The tentative hypothesis that the authors propose twice in the manuscript that "the occurrence of such contrasts in domesticated lines suggest the tentative hypothesis that successful expression of fast-proactive phenotypes depend on the presence of at least a small fraction of passive copers in the population." This does not seem to be a testable hypothesis. You're always going to have a normal distribution of fish weights and a range of behaviours. Can the authors comment/elaborate on this? -With the LM being not smoltified and the UM being smoltified, that would indicate large differences in gill morphology that may influence their hypoxia tolerance. Can the authors address whether how that would impact the interpretation of these results? Minor comments (* indicate my edits): -Add x-axis labelling into Fig.3A -Genus and species need to be italicized throughout the manuscript -"N2" and "O2", 2 needs to be in subscript throughout the manuscript -Figure captions: include sample sizes - Figure 2A: since the x-axis is not continuous, these points shouldn't be connected with lines - Figure 4B: Perhaps different color schemes could be used here to match what is used for LM and UM in Fig.4A, since these are all UM fish and no LM fish are present for this dataset -Page 1, line 35: "This raises the question *of whether* such traits cluster… " -Page 1, line 45: "There was a higher frequency of …. In this fast-developing fraction of fish", higher compared to? -Page 1, line 56: "… showing consistency between context and over time", is this indicating that behavioural traits displayed by organisms are consistent in different context and over the course of an organisms life-time? Phrasing for POLS unclear.
-Page 1, line 59: "behavioural trait correlation", correlation singular to agree with "is" following. -Page 2, line 15: "That developmental ratio is related to stress coping styles is further strengthen *by* a study" -Page 2, line 16: no comma after "O. mykiss, strains" -Page 2, line 21: "Still, *whether* time to reach…" -Page 3, line 23: add year to October and November test times -Page 4, line 46: "… it *became* clear that the fish became increasingly inactive…" -Page 5, line 14: "This is *consistent* to bimodality of life history traits…" -Page 5, line 15: "This smoltification process includes a *suite*… " -Page 5, line 22: "There *was* a higher proportion…" -Page 5, line 28: Any citation for this previous observation that Stayers would rather die of low O2 than escape? -Page 5, line 29: "… the individuals have the option of *moving* to other habitats…" -Page 5, line 37: not sure what you mean here by 'exposed to' higher mortality, "… predicting that proactive/bold personality types *show* higher mortality…"? -Page 5, line 49: "… aquaculture selects for selecting..", awkward phrasing -Page 5, lines 51-53: This hypothesis would not be testable and also seems to be a truism. You're always going to have a normal distribution of fish weights and a range of behaviours. Can the authors comment on this? -Page 5, line 59 to end of page: This sentence is awkwardly long and requires editing: "In line with this,….a sit and wait coping strategy". -Page 6, line 5-6: This sentence requires a citation: "Generally, the proactive behavioural profile… characterized by a high level of active avoidance together with a low HPI axis reactivity …" -Page 6, line 17: "The present *study* shows that active avoidance to hypoxia is associated…" -Page 6, line 20: "… lends support to a relationship between", on comma after "between" -Page 6, line 22: "… sorting fish *with* respect to hypoxia avoidance" -Review form: Reviewer 2 Is the manuscript scientifically sound in its present form? Yes

Is the language acceptable? Yes
Is it clear how to access all supporting data? Not Applicable

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)
Overall I found this paper interesting and easily readable. I thought that the authors outlined the need for further investigation rather well, but it could benefit from a more explicit hypothesis statement in the introduction.
There were a few typos throughout, and I would recommend reading through and fixing as they occur.
I would recommend a justification for using 35g as the split between the LM and UM groups. You noted in the study that there were a limited number of LM individuals in the control treatment that impaired your ability to conduct all statistical tests. Is there a biological or observational reason for this cutoff, or simply numeric?
Decision letter (RSOS-181859.R0) 30-Nov-2018 Dear Professor Damsgård, The editors assigned to your paper ("Proactive avoidance behaviour and pace-of-life syndrome in Atlantic salmon") 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 23-Dec-2018. 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.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response.
In addition to addressing all of the reviewers' and editor's comments please also ensure that your revised manuscript contains the following sections as appropriate before the reference list: • Ethics statement (if applicable) 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.
• 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 have 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 have 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.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-181859 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• 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.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Please note that Royal Society Open Science charge article processing charges for all new submissions that are accepted for publication. Charges will also apply to papers transferred to Royal Society Open Science from other Royal Society Publishing journals, as well as papers submitted as part of our collaboration with the Royal Society of Chemistry (http://rsos.royalsocietypublishing.org/chemistry). If your manuscript is newly submitted and subsequently accepted for publication, you will be asked to pay the article processing charge, unless you request a waiver and this is approved by Royal Society Publishing. You can find out more about the charges at http://rsos.royalsocietypublishing.org/page/charges. Should you have any queries, please contact openscience@royalsociety.org.
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. We have received the feedback from two reviewers that agreed on the merits of the paper. Before the paper can be accepted for publications, the authors should address the constructive feedback from reviewer 1.

Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) I have reviewed the manuscript "Proactive avoidance behaviour and pace of life syndrome in Atlantic salmon". The goal of this study was to investigate whether there is a relationship between growth and development rate and boldness in behaviour (as assessed by hypoxia avoidance and plasma cortisol levels) in hatchery bred Atlantic salmon.
I think the results of the study do address the hypothesis at hand, although it seems to be spread across several figures. Thus, I have comments/suggestions regarding data presentation and several major questions regarding the experiments. I also have more minor editorial comments for the authors. Major questions and comments: -Seeing that the goal was to investigate a possible relationship between growth and boldness in behaviour, and also that for both are continuous measurements, it should be possible to relate fish weight to frequency of Stayers and Leavers (Fig.4A measurement) and/or to plasma cortisol concentration (Fig.4B measurement) and to look at the correlation between the two variables? This way of data presentation would presumably directly address the hypothesis instead of having weight distribution in one set of figures (Fig.2) and the response variable in 2-3 other figures (Fig.3-4).
- Figure 3C: It's not clear which of data points in here are from fish that were from UM and LM groups. Was there a difference in swimming depth between the two groups? Could the fish classified in the two different groups (if there was a spread) be indicated in different colors to indicate whether there was a difference in swimming depth? - Figure 4: Due to the distribution of fish weights, is there a size-dependency of plasma cortisol concentration at all? - Figure 4: Can the authors discuss whether because the frequency of LM fish was lower (Fig.2B) whether that created a bias in selecting fish with certain personalities for the hypoxia avoidance experiment? -The tentative hypothesis that the authors propose twice in the manuscript that "the occurrence of such contrasts in domesticated lines suggest the tentative hypothesis that successful expression of fast-proactive phenotypes depend on the presence of at least a small fraction of passive copers in the population." This does not seem to be a testable hypothesis. You're always going to have a normal distribution of fish weights and a range of behaviours. Can the authors comment/elaborate on this? -With the LM being not smoltified and the UM being smoltified, that would indicate large differences in gill morphology that may influence their hypoxia tolerance. Can the authors address whether how that would impact the interpretation of these results? Minor comments (* indicate my edits): -Add x-axis labelling into Fig.3A -Genus and species need to be italicized throughout the manuscript -"N2" and "O2", 2 needs to be in subscript throughout the manuscript -Figure captions: include sample sizes - Figure 2A: since the x-axis is not continuous, these points shouldn't be connected with lines - Figure 4B: Perhaps different color schemes could be used here to match what is used for LM and UM in Fig.4A, since these are all UM fish and no LM fish are present for this dataset -Page 1, line 35: "This raises the question *of whether* such traits cluster… " -Page 1, line 45: "There was a higher frequency of …. In this fast-developing fraction of fish", higher compared to? -Page 1, line 56: "… showing consistency between context and over time", is this indicating that behavioural traits displayed by organisms are consistent in different context and over the course of an organisms life-time? Phrasing for POLS unclear.
-Page 1, line 59: "behavioural trait correlation", correlation singular to agree with "is" following. -Page 2, line 15: "That developmental ratio is related to stress coping styles is further strengthen *by* a study" -Page 2, line 16: no comma after "O. mykiss, strains" -Page 2, line 21: "Still, *whether* time to reach…" -Page 3, line 23: add year to October and November test times -Page 4, line 46: "… it *became* clear that the fish became increasingly inactive…" -Page 5, line 14: "This is *consistent* to bimodality of life history traits…" -Page 5, line 15: "This smoltification process includes a *suite*… " -Page 5, line 22: "There *was* a higher proportion…" -Page 5, line 28: Any citation for this previous observation that Stayers would rather die of low O2 than escape? -Page 5, line 29: "… the individuals have the option of *moving* to other habitats…" -Page 5, line 37: not sure what you mean here by 'exposed to' higher mortality, "… predicting that proactive/bold personality types *show* higher mortality…"? -Page 5, line 49: "… aquaculture selects for selecting..", awkward phrasing -Page 5, lines 51-53: This hypothesis would not be testable and also seems to be a truism. You're always going to have a normal distribution of fish weights and a range of behaviours. Can the authors comment on this? -Page 5, line 59 to end of page: This sentence is awkwardly long and requires editing: "In line with this,….a sit and wait coping strategy".
-Page 6, line 5-6: This sentence requires a citation: "Generally, the proactive behavioural profile… characterized by a high level of active avoidance together with a low HPI axis reactivity …" -Page 6, line 17: "The present *study* shows that active avoidance to hypoxia is associated…" -Page 6, line 20: "… lends support to a relationship between", on comma after "between" -Page 6, line 22: "… sorting fish *with* respect to hypoxia avoidance" -Reviewer: 2 Comments to the Author(s) Overall I found this paper interesting and easily readable. I thought that the authors outlined the need for further investigation rather well, but it could benefit from a more explicit hypothesis statement in the introduction.
There were a few typos throughout, and I would recommend reading through and fixing as they occur.
I would recommend a justification for using 35g as the split between the LM and UM groups. You noted in the study that there were a limited number of LM individuals in the control treatment that impaired your ability to conduct all statistical tests. Is there a biological or observational reason for this cutoff, or simply numeric?
Author's Response to Decision Letter for (RSOS-181859.R0) See Appendix A.

Is the language acceptable? Yes
Is it clear how to access all supporting data? Not Applicable

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

Comments to the Author(s)
The revisions greatly improve the clarity of hypotheses and how this study fits into the broader scope of the field. A few comments are listed below: Page 8, Line 15: "where proactive individuals are being characterized...." being should be removed Page 8, Line 16-20: Reword the sentence that begins "In addition, the stress response in.....". In its current state, it is a bit confusing, especially the portion about the low HPA or HPI axis. I know what it means, just rework for clarity.
Page 15, first sentence: "... reactivity axis of the pro-and reactive coping styles /is/ ;ess rigid tha(n) previously though." Page 15, final sentence of conclusion: occurring should be changed to either "that is occurring" or "that occurs" Decision letter (RSOS-181859.R1)

15-Feb-2019
Dear Professor Damsgård: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-181859.R1 entitled "Proactive avoidance behaviour and pace-of-life syndrome in Atlantic salmon" 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.
• 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.
• 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.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-181859.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• 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.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Please note that we cannot publish your manuscript without these end statements included. We have included a screenshot example of the end statements for reference. If you feel that a given heading is not relevant to your paper, please nevertheless include the heading and explicitly state that it is not relevant to your work.
Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript before 24-Feb-2019. Please note that the revision deadline will expire at 00.00am on this date. If you do not think you will be able to meet this date please let me know immediately.
To revise your manuscript, log into https://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions". Under "Actions," click on "Create a Revision." You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you will be able to respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". You can use this to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the referees.
When uploading your revised files please make sure that you have: 1) A text file of the manuscript (tex, txt, rtf, docx or doc), references, tables (including captions) and figure captions. Do not upload a PDF as your "Main Document". 2) A separate electronic file of each figure (EPS or print-quality PDF preferred (either format should be produced directly from original creation package), or original software format) 3) Included a 100 word media summary of your paper when requested at submission. Please ensure you have entered correct contact details (email, institution and telephone) in your user account 4) Included the raw data to support the claims made in your paper. You can either include your data as electronic supplementary material or upload to a repository and include the relevant doi within your manuscript 5) All supplementary materials accompanying an accepted article will be treated as in their final form. Note that the Royal Society will neither edit nor typeset supplementary material and it will be hosted as provided. Please ensure that the supplementary material includes the paper details where possible (authors, article title, journal name).
Supplementary files will be published alongside the paper on the journal website and posted on the online figshare repository (https://figshare.com). The heading and legend provided for each supplementary file during the submission process will be used to create the figshare page, so please ensure these are accurate and informative so that your files can be found in searches. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI.
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. The revised version of the manuscript addressed the reviewers concerns, and this paper can be accepted for publication upon minor revisions.
Reviewer comments to Author: Reviewer: 2 Comments to the Author(s) The revisions greatly improve the clarity of hypotheses and how this study fits into the broader scope of the field. A few comments are listed below: Page 8, Line 15: "where proactive individuals are being characterized...." being should be removed Page 8, Line 16-20: Reword the sentence that begins "In addition, the stress response in.....". In its current state, it is a bit confusing, especially the portion about the low HPA or HPI axis. I know what it means, just rework for clarity.
Page 15, first sentence: "... reactivity axis of the pro-and reactive coping styles /is/ ;ess rigid tha(n) previously though." Page 15, final sentence of conclusion: occurring should be changed to either "that is occurring" or "that occurs" Author's Response to Decision Letter for (RSOS-181859.R1)

18-Feb-2019
Dear Professor Damsgård, I am pleased to inform you that your manuscript entitled "Proactive avoidance behaviour and pace-of-life syndrome in Atlantic salmon" 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. Our response: We have looked into the suggestions made by the reviewer.

Major questions and comments:
2. Seeing that the goal was to investigate a possible relationship between growth and boldness in behaviour, and also that for both are continuous measurements, it should be possible to relate fish weight to frequency of Stayers and Leavers (Fig.4A measurement) and/or to plasma cortisol concentration (Fig.4B measurement) and to look at the correlation between the two variables? This way of data presentation would presumably directly address the hypothesis instead of having weight distribution in one set of figures (Fig.2) and the response variable in 2-3 other figures (Fig.3-4).
Our response: The issue raised by the reviewer is interesting, but it is not a question of continuous measurements and thus not an expected correlation between size and any variable. Any behavioural differences that can be explained in terms of unimodal size differences may simply be part of a proximate physiological explanations, rather than POLS. In anadromous salmonid fishes the life history choice of sea migration related to the development the year before smoltification. In line with this, the seawater challenge revealed the fast-growing upper mode fish smoltified, while the lower mode group did not. It is well known that growth thus is a consequence of the life history decision to become a smolt, and smoltification per se is not a consequence of size. Accordingly, we treated UM and LM as grouping variables. Like vice, the hypoxia test revealed two contrasting behavioural responses to this challenge; leavers and stayers. A confinement stress test confirmed that these two contrasting behavioural reactions to hypoxia corresponded to the proactive and reactive stress coping styles respectively. Thus, the bimodality of growth/developmental trajectories and the contrasting stress coping styles suggests that that the data should be treated as categorical rather that continuous.
We do however see from the reviewers' concerns that the aim of the study needs to be clarified. To further clarify this, we have changed the last paragraph in the introduction to clarify that it is a question of bimodality due to the growth pattern based on the life history decision to reach smoltification.
3. Figure 3C Figure legends to clarify this. Our study provides a very detailed and consistent way of measuring the responses to a decline in oxygen, and we felt it was necessary to show the changes in oxygen (Fig. 3 a) and swimming depth (Fig. 3 b) from 80 minutes before to 80 minutes after the onset of hypoxia. These two figures do however not show the relationship between the saturation and the swimming depth, and the data point were thus presented in Fig. 3 C, because it is not the time per se in the system that trigger the behavioural changes, but a threshold oxygen saturation. The data points in Fig. 3 C are not fish individuals, but measurements of swimming depths (based on triggering of infrared sensors), and it is thus not possible to separate between the two groups. It is however a very good idea to develop such method in the future. The identification of the individual fish could first be done when they left hypoxia and the tag number could be recorded by the PitTag antenna.

Figure 4: Due to the distribution of fish weights, is there a size-dependency of plasma cortisol concentration at all?
Our response: The main difference in the plasma cortisol was between the two groups of UM and LM, and there was only a weak (R-square=0.03) relationship in the model. It is thus not a size-dependency in our data, but a group dependency. Figure 4: Can the authors discuss whether because the frequency of LM fish was lower (Fig.2B) whether that created a bias in selecting fish with certain personalities for the hypoxia avoidance experiment?

5.
Our response: All fish that were used in all tests were un-selected and due to this and a very low mortality since hatching we might expect the fish to represent a whole un-biased population. The hypoxia tests were done a time period before the stress tests in order to avoid effects of the tests, and the stress tests was done in un-selected groups independent of the result in the hypoxia test. This protocol was used to avoid biased results, but the downside of that is of course that the numbers of individual in the end result will differ. The chisquare test does not require equal group sizes, so a lower number of individuals in the LM group are not expected to induce a type I error.
6. The tentative hypothesis that the authors propose twice in the manuscript that "the occurrence of such contrasts in domesticated lines suggest the tentative hypothesis that successful expression of fast-proactive phenotypes depend on the presence of at least a small fraction of passive copers in the population." This does not seem to be a testable hypothesis. You're always going to have a normal distribution of fish weights and a range of behaviours. Can the authors comment/elaborate on this?
Our response: We understand the concern of the reviewer, and this statement was not meant as conclusions from our study, but rather as a hypothesis that might be tested in future studies. Our study demonstrates the occurrence of behavioural traits linked to distinct developmental/growth trajectories, but it is still an open question why such differences exist, and how domestication in aquaculture should take that into account. We have omitted the statement from the Conclusion and changed it in the Discussion to avoid misunderstandings.
7. With the LM being not smoltified and the UM being smoltified, that would indicate large differences in gill morphology that may influence their hypoxia tolerance. Can the authors address whether how that would impact the interpretation of these results?
Our response: That is a good question that unfortunately cannot be tested in our setup, but needs a completely different physiological approach. However, size differences could not explain the hypoxia response, but there might of course be other, not size dependent, physiological mechanisms explaining why the UM group have a lower threshold for hypoxia. In the gills, the smoltification process mostly affects the development of the chloride cells, and not the gill morphology. A study of Jenjan et al. 2013 (Respiratory function in common carp…, Animal Behaviour 85, 1245-1249) demonstrated a larger gill surface in proactive fish. Since the proactive fish were the first to leave the hypoxia, a different oxygen threshold cannot be explained as a larger gill surface. Such mechanisms have not been studied in salmon smolts, and we cannot speculate based upon our study.

Minor comments (* indicate my edits):
8. Add x-axis labelling into Fig.3A Our response: The x-axis in Fig. 3 a was omitted because it is the same as in 3 b, and it is stated in the legend that it is the same. Based on the comments we have included it again.
9. Genus and species need to be italicized throughout the manuscript 10. "N2" and "O2", 2 needs to be in subscript throughout the manuscript Our response: Both point 9 and 10 seems to be happen when the document is uploaded to RSOS, including some letters in the title. We will correct it again, and hopefully it will be correct in the next upload.

Figure captions: include sample sizes
Our response: We have included sample size in the text, e.g. in connection with comparisons and statistical tests, and generally think that a full list of sample sizes in the figure captions are making the legends less readable. To meet the reviewer's comment, we have however included the sample sizes when possible in the figure captions.
12. Figure 2A: since the x-axis is not continuous, these points shouldn't be connected with lines Our response: We do not disagree with the reviewer, and included the lines in order to make the figure easier to understand. We have omitted the lines and changed to a bar figure.
13. Figure 4B: Perhaps different color schemes could be used here to match what is used for LM and UM in Fig.4A, since these are all UM fish and no LM fish are present for this dataset Our response: It is right that Fig. 4 b is only UM fish (as stated in the figure legend), but the use of black and grey is consistent in both figures, and we have used black/white figures throughout the whole manuscript.
15. Page 1, line 45: "There was a higher frequency of …. In this fast-developing fraction of fish", higher compared to?
Our response: Corrected.
16. Page 1, line 56: "… showing consistency between context and over time", is this indicating that behavioural traits displayed by organisms are consistent in different context and over the course of an organisms life-time?
Our response: The question raised by the reviewer is interesting and relevant, but beyond the scope of this study, and in this part of the text we are using the normal definitions of this traits. However, the majority of studies regarding To what extent a behavioural trait is life-long will be a bit out of scope of this paper.