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PONE-D-22-23795

Species independence of eye lens dimensions in teleost and elasmobranchs

PLOS ONE

Dear Dr. Leifsdóttir,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Athanassios C. Tsikliras

Academic Editor

PLOS ONE

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Specifically, the study objectives should be clearly defined in Introduction along with a clarification of the hypotheses that were examined, statistical analyses are required in the results to support the findings of the paper (also to be described in materials and methods), some parts of the discussion should be re-written and references updated and the conclusions should be formulated to comply with the findings of the study. 

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: ### Introduction

The authors start with a summary of latest interest regarding vertebrate eye lenses, after which they outline the lens development and an overall structure of a lens. They expand that topic describing three, significant to their research, regions of a fish eye lens: outer cortex, hard core, and embryonic region. This section could use a clarification. On the first pass I misunderstood that the lens capsule was one of the three regions. I suggest either 1) enumerating numerically or textually the regions as they are listed in text (as in this very line) or 2) listing all three regions before discussing them. The authors describe an issue with inconsistency regarding naming in the literature, defining clearly what terms will they use for each region of interest (Hardened lens and central hardened lens, HL and CHL respectively).

The next section the authors start strong claiming HL plays central role in maintaining focused vision. I cannot agree with that statement. HL’s role is as important as the rest of the lens in forming image. In the study the authors refer to (Schartau et al. 2009), the changes in optical properties happen pass the cortex-HL horizon at 70% radius (R) demonstrating that not only the HL plays an important role. The authors pose a question of what the function of such a well-defined cortex-HL horizon is. They suggest that ubiquity of horizon’s location points to optical properties and put in the opposition the transparency caused by the loss of intracellular structures. I am not sure why those two were selected as opposing hypotheses since the intracellular loss happens way pass the 70% R all the way to 92% R. This makes the work’s motivation somewhat weak. Despite that, the authors close the section with clearly defined study objectives: testing for relative location of cortex-HL horizon in different species.

### Materials and methods

The authors provide a detailed description of samples collection: time, location, conditions, number of individuals, and their species. They describe the methodology regarding euthanasia, extraction of lens from a fish eye, extraction of HL from a lens, and measurements of fishes, lenses, and HL. Materials and methods section is solid and detailed. However, “error in identifying the horizon (…) was virtually impossible” has no place in scientific publication. Errors can be highly unlikely but never impossible, especially when the used method involves squashing a gelatinous sphere with fingers. This was particularly surprising to read because the reminder of this sentence describes the best possible approach to the problem: measuring lenses from both eyes and comparing them. Which is exactly what the authors did.

### Results

Firstly, the authors report that lens diameter (LD) and HL diameter (HD) are linearly correlated with total length (TL) of the fish, but the relation is different for different species. The relation between LD and HD is also linear. There is no significant difference between individual species, only when two groups (teleost vs elasmobranchs) were compared. This is likely caused by thorny skate which stands out from the rest. The authors establish combined for all species HD:LD ratio to be 69%. They also provide a breakdown of ratios for both groups (68% and 70%). The ratio is also invariant to TL. The authors back all their findings with statistical analysis. At the end the authors include data from five lenses of species not involved in this study. They find no correlation between DH and fork length, but they point out small sample size and sample size range. This part brings nothing to the study and makes me wonder why mentioning this at all.

### Discussion

The authors discuss that the link between LD and HL agrees with other studies and that HL increases its size to keep the pace with the fish size. The ratio of HD:LD is 69% which also agrees with reports on other species. The authors argue that whereas the relation between TL and LD is different for different species, consistent HD:LD ratio suggest HL increasing size is linked to maintenance of optical quality during the lens growth. Further the authors explain that refractive index gradient (decreasing from the center to the surface) present in most vertebrate eyes increases the optical power and reduces spherical aberration. The refractive index gradient is especially important to spherical lenses (such as in fishes) due to surrounding materials of similar refractive index (water, cornea, intraocular vitreous humor).

Unfortunately, in the next part the authors bring up outdated views regarding the refractive index profile in a fish eye lens. They refer to conclusions drawn by Fernald and Wright (1983) that the HL has a uniform refractive index, that optical properties are maintained during the growth by maintaining the cortex-HL horizon, and that central fibers are compressed due to high refractive index. Continuous gradient of refractive index has been demonstrated in Kröger 2013. A study that involved Fernald himself (Kröger et al. 2001, DOI: 10.1016/s0042-6989(00)00283-2) showed that optical properties are maintained during the growth by maintaining the gradient profile normalized to radius. The compression of fibers has also been disproved relatively recently (Kozłowski and Kröger 2019, DOI: 10.1016/j.visres.2019.06.008). The authors comment on Fernald and Wright’s conclusions by mentioning early studies that disagreed with Fernald and Wright but, as publications I brought up, demonstrate it is far from a debate. The section is closed with a conclusion that HD:LD ratio similar between species is important for maintenance of optical quality.

The authors continue with a summary of lens development leading to the fact that fiber cells lose their organelles and nuclei. They also mention that cells lose the water which is not the case. The authors seem surprised that organelle free zone (OFZ) is reported up to 92% R whereas the HL is only up to 70% R. Presence of water is exactly the reasons. It would be impossible to rub off the gelatinous outer cortex if loss of organelles would go with a loss of water. The authors also believe that 70% R reported by Schartau et al. 2009 is a threshold of optical plasticity. In Schartau’s work one can observe small differences at regions as close as 20% R (Day vs Night). If those differences are too small to be convincing, Jönsson et al. 2014 (DOI: 10.1007/s00359-014-0941-z) showed changes in lenes of Atlantic cod along almost the entire radius below the 70% R. Naturally, based on those wrong assumptions the authors build an incorrect conclusion that optical plasticity is better reflected by the HL rather than by the OFZ, where studies shows neither seem to really affect it.

The discussion is closed with a possible application of back calculations for estimates of fish sizes based on the size of isolated central core.

### Final remarks

I know my review will not make the authors’ day particularly good, but I believe that the experiment was done and reported well, and the findings can contribute to our collective knowledge on lenses. The science is good but it is not a publication material in its current form. The study needs a better motivation and conclusions drawn with better understanding of relation between optics, refractive index, and proteins.

When looking at single species, fixed cortex-HL horizon means similar density of proteins, which in turn means refractive index. The horizon must be maintained in that position throughout the growth because the entire refractive index profile must remain the same. It is very interesting that one specific refractive index (thus protein concentration, thus hardness) always ends up in the same relative position for different species, suggesting that they must have a very similar refractive index profile.

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Reviewer #1: Yes: Tomasz M. Kozłowski

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PONE-D-22-23795R1Species independence of eye lens dimensions in teleost and elasmobranchsPLOS ONE

Dear Dr. Leifsdóttir,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the three minor points raised during the review process.

Please submit your revised manuscript by May 11 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Athanassios C. Tsikliras

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors addressed all the concerns from the original review. I am returning with one minor remark, few mistakes, and a comment. I will discuss them as they appear in text:

Line 79: "...a refractive index profile (...) which are common" - I think it should be "is"

Lines 167-175: The way it is formulated suggests a direct contradiction. Both Kröger and Wang talk about optical properties with lens growth - constant vs changing, but both mean the "lens growth" in a different way. Wang looked at the full growth from larva to adult, whereas Kröger looked at individuals form sexual maturity up to 3 years. Wang's results for similar range show that the changes are very small (117dpf - 880dpf). I leave the clarification at the authors' discretion.

Lines 172-173: "...continuous and declining refractive gradient from the surface towards a plateau near the center of the lens" - Refractive index is the highest in the center, so it should be "increasing" instead of "declining". Alternatively: "...declining refractive gradient from a plateau near the center of the lens towards the surface"

Line 206: "likely in exchange with water" - This part can be removed. The mechanism we suspected was transport of proteins which would dissolve in water rather than exchange it.

Line 177: "both reported a uniform refractive index at the center of the fish eye lens" - I have been thinking about this for a very long time. I disagree with this statement but I have trouble accusing the authors of being incorrect. To me Wang's plateau is not a region of constant refractive index, but rather a region in which the change is very small. However, what does it mean "constant"? What differentiate "constant" from "plateau" by my definition, would be a difference in refractive index several places after comma. Others may look at this with different precision or threshold. As such it is more of an opinion rather than a fact, making it an academic discussion which is outside the scope of a review. Nevertheless I am bringing authors' attention to this detail.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Tomasz M Kozłowski

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Species independence of eye lens dimensions in teleost and elasmobranchs

PONE-D-22-23795R2

Dear Dr. Leifsdóttir,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Athanassios C. Tsikliras

Academic Editor

PLOS ONE

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