-->PONE-D-26-23707-->-->Peripheral anatomy of the dolphin ear and associated nervous structures: insights from macroscopic dissection, DICE-µCT, histology, and confocal microscopy-->-->PLOS One-->-->

Dear Dr. De Vreese,

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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ACADEMIC EDITOR:  Dear Dr. De Vreese,

I am writing to inform you that your manuscript titled Peripheral Anatomy of the Dolphin Ear and Associated Nervous Structures: Insights from Macroscopic Dissection, DICE-µCT, Histology, and Confocal Microscopy was reviewed and that both reviewers suggested minor revisions. The reviewers found it particularly difficult to interpret the figures, figure letterings and caption structures. Fundamentally, I share their opinion. Please, provide a revision of the manuscript by taking care of the reviewers’ suggestions and requests before resubmitting it.

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Michelangelo Bisconti, PhD

Academic Editor

PLOS One

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“Part of this study was funded by the Italian Cassa di Risparmio del Veneto, while another as part of the Project PCI2022-135022-2 financed by MICIU/AEI /10.13039/501100011033 and by the European Union NextGenerationEU/PRTR, supported by JPI Oceans.”

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“Part of this study was funded by the Italian Fondazione Cassa di Risparmio del Veneto e Rovigo, while another as part of the Project PCI2022-135022-2 financed by MICIU/AEI /10.13039/501100011033 and by the European Union NextGenerationEU/PRTR, supported by JPI Oceans. Gmar is funded by a Catalan Government Grups de Recerca Consolidats grant (ref. 2021 SGR 01195). The authors would like to acknowledge the Italian Zooprofilactic Institutes of Venezia, Lazio e Toscana, Lìguria, and Sardinia for collaboration in the sampling network, and Mariano Domingo (Department of Health and Anatomy, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra). Also thanks to Prof. dr. Hansjörg Schröder of the Department II of Anatomy (Neuroanatomy), University of Cologne (50924 Cologne, Germany) for allowing SDV to perform part of the macroscopic studies on site.”

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: I found the paper to be very interesting and a much needed description of the outer ear of odontocetes, which is an anatomical region that has received little attention. I particularly appreciate the discussion of the potential function of the outer ear region, which often is considered functionless.

Overall, the paper is well written and the conclusions are well supported by the observations presented. The figures are good, although I did find some of them a little difficult to interpret. In fact, the vast majority of my comments deal with the figures and associated captions themselves. I have made several comments and suggestions below, both general and specific, all of which I think will strengthen the paper.

A general comment about the figures is that a lot of the anatomy is difficult to interpret. Figures 4 & 5 were probably the most helpful, but I think that an illustrated or schematic figure that represents the 3-dimensional shapes would be very helpful. I also found the numbers in the figure to be a bit distracting and it was a little cumbersome to flick back and forth between the figure and the caption. One option would be to replace them with abbreviations, but that might be a matter of taste. I found the numbering to be especially distracting when they aren’t all listed numerically at the end of the captions, such as in Figure 3 in which some of the structures are numbered in the text of the caption. I would recommend listing all of the numbers at the end of the caption even if they are listed earlier in the caption text.

I also think that more specific citations to the figures would help the reader to follow the anatomical descriptions, especially in the ‘Mandibular fat bodies’ section beginning around page 11. When possible, please cite the figures more frequently.

Page 3, second sentence under ‘Materials and Methods’ - I think that “spotted dolphin foetus" should be plural here. (E.g., “two bottlenose dolphin…and Pantropical spotted dolphin foetuses.”)

Further in the same paragraph, “details” in “details can be found” should be lowercase.

First sentence of third paragraph under ‘Material and Methods’, “from the out ear canal” should be “from the outer ear canal”.

Page 5, first paragraph under ‘Region of the ear canal and middle ear’ - the anatomy described here is difficult to interpret even using the figures. This place in particular would benefit from a schematic or illustrated figure to orient the reader and accentuate the anatomy.

Page 6, Figure 1 caption - In case I missed it, IMFB and EMFB have not been defined by this point in the manuscript. These should be defined (i.e., spelled out) earlier in the manuscript.

Page 6, Figure 2 caption - Define/ spell out “TP-complex”. I am guessing this is the tympanoperiotic complex.

Figure 3 - It could be on account of the resolution for the review, but the yellow asterisk was difficult to see. Perhaps outline the shape in black to make it stand out more?

Page 7, paragraph after Fig. 3 caption - The auriculotemporal branch if V3 is mentioned in the second sentence of the paragraph, but it doesn’t appear to be labeled in Figure 3 unless I am missing it. Maybe move the citation for figure 4 up to this part of the paragraph?

Figure 4 - The numbers in this figure appear to be off. For example, #9 is clearly pointing to the skull and not a nerve as identified in the caption. Also, #15 looks like the tympanic bulla rather than the external ear canal, which is either unlabeled, or perhaps #14? Lastly, I don’t see #16 in the figure, which the caption says should be the tympanic bone.

Figure 5 - Add number citations behind anatomical structures in the text of the caption to follow the style of other figure captions. Regarding the maxillary vein, the muscles are colored red also which makes the maxillary vein difficult to distinguish. Is #15 actually pointing to the tympanic bone? How is this distinguishable from the IMFB vascular plexus (#14)?

Page 9, last sentence of 2nd paragraph after Fig. 5 caption - “…not visibly connect to the main plexus…” should be “…not visibly connected to the main plexus…”

Figure 6 - There don’t appear to be numbered structures in the image although they are referenced in the figure caption. Also, anti-DAP2 is stained blue, but there is a lot of blue in the image. Is it all of the blue? I have to admit that I am not very well versed in confocal microscopy or staining techniques involved, and so I had difficulty following much of this.

Figure 7 - Following my comment above, I was confused by what I was looking at in this figure and so further explanation of what is being presented would be helpful. What are the colors representing? Do each of the panels represent the same corpuscle just at different focal depths? The caption states that there are “five consecutive images”, although I see a total of six (A, B, C, D, E, & F). It appears that we are to compare panels A, C, and E. If so, is there a reason that they aren’t adjacent to one another? This may relate to my ignorance of confocal microscopy techniques, but I was generally confused by this figure.

Figure 8 - This is a cross section through the external ear canal, correct? What is the orientation (anterior, dorsal, etc.)? The red circles around the nervous structures are difficult to see (red on pink) and the individual ID numbers are illegible.

Figure 9 - This is an interesting image! Which direction is external?

Page 13, under ‘1. Accessory sinus system and vascular plexuses’ - are the trabeculated air spaces observable in any figures? If so, please include a figure citation. Also, the “1” in the section heading does not follow the style of other section headings.

Figure 11 - Would it be possible to label some of the “large nerve fibers in the fibrovenous wall?”

Page 15, first paragraph under ‘Discussion’ - It should be noted that the minke whale is a mysticete and that the anatomy observed in that species might be convergent with what is observed in odontocetes.

Reviewer #2: This is an interesting and useful paper with excellent figures supporting a detailed reconstruction of the peripheral auditory region of toothed whales. It corroborates prior research and adds meaningfully to our understanding of the region. I only have a few minor comments to improve clarity, particularly regarding the figures. Please see the attached annotated PDFs for specific notes.

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Reviewer #1: Yes: Eric G. Ekdale

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-26-23707_RR.pdf

Attachment

Submitted filename: PONE-D-26-23707_reviewer-Fig_captions.pdf

Peripheral anatomy of the dolphin ear and associated nervous structures: insights from macroscopic dissection, DICE-µCT, histology, and confocal microscopy

PONE-D-26-23707R1

Dear Dr. De Vreese,

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,

Michelangelo Bisconti, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Dr De Vreese,

I carefully read the revised version of your manuscript and the new illustrations together with the responses you and your co-authors provided to reviewers' questions and comments. I found that your revision is adequate and that the manuscript is now improved by addressing all the reviewers' questions. I therefore recommend that this manuscript can be accepted for publication in Plos ONE.

Congratulations and best regards,

Michelangelo Bisconti

Reviewers' comments: