-->PONE-D-26-12952-->-->Morphology of the light-organ system and bioluminescent blinking in the ponyfish tribe Equulitini (Teleostei: Leiognathidae)-->-->PLOS One

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“The American Museum of Natural History and Regis University provided facilities and equipment in support of this work. This research was funded, in part, by a Regis URSC Grant to M. Ghedotti. D. Catania of CAS, P. Hastings and H.J. Walker of SIO, W. Ludt , J. Seigel & C. Thacker of of LACM, M. McGrouther & Y.-K. Tea of AMS, D. Nelson of UMMZ, L. Parenti of USNM, M. Sabaj Pérez of ANSP,  A. Simons & K. Ford of JFBM, and R. Thoni of AMNH loaned specimens in their care, supported specimen use, allowed histological sampling, and/or allowed CT scanning for this study. P. Fu and K. Hurdle, AMNH Microscopy and Imaging Facility, provided essential support for CT scanning.

We are grateful to S. Panha, Piyoros (Pomme) Tongkerd (Chulalongkorn University, Bangkok), and their students for facilitating our collections and studies in Thailand. We also thank Jirasin Limpichat (Mahidol University, Kanchanaburi), D. Boyd (LSU), and R. Thoni (AMNH) for assistance with packing, curating, and shipping specimens.”

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: N/A

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

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: Abstract

General comments:

- Lines 19 - 23: I recommend designating “communication”, namely intra- or/and inter-specific communication. In addition to these two categories. consider including the ecological role of illumination (of surroundings) as well.

- Lines 31 – 34: Please take caution when stating “novel mechanism for blinking”, since producing flashes via regulation of the chromatophore-filled tissues “shutters” was previously described for ponyfishes in, for example, Haneda (1949).

Reference mentioned: Haneda Y .1950. Luminous organs of fish which emit light indirectly. Pacific Science. 4:214–227.

Introduction

General comments: Please avoid the term “chromatiridophore” in this section and posteriorly in the manuscript, since it is not a recognized category of chromatophore cells. If the authors are reporting a new type of chromatophore, please include in the manuscript the supportive results, as electronic microscopy (cellular arrangement and ultrastructure), immunolabelling of melanin and guanine (pigment identification in histological structures), and Fourier-transformed infrared spectroscopy (FTIR) and/or x-ray diffraction (XRD) (chemical identification). In the case that the authors lack the access to biological samples, equipment or technical infrastructures to perform an accurate report of a new chromatophore type, please provide support information in the manuscript of the properties of the colored structures (e.g., morphological, cellular, behavior to focused light) and describe the structures by using terminology such as “chromatiridophore-like” or “potential chromate-iridophore”. Since these structures are most likely a combination of pigmentary and structural color cells (in this case, melanophores lying above iridophores), similar to lizards (Saenko et al., 2013) and other vertebrates (Shawkey and D’Alba 2017), I recommend using an adequate and accurate terminology such as “combination of iridophore and chromatophore”.

References mentioned:

Saenko S, Teyssier J, Marel D, Milinkovitch MC. 2013. Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern in Phelsuma lizards. BMC Biology. 11:105.

Shawkey MD, D’Alba L. 2017. Interactions between colour-producing mechanisms and their effects on the integumentary colour palette. Philosophical Transactions of the Royal Society B: Biological Sciences. 372:20160536.

- Lines 48 – 50: Same consideration as for Abstract lines 19 – 23.

- Lines 56 – 58: The bibliographic reference 13 details the reflective system of photophores in hatchetfishes. Please rephrase this sentence or replace this reference with another clearly supporting the information mentioned in this phrase.

Materials and Methods

- Lines 155 – 156: Consider mentioning the full designation for the abbreviation “diceCT”.

- Lines 159 – 168: To enhance the clarity for the readers, I suggest presenting in table format the information regarding the specimens used in this study.

- Lines 178 – 179: I recommend describing which type of illumination source (e.g., broad visible-spectra white lights such as tungsten, LED, halogen) was used to observe the reflection and coloration of the chromatophore-filled tissues.

Results

General comments: Consider more caution on the interpretation of morphological characteristics on the anatomical structures and their optical and light modulation properties without supportive data on these properties.

- Lines 224 – 227: Angle-dependent reflection is characteristic of iridophores, and such observation on the tissue suggests that the guanine iridophores have a degree of arrangement to reflect light from and to certain directions (see, for example, Denton and Land,1971; Land 1972). The suggestion that “ the guanine crystals determine the direction of light emission” would require the support of results, at least, regarding the iridophores angular arrangement in the reflective tissue, and angle between the light source and the observation/photograph. Please rephrase this sentence and take care of the interpretation for the observed reflective properties on the external tissues.

Mentioned references:

Denton EJ, Land MF. 1971. Mechanism of reflection in silvery layers of fish and cephalopods. Proceedings of the Royal Society B: Biological Sciences. 178:43-61.

Land MF. 1972. The physics and biology of animal reflectors. Progress of Biophysical and Molecular Biology. 24:75-106.

- Lines 245 – 247: I recommend adding morphological measurements for the length and thickness of the esophagus in the mentioned species.

- Lines 289 – 291: Please rephrase this sentence in a more cautious way when mentioning “through which light would be able to pass”, since this study does not contain or mentions any data on the light transmission properties (mainly spectra and intensity) of this tissue.

Discussion

General comments: Same considerations as for the Results section.

- Lines 379 – 391; 409 – 410; 417 – 422; 435 – 439: Please rephrase this sentence accordingly to the previously commented for lines 224 – 227 and 289 – 291 of Results section.

Conclusion

No comments on this section.

References

No comments on this section.

Figures and Tables

- Figures 1 and 2. Consider indicating the whole area of the LOS windows. These structures are difficult to distinguish in fixed and fresh specimens, so representing the anterior-most extension may not be clear to the readers.

- Figure 5. Please confirm all the abbreviations representing the morphological structures in the figure match the ones in the legend.

-Figure 7. The words in bold are difficult to distinguish for the reader. I suggest adjusting it by enhancing the lettering size or use normal text instead of bold.

Reviewer #2: Comments to authors

PONE-D-26-12952 “Morphology of the light-organ system and bioluminescent blinking in the ponyfish tribe Equulitini (teleostei: Leiognathidae). By Ghedotti et al. This manuscript explains the blinking mechanism of luminescence in ponyfish for the first time. The scientific importance is undoubted. However, for accepting this manuscript, I wish you would please give clear answers to the following questions.

Fig. 2B. The method of photographing this picture should be shown in the Materials and Methods section. Which light source did you use? At which angle is the light irradiated? The interpretation of this picture should also be discussed in the text. Is this specimen an ethanol-fixed specimen? I think ethanol-preserved specimens lose the transparency of the muscle.

Fig. 4. Why is ‘cm’ labeled as striated muscle? It might confuse the readers.

Fig. 5. Where is ‘bc’ located? Is the ‘screening tissue’ a part of the connective tissue?

Fig. 6. Which panels are for Photolaterlis stercorarius? It should be stated clearly in the legend.

It is stated that ‘fully obscure light release’ (Line 418), but in Fig. 5B, guanine particles on the screening tissue look very sparse. I am wondering if it can ‘fully’ obscure the light release?

The authors suggested the similarity between the shutter system of ponyfish and that of Anomalopidae. However, as shown in reference 56, the shutter mechanisms vary among the species of Anomalopidae. This means that the shutter system of Equulites shown in this study might not be able generalizable to all blinking ponyfish species.

The mechanism is complicated, and I recommend the authors include a conclusive schematic drawing to explain the mechanism for blinking (shutter movement).

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Reviewer #1: Yes: José Rui Lima PaitioJosé Rui Lima Paitio

Reviewer #2: Yes: Yuichi ObaYuichi Oba

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<p>Morphology of the light-organ system and bioluminescent blinking in the ponyfish tribe Equulitini (Teleostei: Leiognathidae)

PONE-D-26-12952R1

Dear Dr. Ghedotti,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Caio Santos Nogueira, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Authors,

I am pleased to inform you that both reviewers have evaluated the second version of the manuscript and recommended its acceptance.

I congratulate you on this achievement and thank you for choosing PLOS ONE for the publication of your article.

I wish you success with the publication of your work.

Reviewers' comments:

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-->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: All comments have been addressed

Reviewer #2: All comments have been addressed

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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

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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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.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

Reviewer #2: 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

Reviewer #2: Yes

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-->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 manuscript alterations met the comments to the version previously submitted.

I recommend the publication of the manuscript, and I would like to congratulate the authors for such a nicely done and interesting work, noteworthy for the research on bioluminescence in teleosts.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: Yes: Yuichi ObaYuichi Oba

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