-->PONE-D-25-51753-->-->Pathological, morphological, and molecular analysis of didymozoid trematodes infections in the nasal cavity of orange-spotted grouper (Epinephelus coioides) caught from Arabian Gulf waters-->-->PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions-->

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: Nasal cavity didymozoid trematodes were found exclusively in Epinephelus coioides from the Arabian Gulf, with integrated morphological and molecular analyses confirming their identity as Didymodiclinus and highlighting high host and site specificity. Nasal cavity didymozoid trematodes were found exclusively in Epinephelus coioides from the Arabian Gulf, with integrated morphological and molecular analyses confirming their identity as Didymodiclinus and highlighting high host and site specificity.

Here are comments to the authors

1. Line 128 and 133: Why were the didymozoid cysts fixed in 5% neutral buffered formol saline instead of 10% neutral buffered formalin, as used for the infected tissues?

2. Line165: Please specify the expected and/or obtained PCR product size.

3. Line 196-197: Since no external signs of inflammation or tissue damage were observed around the cyst attachment sites, does the relationship between the didymozoid trematodes and the orange-spotted groupers represent a parasitic, mutualistic, or commensal association?

4. Line 2: Since no external signs of inflammation or tissue damage were observed, the authors may need to reconsider the use of the terms “pathological” and “infection” in the title.

5. Is there any data available on the physiological parameters (weights and lengths) of orange-spotted groupers with and without didymozoid cysts? Such information could help determine whether the presence of didymozoid cysts affects the growth and health of orange-spotted groupers.

6. Line 58-66: Please provide more information about the family Didymozoidae and didymozoid trematodes, including aspects such as their pathogenesis, impact on the host, and associated mortality in host.

Reviewer #2: In general, this is a well-organized and very interesting manuscript. The authors have analyzed three categories of parasitology, i.e. pathological, morphological and molecular level investigations, for Didymozoidae in orange-spotted grouper hosts, and generated a comprehensive report. However, there are few questions need to be answered. If the authors can adequately answer these questions, I will recommend accept the manuscript for publication. In general, this is a well-organized and very interesting manuscript. The authors have analyzed three categories of parasitology, i.e. pathological, morphological and molecular level investigations, for Didymozoidae in orange-spotted grouper hosts, and generated a comprehensive report. However, there are few questions need to be answered. If the authors can adequately answer these questions, I will recommend accept the manuscript for publication.

1. Regarding the histopathological analyses (lines 133-137), when you classified tissues as infected, did you examine the entire nasal cavity, or just specific portions of the nasal chamber? How did you determine which infected tissues for sampling, particularly in terms of their size and shape? What was your protocol for processing these tissue samples for the subsequent histological examinations?

2. For the molecular identification of the parasites, I could not find any information about your biological and technical replicates. To obtain a statistically valid picture of genetic variation, you need to balance breadth (sampling different hosts from different locations) with depth (analyzing multiple parasites from each host). So, how many individual worms did you collect from each host? And, how many hosts in total were sampled for rDNA sequencing? Did you pool samples from multiple worms together, or was each worm sequenced individually? Also, how many sequencing replicates did you perform for each sample to ensure reproducibility?

3. The phylogenetic analysis relies exclusively on the 28S rDNA D1 - D3 regions. However, using solely this molecular marker may be problematic:

1. Many didymozoids have undergone recent divergence as they adapted to specific microhabitats within particular fish hosts, which result in cryptic species complexes (rDNA sequences suggest organisms are the same, while their biological characteristics indicate they are different).

2. If these parasites evolved rapidly, it creates rapid radiation. In such cases, the branches representing different species in a 28S rDNA phylogenetic tree become extremely short, making it difficult to resolve relationships between closely related species.

3. Some didymozoids possess unusually long expansion segments in their 28S rDNA, (especially D2 and D3). In such lineages, these expansion segments can evolve at very slow substitution rates, resulting in highly conserved LSU sequences across multiple species. This conservation means different species may share identical or nearly identical D1 - D3 sequences, limiting resolution to the genus level rather than enabling species level discrimination. Moreover, when LSU sequences are extremely conserved, D1 - D3 sequencing lacks sufficient variation to resolve fine scale evolutionary divergences, such as population level genetic structure.

Given these limitations, what is your rationale for concluding that 28S D1 - D3 rDNA sequencing alone provides sufficient resolution to construct a comprehensive phylogenetic framework for Didymozoidae? Will you consider a multi marker approach, such as ITS2 (rDNA spacer, which help resolving species/sibling species relationships) and COI (mtDNA, which help detecting shallow divergences and identify cryptic populations), to create a concatenated phylogenetic tree would provide a more complete picture of Didymozoidae evolutionary history?

These comments are also included in my attached reviewer report.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: reviewer report.docx

Morphological, molecular, and pathological characterization of didymozoid trematode infection in the nasal cavity of orange-spotted grouper (Epinephelus coioides) from Arabian Gulf waters

PONE-D-25-51753R1

Dear Dr. Attia,

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,

Tzong-Yueh Chen, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

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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: Yes

Reviewer #2: Yes

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-->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.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: 1. All comments have been addressed.

2. Line 160: The description "fixed in . Tissue blocks" should be deleted.

Reviewer #2: Since the authors has adequately answered my questions and provided reasonable rationalizations; and it is a first in class study in certain field, I will recommend accept the manuscript for publication.

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Reviewer #1: No

Reviewer #2: No

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