Intestinal Microeukaryotes in Fish: A Concise Review of an Underexplored Component of the Microbiota

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled "Intestinal Microeukaryotes in Fish: A Concise Review of an Underexplored Component of the Microbiota," provides a comprehensive synthesis of current knowledge on the diversity, ecological roles, and potential functions of microeukaryotes (fungi, protozoa, and other eukaryotes) in the gut microbiota of teleost fish. I compliment the authors on writing a good manuscript. I would support the publication of this manuscript after major revisions as mentioned below -

 

1. While the review describes diversity patterns (dominance of Ascomycota), a meta-analysis or systematic summary of prevalence across studies would strengthen its impact. For example, a table summarizing microeukaryote composition across fish species/habitats could be added.
2. The manuscript briefly mentions challenges (host DNA contamination, low abundance) but could expand on how methodological choices (18S rRNA vs. ITS sequencing, microscopy vs. molecular tools) influence observed diversity.
3. Streamlining some redundancy, such as, reiteration of Ascomycota dominance in multiple sections could improve conciseness.
4. How do microeukaryote-host relationships compare across vertebrates (fish vs. mammals)?
5. Are certain functions (e.g., immune modulation) conserved? A brief discussion would broaden relevance.
6. Some sentences are overly long (line no. 54-58). Break into shorter, clearer statements.
7. A diagram summarizing microeukaryote interactions including fungi-bacteria-protist networks and their hypothesized roles in fish health would visually reinforce key messages.
8. Expand on Applied Implications
9. Discuss how microeukaryotes could be harnessed in aquaculture as probiotics and bioindicators of stress/disease with specific examples.
10. Distinguish between "core" vs. "transient" microeukaryotes more explicitly.
11. Are any taxa consistently associated with fish guts across environments?
12. Ensure inclusion of 2023-2025 studies on fish microeukaryotes (if available) to reflect the latest research.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The review by Guirdao-Glores despite targeting in an under-studied issue, it remains far from being a concise, thorough, comprehensive and insightful review.

The statement in L. 13 is wrong, the papers on fish Bacteria vs. Archaea are several orders of magnitude more abundant. The authors seem to have put together some relevant papers instead of a targeted and organised literature review. For example, in the very much understudied issue of Archaea in fish, the paper [Kormas KA, Meziti A, Mente E, Frentzos A (2014) Dietary differences are reflected on the gut prokaryotic community structure of wild and commercially reared sea bream (Sparus aurata). MicrobiologyOpen 3:718-728] has investigated Archaea in the gilthead sea bream and it is not mentioned. In addition, there are several papers on fungi in fish that are not included.

There are no attempts on trying to categorise the synthesised data presented. For example, are there any patterns/relations any unicellular eukaryotes with gut morphology (see Egerton S, Culloty S, Whooley J, Stanton C, Ross RP (2018) The gut microbiota of marine fish. Frontiers in Microbiology 9,873).

The literature search criteria and strategy are not included ... so how does the reader can be rest assured that this review has extensively included all the available and relevant literature?

Perspectives are far too long and mostly not supported by references, thus, they remain rather mere opinions of the authors.

I believe that the authors are eligible in putting together a much better designed and solid review on this important issue on fish microbiology.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review synthesizes current knowledge on the diversity, ecology, and potential functions of intestinal microbiota of fish, focusing on fungi and protozoa. I think the review article here is well written. The study of gut microbiota has been a hot topic in recent years. Intestinal microbiota is involved in functions such as digestion and immunity.
Some suggestions or questions:
1. The article needs to introduce research techniques and methods related to gut microbiota.
2. How to distinguish whether microeukaryotes settle in the intestines of fish for a long time or are eaten as food in the intestines?
3. The habitat or living environment of fish will have a significant impact on the species, abundance, and diversity of gut microbiota. How to define the function of gut microbiota by comparing and excluding environmental influences?
4. The research outlook of this article mentions the application scenarios of fish gut microbiota. May I ask if there are any successful cases that illustrate the use of microeukaryotes in fish farming for disease resistance and immune regulation?

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

Research on fish gut microbiota has usually predominantly focused on prokaryotes (bacteria and archaea), while largely neglecting the study of eukaryotic microorganisms, such as fungi. The authors address this gap by providing a comprehensive review. They synthesize current knowledge on the diversity and functional roles of these understudied eukaryotic organisms within the fish intestinal microbiota. This review offers a crucial new perspective on fish gut microbiome research by highlighting the significance and potential contributions of the eukaryotic component. The work is assessed as novel, well-structured, well-argued, and nearly ready for publication.

One comment give to authors, that is please summarize protists in the fish intestinal microbiota into a table like in fungi. It could make the review to be more clear. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

Review comments
1. Overall evaluation
This article systematically reviews the diversity, ecological functions, and research challenges of fish gut microbiota (fungi, protozoa, and other eukaryotic groups), filling the current cognitive gap in eukaryotic components in fish microbiome research. The topic is novel, the structure is clear, and it has important reference value for the fields of aquaculture and microbial ecology. Suggest accepting publication after supplementing some content and correcting details.
2. Main advantages
Strong innovation in topic selection: Focusing on the microbiota in fish intestines that have been neglected for a long time (accounting for only 2-5% of the total microbial population), emphasizing their potential functions (such as immune regulation, digestive assistance, and microbial balance), with foresight.
Comprehensive content: covering multiple taxa such as fungi, protozoa, parasitic worms, etc., integrating taxonomic diversity, functional hypotheses, and technical challenges (such as limitations in detection methods).
Clear logic: unfold according to "Introduction → Diversity → Classification of Fungi/Protozoa/Other Eukaryotes → Outlook", with clear hierarchy.
Practical chart: Figure 1 visually displays the interaction network of gut microbiota, and Table 1 summarizes fungal functional cases to improve readability.
Application oriented: The conclusion emphasizes the potential value of microendoeukaryotes in aquaculture (such as probiotic development and health monitoring indicators), which is in line with practical needs.

3. Revision suggestions and issues
(1) Content depth and balance
The fungal part needs to strengthen its mechanism explanation: Pages 3-4 indicate that Ascomycota dominates in various fish species (87.5%), but the specific mechanism of its conservative function (such as whether it secretes specific metabolites?) is not elucidated. Suggest supplementing relevant hypotheses or comparing data across species.

(2) Methodological challenges need to be concretized
The second page points out that eukaryotic research is limited by low abundance, host DNA interference, and database scarcity, but no solutions have been proposed.

(3) Consistency between charts and data

(4) Expansion of Conclusion and Prospect

(5) Language and details, unified terminology, some references are outdated

4. Recommendation Decision
Accept after modification (Minor Revision). This article provides an important perspective for the study of fish microbiome. It is necessary to supplement the content and revise the details to address the above issues, especially to strengthen the discussion of mechanisms and technological prospects.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised paper, although not fully satisfactory inclusive, has been improved.