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

Abstract

The intestinal microbiota of fish is predominantly composed of prokaryotic microorganisms, with research historically focused on bacteria. In contrast, the role of microeukaryotic organisms in the fish gut remains largely unexplored. This review synthesizes current knowledge on the diversity, ecology, and potential functions of intestinal microeukaryotes, particularly fungi and protozoans, in teleost fish. Fungi, especially Ascomycota and Basidiomycota phyla members, are consistently identified across species and may contribute to digestion, immune modulation, and microbial homeostasis. Protists, though often viewed as pathogens, also exhibit potential commensal or immunoregulatory roles, including the modulation of bacterial communities through grazing. Other eukaryotic taxa, including metazoan parasites, microalgae, and zooplankton, are commonly found as transient or diet-derived members of the gut ecosystem. While many of these organisms remain poorly characterized, emerging evidence suggests they may play essential roles in host physiology and microbial balance. The review highlights the need for improved detection methodologies, functional studies using gnotobiotic and in vitro models, and multi-kingdom approaches to uncover fish gut microeukaryotes’ ecological and biotechnological potential.

1. Introduction

The gut microbiota plays a pivotal role in the health and physiology of fish, engaging in complex interactions with the host that can be mutualistic, commensal, or parasitic. These interactions significantly influence key functions as detoxification, energy harvesting, barrier integrity, host development, immune modulation, production of antimicrobial compounds, and nutrient metabolism [1,2,3,4,5,6,7,8,9].

Although relatively resilient, the composition of the gut microbiota can be shaped by a variety of factors, including microorganism-host interactions, host genetics, environmental variations, and diet composition, which is considered the most influential. When detrimental changes occur in intestinal microbiota, hosts may become more susceptible to pathogen proliferation and lose key microbiota-mediated functions, especially under stressful conditions or immune suppression [10,11,12,13]. Because of its broad physiological relevance, gut microbiota is regarded as an auxiliary organ [14].

Most research on fish gut microbiota has focused on prokaryotic microorganisms, which comprise over 95% of the microbial biomass. The core bacterial phyla include Fusobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Cyanobacteria [15,16,17,18,19,20]. These prokaryotes contribute to host development, nutrient absorption, intestinal cell proliferation, disease prevention, and immune system function [21,22]. This core microbiota, or resident microbiota (autochthonous), is characterized by long-term colonization in the fish’s gut, forming a stable community specialized to grow and adhere to the gut mucus lining. It is based on shared microorganisms among comparable and consistently found species, regardless of environmental changes. Meanwhile, transient (allochthonous) microorganisms are short-term colonizers from food items and the surrounding water [23,24].

Archaea, another group of prokaryotes, represent a smaller fraction (1–5%) of the gut microbiota but fulfill specialized functions [25]. Methanogenic archaea are often found in the intestines of herbivorous fish or those consuming carbohydrate-rich diets. Though less abundant, they participate in fermentation and methane production, underscoring their role in digestive processes [26].

In contrast, microeukaryotic microorganisms comprise only 2% to 5% of the gut microbiota [27], and their role in fish remains largely underexplored [28,29]. Several challenges hinder their study, including low abundance relative to prokaryotes [30], their limited representation in genomic databases [28], and the transient nature of many microeukaryotes introduced through diet [31].

Microeukaryotic communities can be examined through sequencing and can be studied using metagenomics or amplicon sequencing. In the last case, sequencing the 18S rRNA and ITS (Internal Transcribed Spacer) gene marker is widely used for microeukaryotic profiling. The chosen gene marker will determine the community profile [32]. For instance, 18S rRNA is well suited for identifying higher-level taxonomic groups and assessing overall eukaryotic diversity [33]. On the other hand, ITS has a better probability of successful fungi identification, particularly the ITS1 and ITS2 regions, which are more variable and provide better resolution for distinguishing between closely related species and for studying intraspecific diversity. Additionally, microscopy provides more accurate quantitative results regarding abundance and biomass, while high-throughput sequencing provides a better estimate of the taxonomic richness of an ecosystem. The chosen biological marker and combining two methodological approaches yield more reliable and accurate results for eukaryotic diversity [34,35].

The presence of host DNA interferes with downstream analysis, and common molecular biomarkers often cross-react with host DNA, creating technical interferences that require refined detection strategies [36]. Among the strategies to overcome this challenge, it is proposed that during DNA extraction and chemical lysis, the detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) increase yield while maintaining the integrity of the DNA in polysaccharide-rich organisms, such as plants or fungi [37]. In addition, the application of host blocking primers in fish samples improved the detection of protozoans and detected only the eukaryotic species of interest by binding to the host DNA [38].

Although some intestinal microeukaryotes are linked to disease, others may serve as commensal or beneficial members of the gut ecosystem [39]. They are hypothesized to contribute to microbiota balance and may engage in symbiotic relationships with their fish host. Therefore, this review aims to synthesize current knowledge on the teleost intestinal microeukaryotes in fish, focusing on their diversity, ecological and physiological functions, and future research directions, to understand their role in fish health and microbial ecology. The applied research in this review was conducted employing the following keywords: “microeukaryotes”, “microeukaryome”, “eukaryotic microorganisms”, and “fish gut microbiota”.

2. Diversity of Microeukaryotes in the Intestinal Microbiota

Microeukaryotic microorganisms inhabiting the intestinal tracts of fish represent a wide array of life forms, including fungi (both yeasts and filamentous types), metazoan parasites (e.g., nematodes, cestodes, helminths), and protozoans [39] (Figure 1). Although they are typically less abundant and diverse than prokaryotes [40], recent advances in sequencing technologies have enabled better characterization of these complex microbial components.

In zebrafish, for example, the fungal phylum Ascomycota has been identified as dominant, accounting for approximately 87.5% of all fungal sequences, followed by Basidiomycota (6.8%), and a smaller proportion of Zygomycota and unclassified fungi (5.7%) [45]. Protozoans have been detected in marine fish; for instance, Blastocystis sp. was detected in herring, whiting, saithe, and mackerel [46]. Other eukaryotic taxa commonly found in the fish gut include crustaceans, mollusks, rotifers, copepods, and diatoms [47,48]. These findings collectively illustrate a surprisingly broad microeukaryotic diversity within the fish gut, many of which are structurally, behaviorally, and evolutionarily more complex than their prokaryote counterparts. Despite this diversity, defining a consistent profile for the fish intestinal microeukaryome remains challenging. The scarcity of reference sequences, variability in sampling and sequencing methods, and limited geographic and taxonomic coverage have prevented large-scale comparative analyses across fish species (Table 1).

A notable microeukaryotic feature is their genomic complexity. Fungal genomes, for example, range from 9 to nearly 180 megabases and encode between 10,000 and 25,000 genes, whereas bacterial genomes typically span less than 1 to 8 megabases and have 600 to ~6000 genes [42,49,50]. This greater complexity may confer broader functional capabilities; however, mutualistic or symbiotic roles in fish are still poorly defined compared to bacteria.

Emerging evidence suggests these microbes are involved in critical physiological processes, including beneficial activities. Their primary beneficial attribute, the probiotic function, has been associated with microeukaryotes from the digestive tract [39,51,52]. Some yeasts isolated from the gut of healthy salmonids, yellowtail, and croakers exhibit antagonistic activities against pathogens, promote intestinal maturation, and modulate antioxidant enzymes [53,54]. High intestinal microeukaryotic diversity has also been associated with reduced disease prevalence in healthy individuals [55,56], suggesting a potential protective role.

Recent studies suggest that certain ecological and immunological roles of gut microeukaryotes may be conserved across vertebrate lineages, including fish and mammals. For example, fungal genera such as Candida and Saccharomyces, commonly found in both mammalian and fish intestines, produce immunomodulatory compounds, including β-glucans and mannans, that can stimulate mucosal immunity and enhance barrier integrity [57,58]. Similarly, protozoans such as Blastocystis (once considered purely pathogenic) have been associated with increased bacterial diversity and reduced inflammation in both human and fish models, indicating a possible symbiotic or regulatory role [59,60]. Furthermore, protozoans have been shown to influence bacterial community structure through selective grazing in both mice and fish, suggesting a conserved top-down ecological impact [61]. Although functional studies in fish remain limited compared to those in mammals, these parallels suggest that microeukaryotes play evolutionarily conserved roles in host immune modulation, microbial homeostasis, and potentially even host metabolism. Integrating findings from both vertebrate groups could provide deeper insights into the ecological significance of microeukaryotes across animal microbiomes.

In summary, the intestinal microeukaryome in fish is taxonomically rich and functionally promising, though significantly underexplored. Future studies will be essential to determine whether observed taxa are transient dietary passengers or consistent, active members of the gut ecosystem, and what roles they may play in host physiology, health, and microbial balance. To distinguish whether microeukaryotes in the fish intestine are long-term residents or transients, researchers examine their location (mucosa-associated vs. gut contents), metabolic activity (via RNA-based sequencing), and persistence over time or diet changes. Microscopy and Fluorescence in situ hybridization (FISH) can reveal whether they adhere to or interact with the intestinal lining, indicating colonization. Experimental approaches, such as controlled feeding trials or gnotobiotic models, further help confirm whether specific taxa can survive and establish in the gut. Together, these methods differentiate active, resident microeukaryotes from transient ones simply passing through the digestive tract.

Table 1. Gut microeukaryote composition in fish species.

Fish Species	Microeukaryote Composition	Habitat and Feeding Habits	Ribosomal Gene Region	Reference
Zebrafish, Danio rerio	87.5% Ascomycota and 6.8% Basidiomycota	Model study fish; omnivorous freshwater species	ITS2	[45]
Amazonian catfish, Panaque nigrolineatus	40% Dothideomycetes and 36% Sordariomycetes	Freshwater, wood-eating fish	ITS1	[62]
Cobia Fish, Rachycentron canadum	88% Ascomycota and 11% Basidiomycota	Carnivorous marine benthopelagic fish, Tropical fish	ITS2	[63]
Grass carp, Ctenopharyngodon idella	68.2% Ascomycota, 13.4% Basidiomycota, 12.1% Mortierellomycota, and 0.5% Chytridiomycota	Aquatic macrophytes, algae, invertebrates, and vertebrates	ITS1 and ITS2	[64]
Black carp, Mylopharyngodon piceus	68.6% Ascomycota, 12.3% Basidiomycota, 9.4% Mortierellomycota, and 1.07% Chytridiomycota	Mollusk-eating cyprinid fish native to eastern Asia	ITS1 and ITS2	[64]
Bighead carp, Aristichthys nobilis	39.3% Ascomycota, 42% Rozellomycota, and 1.4% Basidiomycota	Filter-feeding freshwater fish from Hongchaojiang Reservoir in Guangxi, China	ITS	[65]
Nile tilapia, Oreochromis niluticus	48.9% Ascomycota, 2.4% Basidiomycota, and 0.6% Rozellomycota 90–98% Opisthokonta, 0.3–7.5% Bacillariophyta, and > 0.5% Archaeplastida	Wild tilapia from Lake Nasser, Egypt Feeding on plankton, some aquatic macrophytes, fish larvae, and decaying organic tissue	ITS 18S region V9	[65]
Mullet, Mugil cephalus	Gut content mainly by Rotifera and Copepoda, followed by Bacillariophyceae and Chlorophyceae.	Inhabit coastal temperate and tropical waters	18S

Table 1. Gut microeukaryote composition in fish species.

Fish Species	Microeukaryote Composition	Habitat and Feeding Habits	Ribosomal Gene Region	Reference
Zebrafish, Danio rerio	87.5% Ascomycota and 6.8% Basidiomycota	Model study fish; omnivorous freshwater species	ITS2	[45]
Amazonian catfish, Panaque nigrolineatus	40% Dothideomycetes and 36% Sordariomycetes	Freshwater, wood-eating fish	ITS1	[62]
Cobia Fish, Rachycentron canadum	88% Ascomycota and 11% Basidiomycota	Carnivorous marine benthopelagic fish, Tropical fish	ITS2	[63]
Grass carp, Ctenopharyngodon idella	68.2% Ascomycota, 13.4% Basidiomycota, 12.1% Mortierellomycota, and 0.5% Chytridiomycota	Aquatic macrophytes, algae, invertebrates, and vertebrates	ITS1 and ITS2	[64]
Black carp, Mylopharyngodon piceus	68.6% Ascomycota, 12.3% Basidiomycota, 9.4% Mortierellomycota, and 1.07% Chytridiomycota	Mollusk-eating cyprinid fish native to eastern Asia	ITS1 and ITS2	[64]
Bighead carp, Aristichthys nobilis	39.3% Ascomycota, 42% Rozellomycota, and 1.4% Basidiomycota	Filter-feeding freshwater fish from Hongchaojiang Reservoir in Guangxi, China	ITS	[65]
Nile tilapia, Oreochromis niluticus	48.9% Ascomycota, 2.4% Basidiomycota, and 0.6% Rozellomycota 90–98% Opisthokonta, 0.3–7.5% Bacillariophyta, and > 0.5% Archaeplastida	Wild tilapia from Lake Nasser, Egypt Feeding on plankton, some aquatic macrophytes, fish larvae, and decaying organic tissue	ITS 18S region V9	[65]
Mullet, Mugil cephalus	Gut content mainly by Rotifera and Copepoda, followed by Bacillariophyceae and Chlorophyceae.	Inhabit coastal temperate and tropical waters	18S

3. Fungi in the Intestinal Microbiota of Fish

Fungi are key ecological players across ecosystems, functioning as mutualists, decomposers, and pathogens. They represent one of the most diverse biological kingdoms [66,67]. Within the gut ecosystem, fungi are increasingly recognized as integral microbiota members, contributing to host health through various physiological and immunological mechanisms [68,69]. Although fungal cells constitute a much smaller fraction of the intestinal microbiota than bacteria, they are significantly larger and possess greater genomic complexity [30,32,70].

In fish, intestinal fungi form structured communities interacting with bacteria and other microorganisms through synergistic, antagonistic, or symbiotic relationships (Table 2). These interactions are inferred to help preserve microbiota stability, regulate the mucosal barrier function, and support immune system development [58].

Among fungal phyla, Ascomycota is consistently reported as the dominant group in teleost intestines. For instance, studies on Barramundi (L. calcarifer), Golden Permit (T. blochii), Royal Pleco (Panaque nigrolineatus), the silver pompano (Trachinotus blochii), and Mangrove Red Snapper (Lutjanus argentimaculatus) have reported twelve genera from the Ascomycota phylum (Aspergillus, Penicillium, Talaromyces, Aureobasidium, Cladosporium, Fusarium, Clonostachys, Myrothecium, Parengyodontium, Trichoderma, Hypocrea, Microsphaeropsis) [62]. Three Basidiomycota genera (Schizophyllum, Rigidoporus, and Cutaneotrichosporon) were also identified [71]. In zebrafish, more than 15 fungal classes were detected across Ascomycota, Basidiomycota, and Zygomycota phyla, with Ascomycota comprising 87.5% of the identified sequences [45]. Similar patterns were observed in cobia fish (Rachycentron canadum), where the fungal microbiota consisted mainly of Ascomycota (~88%) and Basidiomycota (~11%), with Ascobulus identified as a novel core genus in fish [63].

Table 2. Fungal activity and function in fish intestinal microbiota.

Fungal Species	Function/Activity	Reference
Debaryomyces hansenii	Gut maturation, increasing amylase secretion, and probiotic potential	[45,72,73]
Candida spp.	Associated with lipid metabolism and fermentation activities	[74]
Aureobasidium pullulans	Active against pathogenic yeast strains	[71,73]
Schizophyllum commune	Isolated from coral reef fish intestines; antimicrobial activity	[71]
Pichia kudriavzevii	Extracellular enzyme-producing (amylase, protease, lipase, cellulase, xylanase, and phytase)	[74]
Yarrowia lipolytica	Directing anti-Vibrio action and modulating the host’s innate immune system	[75]

Table 2. Fungal activity and function in fish intestinal microbiota.

Fungal Species	Function/Activity	Reference
Debaryomyces hansenii	Gut maturation, increasing amylase secretion, and probiotic potential	[45,72,73]
Candida spp.	Associated with lipid metabolism and fermentation activities	[74]
Aureobasidium pullulans	Active against pathogenic yeast strains	[71,73]
Schizophyllum commune	Isolated from coral reef fish intestines; antimicrobial activity	[71]
Pichia kudriavzevii	Extracellular enzyme-producing (amylase, protease, lipase, cellulase, xylanase, and phytase)	[74]
Yarrowia lipolytica	Directing anti-Vibrio action and modulating the host’s innate immune system	[75]

These taxonomic similarities across species suggest the existence of a conserved fungal core microbiota in fish. When the fungal diversity of three coral reef fish (Lates calcarifer, Trachinotus blochii, and Lutjanus argentimaculatus) was compared, similar taxonomic profiles among the three fish species were observed [71]. Further, species from the Ascomycota phylum, Debaryomyces hansenii and Rhodotorula mucilaginosa, are consistently found in salmonids regardless of the rearing condition. Moreover, Sordariomycetes and Tremellomycetes are fungal taxa found in wild and laboratory-reared zebrafish guts [45,76]. In addition, the anatomical location within the gut seems to influence fungal distribution. For example, yeast richness is generally higher in the foregut and midgut than in the hindgut, possibly reflecting the distinct physiological roles of these regions [71].

While the specific functions of many fungal taxa remain uncharacterized, some genera show promising contributions. For instance, Rhodotorula (Basidiomycota) and Debaryomyces (Ascomycota), frequently found in freshwater and marine fish, dominate the rainbow trout gut [77]. These yeasts produce immunostimulatory compounds such as β-glucans, nucleic acids, and mannan oligosaccharides that enhance host immunity and may protect against pathogens [63,78,79].

The enhancement of metabolic capacity is another contribution of fungi. In carnivores such as the red cusk-eel (Genypterus chilensis) and the Chilean hake (Seriolella violacea), fungal isolates have shown enzymatic activities including proteases, lipases, glycosidases, and phosphatases. These isolates included Rhodotorula mucilaginosa, Candida palmioleophila, Candida pseudorugosa, Cystobasidium slooffiae, and a member of the genus Yamadazyma, and their enzymatic profiles are aligned with dietary components such as phospholipids, algae, hydrolyzed marine proteins, and unsaturated fatty acids, suggesting that fungi contribute to digestive efficiency and metabolic capacity [80,81,82]. On the other hand, in the black carp and herbivorous carp, Ascomycota and Basidiomycota were the dominant phyla in the fish gut. Both phyla contain members capable of decomposing complex organic matter [64], providing additional metabolic capabilities to the fish.

Species from the Ascomycota phylum, including Candida, Debaryomyces, and Saccharomyces, are commonly found in freshwater and marine fish and are implicated in carbohydrate fermentation and potential probiotic activities [6,83]. Ascomycota includes species that produce digestive enzymes, which promote optimal growth and development by maximizing nutrient utilization and significantly influencing overall health and metabolism [74,78,84]. The conservative functions of the Ascomycota phylum within the gut in fish could be a result of its stability in the gut despite environmental factors [85]. Also, this group has the capability to modify the composition of lectin in the mucinous content of goblet cells, which plays an important role in gut health and condition [86]. Additionally, they can produce extracellular phytases, which are involved in the degradation of phytate [45]. Finally, it is reported that they are capable of alkaloid biosynthesis. The properties of these organic molecules include antimicrobial and antioxidant activity [87].

Similarly, the Basidiomycota phylum has genera (Cryptococcus and Rhodotorula) also implicated in gut health through their metabolic byproducts and immunological interactions [88]. Although less prevalent, Chytridiomycota contribute to the breakdown of complex polymers, particularly in fish that consume algae or detritus, while Mortierellomycota may influence lipid metabolism and immune responses [89], although further research is needed to fully understand their functions. These fungal communities work synergistically with bacterial populations to enhance digestive efficiency, bolster pathogen resistance, and maintain gut homeostasis.

Hypothesized functions, based on studies in mammals, further suggest that gut fungi may influence bile acid metabolism, barrier integrity, and even neural signaling via the gut–brain axis. For instance, Candida and Saccharomyces can transform bile acids, potentially impacting lipid metabolism and microbial dynamics [90]. Fungal components like mannan and chitin may also interact with host epithelial and immune cells to strengthen mucosal defenses [57], mechanisms critical in fish, particularly under environmental stress or pathogen exposure. Furthermore, fungal dysbiosis has been linked to behavioral and cognitive alterations in rodent models, opening questions about whether similar mechanisms might operate in fish [91].

Although functional evidence in fish is still emerging, these findings collectively support the idea that fungi are not passive gut inhabitants but may actively contribute to host physiology and gut homeostasis. Since most available research focuses on the influence of the fish environment and its modification of the gut microbiota, continued investigation, including gnotobiotic and multi-omics approaches, is essential to elucidate their ecological and metabolic roles. Notably, the use of gnotobiotic models allows the exclusion of environmental factors and their influence on the structure and interactions of fungi, protists, and prokaryotes.

Furthermore, the recurrence of specific fungal taxa such as Debaryomyces and Rhodotorula across diverse fish species suggests a possible conservation of functional traits like immune modulation and digestive enzyme production. Although interspecific variability exists, the consistent detection of these taxa across freshwater and marine environments implies that certain physiological roles, including the stimulation of host immunity and maintenance of gut homeostasis, may be conserved among teleosts.

4. Protists in the Fish Intestinal Microbiota

Protists are a diverse group of unicellular microeukaryotes that inhabit moist and aquatic environments, where they play a crucial role in ecological and trophic networks [92,93]. This group includes both autotrophic microorganisms (e.g., microalgae) [94] and heterotrophic protozoans, which can be classified by their locomotion strategies, such as flagella, cilia, and pseudopodia. Furthermore, among protozoans are the apicomplexans, which are exclusively parasitic and characterized by an apical complex (organelles underlying the oral structure) [93,95]. Additionally, protozoans exhibit a variety of reproductive methods, including the alternation between life stages (trophozoites and cysts), asexual reproduction through binary fission, and sexual reproduction; some protozoans can reproduce sexually and asexually [93]. This complex reproductive strategy allows some ciliate protozoans to modify their reproduction and feeding behavior based on the bacterial community available in the environment, thereby altering the media parameters [96].

Protozoans, in particular, are often overlooked in gut microbiota studies due to their relatively low abundance compared to bacteria. However, they are increasingly recognized as functionally relevant symbionts because of their interactions with bacterial communities [59]. Although many protozoans in fish have been traditionally viewed as pathogens, they have gained positive recognition; Blastocystis sp., a controversial protozoan in mammals, has been proposed as a marker of gut health [97], raising questions about similar roles in fish. Still, there is a lack of information about how protozoans integrate into an ecosystem primarily enriched with commensal bacteria [61].

Microscopy and molecular studies have revealed a variety of protozoan taxa inhabiting the fish gut. Notable examples include Ichthyophthirius multifiliis, Trichodina spp., Hexamita spp., and Spironucleus spp., found across various freshwater and marine species [98,99]. While some of these protozoans are known pathogens, others thrive in a commensal or opportunistic relationship with the host. Their presence suggests potential roles in shaping the gut ecology by directly interacting with host tissues or feeding on bacterial populations, thus shaping microbiota composition and dynamics. For instance, protists can consume up to 1000 bacterial cells per hour, positioning them as top-down regulators of microbial populations in the gut [93]. This grazing behavior, in turn, affects both bacterial abundance and community structure. Conversely, the stability and presence of protozoans are also shaped by bacterial diversity and nutrient availability, highlighting a reciprocal relationship between these microbial groups [61,100].

In Nile tilapia (Oreochromis niloticus) from Lake Nasser, Egypt, gut microbiota analysis revealed that more than 90% of 18S rRNA sequences belonged to the flagellate group Opisthokonta, with diatoms (Bacillariophyta) representing less than 7.5% [47]. In another case, the ciliate Balantidium polyvacuolum was shown to increase bacterial diversity and promote the abundance of phyla such as Fusobacteria and Chloroflexi, associated with immune function, in Xenocyprinae fishes. Interestingly, infected fish were reported to be healthier than their uninfected counterparts [60]. Similar findings were observed with Balantidium ctenopharyngodoni in grass carp, where its presence correlated with increased levels of Clostridium and higher creatine concentration in the hindgut, indicative of enhanced physiological performance [101]. In other vertebrate models, protozoans can trigger mucosal immune responses, such as interleukin-18 release via inflammasome activation, which promotes protective Th1 and Th17 immune responses [102,103].

Nevertheless, many protozoans in fish are still primarily associated with disease [104]. Their presence is often accompanied by increased bacterial diversity and signs of inflammation or tissue disruption, as seen in other host systems [105,106]. Additionally, protozoans may act as vectors for bacterial pathogens [107] or exacerbate dysbiosis by modifying bacterial diversity in the gut microbiota [100]. For example, the ciliate Nyctotherus sp. was reported to reduce Proteobacteria abundance in Mesonauta festivus (Amazonian cichlid), a pattern associated with dysbiosis [108].

Notably, many protozoans in the fish gut may reflect dietary intake rather than established symbiotic relationships. In many cases, they are transient members that do not colonize the gut but instead pass through with ingested material.

Overall, intestinal protists in fish are multifaceted. While some act as pathogens, others may provide immunological benefits, regulate bacterial populations, or enhance gut health (

Table 3. Protists’ activity and function in fish intestinal microbiota.

Protist Species	Function/Activity	Reference
Balantidium polyvacuolum, an obligate ciliate of Xenocyprinae fishes	The presence of B. polyvacuolum increases the diversity of the fish gut microbiota, notably increasing the relative abundance of Fusobacteria and Chloroflex. Increase of total short-chain fatty acids, acetic acid, isovaleric acid, propionic acid, isobutyric and butanoic acid.	[60]
Balantidium ctenopharyngodoni, an obligate intestinal ciliate	Associated with carbohydrate metabolism (hydrolysis of starch and glycogen), related to herbivores diet fish.	[109]
Nyctotherus sp., a ciliate endosymbiont in fish	Decrease the relative abundance of Proteobacteria and promote a high abundance of the methanogenic archaea Halobacterota.	[108]
Pseudocapillaria tomentosa, a helminth of zebrafish	Disruption of the gut microbiome composition. No conclusive explanation for the interaction between the gut microbiome and P. tormentosa.	[110]
Ichthyophthirius multifiliis, a ciliate ectoparasite of grass carp	A decrease of Actinobacteria and an increase of Proteobacteria, which is related to the promotion of opportunistic pathogens like Aeromonas. Functional prediction exhibits a reduction of the branched-chain amino acid ABC transporter, permease component.	[111]

). The dynamic nature of their interactions with the host and bacterial communities underscores the need for further investigation. Deciphering the ecological and functional significance of these protists will be crucial for a comprehensive understanding of gut microbiota in fish.

5. Other Microeukaryotes in the Fish Intestinal Microbiota

Beyond fungi and protozoans, various microeukaryotic organisms have been detected in the gastrointestinal tracts of fish. These include parasitic and free-living members of phyla such as Nematoda, Streptophyta, Platyhelminthes, and Acanthocephala, as well as taxa from mollusks, crustaceans, and zooplanktonic organisms. For instance, in the flag cichlid (Mesonauta festivus), 18S rRNA sequencing revealed a high prevalence of metazoan microeukaryotes in the gut, including Nyctotherus sp., Chromadorea (nematodes), Cestoda, Trematoda, and Neoechinorhynchida (acanthocephalans) [108]. These organisms are often linked to parasitism and are particularly common in detritivorous or omnivorous tropical fish, where trophic networks increase the likelihood of exposure to a wide range of environmental microeukaryotes.

While generally considered pathogenic, some helminths have demonstrated immunomodulatory effects in other hosts. In humans, for example, helminth infections in patients with early-stage multiple sclerosis have been associated with a halt in disease progression and the emergence of myelin-recognizing regulatory T cells (Treg) in the peripheral blood [112]. These findings raise interesting possibilities about the complex and potentially beneficial immunological roles of intestinal helminths in other vertebrates, including fish.

Eukaryotes such as crustaceans, mollusks, and fish larvae have also been found within the fish gut microbiota, often reflecting dietary intake rather than resident microbiota. In Oreochromis niloticus (Nile tilapia), members of the Ostracoda class (small crustaceans) were among the most abundant gut microeukaryotes detected [47]. Similarly, fish larvae such as Gobiocypris rarus and zooplankton like Acanthocyclops vernalis have been recovered from the guts of silver carp, grass carp, bighead carp, and blunt snout bream larvae [113]. In these cases, the shared presence of dietary microeukaryotes across multiple species likely reflects overlapping food sources rather than stable colonization. As such, these organisms are better described as transient microbiota, introduced with the diet and unlikely to contribute significantly to gut homeostasis or host physiology. A key challenge is distinguishing between core microeukaryotic residents and transient taxa derived from diet or environment. While genera like Candida and Debaryomyces appear across diverse fish species and may represent core components, others, such as diatoms or copepods, are likely dietary artifacts. Longitudinal studies and gnotobiotic models are needed to clarify colonization dynamics.

While nematodes, flatworms, and other helminths are often associated with pathogenesis, their broader ecological roles in the gut remain poorly characterized. Some, like Capillaria acanthopagri, have been identified as parasites of Acanthopagrus schlegelii (Blackhead seabream), and cestodes like Khawia sinensis are known to infect carp (Cyprinus carpio) [114,115]. Although these organisms may influence nutrient availability, tissue damage, or immune activation, they generally do not exhibit the mutualistic or commensal properties observed in fungal and protozoan communities. Instead, their presence is often linked to parasitism, resource competition, or diet-derived transience, with unclear or limited contributions to gut microbiota functionality.

In conclusion, while these other microeukaryotic taxa reveal the ecological complexity of the gut environment, their significance appears less critical than that of fungi and protozoans. Nonetheless, they may serve as indicators of environmental exposure, dietary habits, or parasitic burden and merit further study in the context of fish health and aquaculture.

6. Perspectives

Based on the previous research findings, we established the following perspectives. Despite recent advances in fish microbiome research, the microeukaryotic component of the intestinal microbiota remains significantly understudied compared to its prokaryotic counterpart. Understanding the diversity, ecology, and function of intestinal microeukaryotes is a promising research frontier with the potential to uncover new mechanisms of host-microbe interactions, immune modulation, and metabolic integration.

Environmental factors influencing microeukaryotic microbiota require thorough investigation. Factors such as diet, antibiotic use, and fish farming practices may differentially affect the composition and function of gut microeukaryotes, but the mechanisms behind this are not yet understood. Comparative studies across species and populations are essential to determine how these microorganisms vary and which environmental drivers shape these differences. The interactions between microeukaryotes and bacteria play a critical role in gut microbiota. Understanding how these interactions influence gut health and disease could provide novel insights into microbiota dynamics. In this regard, evaluating the in vitro relationship between gut prokaryotic and eukaryotic microbes is crucial for comprehending their ecological interactions and collective impact on host health. These microbes influence each other through competition, predation, and metabolite exchange, shaping community composition and function. In vitro models offer controlled settings to study these dynamics, helping to uncover mechanisms that remain obscured in complex in vivo systems. Such research is especially valuable for developing microbiota-based interventions in aquaculture and other applied settings. Additionally, investigating the evolutionary relationships between these taxonomic groups, their hosts, and their adaptation mechanisms within the gut environment is essential for a comprehensive understanding of microbiota function.

On the other hand, using gnotobiotic models to study the relationship between gut prokaryotic and eukaryotic microbes is essential for uncovering their functional roles in a controlled host context. This approach allows for precise manipulation of microbial communities, providing conditions to isolate and assess specific interkingdom interactions and their effects on host physiology. By eliminating the complexity of natural microbiota, gnotobiotic systems can offer deeper insight into how prokaryotic and eukaryotic members influence immune responses, nutrient absorption, and microbial balance.

Identifying and characterizing gut microeukaryotes presents a significant challenge in research on eukaryotic microbiota due to their greater complexity than that of prokaryotic microbiota. Therefore, developing advanced methodologies and technologies for accurate identification, quantification, and functional analysis is essential. Integrative approaches that combine multi-omics technologies with advanced imaging and in vivo experimentation will be essential for unveiling the ecological roles and metabolic contributions of microeukaryotes within the gut ecosystem. Moreover, interdisciplinary collaborations among parasitologists, microbiologists, immunologists, and aquaculture researchers are needed to bridge existing knowledge gaps and accelerate discovery in this underexplored area. As aquaculture expands, understanding the functional relevance of microeukaryotic communities in fish may contribute to sustainable farming practices. Microeukaryotes could indicate gut health, environmental stress, or even components of next-generation probiotics tailored for aquaculture species. These findings carry practical implications for aquaculture. Understanding the functional roles of gut microeukaryotes opens avenues for their biotechnological application, such as the development of tailored microbial consortia to enhance disease resistance, stress tolerance, and feed efficiency. For instance, probiotic yeasts like Debaryomyces have demonstrated benefits in larval fish by stimulating digestive enzyme activity and promoting immune readiness [45,54]. Additionally, microeukaryotic diversity may serve as an indicator of gut resilience under environmental or dietary challenges, providing a diagnostic tool for health monitoring in aquaculture systems. Therefore, future research must focus on taxonomic identification, functional relevance, and application, positioning microeukaryotes as central players in host–microbiota interactions within aquatic environments.

Hypothetically, intestinal microeukaryotes could be developed as functional components in aquaculture biotechnology. Several studies have demonstrated the probiotic potential of gut-derived yeasts, such as Debaryomyces hansenii and Yarrowia lipolytica, in improving fish immunity and growth. Likewise, protozoans that influence bacterial community composition through grazing or competitive exclusion could, in theory, be used to stabilize gut microbiota under stressful farming conditions. In addition, shifts in the abundance or diversity of specific microeukaryotic taxa might one day serve as early-warning bioindicators of intestinal dysbiosis, disease onset, or environmental stressors. These potential applications, while still speculative, highlight the untapped value of microeukaryotes in promoting sustainable aquaculture and warrant targeted investigation using controlled in vivo and in vitro models. Additionally, they may also serve as bioindicators of gut health or environmental stress, especially in intensive aquaculture systems. Thus, integrating microeukaryotic biomarkers into routine aquaculture diagnostics and exploring targeted microbial supplementation could enhance sustainability and disease prevention strategies.

7. Conclusions

Studying intestinal eukaryotic microorganisms in fish is crucial for understanding their roles in host health and well-being. These microorganisms, including fungi, protists, and other microeukaryotes, play essential roles in nutrient digestion, immune system modulation, and interactions with various gut microbiota components. Fungi significantly contribute to nutrient decomposition and maintain intestinal health while facilitating the production of immunostimulant compounds and digestive processes through specific enzymes. Meanwhile, although often regarded as parasites, protists can also fulfill commensal and mutualistic roles. The diversity and complexity of these microorganisms highlight their importance in the intestinal ecology of fish. Understanding their functions and interactions can enhance aquaculture practices and promote sustainability in the sector. Future research should focus on exploring this diversity and the impacts of these microorganisms on fish health, aiming to optimize aquaculture practices and ensure the health and well-being of fish populations.