Symbionts of Ciliates and Ciliates as Symbionts

Abstract

Endosymbiotic relationships between ciliates and others are critical for their ecological roles, physiological adaptations, and evolutionary implications. These can be obligate and facultative. Symbionts often provide essential nutrients, contribute to the ciliate’s metabolism, aid in digestion, and offer protection against predators or environmental stressors. In turn, ciliates provide a protected environment and resources for their symbionts, facilitating their survival and proliferation. Ultrastructural and full-cycle rRNA approaches are utilized to identify these endosymbionts. Fluorescence in situ hybridization using “species- and group-specific probes” which are complementary to the genetic material (DNA or RNA) of a particular species or group of interest represent convenient tools for their detection directly in the environment. A systematic survey of these endosymbionts has been conducted using both traditional and metagenomic approaches. Ciliophora and other protists have a wide range of prokaryotic symbionts, which may contain potentially pathogenic bacteria. Ciliates can establish symbiotic relationships with a variety of hosts also, ranging from protists to metazoans. Understanding ciliate symbiosis can provide useful insights into the complex relationships that drive microbial communities and ecosystems in general.

Introduction

Ciliates are a diverse group of single-celled organisms belonging to the phylum Ciliophora. They are known for their unique characteristics, such as the presence of hair-like structures called cilia, which they use for locomotion and feeding. While ciliates are typically free-living, they can also form symbiotic associations with other organisms, acting as hosts or serving as symbionts themselves for mutual benefit. Ciliates act as hosts for other protists, viruses, algae, and bacteria [1–3]. Symbiosis is the foundation of many biological systems, demonstrating the interconnection and interdependence of life forms. It can be mutualistic (benefiting both species), parasitic (one benefits at the expense of the other), or commensalistic (one benefits while the other remains unaffected). Most symbioses investigated till date are mutual. The formation of an endosymbiotic relationship appears to be governed by the endosymbiont’s biochemical versatility complementing the host’s limited metabolic capabilities [4]. Endosymbiotic partnerships with bacteria and a variety of symbiotic alliances are preferred by ecological and trophic preferences. In these associations, ciliates can provide services such as nutrient recycling, carbon transfer, or protection against pathogens. Some ciliates engage in mutualistic relationships with photosynthetic algae, forming endosymbiotic partnerships that enable them to acquire energy through photosynthesis. One of the key drivers behind evolution is endosymbiosis as host organisms acquire new features and roles through the absorption of once-independent organisms. This process has been critical in shaping the diversity and complexity of life on our planet. The interaction between ciliates and their symbionts stands as a fascinating and diverse field of study.

More and more species are being described, and there are already over 40 free-living Euplotes species [5, 6]. Several of these endosymbionts were identified and described in the “pre-molecular era” and their functions are still unknown. It is beneficial to locate them once more, examine them using molecular techniques, and look at their interactions with the host cell. According to this viewpoint, new technologies can offer crucial information to elucidate various facets of the subject, such as next-generation sequencing and the utilization of genomics data. The sole exceptions are Caedibacter [7] (five species), Pseudocaedibacter [8] (three species), Lyticum [7] (two species), and Holospora (Hafkine, 1890; [9], and a solitary Euplotes endosymbiont that was later redescribed as Polynucleobacter necessarius [10]. Fewer ciliate bacterial symbionts have available molecular phylogenetic affiliations [11–14]. Metopus palaeformis along with its rod-shaped endosymbiont [15] was the first molecularly investigated symbiosis [16].

Symbiotic systems between ciliates/animals are found across the Animalia kingdom, including chimpanzees buffaloes, cattle, dromedary camels, cuttlefish, great apes, elephants, humans, fishes, frogs, horses, polyps of hydras, mollusks, insects, mammals, nematodes, polychaetes, nemerteans, oligochaetes, sea urchins and many more [17].

This review attempts to dig into the enthralling world of ciliate symbiosis, investigating the vast array of symbionts that seek refuge within ciliate hosts as well as the unique adaptations these ciliates exhibit when functioning as symbiotic partners. We strive to explore the mechanisms underlying these relationships, from molecular complexities to ecological ramifications, through an examination of many cases from varied habitats and locations. This study aims to add to our greater understanding of symbiosis within the microcosm by synthesizing current research and putting light on new advances, providing insights into the intricate fabric of life's interdependencies at the molecular level. Some of the primary objectives of studying symbionts of ciliates and ciliates as symbionts include understanding their ecological significance, evolutionary dynamics and applications in various fields, including biotechnology, environmental science, and medicine. For instance, understanding symbiont-ciliate interactions might provide insights into natural mechanisms for disease control or offer potential avenues for biotechnological innovations.

Bacterial Symbionts

In 1890, Waldemar Mordechai Wolff A. Hafkine (1860–1930) reported three intranuclear bacteria in Paramecium caudatum. Holospora obtusa infects the ciliate's macronucleus, Holospora elegans and Holospora undulata infects the micronucleus [18]. These bacteria form symbiotic connections with their host ciliates and can impact many cellular processes and functions. Most of the studied symbionts belong to Alphaproteobacteria [19–24].The non-motile Alphaproteobacteria species holospora and holospora-like bacteria (HLB), exhibit a distinctive life cycle with reproductive and infectious forms, localize and establish themselves primarily in the nuclear apparatus of the host ciliate, Oligohymenophorea, Armophorea, Heterotrichea, Phyllopharyngea, and Prostomatea, modify the host's nuclear material, undergo replication, leading to an increase in their population and producing new infectious particles [22, 25].The majority of Holospora genus members were found only in Paramecium. To date, at least 13 symbionts associated with HLB have been identified in Paramecium species [21, 25, 26], though only a subset of them have been characterized at the molecular level [22, 27, 28]. HLBs from P. putrinum [29], P. jenningsi [30], P. chlorelligerum [31], and P. multimicronucleatum [32] have recently been described. None have been found in other Paramecium species (such as P. duboscqui, P. woodruffi, P. polycaryum). Ca. Trichorickettsia mobilis, a strange flagellated endosymbiont, has been discovered in the macronucleus of the ciliate Paramecium multimicronucleatum. These Rickettsiaceae family members are ausative agents of serious human diseases, such as Rocky Mountains spotted fever and epidemic typhus [33].

To the best of our knowledge, Rickettsiales are all obligate intracellular bacteria. With such discrepancies in the evolutionary histories of symbionts and hosts, the discovery of so many Rickettsiales species in phylogenetically distant eukaryotes like ciliates clearly supports many occurrences of horizontal transmission. Furthermore, the habitats of ciliates, as well as some sponges and cnidarians hosting bacteria from the "Midichloria clade," show that Rickettsiales bacteria, potentially dangerous ones, are more reliant on water as a vector. The possibility that some of these Rickettsiales representatives can transmit horizontally.

Oligohymenophorea and Spirotrichea (especially Euplotes) are two most promising subgroups for studying prokaryotic symbioses in Ciliophora. Polynucleobacter necessarius, a betaproteobacterium that forms an obligatory mutualistic interaction with the host, is the most well-known symbiont of Euplotes [10, 34, 35]. The initiation of the obligate symbiosis between some phylogenetically related species of Euplotes and Polynucleobacter necessaries or Polynucleobacter spp. (Burkholderiales, Betaproteobacteria), initially known as omicron or omicron-like particles, was a significant development in the evolutionary history of the genus [10]. Bacteria from at least three classes of the phylum Proteobacteria (Alpha-[11, 13, 19, 36, 37], Beta-[14, 35], and Gammaproteobacteria [12, 19, 38], as well as the phyla Firmicutes [39] and Verrucomicrobia [40], have been characterized using the modern molecular approach. Bacteria-ciliate symbiosis appears to have evolved on multiple occasions.

However, endosymbiotic relationships between prokaryotes and Euplotes are not limited to only Polynucleobacter. A subset of populations relies on bacteria from the genera Devosia [41] and 'Candidatus Protistobacter' [35, 42]. In addition to these essential symbionts, many Euplotes harbor 'accessory' bacteria that are unlikely to be essential to the host's survival because they are not found in all strains of the host species, mostly co-occur with known essential symbionts, and typically belong to intracellular parasite groups [19, 30, 37, 43, 44]. The unusual ability of "Candidatus Midichloria mitochondrii" to infect mitochondria [43, 45–47], its sex-based prevalence in ticks, its predilection for ovarian tissues, and its vertical transmission distinguish it [11, 46]. More research is required to fully comprehend the mechanisms underlying these characteristics, as well as the bacterium's potential impact on the biology and health of its metazoan hosts, particularly ticks. Endosymbiotic bacteria can be transferred from one Euplotes species to another using microinjection [48, 49].

Metopus palaeformis, Plagiopyla frontata and Metopus contortus contain fluorescing bacteria, which produce methane and help ciliates to survive in anaerobic conditions [50]. Cyclidiumporcatum, Methanoplanus endosymbiosus from the Methanomicrobiales and M. formicicum in the Methanobacteriales are a few examples of this category [51]. In Daytricba rzlminantizlm, the hydrogenosomes oxidize pyruvate to produce ATP, H₂, CO₂, and acetate [52]. The presence of ferredoxin oxidoreductase and hydrogenase in the symbiotic connection of endosymbiotic methanogens and anaerobic ciliates promotes the H₂-evolving fermentation process. This mechanism is essential for the survival of the symbiosis since it allows for the production and utilisation of hydrogen gas, which eventually leads to the generation of energy via methanogenesis [53, 54].

A non-sulfur purple bacteria capable of anoxygenic photosynthesis is found in Strombidium purpureum, and Codonella sp. is the only marine ciliate known to harbor a cyanobacterium [55]. Candidatus Thiobios zoothamnicoli in Zoothamnium niveumisis also an example of sulfur-oxidizing bacterium [56].

Although symbioses between eukaryotes and Bacteria are ubiquitous and extensively established, anaerobic ciliates are the only eukaryotes known to contain endosymbiotic Archaea.

Genomes of Bacterial Symbionts

Second and third-generation sequencing techniques can be used to isolate and study the genomes of ciliate symbionts. To date, roughly 20 complete genome sequences of bacterial endosymbionts have been sequenced and annotated (Table 1). Although the majority of endosymbionts were Proteobacteria, notably Alphaproteobacteria and Gammaproteobacteria, two methanogenic archaeal endosymbiont genomes have been published [57]. The genome size of ciliate symbionts range from 158 and 163 kbp of "Ca. Organicella extenuata", an endosymbiont of Euplotes sp. and the E. vanleeuwenhoeki’s endosymbiont, "Ca. Pinguicoccus supinus" respectively [5], to 3.31 and 5.02 Mbp of Kentrophoros symbionts [58]. The entire genome assembly of "Ca. Gromoviella agglomerans" (589.967 bp) is the smallest documented genome in the order Holosporales and demonstrates substantially decreased metabolism in terms of biochemical pathways as well as energy production and conversion [59]. Certain obligate intracellular symbionts go through genomic reduction, which can include substantial gene loss, pseudogenization, a high mutation rate, and a low GC content [57, 60, 61]. The severely shortened genome of "Ca. Azoamicus ciliaticola," an unnamed plagiopylid, appears to have conserved host-beneficial features as well as anaerobic respiration energy. Indeed, this symbiont is an obligate endosymbiont with cellular functions that are strikingly similar to those of mitochondria, despite the fact that it did not descend from the mitochondrial line of descent [62]. P. necessaries, on the other hand, has a genome (1.56 Mbp long) that is nearly the same size as its free-living cousins [63]. Intracellular bacteria use strategies such as secretion systems and effectors such as Type IV and Type VI secretion systems [64], proteins with repeat motifs such as ankyrin repeat motifs [60], and ADP/ATP translocase to interact with, invade, and exploit their host cell [27, 61, 65, 66]. This is demonstrated by the genomes of two anaerobic ciliate methanogenic endosymbionts, Nyctotherus ovalis and Metopus contortus. Their genomes are in an early stage of adaptation for endosymbiosis, as evidenced by the significant number of genes undergoing pseudogenization [57].

Table 1.

List of bacterial symbionts

Symbiont	Host	References
Holospora curviuscula	Paramecium bursaria	[61]
Holospora obtusa	Paramecium caudatum	[27]
Holospora elegans	Paramecium caudatum	[27]
Holospora undulata	Paramecium caudatum	[27]
Caedimonas varicaedens	Paramecium biaurelia	[67]
Candidatus Fokinia solitaria	Paramecium sp.	[60]
Candidatus Deianiraea vastatrix	Paramecium primaurelia	[64]
Candidatus Sarmatiella mevalonica	Paramecium tredecaurelia	[65]
Candidatus Gromoviella agglomerans	Paramecium polycaryum	[65]
Candidatus Trichorickettsia mobilis	Paramecium multimicronucleatum	[33]
Francisella adeliensis	Euplotes petzi	[66]
Candidatus Azoamicus ciliaticola	Undescribed Plagiopylea	[62]
Candidatus Kentron clade	Kentrophoros sp.	[58]
Caedibacter taeniospiralis	Paramecium tetraurelia	[68]
Candidatus Thiodictyon intracellulare	Pseudoblepharisma tenue	[69]
Unnamed, strain TC1	Trimyema compressum	[39]
Polynucleobacter spp. including Polynucleobacter necessarius	Euplotes spp.	[30]
Candidatus Organicella extenuata	Euplotes sp.	[70]
Candidatus Pinguicoccus supinus	Euplotes vanleeuwenhoeki	[5]
Candidatus Protistobacter	Euplotes sp.	[42, 49]
Candidatus Thiobios zoothamnicoli	Zoothamnium niveumis	[56]
Methanocorpusculum sp.	Metopus contortus	[57]
Methanogenic archaea	Trimyema sp.	[71]
Methanobacterium formicicum	Trimyema sp.	[72]
Methanogenic archaea	Cyclidium porcatum	[15]
Caedibacter sp.	Paramecium sp.	[7]
Pseudocaedibacter sp.	Paramecium sp.	[8]
Lyticum sp.	Paramecium sp.	[7]
Metopus palaeformis	Paramecium sp.	[15]
Rickettsiales	Paramecium sp.	[73]
Firmicutes	Euplotes sp.	[39]
Verrucomicrobia	Euplotes sp.	[40]
Proteobacteria	Euplotes sp.	[19]
Devosia	Euplotes sp.	[41]
Cyanobacterium	Codonella sp.	[55]
Non-sulfur purple bacterium	Strombidium purpureum	[55]
Chemoautotrophic bacteria	Zoothamnium niveum	[74]

Algal Symbionts

A phagotrophic protist may consume an alga as a prey, which could lead to the onset of endosymbiosis, which is known as mixotrophy. The temporary symbiont subsequently develops into a persistent symbiont when the host’s and the symbiont's cell cycles are synchronized. Next, there is a lateral gene transfer from the symbiont’s nucleus to the host’s nucleus, which results in less autonomy for the symbiont and more control by the host. The symbiont's organelles are then reduced one by one, but a relict symbiont nucleus is preserved as nucleomorph for a particular time period until full host control is established (cryptophytes, chlorarachniophytes). These protists, which typically contain hundreds of algae within a host cell, are known as ‘multi-algae retaining protists' (MARP). The degree of integration of symbionts in MARP symbioses, as represented by their or their progeny' retention time, is little understood [75]. Chlorella-like green algae or Chlorella are found as autotrophic eukaryotic symbionts in two Paramecia species, P. Chlorelligerum and P. bursaria.

Green algal endosymbionts help the ciliate to withstand the anoxic and nutrient-rich environment. The ciliates lose their endosymbionts and change to a heterotrophic lifestyle when grown outside of the traps in an oxygen-rich environment. This demonstrated unequivocally that the green algal endosymbiont Micractinium in Tetrahymena serves a unique purpose of giving oxygen to its hosts [76]. Prokaryotic endosymbionts incorporate various new biochemical properties into protist hosts, such as nitrogen fixation and recycling, photosynthesis, and methanogenesis [4]. Endosymbiotic algae have attracted particular attention in phycology, zoology, microbiology, and virology; a majority of them are members of the Chlorella clade of the Trebouxiophyceae group [77]. The various ciliate groups differ in their capacity to acquire and maintain symbionts.

Ciliates may feast on diatoms, although there are also some cases of diatom-ciliate symbiotic partnerships. The diatoms Chaetoceros dadayi and C. Tetrastichon linked to the lorica of species of the tintinnid ciliate genus Eutintinnus, as well as the consortium of Chaetoceros coarctatus and the peritrich ciliate Vorticella oceanica, are the most common consortia. The consortia of the peritrich ciliate Pseudovorticella coscinodisci connected to several big pelagic diatoms and the consortium of the diatom Fragilariopsis doliolus and many tintinnid species are less common instances [78].

Only one freshwater ciliate species has the photoautotrophic phytoflagellate Chlamydomonas sp. as an endosymbiont, while others have kleptochloroplasts [55]. However, whether the inner compartments are generated by endosymbiotic algae or chloroplasts is unknown. There are few reports on the presence of symbiotic chloroplasts in Prorodon, Strombidium, and Mesodinium rubrum [1]. Laboea strobila and Strombidium often contain endosymbiotic algae as reported for the Baltic Sea [79]. Maristentor dinoferus contains 500–800 symbiotic algal cells, all of which are phylogenetically connected to the dinoflagellate Symbiodinium [80]. M. dinoferus symbionts produce mycosporine-like amino acids and protect the host against UV irradiation [81].

Genomes of Algal Symbionts

Algal symbionts are algae that have a symbiotic connection with other creatures such as corals, fungus, or ciliates (Table 2). Algal symbiont genomes can be extremely varied, even within the same group or species based on the species participating in the symbiosis. The Symbiodiniaceae family, formerly known as Zooxanthellae, is a well-known group of algae symbionts. These dinoflagellates are frequently found symbiotically. The genomes of the Symbiodiniaceae are relatively large and complex, ranging from 1.5 to 3.5 gigabases (billions of base pairs). They have a high gene density and a diverse set of functional genes, including photosynthesis, food absorption, and stress response [82, 83].

Table 2.

List of algal symbionts

Symbiont	Host	References
Chlorella-like	Acaryophyra sp.	[84, 85]
	Climacostomum virens	[86]
	Colepshirtus	[86]
	Disematostoma butschlii	[85]
	Euplotes daidaleos	[86]
	Frontonia leucas	[85, 87]
	Holosticha viridis	[87]
	Malacophrys sphagni	[81]
	Ophrydium versatile	[88]
	Prorodon viridis	[87]
	Prorodon ovum	[87]
	Spirostomim viridis	[88]
	Stentor niger	[89]
	Stentor polymorphus	[86]
	Stentor roeseli	[90]
	Tutorials tricycle	[88]
	Vorticella chlorellata	[91]
	Vorticella sp.	[91]
Chlorella vulgaris	Paramecium bursaria	[79]
Chlorella variabilis	Paramecium bursaria	[92]
Dinoflagellate symbiodinium	Maristentor dinoferus	[73]
Dinophlagellates, or chloroplast from Cryptophytes	Mesodinium rubrum	[93]
Micractinium reisseiri	Paramecium bursaria	[94]
Micractinium conductrix	Paramecium bursaria	[95]
Micractinium sp.	Tetrahymena thermophila	[96, 97]
Chloroplast	Strombidium sp.	[98]
Chloroplast from Chlorophycea	Strombidium acutum (S. Rassoulzadegani)	[98]
Chloroplast from Chromophyte alga	Tontonia spp.	[98, 99]
Choricystis parasitica	Paramecium bursaria	[77]
Chlorb S-type	Stokesiavernalis, Didinium sp.	[100]
Chlorb P-type	Pelagodileptus trachelioides, Didinium sp.	[100]
Chlorb B-type	Bursellopsis spumosa	[100]
Chlorb C-type	Cyclotrichium viride	[100]
Coccomyxa	Didinium sp.	[100]
Yamagishiella	Didinium sp.	[100]
Chaetoceros sp.	Eutintinnus sp.	[78]
Hemiaulus spp.	Eutintinnus lususundae	[78]
Richelia intracelularis	Eutintinnus lususundae	[78]
Fragilariopsis doliolus	Salpingella spp.	[78]
Thalassionema sp.	Eutintinnus lususundae	[78]
Licmophora sp.	Zoothamnium pelagicum	[78]
Ectobacteria	Zoothamnium pelagicum	[78]
Chaetoceros densus	Vorticella sp.	[78]
Coscinodiscus spp.	Pseudovorticella coscinodisci	[78]
Chaetoceros coarctatus	Vorticella oceanica	[78]
Symbiodinium sp.	Maristentor dinoferus	[80]

Viral Symbionts

In contrast, there have been few reports of viruses or plasmids in ciliates, and all have been found in bacterial or algal endosymbionts. Viruses discovered in P. aurelia are found within endosymbiotic killer bacteria [101]and virus-like particles within P. bursaria endosymbiotic chlorella [102]. Ciliates are also known to contain “spheres”, “helical fibrils” (or simply “helices”), and “crystalloids” in their macro- and micronuclei, as well as in their cytoplasm [103]. Euplotes include helical fibres [104]. Fibrillar "spheres" have been seen in a variety of ciliates’ micronuclei, including Loxodes, Ichthyophthirius, Didinium, and Stentor. Crystalloids were found in Tracheloruphis' macronucleus. Helices are found in the nucleus of some amoebae also [105] and in the macronucleus of the hypotrich ciliate Euplotes eurystomus [104]. Tightly coiled microfibrils that resemble helices have also been observed in the macronucleus of P. aurelia and the macronuclear primordium of exconjugant cells of Stylonychia mytilus [106]. The macronucleus of P. caudatum had fibrillar bundles. Tetrahymena micronuclei include nucleolus-like structures associated with chromosomes. The nuclei of the amoeba Acanthamoeba pafestinesis form dense spheres. These structures likely serve important roles in genetic regulation and cellular processes within these respective organisms [107].

The macronuclear inclusion bodies identified in Paramecium caudatum are a non-bacterial endosymbiont that can proliferate autonomously within macronuclei and infect other cells through cell-to-cell contact (conjugation). Plasmids were discovered in the P. aurelia complex's bacterial endosymbiont kappa (Caedibacter taeniospirafis) [108]. Similar structures have been discovered in the macronucleus of P. caudatum's kappa-like bacterial endosymbionts. Virus-like particles were also discovered among the intracellular symbiotic algae of P. bursaria [102].

Genomes of Viral Symbionts

Endogenous viral elements (EVEs) or viral relics are viral DNA sequences that have gotten incorporated into the genomes of host species as a result of previous viral infections. These viral sequences are frequently passed down from generation to generation and can be discovered in the genomes of a variety of organisms, including bacteria, plants, and mammals [109].

The genome size of viral endosymbionts can vary greatly depending on the viral group and the host organism. Virus endosymbiont genome sizes can range from tiny to huge, depending on factors such as virus complexity and degree of integration into the host genome [110].

Due to the loss of non-essential genes and genetic material during integration and evolution within the host genome, viral endosymbionts' genomes can be drastically reduced in some situations. These shortened viral genomes could be as little as a few kilobases (thousands of base pairs). Some viral endosymbionts, on the other hand, may have larger genomes if they have kept more of their original genetic content or have integrated additional genetic material into the host genome. These genomes can range in size from a few kilobases to millions of base pairs [111].

Ciliates as Symbionts

According to Corliss [112], 2600 species of ciliates have been described as symbionts. This equates to 33% of all known species in the phylum. Armophorea, Heterotrichea, Phyllopharyngea, Nassophorea, Oligohymenophorea, Litostomatea, Plagiopylea, and Spirotrichea are the eight classes, 31 orders, 151 families, and over 700 genera [113] (Table 3). These symbiotic ciliates have been found in anaerobic and aerobic conditions, as well as in aquatic and terrestrial environments [114].

Table 3.

List of Ciliates as symbionts

Symbionts	Host	References
Folliculinids	Integument of invertebrates	[115]
Euplotes balteatus	Intestinal tracts of Sea urchins	[116]
Plagiotoma lumbrici	Oligochaetes	[117]
Stichotrichids	Oligochaetes	[117]
Nyctotheridae	Invertebrates and vertebrates	[113]
Balantidium coli	Digestive systems of Mollusks, arthropods, fishes, reptiles, birds, and mammals	[119]
Charonina ventriculi	Digestive systems of Mollusks, arthropods, fishes, reptiles, birds, and mammals	[120]
Dasytricha ruminantium	Digestive systems of Mollusks, arthropods, fishes, reptiles, birds, and mammals	[121]
Entodinium spp.	Digestive systems of Mollusks, arthropods, fishes, reptiles, birds, and mammals	[121]
Ophryoscolecidae	Ruminants	[123]
Cycloposthiidae	Equids	[123]
Triplumaria sp.	Intestines of Elephants and Rhinoceroses	[124]
Entodiniomorphida	Non-ruminant mammals	[125]
Chonotrichs	Crustaceans	[127]
Ichthyophthirius multifiliis	Fish
Mesanophrys pugettensis	Dungeness crab	[128]
Conchophthirus sp.	Freshwater clams or mussels	[129]
Hemispeiridae	Mollusks	[17]
Ancistrocomidae	Mollusks	[17]
Sphenopryidae	Mollusks	[17]
Thigmophryidae	Mollusks	[17]
Vampyrophrya pelagica	Acartiatonsa A. longiremis Eucalanus sp. Centropages hamatus C. typicus Corycaeus sp. Eucalanus sp. Eurytemora sp. Oncaea minuta Labidocera aestiva, Paracalanus sp.	[130]
Gymnodinioides	Crustacean	[132]
Ambiphrya sp.	Zooplanktonic invertebrates, larval stages of aquatic insects, aquatic mollusks, crustaceans, fish, amphibians, and reptiles	[133]
Epistylis sp.	Zooplanktonic invertebrates, larval stages of aquatic insects, aquatic mollusks, crustaceans, fish, amphibians, and reptiles	[133]
Epistylis sp.	Metazoans	[134]
Heteropolaria sp.	Zooplanktonic invertebrates, larval stages of aquatic insects, aquatic mollusks, crustaceans, fish, amphibians, and reptiles	[133]
Rhabdostyla sp.	Zooplanktonic invertebrates, larval stages of aquatic insects, aquatic mollusks, crustaceans, fish, amphibians, and reptiles	[133]
Zoothamnium sp.	Zooplanktonic invertebrates, larval stages of aquatic insects, aquatic mollusks, crustaceans, fish, amphibians, and reptiles	[133]
Lagenophrys sp.	Crabs	[135]
Trichodina sp.	Fish, Limpet gills	[136, 138]
Urceolaria sp.	Freshwater turbellarians, marine polychaetes, and mollusks	[140]
Leiotrocha sp.	Marine mollusks	[140]
Polycycla sp.	Holothuroidea	[140]
Nyctotherus ovalis	Termites’ hindgut	[143]
Tetrahymena sp.	Frogs’ digestive system	[141]
Colpoda sp.	Human urine	[142]
Colpodella spp.	Human urine	[142]

Ciliate taxa that include symbiotic species are as follows:

Heterotrichea: Folliculinids adhere to the integument of a diverse range of invertebrates, including bivalve shells, crustacean exoskeletons, hydroid perisarcs, polychaete tubes, and bryozoan tests; their life cycle includes a migratory swimming stage [115].

Spirotrichea: Although hypotrichs are mostly known as free-living animals, some species, such as Euplotes balteatus, have been found in the intestinal tracts of sea urchins [116]. Plagiotoma lumbrici and other stichotrichids are endosymbionts of oligochaetes [117].

Armophorea: includes clevellandellids as Nyctotheridae, with obligate endosymbionts commonly as commensals of invertebrates and vertebrates; life cycles include a cyst phase [113].

Litostomatea: Trichostomes are ruminant and foregut fermenter symbionts [113], including the human pathogen, Balantidium coli, with a life cycle that includes two phases: trophozoites and cysts [118]. This species is thought to belong to a new genus, Neobalantidium coli. Balantidium contains a larger number of species that have been documented as endocommensals in the digestive systems of a wide variety of metazoans, including mollusks, arthropods, reptiles, birds, fishes, and mammals [119]. Ciliates can contribute for up to 50% of total microbial nitrogen in the rumen environment, reaching concentrations of 105 to 106 cells/ml rumen fluid, with Charonina ventriculi being one of the smallest rumen ciliates [120]. Dasytricha ruminantium and Entodinium spp., which together accounted for more than 90% of all rumen ciliates, harboured intracellular methanogenic bacteria living freely in the cytoplasm [121]. They digest starch and proteins and produce hydrogen, amino acids, volatile fatty acids, ammonia, and sugars in their food vacuoles [122].

The families Ophryoscolecidae and Cycloposthiidae contain species that are endosymbionts of ruminants and equids, respectively [123]. Triplumaria ciliates have been discovered in the intestines of rhinoceroses and elephants [124]. Entodiniomorphida do not produce cysts, and coprophagy is used to infect hosts in non-ruminant mammals [125].

Phyllopharyngea: Chonotrichs live on both freshwater and marine hosts and divide by generating external or internal buds [126], with a dimorphism in which the adults reside connected to multiple appendages of crustaceans while the larva is free and swims to a new host [127]. Suctorians, generally, reproduce via diverse budding mechanisms, generating one to several larvae that swim briefly before shedding their cilia and metamorphosing into adults or trophonts [126].

Oligohymenophorea: Ichthyophthirius multifiliis is a parasite of fish. Mesanophrys pugettensis, a scuticociliatethata, is a facultative parasite of the Dungeness crab [128]. Conchophthirus species inhabit the mantle cavity of freshwater clams or mussels [129].

Raabe studied higmotrichids from several families, finding that Hemispeiridae species are molluscan symbionts of the mantle cavity and nephridia, Ancistrocomidae, Sphenopryidae, and Thigmophryidae species are molluscan ectosymbionts of the mantle cavity and gills [17].

Ciliates are frequent protozoan symbionts found on copepods, with the most prevalent lineages being Apostomatida, Peritrichia, and Suctoria. Vampyrophrya pelagica has been found on Acartiatonsa, A. longiremis, Eucalanus sp., Eurytemora sp., Centropages hamatus, C. typicus, Labidocerca aestiva, Corycaeus sp., Eucalanus sp., Oncaea minuta, and Paracalanus sp. in North Carolina [130].

Apostomes are found in the renal organs and opalinopsids found in the liver and intestines of cephalopods ingesting fluids and cells such as Vampyrophrya [131]. Apostome ciliates have life cycles that typically involves crustaceans [126]. Collinia are endoparasites that multiply fast within the host and invariably kill the euphausiid within 40 h of infection; The genus Gymnodinioides contains exuviotrophic organisms that feed on the fluid within crustacean hosts’ exuviae [132].

Species of sessile peritrichs genera such as Ambiphrya, Epistylis, Rhabdostyla, Heteropolaria, and Zoothamnium are epibionts of zooplanktonic invertebrates, larval stages of aquatic insects, amphibians, aquatic mollusks, crustaceans, fish, and reptiles [133].

Epistylis members have been identified as epibionts in a variety of metazoans, as well as an important fish ectoparasite that is considered an emerging disease [134]. The genus Lagenophrys contains exclusively symbiotic freshwater and marine crabs [135]. Trichodinids are the most damaging ectoparasites of cultured fish, causing significant harm [136], and over 300 species of Trichodina have been reported, predominantly from freshwater habitats [137]. There have also been reports of trichodinids from limpet gills [138], which have been recorded as symbionts of a diverse range of aquatic and terrestrial invertebrates and vertebrates hosts. Trichodinella epizootica is a common freshwater trichodinid in Europe and Asia, but it has also been found in Africa, the Pacific area, and North America [139]. Urceolaria species are ectosymbionts of freshwater turbellarians, marine polychaetes, and mollusks; Leiotrocha species are ectocommensals and endocommensals of marine mollusks; and Polycycla sp. are endocommensals of Holothuroidea [140].

Ciliate infections in frogs are typically caused by pathogenic species of ciliates. Tetrahymena, a ciliate genus that contains species such as Tetrahymena pyriformis and Tetrahymena corlissi, is one example. These ciliates can infect frogs' digestive systems, causing health problems. Ciliate infections in frogs can induce diarrhoea, weight loss, lethargy, and a loss of appetite. These infections can be fatal in severe situations, especially if left untreated [141].

Colpoda sp. and Colpodella spp. are also detected in human urine. The presence of these parasites was not associated with any clinical signs of urinary disease [142].

Genomes of Ciliates as Symbionts

Symbiont ciliate genomes can differ based on the species and their biological context. They do, however, have relatively tiny genomes as compared to free-living ciliates. Symbiont ciliate genomes typically range from a few million to tens of millions of base pairs. Holospora spp., an endosymbiont in freshwater ciliates, is one well-studied example of a symbiotic ciliate. Holospora's genome is quite short, ranging from 1.3 to 2.6 million base pairs. It has a smaller gene set than free-living ciliates and is highly dependent on the host for crucial nutrients and cellular processes [25].

Nyctotherus ovalis, an endosymbiont in termites' hindgut, is another well-known example of an endosymbiotic ciliate. The genome of N. ovalis is quite tiny, containing between 12 and 15 million base pairs. It has been used as a model organism to study the evolution of endosymbiotic ciliates' adaption to their specialised ecological niche [143].

Conclusion

The study is a multifaceted field that provides remarkable insights into the intricate web of connections within microbial ecosystems. The enormous spectrum of bacterial, algal, viral, and ciliate groups that have established endosymbiotic partnerships demonstrates the evolutionary success of endosymbioses. Endosymbiosis indicates a common evolutionary strategy used by eukaryotes to acquire fresh biochemical capabilities and compartments to carry out their separate functions including photosynthesis, nitrogen fixation, nitrogen recycling, methanogenesis, and sulfide oxidation.

Characterization of new symbiotic systems, which may contain a considerable fraction of the bacterial biodiversity that is currently unknown, is crucial. Further research into the ecological significance of symbionts is also required. Different aspects of this ciliate-green algal association can be studied in depth as these species are available in culture form. Nothing is known about the uniqueness of this symbiotic relationship. This ciliate and its endosymbiont are ideal model organisms for studying ciliate-green interactions as they can be cultivated easily.

Key problems that remain unanswered in most endosymbiotic interactions are how the host cell controls endosymbiont division and growth, what modes of communication exist between the symbiotic partners, and how metabolites, or even many proteins, are transferred. A solution to these questions could also aid in understanding the intricate process of organelle evolution that once and for all shaped life on this planet.

Understanding these symbiotic interactions may inspire novel solutions in disease control, biotechnological developments, and ecosystem management and preservation techniques. Continued research in this area is critical for unravelling the complicated fabric of symbiotic interactions, which provides a window into the natural world and its diverse ecological linkages.

Future Perspectives

Genomic investigations of ciliate symbionts and the role of ciliates as symbionts provide a good opportunity for further understanding of symbiotic mechanisms, which is crucial for comprehending the ecological and evolutionary implications of these relationships and further manipulating them for various applications. These relationships have important implications for the nutrient cycle, energy flow, and ecosystem stability. Predicting how alterations in ecosystems affect these symbiotic associations and subsequently impact ecosystem functions remains an essential area for future research. Furthermore, examining ciliates as symbionts helps us understand the evolution and diversity of symbiotic interactions across taxa with advanced molecular tools and techniques, such as metagenomics, transcriptomics, and single-cell sequencing. This area of inquiry is currently underexplored due to a lack of significant molecular data. As a result, a crucial challenge will be to expand the number of entire genomes, which, in conjunction with comparative genomics investigations, will offer light on specific symbiotic adaptations. Further targeted genomic investigations will be required to have a better understanding of host-symbiont interactions. The combination of multi-omics data, ecological modelling, and experimental techniques will very certainly improve our understanding of symbiont-ciliate systems. Collaboration across disciplines could lead to a comprehensive understanding of the dynamics and functions of these symbiotic connections. The potential for using symbiont-ciliate interactions in biotechnological disciplines such as bioengineering, bioremediation, and medicines is largely untapped. Understanding these interactions could lead to breakthrough biotechnology solutions ranging from novel bioactive chemicals to eco-friendly ways to environmental repair.

Declarations

Conflict of interest

The authors declare no conflict of interest.