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A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales ); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria , which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, " Candidatus Hydrogenosomobacter endosymbioticus." IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates., (Copyright © 2019 American Society for Microbiology.)

Evolutionary theory predicts potential shifts between cooperative and uncooperative behaviour under fluctuating environmental conditions. This leads to unstable benefits to the partners and restricts the evolution of dependence. High dependence is usually found in those hosts in which vertically transmitted symbionts provide nutrients reliably. Here we study host dependence in the marine, giant colonial ciliate Zoothamnium niveum and its vertically transmitted, nutritional, thiotrophic symbiont from an unstable environment of degrading wood. Previously, we have shown that sulphidic conditions lead to high host fitness and oxic conditions to low fitness, but the fate of the symbiont has not been studied. We combine several experimental approaches to provide evidence for a sulphide-tolerant host with striking polyphenism involving two discrete morphs, a symbiotic and an aposymbiotic one. The two differ significantly in colony growth form and fitness. This polyphenism is triggered by chemical conditions and elicited by the symbiont's presence on the dispersing swarmer. We provide evidence of a single aposymbiotic morph found in nature. We propose that despite a high fitness loss when aposymbiotic, the ciliate has retained a facultative life style and may use the option to live without its symbiont to overcome spatial and temporal shortage of sulphide in nature.

Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO 2 by the symbionts drives these nutritional associations. In this study, we investigated " Candidatus Kentron," the clade of symbionts hosted by Kentrophoros , a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron bacteria lack the key canonical genes for any of the known pathways for autotrophic carbon fixation and have a carbon stable isotope fingerprint that is unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to gain energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. Here we show that Kentron bacteria are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or the reverse tricarboxylic acid cycle for autotrophy. IMPORTANCE Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO 2 , serving as primary producers for their hosts. Here we describe a clade of nonautotrophic sulfur-oxidizing symbionts, " Candidatus Kentron," associated with marine ciliates. They lack genes for known autotrophic pathways and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead, they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely., (Copyright © 2019 Seah et al.)

The single-celled ciliate Paramecium bursaria is an indispensable model for investigating endosymbiosis between protists and green-algal symbionts. To elucidate the mechanism of this type of endosymbiosis, we combined PacBio and Illumina sequencing to assemble a high-quality and near-complete macronuclear genome of P. bursaria. The genomic characteristics and phylogenetic analyses indicate that P. bursaria is the basal clade of the Paramecium genus. Through comparative genomic analyses with its close relatives, we found that P. bursaria encodes more genes related to nitrogen metabolism and mineral absorption, but encodes fewer genes involved in oxygen binding and N-glycan biosynthesis. A comparison of the transcriptomic profiles between P. bursaria with and without endosymbiotic Chlorella showed differential expression of a wide range of metabolic genes. We selected 32 most differentially expressed genes to perform RNA interference experiment in P. bursaria, and found that P. bursaria can regulate the abundance of their symbionts through glutamine supply. This study provides novel insights into Paramecium evolution and will extend our knowledge of the molecular mechanism for the induction of endosymbiosis between P. bursaria and green algae.

Members of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont "Candidatus Megaira", which is phylogenetically closely related to the pathogen Rickettsia. "Candidatus Megaira" was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species "Candidatus Megaira polyxenophila", and the multidisciplinary description of a novel species, "Candidatus Megaira venefica", presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of "Candidatus Megaira", and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.

Endosymbiosis is a widespread phenomenon in the microbial world and can be based on diverse interactions between endosymbiont and host cell. The vast majority of the known endosymbiotic interactions involve bacteria that have invaded eukaryotic host cells. However, methanogenic archaea have been found to thrive in anaerobic, hydrogenosome-containing protists and it was suggested that this symbiosis is based on the transfer of hydrogen. Here, we used culture-independent genomics approaches to sequence the genomes of two distantly related methanogenic endosymbionts that have been acquired in two independent events by closely related anaerobic ciliate hosts Nyctotherus ovalis and Metopus contortus, respectively. The sequences obtained were then validated as originating from the ciliate endosymbionts by in situ probing experiments. Comparative analyses of these genomes and their closest free-living counterparts reveal that the genomes of both endosymbionts are in an early stage of adaptation towards endosymbiosis as evidenced by the large number of genes undergoing pseudogenization. For instance, the observed loss of genes involved in amino acid biosynthesis in both endosymbiont genomes indicates that the endosymbionts rely on their hosts for obtaining several essential nutrients. Furthermore, the endosymbionts appear to have gained significant amounts of genes of potentially secreted proteins, providing targets for future studies aiming to elucidate possible mechanisms underpinning host-interactions. Altogether, our results provide the first genomic insights into prokaryotic endosymbioses from the archaeal domain of life.

Symbiosis is a diverse and complex phenomenon requiring diverse model systems. The obligate relationship between a monophyletic group of Euplotes species ("clade B") and the betaproteobacteria Polynucleobacter and "Candidatus Protistobacter" is among the best-studied in ciliates, and provides a framework to investigate symbiont replacements. Several other Euplotes-bacteria relationships exist but are less understood, such as the co-dependent symbiosis between Euplotes magnicirratus (which belongs to "clade A") and the alphaproteobacterium "Candidatus Devosia euplotis". Here we describe a new Devosia inhabiting the cytoplasm of a strain of Euplotes harpa, a clade B species that usually depends on Polynucleobacter for survival. The novel bacterial species, "Candidatus Devosia symbiotica", is closely related to the symbiont of E. magnicirratus, casting a different light on the history of bacteria colonizing ciliates of this genus. The two Devosia species may have become symbionts independently or as the result of a symbiont exchange between hosts, in either case replacing a previous essential bacterium in E. harpa. Alternatively, both may be remnants of an ancient symbiotic relationship between Euplotes and Devosia, in which case Polynucleobacter and "Ca. Protistobacter" are recent invaders. Either way, symbiont replacement between bacteria belonging to different classes must be evoked to explain this fascinating system., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Symbioses between eukaryotes and sulfur-oxidizing (thiotrophic) bacteria have convergently evolved multiple times. Although well described in at least eight classes of metazoan animals, almost nothing is known about the evolution of thiotrophic symbioses in microbial eukaryotes (protists). In this study, we characterized the symbioses between mouthless marine ciliates of the genus Kentrophoros , and their thiotrophic bacteria, using comparative sequence analysis and fluorescence in situ hybridization. Ciliate small-subunit rRNA sequences were obtained from 17 morphospecies collected in the Mediterranean and Caribbean, and symbiont sequences from 13 of these morphospecies. We discovered a new Kentrophoros morphotype where the symbiont-bearing surface is folded into pouch-like compartments, illustrating the variability of the basic body plan. Phylogenetic analyses revealed that all investigated Kentrophoros belonged to a single clade, despite the remarkable morphological diversity of these hosts. The symbionts were also monophyletic and belonged to a new clade within the Gammaproteobacteria, with no known cultured representatives. Each host morphospecies had a distinct symbiont phylotype, and statistical analyses revealed significant support for host-symbiont codiversification. Given that these symbioses were collected from two widely separated oceans, our results indicate that symbiotic associations in unicellular hosts can be highly specific and stable over long periods of evolutionary time., (© 2017 The Authors.)

Large-scale studies on obligate bacterial endosymbionts may frequently require preliminary purification and enrichment protocols, which are often elaborate to set up and to evaluate, especially if the host organism is a protist. The purpose of this study was to develop a real-time PCR-based strategy and employ it for assessing two of such enrichment protocols for Holospora caryophila, hosted by the ciliate Paramecium. Four SSU rRNA gene-targeted real-time PCR assays were designed, which allowed to compare the amount of H. caryophila to other organisms, namely the host, its food bacterium (Raoultella planticola), and free-living bacteria present in the culture medium. By the use of the real-time PCR assays in combination, it was possible to conclude that the "cell fractionation" protocol was quite successful in the enrichment of the symbiont, while the "Percoll gradient" protocol will need further refinements to be fully repeatable. The proposed approach has the potential to facilitate and encourage future studies on the yet underexplored field of bacterial endosymbionts of ciliates and other protists. It can also find valuable applications for experimental questions other than those tested, such as fast and precise assessment of symbiont abundance in natural populations and comparison among multiple coexisting symbionts.

The aim of the present study was to investigate the prokaryotic community structure of the anaerobic ciliate, Metopus sp. using rRNA sequencing, fluorescence in situ hybridization (FISH), and transmission electron microscopy (TEM). Metopus sp. was physically separated from anaerobic granular sludge in a domestic wastewater treatment plant and anoxically cultivated for 7 d. 16S rRNA gene sequences from the prokaryotes Methanoregula boonei and Clostridium aminobutyricum were abundantly detected in Metopus ciliates. The FISH analysis using the oligonucleotide probes Mg1200b and Cla568 demonstrated that these prokaryotes were localized within Metopus cells. These results identify M. boonei- and C. aminobutyricum-like prokaryotes as novel endosymbionts of Metopus ciliates.

Representatives of all classes of Ciliophora have been studied for the detection and investigation of both prokaryotic and eukaryotic (not algal) endo- (EnS) and ectosymbionts (EcS). Different methods including transmission electron microscopy (TEM) and fluorescence in situ hybridisation (FISH) have been used. Apparently, the capability of keeping symbionts varies among the different ciliate groups as it generally is the case in different protist taxa. Most of the prokaryotic EnSs detected belong to Alphaproteobacteria. Holospora or Holospora-like infectious bacteria of this group were found in representatives of Heterotrichea, Armophorea, Phyllopharyngea, Prostomatea and mainly of Oligohymenophorea. Bacteria associated with bacteriophages were found in species of Heterotrichea and Oligohymenophorea. This holds true also for bacteria with R-bodies. A quite rare type of EnS - motile bacteria - was found in ciliates of the same two classes as well, either in the cytoplasm (Heterotrichea) or in the macronucleus and its perinuclear space (Oligohymenophorea). EcSs are more common in Heterotrichea, Armophorea and Plagiopylea, but were never found in other groups. Among the eukaryotic EnSs of ciliates, very few representatives of Microsporidia and Trypanosomatidae were recorded. In conclusion, heterotrichs and oligohymenophoreans are the most promising groups of Ciliophora for the investigation of symbiosis., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an approximately 1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4',6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.

Epifluorescence microscopy and whole cell in situ hybridization analysis revealed the presence of Methanosaeta sp. as endosymbionts in Metopus es. Direct microscopic observation under epifluorescent microscope showed the presence of long slender rods with an average length of 3.4 microm. The number of methanogenic rods varied from 792 +/- 12 in a single M. es cell with a biovolume of 3.4 x 10(5) microm(3). At the exponential growth stage, a single symbiotic methanogen in M. es produced about 1 fmol methane/h leading to a methane production rate of 0.85 pmol/ciliate/h. The presence of endosymbiotic methanogens in the domain archaea and Methanosaeta sp. was confirmed by FISH with ARCH 915 and MX 825 oligonucleotide probes specific to domain archaea and Methanosaeta respectively. The homogenized cells of M. es also showed bright fluorescing rods with MX 825 hybridization. The culture obtained on inoculation of the released endosymbiotic organisms on Methanosaeta-specific medium lent support to the growth of long slender rods having the same range of mean length (3.6 microm) as that of the endosymbiotic methanogens observed. Both intra- and extracellular production of acetate was detected in M. es culture.

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 microg ml(-1)), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host's fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.

It is well documented that organelles can be retained and used by predatory organisms, but in most cases such sequestrations are limited to plastids of algal prey. Furthermore, sequestrations of prey organelles are typically highly ephemeral as a result of the inability of the organelle to remain functional in the absence of numerous nuclear-encoded genes involved in its regulation, division and function. The marine photosynthetic ciliate Myrionecta rubra (Lohmann 1908) Jankowski 1976 (the same as Mesodinium rubrum) is known to possess organelles of cryptophyte origin, which has led to debate concerning their status as permanent symbiotic or temporary sequestered fixtures. Recently, M. rubra has been shown to steal plastids (that is, chloroplasts) from the cryptomonad, Geminigera cryophila, and prey nuclei were observed to accumulate after feeding. Here we show that cryptophyte nuclei in M. rubra are retained for up to 30 days, are transcriptionally active and service plastids derived from multiple cryptophyte cells. Expression of a cryptophyte nuclear-encoded gene involved in plastid function declined in M. rubra as the sequestered nuclei disappeared from the population. Cytokinesis, plastid performance and their replication are dependent on recurrent stealing of cryptophyte nuclei. Karyoklepty (from Greek karydi, kernel; kleftis, thief) represents a previously unknown evolutionary strategy for acquiring biochemical potential.

Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status "Candidatus Thiobios zoothamnicoli" is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence.

Methanogenesis in rice field soils starts soon after flooding while potentially competing processes like reduction of sulphate and iron take place. Early methanogenesis is mainly driven by hydrogen, while later in the season acetate tends to become more important. Anaerobic ciliates are abundant during this period, and their endosymbionts use hydrogen produced by the ciliates to reduce carbon dioxide to methane. These endosymbiotic methanogens are protected from the competition for substrates with other bacteria that may control methanogenesis outside the protozoan cells. Thus, we focussed on early methanogenesis and on the potential contribution from ciliates and their endosymbionts. Only ciliates of the genus Metopus were found to harbour methanogens, as identified by the F(420)-fluorescence of the endosymbionts. We followed the population dynamics of the ciliates with time, and calculated the ratio of symbiotic methane production to overall methanogenesis. Symbiotic methane production was calculated from the species-specific numbers of methanogenic endosymbionts times the cell-specific methane production of the symbionts. According to this calculation, the symbionts' contribution to overall methane production was only 6.4% at the beginning and decreased with time. In a second experiment, colchicine and cycloheximide were used to inhibit all eukaryotes, comparing the remaining methane production rate to a control without inhibitors. In the inhibition experiment, the contribution from symbionts decreased from 40% to 6% during the first days after flooding, and dropped to near zero within 2 weeks. However, nearly all methane produced from H(2)/CO(2) could be attributed to the ciliates' symbionts between days 5 and 10 after flooding. Both experiments showed that the contribution of methanogenic symbionts to overall methane production is a transient phenomenon, restricted to the first 2 weeks.

Ciliates may be hosts for numerous bacteria, which can occupy almost all cellular compartments of the protists. About 200 ciliate species are recorded as hosts of different intracellular bacteria, being a small part of the diversity for such types of endocytobiosis in nature. In the Paramecium genus alone close to 60 types of bacteria adapted for intracellular life are known. In this review extensive material concerning the variety of endocytobionts, their categories, and their interaction with host cells is presented. Special attention is paid to endocytobiosis in Paramecium with highly infectious bacteria Holospora, bacteria of the Caedibacter and Polynucleobacter genera, methanogenic bacteria, and "xenosomes" as well as to life cycles and strategies of bacterial endonucleobionts. The above model bacteria and their interactions with hosts have not been exhaustively studied. A number of unsolved problems concerning their interactions within an endocytobiotic system and their ecological implications remain to be studied.

Structure of cytoplasmic bacterial symbionts of chlorella-free ciliate Climacostomum virens has been investigated. It is shown that ciliates are not able to support simultaneously growth and duplication of two different symbionts--bacteria and chlorella. Cells of C. virens lost bacterial symbionts after an artificial infection with chlorella by microinjection. Competitive relationships between two endopionts are discussed.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

The polymerase chain reaction was used to amplify and partially sequence the 16S ribosomal RNA genes of symbiotic bacteria within the anaerobic ciliate Metopus contortus. In situ probing with fluorescent oligonucleotides showed that the amplified sequences originated from a single species of archaebacterium which is closely related to Methanocorpusculum parvum. The probed symbionts exhibited a variety of shapes and sizes. These data support the hypothesis, first proposed on the basis of electron microscopy, that the symbionts undergo a morphological transformation as part of the symbiotic process.

The polymerase chain reaction (PCR) was used to amplify small-subunit ribosomal DNA from the anaerobic ciliated protozoon Metopus palaeformis, and from its uncultured endosymbiotic bacteria. This was accomplished directly from total DNA extracted from protozoa without prior isolation or enrichment for symbiont cells. The double-stranded amplification products were precipitated and directly sequenced using the linear PCR reaction. Fluorescent oligonucleotide probes were designed and used in whole-cell hybridizations to provide direct visual evidence that the sequences originated from the host ciliate and from the endosymbiont. Phylogenetic analysis of the Metopus palaeformis sequence consistently placed it as a deep-branching lineage near the root of the ciliate tree. However, the present data were insufficient to resolve the detailed relationship between Blepharisma and Metopus and thus to determine if the heterotrichs are mono- or paraphyletic. Phylogenetic analysis of the symbiont partial sequence clearly demonstrated that it is an archaeobacterium and that it is closely related to, but distinct from, Methanobacterium formicicum.

The identity of ectosymbiotic bacteria of some marine, free-living anaerobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.

Production of methane in the hindgut of the cockroach Periplaneta americana was found to vary, depending on the feeding regimen. Methane production was positively correlated with the numbers of the ciliate Nyctotherus ovalis living in the cockroach hindgut. Defaunation of the cockroaches by means of low concentrations of metronidazole (Flagyl) resulted in a quick drop of methane production. Addition of the methanogenic substrates acetate and formate to isolated hindguts stimulated methane production. Inside the ciliate cells, autofluorescing bacteria could be demonstrated which were presumed to be methanogens. Electron microscopy revealed that the bacteria resembled Methanobrevibacter and that they were closely associated with organelles which contained infolded membranes and which were presumably hydrogenosomes.