Anaerobiosis and Symbiosis with Bacteria in Free-living Ciliates

SYNOPSIS. Marine, sediment-dwelling ciliates were examined for cytochrome oxidase activity by a cytochemical method and for fine structural details. Species of Plagiopylidae (Trichostomatida), i.e. Plagiopyla frontata, Sonderia vorax and Sonderia sp., and of Heterotrichida, i.e., Parablepharisma pellitum, Parablepharisma sp., Metopus contortus, Metopus vestitus and Caenomorpha capucina ; previously considered to be obligate anaerobes because of their sulfide-containing habitat, do not have cytochrome oxidase activity or mitochondria with cristae or tubuli. The evolutionary origin and significance of anaerobic ciliates is discussed. Most of the anaerobic ciliates harbor a flora of ecto- and endosymbiotic bacteria as demonstrated by transmission and scanning electron micrographs. It is speculated that the bacteria may utilize the metabolic end products of the protozoa for growth and energy yielding processes. These associations are also compared with other, previously described cases of symbiosis involving prokaryotes and protozoa.     

Anaerobiosis and symbiosis with bacteria in free-living ciliates.

Marine, sediment-dwelling ciliates were examined for cytochrome oxidase activity by a cytochemical method and for fine structural details. Species of Plagiopylidae (Trichostomatida), i.e. Plagiopyla frontata, Sonderia vorax and Sonderia sp., and of Heterotrichda, i.e., Parablepharisma pellitum, Parablepharisma sp., Metopus contortus, Metopus vestitus and Caenomorpha capucina; previously considered to be obligate anaerobes because of their sulfide-containing habitat, do not have cytochrome oxidase activity or mitochondria with cristae or tubuli. The evolutionary origin and significance of anaerobic ciliates is discussed. Most of the anaerobic ciliates harbor a flora of ecto- and endosymbiotic bacteria as demonstrated by transmission and scanning electron micrographs. It is speculated that the bacteria may utilize the metabolic end products of the protozoa for growth and energy yielding processes. These associations are also compared with other, previously described cases of symbiosis involving prokaryotes and protozoa.     

Systematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoaMetopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. andPelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells ofMetopus palaeformis, Metopus contortus, Trimyema sp. andCyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera;Methanobacterium, Methanocorpusculum andMethanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. andMetopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis andM. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses inMetopus must postdate the speciation ofM. palaeformis andM. contortus. Despite this,Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.     

Production of methane and hydrogen by anaerobic ciliates containing symbiotic methanogens

Rates of methane production by three anaerobic ciliates containing symbiotic methanogens (the marine Metopus contortus and Plagiopyla frontata, and the limnic Metopus palaeformis) were quantified. Hydrogen production by normal (containing active symbionts), aposymbiotic and BES-treated cells was also measured in the case of the marine species. Methanogenesis was closely coupled to host metabolism and growth; at maximum ciliate growth rates (20°C) each methanogen produced about 1 fmol CH₄ per hour corresponding to about 7, 4 and 0.35 pmol per ciliate per hour for M. contortus, P. frontata and M. palaeformis, respectively. Normal cells produced traces of H₂. Hydrogen production by BES-treated or aposymbiotic cells accounted for 75 and 45% of the methane production of normal M. contortus and P. frontata cells, respectively. However, it is possible that hydrogen production was partly inhibited in the absence of methanogens. Theoretical considerations suggest that hydrogen transfer is significant to the metabolism of larger anaerobic ciliates. Ciliates with methanogens produced CH₄ under microaerobic conditions due to their ability to maintain an anoxic intracellular environment at low external oxygen tensions. Methanogenesis was still detectable at a pO₂ of 0.63 kPa (3 %atm sat).     

Some rumen ciliates have endosymbiotic methanogens

Abstract 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.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.      

Role of anaerobic ciliates in planktonic food webs: abundance, feeding, and impact on bacteria in the field

We studied the dynamics of two populations of anaerobic ciliates, Plagiopyla sp. and Metopus sp., and of their potential prey, heterotrophic and phototrophic purple bacteria, in Lake Cisó throughout a 1-year cycle. The abundance of both ciliates was very low (less than 2 individuals per ml). During mixing, Plagiopyla ciliates exhibited high clearance rates (about 100 nl ciliate h), its integrated abundance increased with a net doubling time of 47 days, and its potential doubling times, as calculated from the number of bacteria consumed, ranged between 5 and 8 days. During stratification, the activity of Plagiopyla ciliates was reduced and the population decreased; this was related to the higher amounts of sulfide present. The impact of predation by the Plagiopyla population on bacterioplankton was found to be insignificant, less than 0.1% of bacterial biomass consumed per day. Thus, anaerobic ciliates cannot control the bacterioplankton in Lake Cisó because of both the low abundance over the period studied and the low feeding rates during certain periods. A review of available field studies suggests that this conclusion can be extrapolated to most other anoxic systems.     

The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont.

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.     

Effects of temperature, sulfide, and food abundance on growth and feeding of anaerobic ciliates

The trophic role of ciliates in anaerobic food webs has not been assessed experimentally. In order to obtain basic information necessary to interpret field situations, we studied the effects of temperature, sulfide concentration, and food abundance on the growth and feeding activities of two anaerobic ciliates, Plagiopyla nasuta and Metopus es. The growth rate of P. nasuta increased with temperature from 8 to 18 degrees C (Q(10) = 2.0) and remained constant in the range between 18 and 24 degrees C (0.22 day). Sulfide concentrations of between 0 and 1 mM did not affect the feeding activities, but concentrations greater than 2 mM were inhibitory. The functional response of P. nasuta feeding on fluorescently labeled heterotrophic and phototrophic bacteria was investigated. In both cases, the parameters of the functional response were almost identical when expressed in terms of biovolume: the maximal uptake rate (U(m)) was 1,800 mum ciliate h and the half-saturation constant for ingestion (k) was 1.5 x 10 mum ml. The functional response of M. es feeding on heterotrophic bacteria was found to be similar to that of P. nasuta. These ciliates needed high bacterial abundances in order to maintain their growth (k of about 4 x 10 bacteria ml), implying that they will frequently be food limited in planktonic environments. Both the maximal uptake rates and the maximal clearance rates were comparable to those of aerobic ciliates. By combining the growth and feeding data, we estimated gross growth efficiencies of 12 and 13% for P. nasuta and M. es, respectively. These results indicate that the feeding rates of anaerobic ciliates are similar to those of aerobic ciliates. Their slower growth must, therefore, be due to the lower gross growth efficiency (likely due to anaerobic metabolism).     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Abundance and distribution of anaerobic protozoa and their contribution to methane production in Lake Cadagno (Switzerland)

Abstract In the uupermost layers of the anoxic sediment in Lake Cadagno, 9 different species of anaerobic protozoa were identified. The total number of these organisms was about 580 cells·ml⁻¹ sediment. Most pf these protozoa contained endosymbiotic methanogenic bacteria which in total amounted to 10⁶ methanogens·ml⁻¹ sediment. In addition to the methanogenic endosymbionts, cells of Metopus setosus and Caenomorpha lata also contained a non-fluorescent bacterial rod inside the cytoplasm. In some individual cells of C. lata this second type of endosymbiotic bacterium was sometimes the only endosymbiont observed. Contrary to earlier suggestions, anaerobic protozoa do not seem to play a major role in methane production at least in Lake Cadagno. No significant methane production due to the anaerobic protozoa and their methanogenic endosymbionts was found in situ. Isolated ciliates and amoebae produced methane at 12°C, but not at 6°C, probably as a result of temperature limitation. In the sediment of Lake Cadagno sulfate reduction seemed to be the dominant terminal degradation process.     

Electromigration, a tool for studies on anaerobic ciliates

Abstract By applying electric current, anaerobic ciliates could be extracted from sludge samples. All the freshwater ciliates tested had one point of optimal current and voltage where they reached the highest net speed of migration in the electric field. The swimming behavior of Metopus es was tested under different current and voltage conditions. During electromigration the freshwater, marine and rumen ciliates moved from the anode to the cathode. In the anaerobic freshwater ciliates high numbersof methanogenic bacteria of different size were present. At the depth in the sludge with the highest number of Metopus minimus (660 cells ·ml⁻¹, a peak of methane production also occurred.     

Chemosensory Behaviour of Strombidium purpureum, an Anaerobic Oligotrich with Endosymbiotic Purple Non-Sulphur Bacteria

ABSTRACT. Strombidium purpureum Kahl, is an anaerobic oligotrichous ciliate with endosymbiotic phototrophic bacteria. Like other anaerobic ciliates, S. purpureum reacts to O₂-pressure. In the light, the ciliates avoid even traces of O₂ (< 1% atmospheric saturation). In the dark, however, the ciliates accumulate in water with a pO₂ of 1–4% atmospheric saturation. Experiments show that ciliates which have accumulated in the dark under microaerophilic conditions react when the light is turned on, and their ability to escape is enhanced by steep O₂-gradients. The ciliates orient themselves in O₂-gradients by a series of phobic responses. They tumble whenever they swim towards a higher O₂-tension and thus they eventually all swim in the direction of lower pO₂. The ecological implications of such behaviour are discussed.     

Soil type links microbial colonization of rice roots to methane emission

Most of the methane (CH₄) emission from rice fields is derived from plant photosynthates, which are converted to CH₄. Rice cluster I (RC-1) archaea colonizing the rhizosphere were found to be the methanogens responsible for this process. Hence, RC-1 methanogens seem to play a crucial role in emission of the greenhouse gas CH₄. We determined the community composition and activity of methanogens colonizing the roots of eight different rice cultivars after growth on both Italian rice soil and river bank soil, which contained different communities of methanogenic archaea. The community composition was analyzed by terminal restriction fragment length polymorphism and cloning/sequencing of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase. When grown on rice field soil, the methanogenic community of the different rice cultivars was always dominated by RC-1 methanogens. In contrast, roots were colonized by Methanomicrobiales when grown on river bank soil, in which RC-1 methanogens were initially not detectable. Roots colonized with Methanomicrobiales compared with RC-1 exhibited lower CH₄ production and CH₄ emission rates. The results show that the type of methanogens colonizing rice roots has a potentially important impact on the global CH₄ cycle.     

An anaerobic protozoon, with symbiotic methanogens, living in municipal landfill material

Abstract We have established that anaerobic protozoa do live in municipal landfill material although they probably spend much of the time encysted, especially in the drier (< 40% water) site. At least eight species were observed; they were readily isolated by adding anoxic water to dry landfill samples. The ciliate Metopus palaeformis was frequently i isolated; it appears to be ubiquitous in anaerobic landfills. It has a polymorphic life cycle, it is positive for hydrogenase, each ciliate contains about 500 bromoethanesulfonate-sensitive methanogen symbionts (probably Methanobacterium formicicum ), and maximum cell densities in culture exceed 3000 per ml. The methanogens are not attached to the hydrogenosomes, neither do they undergo morphological transformation; the ciliate receives no measurable energetic advantage from its symbionts. The ciliate encysts in response to a shortage of food or water, and the methanogens remain viable within the cysts. When the protozoon excysts, the methanogens resume growth and cell division within the trophic form of the ciliate. Unlike free-living methanogens, the M. palaeformis -methanogen consortium is not particularly sensitive to oxygen; the symbiotic methanogens remain viable following exposure of the consortium to atmospheric oxygen for several days. Dispersal of methanogen-bearing protozoan cysts through oxygenated environments is a potential mechanism of transfer between landfill sites and other anaerobic environments. Anaerobic protozoan consortia are theoretically capable of making a significant contribution to methane generation from wet landfill sites.     

Synchronous Division of an Endosymbiotic Methanogenic Bacterium In the Anaerobic Ciliate Plagiopyla Frontata Kahl

ABSTRACT The life cycle of a methanogenic bacterium, symbiotic within the marine, free-living anaerobic ciliate Plagiopyla frontata , was studied using light microscopy and transmission electron microscopy (TEM). the bacteria are disc-shaped. During the growth phase of the host, bacteria and hydrogenosomes (organelles which ferment pyruvate into acetate and hydrogen) are arranged in conglomerates resembling stacks of coins in which bacteria and hydrogenosomes alternate; hydrogenosomes always cap the ends of the stacks. During the growth phase, numbers of hydrogenosomes and bacteria remain constant (about 5,000 and 3,500 per cell, respectively). Hydrogenosomes increase in volume shortly after cell division. Methanogens increase in volume slowly during the growth phase of the ciliate and rapidly when the ciliate begins to divide. the hydrogenosomes divide mainly during the initial phases of cell division while the methanogens divide synchronously during the last phase of ciliate division. the timing of reproduction of the symbionts is controlled by the host-cell cycle. the ciliate is known to receive an energetic advantage from its symbionts. the suppression of continuous bacterial reproduction may trigger the secretion of excess bacterial production as soluble organic compounds, for use by the ciliate.     

The microplankton organisms at the oxic-anoxic interface in the pelagial of the Black Sea

Abstract The semi-permanent anoxia in the deep-waters of the Black Sea supported the formation of a unique microplankton community at the oxicanoxic interface. One group of ciliates ( Pleuronema marinum, Askenasia sp., species of the families Tracheliidae, Holophryidae and Amphileptidae) inhabited the water layer just above the upper boundary of H₂S, and was spatially associated with and fed on large colorless sulphur bacteria ( Thiovulum spp.). Other ciliates (species of order Scuticociliatida) populated the upper layer of the H₂S zone. A significant part of them possessed ectosymbiotic bacteria. Since the metazoans were not found in the O₂ and H₂S boundary layer, the protist community is considered to be the main factor in utilization of chemotrophic bacterial production.     

Cultivation of the sapropelic ciliate Plagiopyla nasuta Stein and isolation of the endosymbiont Methanobacterium formicicum

The sapropelic ciliate Plagiopyla nasuta was isolated and cultured in monoculture. Optimal conditions for growth were: 15–20°C, pH about 7, and about 2% of oxygen in the headspace. Cultures of P. nasuta produced methane. Epifluorescence microscopy revealed the presence of methanogenic bacteria as endosymbionts. An endosymbiont of the ciliate was isolated and identified as Methanobacterium formicicum. In the ciliate cell these methanogens were found to be closely associated with microbody-like organelles. No mitochondria could be detected.     

Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost

Abstract During the first stage of the preparation of mushroom compost oxygen is believed to be readily available. However we measured methane in the evoking air above the compost piles and were able to isolate thermophilic methanogenic bacteria from this compost. The isolates grow only on H₂ and CO₂ as energy and carbon source and do not require complex factors for growth. On the basis of nutritional and morphological characteristics these methanogens were identified as strains of Methanobacterium thermoautotrophicum .     

Isolation and characterization of refuse methanogens

Four mesophilic, irregular, rod-shaped methanogenic bacteria were isolated from decomposing refuse recovered from laboratory-scale reactors and a municipal solid waste landfill. H₂/CO₂ was the only substrate on which the isolates could grow in a complex medium. Isolates grew between either 25° or 30° and 45°C and between pH 6 and 8. One isolate exhibited growth at pH 5. Growth of each isolate was enhanced by yeast extract and inhibited by anaerobic sewage sludge supernatant fluid. No isolate showed greater than 25% lysis on exposure to 1% sodium dodecyl sulphate (SDS) for 24 h, as is typical of methanogens with a proteinaceous cell wall. The physiological traits of the methanogens isolated here are similar to many previously characterized isolates.