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).     

Endosymbiotic Methanogenic Bacteria In Anaerobic Ciliates: Significance For the Growth Efficiency of the Host

ABSTRACT Endosymbiotic methanogenic bacteria of three species of anaerobic ciliates (Plagiopyla frontata, Metopus conforms , and M. palaeformis) were inactivated with the specific methanogen inhibitor 2-bromoethanesulfonic acid. the absence of endosymbiont methanogens reduced growth rate and growth yield by about 30% in P. frontata and M. contortus , while no significant change in fitness was observed in M. palaeformis. In Plagiopyla the growth rate constant is not affected by an artificially increased pH₂ neither in normal nor in methanogen-free ciliates. the energetic advantage conferred by endosymbiont methanogens in Plagiopyla and in Metopus contortus probably is due to excretion of organic material from the bacteria at the expense of bacterial reproduction. It is unlikely that the maintenance of a low pH₂ within the cells due to H₂-consumption by the bacteria is important to the ciliates.     

Metopus, Cyclidium and Sonderia: ciliates enriched and cultured from sulfureta of a microbial mat community

Metopus contortus (Class Spirotrichea), Cyclidium borrori, Cyclidium sp., Sonderia tubigula, and two other species of Sonderia (Class Oligohymenophorea) were enriched from the sulfureta of microbial mat communities at Laguna Figueroa near San Quintin, Baja California, Mexico. The ability of some of these species to grow under anaerobic, high-sulfide conditions was demonstrated. The ultrastructural morphology of Metopus and Cyclidium revealed adaptations to anaerobic, high sulfide niches. Metopus lacks typical mitochondria and has structures which may be intracellular bacterial symbionts. Cyclidium borrori, when grown aerobically, has mitochondria with tubular cristae. When it is grown anaerobically in the presence of high amounts of hydrogen sulfide the number of mitochondria remains the same but the mitochondria dedifferentiate, becoming smaller with less well-defined cristae.     

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.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

RNA sequence analysis shows that the symbionts in the ciliate Metopus contortus are polymorphs of a single methanogen species

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.     

Recent developments inRhizobium taxonomy

Recent developments inRhizobium taxonomy are presented from a molecular and evolutionary point of view. Analyses of ribosomal RNA gene sequences provide a solid basis to infer phylogenies in the Rhizobiaceae family. These studies confirmed thatRhizobium andBradyrhizobium are only distantly related and showed thatRhizobium andBradyrhizobium are related to other groups of bacteria that are not plant symbionts.Rhizobium andAgrobacterium species are intermixed. Differences in plasmid content may explain to a good extent the different behavior ofRhizobium andAgrobacterium as symbionts or pathogens. Other approaches to identify and classify bacteria such as DNA-DNA hybridization, fatty acid analysis, RFLP and RPD-PCR techniques and phylogenies derived from other genes are in general agreement to the groupings derived by ribosomal sequences. Only a small proportion of nodulated legumes have been sampled for their symbionts and more knowledge is required on the systematics and taxonomy ofRhizobium andBradyrhizobium species.     

Phylogenetic Analysis and Fluorescence <Emphasis Type="Italic">In Situ</Emphasis> Hybridization Detection of Archaeal and Bacterial Endosymbionts in the Anaerobic Ciliate <Emphasis Type="Italic">Trimyema Compressum</Emphasis>

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 μg 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.     

Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora,Metopida), with Identification of the Intracellular Bacteria

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18–21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21–29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion‐resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).     

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.     

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.      

Methanogenic symbionts of anaerobic ciliates and their contribution to methanogenesis in an anoxic rice field soil

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.     

Association of methanogenic bacteria with flagellated protozoa from a termite hindgut

Epifluorescence microscopy was used to examine hindgut contents ofZootermopsis angusticollis (Hagen) termites for the presence of methanogenic bacteria, which can be identified on the basis of the fluorescence of the novel cofactors F₄₂₀ and F₃₅₀. Small, autofluorescent, rod-shaped bacteria of theMethanobrevibacter sp. morphotype were observed associated with three flagellates tentatively identified asTrichomitopsis termopsidis (Cleveland),Tricercomitus termopsidis Kirby andHexamastix termopsidis Kirby. Methanogens were not observed associated with any other protozoal morphotypes and were not numerous in the free-living state inZ. angusticollis hindgut fluid. Electron micrographs of thin sections of hindgut protozoa suggest methanogens are endosymbionts in the small trichomonad protozoa. Our observations are consistent with the finding of Odelson and Breznak that methane is a minor endproduct of the metabolism of termite gut microbiota.Deceased.     

Bacterial symbionts as mediators of ecologically important traits of insect hosts

1. Bacterial symbionts play a prominent role in insect nutritional ecology by aiding in digestion of food or providing nutrients that are limited or lacking in the diet. Thereby, endosymbionts open niches to their insect host that would otherwise be unavailable. 2. Currently, several other ecologically relevant traits mediated by endosymbionts are being investigated, including enhanced parasite resistance, enhanced heat tolerance, and influences on insect–plant interactions such as manipulation of plant physiology to the benefit of the insect. 3. Traits mediated by endosymbionts are often identified by correlative studies where traits are found to be altered in the presence of a particular symbiont. Recent developments in genomic tools offer the opportunity for studying the impact of bacteria–insect symbioses under natural conditions in a population and community ecology context. In vivo experiments specifically testing putative functions of endosymbionts in parallel to population‐level studies on the prevalence of endosymbionts allow disentangling host versus symbiont contribution to phenotypic variability observed in individuals. Effects of symbionts on host phenotype are often large and relevant to host fitness, e.g. by significantly enhancing survival or fecundity in a context‐dependent manner. 4. Predominantly vertically transmitted endosymbionts contribute to the heritable genetic variation present in a host species. Phenotypic variation on which selection can act may be due to differences either among host genomes, symbiont genomes, or genotype × genotype interactions. Therefore the holobiont, i.e. the host including all symbionts, should be regarded as the unit of selection as the association between host and symbionts may affect the fitness of the holobiont depending on the environment.     

The eubacterial endosymbionts of whiteflies (homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs

Whiteflies (superfamily Aleyrodoidea) contain eubacterial endosymbionts localized within host cells known as mycetocytes. Sequence analysis of the genes for the 16S rRNA of the endosymbionts ofBemisia tabaci, Siphoninus phillyreae, andTrialeurodes vaporariorum indicates that these organisms are closely related and constitute a distinct lineage within the -subdivision of theProteobacteria. The endosymbionts of whiteflies are unrelated to the endosymbionts of aphids and mealybugs, which are in two separate lineages.     

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.     

Bacterial symbionts in insects: balancing life and death

Arthropods, particularly insects, form successful long-term symbioses with endosymbiotic bacteria. The associations between insects and endosymbionts are remarkably stable; many stretch back several hundred million years in evolutionary time. With the exception, perhaps, of the filarial nematodes no other group of metazoans shows such a proclivility for their intracellular symbionts. The identification and classification of bacterial symbionts and hosts has grown rapidly over the last two decades and these relationships form a continuum from classical mutualism to parasitism. Complete genomes have been sequenced for many of these bacteria and some of their hosts. Now more intractable questions regarding endosymbiosis are being addressed. Investigations on the role of the host immune system in the maintenance of symbiosis, the nature of bacteriophages and transposable elements found in the genomes of many bacterial symbionts, and the molecular mechanisms involved in establishing reproductive phenotypes such as parthenogenesis, male killing, cytoplasmic incompatibility and feminization have been recently reported. This review will focus on the impact of the secondary endosymbionts Wolbachia, Cardinium, and Spiroplasma on host fitness and immunity and will revisit the question of whether these bacteria are friend or foe from an insect’s point of view.     

Micrometeorological factors involved in development and survival of free-living stages of the sheep nematodesHaemonchus contortus andTrichostrongylus colubriformis. A review

Temperature and soil moisture are the most important factors affecting the development and survival ofHaemonchus contortus andTrichostrongylus columbriformis eggs and larvae on pasture. More than half of the eggs develop into infective larvae in the laboratory, but a very low percentage (0.03% forH. contortus) does so on pasture. There is a marked difference betweenH. contortus andT. colubriformis in survival of infective larvae.H. contortus larvae survived in the winter at Urbana poorly, whereasT. colubriformis did well. The former survived better than the latter in the spring and worse in the summer, while both survived equally well in the fall. Technics for larval recovery from pasture are not very efficient. Meteorologic conditions at ground level where the larvae exist are quite different from those in a standard weather shelter 1.6 m above the ground. Bioclimatographs in which mean monthly maximum temperatures are plotted against total monthly precipitation are fairly good in predicting the type of nematode liable to be important in a given region, but they are too simplistic to be relied on for more than approximations.     

Bacterial Endosymbioses of Gutless Tube-Dwelling Worms in Nonhydrothermal Vent Habitats

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to γ-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of α-, β-, γ-, and ε-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to β-proteobacteria. An unidentified pogonophoran from the world’s deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.     

The symbiotic performance of lupin bacteria under glasshouse and field conditions

Summary Bacteria from the nodules ofLupinus angustifolius L.,L. digitatus Forsk., andL. luteus L. have been isolated, and the symbiotic relationships of nine of these strains and the three lupin species investigated in a glasshouse experiment. All symbioses were effective and no interaction or specificity was detected among the symbionts.Soil factors modified expression of the strains' symbiotic abilities in the field, but several strains have shown successful symbiosis with all three lupin species.One lupin species,L. digitatus, nodulates freely under natural conditions in Western Australia, whereasL. luteus andL. angustifolius do not. This lupin is more readily infected by native strains of rhizobia than the others.