Endosymbiotic purple non-sulphur bacteria in an anaerobic ciliated protozoon

Abstract The marine ciliate Strombidium purpureum Kahl harbours endosymbiotic purple non-sulphur bacteria. The bacteria contain bacteriochlorophyll a and the carotenoid spirilloxanthin, and they have photosynthetic membranes and cell walls. The ciliates require light for survival and growth under anaerobic conditions; in the dark the cells prefer microaerobic conditions. The ciliates show a photosensory behaviour, and they accumulate in light at wave lenghts corresponding to the absorption spectrum of the symbionts. The findings are discussed in terms of theories on the endosymbiotic origin of mitochondria.     

Anti‐helminth‐induced changes in the prevalence of entodiniomorphid ciliates in semi‐captive chimpanzees

Ciliates are some of the most abundant gut fauna in wild chimpanzees. However, their presence in captive populations is usually low presumably due to anti‐helmintic prophylaxis or feeding on low fibre diet. We studied a semi‐captive colony of chimpanzees at the Sweetwaters Sanctuary in Kenya subject to routine prophylactic dose of albendazole to clear chimpanzees of parasitic helminths. Fresh faecal samples from known individuals were collected before and subsequently after prophylaxis. The samples were fixed in 10% formalin and examined by the sedimentation method. Troglodytella abrassarti had 42.5% prevalence whereas other ciliates had 65% prevalence. The prevalence of the T. abrassarti and other ciliates significantly declined immediately after prophylaxis and then rose slowly thereafter. Our results suggest that ciliates are susceptible to anti‐helmintic prophylaxis and that treatment may eliminate ciliates or inhibit their proliferation within the host subsequently lowering their prevalence in the population. Variation in prevalence was not influenced by the age of the host. However, a steady recovery of ciliate prevalence was lower for male compared to female hosts. Our results imply that the intervals between prophylactic regimes could be prolonged differently for males and females to increase the prevalence of ciliates in captive populations.     

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.     

Population Dynamics of Active and Total Ciliate Populations in Arable Soil Amended with Wheat

Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.     

Population dynamics of active and total ciliate populations in arable soil amended with wheat

Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.     

Doublet Formation and Cytoplasmic Symbionts in the Ciliate Urostyla grandis

The formation of homopolar doublets can be induced by the action of antibiotics which inhibit the growth of cytoplasmic bacterial symbionts whose cell cycle appears to be controlled by the host. They multiply during the macronuclear S-phase of the ciliate and become enclosed in vesicles and are largely destroyed just before cell division is completed. Since doublet formation is due to an incomplete cell division, and because experimental disturbances at the cell cortex of dividing ciliates also lead to doublets, the symbionts are thought to contribute some factor which is essential for normal cytokinesis of the host cell.     

The Molecular Biology of a Bacterial Endosymbiont1

ABSTRACT. The marine ciliate Parauronema acutum harbors a group of symbiotic bacteria, termed xenosomes, which reside exclusively in the cytoplasm where they grow and divide in a remarkable synchronism with the host. When released into the ambient culture medium by gentle rupture of the protozoans, the symbionts can infect homologous as well as heterologous Parauronema stocks and, oddly, Miamiensis avidus, a distantly related marine ciliate. Xenosomes from certain Parauronema stocks can kill other marine ciliates, particularly those of the genus Uronema. Our principal aims with this host-symbiont system have been to study, at the molecular level, the nature of the interaction of the xenosome with the host, infection and the killer effect. Our most recent investigations have been directed toward establishing the phylogenetic origins of the symbionts using molecular approaches. This paper summarizes our previous work and updates our more recent studies on the association of a bacterial symbiont with its protozoan host. The reader is referred to previous reviews for more details on the subject [4, 5].     

Ecological significance of endosymbionts in a mixotrophic ciliate--an experimental test of a simple model of growth coordination between host and symbiont

Ciliates with endosymbiotic algae (green ciliates) have oftenbeen found to be more viable than aposymbiotic (without endosymbionts)counterparts during periods of starvation. However, the possiblebenefit of algal endosymbionts to the growth of ciliate hostshas rarely been quantified. Growth coordination between hostand symbionts is essential to maintain the symbiosis, but themechanism behind this is also uncertain. Our hypothesis is thatthe growth rate of the symbionts is always close to its maximum,irrespective of the nutritional status of the host. To testthis hypothesis we built a model based on a constant symbiontgrowth rate, and performed an experiment where we observed thegrowth rate of aposymbiotic and green Coleps cells under differentlight conditions and food concentrations. The results were ingood agreement with the model, and showed that at low food concentrationthe growth rate of green Coleps was clearly higher than thatof aposymbiotic Coleps, while there were no significant differenceswhen food was abundant. Our results indicate that algal grossgrowth rate is always close to maximum, and that growth coordinationbetween host and symbiont is obtained by a variable degree ofleakage of photosynthetic products from the symbionts to theciliate host.     

Grazing protozoa and magnetosome dissolution in magnetotactic bacteria

Magnetotactic bacteria show an ability to navigate along magnetic field lines because of magnetic particles called magnetosomes. All magnetotactic bacteria are unicellular except for the multicellular prokaryote (recently named 'Candidatus Magnetoglobus multicellularis'), which is formed by an orderly assemblage of 17-40 prokaryotic cells that swim as a unit. A ciliate was used in grazing experiments with the M. multicellularis to study the fate of the magnetosomes after ingestion by the protozoa. Ciliates ingested M. multicellularis, which were located in acid vacuoles as demonstrated by confocal laser scanning microscopy. Transmission electron microscopy and X-ray microanalysis of thin-sectioned ciliates showed the presence of M. multicellularis and magnetosomes inside vacuoles in different degrees of degradation. The magnetosomes are dissolved within the acidic vacuoles of the ciliate. Depending on the rate of M. multicellularis consumption by the ciliates the iron from the magnetosomes may be recycled to the environment in a more soluble form.     

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).     

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.     

Epibiotic Bacteria on Several Ciliates from Marine Sediments

ABSTRACT Bacterial epibionts were observed on the surface of the marine sediment ciliate Geleia fossata. Rod-shaped bacteria, from 2-10 X10³ per ciliate, were universally positioned in ciliated grooves, in apparent spatial association with dikinetids. SEM and TEM examination of the ciliates confirmed that a tight affiliation exists between the epibiotic bacteria and ciliate cortex infrastructures. These observations, as well as the distinct bacterial distribution pattern over ciliate surface, suggest that there is a close epibiont/host physiological integration. Epibiotic bacteria were also observed on the surfaces of other sediment ciliates from the genera Loxophyllum, Tracheloraphis, Geleia, Paraspathidium , and Cyclidium. These findings indicate that the bacterial/protozoa associations are widespread in the marine benthic environment. The potential benefits for both epibionts and their hosts are discussed.     

Acquired phototrophy in ciliates: a review of cellular interactions and structural adaptations

Many ciliates acquire the capacity for photosynthesis through stealing plastids or harboring intact endosymbiotic algae. Both phenomena are a form of mixotrophy and are widespread among ciliates. Mixotrophic ciliates may be abundant in freshwater and marine ecosystems, sometimes making substantial contributions toward community primary productivity. While mixotrophic ciliates utilize phagotrophy to capture algal cells, their endomembrane system has evolved to partially bypass typical heterotrophic digestion pathways, enabling metabolic interaction with foreign cells or organelles. Unique adaptations may also be found in certain algal endosymbionts, facilitating establishment of symbiosis and nutritional interactions, while reducing their fitness for survival as free-living cells. Plastid retaining oligotrich ciliates possess little selectivity from which algae they sequester plastids, resulting in unstable kleptoplastids that require frequent ingestion of algal cells to replace them. Mesodinium rubrum (=Myrionecta rubra) possesses cryptophyte organelles that resemble a reduced endosymbont, and is the only ciliate capable of functional phototrophy and plastid division. Certain strains of M. rubrum may have a stable association with their cryptophyte organelles, while others need to acquire a cryptophyte nucleus through feeding. This process of stealing a nucleus, termed karyoklepty, was first described in M. rubrum and may be an evolutionary precursor to a stable, reduced endosymbiont, and perhaps eventually a tertiary plastid. The newly described Mesodinium"chamaeleon," however, is less selective of which cryptophyte species it will retain organelles, and appears less capable of sustained phototrophy. Ciliates likely stem from a phototrophic ancestry, which may explain their propensity to practice acquired phototrophy.     

Frequency and biodiversity of symbionts in representatives of the main classes of Ciliophora

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.     

From extracellular to intracellular: the establishment of a symbiosis.

The colonization of host cells by modern symbionts is surveyed. The morphological distinction between extracellular and intracellular symbionts is not sharp, and the various kinds of association can be arranged in a graded series of increasing morphological integration of the symbiont into the host cell. Apart from some aggressive parasitic infections, the great majority of symbionts are enclosed by a host membrane in a vacuole. Those not enclosed in a host vacuole usually cannot be cultivated outside the cell. It is therefore surmised that encirclement by a vacuolar membrane would only disappear, if at all, in the later stages of the evolution of intracellular symbiosis. Recognition mechanisms between host and symbiont occur, but have been little studied. In some associations, recognition at surface contact occurs, and there is evidence for the involvement of lectins in certain cases. In other associations, recognition may occur wholly or in part after the entry of symbiont into host cells. After entry, special mechanisms for the biotrophic transfer of nutrients from symbiont to host develop. Both the symbiont population size and its rate of increase are strictly regulated by the host cell; symbiont metabolism may be controlled likewise. Rates of evolution of intracellular symbionts are probably very rapid, owing in part to responses of the host cell to its symbiont.     

Radiolaria associated with large diversity of marine alveolates

We have isolated cells of unculturable radiolarians from marine coastal waters. Individual cells were subjected to single cell whole genome amplification (SCWGA) and gene-targeted PCR. Using this approach we recover a surprisingly large diversity of sequences related to the enigmatic marine alveolate groups 1 and 2 (MALV I and MALV II) that most likely represent intracellular symbionts or parasites of the radiolarian cells. 18S rDNA phylogeny of the MALV sequences reveals 4 distinct clades of radiolarian associates here named Radiolarian Associated Sequences (RAS) 1-4. One clade of both phaeodarian and radiolarian associates and one clade of only phaeodarian associates are also identified. The MALV sequences cluster according to host type, i.e. sequences from associates identified in radiolarians, fish, copepods, ciliates or dinoflagellates are not intermixed but separated into distinct clades. This implies several independent colonizations of host lineages and links a large diversity of MALV to radiolarian-associated species. This demonstrates that radiolarians may be an important reservoir for MALV, making them a key group for understanding the impact of intracellular symbionts on the marine ecosystem. This study shows that applying SCWGA on unculturable cells is a promising approach to study the vast diversity and interactions of intracellular eukaryote organisms.     

Accumulation of selenium in a model freshwater microbial food web.

The transfer of selenium between bacteria and the ciliated protozoan, Paramecium putrinum, was examined in laboratory cultures. The population growth of the ciliate was not inhibited in the presence of the highest concentrations of dissolved selenite or selenate tested (10(3) micrograms liter-1). Experiments with radioactive 75selenite or 75selenate indicated that accumulation of selenium by ciliates through time was low when feeding and metabolism were reduced by incubating at 0 degrees C. However, selenium accumulated in ciliate biomass during incubation with dissolved 75Se and bacteria at 24 degrees C and also when bacteria prelabeled with 75Se were offered as food in the absence of dissolved selenium. When 75Se-labeled bacterial food was diluted by the addition of nonradioactive bacteria, the amount of selenite and selenate in ciliates decreased over time, indicating depuration by the ciliates. In longer-term (> 5-day) fed-batch incubations with 75selenite-labeled bacteria, the selenium concentration in ciliates equilibrated at approximately 1.4 micrograms of Se g (dry weight)-1. The selenium content of ciliates was similar to that of their bacterial food on a dry-weight basis. These data indicate that selenium uptake by this ciliate occurred primarily during feeding and that biomagnification of selenium did not occur in this simple food chain.     

Predation rates of flagellate and ciliated protozoa on bacterioplankton in a river

Abstract Predation rates of flagellate and ciliate protozoa on the bacterioplankton of Butrón River (Spain) were determined from FLB (fluorescently labelled bacteria) uptake rates. Bacterial and ciliate protozoa counts were higher when higher water temperature was recorded. Flagellate counts did not show this pattern, which suggested predation of flagellates by other organisms, or some other different nutritional mode besides phagotrophy. Average individual ciliate predation rates were up to 40-times higher than those of flagellates. These results were compared with similar data obtained from other authors in several aquatic systems. However, the population predation rates of flagellate protozoa were on average 6-times higher than that of ciliate protozoa, due to the low population numbers of the latter. Thus, flagellate protozoa can be considered as more important bacterial consumers than ciliates in this aquatic system.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Identification of Intracellular Bacteria in the Ciliate Balantidium ctenopharyngodoni (Ciliophora,Litostomatea)

The ciliate Balantidium ctenopharyngodoni is the most prominent protist in the guts of grass carp, where it mainly inhabits the creamy luminal contents of the hindgut. Ciliates are generally colonized by microorganisms via phagotrophic feeding. In order to study the intracellular bacteria in this ciliate, we have successfully established it in in vitro culture. Herein, we investigated and compared the bacterial community structures of cultured and freshly collected B. ctenopharyngodoni. The results showed that these two groups exhibited different bacterial communities. The most abundant bacterial family in freshly collected samples was Enterobacteriaceae, while in cultured samples it was Fusobacteriaceae. In addition, a key intracellular bacterium, Cetobacterium somerae, was identified in the cytoplasm of cultured ciliates using fluorescence in situ hybridization (FISH). This study shows that ciliates can retain the intracellular bacteria acquired in the natural habitat for quite a long time, but the bacterial community structure of ciliates eventually changes after a long period of cultivation.