α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Metabolic influence of core ciliates within the rumen microbiome.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

Bioactive molecules from ciliates: Structure, activity, and applicative potential.

Ciliates are a rich source of molecules synthesized to socialize, compete ecologically, and interact with prey and predators. Their isolation from laboratory cultures is often straightforward, permitting the study of their mechanisms of action and their assessment for applied research. This review focuses on three classes of these bioactive molecules: (i) water-borne, cysteine-rich proteins that are used as signaling pheromones in self/nonself recognition phenomena; (ii) cell membrane-associated lipophilic terpenoids that are used in interspecies competitions for habitat colonization; (iii) cortical granule-associated molecules of various chemical nature that primarily serve offence/defense functions.

Early signaling pathways mediating dormant cyst formation in terrestrial unicellular eukaryote Colpoda.

Dormant (resting) cyst formation (encystment) in unicellular eukaryotes is the process of a large-scale digestion of vegetative cell structures and reconstruction into the dormant form, which is performed by cell signaling pathways accompanied by up- or down-regulation of protein expression, and by posttranslational modification such as phosphorylation. In this review, the author describes the morphogenetic events during encystment of Colpoda and the early molecular events in the Ca2+/calmodulin-triggered signaling pathways for encystment, based mainly on our research results of the past 10 years; especially, the author discusses the role of c-AMP dependently phosphorylated proteins (ribosomal P0 protein, ribosomal S5 protein, Rieske iron-sulfur protein, actin and histone H4) and encystment-dependently upregulated (EF-1α-HSP60, actin-related protein) and downregulated proteins (ATP synthase ß-chain). In addition, the roles of AMPK, a key molecule in the signaling pathways leading to Colpoda encystment, and differentially expressed genes and proteins during encystment of other ciliates are discussed.

Anaerobic endosymbiont generates energy for ciliate host by denitrification.

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe 'Candidatus Azoamicus ciliaticola', which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. 'Candidatus A. ciliaticola' contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron-sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. 'Candidatus A. ciliaticola' and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.

Preliminary Data Related to the Effect of Climacostol Produced by the Freshwater Ciliate Climacostomum virens on Human Adenovirus.

The new epidemiological scenario has so far focused on the environmental circulation of human viral pathogens. Owing to the side effects of chemical disinfectants, there is an increasing need for knowledge on the use of virucidal compounds, especially those of a natural origin. Climacostol is a molecule produced by a freshwater ciliate and it exhibits activity against bacterial and fungal pathogens. We thus also speculated that there might be an effect on viral viability, which has never been tested. To evaluate such activity, we chose human adenovirus (HAdV), which is representative of waterborne viruses. We conducted experiments using HAdV serotype 5, whose titer was determined by infecting HeLa cell cultures. HAdV5 was shown to be sensitive to climacostol at a concentration of 0.0002 mg/mL, with an approximate 3 Log10 reduction when the initial titer of HAdV5 was approximately 104 and 103 TCID50/mL. These preliminary results could be an important starting point for further research aimed at improving the characterization of climacostol activity under different experimental conditions and against various viruses, including enveloped ones (i.e., the coronavirus). The production of climacostol by a protist living in fresh water also suggests a possible application in the activated sludge of wastewater treatment plants.

Novel insights into molecular mechanisms of Pseudourostyla cristata encystment using comparative transcriptomics.

The encystment of many ciliates is an advanced survival strategy against adversity and the most important reason for ciliates existence worldwide. However, the molecular mechanism for the encystment of free-living ciliates is poorly understood. Here, we performed comparative transcriptomic analysis of dormant cysts and trophonts from Pseudourostyla cristata using transcriptomics, qRT-PCR and bioinformatic techniques. We identified 2565 differentially expressed unigenes between the dormant cysts and the trophonts. The total number of differentially expressed genes in GO database was 1752. The differential unigenes noted to the GO terms were 1993. These differential categories were mainly related to polyamine transport, pectin decomposition, cytoplasmic translation, ribosome, respiratory chain, ribosome structure, ion channel activity, and RNA ligation. A total of 224 different pathways were mapped. Among them, 184 pathways were upregulated, while 162 were downregulated. Further investigation showed that the calcium and AMPK signaling pathway had important induction effects on the encystment. In addition, FOXO and ubiquitin-mediated proteolysis signaling pathway jointly regulated the encystment. Based on these findings, we propose a hypothetical signaling network that regulates Pseudourostyla cristata encystment. Overall, these results provide deeper insights into the molecular mechanisms of ciliates encystment and adaptation to adverse environments.

Selection for tandem stop codons in ciliate species with reassigned stop codons.

The failure of mRNA translation machinery to recognize a stop codon as a termination signal and subsequent translation of the 3' untranslated region (UTR) is referred to as stop codon readthrough, the frequency of which is related to the length, composition, and structure of mRNA sequences downstream of end-of-gene stop codons. Secondary in-frame stop codons within a few positions downstream of the primary stop codons, so-called tandem stop codons (TSCs), serve as backup termination signals, which limit the effects of readthrough: polypeptide product degradation, mislocalization, and aggregation. In this study, ciliate species with UAA and UAG stop codons reassigned to code for glutamine are found to possess statistical excesses of TSCs at the beginning of their 3' UTRs. The overrepresentation of TSCs in these species is greater than that observed in standard code organisms. Though the overall numbers of TSCs are lower in most species with alternative stop codons because they use fewer than three unique stop codons, the relatively great overrepresentation of TSCs in alternative-code ciliate species suggests that there exist stronger selective pressures to maintain TSCs in these organisms compared to standard code organisms.

Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 µm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Response of Anaerobic Protozoa to Oxygen Tension in Anaerobic System.

The effect of oxygen on anaerobic protozoa was studied in anaerobic batch reactors inoculated with sludge and protozoa cultures. Among the protozoa genera, Metopus, Brachonella, Plagiopyla, Trepomonas, and Vanella were more sensitive to oxygen compared to other genera. Protozoa genera Menoidium, Rhynchomonas, Cyclidium, Spathidium, and Amoeba were found to survive under aerobic conditions, and the growth rate was slightly higher or similar to anaerobic condition. O2 tension resulted in the loss of free and endosymbiotic methanogens in anaerobic system, while methanogens were observed inside the protozoan cysts. Survival of anaerobic protozoa declined considerably when the O2 tension exceeded 1% atm. sat. and showed chemosensory behavior in response to O2 exposure. Superoxide dismutase activity was detected in survived protozoa cells under O2 tension. Facultative anaerobic protozoa with SOD activity can provide a mechanism to overcome possible occurrence of oxygen toxicity in the treatment of wastewater in anaerobic reactor.

Glycine Betaine and Ectoine Are the Major Compatible Solutes Used by Four Different Halophilic Heterotrophic Ciliates.

One decisive factor controlling the distribution of organisms in their natural habitats is the cellular response to environmental factors. Compared to prokaryotes, our knowledge about salt adaptation strategies of microbial eukaryotes is very limited. We, here, used a recently introduced approach (implementing proton nuclear magnetic resonance spectroscopy) to investigate the presence of compatible solutes in halophilic, heterotrophic ciliates. Therefore, we isolated four ciliates from solar salterns, which were identified as Cyclidium glaucoma, Euplotes sp., Fabrea salina, and Pseudocohnilembus persalinus based on their 18S rRNA gene signatures and electron microscopy. The results of 1H-NMR spectroscopy revealed that all four ciliates employ the "low-salt-in" strategy by accumulating glycine betaine and ectoine as main osmoprotectants. We recorded a linear increase of these compatible solutes with increasing salinity of the external medium. Ectoine in particular stands out as its use as compatible solute was thought to be exclusive to prokaryotes. However, our findings and those recently made on two other heterotroph species call for a re-evaluation of this notion. The observation of varying relative proportions of compatible solutes within the four ciliates points to slight differences in haloadaptive strategies by regulatory action of the ciliates. Based on this finding, we provide an explanatory hypothesis for the distribution of protistan diversity along salinity gradients.

Transcriptional profiling of antioxidant defense system and heat shock protein (Hsp) families in the cadmium- and copper-exposed marine ciliate Euplotes crassu.

To understand the transcriptional response of antioxidant defense system and heat shock protein (Hsp) families of the marine ciliate Euplotes crassus, we analyzed the transcriptome profile using RNA-seq technology after exposure to cadmium (Cd) and copper (Cu). De novo sequence assembly produced 61,240 unigenes with 21,330 BLAST hits and showed high sequence orthology with transcriptomes of other ciliates. Gene annotation and gene ontology (GO) comparison revealed that E. crassus expressed highly diversified but conserved stress-responsive gene families of the antioxidant defense system and Hsps. After waterborne exposure to 250 µg/L of Cd and 25 µg/L of Cu, transcriptional responses of the gene families were significantly modulated, suggesting that even the unicellular E. crassus has a conserved molecular defense mechanism, such as modulating mRNA expression, for homeostasis. These transcriptional responses make E. crassus a potential model for understanding the molecular response of single cell ciliates to heavy metal contamination.

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum.

Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result in an increase in cytoplasmic sodium concentration in S. salinarum. This, together with the decrease in the catalytic activities of MDH and ICDH at high salt concentration, demonstrates that S. salinarum employs the salt-out strategy for haloadaptation.

Resting Cysts of the Pigmented Ciliate Blepharisma sinuosum Sawaya, 1940 (Ciliophora: Heterotrichea).

The morphology of Blepharisma sinuosum resting cysts and the dynamics of pigmentation at different stages of encystment are presented for the first time. Cyst morphometrics are similar to other Blepharisma species, with three-wall layers, bacteria surrounding the ectocyst, a conical plug, and wrinkly surface toward the plug in mature stages. The vegetative moniliform macronucleus changes to a horseshoe shape, and at early stages, the cystic cytoplasm is homogeneously pigmented, comprising a contractile vacuole; later, pigments polarize toward the plug, decorate the cortical layer, and become brownish. This work reinforces the potential role of pigment dynamics on cyst biology.

The Green Tetrahymena utriculariae n. sp. (Ciliophora, Oligohymenophorea) with Its Endosymbiotic Algae (Micractinium sp.), Living in Traps of a Carnivorous Aquatic Plant.

The genus Tetrahymena (Ciliophora, Oligohymenophorea) probably represents the best studied ciliate genus. At present, more than forty species have been described. All are colorless, i.e. they do not harbor symbiotic algae, and as aerobes they need at least microaerobic habitats. Here, we present the morphological and molecular description of the first green representative, Tetrahymena utriculariae n. sp., living in symbiosis with endosymbiotic algae identified as Micractinium sp. (Chlorophyta). The full life cycle of the ciliate species is documented, including trophonts and theronts, conjugating cells, resting cysts and dividers. This species has been discovered in an exotic habitat, namely in traps of the carnivorous aquatic plant Utricularia reflexa (originating from Okavango Delta, Botswana). Green ciliates live as commensals of the plant in this anoxic habitat. Ciliates are bacterivorous, however, symbiosis with algae is needed to satisfy cell metabolism but also to gain oxygen from symbionts. When ciliates are cultivated outside their natural habitat under aerobic conditions and fed with saturating bacterial food, they gradually become aposymbiotic. Based on phylogenetic analyses of 18S rRNA and mitochondrial cox1 genes T. utriculariae forms a sister group to Tetrahymena thermophila.

Natural variation in methane emission of sheep fed on a lucerne pellet diet is unrelated to rumen ciliate community type.

Only limited information is available on the roles of different rumen ciliate community types, first described by Eadie in 1962, in enteric methane (CH4) formation by their ruminant hosts. If the different types were differentially associated with CH4 formation, then ciliate community typing could be used to identify naturally high and low CH4-emitting animals. Here we measured the CH4 yields [g CH4 (kg feed dry matter intake, DMI)(-1)] of 118 sheep fed a standard pelleted lucerne diet at two different times, at least 2 weeks apart. There were significant differences (P < 2.2 × 10(-16), Wilcoxon rank sum test) in the CH4 yields (± sd) from sheep selected as high [16.7 ± 1.5 g CH4 (kg DMI)(-1)] and low emitters [13.3 ± 1.5 g CH4 (kg DMI)(-1)]. A rumen sample was collected after each of the two measurements, and ciliate composition was analysed using barcoded 454 Titanium pyrosequencing of 18S rRNA genes. The genera found, in order of mean relative abundance, were Epidinium, Entodinium, Dasytricha, Eudiplodinium, Polyplastron, Isotricha and Anoplodinium-Diplodinium, none of which was significantly correlated with the CH4 emissions ranking associated with the rumen sample. Ciliate communities naturally assembled into four types (A, AB, B and O), characterized by the presence and absence of key genera. There was no difference in CH4 yield between sheep that harboured different ciliate community types, suggesting that these did not underlie the natural variation in CH4 yields. Further research is needed to unravel the nature of interactions between ciliate protozoa and other rumen micro-organisms, which may ultimately lead to contrasting CH4 emission phenotypes.

Evolution of the Dynein Heavy Chain Family in Ciliates.

Dynein heavy chains are motor proteins that comprise a large gene family found across eukaryotes. We have investigated this gene family in four ciliate species: Ichthyophthirius, Oxytricha, Paramecium, and Tetrahymena. Ciliates appear to encode more dynein heavy chain genes than most eukaryotes. Phylogenetic comparisons demonstrated that the last common ancestor of the ciliates that were examined expressed at least 14 types of dynein heavy chains with most of the expansion coming from the single-headed inner arm dyneins. Each of the dyneins most likely performed different functions within the cell.

Large-scale identification of encystment-related proteins and genes in Pseudourostyla cristata.

The transformation of a ciliate into cyst is an advance strategy against an adverse situation. However, the molecular mechanism for the encystation of free-living ciliates is poorly understood. A large-scale identification of the encystment-related proteins and genes in ciliate would provide us with deeper insights into the molecular mechanisms for the encystations of ciliate. We identified the encystment-related proteins and genes in Pseudourostyla cristata with shotgun LC-MS/MS and scale qRT-PCR, respectively, in this report. A total of 668 proteins were detected in the resting cysts, 102 of these proteins were high credible proteins, whereas 88 high credible proteins of the 724 total proteins were found in the vegetative cells. Compared with the vegetative cell, 6 specific proteins were found in the resting cyst. However, the majority of high credible proteins in the resting cyst and the vegetative cell were co-expressed. We compared 47 genes of the co-expressed proteins with known functions in both the cyst and the vegetative cell using scale qRT-PCR. Twenty-seven of 47 genes were differentially expressed in the cyst compared with the vegetative cell. In our identifications, many uncharacterized proteins were also found. These results will help reveal the molecular mechanism for the formation of cyst in ciliates.

Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans.

Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and α-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit α, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while ß-tubulin, V-type ATPase α subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, ß-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75 kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit α, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development.

The role of microorganisms in a full-scale sequencing batch reactor under low aeration and different cycle times.

This study describes the role of microorganisms in a full-scale step-feed sequencing batch reactor (SBR) system for urban wastewater treatment. Chemical profiles for three different cycle times were measured under low aeration conditions with a high carbon-to-nitrogen ratio. The applied organic load was above 1.0 g chemical oxygen demand (COD)/L x d. The removal efficiencies were higher than 81%, 93%, and 76% for soluble COD, N-NH4+, and total Kjeldahl nitrogen, respectively. The ratio of volatile suspended solids (VSS) to total suspended solids was 78%, and the food-to-microorganism ratio was an average of 1.41 g COD/g VSS x d. The active biomass was comprised of 87.8% heterotrophic and 12.2% autotrophic organisms. Nitrifying organisms were found with a low amount of ammonia-oxidizing bacteria (5%) and a much higher amount of nitrite-oxidizing bacteria. Polyphosphate-accumulating organisms (PAOs) were found at high amounts (25%) compared to glycogen-accumulating organisms, even in a system with a high carbon to phosphorus ratio. The activity of denitrifying PAOs was 72%.