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.

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.

Soil Protists Can Actively Redistribute Beneficial Bacteria along Medicago truncatula Roots.

The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria: all play significant roles in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a bioavailable form. In soil, S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system, able to travel quickly along roots and water films, who prey on soil bacteria and have been known to egest undigested phagosomes. We show that a soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. Using model soil microcosms, we directly observed fluorescently labeled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal extended 52 mm farther down plant roots when Colpoda sp. was also present versus treatments that contained bacteria but not protists. Direct counts also showed protists are required for viable bacteria to reach the deeper sections of our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. IMPORTANCE Soil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here, we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen-fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

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.

The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment.

Entodinium caudatum is an anaerobic binucleated ciliate representing the most dominant protozoal species in the rumen. However, its biological features are largely unknown due to the inability to establish an axenic culture. In this study, we primally sequenced its macronucleus (MAC) genome to aid the understanding of its metabolism, physiology, ecology. We isolated the MAC of E. caudatum strain MZG-1 and sequenced the MAC genome using Illumina MiSeq, MinION, and PacBio RSII systems. De novo assembly of the MiSeq sequence reads followed with subsequent scaffolding with MinION and PacBio reads resulted in a draft MAC genome about 117 Mbp. A large number of carbohydrate-active enzymes were likely acquired through horizontal gene transfer. About 8.74% of the E. caudatum predicted proteome was predicted as proteases. The MAC genome of E. caudatum will help better understand its important roles in rumen carbohydrate metabolism, and interaction with other members of the rumen microbiome.

On the power per mitochondrion and the number of associated active ATP synthases.

Recent experiments and thermodynamic arguments suggest that mitochondrial temperatures are higher than those of the cytoplasm. A "hot mitochondrion" calls for a closer examination of the energy balance that endows it with these claimed elevated temperatures. As a first step in this effort, we present here a semi-quantitative bookkeeping whereby, in one stroke, a formula is proposed that yields the rate of heat production in a typical mitochondrion and a formula for estimating the number of "active" ATP synthase molecules per mitochondrion. The number of active ATP synthase molecules is the equivalent number of ATP synthases operating at 100% capacity to maintain the rate of mitochondrial heat generation. Scaling laws are shown to determine the number of active ATP synthase molecules in a mitochondrion and mitochondrial rate of heat production, whereby both appear to scale with cell volume. Four heterotrophic protozoan cell types are considered in this study. The studied cells, selected to cover a wide range of sizes (volumes) fromca.100µm3to 1 millionµm3, are estimated to exhibit a power per mitochondrion ranging fromca.1 pW to 0.03 pW. In these cells, the corresponding number of active ATP synthases per mitochondrion ranges from 5000 to just about a hundred. The absolute total number of ATP synthase molecules per mitochondrion, regardless of their activity status, can be up to two orders of magnitudes higher.

Barcode sequence could be a good target for developing a species-specific anti-parasite agent based on CRISPR-Cas9.

Parasitic infections are a severe issue in many regions of the world. We assume that if a chemical can destroy a DNA barcode sequence, then this chemical could be developed as a species-specific parasiticidal agent. To test this hypothesis, we designed sgRNAs that target the sequences of both a DNA barcode (ITS-2) and a control (5.8S rDNA) in Cryptocaryon irritans. In in vivo tests, we found that exposure to Cas9 mRNA mixed with sgRNAs was able to significantly reduce the hatching rate of tomont and the survival rate of theront. Quantitative Real-time PCR demonstrated that the DNAs of tomont and theront exposed to sgRNAs and Cas9 mRNA were significantly disrupted, no matter whether they were exposed to a single sgRNA or a mixture of two sgRNAs. DNA sequencing also suggested the test group that was exposed to a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments and the test group exposed to two sgRNAs combined with Cas9-induced deletion of large pieces. The findings and principles provided by this study contribute to the development of novel nucleic acid therapeutic drugs for cryptocaryoniasis and other parasitic diseases and provide insight into the development of species-specific parasiticidal agents.

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.

Scripting a place in time.

Evidence in many experimental systems supports the idea that non-uniform distributions of morphogen proteins encode positional information in developing tissues. There is also strong evidence that morphogen dispersal is mediated by cytonemes and that morphogen proteins transfer from producing to receiving cells at morphogenetic synapses that form at sites of cytoneme contacts. This essay considers some implications of this mechanism and its relevance to various contexts including large single cells such as the pre-cellular Drosophila embryo and the ciliate Stentor.

Hysteretic response of Microbial Eukaryotic Communities to Gradually Decreased Nutrient Concentrations in Eutrophic Water.

External environments to microbial eukaryotic communities often change gradually with time. However, whether the responses of microbial eukaryotic communities to these gradually changed environments are continuous or hysteretic and the mechanisms underlying these responses are largely unknown. Here, we used a microcosm to investigate the temporal variation of microbial eukaryotic communities with the gradually decreased nutrient concentrations (nitrogen and phosphorus). We found the differences of microbial eukaryotic community composition and species richness between the control and treatment groups were low during the days 0 to 12, although the nutrient concentrations decreased rapidly during this period in treatment group. However, these differences were clear during the days 14 to 18, although the nutrient concentrations decreased slowly during this period in treatment group. The mechanisms for these results are that the strong homogenous selection (perhaps due to the biotic factors) during the days 8 to 10 in treatment group might enhance the stability of microbial eukaryotic communities. However, the continuously decreased nutrient concentrations weakened the homogenous selection and promoted the strength of environmental filtering, and therefore resulted in the distinct change of microbial eukaryotic communities during the days 14 to 18 in treatment group. Fungi, Chlorophyta and Chrysophyta which associated with the nutrient removal played important roles in this hysteretic change of microbial eukaryotic communities. Overall, our findings suggest that disentangling the non-linear response of communities to gradual environmental changes is essential for understanding ecosystem restoration and degradation in future.

DNA elimination in ciliates: transposon domestication and genome surveillance.

Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.

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.

Expression and molecular characterization of stress-responsive genes (hsp70 and Mn-sod) and evaluation of antioxidant enzymes (CAT and GPx) in heavy metal exposed freshwater ciliate, Tetmemena sp.

Response of heavy metals namely cadmium (Cd) and copper (Cu) on the expression of stress responsive genes in the fresh water ciliate, Tetmemena sp. (single cell eukaryote) was studied. Stress responsive genes include heat shock protein genes and genes involved in antioxidant defence system. Quantitative real time PCR (qRT-PCR) was employed to evaluate the effects of Cd and Cu on the expression of cytosolic hsp70 and Mn-sod genes. Increase in the expression of these genes was observed after exposure with the heavy metals. The macronuclear cytosolic hsp70 and Mn-sod (SOD2) genes were also sequenced and characterized using various bioinformatics tools. In antioxidant defence system, the superoxide dismutase (SOD) family is a first line antioxidant enzyme group involved in catalysing reactive oxygen species (ROS) to hydrogen peroxide and molecular oxygen. Influence of Cd and Cu on the activity of SOD has already been reported by our group. Therefore, the enzymatic activities of antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx) were studied in the presence of Cd and Cu and there was significant increase in activity of these enzymes in concentration dependent manner. This study suggests that cytosolic hsp70, Mn-sod and the antioxidant enzymes such as CAT and GPx can be used as effective molecular biomarkers for heavy metal toxicity and Tetmemena sp. can be used as potential model for understanding the molecular response to heavy metal contamination in aquatic ecosystems.

Rumen microbial responses to supplemental nitrate. I. Yeast growth and protozoal chemotaxis in vitro as affected by nitrate and nitrite concentrations.

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH4 emissions. In the NO3- reduction process, NO2- can accumulate, which could directly inhibit methanogens and some bacteria. However, little information is available on eukaryotic microbes in the rumen. Protozoa were hypothesized to enhance nitrate reductase but also have more circling swimming behavior, and the yeast Saccharomyces cerevisiae was hypothesized to lessen NO2- accumulation. In the first experiment, a culture of S. cerevisiae strain 1026 was evaluated under 3 growth phases: aerobic, anoxic, or transition to anoxic culture. Each phase was evaluated with a control or 1 of 3 isonitrogenous doses, including NO3-, NO2-, or NH4+ replacing peptone in the medium. Gas head phase, NO3-, or NH4+ did not influence culture growth, but increasing NO2- concentration increasingly inhibited yeast growth. In experiment 2, rumen fluid was harvested and incubated for 3 h in 2 concentrations of NO3-, NO2-, or sodium nitroprusside before assessing chemotaxis of protozoa toward glucose or peptides. Increasing NO2- concentration decreased chemotaxis by isotrichids toward glucose or peptides and decreased chemotaxis by entodiniomorphids but only toward peptides. Live yeast culture was inhibited dose-responsively by NO2- and does not seem to be a viable mechanism to prevent NO2- accumulation in the rumen, whereas a role for protozoal nitrate reductase and NO2- influencing signal transduction requires further research.

Identifying protist consumers of photosynthetic picoeukaryotes in the surface ocean using stable isotope probing.

Photosynthetic picoeukaryotes contribute a significant fraction of primary production in the upper ocean. Micromonas pusilla is an ecologically relevant photosynthetic picoeukaryote, abundantly and widely distributed in marine waters. Grazing by protists may control the abundance of picoeukaryotes such as M. pusilla, but the diversity of the responsible grazers is poorly understood. To identify protists consuming photosynthetic picoeukaryotes in a productive North Pacific Ocean region, we amended seawater with living 15 N, 13 C-labelled M. pusilla cells in a 24-h replicated bottle experiment. DNA stable isotope probing, combined with high-throughput sequencing of V4 hypervariable regions from 18S rRNA gene amplicons (Tag-SIP), identified 19 operational taxonomic units (OTUs) of microbial eukaryotes that consumed M. pusilla. These OTUs were distantly related to cultured taxa within the dinoflagellates, ciliates, stramenopiles (MAST-1C and MAST-3 clades) and Telonema flagellates, thus, far known only from their environmental 18S rRNA gene sequences. Our discovery of eukaryotic prey consumption by MAST cells confirms that their trophic role in marine microbial food webs includes grazing upon picoeukaryotes. Our study provides new experimental evidence directly linking the genetic identity of diverse uncultivated microbial eukaryotes to the consumption of picoeukaryotic phytoplankton in the upper ocean.

Insights into the metabolic functioning of a multipartner ciliate symbiosis from oxygen-depleted sediments.

Symbioses between anaerobic or microaerophilic protists and prokaryotes are common in anoxic and oxygen-depleted habitats ranging from marine sediments to gastrointestinal tracts. Nevertheless, little is known about the mechanisms of metabolic interaction between partners. In these putatively syntrophic associations, consumption of fermentative end products (e.g., hydrogen) by the prokaryotic symbionts is thought to facilitate protistan anaerobic metabolism. Here, we employed metagenomic and metatranscriptomic sequencing of a microaerophilic or anaerobic karyorelictid ciliate and its prokaryotic symbionts from oxygen-depleted Santa Barbara Basin (CA, USA) sediments to assess metabolic coupling within this consortium. This sequencing confirmed the predominance of deltaproteobacterial symbionts from the Families Desulfobacteraceae and Desulfobulbaceae and suggested active symbiont reduction of host-provided sulphate, transfer of small organic molecules from host to symbionts and hydrogen cycling among the symbionts. In addition, patterns of gene expression indicated active cell division by the symbionts, their growth via autotrophic processes and nitrogen exchange with the ciliate host. Altogether, this research underscores the importance of symbiont metabolism to host fermentative metabolism and, thus, likely its success in anoxic and low-oxygen habitats, but also suggests ciliate-associated prokaryotes play a role in important biogeochemical processes.

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.

Tracking a century of changes in microbial eukaryotic diversity in lakes driven by nutrient enrichment and climate warming.

High-throughput sequencing of sedimentary DNA (sed-DNA) was utilized to reconstruct the temporal dynamics of microbial eukaryotic communities (MECs) at a centennial scale in two re-oligotrophicated lakes that were exposed to different levels of phosphorus enrichment. The temporal changes within the MECs were expressed in terms of richness, composition and community structure to investigate their relationships with two key forcing factors (i.e., nutrient enrichment and climate warming). Various groups, including Apicomplexa, Cercozoa, Chrysophyceae, Ciliophora, Chlorophyceae and Dinophyceae, responded to phosphorus enrichment levels with either positive or negative impacts on their richness and relative abundance. For both lakes, statistical modelling demonstrated that phosphorus concentration ([P]) was a dominant contributor to MECs modifications before the 1980s; after the mid-80s, the contribution of air temperature changes increased and potentially surpassed the contribution of [P]. Co-occurrence network analysis revealed that some clusters of taxa (i.e., modules) composed mainly of Dinophyceae and unclassified Alveolata were strongly correlated to air temperature in both lakes. Overall, our data showed that sed-DNA constitutes a precious archive of information on past biodiversity changes, allowing the study of the dynamics of numerous eukaryotic groups that were not traditionally considered in paleo-reconstructions.

The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.