Coupling between Ribotypic and Phenotypic Traits of Protists across Life Cycle Stages and Temperatures.

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

Characterization of a green Stentor with symbiotic algae growing in an extremely oligotrophic environment and storing large amounts of starch granules in its cytoplasm.

The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor, but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis. The cytoplasm of S. piriformis contains a large number of 1-3 µm refractive granules that turn brown by Lugol's staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.

Infectivity and genes differentially expressed between young and aging theront cells of the marine fish parasite Cryptocaryon irritans.

The ciliated protozoan Cryptocaryon irritans infects a wide range of marine fish and causes the highly lethal white spot disease. This parasite possesses three morphologically and physiologically distinct life stages: an infectious theront, a parasitic trophont, and an asexually reproductive tomont. In the past few years, several attempts have been made to help elucidate how C. irritans transforms from one stage to another using transcriptomic or proteomic approaches. However, there has been no research studying changes in transcription profiles between different time points of a single C. irritans life stage-the development of this parasite. Here we use RNA-seq and compare gene expression profiles of theront cells collected by 1 and 10 hrs after they emerged from tomonts. It has been shown that infectivity of theront cells declines 6-8 hours post-emergence, and we used this characteristic as a physiological marker to confirm the aging of theront cells. We identified a total of 41 upregulated and 90 downregulated genes that were differentially expressed between young and aging theront cells. Using Blast2Go to further analyze functions of these genes, we show that genes related to energy production are downregulated, but quite surprisingly many genes involved in transcription/translation processes are upregulated. We also show that expression of all nine detectable agglutination/immobilization antigen genes, with great sequence divergence, is invariably downregulated. Functions of other differentially expressed genes and indications are also discussed in our study.

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.

The Encystment-Related MicroRNAs and Its Regulation Molecular Mechanism in Pseudourostyla cristata Revealed by High Throughput Small RNA Sequencing.

MicroRNAs (miRNAs) regulate the expression of target genes in diverse cellular processes and play important roles in different physiological processes. However, little is known about the microRNAome (miRNAome) during encystment of ciliated protozoa. In the current study, we first investigated the differentially expressed miRNAs and relative signaling pathways participating in the transformation of vegetative cells into dormant cysts of Pseudourostyla cristata (P. cristata). A total of 1608 known miRNAs were found in the two libraries. There were 165 miRNAs with 1217 target miRNAs. The total number of differential miRNAs screened between vegetative cells and dormant cysts databases were 449 with p < 0.05 and |log2 fold changes| > 1. Among them, the upregulated and downregulated miRNAs were 243 and 206, respectively. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that some of the differentially expressed miRNAs were mainly associated with oxidative phosphorylation, two-component system, and biosynthesis of amino acids. Combining with our bioinformatics analyzes, some differentially expressed miRNAs including miR-143, miR-23b-3p, miR-28, and miR-744-5p participates in the encystment of P. cristata. Based on these findings, we propose a hypothetical signaling network of miRNAs regulating or promoting P. cristata encystment. This study shed new lights on the regulatory mechanisms of miRNAs in encystment of ciliated protozoa.

Morphology, Morphogenesis, and Molecular Phylogeny of Neobakuella aenigmatica n. sp. (Ciliophora, Spirotrichea, Bakuellidae).

The morphology and morphogenesis of a new ciliate species, Neobakuella aenigmatica n. sp., which was discovered in an estuary in Korea, were investigated, using live observation, protargol impregnation, and scanning electron microscopy. This new species is characterized by a large (185-300 × 55-105 µm in vivo), elongate-ellipsoidal, flexible but not contractile body. It has ellipsoidal, yellowish cortical granules, 1.3 × 1.0 µm in size. The species has invariably 3 frontal and 2 frontoterminal cirri, about 5-10 buccal and 1-6 parabuccal cirri, 7 midventral rows, and 1 right and 2-4 left marginal rows. The outer left marginal row(s) consists of 1-7 short rows of cirri. The nuclear apparatus comprises 130 macronuclear nodules and 2 spherical micronuclei on average. The dorsal ciliature consists of 3 dorsal kineties. The leftmost left marginal row(s) likely develops from anlagen originating from both the rightmost and leftmost left marginal row(s). The molecular phylogenetic tree based on SSU rDNA suggests the nonmonophyly of the genus Neobakuella.

Reorganization of complex ciliary flows around regenerating Stentor coeruleus.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such as Chlamydomonas. Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliate Stentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring in Stentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Thiotrophic bacterial symbiont induces polyphenism in giant ciliate host Zoothamnium niveum.

Evolutionary theory predicts potential shifts between cooperative and uncooperative behaviour under fluctuating environmental conditions. This leads to unstable benefits to the partners and restricts the evolution of dependence. High dependence is usually found in those hosts in which vertically transmitted symbionts provide nutrients reliably. Here we study host dependence in the marine, giant colonial ciliate Zoothamnium niveum and its vertically transmitted, nutritional, thiotrophic symbiont from an unstable environment of degrading wood. Previously, we have shown that sulphidic conditions lead to high host fitness and oxic conditions to low fitness, but the fate of the symbiont has not been studied. We combine several experimental approaches to provide evidence for a sulphide-tolerant host with striking polyphenism involving two discrete morphs, a symbiotic and an aposymbiotic one. The two differ significantly in colony growth form and fitness. This polyphenism is triggered by chemical conditions and elicited by the symbiont's presence on the dispersing swarmer. We provide evidence of a single aposymbiotic morph found in nature. We propose that despite a high fitness loss when aposymbiotic, the ciliate has retained a facultative life style and may use the option to live without its symbiont to overcome spatial and temporal shortage of sulphide in nature.

Specific inhibitors of lysozyme and peptidases inhibit the growth of the rumen protozoan Entodinium caudatum without decreasing feed digestion or fermentation in vitro.

AIMS: Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases on rumen protozoa and the associated prokaryotes, and in vitro fermentation using Entodinium caudatum as a model protozoan species. METHODS AND RESULTS: Imidazole (a lysozyme inhibitor), phenylmethylsulphonyl fluoride (PMSF, a serine peptidase inhibitor) and iodoacetamide (IOD, a cysteine peptidase inhibitor) were evaluated in vitro both individually and in two- and three-way combinations using E. caudatum monocultures with respect to their ability to inhibit the protozoan and their effect on feed digestion, fermentation and the microbiota. All the three inhibitors, both individually and in combination, decreased E. caudatum counts (P < 0·001), and IOD and its combinations with the other inhibitors significantly (P < 0·01) decreased ammonia concentration, with the two- and three-way combinations showing additive effective. Feed digestion was not affected, but fermentation and microbial diversity were affected mostly by PMSF, IOD and their combinatorial treatments potentially due to the overgrowth of Streptococcus luteciae accompanying with the disappearance of host ciliates. CONCLUSIONS: Entodinium caudatum depends on lysozyme and peptidase for digestion and utilization of the engulfed microbes and specific inhibition of these enzymes can inhibition E. caudatum without adversely affecting feed digestion or fermentation even though they changed the microbiota composition in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The peptidase inhibitors may have the potential to be used in controlling rumen protozoa to improve ruminal nitrogen utilization efficiency.

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.

Morphology and Molecular Phylogeny of Two New Terrestrial Ciliates, Australocirrus rubrus n. sp. and Notohymena gangwonensis n. sp. (Ciliophora: Oxytrichidae), from South Korea.

Two new soil oxytrichids, Australocirrus rubrus n. sp. and Notohymena gangwonensis n. sp., were discovered from South Korea. Morphologically, A. rubrus shares many features with A. australis, and these two species form a single clade in a molecular tree based on nuclear small subunit ribosomal RNA (SSU rRNA) gene sequences. Australocirrus rubrus mainly differs from A. australis in the color (citrine color vs. reddish) and distribution of the cortical granules. Additionally, we confirm that the genus Australocirrus is not a monophyletic group, as A. shii is separated from the clade comprising the other Australocirrus species, being clustered instead with other taxa. Notohymena gangwonensis n. sp. mainly differs from its congeners by the following combination of features: irregularly distributed cortical granules (vs. arranged in groups associated with cirri and dorsal kineties), variable four or five (usually four) transverse cirri (vs. invariable five), and the anteriormost pretransverse cirrus V/2 on 13.2-16.1% of cell length (vs. on or above 18.9% of cell length). Currently, there are no available gene sequences for members of the genus Notohymena, thus we provide SSU rRNA gene sequences from the new species of Notohymena, as well as detailed morphological descriptions of the novel species.

Morphology, Morphogenesis and Molecular Phylogeny of a New Obligate Halophile Ciliate, Schmidtiella ultrahalophila gen. nov., spec. nov. (Ciliophora, Hypotrichia) Isolated from a Volcanic Crater on Sal (Cape Verde Islands).

A new hypotrichous ciliate, Schmidtiella ultrahalophila gen. nov., spec. nov., was isolated from a solar saltern on the island of Sal, Cape Verde. The possession of only one short dorsal kinety clearly distinguishes S. ultrahalophila from other known hypotrichous genera and species. Further diagnostic characters include: a flexible and slender body, an average size of 85 × 15 µm in vivo; a bipartite adoral zone with two hypertrophied frontal adoral membranelles and nine to twelve ventral adoral membranelles; three frontal, one parabuccal, two frontoventral, two or three postoral ventral, and two or three frontoterminal cirri; and marginal cirral rows variable in number, usually one on each side. Ontogenetic data indicate the following: the frontal-ventral cirri originate from six or five anlagen; the proter inherits the parental adoral zone; the frontal and ventral cirri originate from five or six anlagen; and the marginal cirral rows and the dorsal kinety tend to originate intrakinetally. Additional marginal rows are rarely derived from de novo anlagen. Based on its morphology, morphogenesis and its SSU rRNA phylogenetic placement, the new species should be assigned to the order Sporadotrichida Fauré-Fremiet, 1961. Due to low taxon sampling, however, its exact position in this order remains enigmatic.

Spatial variations in trophic-functional patterns of periphytic ciliates and indications to water quality in coastal waters of the Yellow Sea.

To evaluate the water quality status using ecological features of the periphytic ciliate communities, a 1-year (Jan. to Dec., 2016) investigation was conducted in coastal waters of the Yellow Sea, northern China. Four trophic-functional groups (TFgrs) were recorded from a total of 141 species-abundance dataset: algivores (A); bacterivores (B); non-selectives (N); and predators (R), comprising of 65, 34, 26, and 16 species, respectively. In terms of species number, TFgr A was predominant in clean areas while TFgrs B and N were dominant in heavy polluted areas and TFgr R was dominant in slightly polluted area. The trophic-functional patterns of the periphytic ciliate communities showed a clear spatial variation within the pollution gradient. Trophic-functional trait diversity measures represented a clear increasing trend from polluted stations to the clean area regarding the pollution gradients. Multivariate correlation and best matching analysis revealed that the spatial pattern of the trophic-functional groupings were significantly shaped by environmental variable nutrients and chemical oxygen demand, alone or in combination with pH, dissolved oxygen, salinity, and transparency. Thus, we suggest that the ecological features based on the trophic-functional patterns of periphytic ciliate communities might be used for bioassessment of water quality in marine ecosystems.

Antiparasitic Effect of Copper Alloy Surface on Cryptocaryon irritans in Aquaculture of Larimichthys crocea.

Copper and alloys containing >60% copper by weight are antimicrobial. In aquaculture, copper alloys are used as part of corrosion-resistant cages or as part of copper coating. To test whether a copper alloy surface prevents the outbreak of parasitosis in the aquaculture of Larimichthys crocea, we covered the bottom of the aquaculture tank with sheets of copper alloy containing 74% to 78% copper, and we cultured L. crocea juveniles that had been artificially infected with the protozoan parasite Cryptocaryon irritans Our results showed that these copper alloy sheets effectively blocked the infectious cycle of C. irritans within a 1-week period and significantly reduced the number of C. irritans trophonts and tomonts, thereby decreasing the mortality rate of L. crocea In in vitro assays, the cytoplasmic membranes of protomonts disintegrated and the cytoplasm overflowed after just 5 minutes of contact with copper alloy surfaces. Although the same cytoplasmic membrane disintegration was not observed in tomonts, the tomonts completely lost their capacity for proliferation and eventually died following direct contact with copper alloy sheets for 1 h; this is likely because C. irritans tomonts took in >100 times more copper ions following contact with the copper alloy sheets than within the control aquaculture environment. Exposure to copper alloy sheets did not lead to excessive heavy metal levels in the aquacultured fish or in the culture seawater.IMPORTANCECryptocaryon irritans, a parasitic ciliate that penetrates the epithelium of the gills, skin, and fins of marine fish, causes acute suffocation and death in cultured fish within days of infection. Much of the existing research centers around the prevention of C. irritans infection, but no cure has been found. Studies demonstrate that copper has strong antimicrobial properties, and fish grown in copper-containing cages have lower rates of C. irritans infection, compared to those grown in other currently used aquaculture cages. In this study, we found that an alloy containing 74% to 78% copper by weight effectively killed C. irritans cells and prevented cryptocaryoniasis outbreaks within a 1-week period. These findings offer a new perspective on the prevention and control of cryptocaryoniasis.

Notes on the Cultivation of Two Mixotrophic Dinophysis Species and Their Ciliate Prey Mesodinium rubrum.

Kleptoplastic mixotrophic species of the genus Dinophysis are cultured by feeding with the ciliate Mesodinium rubrum, itself a kleptoplastic mixotroph, that in turn feeds on cryptophytes of the Teleaulax/Plagioselmis/Geminigera (TPG) clade. Optimal culture media for phototrophic growth of D. acuminata and D. acuta from the Galician Rías (northwest Spain) and culture media and cryptophyte prey for M.rubrum from Huelva (southwest Spain) used to feed Dinophysis, were investigated. Phototrophic growth rates and yields were maximal when D. acuminata and D. acuta were grown in ammonia-containing K(-Si) medium versus f/2(-Si) or L1(-Si) media. Dinophysis acuminata cultures were scaled up to 18 L in a photobioreactor. Large differences in cell toxin quota were observed in the same Dinophysis strains under different experimental conditions. Yields and duration of exponential growth were maximal for M. rubrum from Huelva when fed Teleaulax amphioxeia from the same region, versus T. amphioxeia from the Galician Rías or T. minuta and Plagioselmis prolonga. Limitations for mass cultivation of northern Dinophysis strains with southern M. rubrum were overcome using more favorable (1:20) Dinophysis: Mesodinium ratios. These subtleties highlight the ciliate strain-specific response to prey and its importance to mass production of M. rubrum and Dinophysis cultures.

Seasonal succession of ciliate Mesodinium spp. with red, green, or mixed plastids and their association with cryptophyte prey.

Mesodinium spp. are commonly found in marine and brackish waters, and several species are known to contain red, green, or both plastids that originate from cryptophyte prey. We observed the seasonal succession of Mesodinium spp. in a Japanese brackish lake, and we analysed the origin and diversity of the various coloured plastids within the cells of Mesodinium spp. using a newly developed primer set that specifically targets the cryptophyte nuclear 18S rRNA gene. Mesodinium rubrum isolated from the lake contained only red plastids originating from cryptophyte Teleaulax amphioxeia. We identified novel Mesodinium sp. that contained only green plastids or both red and green plastids originating from cryptophytes Hemiselmis sp. and Teleaulax acuta. Although the morphology of the newly identified Mesodinium sp. was indistinguishable from that of M. rubrum under normal light microscopy, phylogenetic analysis placed this species between the M. rubrum/major species complex and a well-supported lineage of M. chamaeleon and M. coatsi. Close associations were observed in cryptophyte species composition within cells of Mesodinium spp. and in ambient water samples. The appearance of suitable cryptophyte prey is probably a trigger for succession of Mesodinium spp., and the subsequent abundance of Mesodinium spp. appears to be influenced by water temperature and dissolved inorganic nutrients.

Effect of ciliate strain, size, and nutritional content on the growth and toxicity of mixotrophic Dinophysis acuminata.

Previous studies indicate differences in bloom magnitude and toxicity between regional populations, and more recently, between geographical isolates of Dinophysis acuminata; however, the factors driving differences in toxicity/toxigenicity between regions/strains have not yet been fully elucidated. Here, the roles of prey strains (i.e., geographical isolates) and their associated attributes (i.e., biovolume and nutritional content) were investigated in the context of growth and production of toxins as a possible explanation for regional variation in toxicity of D. acuminata. The mixotrophic dinoflagellate, D. acuminata, isolated from NE North America (MA, U.S.) was offered a matrix of prey lines in a full factorial design, 1 × 2 × 3; one dinoflagellate strain was fed one of two ciliates, Mesodinium rubrum, isolated from coastal regions of Japan or Spain, which were grown on one of three cryptophytes (Teleaulax/Geminigera clade) isolated from Japan, Spain, or the northeastern USA. Additionally, predator: prey ratios were manipulated to explore effects of the prey's total biovolume on Dinophysis growth or toxin production. These studies revealed that the biovolume and nutritional status of the two ciliates, and less so the cryptophytes, impacted the growth, ingestion rate, and maximum biomass of D. acuminata. The predator's consumption of the larger, more nutritious prey resulted in an elevated growth rate, greater biomass, and increased toxin quotas and total toxin per mL of culture. Grazing on the smaller, less nutritious prey, led to fewer cells in the culture but relatively more toxin exuded from the cells on per cell basis. Once the predator: prey ratios were altered so that an equal biovolume of each ciliate was delivered, the effect of ciliate size was lost, suggesting the predator can compensate for reduced nutrition in the smaller prey item by increasing grazing. While significant ciliate-induced effects were observed on growth and toxin metrics, no major shifts in toxin profile or intracellular toxin quotas were observed that could explain the large regional variations observed between geographical populations of this species.

Bacterial and ciliate biofilm community structure at different spatial levels of a salt lake meta-community.

Meta-communities are assembled along an ecological scale that determines local and regional diversity. Spatial patterns have been detected in planktonic bacterial communities at distances <20 m, but little is known about the occurrence of similar variation for other microbial groups and changes in microbial meta-community assembly at different levels of a meta-community. To examine this variation, the biofilm of eight saline ponds were used to investigate processes shaping diversity within ponds (ß) and between ponds (δ). Bacterial and ciliate communities were assessed using ARISA and T-RFLP respectively, while diversity partitioning methods were used to examine the importance of taxonomic turnover and variation partitioning was used to distinguish spatial from environmental determinants. The results show that turnover is important for determining ß- and δ-diversity of biofilms. Spatial factors are important drivers of bacterial ß-diversity but were unimportant for ciliate ß-diversity. Environmental variation was a strong determinant of bacterial and ciliate δ-diversity, suggesting sorting processes are important for assembling pond communities. Determinants of diversity in bacteria are not universal for ciliates, suggesting higher functional redundancy of bacteria or the greater niche breadth of ciliates may be important in discriminating assembly processes between the two organisms.

Aerobic cultivation of anaerobic rumen protozoa, Entodinium caudatum and Epidinium caudatum.

Rumen protozoa, primarily ciliates, are one of the important groups of strictly anaerobic microbes living in the rumen. Despite their ubiquitous occurrence in the rumen and significant contribution to host animals, it is still poorly understood why they live only in the rumen and similar environment. Because rumen protozoa require strict anaerobic conditions to sustain their viability and grow, only a few laboratories equipped with protozoology expertise and anaerobic facilities can grow rumen protozoa in laboratory. Also for the same reason, only a few species have been grown and maintained as laboratory cultures for research. Prompted by a recent study, we hypothesized that anaerobic rumen protozoa could also be cultivated aerobically if antioxidants were included in the media. Indeed, our experiments showed that the cultures of both Entodinium caudatum and Epidinium caudatum, two major rumen protozoal species, could be cultured successfully in aerobic media supplemented with ascorbic acid, glutathione and α-ketoglutarate as antioxidants. Anaerobic fermentation was maintained through the fermentation characteristics and microbial populations were altered to some extent under aerobic conditions. The antioxidants also enhanced the revival of cryopreserved stock cultures of both rumen protozoal species. The results of this study may facilitate and promote future research in which rumen protozoa need to be cultured in laboratory.

Methods for the Study of Regeneration in Stentor.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.