Ciliated Research Articles

Using galvanized materials to control Cryptocaryon irritans infestation

Infection by Cryptocaryon irritans, a ciliated protozoa parasite of marine fish, causes mass mortality and economic loss to the mariculture industry. In this study, plate and mesh galvanized zinc were embedded on the bottom of seawater to evaluate the protection to susceptible host Trachinotus ovatus from C. irritans reinfection at various seawater exchange rates and dosages. Furthermore, an evaluation of the zinc ion released from galvanized materials was carried out through detection of the zinc ion content in seawater and muscle tissue, and the observed pathological changes of liver. The results showed that, at all flow exchange rates (0, 50, or 1200%), the parasite abundance of infected T. ovatus were significantly decreased with galvanized materials. Survival rates were 100% at 14 days post infection when 56.25% of the tank was covered with galvanized iron plates or galvanized iron mesh at a dose of 0.2 m2/m3. The zinc ion concentrations in seawater after using galvanized materials increased continuously. However, residual zinc ion in the muscle of T. ovatus were not significantly different to the control group. Furthermore, no obvious pathological change was observed in the liver of T. ovatus. Consequently, using galvanized materials can control the reinfection of C. irritans and poses no threat to protected fish. Therefore, the use of galvanized materials could be a promising strategy to control cryptocaryoniasis.

Ecotoxicological effect of heavy metals in free-living ciliate protozoa of Lake Maracaibo, Venezuela

Ecotoxicological effect of heavy metals in free-living ciliate protozoa of Lake Maracaibo, Venezuela

Delete and survive: strategies of programmed genetic material elimination in eukaryotes.

ABSTRACTGenome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.

Infection of Slugs with Theronts of the Ciliate Protozoan, Tetrahymena rostrata.

Tetrahymena rostrata is a free-living ciliated protozoan and is a facultative parasite of some species of terrestrial mollusks. It is a potential biopesticide of pest slugs, such as the grey field slug, which cause considerable damage to crops. T. rostrata has several developmental forms. Homogeneous preparations of the feeding stage cells (trophonts) and excysted stage cells (theronts) were compared for their ability to infect and kill Deroceras reticulatum slugs. Theronts were more effective and remained viable and infective, even after prolonged starvation.

Crude coriander oil in the diet of lactating goats enhanced lactational performance, ruminal fermentation, apparent nutrient digestibility, and blood chemistry

The use of safe feed additives in diets of lactating animals has gained increasing interest. The effects of feeding lactating goats on diet supplemented with crude coriander oil at different levels were evaluated. Thirty multiparous lactating Damascus × Baladi goats (27.2 ± 1.0 kg BW) were used in 3 × 3 Latin square design (3 treatments, 3 periods, and 10 does per treatment). Each experimental period lasted 30 d (comprising 20 d of treatments adaptation and 10 d of sampling). The treatments consisted of the control basal diet with no crude coriander oil, the control basal diet supplemented daily with 0.95 g crude coriander oil/kg DM feed, or the control basal diet supplemented with 1.9 g crude coriander oil/kg DM feed. Crude coriander oil supplementation linearly increased (P < 0.05) daily milk production, yields of total solids, fat and lactose, and concentration of milk fat (P = 0.022) as well as feed conversion ratio (P = 0.021). Crude coriander oil supplementation linearly enhanced (P < 0.05) the apparent digestibilities of dry matter, organic matter, non-structural carbohydrates, and fibers, while the high crude coriander oil supplementation level increased apparent crude protein digestibility. Crude coriander oil supplementation linearly decreased ruminal pH and ciliate protozoal counts, and increased the concentrations (P < 0.05) of total volatile fatty acids, acetate, propionate and valerate. Crude coriander oil supplementation linearly increased (P < 0.05) the concentration of blood glucose and linearly decreased (P < 0.05) the serum levels of alanine transaminase and aspartate aminotransferase, while the high coriander oil treatment decreased the concentrations of total lipids (P = 0.038) and cholesterol (P = 0.021). In conclusion, crude coriander oil supplementation at 0.95 or 1.9 g crude coriander oil/kg DM feed improved nutrient utilization, milk production, and milk nutritive value. The high level of supplementation is recommended.

The effects of feeding rations that differ in neutral detergent fiber and starch within a day on the daily pattern of key rumen microbial populations

The effect of feeding a single TMR versus multiple rations across the day that differ in concentrations of neutral detergent fiber (NDF) and starch on the daily pattern of rumen microbial populations was characterized. Diets included a control total mixed ration (CON; 33.3% NDF), a low-fiber diet (LF; 29.6% NDF), and a high-fiber diet (HF; 34.8% NDF). Nine cannulated Holstein cows were assigned to 1 of 3 treatment sequences in a 3 × 3 Latin square design. Treatments included feeding CON ad libitum at 0900 h, feeding HF at 70% of daily offering at 0900 h and LF at 30% of daily offering at 2200 h (H/L), and feeding LF at 30% of daily offering at 0900 h and HF at 70% of daily offering at 1300 h (L/H). Rumen digesta was collected to represent every 3 h across the day, microbial DNA was extracted, and real-time quantitative PCR was used to determine the relative abundances of total bacteria, total fungi, total protozoa, Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Fibrobacter succinogenes, Megasphaera elsdenii, Prevotella bryantii, Ruminococcus albus, Selenomonas ruminantium, and Streptococcus bovis. The relative abundances of total bacteria, total ciliated protozoa, F. succinogenes, P. bryantii, R. albus, S. ruminantium, and Strep. bovis were affected by time of day. Additionally, treatment affected the relative abundance of certain microbial groups at specific times of day. Notably, H/L treatment dramatically increased the relative abundances of B. fibrisolvens, B. hungatei, and Strep. bovis at 0900 h, by 2.5-, 5.4-, and 4.4-fold, respectively. Furthermore, the relative abundances of B. hungatei (3.9-fold), M. elsdenii (3.9-fold), R. albus (1.3-fold), S. ruminantium (1.3-fold), and Strep. bovis (4.5-fold) were greatly increased by L/H at 0900 h. At 0600 h, the relative abundance of F. succinogenes was 58% greater in L/H than H/L and the relative abundance of P. bryantii was 49% greater in H/L than L/H. Results suggest that there is a daily pattern of selected microbial populations that is altered by feeding rations that differ in NDF and starch within a day, with the greatest difference occurring before morning feeding.

Leukocyte numbers and intestinal mucosal morphometrics in horses with no clinical intestinal disease.

Healthy horses and other animals have large numbers of resident leukocytes in the intestinal wall, but there is scant information regarding which and how many leukocytes are normally present in the equine intestinal wall. Our aim was to provide a reference range of leukocytes in the intestinal mucosal and submucosal propria of normal horses. We included in our study intestinal tissues from 22 Thoroughbred racehorses with no clinical intestinal disease, which had been euthanized because of catastrophic musculoskeletal injuries. Neutrophils, lymphocytes, eosinophils, macrophages, and plasma cells were counted in 5 random 17,600-µm2 areas of villus lamina propria of the duodenum, jejunum, and ileum, and deep lamina propria of the duodenum, jejunum, ileum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and small colon. Other features investigated in the same intestinal segments included villus height and width (small intestine), presence of ciliated protozoa, Paneth cells number, subcryptal leukocyte layers (number of leukocyte layers between the bottom of the crypts and the muscularis mucosae), and submucosal leukocytes. Lymphocytes were the most numerous cells in all segments analyzed, followed by plasma cells, eosinophils, macrophages, and neutrophils. Eosinophil numbers were significantly higher in both lamina propria and submucosa of the large intestine than in the small intestine. The duodenum had shorter and thinner villi than either jejunum or ileum. The data provided from our study will be useful for diagnosticians examining inflammatory processes in the intestinal tract of horses.

Rumen Ciliated Protozoa of the Free-Living European Bison (Bison bonasus, Linnaeus).

This study aims to perform population analysis of the rumen ciliated protozoa of the free-living European bison (wisent, Bison bonasus, Linnaeus). The samples of the rumen fluid from the 18 bison subjected to the controlled culls within the free-ranging population in the Bialowieza primeval forest in Poland were collected and examined. The examined ciliates population consisted of the species of the families Isotrichidae and Ophryoscolecidae. There were 12 genera (Isotricha, Dasytricha, Diplodinium, Elytroplastron, Entodinium, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium, Ophryoscolex, and Ostracodinium) and 32 morphospecies of the ciliates. We observed the prevalence of a type B protozoan population (56% animals) with the typical Epidinium and Eudiplodinium genera members. Other examined animals possessed the mixed A–B population with Ophryoscolex genus, distinct for type A ciliate population. The average total ciliates count was 2.77 ± 1.03 × 105/ml (mean ± SD). The most abundant genera were Entodinium, 83%, and Dasytricha, 14%. The abundance of other genera was <1% of the total count. Within the 16 Entodinium species determined, the most abundant species was Entodinium nanellum (16.3% of total ciliates count). The average Shannon–Wiener diversity index was 2.1 ± 0.39, evenness was 0.7 ± 0.11, and species richness was 24 ± 3.0 (mean ± SD). Our study is the first report on the population composition and diversity of rumen ciliates of European bison. The composition and counts of ciliate genera and species were similar to the composition and counts of the rumen ciliated protozoa of American bison and many other kinds of free-living and domestic ruminants. Our European bison ciliate population analysis has shown medium ciliate density and high diversity typical for large free-living ruminants with mixed feeding behavior.

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

WITHDRAWN: First record of ciliophora (Orchitophryidae) isolated from CSF samples of patients with meningitis in Thi-Qar province south of Iraq

In September 2020 when we were investigating about opportunistic free living amoeba in patients with meningitis in Thi –Qar province south of Iraq, throughout examination the CSF which was cultured on NN Agar medium. It was surprisingly we found the ciliates protozoa (Orchitophryidae) in both stages, vegetative and cystic, after that the ciliates were isolated and cultured in vitro and then morphological, molecular and virulence characterizations were studies. The results of morphological study revealed that these ciliates were similar to Mesanophrys sp. whereas the sequence of rDNA of studied ciliate with accession numbers being given. in NCBI LC621170.1 was show (86.04%) homology identity to Mesanophrys sp. 18S rRNA gene (accession number MN260367.1). but show (98.56%) and (97.92%) homology identity to Orchitophrydiae 18S rRNA gene (accession number EF023919.1 and EF023549.1), that may be this ciliates in our study is a new species for Mesanophrys . In addition experimental infection confirmed that Mesanophrys like ciliates was the pathogen that infected human. In summary this study represent the first record of the ciliophora Orchitophryidae ( Mesanophrys like ciliates) as a human pathogen from Thi-Qar province / Iraq.

Ruminal Protozoal Populations of Angus Steers Differing in Feed Efficiency.

Simple SummaryThe rumen protozoa have been demonstrated to enhance methanogenesis and impact intraruminal recycling of microbial protein. However, they are also known to contribute to fiber degradation and the stabilization of ruminal pH changes. The apparent metabolic impact of ciliated protozoa in the rumen may contribute to the variation in feed efficiency. This study examined the relationship between the rumen protozoa and feed efficiency in beef steers. We monitored feed intake and body weight for a 70-day feed efficiency trial. Following the trial, rumen content was collected, protozoal DNA was extracted from the content, and the relationship between protozoa community diversity and species abundance with feed efficiency was examined. The ciliated protozoal community diversity differed between low- and high-feed efficient steers. Greater abundances of unidentified protozoa genera were detected in the low-feed efficient steers. These data suggest that unidentified protozoa and ciliated protozoal community diversity influence feed efficiency in beef steers. Feed accounts for as much as 70% of beef production costs, and improvement of the efficiency with which animals convert feed to product has the potential to have substantial financial impact on the beef industry. The rumen microbiome plays a key role in determining feed efficiency; however, previous studies of rumen microbiota have not focused on protozoal communities despite the estimation that these organisms represent approximately 50% of rumen content biomass. Protozoal communities participate in the regulation of bacterial populations and nitrogen cycling—key aspects of microbiome dynamics. The present study focused on identifying potential associations of protozoal community profiles with feed efficiency. Weaned steers (n = 50) 7 months of age weighing approximately 260 kg were adapted to a growing ration and GrowSafe for 2 weeks prior to a 70-day feed efficiency trial. The GrowSafe system is a feeding system that monitors feed intake in real time. Body weights were collected on the first day and then every 7 days of the feed efficiency trial, and on the final day, approximately 50 mL of rumen content were collected via orogastric tubing and frozen at −80 °C. Body weight and feed intake were used to calculate residual feed intake (RFI) as a measure of feed efficiency, and steers were categorized as high (n = 14) or low (n = 10) RFI based on ±0.5 standard deviations about the mean RFI. Microbial DNA was extracted, and the eukaryotic component profiled by amplification and sequencing of 18S genes using degenerate primers that can amplify this locus across a range of protists. The taxonomy of protozoal sequences was assigned using QIIME 1.9 and analyzed using QIIME and SAS 9.4 with significance determined at α ≤ 0.05. Greater abundances of unassigned taxa were associated with high-RFI steers (p = 0.03), indicating a need for further study to identify component protozoal species. Differences were observed between low- and high-RFI steers in protozoal community phylogenetic diversity, including weighted beta-diversity (p = 0.04), Faith’s phylogenetic diversity (p = 0.03), and observed Operational taxonomic unit (OTU) (p = 0.03). The unassigned taxa and differences in phylogenetic diversity of protozoal communities may contribute to divergences observed in feed efficiency phenotypes in beef steers.

Signaling in temperature-induced resting cyst formation in the ciliated protozoan Colpoda cucullus

The terrestrial ciliated protozoan Colpoda cucullus inhabits soil. When the habitat conditions become unfavorable, the vegetative cells of C. cucullus quickly transform into resting cysts. C. cucullus culture is established in our laboratory, and encystment is routinely induced by the addition of Ca2+ to overpopulated vegetative cells. However, an increase in Ca2+ concentration and overpopulation of vegetative cells do not always occur in natural. We investigated the effect of temperature and found that cyst formation was induced by a rapid increase of 5 °C within 2 min but not by a decrease. Moreover, an increase in intracellular Ca2+ concentrations is essential, but Ca2+ inflow does not necessarily occur during encystment. Ca2+ image analysis showed that Ca2+ is stored in vesicular structures and released into the cytoplasm within 60 s after temperature stimulation. Multiple signaling pathways are activated after the release of Ca2+ from vesicles, and cAMP is a candidate second messenger with a crucial role in the process of temperature-induced encystment. Further studies are needed to clarify the mechanism underlying the sensing of temperature and release of Ca2+ from vesicles.

Scuticociliate (Philasterides dicentrarchi) infection cluster in a multispecies marine aquarium system.

Scuticociliatosis, caused by ciliated protozoa of the subclass Scuticociliatia, has been associated with high mortalities in marine fish. Environmental factors such as an increase in water temperature can enhance this disease. The aim of the present report is to describe the occurrence of a cluster of cases of scuticociliatosis in a multispecies marine cold-water system in a public aquarium. Philasterides dicentrarchi was identified by PCR in formalin-fixed tissues of some of the fish showing meningitis or meningoencephalitis, dermatitis and myositis with intralesional protozoa. An increase in water temperature of approximately 2°C was identified as a potential contributing factor for this cluster of infections. Higher temperature may have enhanced the propagation or pathogenicity of scuticociliates or increased host susceptibility of some species of fish, especially wolf-eel Anarrhichthys ocellatus and spotted ratfish Hydrolagus colliei. This report also highlights the complexity of dealing with mixed species systems housing fish from different natural ecozones.

Transcriptome analysis reveals the encystment-related lncRNA expression profile and coexpressed mRNAs in Pseudourostyla cristata

Ciliated protozoans form dormant cysts for survival under adverse conditions. The molecular mechanisms regulating this process are critical for understanding how single-celled eukaryotes adapt to the environment. Despite the accumulated data on morphology and gene coding sequences, the molecular mechanism by which lncRNAs regulate ciliate encystment remains unknown. Here, we first detected and analyzed the lncRNA expression profile and coexpressed mRNAs in dormant cysts versus vegetative cells in the hypotrich ciliate Pseudourostyla cristata by high-throughput sequencing and qRT-PCR. A total of 853 differentially expressed lncRNAs were identified. Compared to vegetative cells, 439 and 414 lncRNAs were upregulated and downregulated, respectively, while 47 lncRNAs were specifically expressed in dormant cysts. A lncRNA-mRNA coexpression network was constructed, and the possible roles of lncRNAs were screened. Three of the identified lncRNAs, DN12058, DN20924 and DN30855, were found to play roles in fostering encystment via their coexpressed mRNAs. These lncRNAs can regulate a variety of physiological activities that are essential for encystment, including autophagy, protein degradation, the intracellular calcium concentration, microtubule-associated dynein and microtubule interactions, and cell proliferation inhibition. These findings provide the first insight into the potentially functional lncRNAs and their coexpressed mRNAs involved in the dormancy of ciliated protozoa and contribute new evidence for understanding the molecular mechanisms regulating encystment.

Microfluidic guillotine reveals multiple timescales and mechanical modes of wound response in Stentor coeruleus

BackgroundWound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism.ResultsHere we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response in Stentor. We found that Stentor requires ~ 100–1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~ 8–80 μm2/s, faster than most other single cells reported in the literature. Further, we characterized three distinct mechanical modes of wound response in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others.ConclusionsWhile events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.

When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast.

Telomeres, repetitive sequences located at the ends of most eukaryotic chromosomes, provide a mechanism to replenish terminal sequences lost during DNA replication, limit nucleolytic resection, and protect chromosome ends from engaging in double-strand break (DSB) repair. The ribonucleoprotein telomerase contains an RNA subunit that serves as the template for the synthesis of telomeric DNA. While telomere elongation is typically primed by a 3′ overhang at existing chromosome ends, telomerase can act upon internal non-telomeric sequences. Such de novo telomere addition can be programmed (for example, during chromosome fragmentation in ciliated protozoa) or can occur spontaneously in response to a chromosome break. Telomerase action at a DSB can interfere with conservative mechanisms of DNA repair and results in loss of distal sequences but may prevent additional nucleolytic resection and/or chromosome rearrangement through formation of a functional telomere (termed “chromosome healing”). Here, we review studies of spontaneous and induced DSBs in the yeast Saccharomyces cerevisiae that shed light on mechanisms that negatively regulate de novo telomere addition, in particular how the cell prevents telomerase action at DSBs while facilitating elongation of critically short telomeres. Much of our understanding comes from the use of perfect artificial telomeric tracts to “seed” de novo telomere addition. However, endogenous sequences that are enriched in thymine and guanine nucleotides on one strand (TG-rich) but do not perfectly match the telomere consensus sequence can also stimulate unusually high frequencies of telomere formation following a DSB. These observations suggest that some internal sites may fully or partially escape mechanisms that normally negatively regulate de novo telomere addition.

Effect of habitat quality and phenotypic variation on abundance‐ and trait‐based early warning signals of population collapses

Loss of resilience in population numbers in response to environmental perturbations may be predicted with statistical metrics called early warning signals (EWS) that are derived from abundance time series. These signals, however, have been shown to have limited success, leading to the development of trait‐based EWS that are based on information collected from phenotypic traits such as body size. Experimental work assessing the efficacy of EWS under varying ecological and environmental factors are rare. In addition, disentangling how such warning signals are affected under varying ecological and environmental factors is key to their application in biological conservation. Here, we experimentally test how different rates of environmental forcing (i.e. warming) and varying ecological factors (i.e. habitat quality and phenotypic diversity) affected population stability and predictive power of early warning signals of population collapse. We analyzed population density and body size time series data from three phenotypically different populations of a protozoan ciliate Askenasia volvox in two levels of habitat quality subjected to three different treatments of warming (i.e. no warming, fast warming and slow warming). We then evaluated how well abundance‐ and trait‐based EWS predicted population collapses under different levels of phenotypic diversity, habitat quality and warming treatments. Our results suggest that habitat quality and warming treatments had more profound effects than phenotypic diversity had on both population stability and on the performance of abundance‐based signals of population collapse. In addition, trait‐based EWS generally performed well, were reliable and more robust in forecasting population collapse than abundance‐based EWS, regardless of variation in environmental and ecological factors. Our study points towards the development of a predictive framework that includes information from phenotypic traits such as body size as an indicator of loss of resilience of ecological systems in response to environmental perturbations.

Ciliates as bioindicators of environmental pressure in a karstic river

Ciliates (protozoa) are a very large and diverse group of microeukaryotes that occupy a central position in the trophic web of freshwater ecosystems. Ciliates exhibit high ecological sensitivity and have tremendous bioindicator potential, but they are largely ignored in routine biomonitoring, mainly due to limitations in morphological diagnosis, which is both time-consuming and costly. In this study, we combined molecular (Illumina sequencing of the hypervariable V9 region of the SSU rRNA gene) and morphological approaches to get a detailed insight into the periphytic ciliate community structure and function within a karst, tufa-precipitating hydrosystem (National Park Krka, Croatia). Periphyton (biofilm) was sampled from light- and dark-exposed lithified tufa/stones at four representative locations within the Krka River, including upstream, midstream, and downstream river sections. We identified hydrological parameters and saprobiological classification of the sampling locations as the main structuring factors for ciliate communities. The molecular approach showed a clear separation of sampling locations in beta diversity analyses, as confirmed by PERMANOVA test. In contrast, alpha diversity was mostly affected by light exposition of the lithified tufa/stones, as confirmed by morphological approach. Environmental conditions consistently affected ciliate community composition at all locations, resulting in clear separation of periphyton samples. Our results indicate that ciliates should be considered as important bioindicators in monitoring tufa-forming rivers and streams, and the molecular approach (V9 region) proved to be applicable because as it efficiently captured the diversity of ciliates in periphyton. Validation of such an approach would lead to significant progress in extending monitoring to a broader range of indicator organisms than those now included in standard monitoring.

Brief description of 18 newly recorded ciliate species from soil and inland waters(Protozoa, Ciliophora) in South Korea

Brief description of 18 newly recorded ciliate species from soil and inland waters(Protozoa, Ciliophora) in South Korea

Association of ionophores, yeast, and bacterial probiotics alters the abundance of ruminal microbial species of pasture intensively finished beef cattle

The effect of the association of non-protein nitrogen, yeast, and bacterial probiotics on the ruminal microbiome of beef cattle intensively finished on pasture was evaluated. The experiment was carried out in a completely randomized design with five treatments and four replications. The treatments consisted of a group of animals kept on pasture that received low consumption supplementation (LS) and four groups that received for 98 days, 17.5 g concentrate kg-1 body weight. The supplements were composed of the association of additives: urea (U), slow-release non-protein nitrogen (U+SRN), yeast (Saccharomyces cerevisiae; U+SRN+Y), and bacterial probiotics (live strains of bacteria; U+SRN+Y+BP). All supplements also contained salinomycin and virginiamycin. After slaughtering the animals, samples of ruminal content were collected to quantify groups of fibrolytic bacteria (Ruminococcus albus and Fibrobacter succinogenes), non-fibrolytic (Prevotella ruminicola, Selenomonas ruminantium, and Streptococcus bovis), Archaea, and ciliate protozoa, using the qPCR technique. The abundance of F. succinogenes was the same for the LS animals and those that received the supplement U+SRN+Y (1.42×108 copies mL-1) but higher than the other treatments. Supplementation reduced by 90% the abundance of S. bovis compared to the LS. The inclusion of yeast increased the abundance of fibrolytic bacteria by 2.2-fold. For animals that received the supplement U+SRN+Y+BP and the LS, there was no difference for non-fibrolytic bacteria (3.07×109 copies mL-1). The use of yeasts and sources of non-protein nitrogen in high-concentrate diets for beef cattle stimulates the growth of fibrolytic bacteria, which can contribute to the reduction of digestive disorders and metabolic diseases in animals that receive diets with high concentrate in pasture intensive termination systems.