Ciliate Population Research Articles

A Single-Cell Atlas of the Murine Pancreatic Ductal Tree Identifies Novel Cell Populations With Potential Implications in Pancreas Regeneration and Exocrine Pathogenesis

Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells; terminal, intercalated, intracalated ducts; and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. We used small conditional RNA sequencing to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models, and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential invitro, including a Wnt-responsive population, a ciliated population, and FLRT3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples. The expression of WNT-responsive, interferon-responsive, and epithelial-to-mesenchymal transition population markers increases in chronic pancreatitis and tumor samples. In light of our discovery of previously unidentified ductal populations, we unmask potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis. Thus, novel lineage-tracing models are needed to investigate ductal-specific populations invivo.

Using digital PCR to predict ciliate abundance from ribosomal RNA gene copy numbers

AbstractCiliates play a key role in most ecosystems. Their abundance in natural samples is crucial for answering many ecological questions. Traditional methods of quantifying individual species, which rely on microscopy, are often labour‐intensive, time‐consuming and can be highly biassed. As a result, we investigated the potential of digital polymerase chain reaction (dPCR) for quantifying ciliates. A significant challenge in this process is the high variation in the copy number of the taxonomic marker gene (ribosomal RNA [rRNA]). We first quantified the rRNA gene copy numbers (GCN) of the model ciliate, Paramecium tetraurelia, during different stages of the cell cycle and growth phases. The per‐cell rRNA GCN varied between approximately 11,000 and 130,000, averaging around 50,000 copies per cell. Despite these variations in per‐cell rRNA GCN, we found a highly significant correlation between GCN and cell numbers. This is likely due to the coexistence of different cellular stages in an uncontrolled (environmental) ciliate population. Thanks to the high sensitivity of dPCR, we were able to detect the target gene in a sample that contained only a single cell. The dPCR approach presented here is a valuable addition to the molecular toolbox in protistan ecology. It may guide future studies in quantifying and monitoring the abundance of targeted (even rare) ciliates in natural samples.

Physiological mortality of planktonic ciliates: Estimates, causes, and consequences

AbstractContrasting physiological mortality with predator‐induced mortality is of tremendous importance for the population dynamics of many organisms but is difficult to assess. This is especially true for tiny organisms, such as most protists, which do not leave any carcasses behind. I performed a meta‐analysis using planktonic ciliates as model organisms to estimate the maximum physiological mortality rates (δmax) across pelagic ecosystems in relation to environmental and biotic factors. The median δmax of planktonic ciliates was 0.62 d−1 and did not differ between marine and freshwater species. Maximum ciliate mortality rates were species‐specific and affected by their specific growth rates (rmax), cell volume, and ability to encyst. Cyst‐forming species had, on average, higher δmax than species unable to encyst. Maximum mortality rates were positively related to rmax, but, in contrast to the latter, δmax appeared unaffected by temperature. I conclude that (1) in the ocean, physiological mortality is more critical for controlling ciliate population size than ciliate losses imposed by microcrustacean predation, but (2) in many lakes, the opposite holds; (3) cyst formation is an effective ciliate trait to cope with the high mortality of motile cells upon starvation. The lack of a temperature effect on δmax deserves further study; if correct, planktonic ciliates may take advantage of rising ocean and lake temperatures, with important implications for the pelagic food web.

Эффективность применения целлюлозолитического ферментного комплекса в составе энергокорма в питании молочных коров

The research was carried out on 2 groups of dairy cows of 30 heads each on a farm with the formation of technological groups according to calving dates and milking on a unit with individual milk yield accounting. The cows of the experimental group, in the period two weeks before calving and for 2 months after calving, were added to the diet, respectively, 150 and 300 ml/head/day of energy feed based on propylene glycol and glycerol with a complex of cellulase, xylase, β-gluconase and glucoamylase enzymes. activity (EFEC). The main diet consisted of traditional feed and was designed to produce an average daily milk yield of 25 kg of milk. EFEA had virtually no effect on the pH and ammonia content in rumen chyme, but increased the VFA content in it by 3.6 %. It had no effect on the total microbial mass, but reduced the ciliate population by 23.5 % and increased the bacterial population by 27.8 %. EFEC had a positive effect on protein-nitrogen metabolism, functional state of the liver, antioxidant activity, hormonal status and hematology. As a result, during 3 months of lactation, the average daily milk yield of cows in the experimental group was 19.9, 20.9 and 21.1 kg of milk (P<0.001), which was 13.0 higher than in the control. 9.4 and 16.5 %, respectively, but at the same time there was a decrease in fat and protein content in milk.

Temperature-dependent resistance to starvation of three contrasting freshwater ciliates

We investigated the temperature-dependent response to starvation of three contrasting freshwater ciliates (Ciliophora). The cyst-forming algivorous species Meseres corlissi and the bactivorous species Glaucomides bromelicola, which cannot form cysts, co-occur in the reservoirs (tanks) of tree bromeliads. The mixotrophic species Coleps spetai is common in many lakes. We hypothesized that the ciliates’ different traits and life strategies would affect their survival rates and temperature sensitivity under food depleted conditions. We measured the decline of the ciliate populations in microcosm experiments at different temperatures for several days. We used an imaging flow cytometer to size the ciliates and documented their morphological and physiological changes in response to starvation. We found that the cyst-forming species had the highest mortality rates but may endure long-term starvation by encystment. The sympatric, non-encysting species suffered the lowest mortality rates and could survive for more than three weeks without food. The mixotrophic species had intermediate mortality rates but showed the highest phenotypic plasticity in response to starvation. A significant fraction of the C. spetai population appeared unaffected by starvation, suggesting that the endosymbionts provided some resources to the host cells. The mean mortality rate per day of all three species increased with temperature by 0.09 °C−1.

Functional ecology of planktonic ciliates: Measuring mortality rates in response to starvation.

Population dynamics of aquatic ciliates are controlled "bottom-up" via food supply and "top-down" by grazing and parasitism. While intrinsic growth rates of ciliates under saturating food conditions have been studied in some detail, mortality rates induced by starvation have received little attention thus far. To this end, we examined the response of three algivorous freshwater ciliate species to starvation using three different optical methods. Two of these methods, i.e. ciliate mortality rates (δ) estimated from (i) numerical response experiments and (ii) the rate of decline (ROD) in cell numbers, investigated the response of the ciliate population using conventional light microscopy. The third method, imaging cytometry using a FlowCAM instrument, monitored single cells during the starvation experiment. Like light microscopy, the FlowCAM approach estimated δ based on ROD in the experimental containers. However, imaging cytometry also measured the relative cellular chlorophyll a content in the ciliates' food vacuoles as a proxy for the nutritional status of the cells. The linear decline of the cellular chl. a yielded an independent estimate of δ that was similar to δ calculated from ROD. Additionally, the FlowCAM measurements revealed a high degree of phenotypic plasticity of the ciliates when exposed to starvation.

Protists’ microbiome: A fine-scale, snap-shot field study on the ciliate Euplotes

Host-microbiome relationships play a fundamental role in the evolution and ecology of any living being. As unicellular organisms, protists represent a unique eukaryotic model to investigate selection mechanisms of the prokaryotic microbiome at the cellular level. Field investigations are central to disentangle relative importance of selective drivers in nature. Here we performed an analysis on data from a snap-shot field study reported previously on bacterial microbiomes associated to natural populations of protist ciliates of the genus Euplotes to detect at a fine scale any influence of habitat and/or host identity in microbiome selection. Comparative analyses revealed environment at a relatively large scale (sampling area) as the main driving factor in shaping prokaryotic communities’ structures. No evidence of habitat as key-factor emerged when a smaller spatial scale was considered (pond/channel or site). When only microbiomes of ciliates from the same site were compared, a clear assessment on the influence of host identity at the species level was not achieved, probably due to the small and unbalanced number of individuals for the two considered host species. Starting from this point, wider sampling campaigns will contribute in the future to depict a general view of the drivers influencing the prokaryotic microbiomes of natural protist populations.

First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora).

Hybridization is an important evolutionary process that can fuel diversification via formation of hybrid species or can lead to fusion of previously separated lineages by forming highly diverse species complexes. We provide here the first molecular evidence of hybridization in wild populations of ciliates, a highly diverse group of free-living and symbiotic eukaryotic microbes. The impact of hybridization was studied on the model of Plagiotoma, an obligate endosymbiont of the digestive tube of earthworms, using split decomposition analyses and species networks, 2D modeling of the nuclear rRNA molecules and compensatory base change analyses as well as multidimensional morphometrics. Gene flow slowed down and eventually hampered the diversification of Lumbricus-dwelling plagiotomids, which collapsed into a single highly variable biological entity, the P. lumbrici complex. Disruption of the species boundaries was suggested also by the continuum of morphological variability in the phenotypic space. On the other hand, hybridization conspicuously increased diversity in the nuclear rDNA cistron and somewhat weakened the host structural specificity of the P. lumbrici complex, whose members colonize a variety of phylogenetically closely related anecic and epigeic earthworms. By contrast, another recorded species, P. aporrectodeae sp. n., showed no signs of introgression, no variability in the rDNA cistron, and very high host specificity. These contrasting eco-evolutionary patterns indicate that hybridization might decrease the alpha-diversity by dissolving species boundaries, weaken the structural host specificity by broadening ecological amplitudes, and increase the nuclear rDNA variability by overcoming concerted evolution within the P. lumbrici species complex.

Top-down control of planktonic ciliates by microcrustacean predators is stronger in lakes than in the ocean

Planktonic ciliates are major components of pelagic food webs in both marine and freshwaters. Their population dynamics are controlled ‘bottom-up’ by prey availability and ‘top-down’ by microcrustacean predators. In oceans, copepods are the main ciliate predators while in lakes cladocerans are the typical predators. The efficacy by which these functionally different predators control ciliate population dynamics is debated. We, therefore, investigated experimentally the grazing of three microcrustacean predators with different feeding modes on five freshwater ciliates. We then performed a meta-analysis to assess if our findings can be generalised for aquatic ecosystems. We hypothesized that top-down control is stronger in lakes than in the ocean. We find that: (i) average ingestion rates of marine and freshwater microcrustaceans do not differ; (ii) clearance rates of freshwater cladocerans decrease with ciliate size but increase with ciliate size in freshwater copepods; (iii) clearance rates of the marine microcrustaceans is unrelated to ciliate cell size. These findings have implications for the functioning of freshwater and marine food webs: (i) the ciliate—microcrustacean link is stronger in lakes than in the ocean, and (ii) globally top-down control of ciliates is unlikely in the ocean.

The combined effects of microplastics and the heavy metal cadmium on the marine periphytic ciliate Euplotes vannus

Microplastics could be grazed by marine organisms and possibly transferred to higher trophic levels along the microbial loop. Due to their size and capacity to concentrate heavy metals that trigger joint toxic effects, microplastics (MPs) have already become a severe threat to marine organisms. The detrimental effects of MPs on large marine organisms have been studied, but the combined toxicity of MPs and cadmium (Cd) on protozoan ciliates remains unclear. In the present study, we selected different diameters and concentrations of polystyrene microspheres (PS-MPs) and Cd2+ as model MPs and heavy metals to evaluate their single and combined effects on the periphytic marine ciliate Euplotes vannus in relation to carbon biomass and oxidative stress. The MPs were indeed ingested by Euplotes vannus and significantly reduced the abundance and carbon biomass of ciliate populations. Combined exposure to MPs and Cd2+ not only increased the bioaccumulation of Cd2+ in ciliates but also exacerbated the decrease in ciliate biomass by increasing oxidative stress and membrane damage. In comparison, the effects of nano-sized plastics (0.22 μm) were more harmful than those of micro-sized plastics (1.07 μm, 2.14 μm and 5.00 μm). A smaller size represents a higher potential for penetrating biological members and a stronger adsorption capacity for cadmium. These results provide new insight into the combined toxicity of microplastics and heavy metals on ciliated protozoa and lay a foundation for higher trophic levels and ecosystems.

Amitosis as a strategy of cell division—Insight from the proliferation of Tetrahymena thermophila macronuclei

Cell division is a necessity of life which can be either mitotic or amitotic. While both are fundamental, amitosis is sometimes considered a relic of little importance in biology. Nevertheless, eukaryotes often have polyploid cells, including cancer cells, which may divide amitotically. To understand how amitosis ensures the completion of cell division, we turn to the macronuclei of ciliates. The grand scheme governing the proliferation of the macronuclei of ciliate cells, which involves chromosomal replication and amitosis, is currently unknown, which is crucial for developing population genetics model of ciliate populations. Using a novel model that encompasses a wide range of mechanisms together with experimental data of the composition of mating types at different stages derived from a single karyonide of Tetrahymena thermophila, we show that the chromosomal replication of the macronucleus has a strong head-start effect, with only about five copies of chromosomes replicated at a time and persistent reuse of the chromosomes involved in the early replication. Furthermore the fission of a fully grown macronucleus is non-random with regard to chromosome composition, with a strong tendency to push chromosomes and their replications to the same daughter cell.

How do microalgae in response to biological pollution treat in cultivation? A case study investigating microalgal defense against ciliate predator Euplotes vannus.

Microalgae have significant amounts of proteins, lipids, carotenoids, vitamins, minerals, and unique pigments. However, with the gradual expansion of microalgae cultivation, hostile biological pollution seriously restricted the large-scale microalgae cultivation and limited the exploitation of its biological resources. Moreover, protozoan poses the greatest threat to microalgae cultivation. Here, the relationship between six marine economic microalgae populations and their ciliate predator Euplotes vannus was examined. And four concentrations were designed for each type of microalgae to carry out the experiment. It was revealed that four species of microalgae inhibit the ciliate population growth at high density. Furthermore, the experiment which was the influence of microalgae at three different growth stages on the growth of the ciliates for these four kinds of high-density inhibitory microalgae was designed. The microalgae inhibitory effects were already exhibited at the end of the exponential growth phase, and it was significantly inhibited during the stationary growth phase. As the microalgae concentration increased, the inhibitory effect became more pronounced. This study provides fundamental data for screening protozoan-inhibiting microalgae and shows potential to be used in algae cultivation.

Chronic toxicity of new pyridine-n-oxide derivatives for tetrahymena pyriformis w, determination of “structure-toxicity”, “concentration-time-effect” correlation

There is a considerable amount of work in the scientific literature on nonlinear (“paradoxical”) effects, but their toxicological significance for the body is still poorly understood. Plant growth regulators are biologically active substances at the level of low and ultra-low doses and concentrations, they are widely used in agriculture. To date, the mechanisms of their toxic and specific effects on the body, the presence of “paradoxical” effects at low doses have not been sufficiently studied. Thus, it is important to further indepth study of the “concentration-time-effect” relationship under chronic exposure to the body, which will be important for understanding the general biological processes, developing approaches to hygienic regulation and risk assessment for human health and non-target environmental objects. The Aim of the Research. Determine the dependence of “structure-toxicity”, “concentration-time-effect” of plant growth regulators – pyridine-N-oxide derivatives under conditions of chronic effects on the body of ciliates Tetrahymena pyriformis W. Materials and Methods. The object of research was complexes of methyl derivatives of pyridine-N-oxide with organic acids and metal salts. Studies were performed on ciliates of Tetrahymena pyriformis W in the stationary phase of growth. The chronic effect of plant growth regulators on ciliates was studied in a 96-hour experiment in the concentration range from 1×10 -2 M to 1×10-28 M. The initial number of ciliates in the sample was 20,000 cells/ml of culture medium. The samples were incubated in a dry air thermostat at 25°C. After 24, 48, 72, 96 hours, the number of ciliates in different growth phases was determined. The experiments were performed in four replicates. The results of the research were subjected to mathematical processing by methods of variation statistics using the standard software package Microsoft® Office Excel 2010. Results. It is shown that at a concentration of 1×10-2 M investigated complexes of methyl derivatives of N-oxidepyridine with metal salts are more toxic than with organic acids. In the range of concentrations from 1×10-4 M to 1×10-28 M dependence "concentration-timeeffect" was not detected. Regardless of the concentration and duration of action, both inhibition and induction of ciliate population growth were observed. The change in the direction of the effect was observed at the level of high, low and ultralow concentrations. Conclusions. 1. Under conditions of chronic exposure to the population of ciliates Tetrahymena pyriformis W, the rate of growth inhibition at a concentration of 1×10-2 M, studied complexes DG-377 (Di-2,6-dimethylpyridine-N-oxide with ZnCl2), DG-380 (Di-2,6-dimethylpyridine-N-oxide with ZnI2), DG-387 (Di-2,6-dimethylpyridine-N-oxide with CoCl2), DG-480 (2-methylpyridineN-oxide with CoCl2), DG-471 (Di-2-methylpyridine-N-oxide with CoI2) are more toxic than similar complexes with organic acids – DG-361a (2,6-dimethylpyridine-N-oxide with succinic acid), DG-361 (Di-2,6-dimethylpyridine-N- with succinic acid), DG-362 (2,6-dimethylpyridine-N-oxide with maleic acid), DG-349a (2-methylpyridine-N-oxide with succinic acid). 2. In the range of concentrations from 1×10-4 M to 1×10 -28 M, regardless of the concentration and time of action, there is both inhibition and induction of growth of the ciliate population. The change in the direction of the effect was observed at the level of high, low and ultralow concentrations. The growth of the ciliate population, depending on the concentrations of the studied substances and the time of exposure, was polymodal in nature. 3. According to the direction and severity of the effect on the growth of the ciliate population for DG-361a, DG-361, DG-377, DG-387, DG-349a and DG-480 is characterized mainly by an inhibitory effect. The stimulating effect was observed in some phases of growth and was moderate or weak. For DG-362, DG-380 and DG-471 is characterized by a pronounced stimulation of population growth of ciliates. Key Words: methyl derivatives of pyridine-N-oxide, ciliates, “structure-toxicity”, “concentration-time-effect”.

Potential to resist biological contamination in marine microalgae culture: Effect of extracellular substances of Nannochloropsis oceanica on population growth of Euplotes vannus and other protozoa

The commercially important marine microalgae Nannochloropsis oceanica is easily ingested by protozoan predators during large-scale cultivation. However, investigations into the effect of microalgae on the growth of protozoa are scant. A feeding experiment was conducted with Euplotes vannus grazing on different concentrations of N. oceanica. The ciliate population was significantly lower in the high concentration of algae than that in the low or medium algal concentration treatments. The density of ciliates cultured in algae filtrate media was significantly lower than that in lysate media and the blank control. Furthermore, the algal cell filtrate was added to three other protozoan populations, and they all gradually lost their ability to move and their body shape changed. This study investigated the interactions between N. oceanica and protozoan predators and provides insight on using microalgal extracellular substances to control biological contamination in the future.

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.

Ecophysiological traits of mixotrophic Strombidium spp

Abstract Ciliates represent an important trophic link between nanoplankton and mesoplankton. Many species acquire functional chloroplasts from photosynthetic prey, being thus mixotrophs. Little is known about which algae they exploit, and of the relevance of inorganic carbon assimilation to their metabolism. To get insights into these aspects, laboratory cultures of three mixotrophic Strombidium spp. were established and 35 photosynthetic algal species were tested as prey. The relative contributions of ingestion and photosynthesis to total carbon uptake were determined, and responses to prey starvation were studied. Ciliate growth was supported by algal species in the 2–12 μm size range, with cryptophytes and chlorophytes being the best prey types. Inorganic carbon incorporation was only quantitatively important when prey concentration was low (3–100 μgCL−1), when it led to increased gross growth efficiencies. Chla specific inorganic carbon uptake rates were reduced by 60–90% compared to that of the photosynthetic prey. Inorganic carbon uptake alone could not sustain survival of cultures and ciliate populations declined by 25–30% during 5 days of starvation. The results suggest that mixotrophy in Strombidium spp. may substantially bolster the efficiency of trophic transfer when biomass of small primary producers is low.

Community structure and vertical distribution of planktonic ciliates in the saline meromictic lake Shira during breakdown of meromixis

The study deals with the vertical distribution and seasonal dynamics of planktonic ciliates in the pelagic and littoral zones of a saline meromictic Lake Shira. Fourteen species of free-living ciliates have been found in the Lake, seven of them inhabiting the pelagic zone. The richness of ciliates is higher both in terms of the number of species and biomass in the littoral zone compared to the pelagic zone. Although the ciliate species diversity is low in the pelagic zone, in certain seasons, the biomass of some of the species may reach considerable values, up to 3.4 g m−2 in the water column. The biomass of ciliates in Lake Shira is generally comparable to the average values for other mesotrophic lakes. The abundance and composition of ciliate populations in Lake Shira vary considerably with depth.Changes in the Lake ecosystem caused by meromixis breakdown in 2015-2016, which induced considerable variations in many Lake components, affected the vertical distribution of planktonic ciliates. However, the annual average biomass of the species that inhabited the Lake before meromixis breakdown remained unchanged. The total ciliate biomass increased due to the presence of the new species.

Oxygen Gradients and Structure of the Ciliate Assemblages in Floodplain Lake

This paper presents the results of studies on the structure of the ciliate population in a freshwater lake. The classification of the ciliated communities based on the analysis of the distribution of ciliate population density in the lake along the oxygen gradients, taking into account their oxygen preferences, was proposed. It was shown that the distribution of ciliated protozoa in the space of a reservoir is determined not by such spatial units as the water column, bottom, and periphytal, but by the oxygen gradients. Four types of habitats with different oxygen regimes were distinguished: With stably high oxygen concentration, stably low oxygen concentration, stably oxygen-free conditions, and conditions with a high amplitude of diurnal oxygen variations. The location of these habitats in the space of the lake and their seasonal changes were determined. On the basis of the quantitative development of ciliate populations, zones of optima and tolerance ranges of some ciliate species in the oxygen gradient were established. The oxygen preferences were established for the species from four distinguished assemblages: Microoxyphilic, oxyphilic, euryoxyphilic, and anoxyphilic (anaerobic). The presence or the absence of a certain type of assemblage in the reservoirs depends solely on the parameters of the oxygen gradients. The diversity of the ciliated protozoa in water bodies also depends on the stability and diversity of the oxygen gradients.

Abstract B09: Single-cell RNA sequencing of normal endometrial organoids uncovers novel cell-type markers for prognostication of primary tumor samples

Abstract Endometrial epithelium gives rise to both endometrial and ovarian cancers (of clear-cell and endometrioid subtypes), the latter arising from ectopic endometrium (endometriosis). Endometrial epithelium comprises mainly secretory cells, with a minor ciliated cell population. Due to their scarcity, little is known about the biology or function of endometrial ciliated cells. To understand the biology of endometrial epithelium, and by extension the cancers that arise from it, organoids derived from normal endometrial tissue were cultured. Notch signaling inhibitors were used to induce ciliated cell differentiation. Through single-cell RNA sequencing, distinct secretory and ciliated cell populations were observed, with the ciliated cell population increasing with Notch signaling inhibition. Many novel markers of ciliated cells were observed, but no highly specific markers of secretory cell differentiation. A marker of secretory cells (MST) and several markers of ciliated cells (FAM92B, WDR16 and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. In endometrial tumors, both MST and FAM92B exhibited diffuse staining and were markers of better prognosis. This suggests that tumors expressing differentiation markers have better prognosis, whether it is a marker of secretory or ciliated cells. Interestingly, a small number of endometrial tumors stained positive for DYDC2; however, these tumors exhibited a variable staining pattern with 25-50% tumor cells staining intensely, and the remaining tumor cells not staining at all. A similar variable staining pattern had been observed previously with CTH, another ciliated cell marker. Endometrial and ovarian tumor tissue microarrays were stained with DYDC2, CTH and two ciliated cell markers, FOXJ1 and p73. For all these markers, a subset of tumors displayed a variable staining pattern and for endometrial cancers, the variable staining was a good prognostic indicator. Single-cell sequencing of endometrial tumors has been able to capture these two populations of tumor cells. In ovarian tumors, only variable CTH staining was a significant prognostic indicator. Normal endometrial secretory cells are able to differentiate into ciliated cells, and the variable staining pattern suggests that a subset of tumors retains this ability, and these are clinically less aggressive. Using single-cell sequencing technology on normal tissues to guide development of prognostic markers and provide insight into the biology of the tumors arising from these tissues may be useful for many other tumor types. Citation Format: Dawn R. Cochrane, Kieran R. Campbell, Kendall Greening, Germain C. Ho, James Hopkins, Minh Bui, Vassilena Sharlandjieva, Daniel Lai, Maya DeGrood, Evan W. Gibbard, Samuel Leung, Angela S. Cheng, Jamie L.P. Lim, Samantha Neilson, David Farnell, Friedrich Kommoss, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. Single-cell RNA sequencing of normal endometrial organoids uncovers novel cell-type markers for prognostication of primary tumor samples [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B09.

Predation on planktonic ciliates in Kenyan soda lakes

Ciliated protozoa are important components of the plankton of aquatic systems with ability to recycle nutrients and to link microbial food webs to metazoans. This is because they are numerous and have higher growth rates compared to other unicellular eukaryotes of similar size. Predation and competition are some of the factors that negatively affect their abundance and growth. Studies documenting the impact of predation by zooplankton on ciliates have been conducted in marine and freshwaters waters but are lacking in tropical waters especially soda lakes in Kenya. In this study, growth rates of planktonic ciliates were estimated in four soda lakes, Lakes Bogoria, Elmenteita, Simbi and Sonachi, using predator exclusion experiments. In the experiments, ciliate populations increased faster in incubations of lake water with the &amp;gt;40 µm fraction removed than in untreated controls. The difference in these rates was taken as an estimate of predation by zooplankton &amp;gt;40 µm (Mz), and ranged from 0.091 to 3.171 d−1. Our best estimates of growth rates for particular ciliates abundant enough to derive estimates for ranged from 0.18 to 4.78 d−1. When removing the &amp;gt;40 µm fraction did not result in increased numbers of ciliates relative to controls, we hypothesized that this was due to predators within the &amp;lt;40 µm fraction (Mc) and their release from zooplankton predators. We assessed and supported this hypothesis by looking at the production of ciliates in different feeding guilds. The significance of predation on ciliates to the food webs of soda lakes is discussed.