First record of Colpodidium caudatum (Ciliophora, Colpodidiidae) in Europe.

The present study documents the discovery of the first European population of Colpodidium caudatum (Ciliophora, Colpodidiidae) in a water drain in a school playground in Manzanares (Ciudad Real, Spain). This species has been documented on every continent except Antarctica and Europe, until now. The ciliate was isolated from wet run-off soil collected from the water drain and was grown in semi-permanent cultures in the laboratory. The infraciliature of the ciliate was revealed using silver carbonate impregnation and cell measurements were taken from living and silver-impregnated specimens. A comparative analysis of published data from various populations of C. caudatum across the globe showed high intraspecific morphological variability in this species. To differentiate between species within the Colpodidium genus, a dichotomous key is presented. This investigation shows that C. caudatum is a ciliate that is found all over the world and is particularly associated with terrestrial habitats that are periodically flooded.

The Extraordinarily Rare Ciliate Legendrea loyezae Fauré-Fremiet, 1908 (Haptoria, Ciliophora).

Diverse and dynamic communities of ciliates and other microbes thrive in the natural environment, driving the functioning of aquatic ecosystems. Many microbes are present in very low numbers or are dormant in the 'seedbank', escaping detection in environmental surveys and, consequently, remaining underexplored. Here, we report an extraordinarily rare ciliate that was discovered after persistent exploration of freshwater anoxic sediments - Legendrea loyezae Fauré-Fremiet, 1908, a member of the Family Spathidiidae, Order Haptorida. In this study, we present the sixth account of the ciliate since 1908 and reveal its phylogenetic position with the first 18S rRNA data for the genus. We explain the key morphological features of the species, describing a remarkable behaviour in which the ciliate "shapeshifts'' due to its ability of controlled full extension and retraction of its tube-like tentacles. Our results shed light on the similarity of L. loyezae to another ciliate that was first described as Legendrea bellerophon, later moved under a new genus and named Thysanomorpha bellerophon. We question the validity of this taxonomic decision and, based on morphological characters and tentacle movement, we propose moving T. bellerophon back under Legendrea. This study demonstrates how continued and persistent exploration of natural habitats lead to the discovery of microbial communities and species.

Effects of Copper and the Insecticide Cypermethrin on a Soil Ciliate (Protozoa: Ciliophora) Community.

Ciliated protozoa play important ecological roles in soils, yet few studies have investigated the effect of soil pollution on them. We determined the effect of copper (Cu) and cypermethrin on a soil ciliate community under microcosm conditions. Soils were treated with Cu or cypermethrin and the abundance and species richness of ciliates determined 15 days later. Cu treatment increased soil ciliates abundance at the highest concentration (960 mg kg-1), as did cypermethrin at a treatment of 160 mg kg-1. No negative effect on ciliate abundance was found for either substance due to increased numbers of tolerant species, particularly Homalogastra setosa and Chilodonella uncinata in the case of Cu and Colpoda stenii and Colpoda inflata for cypermethrin treatments. However, several species were absent at high treatment levels. Notably, Halteria grandinella was not found in Cu treatments above 240 mg kg-1, whilst Oxytricha setigera was not found in cypermethrin treatments above 160 mg kg-1. For Homalogastra setosa, there was an initial positive response to cypermethrin, but abundance then decreased at a treatment of 320 mg kg-1, and treatment at 640 mg kg-1 eradicated the species from the microcosms. Accordingly, both substances affected the structure of the soil ciliate community at high concentrations.

Cyanobacterial Abundance and Microcystin Profiles in Two Southern British Lakes: The Importance of Abiotic and Biotic Interactions.

Freshwater cyanobacteria blooms represent a risk to ecological and human health through induction of anoxia and release of potent toxins; both conditions require water management to mitigate risks. Many cyanobacteria taxa may produce microcystins, a group of toxic cyclic heptapeptides. Understanding the relationships between the abiotic drivers of microcystins and their occurrence would assist in the implementation of targeted, cost-effective solutions to maintain safe drinking and recreational waters. Cyanobacteria and microcystins were measured by flow cytometry and liquid chromatography coupled to tandem mass spectrometry in two interconnected reservoirs varying in age and management regimes, in southern Britain over a 12-month period. Microcystins were detected in both reservoirs, with significantly higher concentrations in the southern lake (maximum concentration >7 µg L-1). Elevated microcystin concentrations were not positively correlated with numbers of cyanobacterial cells, but multiple linear regression analysis suggested temperature and dissolved oxygen explained a significant amount of the variability in microcystin across both reservoirs. The presence of a managed fishery in one lake was associated with decreased microcystin levels, suggestive of top down control on cyanobacterial populations. This study supports the need to develop inclusive, multifactor holistic water management strategies to control cyanobacterial risks in freshwater bodies.

Morphology and Phylogeny of the Ciliate Psilotricha silvicola n. sp. (Alveolata, Ciliophora) from Woodland Soils in the United Kingdom.

The genus Psilotricha was established by Stein in 1859, with P. acuminata as the type species within the family Oxytrichidae. This species lacked a full description until it was re-discovered in 2001, showing that its morphological and morphogenetic characters confirmed the inclusion in the family Oxytrichidae. Since then, the genus Psilotricha has had a convoluted taxonomy despite the morphological evidence available. In this paper, we describe a new Psilotricha species, Psilotricha silvicola n. sp., from woodland soils in Southern England (United Kingdom). The morphology was investigated in live and protargol-impregnated specimens. Our findings show that P. silvicola n. sp. shares morphological characteristics with P. acuminata, including the distinctive cell shape and the long and sparse cirri. Phylogenetic analysis of the 18S rRNA gene places this new species within the family Oxytrichidae, nested apart from the family Psilotrichidae (which includes the genera Urospinula, Psilotrichides and Hemiholosticha), in a clade containing species of the family Oxytrichidae. Furthermore, the morphology of another Psilotricha species, P. viridis, found in a freshwater pond in the same woodland area, is also here described, bringing additional insight into the taxonomy of the genus. Our findings provide further evidence for inclusion of the genus Psilotricha within the oxytrichids.

Protistological science dissemination.

It is rare to meet protistologists who are not passionate about their study subject. The vast majority of people, however, never get the chance to hear about the work of these researchers. Although every researcher working on protists is likely to be aware of this situation, efforts made and tools employed for dissemination of knowledge are rarely documented. Following a proposal by the Italian Society of Protistology, a workshop at the 2019 VIII European Congress of Protistology in Rome, Italy, was dedicated to protistological knowledge dissemination. Through the many interventions, we discovered the diversity of efforts to reveal the protistan world to the general public, including museum exhibitions and activities, public understanding of science events, citizen science projects, specific book publications, the use of protists in teaching at all levels from primary school children to university undergraduate students, and to a global audience via social media. The participation of the workshop delegates in the discussions indicated that presentations on the wonderful world of protists to the public not only increase the visibility and accessibility of protistology research but are also very important for the scientific community. Here we report on some of the key aspects of the presentations given in the dissemination workshop.

First Records of 'Flagship' Soil Ciliates in North America.

'Flagship' ciliates were investigated from soil samples collected in Florida, USA. This was undertaken to determine if species thought to be restricted to a given world region could be uncovered from similar habitats in a novel location, e.g. another continent. Two species of Condylostomides were discovered, and recorded from the North American continent for the first time. Condylostomides etoschensis was known only from Africa, but was found to be thriving in a Florida study site. An 18S rDNA sequence for this species was determined for the first time. Also discovered from the same study site was the ciliate Condylostomides coeruleus, previously known only from Central and South America. These two 'flagship' ciliates were found in the same habitat, from a continent well outside of their previously recorded biogeographies. Molecular sequencing and microscopy investigations were conducted to form the baseline for future work within this genus. Soil ciliates can obtain large population numbers and form cysts and are therefore likely able to disperse globally. These new records provide additional evidence that large distances, even between continents, do not hinder microbes from thriving globally. The absence of these conspicuously-colored gold and blue ciliates from previous studies is likely due to undersampling, rather than to any physical barriers.

Convergent Evolution of Hydrogenosomes from Mitochondria by Gene Transfer and Loss.

Hydrogenosomes are H2-producing mitochondrial homologs found in some anaerobic microbial eukaryotes that provide a rare intracellular niche for H2-utilizing endosymbiotic archaea. Among ciliates, anaerobic and aerobic lineages are interspersed, demonstrating that the switch to an anaerobic lifestyle with hydrogenosomes has occurred repeatedly and independently. To investigate the molecular details of this transition, we generated genomic and transcriptomic data sets from anaerobic ciliates representing three distinct lineages. Our data demonstrate that hydrogenosomes have evolved from ancestral mitochondria in each case and reveal different degrees of independent mitochondrial genome and proteome reductive evolution, including the first example of complete mitochondrial genome loss in ciliates. Intriguingly, the FeFe-hydrogenase used for generating H2 has a unique domain structure among eukaryotes and appears to have been present, potentially through a single lateral gene transfer from an unknown donor, in the common aerobic ancestor of all three lineages. The early acquisition and retention of FeFe-hydrogenase helps to explain the facility whereby mitochondrial function can be so radically modified within this diverse and ecologically important group of microbial eukaryotes.

Cosmopolitan Metapopulations?

A "metapopulation" is a group of populations of the same species separated by space but linked by dispersal and migration. Metapopulations of macroscopic organisms tend to have geographically-restricted distributions, but this does not seem to be the case in microbial eukaryotes due to their astronomical abundance. The term "metapopulation" was first applied to protists' biogeography in the article Finlay and Fenchel (2004), published in PROTIST, which contributed to the popularity of the paper. The article considered protist species as consisting of a single, cosmopolitan population. Here, we recall this paper, and assess developments during the last 15 years with respect to the question of protist species distribution on the surface of the earth.

Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records.

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1mm in size, could have a global biogeographical distribution.

Morphology and Phylogeny of a New Species of Anaerobic Ciliate, Trimyema finlayi n. sp., with Endosymbiotic Methanogens.

Many anaerobic ciliated protozoa contain organelles of mitochondrial ancestry called hydrogenosomes. These organelles generate molecular hydrogen that is consumed by methanogenic Archaea, living in endosymbiosis within many of these ciliates. Here we describe a new species of anaerobic ciliate, Trimyema finlayi n. sp., by using silver impregnation and microscopy to conduct a detailed morphometric analysis. Comparisons with previously published morphological data for this species, as well as the closely related species, Trimyema compressum, demonstrated that despite them being similar, both the mean cell size and the mean number of somatic kineties are lower for T. finlayi than for T. compressum, which suggests that they are distinct species. This was also supported by analysis of the 18S rRNA genes from these ciliates, the sequences of which are 97.5% identical (6 substitutions, 1479 compared bases), and in phylogenetic analyses these sequences grouped with other 18S rRNA genes sequenced from previous isolates of the same respective species. Together these data provide strong evidence that T. finlayi is a novel species of Trimyema, within the class Plagiopylea. Various microscopic techniques demonstrated that T. finlayi n. sp. contains polymorphic endosymbiotic methanogens, and analysis of the endosymbionts' 16S rRNA gene showed that they belong to the genus Methanocorpusculum, which was confirmed using fluorescence in situ hybridization with specific probes. Despite the degree of similarity and close relationship between these ciliates, T. compressum contains endosymbiotic methanogens from a different genus, Methanobrevibacter. In phylogenetic analyses of 16S rRNA genes, the Methanocorpusculum endosymbiont of T. finlayi n. sp. grouped with sequences from Methanomicrobia, including the endosymbiont of an earlier isolate of the same species, 'Trimyema sp.,' which was sampled approximately 22 years earlier, at a distant (∼400 km) geographical location. Identification of the same endosymbiont species in the two separate isolates of T. finlayi n. sp. provides evidence for spatial and temporal stability of the Methanocorpusculum-T. finlayi n. sp. endosymbiosis. T. finlayi n. sp. and T. compressum provide an example of two closely related anaerobic ciliates that have endosymbionts from different methanogen genera, suggesting that the endosymbionts have not co-speciated with their hosts.

Trophic positioning of meiofauna revealed by stable isotopes and food web analyses.

Despite important advances in the ecology of river food webs, the strength and nature of the connection between the meio- and macrofaunal components of the web are still debated. Some unresolved issues are the effects of the inclusion of meiofaunal links and their temporal variations on the overall river food web properties, and the significance of autochthonous and allochthonous material for these components. In the present study, we conducted analyses of gut content of macro- and meiofauna and stable isotope analyses of meiofauna to examine seasonal food webs of a chalk stream. The results of the gut content analyses, confirmed by the δ13 C signatures, revealed a seasonal shift from a dependence on autochthonous (biofilm) to allochthonous food sources. Here, we demonstrate that aggregating basal or meiofaunal species into single categories affects key web properties such as web size, links, linkage density, and predator-prey ratios. More importantly, seasonal variation in attributes characterized the entire web and these changes persist regardless of taxonomic resolution. Furthermore, our analyses evidenced discrete variations in δ15 N across the meiofauna community with a trophic structure that confirms gut content analyses, placing the meiofauna high in the food web. We, therefore, conclude that small-body-sized taxa can occur high in dynamic river food webs, questioning assumptions that trophic position increases with body size and that webs are static.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry.

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 µM after 30 min of incubation and the orange nucleic acid probe at 1 µM after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 µM concentrations and higher in both probes exhibited a type of non-specific staining, whereby 'live' cells produced a target fluorescence, leading to an over representation of 'non-viable' cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Exploiting eco-physiological niche to facilitate the separation of the freshwater cyanobacteria Microcystis sp. and Synechococcus sp.

In a novel approach to separate the co-occurring freshwater cyanobacteria Microcystis and Synechoccous, published ecological characteristics are used to manipulate temperature and nutrient concentrations to successfully establish a unialgal Microcystis strain. The simple protocol has implications for future cyanobacterial culturing approaches and the establishment of new cyanobacteria strains.

The First Record for the Americas of Loxodes rex, a Flagship Ciliate with an Alleged Restricted Biogeography.

As the foundations of food webs, protozoa are essential to the success of an ecological system. These organisms are often overlooked, and research in the Americas is sparse. Recent samplings conducted in freshwater canals and ponds in Florida, USA, have revealed Loxodes rex, an alleged endemic ciliate species. Originally described as endemic to tropical Africa, L. rex has been considered a prime candidate for proof of microbial endemism. Our studies have shown this giant, non-encysting ciliate to be thriving in subtropical Florida. Our observations are novel and include both the first record of occurrence for the Americas and the first high-quality in vivo images for this charismatic species.

Local ciliate communities associated with aquatic macrophytes.

This study, based within the catchment area of the River Frome, an important chalk stream in the south of England, compared ciliated protozoan communities associated with three species of aquatic macrophyte common to lotic habitats: Ranunculus penicillatus subsp. pseudofluitans, Nasturtium officinale and Sparganium emersum. A total of 77 ciliate species were counted. No species-specific ciliate assemblage was found to be typical of any one plant species. Ciliate abundance between plant species was determined to be significantly different. The ciliate communities from each plant species were unique in that the number of species increased with ciliate abundance. The community associated with R. penicillatus subsp. pseudofluitans showed the highest consistency and species richness whereas S. emersum ciliate communities were unstable. Most notably, N. officinale was associated with low ciliate abundances and an apparent reduction in biofilm formation, discussed herein in relation to the plant's production of the microbial toxin phenethyl isothiocyanate. We propose that the results reflect differences in the quantity and quality of biofilm present on the plants, which could be determined by the different plant morphologies, patterns of plant decay and herbivore defense systems, all of which suppress or promote the various conditions for biofilm growth.

Achieving temperature-size changes in a unicellular organism.

The temperature-size rule (TSR) is an intraspecific phenomenon describing the phenotypic plastic response of an organism size to the temperature: individuals reared at cooler temperatures mature to be larger adults than those reared at warmer temperatures. The TSR is ubiquitous, affecting >80% species including uni- and multicellular groups. How the TSR is established has received attention in multicellular organisms, but not in unicells. Further, conceptual models suggest the mechanism of size change to be different in these two groups. Here, we test these theories using the protist Cyclidium glaucoma. We measure cell sizes, along with population growth during temperature acclimation, to determine how and when the temperature-size changes are achieved. We show that mother and daughter sizes become temporarily decoupled from the ratio 2:1 during acclimation, but these return to their coupled state (where daughter cells are half the size of the mother cell) once acclimated. Thermal acclimation is rapid, being completed within approximately a single generation. Further, we examine the impact of increased temperatures on carrying capacity and total biomass, to investigate potential adaptive strategies of size change. We demonstrate no temperature effect on carrying capacity, but maximum supported biomass to decrease with increasing temperature.

New records of the ectoparasitic flagellate Colpodella gonderi on non-Colpoda ciliates.

Colpodella gonderi is the only ectoparasitic flagellate of ciliated protozoa described thus far. This investigation reveals new records of C. gonderi retrieved from soil samples in southern Scotland, UK. Of fourteen ciliates species identified in one single occasion, three of them, Colpoda steinii, Pseudoplatyophrya nana and Grossglockneria acuta, were infested with the parasite. These results provide further evidence that C. gonderi is not host-specific of the ciliate genus Colpoda.