Pseudovampyrella gen. nov.: A genus of Vampyrella-like protoplast extractors finds its place in the Leptophryidae.

Vampyrellid amoebae are predatory protists, which consume a variety of eukaryotic prey and inhabit freshwater, marine and terrestrial ecosystems. Although they have been known for almost 150 years, much of their diversity lacks an in-depth characterization. To date, environmental sequencing data hint at several uncharacterized lineages, to which no phenotype is associated. Furthermore, there are numerous historically described species without any molecular information. This study reports on two new vampyrellid strains from moorlands, which extract the protoplasts of Closterium species (Zygnematophyceae). Our data on morphology, prey range specificity and feeding strategy reveal that the studied vampyrellids are very similar to the historically described Vampyrella closterii. However, phylogenetic analyses demonstrate that the two strains do not belong to the genus Vampyrella and, instead, form a distinct clade in the family Leptophryidae. Hence, we introduce a new genus of algivorous protoplast extractors, Pseudovampyrella gen. nov., with the species P. closterii (= V. closterii) and P. minor. Our findings indicate that the genetic diversity of morphologically described vampyrellid species might be hugely underrated.

DNA metabarcoding focused on difficult-to-culture protists: An effective approach to clarify biological interactions.

DNA metabarcoding on a single organism is a promising approach to clarify the biological interactions (e.g., predator-prey relationships and symbiosis, including parasitism) of difficult-to-culture protists. To evaluate the effectiveness of this method, Radiolaria and Phaeodaria, which are ecologically important protistan groups, were chosen as target taxa. DNA metabarcoding on a single organism focused on the V9 region of the 18S rRNA gene revealed potential symbionts, parasites and food sources of Radiolaria and Phaeodaria. Previously reported hosts and symbionts (parasites) were detected, and newly recognized combinations were also identified. The contained organisms largely differed between Radiolaria and Phaeodaria. In Radiolaria, members of the same order tended to contain similar organisms, and the taxonomic composition of possible symbionts, parasites, and food sources was fixed at the species level. Members of the same phaeodarian family, however, did not contain similar organisms, and body part (i.e., the central capsule or the phaeodium) was the most important factor that divided the taxonomic composition of detected organisms, implying that the selection of appropriate body part is important when trying to ascertain contained organisms, even for unicellular zooplankton. Our results show that DNA metabarcoding on a single organism is effective in revealing the biological interactions of difficult-to-culture protists.

Metagenomic profiling pipelines improve taxonomic classification for 16S amplicon sequencing data.

Most experiments studying bacterial microbiomes rely on the PCR amplification of all or part of the gene for the 16S rRNA subunit, which serves as a biomarker for identifying and quantifying the various taxa present in a microbiome sample. Several computational methods exist for analyzing 16S amplicon sequencing. However, the most-used bioinformatics tools cannot produce high quality genus-level or species-level taxonomic calls and may underestimate the potential accuracy of these calls. We used 16S sequencing data from mock bacterial communities to evaluate the sensitivity and specificity of several bioinformatics pipelines and genomic reference libraries used for microbiome analyses, concentrating on measuring the accuracy of species-level taxonomic assignments of 16S amplicon reads. We evaluated the tools DADA2, QIIME 2, Mothur, PathoScope 2, and Kraken 2 in conjunction with reference libraries from Greengenes, SILVA, Kraken 2, and RefSeq. Profiling tools were compared using publicly available mock community data from several sources, comprising 136 samples with varied species richness and evenness, several different amplified regions within the 16S rRNA gene, and both DNA spike-ins and cDNA from collections of plated cells. PathoScope 2 and Kraken 2, both tools designed for whole-genome metagenomics, outperformed DADA2, QIIME 2 using the DADA2 plugin, and Mothur, which are theoretically specialized for 16S analyses. Evaluations of reference libraries identified the SILVA and RefSeq/Kraken 2 Standard libraries as superior in accuracy compared to Greengenes. These findings support PathoScope and Kraken 2 as fully capable, competitive options for genus- and species-level 16S amplicon sequencing data analysis, whole genome sequencing, and metagenomics data tools.

Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae).

BACKGROUND: Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents. These phagotrophic "protoplast feeders" represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. Although their fascinating feeding behaviour has been observed for the last 150 years, it is still unknown how protoplast feeders produce the well-defined and species-specific perforations in biochemically diverse cell walls. Differential expression analyses of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria) suggested the involvement of a highly expressed putative glycoside hydrolase of family GH5_5. To assess the importance of this carbohydrate-active enzyme in the feeding act of Orciraptor, we recombinantly produced its catalytic domain and studied the enzymatic activity, cellular localisation and function. RESULTS: The GH5_5 catalytic domain from Orciraptor showed pronounced activity on soluble cellulose derivatives and mixed-linkage glucans, with reaction optima comparable to known GH5_5 representatives. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. Immunocytochemistry with a polyclonal antibody raised against the GH5_5 domain revealed that the native endocellulase localises to the contact zone of Orciraptor and the algal cell wall (= perforation zone) and to intracellular granules, which were enriched during attack. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. CONCLUSIONS: Our experimental data from enzymatic assays, immunocytochemistry and inhibition experiments strongly suggest a key role of the GH5_5 endocellulase in cell wall dissolution by Orciraptor agilis. With that, we provide evidence that the well-defined perforations produced by protoplast feeders are caused by extracellular carbohydrate-active enzymes and made a first step towards establishing the molecular basis of a fascinating, yet poorly understood microbial feeding strategy.

Kinopus chlorellivorus gen. nov., sp. nov. (Vampyrellida, Rhizaria), a New Algivorous Protist Predator Isolated from Large-Scale Outdoor Cultures of Chlorella sorokiniana.

The large-scale culture of low-cost algal biomass can be significantly affected by microbial grazing on the algae. To minimize the impact, it is necessary to manage the predators. In this study, we describe a new genus and species of vampyrellid amoeba, Kinopus chlorellivorus, which caused the loss of Chlorella sorokiniana in large-scale cultures. We assigned it to the family Leptophryidae (Vampyrellida) based on morphology and small-subunit (SSU) rRNA gene sequence comparisons. Using transmission electron microscopy, we found spherical lucent inclusions, which have not been reported for any leptophryids or other vampyrellids. The gene sequence of SSU rRNA did not match any recognized genera or species and contained four characteristic regions. K. chlorellivorus preys on algae by engulfment. Laboratory feeding experiments confirmed that its grazing rate was as high as 131 Chlorella cells day-1 individual-1. Results of prey-range experiments demonstrated that it could consume other chlorophyte microalgae (e.g., Scenedesmus, Coelastrella, and Haematococcus) but with a strong feeding ability on Chlorella spp., with ingestion rates ranging from 2.67 to 3.15 prey predator-1 h-1 and growth rates of the amoeba ranging from 0.039 to 0.045 h-1. On the basis of its high grazing ability on Chlorella, capacity to form large populations in a short period of time, and capacity to form resistant resting stages, this contaminant has the potential to cause serious problems in large-scale Chlorella culture and should be of concern to operators of algal production facilities. IMPORTANCE The vampyrellids (Vampyrellida, Rhizaria) are a major group of predatory amoebae that have attracted significant attention because of their diversity of feeding strategies. The crucial roles they play in important processes such as suppressing soil disease and controlling aquatic algae, and as microbial contaminants in outdoor large-scale algal cultures, have also received increasing attention. In this study, a new genus and species of algivorous vampyrellid amoeba, Kinopus chlorellivorus, is described as a significant grazer responsible for losses in outdoor industrial Chlorella cultures. We found that the amoeba's detrimental effects on Chlorella cultures may be related to its specific feeding characteristics. This study provides phenotypic and genetic information on a previously unknown vampyrellid, emphasizes the impact of contaminating vampyrellids in commercial microalgal cultures, and will contribute to the development of management strategies for predicting this kind of contaminant in large-scale microalgal cultivation.

What Drives the Assembly of Plant-associated Protist Microbiomes? Investigating the Effects of Crop Species, Soil Type and Bacterial Microbiomes.

In a field experiment we investigated the influence of the environmental filters soil type (i.e. three contrasting soils) and plant species (i.e. lettuce and potato) identity on rhizosphere community assembly of Cercozoa, a dominant group of mostly bacterivorous soil protists. Plant species (14%) and rhizosphere origin (vs bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as 'protist microbiomes' in analogy to 'bacterial microbiomes'.

Hierarchical phylogenetic community assembly of soil protists in a temperate agricultural field.

Protists are abundant, diverse and perform essential functions in soils. Protistan community structure and its change across time or space are traditionally studied at the species level but the relative importance of the processes shaping these patterns depends on the taxon phylogenetic resolution. Using 18S rDNA amplicon data of the Cercozoa, a group of dominant soil protists, from an agricultural field in western Germany, we observed a turnover of relatively closely related taxa (from sequence variants to genus-level clades) across soil depth; while across soil habitats (rhizosphere, bulk soil, drilosphere), we observed turnover of relatively distantly related taxa, confirming Paracercomonadidae as a rhizosphere-associated clade. We extended our approach to show that closely related Cercozoa encounter divergent arbuscular mycorrhizal (AM) fungi across soil depth and that distantly related Cercozoa encounter closely related AM fungi across soil compartments. This study suggests that soil Cercozoa community assembly at the field scale is driven by niche-based processes shaped by evolutionary legacy of adaptation to conditions primarily related to the soil compartment, followed by the soil layer, giving a deeper understanding on the selection pressures that shaped their evolution.

Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation.

Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialized parasites. The unicellular "protoplast feeders" represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae and fungi) like some parasites but then devour their cell contents by phagocytosis.1 Besides prey recognition and attachment, this complex behavior involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure.2 Yet the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g., locomotion and cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g., lytic polysaccharide monooxygenases) belong to families that are largely uncharacterized, emphasizing the potential of non-fungal microeukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor agilis, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behavior and new directions for cell biology research on non-model eukaryotes.

Phylogenetic analysis confirms the taxonomic placement of the marine flagellate Hermesinum adriaticum (Thecofilosea, Cercozoa, Rhizaria).

Hermesinum adriaticum is a rare marine and brackish flagellate that is of considerable interest due to its markable and fossilizable siliceous skeleton. Based on this skeleton, Hermesinum was initially considered a microalga of the Dictyochophyceae (Ochrophyta, Stramenopiles). Later on, it was assigned to the Ebriida due to its similarity to Ebria tripartita. The taxonomic assignment of the Ebriida, however, changed several times until it was placed within the Thecofilosea (Cercozoa, Rhizaria), based on genetic data of E. tripartita. We sequenced the 18S marker gene sequence of Hermesinum and confirm the close relationship of Ebria and Hermesinum.

The Vampyrellid Amoebae (Vampyrellida, Rhizaria).

The Vampyrellida (Endomyxa, Rhizaria) is a group of free-living, predatory amoebae, which is most closely related to the Phytomyxea (plasmodiophorids and phagomyxids). It encompasses about 50 credibly described species that have a characteristic life history with the regular alternation of trophic amoebae and immobile digestive cysts. All known vampyrellid amoebae are naked and filose, but the different species display a broad morphological variety. Vampyrellids also vary greatly in their feeding habits, and range from generalist predators to specialized 'protoplast feeders' that exclusively feed on the cell contents of eukaryotic prey. They can be found in freshwater, soil and marine habitats, and appear to be globally distributed. Yet, the phenotypic diversity and ecological roles of the Vampyrellida are still poorly explored. Currently, there are eight well-recognized subclades that comprise four families (Vampyrellidae, Leptophryidae, Placopodidae and Sericomyxidae) as well as some lineages without any phenotypic information. Research on vampyrellids is challenging due to their cryptic occurrence in nature, intricate feeding habits that complicate cultivation, and a convoluted taxonomic history. Here, we review available information about cell structure, diversity, ecology, taxonomy and phylogenetics, and provide an up-to-date introduction to the Vampyrellida that may facilitate future research.

Transfer of the thecate amoebae Lecythium spinosum and Pamphagus armatus to Rhizaspis (Thecofilosea, Cercozoa, Rhizaria).

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. The captivating spine-bearing taxa in Thecofilosea were not yet molecularly characterized due to being very rare. There are only two known spine-bearing species, Pamphagus armatus and Lecythium spinosum, which were synonymized by Penard in 1902. Due to a morphological difference of those taxa, we discuss here that we disagree with this taxonomical act. We further isolated single cells of Pamphagus armatus directly from their habitat and successfully sequenced their SSU rDNA, which we subjected to phylogenetic analyses. We show that Pamphagus armatus branches within the Rhizaspididae (Tectofilosida, Thecofilosea). Accordingly, we transfer Pamphagus armatus and the assumingly closely related species Lecythium spinosum to Rhizaspis.

The wastewater protist Rhogostoma minus (Thecofilosea, Rhizaria) is abundant, widespread, and hosts Legionellales.

Wastewater is treated by concerted actions of the microbial communities within bioreactors. Although protists (unicellular eukaryotes) are good bioindicators and important players influencing denitrification, nitrification, and flocculation, they are the least known organisms in WWTPs. The few recent environmental surveys of the protistan diversity in WWTPs show that the most abundant protistan sequences in WWTPs belong to Thecofilosea (Rhizaria). We re-investigated previously published environmental sequencing data and gathered strains from seven WWTPs to determine which species dominate WWTPs worldwide. We found that all highly abundant thecofilosean sequences represent a single species - Rhogostoma minus. Considering that Thecofilosea are frequent hosts for Legionellales, i.e. bacteria linked to waterborne diseases, we confirm that Rhogostoma minus functions as a host for Legionellales in WWTPs. Whether the highly abundant Rhogostoma minus also serves as a host for known human pathogenic Legionellales requires further attention.

Description of the marine predator Sericomyxa perlucida gen. et sp. nov., a cultivated representative of the deepest branching lineage of vampyrellid amoebae (Vampyrellida, Rhizaria).

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D'Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage ("clade C") of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for "clade C."

On the phenology of protists: recurrent patterns reveal seasonal variation of protistan (Rhizaria: Cercozoa and Endomyxa) communities in tree canopies.

Tree canopies are colonized by billions of highly specialized microorganisms that are well adapted to the highly variable microclimatic conditions, caused by diurnal fluctuations and seasonal changes. In this study, we investigated seasonality patterns of protists in the tree canopies of a temperate floodplain forest via high-throughput sequencing with group-specific primers for the phyla Cercozoa and Endomyxa. We observed consistent seasonality, and identified divergent spring and autumn taxa. Tree crowns were characterized by a dominance of bacterivores and omnivores, while eukaryvores gained a distinctly larger share in litter and soil communities on the ground. In the canopy seasonality was largest among communities detected on the foliar surface: In spring, higher variance within alpha diversity of foliar samples indicated greater heterogeneity during initial colonization. However, communities underwent compositional changes during the aging of leaves in autumn, highly reflecting recurring phenological changes during protistan colonization. Surprisingly, endomyxan root pathogens appeared to be exceptionally abundant across tree canopies during autumn, demonstrating a potential role of the canopy surface as a physical filter for air-dispersed propagules. Overall, about 80% of detected OTUs could not be assigned to known species-representing dozens of microeukaryotic taxa whose canopy inhabitants are waiting to be discovered.

Identifying Potential Hosts of Short-Branch Microsporidia.

Microsporidia are obligate parasites that are closely related to Fungi. While the widely known "long-branch" Microsporidia infect mostly metazoans, the hosts of "short-branch" Microsporidia are only partially characterized or not known at all. Here, we used network analyses from Neotropical rainforest soil metabarcoding data, to infer co-occurrences between environmental lineages of short-branch microsporidians and their potential hosts. We found significant co-occurrences with several taxa, especially with Apicomplexa, Cercozoa, and Fungi, as well as some Metazoa. Our results are the first step to identify potential hosts of the environmental lineages of short-branch microsporidians, which can be targeted in future molecular and microscopic studies.

Saccharomycomorpha psychra n. g., n. sp., a Novel Member of Glissmonadida (Cercozoa) Isolated from Arctic and Antarctica.

A novel genus and species within the order Glissmonadida (Cercozoa, Rhizaria), Saccharomycomorpha psychra n. g., n. sp., is described from lichen in the Ny-Ålesund region (High Arctic) and moss in the Fildes peninsula of King George Island (Maritime Antarctica). Cells were spherical and did not appear to present flagella in organic-rich Potato Dextrose Agar medium where they were able to feed osmotrophically. Molecular phylogenetic analyses based on 18S rRNA gene sequence demonstrated that Saccharomycomorpha psychra belong to "clade T" within the order Glissmonadida (Cercozoa, Rhizaria). All three investigated strains could grow at 4 °C and had an optimum growth temperature of 12 °C, 20 °C, and 20 °C, while a maximum growth temperature of 20 °C, 20 °C, and 25 °C, respectively. In conclusion, we established the phenotypic identity of "clade T," which until now was exclusively detected by environmental sequences, and erect a new family Saccharomycomorphidae for "clade T." Nomenclatural, morphological and ecological aspects of this novel species are discussed.

Description of Phaeobola aeris gen. nov., sp. nov (Rhizaria, Cercozoa, Euglyphida) Sheds Light on Euglyphida's Dark Matter.

The majority of Euglyphida species are characterised by shells with imbricated silica scales. Environmental surveys indicate a large unexplored diversity and recent efforts hinted at a certain diversity of yet undescribed, inconspicuous, scale-lacking Euglyphida. Here we describe Phaeobola aeris gen. nov., sp. nov. that shows a variety of morphological characters typical for the Euglyphida but lacks silica scales-instead, this species bears an agglutinated test. Neither its morphology nor phylogenetic placement allows its assignment to any currently described family. We erected the yet monospecific genus Phaeobola gen. nov., which with yet available data remain Euglyphida incertae sedis.

The Photosynthetic Adventure of Paulinella Spp.

Paulinella photosynthetic species are unicellular, silica shell-forming amoebas classified into the supergroup Rhizaria. They crawl at the bottom of freshwater and brackish environments with the help of filose pseudopodia. These protists have drawn the attention of the scientific community because of two photosynthetic bodies, called chromatophores, that fill up their cells permitting fully photoautotrophic existence. Paulinella chromatophores, similarly to primary plastids of the Archaeplastida supergroup (including glaucophytes, red algae as well as green algae and land plants), evolved from free-living cyanobacteria in the process of endosymbiosis. Interestingly, these both cyanobacterial acquisitions occurred independently, thereby undermining the paradigm of the rarity of endosymbiotic events. Chromatophores were derived from α-cyanobacteria relatively recently 60-140 million years ago, whereas primary plastids originated from ß-cyanobacteria more than 1.5 billion years ago. Since their acquisition, chromatophore genomes have undergone substantial reduction but not to the extent of primary plastid genomes. Consequently, they have also developed mechanisms for transport of metabolites and nuclear-encoded proteins along with appropriate targeting signals. Therefore, chromatophores of Paulinella photosynthetic species, similarly to primary plastids, are true cellular organelles. They not only show that endosymbiotic events might not be so rare but also make a perfect model for studying the process of organellogenesis. In this chapter, we summarize the current knowledge and retrace the fascinating adventure of Paulinella species on their way to become photoautotrophic organisms.

Haplosporosomes, sporoplasmosomes and their putative taxonomic relationships in rhizarians and myxozoans.

This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa - Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.