Demystifying biotrophs: FISHing for mRNAs to decipher plant and algal pathogen-host interaction at the single cell level.

Plant-pathogen interactions follow spatial and temporal developmental dynamics where gene expression in pathogen and host undergo crucial changes. Therefore, it is of great interest to detect, quantify and localise where and when key genes are active to understand these processes. Many pathosystems are not accessible for genetic amendments or other spatially-resolved gene expression monitoring methods. Here, we adapt single molecule FISH techniques to demonstrate the presence and activity of mRNAs at the single-cell level using phytomyxids in their plant and algal host in lab and field material. This allowed us to monitor and quantify the expression of genes from the clubroot pathogen Plasmodiophora brassicae, several species of its Brassica hosts, and of several brown algae, including the genome model Ectocarpus siliculosus, infected with the phytomyxid Maullinia ectocarpii. We show that mRNAs are localised along a spatiotemporal gradient, thus providing a proof-of-concept of the usefulness of single-molecule FISH to increase knowledge about the interactions between plants, algae and phytomyxids. The methods used are easily applicable to any interaction between microbes and their algal or plant host, and have therefore the potential to rapidly increase our understanding of key, spatially- and temporally-resolved processes underpinning complex plant-microbe interactions.

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.

Marteilia cochillia is released into seawater via cockle Cerastoderma edule faeces.

Outbreaks of Marteilia cochillia have caused massive mortalities of common cockle, Cerastoderma edule, in some natural beds in Galicia (NW Spain) since 2012. The life cycle of Marteilia spp. is still unresolved and the most accepted hypothesis suggests that an additional host is involved. Researchers have assumed that sporangia are shed into the environment in the faeces, but details about this process have not been reported previously. Here, we report the massive liberation of Marteilia cochillia sporangia through the exhalant siphon into the environment, packaged as faeces. Using light microscopy observations on fresh samples, imprints and histology, we also describe a thick (ca. 5 µm) transparent envelope covering the sporangia that has not been reported previously. The massive release of encapsulated sporangia reported here ensures that millions of infective stages of M. cochillia cycle through the environment and become available for infection. The elucidation of the role played by the sporangia envelope would be of utmost importance for the understanding M. cochillia life cycle.

Enigmatic Phytomyxid Parasite of the Alien Seagrass Halophila stipulacea: New Insights into Its Ecology, Phylogeny, and Distribution in the Mediterranean Sea.

Marine phytomyxids represent often overlooked obligate biotrophic parasites colonizing diatoms, brown algae, and seagrasses. An illustrative example of their enigmatic nature is the phytomyxid infecting the seagrass Halophila stipulacea (a well-known Lessepsian migrant from the Indo-Pacific to the Mediterranean Sea). In the Mediterranean, the occurrence of this phytomyxid was first described in 1995 in the Strait of Messina (southern Italy) and the second time in 2017 in the Aegean coast of Turkey. Here we investigated, using scuba diving, stereomicroscopy, light and scanning electron microscopy, and molecular methods, whether the symbiosis is still present in southern Italy, its distribution in this region and its relation to the previous reports. From the total of 16 localities investigated, the symbiosis has only been found at one site. A seasonal pattern was observed with exceptionally high abundance (> 40% of the leaf petioles colonized) in September 2017, absence of the symbiosis in May/June 2018, and then again high infection rates (~ 30%) in September 2018. In terms of anatomy and morphology as well as resting spore dimensions and arrangement, the symbiosis seems to be identical to the preceding observations in the Mediterranean. According to the phylogenetic analyses of the 18S rRNA gene, the phytomyxid represents the first characterized member of the environmental clade "TAGIRI-5". Our results provide new clues about its on-site ecology (incl. possible dispersal mechanisms), hint that it is rare but established in the Mediterranean, and encourage further research into its distribution, ecophysiology, and taxonomy.

Food Choice Experiments Indicate Selective Fungivorous Predation in Fisculla terrestris (Thecofilosea, Cercozoa).

Thecofilosea is a class in Cercozoa comprising mainly freshwater inhabiting algivores. Since direct observation of amoeboid protists in soil is not possible, the prey spectra of their terrestrial relatives remain obscure. To test for grazing selectivity and the preferred prey of terrestrial thecofiloseans, we conducted a food choice experiment including yeasts and algae as prey. When being offered all food sources at once, the yeast cells were strongly reduced, whereas the abundance of the algae only slightly decreased. Since Fisculla terrestris thrives with fungal prey, it must be considered as a predator of eukaryotes with high preference for fungal cells.

Transcriptome Profiling of Bigelowiella natans in Response to Light Stress.

Bigelowiella natans is a marine chlorarachniophyte whose plastid was acquired secondarily via endosymbiosis with a green alga. During plastid evolution, the photosynthetic endosymbiont would have integrated with the host metabolic pathways. This would require the evolution and coordination of strategies to cope with changes in light intensity that includes changes in the expression of both endosymbiont and host-derived genes. To investigate the transcriptional response to light intensity in chlorarachniophytes, we conducted an RNA-seq experiment to identify differentially expressed genes following a 4-h shift to high or very-low light. A shift to high light altered the expression of over 2,000 genes, many involved with photosynthesis, PSII assembly, primary metabolism, and reactive-oxygen scavenging. These changes are an attempt to optimize photosynthesis and increase energy sinks for excess reductant, while minimizing photooxidative stress. A transfer to very-low light resulted in a lower photosynthetic performance and metabolic alteration, reflecting an energy-limited state. Genes located on the nucleomorph, the vestigial nucleus in the plastid, had few changes in expression in either light treatment, indicating this organelle has relinquished most transcriptional control to the nucleus. Overall, during plastid origin, both host and transferred endosymbiont genes evolved a harmonized transcriptional network to respond to a classic photosynthetic stress.

First report of vampyrellid predator-prey dynamics in a marine system.

We report for the first time the in situ dynamics of a vampyrellid in a marine system. A high sampling frequency (twice-weekly) was applied in a tropical eutrophic lagoon (Rio de Janeiro, Brazil) for 5 years (2012-2016). The vampyrellid Hyalodiscus sp. specifically fed on the diatom Chaetoceros minimus during a short time window (~3 months), although the prey was intermittently detected as the dominant phytoplanktonic species over a longer period (~1 year). A classic Lotka-Volterra predator-prey dynamic was observed between the two partners, with a significant modification of the short-term oscillations of the prey. Specific abiotic preferences (i.e., relatively low temperature, intermediate salinity, and stratified conditions) associated with prey availability seemed to define this narrow temporal window of occurrence. Our results suggest that vampyrellids can be ecologically relevant in marine pelagic systems, with their impact on planktonic dynamics strongly depending on complex interactions between both biotic and abiotic factors.

Two New Marine Species of Placopus (Vampyrellida, Rhizaria) That Perforate the Theca of Tetraselmis (Chlorodendrales, Viridiplantae).

Vampyrellids (Vampyrellida, Rhizaria) are a major group of predatory amoebae known primarily from freshwater and soil. Environmental sequence data indicate that there is also a considerable diversity of vampyrellids inhabiting marine ecosystems, but their phenotypic traits and ecology remain largely unexplored. We discovered algivorous vampyrellids of the filoflabellate morphotype in coastal habitats in Atlantic Canada, established cultures by single-cell isolation, and characterised three strains using light microscopy, SSU rRNA gene sequencing, feeding experiments and growth experiments at various salinities. These strains exhibit orange, discoid trophozoites with ventral filopodia, moving granules ("membranosomes"), and rolling locomotion, similar to freshwater species previously assigned to Hyalodiscus Hertwig & Lesser, but here moved to Placopus Schulze (due to homonymy with Hyalodiscus Ehrenberg). SSU rRNA gene phylogenies place our strains in two distinct positions within "lineage B3" (here referred to as Placopodidae). Based on these morphological, habitat and molecular data, we describe two new species, Placopus melkoniani sp. nov. and Placopus pusillus sp. nov., both of which feed on chlorophyte flagellates (Tetraselmis, Pyramimonas) and the cryptophyte Chroomonas. They perforate the theca of Tetraselmis to extract the protoplast, and thereby represent the first vampyrellids known to degrade the biochemically exotic cell wall of the Chlorodendrales (Chlorophyta, Viridiplantae).

Gazelletta kashiwaensis sp. nov. (Medusettidae, Phaeodaria, Cercozoa), Its Morphology, Phylogeny, Distribution, and Feeding Behavior.

A phaeodarian morphotype, characterized by the feet surrounded with forked pedal spines with anchor-like structures, was collected in the subtropical North Pacific. Considering the morphological and phylogenetic uniqueness, this morphotype is described as Gazelletta kashiwaensis sp. nov. The distribution of this new species is possibly affected by the Kuroshio Current. The feeding behavior of living phaeodarians was first filmed: the present new species floated in the water column stretching "protoplasmic webs" and collected diatoms by repeating the expansion and retraction of "pseudopodium-like tentacles".

Massive Protein Import into the Early-Evolutionary-Stage Photosynthetic Organelle of the Amoeba Paulinella chromatophora.

The endosymbiotic acquisition of mitochondria and plastids more than 1 Ga ago profoundly impacted eukaryote evolution. At the heart of understanding organelle evolution is the re-arrangement of the endosymbiont proteome into a host-controlled organellar proteome. However, early stages in this process as well as the timing of events that underlie organelle integration remain poorly understood. The amoeba Paulinella chromatophora contains cyanobacterium-derived photosynthetic organelles, termed "chromatophores," that were acquired more recently (around 100 Ma ago). To explore the re-arrangement of an organellar proteome during its integration into a eukaryotic host cell, here we characterized the chromatophore proteome by protein mass spectrometry. Apparently, genetic control over the chromatophore has shifted substantially to the nucleus. Two classes of nuclear-encoded proteins-which differ in protein length-are imported into the chromatophore, most likely through independent pathways. Long imported proteins carry a putative, conserved N-terminal targeting signal, and many specifically fill gaps in chromatophore-encoded metabolic pathways or processes. Surprisingly, upon heterologous expression in a plant cell, the putative chromatophore targeting signal conferred chloroplast localization. This finding suggests common features in the protein import pathways of chromatophores and plastids, two organelles that evolved independently and more than 1 Ga apart from each other. By combining experimental data with in silico predictions, we provide a comprehensive catalog of almost 450 nuclear-encoded, chromatophore-targeted proteins. Interestingly, most imported proteins seem to derive from ancestral host genes, suggesting that the re-targeting of nuclear-encoded proteins that resulted from endosymbiotic gene transfers plays only a minor role at the onset of chromatophore integration.

Grazing of leaf-associated Cercomonads (Protists: Rhizaria: Cercozoa) structures bacterial community composition and function.

Preferential food selection in protists is well documented, but we still lack basic understanding on how protist predation modifies the taxonomic and functional composition of bacterial communities. We conducted feeding trials using leaf-associated cercomonad Cercozoa by incubating them on a standardized, diverse bacterial community washed from plant leaves. We used a shotgun metagenomics approach to investigate the taxonomic and functional changes of the bacterial community after five days protist predation on bacteria. Predation-induced shifts in bacterial community composition could be linked to phenotypic protist traits. Protist reproduction rate, morphological plasticity and cell speed were most important in determining bacterial community composition. Analyses of co-occurrence patterns showed less complex correlations between bacterial taxa in the protist-grazed treatments with a higher proportion of positive correlations than in non-grazed controls, suggesting that predation reduced the influence of strong competitors. Protist predation influenced 14 metabolic core functions including membrane transport from which type VI secretion systems were in particular upregulated. In view of the functional importance of bacterial communities in the phyllosphere and rhizosphere of plants, a more detailed understanding of predator-prey interactions, changes in microbial composition and function, and subsequent repercussions on plant performance are clearly required.

Natural selection drove metabolic specialization of the chromatophore in Paulinella chromatophora.

BACKGROUND: Genome degradation of host-restricted mutualistic endosymbionts has been attributed to inactivating mutations and genetic drift while genes coding for host-relevant functions are conserved by purifying selection. Unlike their free-living relatives, the metabolism of mutualistic endosymbionts and endosymbiont-originated organelles is specialized in the production of metabolites which are released to the host. This specialization suggests that natural selection crafted these metabolic adaptations. In this work, we analyzed the evolution of the metabolism of the chromatophore of Paulinella chromatophora by in silico modeling. We asked whether genome reduction is driven by metabolic engineering strategies resulted from the interaction with the host. As its widely known, the loss of enzyme coding genes leads to metabolic network restructuring sometimes improving the production rates. In this case, the production rate of reduced-carbon in the metabolism of the chromatophore. RESULTS: We reconstructed the metabolic networks of the chromatophore of P. chromatophora CCAC 0185 and a close free-living relative, the cyanobacterium Synechococcus sp. WH 5701. We found that the evolution of free-living to host-restricted lifestyle rendered a fragile metabolic network where >80% of genes in the chromatophore are essential for metabolic functionality. Despite the lack of experimental information, the metabolic reconstruction of the chromatophore suggests that the host provides several metabolites to the endosymbiont. By using these metabolites as intracellular conditions, in silico simulations of genome evolution by gene lose recover with 77% accuracy the actual metabolic gene content of the chromatophore. Also, the metabolic model of the chromatophore allowed us to predict by flux balance analysis a maximum rate of reduced-carbon released by the endosymbiont to the host. By inspecting the central metabolism of the chromatophore and the free-living cyanobacteria we found that by improvements in the gluconeogenic pathway the metabolism of the endosymbiont uses more efficiently the carbon source for reduced-carbon production. In addition, our in silico simulations of the evolutionary process leading to the reduced metabolic network of the chromatophore showed that the predicted rate of released reduced-carbon is obtained in less than 5% of the times under a process guided by random gene deletion and genetic drift. We interpret previous findings as evidence that natural selection at holobiont level shaped the rate at which reduced-carbon is exported to the host. Finally, our model also predicts that the ABC phosphate transporter (pstSACB) which is conserved in the genome of the chromatophore of P. chromatophora strain CCAC 0185 is a necessary component to release reduced-carbon molecules to the host. CONCLUSION: Our evolutionary analysis suggests that in the case of Paulinella chromatophora natural selection at the holobiont level played a prominent role in shaping the metabolic specialization of the chromatophore. We propose that natural selection acted as a "metabolic engineer" by favoring metabolic restructurings that led to an increased release of reduced-carbon to the host.

A Bowl with Marbles: Revision of the Thecate Amoeba Genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa, Rhizaria) Including a Description of Four New Species and an Identification Key.

Although testate amoebae have attracted interest of protistologists for more than 150 years, some groups especially those with a hyaline, organic test (= theca) are still poorly known. One of those is the genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa, Rhizaria), first described by Hertwig and Lesser in 1874. Only old, sometimes obscure, species descriptions were available until only recently a new species of Lecythium was described and a small ribosomal subunit RNA gene (SSU) sequence was provided. To shed light on the phylogeny and taxonomy of Lecythium, we (a) cultured six isolates of five Lecythium species and provide morphological as well as ecological observations, (b) obtained six new SSU sequences and conducted phylogenetic analyses of the Tectofilosida, showing that Lecythium splits into terrestrial and freshwater clades, and (c) did an intensive literature research on testate amoebae with a theca and provide an illustrated identification key focusing on Lecythium. For the first time, the presence of cysts in the genus Lecythium is reported and we compared those to the cysts of the presumed closely related Chlamydophrys stercorea. Our results suggest that still many undescribed Lecythium species will be found in terrestrial and freshwater habitats.

The Earliest Stages of Mitochondrial Adaptation to Low Oxygen Revealed in a Novel Rhizarian.

Mitochondria exist on a functional and evolutionary continuum that includes anaerobic mitochondrion-related organelles (MROs), such as hydrogenosomes. Hydrogenosomes lack many classical mitochondrial features, including conspicuous cristae, mtDNA, the tricarboxylic acid (TCA) cycle, and ATP synthesis powered by an electron transport chain (ETC); instead, they produce ATP anaerobically, liberating H2 and CO2 gas in the process. However, our understanding of the evolutionary transformation from aerobic mitochondria to various MRO types remains incomplete. Here we describe a novel MRO from a cercomonad (Brevimastigomonas motovehiculus n. sp.; Rhizaria). We have sequenced its 30,608-bp mtDNA and characterized organelle function through a combination of transcriptomic, genomic, and cell biological approaches. B. motovehiculus MROs are metabolically versatile, retaining mitochondrial metabolic pathways, such as a TCA cycle and ETC-driven ATP synthesis, but also possessing hydrogenosomal-type pyruvate metabolism and substrate-level phosphorylation. Notably, the B. motovehiculus ETC is degenerate and appears to be losing cytochrome-based electron transport (complexes III and IV). Furthermore, the F1Fo ATP synthase (complex V) is unique, with the highly conserved Atpα subunit fragmented into four separate pieces. The B. motovehiculus MRO appears to be in the process of losing aerobic metabolic capacities. Our findings shed light on the transition between organelle types, specifically the early stages of mitochondrial adaptation to anaerobiosis.

A novel paramyxean parasite, Marteilia octospora n. sp. (Cercozoa) infecting the Grooved Razor Shell clam Solen marginatus from Galicia (NW Spain).

Protozoan parasites of the genus Marteilia have been detected in marine bivalves and other invertebrates around the world, associated in some cases with mass mortalities. The present paper reports the characterization of the Marteilia sp. protozoan infecting the digestive gland of the Grooved Razor Shell clam (Solen marginatus) from Galicia (NW Spain), proposing a novel species in the genus: Marteilia octospora n. sp. Morphological and molecular techniques were used for the description of this parasite. Tissue imprints were essential in the study to confirm the presence of 8 spores per sporangium, a number never reported in other species from this genus. An ultrastructural study revealed that the size and number of dense granules, free in the mature sporangia, were quite different from granules in other Marteilia spp. Another morphological difference is the absence of a layer of concentric membranes found surrounding the mature spore in other species. In addition, concentric membranous structures observed in the different stages of the parasite have never been mentioned in other species of genus Marteilia. Moreover, molecular analysis of the rDNA intergenic spacer (IGS) and the internal transcribed spacer (ITS-1) showed differences with the sequences available for other Marteilia spp.

"Candidatus Finniella" (Rickettsiales, Alphaproteobacteria), Novel Endosymbionts of Viridiraptorid Amoeboflagellates (Cercozoa, Rhizaria).

The Rickettsiales (Alphaproteobacteria) are obligate intracellular bacteria that colonize a wide range of eukaryotic hosts, including diverse metazoa and protists. Here, we characterize rickettsial endosymbionts discovered in the cytoplasm of the algivorous amoeboflagellates Viridiraptor invadens and Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria), supplying evidence of free-living, phagotrophic members of the Cercozoa serving as hosts for Rickettsiales. According to 16S rRNA gene phylogenies, the bacteria represent two closely related but distinct genotypes within a deep-branching rickettsial clade, which contains the genera "Candidatus Odyssella," "Candidatus Paracaedibacter," and "Candidatus Captivus." Using the full-cycle rRNA approach, we detected the novel bacteria in four of nine viridiraptorid strains tested. Furthermore, two specific oligonucleotide probes with a single-nucleotide-difference discriminated both bacterial genotypes by fluorescence in situ hybridization (FISH). We establish the candidate species "Candidatus Finniella inopinata" (found in Viridiraptor invadens) and "Candidatus Finniella lucida" (found in Orciraptor agilis) for the novel bacteria and propose a new, provisional family of Rickettsiales, "Candidatus Paracaedibacteraceae."

Testate amoeba Rhogostoma minus Belar, 1921, associated with nodular gill disease of rainbow trout, Oncorhynchus mykiss (Walbaum).

The case study targeted to determine the aetiology of nodular gill disease (NGD) of farmed rainbow trout. The methods included microscopical examination of gill material in fresh, culturing of isolated organisms, histology, transmission electron microscopy and molecular biology identification. The results revealed an intravital colonization of fish gills by the testate amoeba Rhogostoma minus Belar, 1921. Rhogostoma infection was found in all fish examined microscopically (15/15); in contrast, naked amoebae related to fully developed NGD lesions were found in minority of these fish (5/15). They belonged to four genera, Acanthamoeba, Vermamoeba, Naegleria and Vannella. Results presented in this study contribute to the mosaic of findings that contrary to amoebic gill disease of marine fish turn attention to the possibility of the heterogeneous, multi-amoeba-species and multifactorial aetiology of NGD.

Shifts in soil testate amoeba communities associated with forest diversification.

We studied changes of testate amoeba communities associated with the conversion of spruce monocultures into mixed beech-fir-spruce forests in the Southern Black Forest Mountains (Germany). In this region, forest conversion is characterized by a gradual development of beech undergrowth within thinned spruce tree stands leading to multiple age continuous cover forests with a diversified litter layer. Strong shifts in the abundance of testate amoeba observed in intermediate stages levelled off to monoculture conditions again after the final stage of the conversion process had been reached. The average number of species per conversion stage (i.e., local richness) did not respond strongly to forest conversion, but the total number of species (i.e., regional richness) was considerably higher in the initial stage than in the mixed forests, due to the large number of hygrophilous species inhabiting spruce monocultures. Functional diversity of the testate amoeba community, however, significantly increased during the conversion process. This shift was closely associated with improved C and N availability as well as higher niche diversity in the continuous cover stands. Lower soil acidity in these forests coincided with a higher relative abundance of eurytopic species. Our results suggest that testate amoeba communities are much more affected by physicochemical properties of the soil than directly by litter diversity.