Rhabdamoeba marina is a heterotrophic relative of chlorarachnid algae.

Rhabdamoeba marina is a unique and poorly reported amoeba with an uncertain phylogenetic position. We successfully cultured R. marina from coastal seawater in Japan and performed a molecular phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) gene sequence. Our phylogenetic analysis showed that R. marina branched as a basal lineage of Chlorarachnea, a group of marine photosynthetic algae belonging to the phylum Cercozoa within the supergroup Rhizaria. By comparing the ecological and morphological characteristics of R. marina with those of photosynthetic chlorarachneans and other cercozoans, we gained insight into the evolution and acquisition of plastids in Chlorarachnida.

Molecular and morphological characterization of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater, and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching "orphan" lineage that is considered key to a better understanding of the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to characterize ancyromonads better by integrating environmental surveys with behavioral observation and description of cell morphology, for which sample isolation and culturing are indispensable. We studied 18 ancyromonad strains, including 14 new isolates and seven new species. We described three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas, together encompassing four species. The remaining three new species belong to the already-known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, on morphological and phylogenetic grounds. We studied the morphology of diverse ancyromonads under light and electron microscopy and carried out molecular phylogenetic analyses, also including 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis.

Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants.

An Orphan Protist Quadricilia rotundata Finally Finds Its Phylogenetic Home in Cercozoa.

Quadricilia rotundata is a heterotrophic flagellate with four flagella. However, because this species has no clear morphological characteristics or molecular data affiliating it with any known group, Q. rotundata has been treated as a protist incertae sedis, for a long time. Here, we established a clonal culture of Q. rotundata and sequenced its 18S rDNA sequence. Molecular phylogenetic analysis successfully placed Q. rotundata in an environmental clade within Cercozoa, which contributes to expand the morphological and species diversity within Cercozoa. We also discuss morphological evolution within Cercozoa based on this finding.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Effects of Radiation From Contaminated Soil and Moss in Fukushima on Embryogenesis and Egg Hatching of the Aphid Prociphilus oriens.

Radiation-contaminated soils are widespread around the Fukushima Daiichi Nuclear Power Plant, and such soils raise concerns over its harmful effect on soil-dwelling organisms. We evaluated the effects of contaminated soil and moss sampled in Fukushima on the embryogenesis and hatching of aphid eggs, along with the measurement of the egg exposure dose. Cs-137 concentration in soil and moss from Fukushima ranged from 2200 to 3300 Bq/g and from 64 to 105 Bq/g, respectively. Eggs of the eriosomatine aphid Prociphilus oriens that were collected from a non-contaminated area were directly placed on the soil and moss for 4 or 3 months during diapause and then incubated until hatching. The total exposure dose to the eggs was estimated as ca. 100-200 mGy in the 4-month soil experiment and 4-10 mGy in the 4-month moss experiment. There was no significant difference in egg hatchability between the contaminated soil treatment and the control. No morphological abnormalities were detected in the first instars that hatched from the contaminated soil treatment. However, we found weak effects of radiation on egg hatching; eggs placed on the contaminated moss hatched earlier than did the control eggs. On the contaminated soil, the effects of radiation on egg hatching were not obvious because of uncontrolled environmental differences among containers. The effects of radiation on egg hatching were detected only in containers where high hatchability was recorded. Through the experiments, we concluded that the aphid eggs responded to ultra-low-dose radiation by advancing embryogenesis.

Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: implications for the induction of biomineralization.

Dmp1 is an acidic phosphoprotein that is specifically expressed in osteocytes. During the secretory process, the full-length, precursor Dmp1 is cleaved into N- and C-terminal fragments. C-terminal Dmp1 is phosphorylated, becoming a highly negatively charged domain that may assist in bone mineralization by recruiting calcium ions and influencing subsequent mineral deposition. It has been recently reported that the Golgi-localized protein kinase Fam20C phosphorylates Dmp1 in vitro. To investigate this phosphorylation in situ, we determined the locations of phosphorylated Dmp1 and Fam20C in rat bones using immunohistochemistry. During osteocytogenesis, osteoblastic, osteoid, and young osteocytes (but not old osteocytes) express Dmp1 mRNA and contain Dmp1 protein in the Golgi apparatus. These Dmp1-producing cells were distributed across the surface layer of cortical bone. Using immunofluorescence, we found that N- and C-terminal Dmp1 fragments were predominantly distributed along the lacunar walls and canaliculi of mineralized bone, respectively, but were not present in the osteoid matrix. We also found that Fam20C and its substrate, C-terminal Dmp1, colocalized in the Golgi of osteoblastic, osteoid, and young osteocytes. Furthermore, phosphorylated C-terminal Dmp1 was present in the Golgi of young osteocytes. Double-labeling immunoelectron microscopy revealed that phosphorylated C-terminal Dmp1 localized to the canalicular wall in mineralized bone. These findings suggest that C-terminal Dmp1 is phosphorylated within osteocytes and then secreted into the pericanalicular matrix of mineralized bone. Phosphorylated, negatively charged C-terminal Dmp1 in the pericanalicular matrix may play an important role in bone mineralization by recruiting calcium ions.

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte.

A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage of V. adhaerens consisted of unicellular coccoid cells with cell walls, although sarcinoid colonies and uniflagellate zoospores were also observed. V. adhaerens had chloroplasts with nucleomorphs and pyrenoids that were completely embedded in the chloroplast. A deep plate-like invagination of the periplastidal compartment (PPC) almost partitioned the pyrenoid and chloroplast components, which were surrounded by two membranes. The nucleomorph was positioned in the base of the invagination of the PPC. Molecular phylogenetic analyses using rRNA genes showed that V. adhaerens branched as a sister lineage of the Amorphochlora clade. The sarcinoid colony, pyrenoid embedded in the chloroplast, and nucleomorph located at the base of the deep invagination of the PPC have not been reported in other chlorarachniophytes. Based on these morphological and ultrastructural characteristics and the results of the molecular phylogenetic analyses, we propose V. adhaerens as a new genus and species of chlorarachniophyte.

[Oral care for elderly people.]

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Proposal of a Twin Arginine Translocator System-Mediated Constraint against Loss of ATP Synthase Genes from Nonphotosynthetic Plastid Genomes. [Corrected].

Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes-such as those coding for ATP synthase-remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Directing positional specificity in enzymatic synthesis of bioactive 1-phosphatidylinositol by protein engineering of a phospholipase D.

Phosphatidylinositol (PI) holds a potential of becoming an important dietary supplement due to its effects on lipid metabolism in animals and humans manifested as a decrease of the blood cholesterol and lipids, and relief of the metabolic syndrome. To establish an efficient, enzymatic system for PI production from phosphatidylcholine and myo-inositol as an alcohol acceptor, our previous study started with the wild-type Streptomyces antibioticus phospholipase D (SaPLD) as a template for generation of PI-synthesizing variants by saturation mutagenesis targeting positions involved in acceptor accommodation, W187, Y191, and Y385. The isolated variants generated PI as a mixture of positional isomers, among which only 1-PI exists in nature. Thus, the current study has focused to improve positional specificity of W187N/Y191Y/Y385R SaPLD (NYR) which generates PI as a mixture of 1-PI and 3-PI in the ratio of 76/24, by subjecting four residues of its acceptor-binding site to saturation mutagenesis. Subsequent screening pointed at NYR-186T and NYR-186L as the most improved variants producing PI with a ratio of 1-/3-PI = 93/7 and 87/13, respectively, at 37°C. Lowering the reaction temperature further improved the specificity of both variants to 1-/3-PI > 97/3 at 20°C with no change in total PI yield. Structure model analyses imply that G186T and G186L mutations increased rigidity of the acceptor-binding site, thus limiting the possible orientations of myo-inositol. The two newly isolated PLDs are promising for future application in large-scale 1-PI production.

Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 µm in length and 6.8-15.9 µm in width, whereas the cysts were 8.9-15.8 µm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

A New Heterotrophic Cryptomonad: Hemiarma marina n. g., n. sp.

We report a new heterotrophic cryptomonad Hemiarma marina n. g., n. sp. that was collected from a seaweed sample from the Republic of Palau. In our molecular phylogenetic analyses using the small subunit ribosomal RNA gene, H. marina formed a clade with two marine environmental sequences, and the clade was placed as a sister lineage of the freshwater cryptomonad environmental clade CRY1. Alternatively, in the concatenated large and small subunit ribosomal RNA gene phylogeny, H. marina was placed as a sister lineage of Goniomonas. Light and electron microscopic observations showed that H. marina shares several ultrastructural features with cryptomonads, such as flattened mitochondrial cristae, a periplast cell covering, and ejectisomes that consist of two coiled ribbon structures. On the other hand, H. marina exhibited unique behaviors, such as attaching to substrates with its posterior flagellum and displaying a jumping motion. H. marina also had unique periplast arrangement and flagellar transitional region. On the basis of both molecular and morphological information, we concluded that H. marina should be treated as new genus and species of cryptomonads.

Trachyrhizium urniformis n. g., n. sp., a Novel Marine Filose Thecate Amoeba Related to a Cercozoan Environmental Clade (Novel Clade 4).

A novel cercozoan filose thecate amoeba, Trachyrhizium urniformis n. g., n. sp., was isolated from a marine sediment sample collected at Agenashiku Island, Okinawa, Japan. We performed light and electron microscopic observations, and a molecular phylogenetic analysis using the small subunit ribosomal RNA gene of the isolate. Cells of T. urniformis are spherical in shape and are covered by a thin theca possessing a wide rounded aperture. Branching and occasionally anastomosing filopodia with small granules emerge from the aperture. The granules are transported in the filopodia bidirectionally. Transmission electron microscopy showed that cells of T. urniformis possess nucleus with permanently condensed chromatin, Golgi apparatuses, microbodies, mitochondria with tubular cristae, and extrusomes. Several morphological and ultrastructural features of T. urniformis (the presence of thecae and nucleus with permanently condensed chromatin) show similarities with those of Thecofilosea. In a phylogenetic analysis, T. urniformis included in Thecofilosea with weak statistical supports and formed a clade with two sequences that constitutes a cercozoan environmental clade, novel clade 4. On the basis of morphological and ultrastructural information and the results of the phylogenetic analysis, we propose T. urniformis as a new member of class Thecofilosea.

Plastid genome sequences of Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia reveal remarkable structural conservation among chlorarachniophyte species.

Chlorarachniophyte algae have complex plastids acquired by the uptake of a green algal endosymbiont, and this event is called secondary endosymbiosis. Interestingly, the plastids possess a relict endosymbiont nucleus, referred to as the nucleomorph, in the intermembrane space, and the nucleomorphs contain an extremely reduced and compacted genome in comparison with green algal nuclear genomes. Therefore, chlorarachniophyte plastids consist of two endosymbiotically derived genomes, i.e., the plastid and nucleomorph genomes. To date, complete nucleomorph genomes have been sequenced in four different species, whereas plastid genomes have been reported in only two species in chlorarachniophytes. To gain further insight into the evolution of endosymbiotic genomes in chlorarachniophytes, we newly sequenced the plastid genomes of three species, Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia. Our findings reveal that chlorarachniophyte plastid genomes are highly conserved in size, gene content, and gene order among species, but their nucleomorph genomes are divergent in such features. Accordingly, the current architecture of the plastid genomes of chlorarachniophytes evolved in a common ancestor, and changed very little during their subsequent diversification. Furthermore, our phylogenetic analyses using multiple plastid genes suggest that chlorarachniophyte plastids are derived from a green algal lineage that is closely related to Bryopsidales in the Ulvophyceae group.

Overexpression of molecular chaperone genes in nucleomorph genomes.

Chlorarachniophytes and cryptophytes possess complex plastids that were acquired by the ingestion of a green and red algal endosymbiont, respectively. The plastids are surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment, which corresponds to the remnant cytoplasm of the endosymbiont. Nucleomorphs contain a greatly reduced genome that possesses only several hundred genes with high evolutionary rates. We examined the relative transcription levels of the genes of all proteins encoded by the nucleomorph genomes of two chlorarachniophytes and three cryptophytes using an RNA-seq transcriptomic approach. The genes of two heat shock proteins, Hsp70 and Hsp90, were highly expressed under normal conditions. It has been shown that molecular chaperone overexpression allows an accumulation of genetic mutations in bacteria. Our results suggest that overexpression of heat shock proteins in nucleomorph genomes may play a role in buffering the mutational destabilization of proteins, which might allow the high evolutionary rates of nucleomorph-encoded proteins.

Prospective function of FtsZ proteins in the secondary plastid of chlorarachniophyte algae.

BACKGROUND: Division of double-membraned plastids (primary plastids) is performed by constriction of a ring-like division complex consisting of multiple plastid division proteins. Consistent with the endosymbiotic origin of primary plastids, some of the plastid division proteins are descended from cyanobacterial cell division machinery, and the others are of host origin. In several algal lineages, complex plastids, the "secondary plastids", have been acquired by the endosymbiotic uptake of primary plastid-bearing algae, and are surrounded by three or four membranes. Although homologous genes for primary plastid division proteins have been found in genome sequences of secondary plastid-bearing organisms, little is known about the function of these proteins or the mechanism of secondary plastid division. RESULTS: To gain insight into the mechanism of secondary plastid division, we characterized two plastid division proteins, FtsZD-1 and FtsZD-2, in chlorarachniophyte algae. FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal. Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans. The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes. Furthermore, gene expression analysis confirmed that transcripts of ftsZD genes were periodically increased soon after cell division during the B. natans cell cycle, which is not consistent with the timing of plastid division. CONCLUSIONS: Our findings suggest that chlorarachniophyte FtsZD proteins are involved in partial constriction of the inner pair of plastid membranes, but not in the whole process of plastid division. It is uncertain how the outer pair of plastid membranes is constricted, and as-yet-unknown mechanism is required for the secondary plastid division in chlorarachniophytes.

Cultivation and Characterisation of New Species of Apusomonads (the Sister Group to Opisthokonts), Including Close Relatives of Thecamonas (Chelonemonas n. gen.).

Apusomonads comprise an understudied and undersampled group of heterotrophic flagellates that is closely related to opisthokonts, the supergroup containing animals and fungi. We cultured representatives of a new clade of apusomonads, Chelonemonas n. gen., which is sister to marine forms of Thecamonas in SSU rRNA gene phylogenies. Scanning electron microscopy shows that members of Chelonemonas have a hexagonal patterning to their submembranous pellicle, which is not known to exist in other apusomonads. We propose that the subfamily Thecamonadinae refer to the marine Thecamonas/Chelonomonas clade. We also report two new strains of Multimonas, one of which is genetically divergent from previously described strains, and here described as a new species, Multimonas koreensis. Both strains of Multimonas have appendages on their dorsal surface that could be extrusomes, and a frilled appearance to the border of their pellicle. Explorations of taxon sampling in SSU rRNA gene phylogenies confirm the new strains' evolutionary affinities, but do not resolve relationships among the five main apusomonad clades. These phylogenies also separate the freshwater species "Thecamonas" oxoniensis from the marine members of the genus Thecamonas. The new strains described here may provide valuable genetic and morphological data for evaluating the relationships and evolution of apusomonads.