Characterization of a green Stentor with symbiotic algae growing in an extremely oligotrophic environment and storing large amounts of starch granules in its cytoplasm.

The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor, but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis. The cytoplasm of S. piriformis contains a large number of 1-3 µm refractive granules that turn brown by Lugol's staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.

Pediludiella daitoensis gen. et sp. nov. (Scenedesmaceae, Chlorophyceae), a large coccoid green alga isolated from a Loxodes ciliate.

Freshwater protists often harbor unicellular green algae within their cells. In ciliates, possibly because of large host cell sizes and the small size of algal coccoids, a single host cell typically contains more than a hundred algal cells. While surveying such algae-bearing protists on Minami Daito Jima Island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained one or two dozens of very large coccoid algae. We isolated one of these algae and analyzed its characteristics in detail. A small subunit (SSU) rDNA phylogeny indicated Pseudodidymocystis species (Scenedesmaceae, Chlorophyceae) to be the taxon closest to the alga, although it was clearly separated from this by 39 or more different sites (inclusive of gaps). SSU rRNA structure analyses indicated that these displacements included eight compensatory base changes (CBCs) and seven hemi-CBCs. We therefore concluded that this alga belongs to a separate genus, and described it as Pediludiella daitoensis gen. et sp. nov. The shape of the isolated and cultured P. daitoensis was nearly spherical and reached up to 30 µm in diameter. Chloroplasts were arranged peripherally and often split and elongated. Cells were often vacuolated and possessed a net-like cytoplasm that resembled a football (soccer ball) in appearance, which was reflected in the genus name.

Endosymbiosis-related changes in ultrastructure and chemical composition of Chlorella variabilis (Archaeplastida, Chlorophyta) cell wall in Paramecium bursaria (Ciliophora, Oligohymenophorea).

Chlorella variabilis, a symbiotic alga, is usually present in the cytoplasm of Paramecium bursaria, although it can be cultured in host-free conditions. Morphological and chemical properties of its cell wall were compared between its free-living and symbiotic states. Transmission electron microscopy (quick-freezing and freeze-substitution methods) revealed that the cell wall thickness of symbiotic C. variabilis was reduced to about half that of the free-living one. Chemical properties of the cell wall were examined by treatment with three fluorescent reagents (calcofluor white M2R, FITC-WGA, and FITC-LFA) having specific binding affinities to different polysaccharides. When the algae were re-introduced into Paramecium host cells, calcofluor fluorescence intensity reduced by about 50%. Calcofluor stains ß-d-glucopyranose polysaccharides such as cellulose, N-acetylglucosamine, sialic acid, and glycosaminoglycans. Because treatment with cellulase showed no effect on calcofluor fluorescence intensity, we consider that cellulose is not majorly responsible for the stainability of calcofluor. Staining intensities of FITC-WGA and FITC-LFA were similar in the free-living and symbiotic conditions, suggesting that N-acetylglucosamine and sialic acid are also not responsible for the reduction in the stainability of calcofluor associated with intracellular symbiosis. The amount of glycosaminoglycans on the cell wall may decrease in C. variabilis present in the cytoplasm of P. bursaria.

De novo assembly of middle-sized genome using MinION and Illumina sequencers.

BACKGROUND: The plastid acquisition by secondary endosymbiosis is a driving force for the algal evolution, and the comparative genomics was required to examine the genomic change of symbiont. Therefore, we established a pipeline of a de novo assembly of middle-sized genomes at a low cost and with high quality using long and short reads. RESULTS: We sequenced symbiotic algae Chlorella variabilis using Oxfofrd Nanopore MinION as the long-read sequencer and Illumina HiSeq 4000 as the short-read sequencer and then assembled the genomes under various conditions. Subsequently, we evaluated these assemblies by the gene model quality and RNA-seq mapping rate. We found that long-read only assembly could not be suitable for the comparative genomics studies, but with short reads, we could obtain the acceptable assembly. On the basis of this result, we established the pipeline of de novo assembly for middle-sized algal genome using MinION. CONCLUSIONS: The genomic change during the early stages of plastid acquisition can now be revealed by sequencing and comparing many algal genomes. Moreover, this pipeline offers a solution for the assembly of various middle-sized eukaryotic genomes with high-quality and ease.

DNA Analysis of Algal Endosymbionts of Ciliates Reveals the State of Algal Integration and the Surprising Specificity of the Symbiosis.

Many freshwater protists harbor unicellular green algae within their cells, but little is known of their degree of integration and specificity. Using algae-targeted PCR of whole ciliate cells collected at irregular intervals over 15 months from Lake Biwa, Japan, we explored the SSU-ITS rDNA of the endosymbiotic algae and its changes over time, obtaining sequences of algal rDNA fragments from four ciliate species. A high proportion of clonal algae was evident within the ciliate cells. The differences observed in those sequences from the SSU through to the ITS region were less than 1%. The name 'Chlorb' is proposed for these algae, with the implication that they represent a single 'species.' The sequences of the algal DNA fragments were identical for any given host species throughout the collection period, thus we conclude that these four ciliates stably retain their algae over long term. In contrast, algal DNA fragments obtained from Didinium sp. were variable within each sample, which indicates that this ciliate only temporarily holds its algal cells. The ITS1 sequences of Chlorb populations are close (at intraspecific level) to those of algae isolated from ciliates in Austria, which raises the possibility that Chlorb algae are universally shared as symbionts among various ciliates.

DNA analyses of a private collection of microbial green algae contribute to a better understanding of microbial diversity.

BACKGROUND: DNA comparison is becoming the leading approach to the analysis of microbial diversity. For eukaryotes, the internal transcribed spacer 2 (ITS2) has emerged as a conspicuous molecule that is useful for distinguishing between species. Because of the small number of usable ITS data in GenBank, ITS2 sequence comparisons have only been used for limited taxa. However, major institutions with planktonic algal culture collections have now released small subunit (SSU) to ITS rDNA sequence data for their collections. This development has uplifted the level of molecular systematics for these algae. RESULTS: Forty-three strains of green algae isolated from German inland waters were investigated by using SSU-ITS rDNA sequencing. The strains were isolated through the direct plating method. Many of the strains went extinct during the years of culture. Thus, it could be expected that the surviving strains would be common, vigorous species. Nevertheless, 12 strains did not match any known species for which rDNA sequences had been determined. Furthermore, the identity of one strain was uncertain even at the genus level. CONCLUSIONS: The aforementioned results show that long-forgotten and neglected collections may be of great significance in understanding microbial diversity, and that much work still needs to be done before the diversity of freshwater green algae can be fully described.

Isolation and characterization of a virus (CvV-BW1) that infects symbiotic algae of Paramecium bursaria in Lake Biwa, Japan.

BACKGROUND: We performed an environmental study of viruses infecting the symbiotic single-celled algae of Paramecium bursaria (Paramecium bursaria Chlorella virus, PBCV) in Lake Biwa, the largest lake in Japan. The viruses detected were all Chlorella variabilis virus (CvV = NC64A virus). One of them, designated CvV-BW1, was subjected to further characterization. RESULTS: CvV-BW1 formed small plaques and had a linear DNA genome of 370 kb, as judged by pulsed-field gel electrophoresis. Restriction analysis indicated that CvV-BW1 DNA belongs to group H, one of the most resistant groups among CvV DNAs. Based on a phylogenetic tree constructed using the dnapol gene, CvV was classified into two clades, A and B. CvV-BW1 belonged to clade B, in contrast to all previously identified virus strains of group H that belonged to clade A. CONCLUSIONS: We conclude that CvV-BW1 composes a distinct species within C. variabilis virus.

Secondary structural analyses of ITS1 in Paramecium.

The nuclear ribosomal RNA gene operon is interrupted by internal transcribed spacer (ITS) 1 and ITS2. Although the secondary structure of ITS2 has been widely investigated, less is known about ITS1 and its structure. In this study, the secondary structure of ITS1 sequences for Paramecium and other ciliates was predicted. Each Paramecium ITS1 forms an open loop with three helices, A through C. Helix B was highly conserved among Paramecium, and similar helices were found in other ciliates. A phylogenetic analysis using the ITS1 sequences showed high-resolution, implying that ITS1 is a good tool for species-level analyses.

Phylogenetically close group I introns with different positions among Paramecium bursaria photobionts imply a primitive stage of intron diversification.

Group I introns are a distinct RNA group that catalyze their excision from precursor RNA transcripts and ligate the exons. Group I introns have a sporadic and highly biased distribution due to the two intron transfer mechanisms of homing and reverse splicing. These transfer pathways recognize assigned sequences even when introns are transferred beyond the species level. Consequently, introns at homologous gene sites between different host organisms are more related than those at heterologous sites within an organism. We describe the subgroup IE introns of two Chlorella species that are symbiotic green algae (photobionts) of a ciliate, Paramecium bursaria. One strain Chlorella sp. SW1-ZK (Csw.) had two IE introns at S651 and L2449, and the other strain Chlorella sp. OK1-ZK (Cok.) had four IE introns at S943, L1688, L1926, and L2184 (numbering reflects their homologous position in Escherichia coli rRNA gene: S = small subunit rRNA, L = large subunit rRNA). Despite locating on six heterologous sites, the introns formed a monophyletic clade independent of other groups. Phylogenetic and structural analyses of the introns indicated that Csw.L2449 has an archaic state, and the other introns are assumed to be originated from this intron. Some of the introns shared common internal guide sequences, which are necessary for misdirected transfer (i.e., transposition) via reverse splicing. Other introns, however, shared similar sequence fragments further upstream, after the insertions. We propose a hypothetical model to explain how these intron transpositions may have occurred in these photobionts; they transposed by a combination of homing-like event requiring relaxed sequence homology of recognition sequences and reverse splicing. This case study may represent a key to describe how group I intron explores new insertion sites.

Multiple origins of the symbioses in Paramecium bursaria.

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.