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. 2021 Feb 3;11(1):2865.
doi: 10.1038/s41598-021-82416-9.

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

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

Ryo Hoshina et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Geographical distribution and habitat of Stentor pyriformis. (A). Latitude with simple map, altitude, pH, and Electric conductivity (EC) of investigation sites are shown. pH and EC were measured only on 29 sites. For pH and EC, there are some extended notations (e.g., EC: 11–14) in “Tsukii note,” which are spotted as two points of those largest and smallest (e.g., 11 and 14). Reference data of EC values for some general waters were quoted from websites of water companies, Merck Millipore (https://www.merckmillipore.com/), Kurita Water Industries (https://kcr.kurita.co.jp/), and Japan Society of Refrigerating and Air Conditioning Engineers (https://www.jsrae.or.jp/). KCM: 1 × KCM medium (see “Methods”). DW: distilled water. RO: reverse osmosis water. The map data was obtained from Silhouette Design (https://kage-design.com/) and simplified using Adobe Illustrator CS5.B. A bog in Hachimantai area (see Fig. 1). (C). Blooming of S. pyriformis on the bottom of the bog. (D). Stentor pyriformis gathering to plant stalks. (E). Living S. pyriformis gathered in plant debris. A movie is available online showing many Stentors on the bottom of the bog at https://1drv.ms/v/s!Aia81H4VPPEYgctoYoHSdbRPYBkvyg?e=KXnnJa/.
Figure 2
Figure 2
Light micrographs of living and crushed cells of Stentor pyriformis. (A). A swimming cell showing short and fat body. (B). A slightly squeezed cell under coverslip. (C). Buccal cavity. (D): Surface vesicles immediately under the cell surface. (E): Symbiotic Chlorella cells as seen in the crushed cytoplasm of S. pyriformis with starch granules. The Chlorella cells appear vividly green, and dividing cells were rarely seen. (F). Symbiotic Chlorella variabilis cells in Paramecium bursaria for comparison. The picture was taken under the same photographic conditions as (E). Cells are pale green and many dividing Chlorella cells are observed (arrows). The cell size variation was larger than that in S. pyriformis. G. Spherical macronuclei (arrows) found with abundant starch granules in the cytoplasm crushed between the slide and the coverslip.
Figure 3
Figure 3
(A) and (B). Chemically-fixed specimen of S. pyriformis. The cytoplasm was observed as highly vacuolated, in which symbiotic Chlorella cells (Ch) were observed to be scattered in a large vacuole without being covered by individual surrounding membranes. Chlorella at the lower left is a rarely seen dividing organism. Many small electron-dense granules of 1–2 μm in size were present in the cytoplasm. (C)–(E)S. Quick-frozen and freeze substituted specimen of S. pyriformis. The cytoplasm was not vacuolated, and each symbiotic Chlorella was surrounded by a symbiosome membrane (arrowheads in D). The symbiosome membrane was closely opposed to the cell wall surface by a distance of less than 50 nm. The cell wall surface of the symbiotic Chlorella was ornamented by fluffy filaments (arrow). In the symbiotic Chlorella, the pyrenoid structure was penetrated by thylakoid membranes (arrow in C), characteristic of the genus Chlorella. (D) is an enlarged view of the area indicated by the rectangle in (C). In the cytoplasm, many multi-vesicular bodies of about 1 μm in diameter with unknown function were observed (mv in C and also in E).
Figure 4
Figure 4
Histochemical localization of starch in Stentor pyriformis. (A). Light micrograph of a thick section of a Spurr’s resin-embedded cell stained with Lugol’s solution. Cytoplasmic granules stained brown, suggesting high content of α-1, 6-linked glucose branch. (B): Potato starch granules stained with Lugol’s solution shown as comparison. Potato starch stains blue due to its high amylose content. (C): A DIC image of the freshly isolated cytoplasmic granules observed under the crossed polarizer condition, showing that the granules have a high refractile index. (D) and (E). Polarization microscopy of starch granules in S. pyriformis. (D). DIC image of compressed and disrupted cytoplasm of S. pyriformis. Symbiotic Chlorella (c) and cytoplasmic granules (g) are shown. (E). Polarization micrograph of the same area as shown in D under the cross-nicol condition. In the crossed polarizer orientation, birefringent anisotropic specimens such as plant starch grains show a characteristic “Maltese cross” pattern (see Olympus website: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/dic/dicphasecomparison/). The cytoplasmic granules of S. pyriformis show a Maltese cross pattern (arrows), indicating that the granules have birefringent properties like plant starch. Arrowheads show birefringence signals of the starch sheath in symbiotic Chlorella cells. (F) and (G): Transmission electron micrographs of a section of S. pyriformis chemically fixed and stained with lead and lanthanoid. Photographs of the same section were taken before (F) and after (G) treatment with Lugol’s iodine solution. Arrows indicate the pyrenoid starch sheath, which was stained with heavy metals in F, but the stain was removed by iodine treatment. Granules in the ciliate cytoplasm (asterisks) were also destained by iodine treatment.
Figure 5
Figure 5
Phylogenetic relationships of Stentor species. (A). Bayesian inference tree for Stentor species based on SSU rDNA sequences. The tree was rooted with Gruberia sp. SUAS-2014. Numbers at the branches correspond to MrBayes posterior probabilities (PP)/maximum likelihood/neighbor-joining bootstrap values (BVs). Hyphens correspond to PP values below 0.70 and BVs below 50%. (B). Summary of the interspecific relationships and their iconic morphocharacters (shapes of macronucleus, presence or absence of cortical pigmentation and symbiotic algae). The figure explaining the varieties of macronuclei was modified from Foissner and Wölfl.
Figure 6
Figure 6
Sequence differences of SSU, ITS1, 5.8S and ITS2 rRNA gene (without group I introns) among Chlorella variabilis. “PbS-gt” indicates Paramecium bursaria symbiont genotypes. Genotype 1 includes SAG 211-6, ATCC 50258 (NC64A), NIES-2541, and some other US and Japanese strains. Genotype 2 is the alga of Chinese P. bursaria strain Cs2, and genotype 3 is the alga of Australian P. bursaria strain MRBG1. For further information, see Hoshina et al.. “SpS” indicates the algal sequence of Stentor pyriformis strains collected from Japan. (A). Different positions. Numerals represent the nucleotide number in aligned sequences (2462 aligned sites). (B). Distance tree of above four types of sequences. (C). E23_2 helix of SSU rRNA structure that includes hemi-CBC at the alignment position 656. (D). Deformation of ITS1 Helix 1 associated with the mutations including several nucleotide insertions.

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