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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. SIMILAR CONTENT BEING VIEWED BY OTHERS PREY PREFERENCE IN A KLEPTOPLASTIC DINOFLAGELLATE IS LINKED TO PHOTOSYNTHETIC PERFORMANCE Article Open

access 30 June 2023 SYMBIOTIC MICROALGAL DIVERSITY WITHIN LICHENICOLOUS LICHENS AND CRUSTOSE HOSTS ON IBERIAN PENINSULA GYPSUM BIOCRUSTS Article Open access 20 August 2020 CONSUMING FRESH

MACROALGAE INDUCES SPECIFIC CATABOLIC PATHWAYS, STRESS REACTIONS AND TYPE IX SECRETION IN MARINE FLAVOBACTERIAL PIONEER DEGRADERS Article Open access 19 May 2022 INTRODUCTION Mixotrophic

protists are reported to live in a wide range of environments1, even in highly oligotrophic environments where other photoautotrophic and heterotrophic organisms cannot survive2,3. Possible

reasons why these protists are adapted to such a harsh environment are (1) there are few large predator animals in such ponds3, (2) high UV resistance due to symbiosis shading effect4, and

(3) mixotrophy allows adaptation to harsh environmental conditions by optimizing the combination of heterotrophic and photoautotrophic organisms in the same organism1. Mixotrophic protists

such as _Stentor pyriformis_ (algae-retaining ciliate) and _Mayorella viridis_ (algae-retaining amoeba) are frequently observed and documented as the dominant protist species in highland

wetlands in Tohoku district, Japan, where average winter temperatures remain below freezing for a few months5. Even in such harsh conditions, these protists survive in non-freezing locations

at the bottom of the pond, but it remains unclear how survival strategies of such protists are related to mixotrophy. The genus _Stentor_ (family Stentoridae, order Heterotrichida) is a

relatively well-known ciliate characterized by its lucid trumpet shape. _S. pyriformis_ is a poorly described species, although _S. pyriformis_ is clearly distinguishable from other

_Stentor_ species (Table S1). The species was first described in 18936 and then appeared in a microbiota report in 19087. However, its next appearance was not until 1994, in the study on

revision of the genus8. As described in the original literature, difficulties in the cultivation of this species6 may have hindered the research on this species. In Japan, _S. pyriformis_

can be found only in highland highly oligotrophic moors, suggesting that intracellular symbiotic algae would help this species of _Stentor_ survive in such a harsh environment. In this

study, we introduce some unique cell morphology of _S. pyriformis_ and the characteristics of symbiotic algae in relation to its life strategy. METHODS SAMPLING Water containing dead leaves,

twigs, or the remnants of submerged plants was sampled from ponds in Japan. The water sample was brought back to the laboratory at Tokyo and was crudely cultured in Petri dishes. A few days

later, _Stentor_ cells containing green coccoids within their bodies were often observed. If the green _Stentor_ was visible, it was directly collected using Komagome pipette or cup

attached to the tip of the rod. We measured hydrogen ion concentration (pH) in some pond samples using URCERI Digital PH Meter (Shenzhenshi Huanhui Dianzishangwu, Shenzhen, China) and

electric conductivity (EC) using AquaPro Water Quality Tester AP-2 (HM Digital, CA, USA). CULTURE Strains of _S. pyriformis_ were cultured in 2% KCM medium (160 µg/L KCl, 260 µg/L CaCl2, 500

 µg/L MgSO4 · 7H2O; pH 6.9) in Petri dishes (diameter, 9 cm; height, 2 cm) under fluorescent (64 W; height, 20 cm) (12L:12D) or LED light conditions at 25 °C. After multiple trials, _S.

pyriformis_ was successfully cultured only in low EC medium such as 2% KCM. Its EC was identified to be 1.5 µS/cm. The culture medium was changed once a week; half the volume of culture

medium (10–15 mL) was discarded and fresh medium was compensated for the shortfall. _S. pyriformis_ were fed a non-photosynthetic cryptophyte, _Chilomonas paramecium_, cultured on _Euglena_

medium (2 g/L tryptone, 1 g/L proteose peptone, 2 g/L yeast extract, 1 g/L sodium acetate, 0.01 g/L CaCl2) in a 50 mL polypropylene tube until stationary phase, which was centrifuged and

washed with pure water or by 2% KCM before feeding. CYTOLOGICAL OBSERVATIONS For electron microscopy, cells were chemically fixed with glutaraldehyde and osmium tetroxide or by metal contact

quick freezing as described previously9,10. After thin sectioning, samples on the grid mesh were stained with a lead citrate stain11 and threefold diluted EM Stainer (a lanthanoid

salts-based stain, Nisshin EM, Tokyo12). The presence of α-1, 4-linked glucose in the cytoplasm of the host _S. pyriformis_ and in the chloroplast of the symbiont was tested using Lugol’s

iodine solution (3% iodine (wt/v), 2% (wt/v) potassium iodine, and 73.4% (v/v) ethanol). Polarized light microscopy using a light microscope (Nikon Eclipse Ni, Nikon, Tokyo) with a set of

orthogonal polarizing filters (Nikon) on both the condenser lens and the CCD camera was used for imaging. For Lugol’s iodine staining, 1-μm-thick sections of chemically fixed, Spurr’s

resin-embedded samples were stained with Lugol’s iodine solution for 1 min and examined under a light microscope. For comparison, potato starch was stained with Lugol’s iodine solution for 1

 min and photographed under the same conditions. For electron microscopy of the iodine reaction, sections were first stained with lead citrate and EM Stainer and then photographed. The

sections were further treated with Lugol’s iodine solution for 30 s, and the same field of view of the same sample was photographed again under an electron microscope. REINFECTION EXPERIMENT

We investigated whether endosymbiotic algal cells isolated from _S. pyriformis_ could also be infected with _Paramecium bursaria_ (strain PbKb1) and coexist in the cytoplasm. The

reinfection experiment was conducted according to Omura et al.13. Aposymbiotic _P. bursaria_ was prepared using the method described by Higuchi et al.14. When endosymbiotic _Chlorella

variabilis_ cells isolated from _P. bursaria_ is mixed with aposymbiotic _P. bursaria_, they re-establish symbiosis within a few days. Therefore, symbiotic algal cells were isolated from _S.

pyriformis_ and fed to aposymbiotic _P. bursaria_. After 30 days, microscopic observation was performed to confirm whether _P. bursaria_ accepted the alga as a symbiont. _P. bursaria_ was

fed with _Chlorogonium capillatum_ (NIES-3374) once every 3 to 4 days as food. DNA EXTRACTION, AMPLIFICATION, AND SEQUENCING _Stentor_ cells in the fresh sample from Toriko-Daira (the day

after the collection) were isolated under a stereoscopic microscope, and each was transferred into a depression slide filled with pure water. Each ciliate was washed through the tip of a

micropipette and transferred into another depression, with this process being repeated twice. Before DNA extraction, we cultured these ‘clean’ (without algae outside) ciliates for 2 days.

The aim of this short-term culture was to prompt the ciliates to digest the algae, which they had taken as food, not as symbionts. Thereafter, the isolated individuals were washed twice, and

then their DNA was extracted. For the cultured _Stentor_, we isolated individuals and washed twice, and then their DNA was extracted. For each strain, about 20 individuals were collected

into one sample. DNA extraction was performed using NucleoSpin Plant II kit (Macherey–Nagel, Düren, Germany) with modified cell fracturing. _Stentor_ cells, each containing many algal cells,

were incubated for 5 min in 400 µL Buffer PL1 with 10 µL RNase A at 65 °C. After adding 400 µL of glass beads (ø 0.1 mm), each sample was homogenized in BeadSmash 12 (WakenBTech, Kyoto,

Japan) at 5,000 rpm for 30 s. The homogenization was repeated five times, and then each sample was again incubated for 10 min at 65 °C. The subsequent procedures were performed according to

the manufacturer’s instructions. PCR was performed to amplify _Stentor_ SSU to internal transcribed spacer (ITS) rDNA region using KOD FX Neo (Toyobo, Osaka, Japan) with the primer pair of

SR-115 (5′ SSU)/Hits5 (5′ LSU; –GGT TCR CTC GCC GTT ACT A–). The PCR conditions were as follows. An initial denaturation step at 94 °C for 2 min was followed by 45 cycles of the following

conditions: 10 s at 98 °C, 30 s at 52 °C, and 90 s at 68 °C. The amplification was completed with a final step of 68 °C for 1 min. The PCR products were verified by agarose gel

electrophoresis, cutting out the shorter band (due to shorter ITSs, a general trend in ciliate, and being intron-less) from the gel and purified using NucleoSpin Gel and PCR Clean-up kit

(Macherey–Nagel). The above primer pair amplifies ciliate DNA well but not algal DNA as it is very thin. Therefore, algae-targeted PCR was separately performed with the primer pairs of

SR-1/INT-5R16 (3′ SSU) and INT-4F16 (3′ SSU)/HLR3R17 (5′ LSU). The PCR conditions were the same as those for _Stentor_. The PCR products were verified by agarose gel electrophoresis and

purified using the NucleoSpin Gel and PCR Clean-up kit. The PCR products for both ciliate and algae were directly sequenced. PHYLOGENETIC ANALYSES OF _S. PYRIFORMIS_ AND THEIR SYMBIOTIC

ALGAE SSU rDNA sequences for the _Stentor_ species were obtained by searching the keywords [stentor + ssu] and [stentor + 18 s] from the NCBI database. After rough alignment using Clustal

X218, the shorter sequences, and sequences including several ‘N’ were removed. Recent phylogenetic analyses including that of _Stentor_ species have indicated stable relationships between

the _Stentor_ species and its sister clades19,20,21,22. Therefore, the _Stentor_ sequences were aligned with a limited number of outgroup taxa. A bootstrap tree was constructed using the

neighbor-joining (NJ) method with default setting in Clustal X2 and examined using 1000 bootstrap replicates. For maximum likelihood (ML) and Bayesian inference (BI) analyses, the best

nucleotide substitution model for the data set was analyzed using the Akaike information criterion (AIC) via MEGA X23, and the GTR + G + I model was selected. ML analyses were performed with

MEGA X using the nearest-neighbor interchange (NNI) branch-swapping algorithm and 1,000 bootstrap replicates were used to estimate node support values. BI analyses were conducted using the

Markov chain Monte Carlo (MCMC) method implemented in MrBayes v3.2.624. MCMC was run for 107 generations with four chains, and trees were sampled every 1000th generation. The fixed number of

samples (25,000) was discarded as burn-in, and convergence was checked by Tracer v1.649. The SSU rDNA sequences of _S. pyriformis_ algae were first checked for group I intron insertions,

following the method described by Hoshina25. The joined exons were then submitted to BLASTN (NCBI), which indicated that the algae are closely related to species of _Chlorella_ clade

(Chlorellaceae). The alignment data of chlorellacean SSU + ITS2 rDNA sequences have been published by Heeg and Wolf26. Based on this, we added several recently described species, symbionts

of some protozoa and sequences obtained here, and then re-aligned them. Tree construction methods (and selected models) were identical to those for the host ciliate, except for MCMC running

for 108 generations. RESULTS DISTRIBUTION AND ENVIRONMENT We investigated 124 ponds and wetlands in Japan and confirmed the presence of _S. pyriformis_ at 23 locations (Fig. 1A).

Distribution areas were somewhat biased into four areas of the middle to Northern part of Japan, which are located at 550 to 2020 m altitude. Water conditions were slightly acidic with a pH

of 3.8 to 6.6 but showed extremely low values of electric conductivity (EC): 6–16 µS/cm. These EC values overlap with those of distilled water or reverse osmosis water (Fig. 1A). Bogs where

_S. pyriformis_ was detected were usually located near the mountain peak or along the ridge (Fig. 1B). Frequently, we encountered blooming of _S. pyriformis_ on the bottom of the bogs (Fig. 

1C). Other times, they were almost all attached to plant stalks or plant debris (Fig. 1D,E). LIGHT MICROSCOPY Cells of _S. pyriformis_ were broadly trumpet-shaped, usually 220–500 × 120–300 

µm. This length–width ratio did not change significantly between the cells attached to something and swimming (Figs. 1E, 2A). The cells were colored green due to their endosymbiotic green

algae that were distributed along the whole body (Fig. 2B,C). A large number of transparent vesicles were present along the ciliary rows immediately under the cell surface (Fig. 2D). To see

the contents, the crushed cells were observed. Symbiotic algae appeared to be typical _Chlorella_-like algae, but no dividing alga was observed (Fig. 2E). The algal cells appeared more

vividly green when compared to those in _P. bursaria_, suggesting that they are richer in photosynthetic pigments (Fig. 2E,F, and Table S2). The symbiotic algae in _S. pyriformis_ had the

same size (Table S2, Fig. S1) and morphology as those in _P. bursaria_, but their biological properties were slightly different. As shown in Table S2, _S. pyriformis_’s algae did not grow on

agar plates, but could only be cultured in well-aerated liquid media. Reinfection experiments showed that _S. pyriformis_’s algae failed to re-infect the aposymbiotic strain of _P.

bursaria_, but those isolated from _P. bursaria_ easily re-infected aposymbiotic _P. bursaria_. Macronuclei were, in general, large and spherical (ø 20–35 µm, Fig. 2G). The average number of

macronuclei was 6.1 (range 4–10, n = 9) for freshly obtained samples, whereas four-year cultured cells (Table 1) contained only one or two. Micronuclei could not be identified. CELLULAR

STRUCTURE OF _S. PYRIFORMIS_ The ultrastructural observations were performed on samples collected on Oct. 28, 2019 at a small pond in Toriko-daira, Japan (37°42′17″N 140°14′53″E). First, the

chemically fixed _S. pyriformis_ was observed with an electron microscope. Large vacuoles were found inside the cells, and the symbiotic algal cells were inside the vacuoles. The symbionts

were found uncovered by individual symbiosome membranes (Fig. 3A,B). Many dark gray stained granules were found in the cytoplasm (asterisks in Fig. 3B). Granules were spherical or oval. The

dyeability was not uniform, and the periphery was dyed more intensely. When the same sample was observed by the quick-freezing and freeze substitution method, the appearance in the cytoplasm

was observed quite differently (Fig. 3C). Large intracellular vacuoles as observed under chemical fixation were not seen. In addition, individual symbiotic algae were covered by a single

symbiosome membrane (Fig. 3C,D). The distance between the symbiosome membrane and the cell wall of alga was extremely close (20–50 nm). Fluffy projections were observed on the cell wall of

the symbiotic algae (arrows in Fig. 3D). Pyrenoids were observed in the chloroplasts of the symbiotic algae, through which thylakoid membranes penetrated (arrow in Fig. 3C). Many

multi-vesicular bodies were observed in the cytoplasm (mv in Fig. 3C,E). The multi-vesicular bodies were not observed at all in the samples prepared by chemical fixation, suggesting that

this structure is very fragile and chemical treatment disintegrates it completely. The number of multi-vesicular bodies per cell was not clear, but there were several granules in each cell.

The maximum size of multi-vesicular bodies was about 1 µm, and the size of small vesicles was 100–400 nm in diameter (Fig. S2). CYTOPLASMIC GRANULES Cytoplasmic granules are colored brown by

Lugol staining, indicating that the granules contained glucans composed of α-1, 4-linked glucose (Fig. 4A). As summarized in Table 2, the stored carbohydrate granules with α-1,4-linked

glucose as a backbone are classified into three types based on their physical and chemical properties, amylose-type starch, amylopectin-type starch, and glycogen. The brown color of the

intracellular granules of _S. pyriformis_ suggests that these granules are rich in amylopectin. For comparison, potato starch, which is an amylose-rich starch, was stained with Lugol under

the same condition and turned blue (Fig. 4B). This indicates that this glucan is of the amylopectin-glycogen type. Observation of the isolated granules with a differential interference

contrast (DIC) microscope revealed that the granules had a strong refractive index (Fig. 4C). Figure 4D,E shows DIC (D) and polarization (E) microscopy of the cytoplasmic granules of _S.

pyriformis_. In the crossed polarizer orientation, each cytoplasmic granule showed a Maltese cross pattern characteristic of starch granules. An ultra-thin section of chemically fixed _S.

pyriformis_ showed that the granules were stained with heavy metals including osmium, lead, and lanthanoid ions (Fig. 4F, asterisks). The starch sheath in the pyrenoid of the symbiont was

also well stained, as shown in Fig. 4F (arrow). After taking the micrograph, the section shown in Fig. 4F was treated with Lugol’s iodine solution, as shown in Fig. 4G. The stain of both the

cytoplasmic granules and the starch sheath was removed by iodine treatment, suggesting that the glucan granules and the starch in symbiotic algae share the same affinity to heavy metals.

HOST RDNA SEQUENCE AND PHYLOGENY SSU, ITS1, 5.8S, ITS2, and 5′ LSU rDNA sequences of four _S. pyriformis_ strains were obtained (Table 1). There were 2049 nucleotides, and all four sequences

were completely identical, including one C/T mixture (Y) at the tetraloop of helix E23_1227 in the SSU rRNA structure (data not shown). _Stentor_ SSU rDNA were collected and aligned with

those of _Blepharisma_ and several outgroup taxa. The phylogenetic tree (Fig. 5A) clearly shows the monophyly of the genus _Stentor_ (the only genus of family Stentoridae). The monophyly of

each species was supported by values of Bayesian posterior probabilities (PP) = 0.99–1 and bootstrap values (BV) = 96–100. For the branching pattern of the relationships of the _Stentor_

species, BI, ML, and NJ analyses showed somewhat different topologies. Here, we provide the species relationships reflecting the differences in these three analyses with iconic morphological

characters (Fig. 5B). The monophyletic relationship of _S. roeseli_ and _S. muelleri_ was perfectly supported. _S. polymorphus_, _S. igneus,_ and _S_. cf. _katashimai_ (see Thamm et al.19)

made a clade, which was placed as a sister to _S. coeruleus_. _S. multiformis_ and _S. elegans_ made a clade. _S. pyriformis_ was clustered with _S. amethystinus_ in all analyses, although

supporting values were not high (PP/MLBV/NJBV = 0.85/74/ < 50). Sequence differences between _S. pyriformis_ and _S. amethystinus_ were 32 substitutions and 5 indels (Y is counted as one

substitution). SYMBIOTIC ALGAL RDNA SEQUENCE AND PHYLOGENY Algal sequences covering SSU, ITS1, 5.8S, ITS2, and 5′ LSU rDNA for four _S. pyriformis_ strains were obtained (Table 1). All

sequences contained group I introns at positions S943, S1367, S1512, and L200 (corresponding to the _Escherichia coli_ SSU and LSU rRNA). Because of this, each sequence reached more than

3,900 bases (L200 introns were not completely determined). The algal sequences among Jodo-daira 1436, Toriko-daira 1256, and fresh samples from Toriko-daira were identical, even including

the introns and the fast evolving ITS rDNA. The algal sequence of Hachimantai 1204 had only one different site from the others. It was at the bulge loop of helix P1 of S1512 intron28, where,

Hachimantai 1204 had A/T mixture (W), whereas the others had A. Search for matching sequences using combined SSU rDNA showed they were closely related to the member of _Chlorella_ clade

(sensu Krienitz et al.29), Chlorellaceae (Trebouxiophyceae). Using SSU + ITS2 rDNA of the member of _Chlorella_ clade, phylogenetic analyses were performed. All tree analyses (only ML tree

is shown) indicated the symbiotic algae of _S. pyriformis_ are clustered with _C. variabilis_, with which monophyletic relationships were fully supported (Fig. S3). DISCUSSION DISTRIBUTION

OF _STENTOR PYRIFORMIS_ IN JAPAN AND ITS OPTIMAL CULTURE CONDITIONS _S. pyriformis_ was described by Johnson in 18936. This algae-bearing _Stentor_ has separated spherical macronuclei

without pigmentation, which certainly differentiates it from other _Stentor_ species (see Table S1, Fig. 5B). While the most common algae-bearing _Stentor_, _S. polymorphus_ assumes a

slender trumpet shape (often shortened), _S. pyriformis_ never resembles such a slender trumpet, but assumes a pear or short conical shape, even when it is swimming6. Presence or absence of

colored pigmentation is also a prominent characteristic for separating _Stentor_ species. Among algae-bearing _Stentor_ spp., _S. polymorphus_ and _S. pyriformis_ only are considered

colorless species, whereas colored species are _S. amethystinus_, _S. fuliginosus_, _S. araucanus,_ and _S. tartari_8 (Table S1). Therefore, _S. pyriformis_ is a clearly discernible species;

however, it remains underexplored. Indeed, we could only find one paper on the new habitats of _S. pyriformis_7, with the exception of the paper of species consolidation of this genus8. We

confirmed the presence of _S. pyriformis_ at 23 locations (Fig. 1A). This indicates that _S. pyriformis_ is by no means a rare organism. We assume one of the reasons why _S. pyriformis_ has

been poorly studied is the difficulty of cultivation. In fact, Johnson6 noted that he could not keep them more than a month and never observed any cells in fission. In addition, after five

years of failure, it was finally possible to culture _S. pyriformis_ for more than several months. Because of objectively unfounded data that we could not include in the distribution data

(Fig. 1A), we noticed the wetlands where we found _S. pyriformis_ were limited to small ponds or bogs locating near the mountain peak or along the ridge (Fig. 1B). That is, the ponds

depending on rainfall without inflowing rivers. Because there is no nutrient flowing in, waters in these ponds showed noticeable oligotrophic tendency, i.e., extremely low electric

conductivity (Fig. 1A), which gave us some clues on culture. The most important point of culture for _S. pyriformis_ was keeping the medium lower electric conductivity. We use the KCM medium

diluted by 2% with Milli-Q water, and changed medium once a week. A non-photosynthetic cryptophyte, _Chilomonas paramecium_ was selected for food. We selected the food so that it would not

itself grow in the culture medium. Growing organisms, like photosynthetic algae, seemed to cause damage to _S. pyriformis_. Using this culture method, _S. pyriformis_ can be maintained for

more than four years (see Table 1). For the organisms not easy to grow in culture, Professor Michael Melkonian mentioned no protist is ‘uncultivable’, there is just human failure30. Here, it

just became possible to culture _S. pyriformis_ 120 years after its discovery; however, this method does not always work. _S. pyriformis_ appears to be extremely fragile and disintegrates

when any variables are unintentionally altered, that is, the culture is still unstable. When its condition deteriorates, the cells divide unevenly in such a way that a part of the cell is

broken. When this happens, the cells become spherical, and the drug drops to the bottom of the dish. It retains this shape for more than a month, but eventually disappears. The doubling time

of _S. pyriformis_ remains 3 to 4 weeks, even under favorable conditions (data not shown). We occasionally encountered the blooming of _S. pyriformis_ all over the bottom of the ponds (Fig.

 1C). _S. pyriformis_, therefore, does not seem to be a particularly slow growing species, but our culture method appears to be far from the optimal culture conditions for them. Three _S.

pyriformis_ strains used in this study are available from the authors upon request. ULTRASTRUCTURE In this study, we compared the conventional chemical fixation method with the

rapid-freezing fixation method for electron microscopic observation. As a result, large vacuoles were observed in the cytoplasm when chemical fixation was used, but not by rapid freezing.

Instead, many multi-vesicular bodies were observed in the cytoplasm. The quick-freezing and freeze-substitution method is considered superior in that it can prevent deformation of the

intracellular structure compared to chemical fixation31. Therefore, it is possible that the originally existing multi-vesicular bodies were artificially disintegrated by chemical fixation,

and the constituent biological membranes fused together, eventually forming large vacuoles. To the authors' knowledge, no intracellular structure similar to the multi-vesicular body in

_S. pyriformis_ has been reported in protists. As multi-vesicular bodies of _S. pyriformis_ could only be observed using the freeze-substitution method, similar granules may also be found in

other protists if the same technique is used for electron microscopy. In animals, on the other hand, aggregates of secretory vesicles resembling the multi-vesicular bodies of _S.

pyriformis_ are present in cardiac telocytes32. The extracellular vesicles form multi-vesicular structures of about 1 μm in diameter and contain materials for intercellular communication

that are involved in cardiac physiology and regeneration. Because _S. pyriformis_ cells often form aggregates at the bottom of the pond, some chemicals may be released from the

multi-vesicular body, attracting nearby cells and forming aggregates. Observation by the freeze-substitution method revealed that the symbiosome membrane was in close contact with the

symbiotic chlorella. Furthermore, fluffy projections were observed on the cell wall of the symbiotic chlorella. These characteristics were consistent with those of _C. variabilis_, which is

symbiotic in the cells of _P. bursaria_9. The only difference was that in _S. pyriformis_, the symbiotic chlorella cells were scattered in the cytoplasm, whereas the symbiotic _Chlorella_ in

_P. bursaria_ were anchored directly below the cell surface. STORAGE GRANULES The iodine in Lugol’s solution selectively binds to α-1, 4-linked glucose found in polysaccharides, such as

starch33 and glycogen34. The color stained with Lugol’s solution reflects the type of glucose polymer. Starches with high amylose content stain blue-violet (cf. Fig. 4B), high amylopectin

stains red–purple, and glycogen stains reddish brown (Table 2). The granules in the cytoplasm of _S. pyriformis_ stained reddish brown with Lugol’s solution (Fig. 4A), suggesting that these

granules are composed of α-1,4-linked glucans with high number of α-1,6-linked branch points, either amylopectin-rich starch or glycogen. The pyrenoid of _Chlorella_ spp. is surrounded by a

starch sheath of two large plates35. As shown in Fig. 4F,G, the image contrast formed by electron staining of the starch granule in the chloroplast (arrow) was lost by treatment with Lugol’s

solution. Although the detailed mechanism is unknown, this observation suggests that electron-stained heavy metals (osmium, lead, and lanthanoid ions) bound to the granules may have been

eliminated by iodine in Lugol’s solution. The cytoplasmic granules of _S. pyriformis_ showed the same staining properties as the starch granules in the chloroplasts of symbiotic chlorella,

suggesting that both types of granules share chemical characteristics as polysaccharides. Alveolates make up one of the most diverse and largest groups of protists. They include three major

taxa: dinoflagellates, ciliates, and apicomplexan protozoa. All three alveolate lineages store glucose in an α-1,4-linked glucose chain with α-1, 6 branches. Ciliates are known to synthesize

glycogen granules. For example, _Tetrahymena_ has glycogen granules between 35 and 40 nm in diameter, each granule being a collection of small γ-granules of 2–3 nm in size36.

Dinoflagellates and apicomplexans typically produce more complex and larger spherical starch particles, usually greater than 1 μm in size37,38. Amylopectin-rich starch and glycogen are very

similar polysaccharides, but they differ in granule size and birefringence (Table 2). Starch granules are large, birefringent, and have a high refractive index, but glycogen does not exhibit

birefringence, and its granules generally have a size of 300 nm or less. When observed with a polarizing microscope, the starch granules show a Maltese cross pattern. This pattern is

derived from the radial arrangement of amylose and amylopectin molecules in granules and is one of the criteria for starch identification. Since the cytoplasmic granules of _S. pyriformis_

are large in size (1–3 μm) and show a typical Maltese cross pattern as shown in Fig. 4E, these granules are likely to be starch granules rich in amylopectin. PHYLOGENY OF _S. PYRIFORMIS_ AND

ITS MORPHOLOGY Relationships of _Stentor_ species were not clearly resolved. BI and ML analyses indicated basal diverging of the _S. pyriformis_ + _S. amethystinus_ clade from others, but

NJ analysis did not indicate so (Fig. 5). Recent phylogenetic analyses inclusive of _Stentor_ species also indicated basal diverging of _S. amethystinus_ from the others; however, the

monophyly of the others is not highly supported21,22. Therefore, the one thing that can be said is that _S. pyriformis_ is closely related to _S. amethystinus_. For the identification of

_Stentor_ species, the shape of macronucleus, presence or absence of cortical pigmentation, and symbiotic algae are very important and iconic characteristics8,19. _S. pyriformis_ and _S.

amethystinus_ share beaded macronuclei and the presence of symbiotic chlorella (Table S1, Fig. 5B). Pigmentation is present in _S. amethystinus,_ but not in _S. pyriformis_. Pigmentation is

a noticeable characteristic, which tinctures the whole body of _Stentor_ cells. The pigment is thought to function as a defense against predators39. However, the kind of pigment compound

depends on the species40, and the relationship between pigment possession and phylogeny is poor (Fig. 5). Of note, colorless vesicles exist in _S. pyriformis_ (Fig. 2D). The short and fat

shape is also a common characteristic for _S. pyriformis_ and _S. amethystinus_, in this genus with many elongated trumpet shape species6,8. SYMBIOTIC ALGAE IN _S. PYRIFORMIS_ Algae-targeted

PCR products from whole cells of _S. pyriformis_ were sequenced directly, and clear peaks were obtained for each. This shows that all or nearly all of the algal symbionts in each _Stentor_

cell are unified, regardless of samples under long-term culture or nature. In addition, all symbionts were closely related to _C. variabilis_ (Fig. S3), which has been known as a

representative symbiont of _P. bursaria_ (Oligohymenophorea), the model organism of multi-algae retaining protists (MARP41) style symbioses. For the chlorellacean species, the diversity of

ITS2 sequence comparisons has often been adopted. For two organisms to compare, ITS2 sequence differences (gaps are counted as a fifth character) usually fall either less than 2% for single

species or more than 10% for different species42,43. This characteristic simply encourages a species concept. The ITS2 sequences of _S. pyriformis_ algae differ only by one nucleotide site

out of 248 sites from those of _P. bursaria_ algae (Fig. 6A), which strongly suggests the symbiotic chlorella of _S. pyriformis_ are also _C. variabilis._ Several _Stentor_ species retain

coccoid green algae8 (Table S1), but only three algal sequences have been published. Two algal sequences from _S. polymorphus_ belonged to different clades from Chlorellaceae44,45. As for

the other algal sequence of _S. amethystinus_, the symbiont may belong to Chlorellaceae46. This sequence (EF589816) is short (991 bp) and only covers a part of SSU rDNA; therefore, it was

not included in our phylogenetic analyses (Fig. S3). The sequence differs from _C. variabilis_ with 10 base changes and 3 indels, indicating that it is not _C. variabilis._ In the case of

_P. bursaria-C. variabilis_ symbiosis, _C. variabilis_ has been shown to be vastly different from other free-living species. _C. variabilis_ demands organic nitrogen compounds47 and leaks

nearly half of the photosynthate to outside algal cells48,49. Furthermore, they are sensitive to the _C. variabilis virus_ (CvV; so-called ‘NC64A virus’), which is abundant in natural

freshwater50,51,52. Therefore, _C. variabilis_ should be considered an already evolved species that is unable to survive without the protection of the host cell53. Four _C. variabilis_ rDNA

sequences obtained from _S. pyriformis_ were identical, with the exception of a nucleotide position in the S1512 intron. Here, the regions without group I introns, i.e., SSU, ITS1, 5.8S, and

ITS2 rDNA, are compared among _C. variabilis_ sequences of _S. pyriformis_ and of _P. bursaria_. Several published sequences cover the above SSU-ITS region, of which varieties are shown as

_P. bursaria_ symbiont genotype (PbS-gt) 1 to 3 (Fig. 6A). Due to the small number of sequences, it is still unknown whether these genotypes depend on (or are related to) their living

regions. Genotype 1 was from USA and Japan, genotype 2 was from China, and genotype 3 was from Australia. All available sequences for _S. pyriformis_ symbionts were obtained in this study,

and they were all from Japan. As a result, all sequences of _S. pyriformis_ symbionts were aggregated into a single genotype SpS, which was distantly related to all _P. bursaria_ symbionts,

including those from Japan (Fig. 6B). Five variable sites are found in SSU rDNA among _C. variabilis_ genotypes, of which four are concentrated to that of the symbionts of _S. pyriformis_

(SpS) (Fig. 6A). C/T substitution at alignment position 656 will be a hemi-compensatory base change (hemi-CBC) at the E23_2 helix of SSU rRNA structure (Fig. 6C), whereas the other four

sites are at single strand regions (data not shown). Mutations (1821–1828) including comparatively large indels were seen in ITS1 region (Fig. 6A). It was found that all these mutations are

assembled in helix 1 (for chlorellacean ITS1 structure, see Bock et al.54,55). Thermodynamic analysis via Mfold56,57 predicted that PbS sequences form linear helix 1 similar to the other

chlorellacean species, but SpS sequences including the additional nucleotides may form a dichotomous branching of helix 1 (Fig. 6D). The group I introns inserted in SSU rDNA of _S.

pyriformis_ symbionts are identical to those of _P. bursaria_ symbionts28,58 in terms of numbers (three introns) and insertion sites (S943, S1367 and S1512). The sequences of S943 and S1512

introns are matched more than 99%. However, with respect to the S1367 intron, a large length gap was found (168 nucleotides) at the tip of P8 (Fig. S4). This section has been indicated as a

homing endonuclease gene remnant28, and those of _S. pyriformis_ symbionts are presumed to be a more degenerated form than those of _P. bursaria_ symbionts. At any rate, the symbiotic algae

of _S. pyriformis_ were found to be _C. variabilis_. Because _S. pyriformis_ never lost the symbiotic algae in four years of culture, and all four algae had nearly identical genetic

characteristics, the symbiotic relationships between _S. pyriformis_ and _C. variabilis_ can be regarded as stable, or permanent. Although _S. pyriformis_ and _P. bursaria_ share _C.

variabilis_ as their endosymbionts, considering the genetic differences depending on their host species, the sharing event has not happened recently. Symbiont sharing among various host

species has also been known for some ciliates41,59 (_Carolibrandtia ciliaticola_ in Fig. S3), and a script to spread a particular algal symbiont has been suggested41. Given the physiological

characters of _C. variabilis_ (mentioned above), this algal species might be an ideal algal symbiont, and it will be no surprise if the other protists also retained _C. variabilis_ as their

algal partners. Research on the symbiotic algae that other _Stentor_ spp. have and on host and regional dependencies are awaited. ADAPTATION OF _S. PYRIFORMIS_ TO OLIGOTROPHIC ENVIRONMENT

IN HIGHLAND MARSH In Japan, _S. pyriformis_ lives only in alpine ponds (Fig. 1), where the winter is cold, and the surface of the pond is always covered with ice. The water in these ponds

has low electrical conductivity (~ 10 μS/cm), and there are few living organisms except _S. pyriformis_, meaning that only little food is available in wintertime. The reason this ciliate is

rich in stored carbohydrate granules may be due to its need for nutrients to survive such harsh winter environments. Preliminary studies suggest that many protists, especially ciliates, may

make starch. Large amounts of cytoplasmic granules that show a Maltese cross were observed in chlorella-bearing ciliates such as _P. bursaria_, while only a small amount of such granules was

observed in _Euplotes aediculatus, Paramecium caudatum, Blepharisma japonicum, and Tetrahymena pyriformis_. Protists with symbiotic algae seem to produce particularly large amounts of

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formation and cannibalism are known as other strategies for protozoans to survive starvation conditions60. This study suggests that the use of carbohydrate granules stored in cells may be

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ACKNOWLEDGEMENTS On 11 February 2018, Professor Dr. Yuuji Tsukii, one of the authors of this article and known as the creator and curator of “Protist Information Server”

(http://protist.i.hosei.ac.jp/), suddenly passed away while preparing for this article. This paper is dedicated to the memory of Dr. Tsukii, who expressed a deep interest in algae-bearing

ciliates in his later years. This work was supported by Japan Society for the Promotion of Science KAKENHI [Grant Number 19K06814]. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Nagahama

Institute of Bio-Science and Technology, Tamura 1266, Nagahama, Shiga, 526-0829, Japan Ryo Hoshina * Laboratory of Biological Science, Hosei University, 2-17-1 Fujimi, Chiyoda-ku, Tokyo,

102-8160, Japan Yuuji Tsukii * Research Group of Biological Sciences, Division of Natural Sciences, Nara Women’s University, Kitauoya-Nishimachi, Nara, 630-8506, Japan Terue Harumoto *

Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, 657-8501, Japan Toshinobu Suzaki Authors * Ryo Hoshina View author publications You can

also search for this author inPubMed Google Scholar * Yuuji Tsukii View author publications You can also search for this author inPubMed Google Scholar * Terue Harumoto View author

publications You can also search for this author inPubMed Google Scholar * Toshinobu Suzaki View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

R.H.: DNA and RNA analyses. Y.T.: Distribution of _Stentor pyriformis_ and establishment of culture method. H.T.: Culture maintenance. T.S.: Detailed observation and analyses of storage

granules. R.H., T.S.: Drafted the manuscript. All authors contributed to preparation of the manuscript. CORRESPONDING AUTHOR Correspondence to Ryo Hoshina. ETHICS DECLARATIONS COMPETING

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_Stentor_ with symbiotic algae growing in an extremely oligotrophic environment and storing large amounts of starch granules in its cytoplasm. _Sci Rep_ 11, 2865 (2021).

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