Favoritos de closterium_mysterium

On the bark of a linden tree, along with patches of Graphis scripta. Photobiont is Trentepohlia - this very humid valley is rich with pure patches of Trentepohlia. The small piece of loose bark I found had no apothecia, but did have two pycnidia. Conidia were hyaline, bacilliform, about 4-5 microns long and 1.2 microns wide.

The 1st and 2nd photos are enlargements of sections of the colony when first encountered, i.e, when the cells were only loosely associated. Photos 3 through 10 capture the relatively rapid aggregation of the cells into a tight cluster; aggregation appeared near maximum within 1.5 minutes after the start of the observation. I suspect exposure to the focused illumination from the scope somehow triggered the aggregation.
Amazing to witness! Interesting (and fast!) signaling biochemistry at play here! ...or is this similar to the coordinated flight of bird flocks, relying on "visual" cues?
Water sample collected from a vernal stream, near moss-covered rocks and leaf-covered bottom; water depth 4-5 inches with very little flow. Air temp 55F

Video: https://youtu.be/sfbkRjOaZI0

Photos are snapshots of the same individual in the video

Cell size: 48 µm in length (excluding flagella)

Site of sample collection: Pavilion, Katsurashima Ryokuchi north pond (a freshwater habitat), Sendai, Japan

Date of sample collection: March 26th 2022

Weather: Cloudy

Water temp.: 9.0°C

pH 6.4

Date of observation: March 27th 2022 (the collected sample in a plastic container was left near a window out of direct sunlight at room temperature until observation)

Bright field observation using a Wraymer microscope (model BX-3500TL, Osaka, Japan) equipped with a Floyd-2 HDMI ethernet digital camera (Wraymer, Osaka, Japan). The accuracy of the scale bar was confirmed by using a stage micrometer glass slide (1 div. = 10 µm; Wraymer, Osaka, Japan) at each magnification.

Mag. 400x
Helical cyanobacteria with crosswalls that are difficult to see, but present (indicated by black arrows where they are distinct). Specie: possibly A. jenneri as seen here https://www.researchgate.net/publication/330579181_Detailed_characterization_of_the_Arthrospira_type_species_separating_commercially_grown_taxa_into_the_new_genus_Limnospira_Cyanobacteria.
A pond edge water sample (freshwater) was taken on 10/1/2021 using a 10 micron dip net to enrich for microorganisms.

Metopus species most consistent with Metopus es (Müller, 1776) Lauterborn, 1916 from the acidic freshwater kettle pond Chatfield's Hole. Culture 2.5 months old, fed with boiled wheat seed. Imaged in Nomarski DIC on Olympus BH2S using SPlan 100 1.25 oil immersion with oiled condenser and Splanapo 40 0.95 objectives plus variable phone camera cropping on Samsung Galaxy S9+. The population has most features consistent with Metopus es except for the thick hyaline striated cortex with perpendicularly arranged rod-shaped extrusomes (or ? ectosymbionts). Such a thick hyaline cortex is not described for M. es but is a feature of M. fuscus and M. ovalis, but these species have markedly different characters from my population. The population here lacks the S-shaped body of M. es, however Bourland et describe loss of this feature in older cultures: " Specimens from older cultures usually broader, lack sigmoid shape, less twisted on long axis,more acuminate posteriorly" (1). Indeed, my culture was 2.5 months old when imaged and had been fed with boiled wheat seed.

The cells measure 140 um in average length. Cells hyaline, dark at anterior pole due to cytoplasmic granule aggregate. Shape usually elongate, slightly sigmoid,distinctly twisted anteriorly and often in posterior third. Specimens from older cultures usually broader, lack sigmoid shape, less twisted on long axis. Preoral dome slightly convex, usually over hangs left margin, less commonly overhangs right margin, inclined 45◦ to long axis, brim rounded. Body posterior to preoral dome ellipsoidal in cross-section, often flattened in posterior part. Macronucleus ovoid and extends only slightly into preoral dome; chromatin coarsely granular, scattered 2.5–5 m nucleoli; no localized perinuclear endobionts. Micronucleus spherical in depression in midportion of macronucleus. Cytopyge not visible. Contractile vacuole terminal, obconical, excretory pore not observed. Satellite vacuoles seen, ? food vacuoles or derived from channels depicted by Kreutz (2). Cortex flexible, appears thick and hyaline with a perpendicular regular row of rod-shaped subpellicular structures ? extrusomes vs symbionts. Prominent kinetal furrows with interkinetal cortical granules (mucocysts) ellipsoidal to globular, i.e., about 1.5 m-long in vivo, arranged in about eight ordinary interkinetal rows; colorless. Anterior pole granule aggregate comprised of densely packed 1.0–1.5 m dark brown spherical granules. Cytoplasmic endosymbionts rod-shaped, conspicuous in vivo under oil immersion. Food vacuoles up to 15 m in diameter. Ordinary somatic cilia about 10 m long in vivo, perizonal stripe cilia about 15 m in vivo, beat in metachronal waves; slightly elongated, i.e., about 13 m-long posterior cilia, inconspicuous in vivo, Swimming pace moderate; rotates on long axis.

1. Redescription and molecular phylogeny of the type species for two main metopid genera, Metopus es (Müller, 1776) Lauterborn, 1916 and Brachonella contorta (Levander, 1894) Jankowski, 1964 (Metopida, Ciliophora), based on broad geographic sampling. William Bourland, Johana Rotterova and Ivan Čepička. European Journal of Protistology Volume 59, June 2017, Pages 133-154
2. https://realmicrolife.com/metopus-es/

Stentor coeruleus Ehrenberg, 1830, showing a rare and unusual bicoloration which is likely environmentally produced from a sample of the riverside benthos of the freshwater portion of the estuarine river Peconic River. The sample was fed with boiled wheat seed. Imaged in Nomarski DIC (and brightfield at 40x and 100 x) on Olympus BH2S using Splan 4, Splan 10, SPlanapo 20 0.70, Splanapo 40 0.95, and SPlan 100 1.25 oil objectives plus variable phone camera cropping on Samsung Galaxy S9+.

The cells so far showed only swimming forms and measure from 520 up to 800 um in length; I have not seen them anchor and fully extend in the manner of S. coeruleus. This size range is rather small for Stentor coeruleus. The spiral of the oral pit appears dark at low magnification and red with higher magnification and stands out starkly from the turquois blue body. At high magnification, one can see mainly turquoise-colored pigmented pellicular granules with fewer smaller red granules between the somatic kineties but red granules are strikingly concentrated in the buccal spiral pit.

I first considered S. introversus Tartar 1958 based on the mentioned close resemblence to S. coeruleus in most characters- turquois blue color without symbiont algae, moniliform macronculeus, smaller size (1,2). My misdiagnosis was largely based on a paper by Kim et al 2020 (3) who redescribed a brackish population which they called S. introversus based largely on the presence of two colors of granules, turquoise blue (somatic) and red (3). However, this feature is not mentioned anywhere with regard to S. introversus. In fact Foissner et al describe S. introversus TARTAR 1958: "which is described in the USA and is identical in granulation and nuclear apparatus, is slightly smaller (up to 450 um), has a brown-yellow cytoplasm and everts its front end characteristically during contraction" (1). Perhaps more importantly, Kim et al 2020 (3) did not illustrate the main defining character of S. introversus in their population as defined by Tartar 1958 (4): the eversion of the buccal field, and that "border of recurved ectoplasm" which he calls "somewhat scalloped". Indeed, the buccal field of my population is that of garden variety S. coeruleus.

So, how to resolve these differential diagnostic discrepancies. Foissner and Wölfl 1994 included S. introversus in their review of the genus: "Stentor introversus Tartar, 1958. This species matches most characters of S.coeruleus. It is, however, smaller [450 um, up to 580 um, according to Jones (1974); weak character] and the entire zone of adoral membranelles cannot only contract, but also retract. We recognize this form as a distinct species because heteroplastic graft combinations between S.introversus and S.coeruleus did not survive (Tartar, 1958), and because Tartar was an expert on S.coeruleus making it unlikely that he was mistaken" (2). Kim et al found Stentor inversus in Korea, ostensibly the first report of the species since Tartar described it in 1958. "Stentor introversus Tartar, 1958 Diagnosis. Extended body size in vivo 500-600×200 250 μm and trumpet-shaped, contracted body size 250 305×200-250 μm in vivo (n=10) and pouch-shaped with retracted buccal field; cytoplasm colorless; 2 kinds of cortical granules densely arranged in between somatic kineties, larger granules turquoise and 0.4-0.5 μm in diameter, smaller granules reddish and 0.2-0.3 μm in diameter; 250 adoral membranelles; 60-73 somatic kineties; 16-20 buccal kineties including 1 peristomial kinety; moniliform macronucleus with 9-14 nodules. Distribution. U.S.A. and Korea. Remarks. Stentor introversus was not reported since the original description by Tartar (1958) from USA. It differs from the most similar species, S. coeruleus (Pallas, 1766) Ehrenberg, 1831, in the extended body size (500-600 μm vs. 1-2 mm), buccal field retracted when contracted (present vs. absent), and the number of types of cortical granules (2 vs. 1) (Foissner et al., 1992; Foissner and Wölfl, 1994) (3).

In evaluating my diagnosis of this population as S. introversus, Bruce Taylor made me aware of some interesting research on S. coeruleus by Tartar 1972 (5), who Foissner acknowledges as an " expert on Stentor coeruleus" (2). In Tartar's experiments, "after a few days in caffeine solution the dark blue-green ciliate Stentor coeruleus loses all visible pigmentation. The cells remain viable and transparent if the solution is then diluted. In time, however, a light pink color is regenerated in maintenance concentration of the drug. In caffeine-free medium the major dark green pigment is regenerated" (5). Tartar feels that the pink coloration was present all the while: "at first colorless, the animals acquired a light pink color, sometimes two or three days after bleaching or sometimes only three or four weeks later, depending on the strain. That this pigment was regenerated and not a new pigment developed under the influence of caffeine is indicated by the fact that at weaker bleaching concentrations of the drug the slower decoloration removed the green but left a pink color from the start, as if a native pink pigment had been unmasked by loss of the green one" (5).

The presence of two native pigments, pink-red and blue, in S. coeruleus is also supported by the work of Moller 1962 (6). Tartar 1972 (5) pointed out: "These two pigments, now demonstrable by separate regeneration of the pink, probably are those discovered by Moller 1962 using extraction procedures on S. coeruleus: a pink one he called stentorin-1 which was alcohol soluble, and a predominating blue-green pigment - stentorin-2, not extractable with alcohol" (5).

So my population is almost certainly an environmentally induced phenotype of Stentor coeruleus which, due to some unknown environmental stimulus, has developed a strikingly visible pink-red coloration of the oral spiral while retaining its usual predominant turquoise granulation elsewhere. The presence of two different colored granules in S. introversus sensu Kim et al 2020 is also explainable in this way. While my population of S. coeruleus is smaller, so far observed at 540-800 um, I have not yet observed sessile feeding forms which may well reach the 1-2 mm length reported for S. coeruleus.

1. Foissner,W., Berger,H. and Kohmann,F. (1992) Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems-Band II: Peritrichia, Heterotrichida, Odontostomatida. Informationsberichte des Bayer. Landesamtes für Wasserwirtschaft, May 1992, pp. 351-4
2. Revision of the genus Stentor Oken (Protozoa, Ciliophora) and description of S.araucanus nov. spec. from South American lakes. W.Foissner and S.Wölfl. Journal of Plankton Research Vol.l6 no.3 pp.255-289. 1994
3. Taxonomy of 16 indigenous ciliate species (Protozoa, Ciliophora) from South Korea. Ji Hye Kim, Atef Omar, Ji Hye Moon and Jae-Ho Jung. Journal of Species Research 9(4):427-442, 2020
4. Tartar, V. (1958) Stentor introversus, n. sp. J. Protozool., 5, 93-95.
5. Caffeine bleaching of Stentor coeruleus. Vance Tartar. Journal of Experimental Zoology Volume181, Issue 2 August 1972 pp 245-25.
6. Moller, K. M. 1962 On the nature of stentorin. C. R. Lab. Carsberg, 32: 471-498.

Second observation of S. niagarae, this time to show the presence of chitin fibrils, just like its close relative in the Stephanodiscaceae, Cyclotella (or Stephanocyclus). The second shot shows a full fibril. It is twice as long as the cell is wide. I can't find literature reports of fibrils on this genus, but maybe the whole family has them.