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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/

A petalomonad flagellate from the benthos of the channel connecting estuary Napeague Bay with Fresh Pond. The salinity of the channel is 30%. Imaged in Nomarski DIC on Olympus BH2 using SPlan 40 0.70 objective plus variable phone camera cropping on Samsung Galaxy S9+. The consensus is that this is a species of Notosolenus. Thanks to Alastair Simpson for confirming that this is Notosolenus ostium.

The cell measures 50 um in length. Originally I did think I was looking at N. ostium Larsen and Patterson 1990. The overall cell shape, size and prominent mouth or ostium are consistent with this as are the delicate ingestion organelle, the position and shape of the nucleus, and the two flagella- the anterior locomotive one as long as the cell and the shorter one only 0.2 times cell length which attaches to the substrate. What troubled me about this diagnosis is I can see no dorsal or ventral grooves. David Patterson feels that these may be less apparent in well-fed individuals (personal communication). Also, there seems to be a double row of round extrusomes adjacent to the delicate rods of the ingestion apparatus which is not described for this species or, I thought, for this genus.

But....thanks are due to Alastair Simpson for pointing out that my observation has extrusomes similar to those he and Won Je Lee 2014 demonstrated in Notosolenus urceolatus Larsen and Patterson, 1990: "a previously undocumented type of large, globular extrusome is present instead of the tubular extrusomes characteristic of Euglenozoa" (1). Lee and Simpson 2014 also noted " refractile granules lying between the left vestibule margin and the anterior of the cell, often along ridges" (3). However, N. urcelolatus differs from my observation in it's size: variously reported as 11–18.8 um (1) and 15-22 um (2) versus 50 um for this observation and 27-56 um for U. ostium (2).
Also, the posterior flagellum of N. urceolatus is reported as 3/4 cell length (3) or 0.2-0.5 um cell length (1) whereas this structure in my observation is 0.2 cell length and in N. ostium it is reported as 0.2-0.6 cell length.

Thus, based mainly on shape, notably in N. urceolatus the
anterior end formed a short narrow neck around the flagellar canal that was bent to the right (1), cell size, and to a lesser extent posterior flagellum length, I feel that my observation is more consistent with N. ostium. Alastair's kind communication regarding the globular extrusomes found in at least one Notosolenus species dispells my concerns at least about genus identification. I note that Lee and Simpson described other workers finding similar putative extrusome structures in two other Notosolenus species (1). I provide below original descriptions of both N. ostium and N. urceolatus.

" Notosolenus ostium Larsen and Patterson , 1990 Description. Cell outline elongate ovate, 27-56 um long, 15-24 mm wide, the ratio of length to width is 1.5 to 3.2. Dorso-ventrally flattened, dorsally with a median longitudinal groove, and ventrally a wide groove and four fine stripes. With a small obliquely oriented ingestion organelle with two fine rods near the anterior. The majority of the cells have a rounded posterior end but some cells have a slightly pointed posterior end. The reservoir is anteriorly situated in the right side of the cell and the nucleus in the left side. Two flagella of unequal length; the anterior flagellum is as long as the cell, held forward in gliding cells. The posterior flagellum is about 0.2-0.6 times the length of the cell. The organism contained eukaryotic algal material up to 8 mm long. Moves by smooth gliding with the anterior flagellum. Common in late culture. Description based on observations of 30 cells" (2).

"Remarks. Previously reported lengths of cells from marine sites (subtropical and tropical Australia, Brazil, Fiji, Hawaii and Panama) range from 24 to 40 mm (Larsen and Patterson, 1990; Ekebom et al., 1996; Patterson and Simpson, 1996 ). Our observations extend the size range. We observed two cells measuring 43 and 56 um which may be assignable to N. ostium. Notosolenus ostium is easily distinguished from other species of Notosolenus by its deep dorsal groove and visible ingestion organelle, which has not been seen in other species of the genus except N. triangularis Larsen and Patterson, 1990. Notosolenus ostium is similar to N. lagenos Skuja, 1948 in length and general appearance and in having a very short recurrent flagellum, but N. ostium can be distinguished by its wide grooves on both faces of the cell" (2).

"Notosolenus urceolatus Larsen and Patterson, 1990. Description. Cell pitcher-shaped, 15- 22µm long, 9- 14µm wide, anteriorly flagella emerge from short protrusion or neck which may or may not lie at a slight angle; posterior broadly obtuse. Ventrally with a shallow median groove and fine distant stripes, dorsally with three ridges. Reservoir in the right hand side of the cell, with refractile granules lying between its left margin and the anterior of the cell, often along ridges. Anterior flagellum slightly longer than the cell, posterior flagellum about 3/4 of the cell length. Nucleus in the left hand side of the cell. Found. Bowling Green Bay, Queensland; Ilha do Fundao, Rio de Janeiro.
Remarks. Notosolenus urceolatus is distinguished by the dorsal ridges which are rare in the genus. N. chelonides Skuja, 1939 has dorsal keels but they are much more conspicuous than in the present species. N. chelonides is twice as large as N. urceolatus.
N. esulcis also has dorsal grooves, but does not have the ventral groove and is more acute anteriorly" (3).

1. Morphological and Molecular Characterisation of Notosolenus urceolatus Larsen and Patterson 1990, a Member of an Understudied Deep-branching Euglenid Group (Petalomonads)
   Won Je Lee & Alastair G. B. Simpson. Journal of Eukaryotic Microbiology 2014, 61, 463–479

2. Heterotrophic flagellates (Protista) from marine sediments of Botany Bay, Australia WON JE LEE and DAVID J. PATTERSON. Journal of Natural History, 2000, 34, 483-562

3. Some flagellates (Protista) from tropical marine sediments
   JACOB LARSEN and DAVID J. PATTERSON. J. Nat. Hist. 1990, 24, 801- 937.

Transverse fission and conjugation in Urocentrum turbo. Imaged in Nomarski DIC using Olympus BH2S under SPlan 40x objective plus phone cropping. From freshwater portion of Peconic River.
Urocentrum Nitzsch, 1827.

Class Oligohymenophora: Subclass Hymenostomata: Order Hymenostomatida
Body cylindrical and ventrally slightly flattened; constricted at the middle; 2 broad girdles of cilia and 1 eccentric posterior tuft; a zone of short cilia in the constricted area; buccal cavity subequatorial with 1 membrane and 2 short undulating membranelles; in the postoral area, a longitudinal zone with small cilia; macronucleus horseshoe-shaped and posteriorly located; a single micronucleus; contractile vacuole terminal, and with 8 collecting canals; large number of trichocysts all over the body.

Heterometopus palaeformis Kahl 1927 Foissner 2016 from decomposing wheat seeds placed in a sample of benthos of the acidic freshwater kettle pond Chatfield's Hole. Imaged in Nomarski DIC on Olympus BH2S using SPlanapo 40 0.95 objective plus variable phone camera cropping on Samsung Galaxy S9+. The cells measure from 90-100 um in length. The body is slender and elongated with roughly parallel sides but occasional individuals are plumper. The peristome roughly 30 % of the cell length and is straight not twisted. The adoral zone is ventrally located and straight, not in the right lateral position and convex as seen in Metopus. The preoral dome is small and does not overhang the body. There is an elongate sausage-shaped macronucleus with a somewhat wavy border in the anterior half of the cell. There is a spherical micronucleus usually overlapping the edge of the macronucleus. There are no caudal cilia or setae. There is a terminal contractile vacuole.

This morphology is that of the common freshwater ciliate Metopus palaeformis. Kahl 1927 as redescribed by Foissner et al 2002 (1). However, Foissner 2016 (2) transferred M. palaeformis to the genus Heterometopus. Foissner writes: " The most conspicuous and diagnostically important features of Heterometopus are the J-shaped zone of adoral polykinetids and the slender, cylindroid body" (2). My population (n=6) differs somewhat in that the long ellipsoid maconucleus is quite anteriorly placed often beginning at the anterior 10% of the cell and seldom extending past the equator. This is in contrast to the postoral position described by Foissner et al 2002 (1). But Esteban et al among others note that the morphology of M. palaeformis is quite variable (3).

From Foissner et al 2002 (1) "Improved diagnosis: Size about 100 x 25 um. Elongate ellipsoidal, hardly spiralized, preoral dome inconspicuous because only slightly curved and flat. Macronucleus postoral in middle body third, oblong. Cortical granules in loosely spaced rows, minute, strongly refractive. All somatic cilia of similar length, arranged in an average of 18 meridional rows, 5 modified to a perizonal stripe about half as long as adoral zone composed of 20 membranelles on average" (1).

"Description of Madagascan neotype population: Size 80-120 x 15-30 um in vivo, in other populations 70-200 x 8-31 um (ESTEBAN et al. 1995); length:width ratio 3.7-5.6:1, on average 4.3:1 in vivo. Shape inconspicuous, that is, elongate ellipsoidal and only indistinctly spiralized and flattened, preoral dome comparatively inconspicuous because only slightly curved and flat. Macronucleus postoral in middle third of cell, globular to very oblong (6:1) and more or less distinctly tortuous. Micronucleus attached to macronucleus at varying positions, about 4 um across in vivo. Contractile vacuole in posterior body end, excretory pore not recognizable. Cortex distinctly ribbed along ciliary rows; cortical granules conspicuous, although colourless and only 0.2-0.3 um across, because strongly refractive and in distinct, occasionally zigzagging rows within and between kineties. Cytoplasm colourless, packed with granules 1-2 um across and 3-5 um long rods,likely methanogenic bacteria, which intensely impregnate with protargol. Likely feeds on bacteria. Movement inconspicuous. Cilia about 12 um long in vivo, no elongated caudal cilia, paired only in dome area, anterior basal body of dikinetids barren and slightly smaller in other regions; arranged in an average of 18 equidistant, almost meridional rows, leaving blank posterior pole centre. Perizonal ciliary stripe of usual structure, short, that is, about half as long as adoral zone of membranelles. Adoral zone inconspicuous because occupying merely one third of body length, only slightly spiralized, and membranellar bases only 4 um long. Paroral membrane short and inconspicuous" (1).

"Occurrence and ecology: Metopus palaeformis is a common freshwater ciliate and rare in terrestrial habitats, where M. hasei is much more frequent. It is an obligate anaerobe lacking conventional mitochondria, like the congeners (Esteban et. al. 1995). Reliable records are known from Europe (Esteban et. al. 1995, KAHI 1932), Namibia and Madagascar. Identification and comparison with related species: Our observations match the original description (Khal 1927a, 1932) and add significant data on the infraciliature. Thus, identification is beyond reasonable doubts, and the population can serve as a neotype. Metopus palaeformis is highly similar to M. hasei, as redescribed by Foissner & Agatha (1999), except for the lacking caudal cilia. Indeed, this is the sole reliable difference, especially when the considerable variation both species exhibit is taken into account (Esteban et al. 1995, Foissner & Agatha 1999). A minor difference concerns the preoral dome, which is more distinctly curved in M. hasei than in M. palaeformis" (1).

"Metopus palaeformis has been kept in culture and studied ecologically for several years by Finlay and co-workers (for a brief review, see Esteban et al. 1995). Detailed morphological data were not provided, although Esteban et al. (1995) (3) documented various cytological features and the morphological variation by excellent micrographs. The strong variation was not seen in the specimens from the non-flooded Petri dish culture and seems to be related mainly to starvation. However, KAHL (1932) observed similar variation in nature and distinguished a forma typica, ovalis, and, attenuatus. Based on the data available, Esteban et al. (1995) suggest the following synonymy, with which we largely agree: M. hyalinus KAHL, 1927a; M. rostratus Kahl, 1927a; M. tenuis KAHL, I927a; and Tesnospira alba Jankowski ,1964b- (for the last two the synonymy is doubtful in our opinion)" (1).

"Heterometopus differs from Metopus s. str. mainly by the shape and location of the adoral zone of polykinetids, a feature used to split Metopus s. l. into several subgenera, especially by Jankowski (1964a,b, 2007). This has been widely acknowledged (Bourland and Wendell 2014; Foissner 2016; Lynn 2008) and the subgenera raised to genera. In Heterometopus, the J-shaped adoral zone extends slightly obliquely on the ventral side and makes a sharp proximal turn when it enters the cell. Indeed, this pattern is highly similar to those found in various heterotrichs, for instance, Blepharisma and Pseudoblepharisma. In Metopus s. str., the adoral zone extends ventrally and right laterally becoming more or less sigmoid and slightly to distinctly convex on the right body side. This causes a very different appearance when Metopus and Heterometopus are seen side by side. The most conspicuous and diagnostically important features of Heterometopus are the J-shaped zone of adoral polykinetids and the slender, cylindroid body" (2).

1. Soil Ciliates (Protozoa) Ciliophora) from Namibia (Southwest Africa), with Emphasis on Two Contrasting Environments, the Etosha Region and the Namib Desert PART I: Text and Line Drawings. Wilhelm Foissner Sabine Agatha and Helmut Berger. Denisia 5 June 2002 , pp. 885-9.
2. Heterometopus meisterfeldi nov. gen., nov. spec. (Protozoa, Ciliophora), a new metopid from Australia. Wilhelm Foissner. European Journal of Protistology Volume 55, Part B, September 2016, Pages 118-127
3. Esteban, G., Fenchel, T., Finlay, B., 1995. Diversity of free-living morphospecies in the ciliate genus Metopus. Arch. Protistenk. 146, 137–164.

Mag. 400x

• A pond-side water sample taken on 02/24/2024 using a small sample bottled attached to an extension pole Air temp 42F (no ice at the shore).

The 3 images derive from 3 slightly different focal planes (looking for flagella). Initially thought this may be Apiocystis brauniana, which has been observed in the area. However the tubular containment of the cells prompted more looking. Palmodictyon varium seems a perfect fit, given its tube-like sheath surrounding the cells and the undulate sheath margin (bulging round cells), characteristics that correspond well with images at -algaebase.org-, https://www.algaebase.org/search/images/detail/?img_id=19805. (also see http://cfb.unh.edu/phycokey/Choices/Chlorophyceae/colonies/colonies_not_flagellated/PALMODICTYON/Palmodictyon_key.htm)

Mastigamoeba species from the intertidal benthos of marine estuary Acabonac Harbopr at Louse Point imaged in Nomarski DIC on Olympus BH2 under SPlan 40x objective plus variable phone cropping on Samsung Galaxy S9+. Folllowing discussion is from: https://de.wikipedia.org/wiki/Mastigamoeba
Mastigamoeba is characterized as a genus of unicellular organisms characterized by an amoeboid (polymorphic) body with hyaline (transparent) cytoplasm and a flagella (amoeboid flagellates). Currently, the World Register of Marine Species lists only two marine species: M. pachyderma Skuja, 1948 and M. schizophrenia Simpson, Bernard, Fenchel & Patterson, 1997.

Due to its similarity to genera such as Mastigella and Mastigina, the genus Mastigamoeba was specified in 1891 to include only organisms with the following characteristics: amoeboid flagellates with hyaline cytoplasm, a direct connection between flagella and nucleus, lateral pseudopodia (pseudopods) and an elongated nucleus. However, the members of the genus change between different morphologies during their life cycle and can exist not only as amoeboid flagellates but also as unflagellated (aflagellate) amoebae, as multinucleated amoebas and as cysts.

During the 20th century, hundreds of species were described under the genus Mastigamoeba solely on the basis of external morphological features. However, recent research on their life cycle has shown that one and the same organism takes on many different morphologies (forms) over the course of its life, calling into question this large number of species described. Currently, there are about nine confirmed and distinguished species of Mastigamoeba, with many more being questioned (see below). Cavalier-Smith described the class Archamoebea in 1983 and included the order Mastigamoebida with the genus Mastigamoeba. Historically, amoeboid flagellates were included in the Pelobionta, with the Mastigamoebidae and the Pelomyxidae (genera Pelomyxa and Mastigella). The naming of the genus is controversial. The genera Mastigamoeba and Phreatamoeba are now considered synonymous by many researchers, although this is questioned by others.

The representatives of the Mastigamoeba are microoxic, i.e. they thrive in environments with low oxygen content (10–20%), e.g. in upper layers of mud or sand or on the sediment surface of shallow ponds. Some have also been found in sewage treatment plants. Such pelobionts are usually found worldwide, studies have confirmed their widespread occurrence in the temperate regions of Europe and North America. Typical habitats include habitats rich in organic matter. Among freshwater rivers and lakes, these organisms are most abundant in stagnant waters where oxygen-poor environments are common. Pelobionts are also found in marine environments. Although most pelobionts are free-living, some representatives are considered endobiotic, i.e. they survive only in the intestines of their hosts. These representatives are completely anoxic and thrive at low pH. They have been found in various vertebrates and invertebrate hosts, especially primates and dogs.

The length of the flagella ranges from 10 μm to 60 μm. In amoeboid locomotion with the help of the pseudopods, the species of clade A have a so-called uroid (also uropodium,typically mulberry-like structure at the rear end, which can appear rounded, rotten and hairy), while the types of clade B instead have a trailing pseudopodium. There is only a single flagellum (flagellum). This consists of the 9+ microtubule structure typical of eukaryotes. The flagellum apparatus consists of a single basal body from which the scourge originates. There is a microtubularcone, a cone-shaped structure that connects the flagellum apparatus directly to the cell nucleus. In the types of clade A, this cone is wide and originates at the base and lateral ends of the basal body. In the species of clade B, however, it is narrow and originates only at the base of this structure. The flagellum apparatus is arranged anteriorly and supports locomotion.

The outside of the cell is covered with a thin, unevenly distributed layer of organic, filamentous (threadlike) material. These threads run parallel to the cell and are 1 μm thick at their thickest point. The chemical composition of this extracellular shell is unknown. Some Mastigamoeba species have spines that are irregularly distributed around the cell. These spines are hollow, and their composition is also unknown. The organic layer sometimes contains prokaryotic symbionts of unknown identity; the exact relationship between the Mastigamoeba sp. and this symbiont is unknown (as of 2011). Another feature of the genus Mastigamoeba is the lack of a Golgi apparatus (dictyosome). However, its central functions are taken over by related elements in the endomembrane system – the endoplasmic reticulum contains some bundled structures and various vesicles that perform the core functions of a Golgi dictyosome. Not all Archamoebae have peroxisomes present. However, studies have shown that some Mastigamoeba species contain at least peroxisomal proteins.

The Archamoebae are all amitochondria, i.e. they have no typical, true mitochondria. The mitochondria in the mastigamoeba have been reduced (in the course of evolution) or transformed into forms that still retain some mitochondrial functions or have altered functions. These are commonly referred to as mitochondria-related organelles (MROs). For example, species like M. balamuthi have MROs called hydrogenosomes. Hydrogenosomes have arisen from mitochondria due to the loss of aerobic life stages. The hydrogenosomes have lost their former genome and the electron transport chain in the course of evolution. However, they continue to be used for ATP production by partial anaerobic oxidation of pyruvate and produce hydrogen gas as a by-product. The biosynthesis of iron-sulfur clusters has passed into a cytosolic function through lateral gene transfer (from the MRO genome to the nuclear genome) and is no longer carried out in the MROs. Other species have reduced mitochondrial organelles called mitosomes. These MROs have been reduced to such an extent that their only function is the biosynthesis of iron-sulfur clusters. They no longer have a function in energy metabolism, so these organisms must obtain their energy in other ways. To compensate for the loss of their own ATP production, these amitochondrial organisms have acquired the ability to import ATP from a host or symbiotic partner.

Several cellular phenotypes are known from many members of the Archamoebae, e.g. (mononucleated) amoeba, multinucleated amoeba, cyst and flagellate. The cyst stage has a single nucleus and is filled with granules and surrounded by a wall of unknown composition (as of 1986). The most important trophic form (growth form, as opposed to resting form) of Mastigamoeba is a mononuclear amoeboid flagellate. However, some species show (at least in some phases of life) a multinucleated morphology: M. schizophrenia has up to 10 nuclei in the multinucleated stage. In M. balamuthi, the predominant trophic form is that of a multinucleated organism that can have up to 46 nuclei. Propagation occurs by mitosis and subsequent budding. In the multinucleated form, this usually results in an unequal number of nuclei in the daughter cells.

Mag. 400x

• A pond-side water sample (retentate) was taken on 01/06/2024 using a 10µ dip net to enrich for microorganisms. (The shoreline had a layer of ice about 1/4" thick that needed to be breached in order to accommodate the dipnet. The retentate contained a lot of sediment due to the shallow depth and disturbance caused by the net.) Air temp 35F.

Smooth dome shape with a turned-up brim (i.e., boarder of the base, like the brim of a hat). The aperture, measured in the first photo, is 30µ. The occupant of the test is not alive. For more information and images, see https://arcella.nl/arcella-vulgaris/.

Mag. 400x

• A pond-side water sample (retentate) was taken on 01/06/2024 using a 10µ dip net to enrich for microorganisms. (The shoreline had a layer of ice about 1/4" thick that needed to be breached in order to accommodate the dipnet. The retentate contained a lot of sediment due to the shallow depth and disturbance caused by the net.) Air temp 35F.

Single specimen. Striae density near center, 8/10µ. As seen here on iNaturalist and here https://diatoms.org/species/gomphonema_acuminatum.

Mag. 400x (1), 200x (2)

• A pond-side water sample (retentate) was taken on 01/06/2024 using a 10µ dip net to enrich for microorganisms. (The shoreline had a layer of ice about 1/4" thick that needed to be breached in order to accommodate the dipnet. The retentate contained a lot of sediment due to the shallow depth and disturbance caused by the net.) Air temp 35F.

Corresponds well with images of Euglena acus found here http://protist.i.hosei.ac.jp/PDB/Images/Mastigophora/Euglena/acus/acus2.html. Inventory: 1 flagellum, 1 eyespot, 2 paramylon granules (rod-shaped, starch storage bodies in the anterior section), discoidal chloroplasts with the appearance, in this specimen, of hexagonal panels in the outer membrane (these are most easily seen, in the central to posterior section, in the middle photo of the composite image and in the last image at lower magnification (but better depth of field).

Cell size: approx. 4–5 µm

Site of collection: Katsurashima Ryokuchi south pond (a freshwater habitat), Sendai, Japan

Date of collection: June 5th, 2021

Weather: Sunny

Water temp.: 22.0°C

pH 6.7
Bright field observation using a Wraymer microscope (model BX-3500TL, Osaka, Japan) equipped with a Floyd-2 HDMI ethernet digital camera (Wraymer, Osaka, Japan).

Movie:
https://youtu.be/8969Rv2i6GA
https://youtu.be/fHdFtUfNSLM

Mag. 400x (1), 200x (2,3)

• A pond-side water sample (retentate) was taken on 01/06/2024 using a 10µ dip net to enrich for microorganisms. (The shoreline had a layer of ice about 1/4" thick that needed to be breached in order to accommodate the dipnet. The retentate contained a lot of sediment due to the shallow depth and disturbance caused by the net.) Air temp 35F.

A euglenid with a single anterior flagellum, hyaline tail section, and a deeply ribbed pellicle (scales!, see comments) reminiscent of euglenoids Lepocinclis fusca and Phacus monilatus. Metabolic movement was not observed. Link to video https://youtu.be/GlnYtTwJ9YI.

Mag. 400x

• A pond-side water sample (retentate) was taken on 10/14/2023 using a 10µ dip net to enrich for microorganisms. Air temp 58F.

Relatively large Galeripora discoides, diameter 130µ; aperture 65µ; ratio of aperture to full diameter 0.5.
Reference: https://arcella.nl/galeripora-discoides/.

Thanks to Ryan Gawryluk for identifying this heliozoan as Ciliophrys species from biofilm that developed in a week old sample from the intertidal benthos of marine estuary Acabonac Harbor at Louse Point launching ramp. The cell body measures 15 um in diameter and has axiopods extending in three dimensions with granular kinetocysts. They taper slightly from base to tip. The long flagellum is held in a double figure 8 pattern. There are peripheral clumps of heterochromatin in the nucleus. These features are all indicative of Ciliophrys azurina Mirkujov and Patterson 2001 (1). In the first video we can see a bacterium being captured in a bag-like pseudopodium as depicted by Siemensma (2).

"Ciliophrys is a naked and heterotrophic pedinellid with either no stalk or a short stubby stalk (unpublished ultrastructural information). It is distinguished from other pedinellid taxa without plastids because the arms radiate from the whole cell surface, and, while in the heliozoan state, has weakly active flagellum held in a figure of 8 configuration. The cell may convert into an arm-less form at which time the flagellum becomes more active and the pseudopodia are withdrawn. These arm-less cells usually swim with the flagellum directed to the front. The fine, non-tapering axopods are supported by single triads of microtubules. As with actinophryids and other pedinellids, the interior ends of these axonemes are associated with nuclei. The composition of the genus was discussed by Larsen and Patterson (1990). We currently admit two species, and here add a third" (1).

"Ciliophrys azurina Patterson, sp. n. Diagnosis. Ciliophrys with tapering arms; nucleus with a central nucleolus and additional peripheral heterochromatin. Description. Cell 15 µm in diameter, with radiating arms with extrusomes. The single flagellum is held in front of swimming cells, and in non-swimming (feeding) cells the flagellum is held tightly curled, typically in a double “figure of 8”. The nucleus is large, prominent and has a nucleolus and clumps of material located around the inner face of the nuclear envelope. Observed consuming diatoms" (1).

"Remarks. Ciliophrys azurina can be distinguished from the other well described species in the genus, C. infusionum, by being considerably larger (15 µm vs 5 µm, although we note that C. infusionum has been reported as up to 20 µm long). More importantly, C. azurina can also be distinguished because the flagellum is longer and held in a double “figure of 8”, because the arms taper from base to tip, and because of the existence of peripheral clumps of heterochromatin in the nuclei. These two characters are held in common with Actinophrys - and there is especial similarity with Actinophrys pontica. We interpret the tapering arms and peripheral heterochromatin as being apomorphic characters for a previously unrecognised clade which includes C. azurina and the two genera of actinophryids and which we here refer to as the heliomonads" (1).

Imaged in Nomarski DIC using Olympus BH2S under SPlan 100x objective and oiled condenser plus variable phone camera cropping on Samsung Galaxy S9 + . One video includes some choanoflagellates just because they are so cute.

1. Taxonomy and Phylogeny of Heliozoa. III. Actinophryids Kirill A. MIKRJUKOV and David J. PATTERSON. Acta Protozool. (2001) 40: 3-25.
2. https://arcella.nl/ciliophrys-infusionum/

Cyclidium glaucoma Möller from a biofilm that formed after a week on a sample of the coarse sand intertidal benthos of marine estuary Acabonac Harbor at Louse Point launching ramp. Imaged in Nomarski DIC on Olympus BH2S using SPlan 40x and 100x oil objective with oiled condenser plus variable phone camera cropping on Samsung Galaxy S9+. The cells measure from 20-24 um in length. The oral apparatus (undulating membrane) occupies from 1/2 up to 3/4 body length. There is a large spherical anterior macronucleus, the micronucleus is not well visualized. There is a long caudal cilium and some cells have a posterior dimple. There is an indistinct cone-shaped frontal plate. There is usually a terminal contractile vacuole and sometimes one or two additional CV in subterminal position are present.

The following is from Taxonomische und ökologische Revision der Ciliaten des Saprobiensytems Band lll Hymenostomata, Prostomatida, Nassulida. W. Foissner, H. Berger, F. Kohmann. Informationsberichte des Bayer. Landesamtes für Wasserwirtschaft, Heft 1/94,548 pp.

Differential diagnosis
1) Size in vivo 14-30 x 6-16 um.
2) Shape barrel-shaped, in poorly fed specimens also spindle-shaped. Front end non-ciliated and slightly cone-shaped (frontal plate), rear end narrow to broadly rounded and, especially in starving cells, with a dimple from which the caudal cilia emerges. Not flattened.
3) Macronucleus spherical in the front half of the body. 1 spherical micronucleus.
4) Contractile vacuole terminal.
5) Resting extrusomes rod-shaped, very inconspicuous.
6) 10 (rarely 11) longitudinal ciliary rows. Cilia in the front half of the body mostly in pairs, at the rear end there is a caudal cilia approximately body-length and terminally curved.
7) Oral apparatus 1/2 body length. 3 very small adoral membranelles arranged one behind the other, right of which an L-shaped undulating membrane, the cilia of which are quite long and when resting. The animal forms a striking sail; detailed structure of the oral cilia only recognizable after silver impregnation, not necessary for the determination).
8) movement jumping and sliding; During the often long breaks the cilia are spread apart undulating membrane is set up like a sail and food is swirled in.

Possible confusion
The following Cyclidium species, some of which are very similar to one another, require a detailed description: C. citrullus (15-16 rows of cilia, --) taxonomy), C. elongatum SCHEWIAKOFF (hind end broadly truncated, oral apparatus about 3/4 body long, usually rests for a very long time and only drives a little back and forth when touched), -+ C. heptatrichum (middle section of body loosely ciliate, hind end with several slightly elongated cilia), C. oblongum BALD (40 pm; mouth on both sides of surrounded by a membrane; check), C. singulare (KAHL) (slender ovoid, contractile vacuole sub terminal, oral apparatus about 1/4 body long), C. versatile PENARD (swims rotating moderately quickly, with the long eyelashes trailing), C. litomesum STOKES (40 pm, dumbbell-shaped shape). -+ Ctedoctema acanthocryptum is significantly slimmer, the pellicle is usually notched and the contractile vacuole is further subterminal. -) Uronema nigricans is in the front third of the oral apparatus slightly dented and has a much more inconspicuous undulating membrane. -+ Pseudocohnilembus species are usually pointed egg-shaped and almost never rest. Features 2, 4, 7, 8 important.

Trepomonas rotans Klebs, 1892 from the northernmost saprobic edge benthos of the spring-fed freshwater coastal pond at Ocean Dunes Apartments in the Atlantic Double Dunes Preserve. This sampling site is situated 250 meters from the edge of the Atlantic Ocean and is rich in decaying organic matter. Imaged in Nomarski DIC using Olympus BH2 under SPlan 40x objective plus variable phone cropping on Samsung Galaxy S9+.

A diplomonad dance party. Today's slide from the northernmost saprobic edge benthos of spring-fed freshwater coastal pond at Ocean Dunes Apartments in the Atlantic Double Dunes Preserve got richer and richer in diplomonad flagellates the longer the slide sat on the stage.

Thanks to Ivan Čepička for identifying this observation. This is the most numerous diplomonad species I observed in a slide that was teeming with diplomonads. The cells measured 10 μm. They were strongly flattened and oval in shape, the rear end was usually wider than
front. Cells moved relatively slowly compared to Trepomonas agilis and T. steinii and changed direction in a calm and fluid manner. The cells rotated vigorously as they moved. These findings were similar to (1).

"Diplomonad flagellates include the genera Trepomonas and Hexamita. Most genera of diplomonads are parasites, and the few genera that are free-living are usually found in organically enriched (and usually anaerobic) sites. The cells are bilaterally symmetrical along their longitudinal axis. There are two anterior nuclei, and associated with each are four flagella which arise at the head of a groove in the body surface. The genera may be distinguished by the relative lengths of the flagella and by the flexibility of the bodies. In both genera, one flagellum of both quartets extends laterally from the head of the groove. The remainder lie within the groove, with those of the more pliable Trepomonas not extending beyond the posterior margin of the cell, as do those of Hexamita . These organisms may feed either by eating bacteria or by pinocytosis" (2).

1. Eva Rmoutilová Thesis: Diversity of free-living diplomonads. Trainer: doc. RNDr. Ivan Čepička, Ph.D.Prague, 2015 Charles University in Prague
   the Faculty of Natural Science.

Mag. 400x

• A pond-side water sample (retentate) was taken on 10/14/2023 using a 10µ dip net to enrich for microorganisms. Air temp 58F.

Pinnularia formica; 120µ L x 18µ. Third encounter with this taxon. There presence appears specific to just 1 of the ~4 local waterbodies I've been monitoring, https://www.inaturalist.org/observations?place_id=any&taxon_id=471765&user_id=mnold1&verifiable=any
Reference: https://diatoms.org/species/pinnularia_formica.

Three posterior spines; . 20 longitudinal rows of plates; body fairly plump; length 50 microns. (Noland, Lowell E. 1925. A Review of the Genus Coleps with Descriptions of Two New Species. Trans. Am. Microsc. Soc. 44(1): 3-13.)
Noland calls them 'micra', an interesting usage which had apparently faded by about 1950.

Mag. 100x (4), 400x (1-3)

• A pond-side water sample (retentate) was taken using a 10µ dip net to enrich for microorganisms. Air temp 58F.

Heliozoan filled with zoochlorella. Note that shorter spine-scales are bifurcated, which is characteristic of A. turfacea. For information and reference images, see https://arcella.nl/acanthocystis-turfacea/.

Mag. 400x

• A pond-side water sample (retentate) was taken using a 10µ dip net to enrich for microorganisms. Air temp 58F.

Flat apex. Widest measure, 85µ; collared, round aperture, 18µ. As seen and described here https://arcella.nl/arcella-costata/.

Mag. 400x

• A water sample was taken on 09/03/2023 from pond-edge using a 10µ dip net to enrich for microbes. Air temp. 80°F.

Amazingly pink ciliate. For a reference see the observation by Bruce Taylor, https://www.youtube.com/watch?v=E0bDRJTc50c.