Volumetric Dynamics of Lorica Forming Material Across the Cell Cycle in the Model Tintinnid Schmidingerella (Alveolata, Ciliophora)

doi:10.21203/rs.3.rs-4641398/v1

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Research Article

Volumetric Dynamics of Lorica Forming Material Across the Cell Cycle in the Model Tintinnid Schmidingerella (Alveolata, Ciliophora)

https://doi.org/10.21203/rs.3.rs-4641398/v1

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Background

Tintinnids are marine planktonic ciliates with tube-shaped or vase-shaped loricae (shells). During cell division, lorica forming material (LFM) is generated and accumulates in the proter (anterior division product). After transverse fission, the proter leaves the lorica and secretes the material, creating its own shell, while the opisthe (posterior division product) retains the parental one. The timing of material production and its final quantity are unknown.

Results

Our study focuses on Schmidingerella Agatha & Strüder-Kypke, 2012, a model tintinnid genus with transparent, champagne flute-shaped loricae. We define five division stages based on the shapes of the opisthe’s newly forming membranellar zone (oral primordium), a feature recognisable both in live and fixed material. In monoclonal, methyl blue-eosin-stained culture material from the Northeast Pacific, the LFM is first perceptible in middle dividers and finally occupies on average 6.7% of the cell volume. The wall volume of the finished lorica estimated by a shape function is at least 4.5-fold larger than the volume of the intracellular material. Protargol-stained field material from the Chesapeake Bay at the Northwest Atlantic provides detailed morphometric data on morphostatic specimens (non-dividers) and dividers. Specimens from our culture and from the field material match in the timing of LFM production and its intracellular distribution.

Conclusions

The LFM is generated only during a certain period of the cell cycle, i.e., in early middle to late dividers. The LFM quantities in the different cell portions (anterior, middle, and posterior portion) suggest an increased production at the level of the oral primordium and the macronuclear nodules prior to its translocation into the anterior portion. The much larger volume of the finished lorica wall indicates a distinct swelling of the material after its secretion by the proter.

Biomaterial

cell division

cell volume

lorica volume

histological stains

marine plankton

Since at least the Jurassic period, tintinnids have inhabited the marine plankton [1]. Vase- or tube-shaped loricae (shells; [2]) are the key apomorphy of tintinnids providing the main feature complex for their taxonomy and classification. Species with hyaline and agglutinated (with foreign particles adhered) loricae are intermixed in molecular phylogenies, indicating homoplasious developments. However, the wall texture (alveolar, solid, monolaminar, bilaminar, or trilaminar) appears to have significant taxonomic importance [3].

Tintinnids divide, like most ciliates, transversely. The first observations published over 230 years ago reported that the opisthe (posterior division product) remains attached to the bottom of the lorica with its contractile peduncle, while the proter (anterior division product) leaves the parental shell [4]. In the historical literature, various modes of lorica construction and sources of the matrix material were discussed: a surface secretion/peeling [5, 6]; a more or less simultaneous transverse split of cell and lorica [7, 8]; and material secretion in a specific region of the proter [9–13] which forms a new lorica autonomously [14] or in collaboration with the opisthe [11].

Campbell (1926) was among the first to document some stages of tintinnid cell division, using mainly iron-haematoxylin for staining the cells of Tintinnopsis nucula (Fol, 1884) Brandt, 1906 [11]. In the following year, he analysed similarly treated material of Schmidingerella serrata (Möbius, 1887) Agatha & Strüder-Kypke, 2012 (reported as Favella serrata) [12]. Later, Biernacka (1952, 1965) studied cell division in Tintinnopsis species, primarily using methyl blue-eosin staining [15, 16]. In his unpublished PhD Thesis, Brownlee (1982) investigated the cell cycles in Schmidingerella (reported as Favella species) and Tintinnopsis species based on protargol-stained material [17]. Consistently, the methods applied in the aforementioned studies revealed the lorica forming material (LFM) as stained granules accumulating in the proter prior to fission (Fig. 1). An involvement of the somatic ciliature in lorica formation has been hypothesised [11, 12, 18], but the process has rarely been observed because of its short duration (a few minutes; [5, 7, 19]) and the ciliates’ sensitivity to increased temperatures and salinities.

Descriptions of tintinnid cell division are usually based on a very limited number of stages, rarely considering the short-lived late dividers. Furthermore, the older publications mentioned above do not provide detailed information on the stomatogenesis (development of the oral ciliature) and the somatic (body) ciliature, as they did not apply appropriate staining methods, except for Brownlee [17]. Recent publications used protargol-stained material, revealing the ciliary pattern and nuclear apparatus but not mentioning the LFM [20–27]. Hence, information on the volumetric dynamics of the LFM during the cell cycle and its intracellular distribution as well as estimates of the total quantity of intracellular LFM and comparisons with the volume of the finished lorica wall are lacking.

Figure 1 Scheme of the cell cycle in Schmidingerella based on data of the present study. Ventral views of the five division stages showing the changes in cell size, the development of the oral primordium as well as the accumulation (increase given in percent) and translocation of the lorica forming material (LFM; marked by magenta). The tintinnid cell divides transversely; thus, the opisthe keeps the parental lorica, while the proter equipped with the material forms a new one. *, postdivider and morphostatic specimen (non-divider) not shown; CM, collar membranelles; L, lorica; OP, oral primordium.

The secreted biomaterial is highly interesting due to its extreme resistance against strong acids and bases [2, 28]; however, its chemical composition has not been elucidated completely, despite some hints for proteinaceous material. After cell death, the nature of the loricae significantly influences their decomposition during and after sedimentation, affects the recycling of the lorica compounds within aquatic ecosystems, and plays a crucial role in fossilisation processes.

The genus Schmidingerella Agatha & Strüder-Kypke, 2012 has been proposed as a model for tintinnid ciliates [29] due to extensive investigations into several aspects of its biology. These studies include: (i) its ultrastructure [3, 29–32], (ii) the general ciliary patterns of morphostatic specimens and some dividers [21], (iii) its genome and transcriptome [33], (iv) its genome architecture [34], and (v) its ecology [35–37]. The study of Smith and collaborators concludes that the genus Schmidingerella comprises two ecologically and morphologically similar species, S. meunieri and S. arcuata, which differ slightly in their 18S rRNA gene sequences (up to 0.5%), significantly in their lorica diameters, and in their micronuclear genomic architecture [34].

This paper on Schmidingerella species addresses several gaps in the current state of knowledge by (i) describing the pattern of cell division based on protargol-stained specimens with unprecedented detail; (ii) providing volumetric data on the intracellular material available for lorica construction; (iii) estimating the timing of material production; (iv) analysing the intracellular material distribution; and (v) calculating the volume of the lorica wall for comparison.

Cell division in Schmidingerella

Cell division patterns were investigated in the field material from the Chesapeake Bay at the Northwest Atlantic, using protargol staining to visualise the most important taxonomic features (Fig. 2 and 'Terminology'). Only properly orientated and sufficiently stained cells were subjected to morphometric analyses. Overall, the results show that cell cycle-induced changes in the oral and somatic ciliature and the nuclear apparatus are not strictly synchronised, as indicated by the considerable variability observed (Figs. 3 and 4). As typical for tintinnids and Oligotrichea in general, (i) the division mode is enantiotropic (with shifting cell main axes and a more or less pronounced inverse orientation of proter and opisthe), (ii) the development of the new oral ciliature takes place in a subsurface pouch without contact to the parental ciliature (hypoapokinetal stomatogenesis), and (iii) mainly the opisthe’s ciliary rows (kineties) are generated by intrakinetal proliferation of basal bodies plus their associated fibrillar structures (kinetids) [38]. To obtain sufficient data for the descriptive statistics, we differentiate only between the three main division stages (early, middle, and late), in addition to morphostatic specimens and post-dividers. In the field material sampled during a single occasion, early dividers were most abundant (n = 210; Figs. 4 and 6) followed by middle dividers (n = 70), late dividers (n = 43), and morphostatic specimens (n = 31). In the supplementary material (Figs. S1–4; Tables S1–5), detailed line drawings and comprehensive morphometric data on morphostatic specimens and dividers are provided.

Morphostatic specimens (Figs. 2, 3 and S1C and D; Table S1). The length of the kineties and the numbers of kinetids in a kinety are highly variable because a differentiation of the morphostatic specimens from very early dividers (start of intrakinetal proliferation) and postdividers with still shorter (opisthe; Figs. S1A and B) or still longer (proter) ciliary rows is frequently difficult (see below). Additionally, deviations from the typical somatic ciliary pattern are occasionally found in form of short kinety fragments in uncommon positions or a somewhat irregular arrangement of the kineties.

Figure 2 Main morphological cell features recognisable in protargol-stained morphostatic specimens of Schmidingerella collected from the Chesapeake Bay at the Northwest Atlantic. Ventral (A) and dorsal (B) views. Asterisk marks position where the oral ciliature of the posterior divider (opisthe) develops in a subsurface pouch without contact to the parental ciliature (hypoapokinetal stomatogenesis). Those basal bodies having associated a distinct cilium are marked by a white dot; for details on the ultrastructure of the oral and somatic ciliature, consult Gruber et al. [29, 30]. MA, macronuclear nodules; PE, contracted peduncle attaching the cell to the bottom of the lorica (not shown); PF, vaulted peristomial field surrounded by the elevated peristomial rim bearing the collar membranelles. Scale bar = 50 µm.

Figure 3 Comparative morphometric analyses (for characters, see Fig. 2) of protargol-stained Schmidingerella specimens collected from the Chesapeake Bay at the Northwest Atlantic. In the case of split kineties (e.g., in late dividers), the sums of the proter’s and opisthe’s kinety lengths and numbers of kinetids are given. The lengths were measured as cord of the organelles. The shorter ciliary fields and lower kinetid numbers indicate that the postdividers studied were formerly opisthes. DK, dorsal kinety; LA, lateral ciliary field; LF, left ciliary field; RF, right ciliary field.

Figure 4 Changes in the shape and arrangement of the macronuclear nodules during the cell cycle in protargol-stained Schmidingerella specimens from the Chesapeake Bay. Both the horizontally orientated nodule and the following L-shaped pattern indicate that the interphase nuclear apparatus with two longitudinally orientated nodules has not been reorganised. After the traverse of the replication bands (sites of DNA replication and histone synthesis), both nodules fuse to one longitudinally orientated mass. 1, one horizontal macronuclear nodule; 2, macronuclear nodule/s arranged in L-shaped pattern; 3, two longitudinal macronuclear nodules; 4, one longitudinal macronuclear nodule; ED, early divider; ELD, early late divider; EMD, early middle divider; LMD, late middle divider; VLD, very late divider.

Morphostatic specimens are about 72 × 61 µm in size in contracted state; their length:width ratio is about 1.2:1. The cell is attached to the bottom of the lorica by a contractile peduncle. The somatic ciliary pattern consists of a right ciliary field with about 16 kineties, a left ciliary field with about 19 kineties, and a lateral ciliary field with about 13 kineties (on average 11 kineties considering also division stages) plus a ventral kinety composed of a monokinetidal anterior and a dikinetidal posterior portion, and usually one dikinetidal dorsal kinety (Fig. 2; [21]); a dikinetidal posterior kinety is lacking. The kineties of the fields are monokinetidal with cilia about 5–6 µm long in the right and left ciliary fields and 2–3 µm in the lateral ciliary field, except for one anterior dikinetid each in the right and left rows with a long (about 17 µm) anterior and a commonly sized posterior cilium. The kineties and kinetids are more densely spaced in the lateral ciliary field than in the right and left ciliary fields, especially, concerning its 5–7 rightmost rows. The ventral kinety is distinctly apart from the right ciliary field, namely, separated by a comparatively broad (about 14 µm) unciliated stripe; thus, it does not commence anteriorly to the right kineties. It extends obliquely and rather parallel to the lateral kineties leftwards to mid-body, where it curves posteriorly and parallels the posterior portion of the dorsal kinety, terminating near the end of cell proper; frequently, it is serpentine due to the cell contraction. The cilia associated with the monokinetids are about 5 µm long, while those associated with the posterior dikinetidal basal bodies increase in length from 6–7 µm in the anterior kinetids to about 10 µm in the posterior ones; the anterior dikinetidal basal bodies are unciliated. The dorsal kinety is distinctly separate from the right and left ciliary fields, extends in a leftwards curvature to the posterior end of cell proper, and is composed of dikinetids having associated a 6–9 µm long cilium with each posterior basal body; frequently, it is accompanied by dikinetidal kinety fragments. The kinetids of a kinety are ostensibly linked by an argyrophilic fibre, corresponding to the long overlapping postciliary ribbons of the kinetids [29]. The oral apparatus occupies the apical cell portion. Even in contracted specimens, the circular adoral zone is perpendicular to the main cell axis and consists of membranelles up to 53 µm long. The about 18 collar polykinetids form a contortus pattern on the peristomial rim and are thus reliably counted only in finished oral primordia; four or five ventral polykinetids extend into the buccal cavity together with invariably one buccal membranelle and the stichomonad (single file of identically orientated basal bodies plus associated fibrillar structures) endoral membrane [30]. The fibrillar system associated with the oral ciliature matches that described in detail by Gruber et al. [30]. Rarely, beaded strands extending about 12 µm beyond the collar membranelles are visible, possibly representing mucocysts extruded from tentaculoids (pin-shaped cytoplasmic extensions; [32]) or striae which are recognisable as longitudinal cytoplasmic stripes of argyrophilic granules on the collar membranelles. About half (56%) of the morphostatic specimens possess two ellipsoidal macronuclear nodules longitudinally orientated in the posterior 75% of the cell; an L-shaped pattern formed by one or two nodules is found in about 33% of specimens, while two horizontal nodules rarely occur (about 11%). One specimen even had replication bands (regions involved in DNA replication and histone synthesis while traversing the macronuclear nodules) in the longitudinally orientated nodules. The two micronuclei (about 3 µm across) were rarely recognisable, probably because they are usually very close to the macronuclear nodules. A low amount of scattered lorica forming material was detected in a single specimen. Food vacuoles contain remains of dinoflagellates (up to 30 × 21 µm in size), centric diatoms (up to 34 µm across), euglenids (up to 45 × 11 µm in size), the silicoflagellate Dictyocha, and coccolithophorids.

Figure S1 Schmidingerella sp., a postdivider (previous opisthe) (A, B), a morphostatic specimen (C, D), and early dividers (E–H) from protargol-stained field material collected in the Chesapeake Bay. Ventral (A, C, E, G) and dorsal (B, D, F, H) views showing the ciliary patterns and nuclear apparatuses. Note that the processes in the somatic ciliature, the nuclear apparatus, and the oral primordium are not completely synchronous, i.e., early dividers might still have not reconstructed the interphasic nuclear apparatus (G). The oral primordia of the early dividers show an anarchic field of basal bodies (E) or the formation of two-rowed membranelles from the anterior right to the posterior left (G). DK, dorsal kinety; LA, lateral ciliary field; LF, LFʻ, proter’s, opisthe’s left ciliary field; MA, macronuclear nodules; OP, oral primordium; RF, RFʻ, proter’s, opisthe’s right ciliary field; VK, ventral kinety. Scale bar = 50 µm.

Figure 5 Protargol-stained Schmidingerella dividers from field material collected in the Chesapeake Bay. Upper row shows the ventral ciliary patterns (A, C, E, G, I). Lower row displays the corresponding optical longitudinal sections demonstrating the accumulation of the lorica forming material and its translocation into the anterior cell portion (B, D, F, H, J). A–D Early dividers. E, F Late middle divider. G, H Early late divider. I, J Very late divider. CM, collar membranelles; LA, LAʻ, proter’s, opisthe’s lateral ciliary field; LF, LFʻ, proter’s, opisthe’s left ciliary field; LFM, lorica forming material; MA, macronuclear nodules; OP, oral primordium; RF, RFʻ, proter’s, opisthe’s right ciliary field; VK, VKʻ, proter’s, opisthe’s ventral kinety. Scale bar = 20 µm.

In the following descriptions of the divisional stages, the increase in length or kinetid numbers refer to the previous stage if not stated otherwise.

Early dividers (Figs. 3, 4, 5A–D, S1E–H and S2A; Table S2). They are on average 81 × 66 µm in size and thus about 12% longer than morphostatic specimens; the length:width ratio is still about 1.2:1. The hypoapokinetal stomatogenesis commences with the formation of an anarchic field of basal bodies left of the ventral kinety’s monokinetidal anterior portion and posteriorly to the lateral ciliary field (Fig. S1E). Soon, the oral primordium sinks into a subsurface pouch and the basal bodies in its middle portion align horizontally. Next, two-rowed membranelle precursors are generated from the anterior right to the posterior left (Figs. 5A and S1G); so far unincorporated basal bodies cluster mainly along the oral primordium’s left and posterior margins. Finally, the parallel membranelle precursors become clockwise inclined (ventral view), and the whole oral primordium commences to spiral, curving its posterior (finally proximal) portion anteriorly and to the cell centre (Figs. 5C and S2A). The portion of the ventral kinety anterior to the oral primordium extends longitudinally and almost parallel to the lateral kineties. At the level of the opisthe’s developing oral apparatus, it arches rightwards and extends as straight oblique row below the oral primordium to the posterior quarter of left cell side, where it curves to the posterior end of cell proper, paralleling the dorsal kinety’s posterior portion. At the level of the oral primordium, a gap between the monokinetidal anterior portion and the mainly dikinetidal posterior portion with potentially some anterior monokinetids widens; in 3% of specimens, overlapping postciliary ribbons no longer connect both portions or are not stained. Single basal bodies in the ventral kinety’s dikinetidal posterior portion as well as paired basal bodies in the ventral kinety’s monokinetidal anterior portion indicate an intrakinetal proliferation of basal bodies (elongation by about 12%). In the right and left ciliary fields, a few kinetids migrate posteriorly, generating a gap between the future proter’s and opisthe’s kinety fragments which remain connected by overlapping postciliary ribbons. Simultaneously, the right and left ciliary fields elongate by about 71% and 15%, respectively (by about 52% and 12% in kinetid number, respectively; always refers to the longest rows). Their posterior fragments are rather similar in length and number of kinetids and soon have one dikinetid at their anterior ends (Figs. 5C, S1G and H and S2A). They are generally shorter and comprise fewer kinetids than the anterior fragments, especially compared to the extremely elongated proter’s right kineties. The kineties in the lateral ciliary field are hardly elongated (by about 14% in length and 24% in kinetid number) and the kinetids remain densely spaced; interestingly, the posteriorly projecting postciliary ribbons of the kineties curve leftwards (Figs. 5C, S1G and S2A). Only in late early dividers, the lateral kineties commence to distinctly proliferate basal bodies and parallel with their posterior portions the upper margin of the oral primordium’s pit (Figs. 5C and S2A). The dorsal kinety is elongated by about 17% due to intrakinetal proliferation as indicated by interspersed single basal bodies (Fig. S1H); hence, the kinetid number has increased by about 38% (rough estimate). By about 30% more specimens have two longitudinally orientated macronuclear nodules, while specimens with different patterns (Figs. 4, 5A and S1G), demonstrating an ongoing reconstruction of the nuclear apparatus, have become fewer. Replication bands traverse the nodules in about 17% of specimens. Lorica forming material (LFM) is not recognisable in early dividers (Figs. 5B and D), except for a single specimen containing very few small granules scattered throughout the cell (Fig. 6B).

Figure S2 Schmidingerella sp., protargol-stained dividers from field material collected in the Chesapeake Bay. Ventral views (A, B, G), optical longitudinal sections (C, H), dorsal view (D), and lateral views (right and left; E, F). A An early divider. The membranelles of the new oral apparatus are still incomplete. B–F Early middle dividers with cylindroidal to funnel-shaped oral primordia. G, H A late middle divider with the new membranelles arranged in a 6-shaped pattern. DK, dorsal kinety; LA, LAʻ, proter’s, opisthe’s lateral ciliary field; LF, LFʻ, proter’s, opisthe’s left ciliary field; LFM, lorica forming material; MA, macronuclear nodules; OP, oral primordium; RB, replication bands; RF, RFʻ, proter’s, opisthe’s right ciliary field; VK, VKʻ, proter’s, opisthe’s ventral kinety. Scale bars = 50 µm.

Middle dividers (Figs. 3, 4, 5E and F and S2B–H; Table S3). They are on average 97 × 71 µm in size and thus about 20% longer than early dividers; the length:width ratio likewise increased to about 1.4:1. The oral primordium of middle dividers comprises the final number of membranelles, which are all three-rowed by now. In early middle dividers, the polykinetids extend on the inner wall of a cylindroidal or funnel-shaped indentation perpendicular to the main cell axis (Figs. 5E and S2B, E and F), and their membranelles commence to grow out. The endoral membrane, which has originated probably de novo, extends at the proximal end of the indentation. In late middle dividers, the oral primordium is roughly 6-shaped in ventral view, i.e., it commences with the distal membranelles directly underneath the ventral kinety’s anterior fragment and performs a clockwise turn (ventral view), plunging into the future buccal cavity, which extends into the ciliate’s right cell half (Figs. 5E and S2G). The gap separating the ventral kinety’s monokinetidal anterior fragment from the dikinetidal posterior fragment with some anterior monokinetids has enlarged and postciliary ribbons connecting both portions are no longer visible. Interspersed single basal bodies indicate an ongoing intrakinetal proliferation in the dikinetidal ventral kinety portion and the dorsal kinety. The total number of kinetids increased by about 21% in the dorsal kinety (rough estimate) and by about 12% in the ventral kinety. In approximately 30% of specimens, the dorsal kinety is accompanied by kinety fragments of varying length (not shown). The corresponding kinety fragments of the long proter’s and short opisthe’s right and left ciliary fields are frequently connected by postciliary ribbons traversing an unciliated horizontal stripe (future division furrow) which gradually broadens. While the right and left ciliary fields have started early to alter and are further elongated by about 25% and 12%, respectively (by about 51% and 41% in kinetid numbers), the developments in the lateral ciliary field are delayed but tremendous. It has elongated by about 137% in length and 128% in kinetid number, particularly in the proter, in which it curves around the oral primordium’s anterior left quarter (Figs. 5E and S2G); the opisthe’s lateral kinety fragments commence to separate and are somewhat shorter than those in the two other fields. The lateral kinetids are less densely spaced than in previous stages, especially in the posterior kinety portions. The number of specimens with replication bands traversing the two longitudinally orientated ellipsoidal macronuclear nodules increased to about 80% (Figs. 4, 5F and S2H); the nodules in about 3% of dividers are in the pre- or post-replication stage. In early middle dividers, most specimens lack LFM, about 27% have a low amount (Fig. S2C), and about 3% have moderate quantities (Figs. 6B and S2H). The granules are usually scattered throughout the cell but show tendencies to enrich in the ventral cell half around the oral primordium (Figs. 5F and S2H). In contrast, all late middle dividers contain LFM: about 25% have low amounts, 73% have moderate amounts (Fig. 5F), and 2% have high amounts which tend to cluster around the opisthe’s membranellar zone (Fig. 6B).

Figure S3 Schmidingerella sp., protargol-stained early late dividers from field material collected in the Chesapeake Bay. Ventral views (A, C) showing the ciliary patterns and the corresponding optical longitudinal sections (B, D) displaying the distribution of the lorica forming material. DK, DKʻ, proter’s, opisthe’s dorsal kinety; LA, LAʻ, proter’s, opisthe’s lateral ciliary field; LF, LFʻ, proter’s, opisthe’s left ciliary field; LFM, lorica forming material; MA, macronuclear nodules; OP, oral primordium; RF, RFʻ, proter’s, opisthe’s right ciliary field; VK, VKʻ, proter’s, opisthe’s ventral kinety. Scale bar = 50 µm.

Late dividers (Figs. 3, 4, 5G–J, S3 and S4; Table S4). They are on average 104 × 73 µm in size and thus about 7% longer than middle dividers and about 45% longer than morphostatic specimens; the length:width ratio is still about 1.4:1. In very late dividers, proter and opisthe have already started to separate and are only connected by a dorsal cytoplasmic bond, while the oblique posterior area of the ventral notch (division furrow) is occupied by the opisthe’s oral primordium (Figs. 5I and S4D and F). The distal collar polykinetids of the opisthe have performed an anti-clockwise rotation, resulting in a circular adoral zone (Figs. 5G, S3A and C and S4A). In early late dividers, the polykinetids are arranged in a contortus pattern and extend across a low peristomial rim parallel to the ventral side (Figs. 5G, S3A and C and S4A). In very late dividers, the ciliated membranellar zone further evaginates and becomes successively perpendicular to the main cell axis with its anterior portion overlaid by the projecting proter’s ventral side bearing the extremely long lateral kineties (Figs. 5I and S4D and F); simultaneously, the peristomial rim bulges, obtaining almost its typical shape. The buccal cavity previously directed obliquely to the right anterior cell portion migrates underneath the lower right quarter of the new adoral zone of membranelles (cp. Fig. S3A and Fig. S3C). The anterior fragment of the ventral kinety is still monokinetidal, except for one specimen with some posterior dikinetids (Fig. S4A), and somewhat longer than in middle dividers, while the posterior fragment consists of several monokinetids followed by many dikinetids and a few interspersed basal bodies. The kinetid number is almost unchanged in the dorsal kinety, which shows a rather indistinct separation of the proter’s and opisthe’s fragments. While the right and left ciliary fields display a moderate increase in length (by about 16% and 14%, respectively) and kinetid numbers (by about 19% and 12%) like the kinetid numbers of the lateral kineties (by about 15%), the elongation of the latter is again more pronounced (by about 63%). Generally, the proter’s fragments contribute distinctly more than the opisthe’s fragments to the total kinety lengths (LF: about 1.8:1; LA: about 4.3:1; RF: about 3.6:1) and total kinetid numbers (LF: about 1.5:1; LA: about 4:1; RF: about 2.3:1) notably in the lateral ciliary field (Figs. 5I, S3C and S4A and D). Whereas the percentage of specimens with replication bands traversing the macronuclear nodules decreased from about 38% in early late to 2% in very late dividers (Fig. 4), the percentage of cells with fused nodules forming a longitudinally orientated elongate ellipsoidal mass increased from about 15% in early late to about 81% in very late dividers (Figs. 5J and S4E and H). In early late dividers, the LFM has arranged around the oral primordium and finally aggregates underneath the proter’s long lateral ciliary field (Figs. 5H and J, S3B and D and S4B, E and G). This large cluster consists of a peripheral longitudinal stripe of small granules embedded in bigger granules (Figs. 5J and S4E and G). Simultaneously, specimens with moderate quantities diminished from early late to very late dividers (from about 35–0%; Fig. 6B).

Postdividers (Figs. 3 and S1A and B; Table S5). Since late dividers are only by 45% longer than morphostatic specimens, a further cell elongation must take place in postdividers, which affects mainly the proter. The comparison of kinety lengths and kinetid numbers between morphostatic specimens and very late dividers (cp. Table S1 and Table S4) indicates likewise a reconstruction of the somatic ciliary pattern after cell division. In the opisthe, the longest field kineties are shorter (by about 17–49%) and comprise fewer kinetids (by about 17–35%) than those in morphostatic specimens, whereas the kineties are longer and have more kinetids in the proter’s right ciliary field (by about 95% and 89%, respectively) and, especially in the lateral ciliary field (by about 257% and 159%, respectively). In the proter’s left ciliary field, however, the rows have almost the same length and number of kinetids as in morphostatic specimen (by about − 5% and + 7%, respectively). The longer right and lateral rows suggest a shortening by kinetid resorption in postdividers representing former proters together with a second round of basal body proliferation in their short dorsal and ventral kineties; apparently, the dikinetidal portion of the ventral kinety is reconstructed mainly after cell division. Similarly, the short kineties of postdividers representing former opisthes necessitate a further basal body proliferation and a concomitant elongation (Figs. S1A and B). The interphase nuclear apparatus, viz., two more or less separated longitudinally orientated nodules, is restricted to 31% of postdividers, while the remaining ones have usually one horizontally orientated (about 50%; Fig. S1B) or inverted L-shaped (about 13%) nodule. A single micronucleus is rarely visible. All available specimens have a complete lorica and do not contain any recognisable granules of LFM.

The present study provides the first volumetric analyses of a tintinnid’s LFM (Figs. 6–9; Tables S6 and S7). Both methyl blue-eosin and protargol stain the intracellular granules of the LFM. Due to a much better differentiation of the granules by the former method than by protargol, which also stains the nuclear apparatus and the ciliature, the quantitative and distributional changes of the intracellular LFM during the cell cycle were analysed in detail only in the monoclonal Schmidingerella from the Northeast Pacific. However, the analysis of the Northwest Atlantic specimens reveals identical processes, allowing a sound generalisation and a more precise localisation of the intracellular material in relation to the ciliary structures.

For describing the LFM accumulation, we define four categories of quantities and distribution patterns each (Figs. 6 and 8; Table S6) and subdivide the cell division in five stages based on the shape of the oral primordium (see 'Terminology’; Figs. 1 and 7). Although the categorisation of the LFM quantities was somehow guided by the division stages, the processes were found to be not entirely synchronous. Anecdotal live observations showed that the cell cycle (Fig. 1) lasts about 24 h. The material was first detected in one early divider (1% of specimens; Figs. 6A and 8). Such small granules which are scattered throughout the cell (Pattern A) and amount up to 1,037 µm³ (category 1) are also detected in roughly half (51%) of the early middle dividers, in which all membranelles already obtained their final three-rowed structure (Figs. 6A, 7A and B, 8 and 9; Table S6). The material further gradually accumulates with a maximum of 4,474 µm³ (category 2) in about 67% of late middle dividers, in which the granules commence to arrange primarily around the oral primordium (Pattern B) (Figs. 6A, 7C and D, 8 and 9; Table S6). In this division stage, the material also starts to aggregate between the opisthe’s and proter’s membranellar zones. An S-shaped pattern dominates in early late dividers (about 46%; Pattern C) (Figs. 6A, 7E and F, 8 and 9; Table S6). Eventually, the material clusters almost exclusively underneath the proter’s lateral ciliary field (see below) with a peripheral longitudinal stripe of small granules embedded in bigger granules (Pattern D) (Figs. 6A, 7G–J, 8 and 9; Table S6). The volume of LFM in very late dividers (n = 5) varies considerably, ranging from moderate (4,098 µm³) to extremely high (21,860 µm³; Table S7); the remaining three very late dividers volumetrically analysed have on average 12,479 µm³ of LFM (Table S7). After separation of both cells, this quantity of LFM is used by the proter to construct its new lorica. To sum up, the LFM accumulation given in percent of its final quantity accelerates during cell division, successively increasing from 8% (early to early middle dividers) via 17% (early to late middle dividers), and 35% (late middle to early late dividers) to 40% (early to very late dividers; Fig. 1; Table S7) in the monoclonal Schmidingerella specimens from the Northeast Pacific.

Figure 6 Bar plots showing the accumulation of lorica forming material (LFM) during cell division in Schmidingerella. A Methyl blue-eosin-stained dividers from monoclonal culture material collected in the Northeast Pacific. B Protargol-stained dividers from field material collected in the Chesapeake Bay at the Northwest Atlantic. 0, no material; 1, low quantity (1–1,037 µm³); 2, moderate quantity (1,038–4,474 µm³); 3, high quantity (4,475–21,860 µm³); ED, early divider; ELD, early late divider; EMD, early middle divider; LMD, late middle divider; n, number of specimens investigated; VLD, very late divider.

Figure 7 Ventral views of monoclonal, methyl blue-eosin-stained Schmidingerella specimens from the Northeast Pacific displaying the purple-coloured lorica forming material (LFM; upper row) and the identically orientated corresponding 3D models of the LFM (lower row). A, B Early middle divider with low quantity (maximum category 1) of LFM scattered throughout the cell (Pattern A). The oral primordium consists of the final number of three-rowed membranelles extending on the inner wall of a funnel-shaped indentation. C, D Late middle divider with moderate quantity of LFM (maximum category 2) mainly arranged around the oral primordium (Pattern B) which forms a 6-shaped pattern. E, F Early late divider with high quantity of LFM (category 3) arranged in an S-shaped pattern around the oral primordium (Pattern C). G, H Very late divider with most material in the anterior cell portion partially covering the obliquely orientated oral primordium (Pattern D). I, J Very late divider just before separation of proter and opisthe. The former contains the entire LFM (Pattern D). The oral primordium is almost perpendicular to the main cell axis. L, lorica; OP, oral primordium. Scale bars = 40 µm.

The disproportionate material accumulation in the middle cell portion probably indicates the main site of its production close to the macronuclear nodules (Fig. 9; Table S7). In late dividers, the LFM generation first stops in the posterior cell portion and finally in the entire cell, while the material is successively translocated anteriorly into the proter (Figs. 7G–J and 9).

To estimate the LFM’s occupancy of the cell volume, the latter was calculated by two different methods: (i) a computer-aided calculation based on the simplified cell outline, and (ii) a geometric calculation, using the cell's dimensions and the formula for a rotational ellipsoid. For determining the finally available LFM quantity, five very late dividers were analysed (Fig. 9; Table S7). In these specimens, the LFM volumes range from 4,098 µm³ to 21,860 µm³, with an average of 12,679 µm³ (without extremes: 12,479 µm³). The cell volumes estimated by the two methods (shape function vs. rotational ellipsoid) deviated somewhat without a clear tendency of higher values generally obtained by one specific method (Table S7). Likewise, the cell volumes do not demonstrate a steady increase during the cell cycle. Comparing the LFM quantities with the corresponding cell volumes estimated by means of the shape function yields occupancies of 2.3–10.3%, on average 6.7% (Table S7).

Comparison of intracellular lorica forming material (LFM) and lorica wall volume

Understanding the behaviour of the LFM from its release by the proter until the formation of the hardened lorica wall requires calculating the volume of the lorica wall and comparing it with the quantity of intracellular material. Based on micrographs displaying optical longitudinal sections of nine typically shaped (excluding Coxliella-shaped ones) Bouin-fixed loricae of Northeast Pacific specimens, the wall volumes were ascertained. They range from 98,655 µm³ to 136,609 µm³ with an average of 120,203 µm³ (M = 120,778 µm³; SD = 11,446 µm³; n = 9). The conservative comparison between the maximum intracellular amount (21,860 µm³) and the minimum wall volume (98,655 µm³) indicates at least a 4.5-fold swelling of the LFM after secretion.

Cell division patterns

Cell division was investigated after protargol staining in only 13 tintinnid species and usually not in detail: Tintinnopsis subacuta, Schmidingerella sp. (reported as Favella sp.), Schmidingerella serrata (reported as Favella serrata) [17, 18], Tintinnopsis cylindrata (with Tintinnidium-like ciliary pattern), Tintinnidium pusillum, T. semiciliatum, Codonella cratera [27], Antetintinnidium mucicola [23], Tintinnopsis cylindrica [20], T. everta [24], T. tubuformis [25], Stenosemella pacifica [22], and Leprotintinnus nordqvisti [26].

Our findings are congruent with previous studies on tintinnid cell division (please, note the differences in terminology). (i) The hypoapokinetal stomatogenesis takes place in a subsurface pouch in the ventral cell half. It comprises a two-tiered circular arrangement of the collar membranelles, namely, a clockwise rotation of the proximal zone portion followed by an anti-clockwise rotation of the distal portion, plus a supposed de-novo development of the endoral membrane (this study; [17, 20, 22, 24, 26, 27, 39]). (ii) The somatic kineties elongate by intrakinetal proliferation of kinetids (this study; [17, 22, 24, 26, 27]), except for the ventral organelles in Tintinnidium species which supposedly originate de novo [27]. (iii) One to several basal bodies migrate posteriorly in each kinety of the ciliary fields, serving as starting points for the opisthe’s kineties (this study; [17, 27]). (iv) The somatic ciliary fields do not elongate and fragment simultaneously, i.e., the right and left fields precede followed by the lateral field (this study; [24, 26]). In contrast, Brownlee [17] described the first signs of proliferation in the lateral field. (v) The opisthe’s right and left kineties soon develop anterior dikinetids and only slightly elongate posteriorly by kinetid proliferation (this study; [27]); in Tintinnidium pusillum, however, a basal body proliferation at the anterior ends of the opisthe’s kineties had been reported [27]. (vi) Just before fission, the opisthe’s ciliary fields are usually shorter and have less kinetids than those of the proter and morphostatic specimens and probably obtain their final length and number of basal bodies only in postdividers (this study; [17, 18, 22, 24, 27]). In contrast, Tintinnidium species (incl. Tintinnopsis cylindrata) perform this second round of proliferation during cell division. (vii) The proter’s ciliary fields are generally longer and have more kinetids than those of the opisthe and morphostatic specimens, particularly the lateral ciliary field, whose kineties extend to the postdivider’s rear end. This suggests a subsequent shortening by kinetid resorption (this study; [17, 18, 20, 22, 24, 40]). (viii) The ventral kinety invariably extends along the right margin of the oral primordium (this study; [17, 18, 20, 22, 24, 27]). In Schmidingerella, this ciliary row is composed of a monokinetidal anterior and a dikinetidal posterior portion. The dikinetidal portion does not represent a separate (posterior) kinety because it extends like the ventral kinety on the oral primordium’s right side, and the ventral kinety displays only a single split (this study; [21]). The ventral kinety of further species (Tintinnopsis cylindrica, T. cf. radix, and T. tocantinensis) has an identical structure but its dikinetidal portion had been misinterpreted as a posterior kinety [20, 41–43]. Interestingly, the formation of dikinetids at the posterior end of the proter’s monokinetidal ventral kinety has rarely been observed, suggesting that this happens mainly in postdividers (this study; [17, 18]). (ix) Regarding dorsal kineties, some studies report an intrakinetal proliferation of basal bodies and a rather clear split of the elongated rows (this study; [17, 18, 27]). Only, Hu et al. (2022) interpreted kinety fragments on the right side of the dorsal kinety as stages of its de-novo origin [26]; however, a more probable explanation might be the occasional occurrence of additional fragments as described in Schmidingerella (this study). (x) In tintinnids with the most complex ciliary pattern, the generation of the proter’s posterior kinety has not satisfactorily been clarified. Gruber et al. (2018) observed a pattern in a single late divider that indicates an unequal split of the posterior kinety [24]. According to Petz and Foissner (1993), its posterior fragment might migrate posteriorly, while its anterior fragment remains near mid-body in a gap between the opisthe’s lateral and left ciliary fields and subsequently migrates anteriorly, shoving through the stripe of the opisthe’s fields [27]. In postdividers, basal bodies might proliferate at the kinety’s anterior end. A de-novo origin of the posterior kinety was postulated by Brownlee (1982) and Hu et al. (2022), however, based on different clues [17, 26]. While Brownlee did not find the proter’s kinety fragment [17], Hu and collaborators interpreted a kinety fragment on the right side of the parental row as the opisthe’s kinety [26]. The latter authors further proposed a fusion of two fragments in postdividers. A more probable explanation might be the occasional occurrence of additional fragments parallel to the posterior kinety. Alternatively, its origin from the dorsal kinety was discussed [17, 18]. (xi) A reorganisation has neither been observed in the proter’s oral nor its somatic ciliature (this study; [22, 26]), except for a supposed renewing of its lateral ciliary field in Codonella cratera [27]. (xii) One replication band each traverses the macronuclear nodules mainly in middle and late dividers. In taxa featuring more than one nodule, these merge into a single mass and then bisect, resulting in one division product each for proter and opisthe (this study; [8, 17, 18, 20, 22, 24, 26, 27, 44, 45]). However, the timing of DNA replication shows an intraspecific variability (this study) and differs between taxa [27, 46]. The reconstruction of the interphase nuclear apparatus takes place in postdividers (this study; [17, 18, 27, 45]), whereas several authors reported it from cells still dividing [8, 12, 15, 44]. Interestingly, the two nodules do not fuse according to Campbell (1927) studying primarily Schmidingerella serrata (reported as Favella serrata; rough lorica wall with a subapical bulge; without canal in process) and occasionally referring to Favella ehrenbergii (smooth lorica wall without subapical bulge; with canal in lorica process) [12]; the observations on both species were confused and are hardly differentiable.

The anterior elongation of a tintinnid’s ciliary row is a rather uncommon morphogenetic process but had been reported for the opisthe’s ventral kinety in Codonella cratera and Tintinnopsis everta [24, 27]. In these taxa, the opisthe’s ciliary row extends longitudinally almost at the same level as the lateral ciliary field, while it is eventually anteriorly curved, commencing above the right ciliary field in morphostatic specimens, necessitating its reshaping and anterior elongation in postdividers [24].

The cell size of late dividers is less than twice the length of morphostatic specimens (this study: 144%; [15]: 145%); thus, also an elongation must take place in postdividers. In our study on Schmidingerella from the North Atlantic, the proter was not distinctly shorter than the opisthe, matching the finding of Laval-Peuto (1981), who calculated the volumes of proter and opisthe to be on average 42% and 58%, respectively [19]; Biernacka (1952) mentioned an uneven division but did not provide details [15].

Lorica forming material (LFM)

So far, only a few anecdotal observations have been published regarding the timing of LFM production and its intracellular translocation. The present study not only provides thorough analyses on these aspects but also presents the first volumetric data, unveiling intriguing insights into lorica formation in tintinnids.

Timing of production. The first granules of LFM were congruently detected in early middle dividers of the Schmidingerella specimens collected at the US west and east coasts (this study; Figs. 6, 7A and B, 8 and S2C). The minor differences between the tintinnids from the two sites emerging in the volumetric analyses (Figs. 6A and B) are attributed to the deviating sampling design (monoclonal cells cumulatively picked during several days vs. one sampling occasion; 'Methods'). Brownlee (1982) depicted the first occurrence of LFM in late middle dividers of S. serrata (reported as Favella serrata) and Tintinnopsis subacuta, while only in early late dividers of Schmidingerella sp. (reported as Favella sp.) [17].

Like the present study, Biernacka (1952) and Laval-Peuto (1981) found LFM only during the process of cell division with a final accumulation anterior to the opisthe’s oral apparatus [15, 19]. According to Biernacka (1965), material not used for lorica formation migrates back into the posterior cell portion where it is resorbed [16]. In other species, material is also present in morphostatic specimens [16, 47]; whether it represents remains of the preceding cell division or is continuously produced is uncertain. Taxon-specific differences in the timing of production and the retention of LFM by the opisthe might affect the ability or inability to generate replacement loricae with a spiralled wall structure and epiloricae (a collar secondarily added to the lorica), respectively. Even, a lorica repair had been suggested [16], yet without convincing evidence.

Translocation of LFM. The findings in the methyl blue-eosin-stained Northeast Pacific specimens match those in the protargol-stained Northwest Atlantic specimens and, in part, previously published data. In early middle dividers, a few, usually small granules of LFM are scattered throughout the cell (Figs. 7A and B, 8 and S2C; Table S6). Transitionally, almost the whole LFM arranges in an irregular ring around the oral primordium (Figs. 5F and H, 7C and D, 8, S2H and S3B; Table S6; [12, 17]). As development progresses, the LFM forms an S-shaped pattern around the oral primordium with a voluminous cluster anteriorly (Figs. 7E and F). In late dividers, most of the LFM aggregate anteriorly to the oral primordium, i.e., in the proter (Figs. 5J, 7G–J, 8, S3D and S4E and G; [15–19, 28, 40, 48]), but does not form a single mass as described by Laval-Peuto in Dictyocysta [18]. While Campbell (1926) was the first to specify the final LFM position underneath the lateral ciliary field [11], the formation of a peripheral longitudinal stripe of small granules embedded in bigger granules is reported here for the first time (Figs. 5J, 7G and I S3D, and S4E and G).

Site of LFM production. The process of LFM generation is unknown and the site of its origin is stated vaguely, namely, the posterior cell portion [15, 17] or the region of the future division furrow [12]. According to our data, however, LFM production is not restricted to a specific cell portion but initiated in the entire cell (Figs. 7A and B and 8 and S2C), and the abovementioned patterns of LFM distribution are best explained by a successive material translocation into the anterior cell half (this study; [15, 17–19]). Since the material quantity in middle cell portions of late middle (LMD) to early late dividers (ELD) is consistently higher than that in the anterior and posterior portions (Fig. 9; Table S7), we suppose that most material is produced in this region, i.e., in proximity to the oral primordium and the nuclear apparatus. Furthermore, the production of LFM accelerates in very late dividers (VLD), especially when the short period of this division stage (about 1 h; own observ.) is considered (Fig. 9; Table S7). In the posterior portions of very late dividers, the LFM production stops and the material is rather completely translocated into the proter (Fig. 9; Table S7; [12, 17, 18]).

Unresolved questions and future directions

Regardless of the staining method used, our Schmidingerella specimens revealed both smaller and larger granules of lorica forming material (LFM) and occasionally displayed internal granule structures. Preliminary ultrastructural studies on the genera Cymatocylis, Cyttarocylis, Parafavella, and Petalotricha identified three types of LFM granules (morula, compact, granular); occasionally, two types co-occurred, potentially representing different stages in the production process [18, 47, 49–52]. Therefore, transmission electron microscopic analyses should be performed to gain insights into the ultrastructure and maturation of the granules, as well as the cellular structures involved in their secretion. Comparative studies on LFM from different tintinnid families will enable the estimation of taxon-specific differences, potentially explaining the variations in lorica structures and textures [3]. These deviations necessitate different behaviours of the released material, such as forming a solid or alveolar wall during the ciliature-mediated self-assembly. Since the presumably proteinaceous lorica is the main tintinnid apomorphy, genes required for lorica formation are likely lineage-specific. Identifying these genes could provide significant insights into the evolution of tintinnids and their adaptation to marine and freshwater environments.

Tintinnids, a fascinating group of predominantly marine planktonic ciliates, have captivated scientists for over 235 years with their elaborate loricae. This study offers the first insights into the volumetric dynamics of lorica forming material (LFM) in the model tintinnid genus Schmidingerella: (i) its quantity produced during the cell cycle, (ii) the timing of its production, and (iii) quantitative comparisons between its final intracellular volume and the cell and lorica wall volumes. We found that LFM is generated only during a specific phase of the cell cycle, viz., from early middle to late division. The distribution of LFM in different cell regions (anterior, middle, and posterior portions) indicates increased production near the oral primordium and macronuclear nodules before its translocation into the anterior portion. The substantially larger volume of the finished lorica wall suggests a significant swelling of the material after its secretion by the proter. Our findings provide crucial insights into the material quantities in the lorica wall, which are vital for ecological studies. Additionally, we introduce an elegant method for calculating lorica wall and cell volumes in further tintinnid taxa, moving beyond the use of simple geometric shapes. This approach can also be applied to volumetric analyses of other organisms with rotational symmetry, improving biovolume estimates. When combined with carbon content conversion factors, it also facilitates the calculation of energy fluxes in food webs. Ultimately, this study advances our understanding of a major aspect of tintinnid biology: how single cells form such a sophisticated structure as the lorica.

The material analysed was collected at the west and east coasts of the USA, namely, in the Northeast Pacific and the Northwest Atlantic. The applied methods are described separately for the specimens from the two locations.

Northwest Atlantic specimens. Field material was taken from the upper 10 m of the Chesapeake Bay (37°07’N, 76°07’W) on 8th July 1987 with a 35 µm-plankton net at a temperature of 24°C and a salinity of 26‰. The specimens were fixed in a modified Bouin’s solution [44] and stained, following the QPS method published by Montagnes and Lynn [53], which reveals not only the nuclear apparatus and ciliary pattern, but also the lorica forming material (LFM). The slides were provided by Wayne Coats (Smithsonian Environmental Research Center, Edgewater, MA, USA). These stained specimens were morphometrically analysed, using an Olympus BX53 microscope equipped with a drawing device. Further, the cell division pattern as well as the accumulation and position of the LFM in the different cell cycle stages were studied.

Northeast Pacific specimens. The monoclonal Schmidingerella strain SPMC 176 was established based on a specimen collected in the Northeast Pacific and provided by Kelley Bright (Western Washington University, WA 98221, Northern USA) in November 2022. In our lab, the culture was maintained for several months in artificial sea water (32‰ salinity; pH 7,8) plus f/2 trace metal solution [54] at 12°C and a light intensity of 1350 lumen of 620–630 nm and 460–470 nm. The cultures were fed with the dinoflagellate Heterocapsa triquetra and the haptophyte Isochrysis galbana (both also provided by K. Bright). In the cultured specimens, the timing of LFM production, the final amount of intracellular LFM, and the volumes of the cell and lorica wall were analysed. We tested three staining procedures to assess which one provides the best differentiation of the intracellular LFM for subsequent volumetric analyses (see 'Supplementary material' for the methods yielding insufficient results). Confocal laser scanning microscopy failed as the LFM had no autofluorescence (for details, see 'Supplementary material').

Methyl blue-eosin stain according to Mann. Staining with the polychromatic solution of methyl blue-eosin provided the best results, namely, the LFM and the loricae tinted pink, the nuclei faintly purple, the cytoplasm blueish, and the ingested dinoflagellate Heterocapsa triquetra orange. Based on the brief information of Biernacka [15] and the protocol of Romeis [55], we developed the following optimal procedure for staining intracellular LFM. The specimens cumulatively collected from the culture over several days were fixed with Bouin’s solution [56] at a sample-to-fixative ratio of 1:2. Next, the cells were washed in distilled water and exposed to a solution consisting of 45 mL 1% a.s. eosin, 35 mL 1% a.s. methyl blue, and 100 mL distilled water. The staining was performed in block dishes for 2 h. After several washing steps in distilled water removing all traces of the dye, the specimens were exposed for 3 min to freshly prepared 0.0005% NaOH in 96% EtOH to remove the excess stain resulting in a clear differentiation of the intracellular LFM. Then, the cells were immediately embedded in albumin on slides, dehydrated in two steps with 100% ethanol for 5 min each, followed by two short exposures to xylol, and finally covered with Eukitt® mounting medium (Merck) and a glass cover slip.

Volumetric analysis of methyl blue-eosin-stained intracellular LFM. In the first phase, the quantity of properly differentiated LFM in each Schmidingerella cell from the Northeast Pacific was categorised into one of four classes: no LFM, low quantity, moderate quantity, or high quantity. In the second phase, the minimum and maximum volumes [µm³] defining the ranges of each class were determined based on volumetric analyses of the categorised specimens. These classes were also applied to the protargol-stained specimens from the Northwest Atlantic.

For volumetric analyses, Z-stack images (140–181 per cell) were captured at intervals of about 500 nm from properly orientated (in ventral or dorsal view) cells. Imaging was performed, using an Olympus BX53 microscope equipped with an Olympus OM-D E M1 Mark II digital camera and the software OM at up to 2000× magnification. Initially, we tested automated pixel classification tools which, however, yielded insufficient results, necessitating manual processing. Therefore, the Z-stack images were processed in Photoshop, using custom scripts, and manually refined to accurately mark only those granules of LFM that were in focus with the focal plane. The processed images were then converted into black (background) and white (LFM) and used as input for Fiji, an ImageJ distribution [57], to generate a 3D surface mesh of the LFM. Subsequently, the LFM mesh was imported into MeshLab [58] for calculation of the total volume. LFM volumes were additionally calculated for three cell portions: the anterior portion (above the oral primordium), the middle portion (at the level of the oral primordium), and the posterior portion (below the oral primordium). These calculations were based on individual meshes generated from the same Z-stack images, where each specific portion was extracted. Consequently, the sum of the volumes of the three portions may slightly differ from the total volume calculated from the entire LFM mesh.

Volumetric analysis of cell volume. Those divider cells, which were previously volumetrically analysed for their LFM (Table S7), were used to assess the cell volumes through two methods: (i) The maximum cell dimensions were input for the formula of a rotational ellipsoid. (ii) A micrograph of an optical longitudinal section depicting the maximum cell dimensions was processed by Photoshop for further analysis of the cell outline with a custom R script (https://github.com/maxganser/tintinnids). In short, the shape of each cell is bipartited along the longitudinal axis, assuming rotational symmetry. Then, the shape is extracted as a black (cell half) and white (background) image. The image is used to acquire x-y coordinates of the cell shape with the R package Momocs [59]. Subsequently, a function calculates the volume, assuming that at each y-coordinate, the shape forms a cylinder with a circular cross-section where the radius is the y-coordinate, and the height is the constant increment in x-coordinates (\({volume}_{xy}= \pi y²x\)). Finally, the total volume is calculated in µm³, using a conversion factor (µm/pixel). In our calculations, we estimated cell volumes, assuming an anterior end truncated at the level of the peristomial rim, thus disregarding the vaulted peristomial field with its marginal furrow, which cannot be analysed by the applied method.

Volumetric analysis of lorica wall. Micrographs of optical longitudinal sections depicting the maximum dimensions of typically shaped Bouin-fixed loricae (excluding Coxliella-shaped ones) were processed as stated in the previous section. However, the final volume of the lorica wall was determined by subtracting the volume enclosed by the inner lorica wall shape from that enclosed by the outer wall shape.

Terminology. The terminology regarding the somatic ciliature follows Agatha and Riedel-Lorjé [20] (Fig. 1) and thus differs from that used earlier by colleagues [17, 27, 60].

Cell division is a continuous process but is usually subdivided in early, middle, and late dividers missing clear definitions in tintinnid ciliates. Those four stages applied by Brownlee [17] differ between the taxa he studied, and clear distinguishing features delimiting these stages are not given. The oral and somatic ciliature, the nuclear apparatus, and the lorica forming material might not develop synchronously within and among species. Further, they are mostly accessible only after application of appropriate staining methods. Thus, we suggest the oral primordium’s shape as sole feature for characterising the division stages; thereby, this terminology is also applicable to aloricate choreotrichids.

Among the methyl blue-eosin-stained specimens from the Northeast Pacific without a discernible oral primordium, small ones with a single horizontally orientated macronucleus are classified as postdividers, while somewhat larger cells with two macronuclear nodules are considered morphostatic specimens. In early dividers (ED), the oral primordium consists of unordered basal bodies or two-rowed membranelles; the elongated proximalmost collar membranelles and the buccal membranelle might lack. In middle dividers, the oral primordium is in a subsurface pouch and complete, i.e., it comprises the final number of membranelles, which are three-rowed. Two substages are distinguished: (i) in early middle dividers (EMD), the membranellar zone extends on the inner wall of a cylindroidal or funnel-shaped indentation perpendicular to the main cell axis, and the membranelles’ distal portions commence to spread; (ii) in late middle dividers (LMD), the membranellar zone forms a 6-shaped pattern, i.e., it commences with the distalmost membranelles just underneath the cell surface and performs a clockwise (in ventral view) curvature, plunging into the deep buccal cavity which extends obliquely to the ciliate’s right side. Late dividers are characterised by a circular oral primordium with a contorted arrangement of the membranelles. Two substages are distinguished: (i) early late dividers (ELD) with the oral primordium parallel to the ventral side and (ii) very late dividers (VLD), in which the new membranellar zone is first obliquely orientated and finally perpendicular to the main cell axis.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this publication and its supplementary information file. The R scripts are available on GitHub (https://github.com/maxganser/tintinnids).

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded in whole or in part by the Austrian Science Fund (FWF) [10.55776/P35736]. For open access purposes, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission.

Authors’ contributions

SA and MHG conceived and guided the study. SA morphometrically analysed the protargol-stained Schmidingerella cells from the Northwest Atlantic. BW tested different staining methods, developed an optimal procedure for staining the intracellular lorica forming material, and wrote the methods section. BW, SA, and LB volumetrically analysed the intracellular lorica forming material in the methyl blue-eosin-stained Schmidingerella cells from the Northeast Pacific. MHG wrote the custom R scripts and calculated the cell and lorica wall volumes. SA and MHG wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are very thankful to Kelley Bright (Shannon Point Marine Center, Western Washington University, USA) for sending the monoclonal culture material of the Northeast Pacific Schmidingerella and Wayne Coats (Smithsonian Environmental Research Center, Maryland, USA) for providing the protargol-stained material of the Northwest Atlantic Schmidingerella specimens.

Lipps JH, Stoeck T, Dunthorn M. Fossil tintinnids. In: Dolan JR, Montagnes DJS, Agatha S, Coats DW, Stoecker DK, editors. The Biology and Ecology of Tintinnid Ciliates: Models for Marine Plankton. Oxford, Chichester: John Wiley & Sons, Ltd; 2013. pp. 186–97.
Agatha S, Simon P. On the nature of tintinnid loricae (Ciliophora: Spirotricha: Tintinnina): a histochemical, enzymatic, EDX, and high-resolution TEM study. Acta Protozool. 2012;51:1–19. 10.4467/16890027AP.12.001.0384.
Agatha S, Bartel H. A comparative ultrastructural study of tintinnid loricae (Alveolata, Ciliophora, Spirotricha) and a hypothesis on their evolution. J Eukaryot Microbiol. 2022;69:e12877. 10.1111/jeu.12877.
Müller OF. Zoologia Danica seu animalium Daniae et Norvegiae rariorum ac minus notorum, descriptiones et historia. Havniae et Lipsiae; 1788.
Entz G. Jr. Studien über Organisation und Biologie der Tintinniden. Arch Protistenk. 1909;15:93–226. VIII-XXI.
Busch W. Beitrag zur Kenntnis der Gehäusebildung bei den Tintinnidae und zur Kenntnis mariner Ciliaten. Arch Protistenk. 1925;53:183–90.
Entz G. Sr. Zur näheren Kenntnis der Tintinnoden. Mitt zool Stn Neapel 1885;6:185–216 XIII, XIV.
Hofker J. Studien über Tintinnoidea. Arch Protistenk. 1931;75:315–402.
Meunier A. Microplankton des Mers de Barents et de Kara. Campagne Arctique de 1907. Bruxelles: C. Bulens; 1910.
Kofoid CA. Some new Tintinnidae from the plankton of the San Diego Region. Univ Calif Publs Zool. 1905;1:287–307.
Campbell AS. The cytology of Tintinnopsis nucula (Fol) Laackmann with an account of its neuromotor apparatus, division, and a new intranuclear parasite. Univ Calif Publs Zool. 1926;29:179–237.
Campbell AS. Studies on the marine ciliate Favella (Jörgensen), with special regard to the neuromotor apparatus and its rôle in the formation of the lorica. Univ Calif Publs Zool. 1927;29:429–53.
Campbell AS. House-forming material in a marine ciliate. Anat Rec. 1929;44:247.
Schweyer A. Zur Kenntnis des Tintinnodeenweichkörpers, nebst einleitenden Worten über die Hülsenstruktur und die Hülsenbildung. Arch Protistenk. 1909;18:134. –189 + Plates X, XI.
Biernacka I. Studia nad rozrodem niektórych gatunków rodzaju Tintinnopsis Stein (Studies on the reproduction of some species of the genus Tintinnopsis Stein). Annls Univ Mariae Curie-Skłodowska, Sec C. 1952;6:211–247.
Biernacka I. Ausscheidung gehäusebildender Substanzen durch reife Formen gewisser Arten der Gattung Tintinnopsis Stein. Acta Protozool. 1965;3:265–8.
Brownlee DC. Measuring the secondary production of marine planktonic tintinnine ciliates. PhD Thesis: University of Maryland, USA; 1982: x + 202.
Laval-Peuto M. Classe des Oligotrichea Bütschli, 1887. Ordre des Tintinnida Kofoid et Campbell, 1929. In: Puytorac P, de, editors. Traité de Zoologie. Anatomie, systématique, biologie. 2. Infusoires ciliés. 2. Systématique. Paris, Milano, Barcelona: Masson; 1994. pp. 181–219.
Laval-Peuto M. Construction of the lorica in Ciliata Tintinnina. In vivo study of Favella ehrenbergii: variability of the phenotypes during the cycle, biology, statistics, biometry. Protistologica. 1981;17:249–72.
Agatha S, Riedel-Lorjé JC. Redescription of Tintinnopsis cylindrica Daday, 1887 (Ciliophora: Spirotricha) and unification of tintinnid terminology. Acta Protozool. 2006;45:137–51.
Agatha S, Strüder-Kypke MC. Reconciling cladistic and genetic analyses in choreotrichid ciliates (Ciliophora, Spirotricha, Oligotrichea). J Eukaryot Microbiol. 2012;59:325–50. 10.1111/j.1550-7408.2012.00623.x.
Agatha S, Tsai S-F. Redescription of the tintinnid Stenosemella pacifica Kofoid and Campbell, 1929 (Ciliophora, Spirotricha) based on live observation, protargol impregnation, and scanning electron microscopy. J Eukaryot Microbiol. 2008;55:75–85; 10.1111/j.1550-7408.2008.00309.x.
Ganser MH, Agatha S. Redescription of Antetintinnidium mucicola (Claparède and Lachmann, 1858) nov. gen., nov. comb. (Alveolata, Ciliophora, Tintinnina). J Eukaryot Microbiol. 2019;66:802–20. 10.1111/jeu.12728.
Gruber MS, Strüder-Kypke M, Agatha S. Redescription of Tintinnopsis everta Kofoid and Campbell 1929 (Alveolata, Ciliophora, Tintinnina) based on taxonomic and genetic analyses — discovery of a new complex ciliary pattern. J Eukaryot Microbiol. 2018;65:484–504. 10.1111/jeu.12496.
He J, Jiang J, Agatha S, Pan H. Taxonomy and phylogeny of the freshwater tintinnid Tintinnopsis tubuformis Chiang, 1956 (Ciliophora, Oligotrichea) and a proposed synonymization of T. longa nom. corr. Chiang, 1956. J Eukaryot Microbiol. 2022;69:e12918. 10.1111/jeu.12918.
Hu T, Wang Z, Liu W, Lin X. Taxonomy and phylogeny of two tintinnid ciliates of Leprotintinnus (Protista, Ciliophora, Choreotrichida) combining the loricae, cytological, ontogenetic features, and barcoding genes. Front Mar Sci. 2022;9:847600. 10.3389/fmars.2022.847600.
Petz W, Foissner W. Morphogenesis in some freshwater tintinnids (Ciliophora, Oligotrichida). Eur J Protistol. 1993;29:106–20. 10.1016/S0932-4739(11)80303-2.
Agatha S, Laval-Peuto M, Simon P. The tintinnid lorica. In: Dolan JR, Montagnes DJS, Agatha S, Coats DW, Stoecker DK, editors. The Biology and Ecology of Tintinnid Ciliates: Models for Marine Plankton. Oxford, Chichester: John Wiley & Sons, Ltd; 2013. pp. 17–41.
Gruber MS, Mühlthaler A, Agatha S. Ultrastructural studies on a model tintinnid - Schmidingerella meunieri (Kofoid and Campbell, 1929) Agatha and Strüder-Kypke, 2012 (Ciliophora). I. Somatic kinetids with unique ultrastructure. Acta Protozool. 2018;57:195–213. 10.4467/16890027AP.18.015.10091.
Gruber MS, Weißenbacher B, Agatha S. Ultrastructural studies on a model tintinnid - Schmidingerella meunieri (Kofoid & Campbell, 1929) Agatha & Strüder-Kypke, 2012 (Ciliophora). II. The oral apparatus. J Eukaryot Microbiol. 2020;67:463–479; 10.1111/jeu.12795.
Ganser MH, Bartel H, Weißenbacher B, Andosch A, Lütz-Meindl U, Radacher P, et al. A light and electron microscopical study on the resting cyst of the tintinnid Schmidingerella (Alveolata, Ciliophora) including a phylogeny-aware comparison. Eur J Protistol. 2022;86:125922. 10.1016/j.ejop.2022.125922.
Ganser MH, Bartel H, Fedrizzi M, Agatha S. A comparative ultrastructural study on the nanoscale extrusomes of tintinnids (Alveolata, Ciliophora, Spirotricha) and their phylogenetic significance. Eur J Protistol. 2023;87:125953. 10.1016/j.ejop.2022.125953.
Smith SA, Maurer-Alcalá XX, Yan Y, Katz LA, Santoferrara LF, McManus GB. Combined genome and transcriptome analyses of the ciliate Schmidingerella arcuata (Spirotrichea) reveal patterns of DNA elimination, scrambling, and inversion. Genome Biol Evol. 2020;12:1616–22. 10.1093/gbe/evaa185.
Smith SA, Santoferrara LF, Katz LA, McManus GB. Genome architecture used to supplement species delineation in two cryptic marine ciliates. Mol Ecol Res. 2022;22:2880–96. 10.1111/1755-0998.13664.
Jung J-H, Choi JM, Coats DW, Kim Y-O. Euduboscquella costata n. sp. (Dinoflagellata, Syndinea), an intracellular parasite of the ciliate Schmidingerella arcuata: morphology, molecular phylogeny, life cycle, prevalence, and infection intensity. J Eukaryot Microbiol. 2016;63:3–15. 10.1111/jeu.12231.
Echevarria ML, Wolfe GV, Strom SL, Taylor AR. Connecting alveolate cell biology with trophic ecology in the marine plankton using the ciliate Favella as a model. FEMS Microbiol Ecol. 2014;90:18–38. 10.1111/1574-6941.12382.
Echevarria ML, Wolfe GV, Taylor AR. Feast or flee: bioelectrical regulation of feeding and predator evasion behaviors in the planktonic alveolate Favella sp. (Spirotrichia). J Exp Biol. 2016;219:445–56. 10.1242/jeb.121871.
Foissner W. Ontogenesis in ciliated protozoa, with emphasis on stomatogenesis. In: Hausmann K, Bradbury PC, editors. Ciliates: Cells as Organisms. Stuttgart: G. Fischer; 1996. pp. 95–177.
Agatha S. Evolution of ciliary patterns in the Oligotrichida (Ciliophora, Spirotricha) and its taxonomic implications. Zoology. 2004;107:153–68. 10.1016/j.zool.2004.02.003.
Brownlee DC. Stomatogenesis in the tintinnine ciliates with notes on lorica formation. J Protozool. 1983;30:1A.
Bai Y, Wang R, Song W, Suzuki T, Hu X. Redescription of five tintinnine ciliates (Alveolata: Ciliophora: Oligotrichea) from coastal waters of Qingdao, China. Mar Life Sci Technol. 2020;2:209–21. 10.1007/s42995-020-00034-2.
Wang R, Bai Y, Hu T, Xu D, Suzuki T, Hu X. Integrative taxonomy and molecular phylogeny of three poorly known tintinnine ciliates, with the establishment of a new genus (Protista; Ciliophora; Oligotrichea). BMC Ecol Evol. 2021;21:115. 10.1186/s12862-021-01831-8.
Coats DW, Kim S, Bachvaroff TR, Handy SM, Delwiche CF. Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an ectoparasite of the ciliate Tintinnopsis cylindrica Daday 1887, and its relationship to Duboscquodinium collini Grassé 1952. J Eukaryot Microbiol. 2010;57:468–82. 10.1111/j.1550-7408.2010.00504.x.
Coats DW, Heinbokel JF. A study of reproduction and other life cycle phenomena in planktonic protists using an acridine orange fluorescence technique. Mar Biol. 1982;67:71–9. 10.1007/Bf00397096.
Laackmann H. Ungeschlechtliche und geschlechtliche Fortpflanzung der Tintinnen. PhD: Christian-Albrechts-Univerity of Kiel, Germany; 1906: 1–25 + Plate explanations + Plates I-III.
Merkle H. Untersuchungen an Tintinnodeen der Ost- und Nordsee. Wiss Meeresunters. Abt Kiel. 1909;11:139–86. II, III.
Hedin H. On the ultrastructure of Favella ehrenbergii (Claparède & Lachmann) and Parafavella gigantea (Brandt), Protozoa, Ciliata, Tintinnida. Zoon. 1975;3:11–8.
Kofoid CA. Factors in the evolution of the pelagic Ciliata, the Tintinnoinea. Contr Mar Biol, Stanford Univ Press. 1930:1–39.
Laval M. Ultrastructure de Petalotricha ampulla (Fol). Comparaison avec d'autres tintinnides et avec les autres ordres de ciliés. Protistologica. 1972;8:369–86.
Laval-Peuto M. Cortex, périlemme et réticulum vésiculeux de Cyttarocylis brandti (Cilié Tintinnide). Les ciliés à périlemme. Protistologica. 1975;11:83–98.
Wasik A, Mikołajczyk E. The morphology and ultrastructure of the Antarctic ciliate, Cymatocylis convallaria (Tintinnina). Acta Protozool. 1992;31:233–9.
Sokolova YY, Gerassimova ZP. The ultrastructure of the ciliates Parafavella denticulata Ehrenberg, 1840. Tsitologiya. 1984;26:1237–45. I-IV.
Montagnes DJS, Lynn DH. A quantitative protargol stain (QPS) for ciliates: Method description and test of its quantitative nature. Mar Microb Food Webs. 1987;2:83–93.
Guillard RRL. Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH, editors. Culture of Marine Invertebrate Animals. New York, London: Plenum; 1975. pp. 29–60.
Romeis B. Mikroskopische Technik. 17th ed. Vienna, Baltimore: Urban & Schwarzenberg;: Munich; 1989.
Foissner W. An update of 'basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa'. Int J Syst Evol Microbiol. 2014;64:271–92. 10.1099/ijs.0.057893-0.
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Meth. 2012;9:676–82. 10.1038/nmeth.2019.
Cignoni P, Callieri M, Corsini M, Dellepiane M, Ganovelli F, Ranzuglia G. MeshLab: an open-source mesh processing tool. In: Sixth Eurographics Italian Chapter Conference. Eurographics; 2008: 129–136.
Bonhomme V, Picq S, Gaucherel C, Claude J. Momocs: outline analysis using R. J Stat Softw. 2014;56:1–24. 10.18637/jss.v056.i13.
Foissner W, Wilbert N, Morphologie. Infraciliatur und Ökologie der limnischen Tintinnina: Tintinnidium fluviatile Stein, Tintinnidium pusillum Entz, Tintinnopsis cylindrata Daday und Codonella cratera (Leidy) (Ciliophora, Polyhymenophora). J Protozool. 1979;26:90–103. 10.1111/j.1550-7408.1979.tb02739.x.

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Volumetric Dynamics of Lorica Forming Material Across the Cell Cycle in the Model Tintinnid Schmidingerella (Alveolata, Ciliophora)

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Abstract

Background

Results

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Background

Results

Comparison of intracellular lorica forming material (LFM) and lorica wall volume

Discussion

Cell division patterns

Lorica forming material (LFM)

Unresolved questions and future directions

Conclusion

Methods

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References

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