Figure 1. Morphology and infraciliature of Neobakuella aenigmatica in vivo (a-d) and after protargol staining (e, f). a Ventral view of a representative individual. b Ventral view, to show cortical granules distributed near marginal cirri. c, d Dorsal views, to show cortical granules near the dorsal cilia (c) and lateral side (d). e, f Ventral (e) and dorsal (f) view of the same specimen showing the infraciliature and nuclear apparatus. In e, arrow shows parabuccal cirri and arrowhead marks the buccal cirri. ADK additional dorsal kineties anterior to right marginal row; AZM adoral zone of membranelles; E endoral membrane; FC frontal cirri; FT frontoterminal cirri; L1-3 the first, second and third left marginal rows, respectively; Ma macronucleus nodules; Mi micronuclei; MP midventral pairs; P paroral membrane; RMR right marginal rows, TC transverse cirri; D1-3 dorsal kineties 1-3; 1-6, midventral rows 1-6. Scale bars = 75 μm (a, e, f)
Figure 2. Photomicrographs of Neobakuella aenigmatica in vivo (a-h) and after protargol staining (i-p). a, b Ventral views of different individuals, showing contractile vacuoles (arrowhead in a). c Ventral view of the anterior portion of cell, showing the adoral zone. d-f Dorsal views, showing arrangement of grass-green colored cortical granules (arrowheads) and midventral cirri (arrows). g, h Ingested algae (arrowheads in g), flagellates (arrows in g) and fungal spores (arrow in h). i, j Ventral views, showing infraciliature and nuclear apparatus. Arrows mark the frontoterminal cirri; arrowhead indicates the third left marginal row. k Ventral view of anterior portion, showing buccal (arrowheads), frontal and parabuccal cirri (in circle) respectively. l Dorsal view of anterior portion, showing dorsal kineties (arrowheads). m-p Ventral views of different individuals, showing different patterns of left marginal rows (arrowheads). Note: m-p are false-colored by inverting color in Photoshop CS5. FC frontal cirri; FT frontoterminal cirri; L1, 2, left marginal rows 1, 2; PBC parabuccal cirri. Scale bars = 85 μm
Character Min Max Mean M SD CV n Body (length) 140.0 250.0 182.8 181.0 29.1 15.9 20 Body (width) 38.0 148.0 70.3 58.5 30.8 43.9 20 Body length:width (ratio) 1.5 4.3 2.9 3.0 0.7 25.0 20 Anterior body end to proximal end of adoral zone (distance) 48.0 93.0 70.2 70.5 11.4 16.2 20 Body length:length of adoral zone (ratio) 2.3 3.1 2.6 2.6 0.3 10.2 20 Anterior body end to anterior end of paroral membrane (distance) 6.0 15.0 11.0 10.5 2.2 19.8 20 Paroral membrane (length) 31.0 63.0 45.6 45.5 8.6 18.8 20 Anterior body end to anterior end of endoral membrane (distance) 8.0 17.0 13.2 13.0 2.8 21.3 20 Endoral membrane (length) 38.0 76.0 55.1 53.5 9.1 16.5 20 Anterior body end to first buccal cirrus (distance) 12.0 31.0 21.0 20.0 4.3 20.6 20 Anterior body end to last buccal cirrus (distance) 31.0 65.0 47.9 45.0 9.5 19.8 20 Anterior body end to posterior end of midventral pairs (distance) 50.0 98.0 69.7 68.0 11.1 16.0 20 Posterior body end to rearmost transverse cirrus (distance) 8.0 48.0 17.9 15.0 10.5 58.6 20 Adoral membranelles (number) 32.0 57.0 42.1 41.5 6.4 15.2 30 Frontal cirri (number) 3.0 3.0 3.0 3.0 0 0 20 Buccal cirri (number) 4.0 10.0 6.4 6.0 1.5 23.4 30 Parabuccal cirri (number) 2.0 3.0 2.1 2.0 0.2 10.9 20 Frontoterminal cirri (number) 2.0 2.0 2.0 2.0 0 0 20 Midventral pairs (number) 8.0 15.0 10.2 10.0 1.7 17.0 20 Midventral rows (number) 4.0 8.0 5.7 6.0 1.0 18.1 20 Cirri of midventral row 1 (number)a 3.0 4.0 3.1 3.0 0.2 7.3 20 Cirri of midventral row 2 (number) 3.0 5.0 3.7 3.5 0.8 21.7 20 Cirri of midventral row n−1 (number)a 8.0 16.0 11.9 12.0 2.2 18.2 20 Cirri of midventral row n (number)a 9.0 18.0 13.1 13.0 2.2 16.9 20 Left marginal rows (number) 2.0 5.0 3.3 3.0 0.7 22.2 20 Cirri of left marginal row 1 (number)b 34.0 68.0 46.4 45.5 8.5 18.4 20 Cirri of left marginal row 2 (number)b 5.0 22.0 11.2 10.5 4.8 43.1 20 Cirri of left marginal row 3 (number)b 2.0 32.0 13.9 13.5 9.6 68.9 20 Cirri of left marginal row 4 (number)b 3.0 16.0 7.8 – – – 5 Cirri of left marginal row 5 (number)b 4.0 9.0 6.5 – – – 2 Cirri of right marginal row (number) 42.0 74.0 53.9 54.5 7.9 14.7 20 Dorsal bristles ahead of right marginal row (number) 2.0 2.0 2.0 2.0 0 0 20 Transverse cirri (number) 8.0 15.0 11.1 10.5 2.3 20.4 20 Dorsal kineties (number) 3.0 3.0 3.0 3.0 0 0 20 Dorsal bristles of dorsal kinety 1 (number) 23.0 47.0 31.2 29.5 7.3 23.3 20 Dorsal bristles of dorsal kinety 2 (number) 25.0 49.0 34.5 34.5 5.8 16.8 20 Dorsal bristles of dorsal kinety 3 (number) 27.0 47.0 35.6 33.5 5.5 15.4 20 Macronuclear nodules (number) 100.0 179.0 127.9 121.0 21.4 16.7 20 CV coefficient of variation in %, M median, Max maximum, Mean arithmetic mean, Min minimum, n number of cells measured, SD standard deviation
aCirral rows with more than two cirri. Midventral row 1 is the anterior most row, midventral row n is the posterior most row
bNumbered from inside to outside
Table 1. Morphometric characterization of Neobakuella aenigmatica based on protargol-stained specimens (measurements in μm)
Body size 150-200 × 50-70 μm in vivo, usually about 170 × 60 μm, ratio of length to width approximately 3:1 in vivo. Outline wide-elliptical, the anterior portion slightly curved rightwards while foraging for food; body slightly fragile, flexible, and contractile i.e., reducing in length by about 10% under mild pressure with cover glass (Fig. 2a, b); ventral side flat, dorsal side usually convex in posterior region, and dorsoventrally flattened at a ratio of approximately 2:1. Macronuclear nodules small and numerous (about 121 nodules), scattered throughout the cell; individual nodules measure about 4-8 × 3-5 μm after protargol staining, usually globular to wide-ellipsoidal, but some are elongate-ellipsoidal, each with several small nucleoli (Figs. 1f, 2i-l). Micronuclei difficult to observe either in vivo or after protargol staining, probably several scattered throughout cell smaller than macronuclear nodules. Contractile vacuole located at the level of buccal vertex near the left cell margin (Figs. 1a, 2a). Cells slightly yellow-brownish at low (× 100) magnification because of cytoplasm and food vacuoles; cortical granules about 1 × 1 μm in size, globular to ellipsoidal, grass-greenish, easy to observe in protargol preparations when the cell is insufficiently bleached (Figs. 1b-d, 2d-f). Cytoplasm is colorless, contains fat globules (ca. 2-3 μm across) and food vacuoles (ca. 10-15 μm across) with compact contents. It is omnivorous and ingests fungal spores, diatoms, flagellates, and small ciliates (Fig. 2g, h). Moves rapidly by crawling over substrate without pause.
Adoral zone palpable, occupying, 32-43% of body length, on average about 38% of body length after protargol staining; composed of 42 membranelles on average, bases of the largest membranelles about 14 μm wide, cilia up to 20 μm long in vivo. Paroral arches strongly, approximately four-fifth the length of endoral which is bow-shaped, anterior ends of each at the same level, paroral and endoral optically intersect in their posterior region (Figs. 1e, 2i-k). Buccal cavity large and deep, the hyaline lip covers the proximal end of the adoral zone of membranelles (Fig. 2c).
Three conspicuously enlarged frontal cirri, about 18 μm long in vivo, the rightmost one near distal end of adoral zone; two or three parabuccal cirri behind right frontal cirrus (Figs. 1e, 2i-k). A row of four to ten buccal cirri at the right of paroral cirri becomes progressively smaller from the anterior to the posterior (Figs. 1e, 2i-k). Constantly two fine frontoterminal cirri are seen which are anterior to the right of the anterior most midventral pair (Figs. 1e, 2i, j). Midventral complex including a row of 8-15 pairs of cirri that terminates at the level of buccal vertex, together with 4-8 oblique, short or long midventral rows; rightmost long midventral row extends to the right of the rightmost transverse cirrus (Figs. 1e, 2i, j). Eight to 15 transverse cirri, with cilia approximately 18 μm long in vivo, closely arranged in oblique rows, either does not project beyond body margin or project only slightly (Fig. 1a, e).
Marginal and midventral cirri 9-14 μm long in vivo. Constantly one right marginal row composed of 42-74 cirri, starting at the right of frontoterminal cirri on dorsolateral surface (with two dorsal bristles ahead), and ending subterminally (Fig. 1e, f). Number of left marginal rows variable, i.e. out of 20 individuals, one had two rows, 14 had three rows, three had four rows and two had five rows. Among these left marginal rows, only left marginal row 1 (the innermost) is a complete row, comprising 34-68 cirri, starting ahead of the level of buccal vertex and terminating at the posterior end of body; left marginal rows, 2-5, commence behind the level of anterior end of innermost row, and comprise variable numbers of sparsely distributed cirri (Figs. 1e, 2i, j, m-p). Three bipolar dorsal rows, with bristles about 5 μm long in vivo; dorsal kineties 1-3 are composed of 31, 35 and 36 dikinetids on average, respectively (Fig. 1f; Table 1).
Figure 3. Morphogenesis of Neobakuella aenigmatica after protargol staining. a, b Ventral views of cells in early stages, to show the opisthe's oral primordium (arrowheads in a) and dedifferentiation of the endoral (arrow in b). c-e Ventral views of an early middle divider, showing the development of frontoventral-transverse cirral anlagen and dedifferentiation of paroral (double-arrowhead). Note d and e, are magnified views of anlagen developing in proter and opisthe as shown in c. In c, the opisthe's oral primordium differentiates into new membranelles (arrow) and proximal membranelles of the proter are in the process of dedifferentiation (arrowhead). In d, arrow indicates that the parental midventral cirri dedifferentiate and join in the formation of the frontoventral-transverse cirral anlagen, arrowhead marks dedifferentiation of proximal membranelles in the proter, and double-arrowhead shows dedifferentiation of paroral. In e, arrow marks the development of opisthe's oral primordium, arrowheads show dedifferentiation of midventral cirri and double-arrowhead points to the appearance of undulating membrane anlage in the opisthe. f, g Ventral and dorsal view of a slightly later divider, to show the formation of dorsal kinety anlagen (arrowheads in g), formation of left and right marginal anlagen, development of frontoventral-transverse cirral anlagen (double-arrows), as well as undulating membrane anlagen starting to split longitudinally and gives rise to the leftmost frontal cirri in both daughter cells (arrows). Note that several marginal anlagen segments form to the left of the innermost left marginal anlagen (arrowheads in f). h, i Ventral and dorsal view of a middle divider, to show the development of dorsal kinety anlagen (arrowheads in i), segmentation of frontoventral-transverse cirral anlagen and the leftmost frontal cirri (arrows in h) and development of the anlagen to form the left marginal segments (arrowheads in h). FVTA frontoventral-transverse cirral anlagen; LMA left marginal anlagen; Ma macronucleus nodules; Mi micronuclei; OP oral primordium; RMA right marginal anlagen. Scale bars = 80 μm (a-c); 15 μm (d, e); 65 μm (f-i)
Stomatogenesis: In the opisthe, stomatogenesis starts with several groups of closely spaced basal bodies originating apokinetally around several of the posterior left cirri of the parental midventral rows (Figs. 3a, 5a). These groups subsequently become larger, merge and form the oral primordium (Figs. 3b, 5c). The oral primordium develops further and the anterior portion then gradually differentiates into new adoral membranelles (Figs. 3c, 5f). By this stage, some posterior left cirri of the midventral complex have been resorbed and others are disintegrating, indicating that the parental basal bodies are incorporated into the oral primordium (Figs. 3c, e, 5f). At the same time, the undulating membrane anlage is formed to the right of the oral primordium (Figs. 3c, e, 5f). Later, the leftmost frontal cirrus is generated from the anterior end of the undulating membrane anlage which splits longitudinally into the endoral and paroral, and the anterior end of the newly built adoral zone of membranelles bends to the right (Figs. 3f, h, 5i, n).
In the proter, the endoral begins to disorganize from anterior to posterior (Figs. 3b, 5b). Very soon, the paroral begins to dedifferentiate (Fig. 5d). As this process continues, the undulating membrane anlage is formed (Figs. 3c, d, 5e). Meanwhile, the posterior end of the parental adoral zone of membranelles begins to disorganize (Figs. 3c, d, 5e). Later several proximal membranelles are replaced by new ones (Figs. 3f, h, 5g, m). The development of the undulating membrane anlage follows a similar pattern to that in the opisthe (Figs. 3f, h, 4a, e, 5g, i, m-p, s).
Figure 4. Morphogenesis (a, b, e, f) and reorganization (c, d) of Neobakuella aenigmatica after protargol staining. a, b Ventral (a) and dorsal (b) view of a late divider, showing that anlage II gives rise to the buccal cirri (arrowhead in a), anlage III produces the parabuccal cirri (arrow), the rightmost anlage forms two frontoterminal cirri (red stars in a), left marginal segments are formed to the left of the innermost left marginal anlagen (double-arrowhead) and the dorsal kinety anlagens are in the process of development (arrowheads in b). c, d Ventral (c) and dorsal (d) view of a reorganizer, showing the frontoventral-transverse cirral anlagen (double-arrowhead), anlagen for left marginal rows (arrows), segments (arrowheads in c), and dorsal kineties (arrowheads in d). e, f Ventral (e) and dorsal (f) view of a very late divider, showing the buccal cirri (arrowheads in e), parabuccal cirri (arrows), frontoterminal cirri (red stars), left marginal segments (double-arrowheads), transverse cirri (in circle) and dorsal kinety anlagen (arrowheads in f). LMA left marginal anlagen; Ma macronucleus nodules; Mi micronuclei; RMA right marginal anlagen; TC transverse cirri. Scale bars = 80 μm
Figure 5. Photomicrographs of Neobakuella aenigmatica during morphogenesis (a-t) and reorganization (u) after protargol staining. a Ventral view of an early divider, to mark the oral primordium (arrowheads). b, c Ventral views of the same early divider, showing the dedifferentiation of endoral (arrows) and buccal cirri (arrowheads), and opisthe's oral primordium. d Ventral view of an early divider, showing the dedifferentiation of the buccal cirri (double-arrowheads), undulating membrane (arrowheads) and frontoventral-transverse cirral anlagen (arrows). e, f Ventral views of the same divider, showing undulating membrane anlage in proter (arrowhead in e), the parental midventral cirri (arrowheads in f) which contributes to the formation of the frontoventral-transverse cirral anlagen and the oral primordium which develops and differentiates into new membranelles (arrow). g-l Ventral (g-k), and dorsal (l) views of the same specimen, to show the undulating membrane anlagen (arrowheads in g and i), the leftmost frontal cirri (arrows in g and i), several marginal anlagen segments formed to the left of the innermost left marginal anlage which then develop into new short left marginal rows (arrows in h, j and k), and the dorsal kinety anlage (arrowheads in l). m, n Ventral views of a middle divider, to mark the leftmost frontal cirri in both daughter cells (arrows). o-r Ventral views of a late divider. o, p showing buccal cirri (double-arrowheads), two frontoterminal cirri (arrows) and transverse cirri (arrowheads). q Demonstrating the micronuclei and fusion of the macronuclear nodules. Arrow in r marks the newly formed left marginal segment. s, t Ventral views of a divider in very late stage, to show the frontoterminal cirri (arrow in s) and left marginal segment (arrow in t). u Ventral view of a middle reorganizer, showing the leftmost frontal cirrus (arrowhead) and the left marginal segment (arrow). Ma macronucleus nodules; Mi micronuclei; OP oral primordium. Scale bars = 25 μm (a-d); 40 μm (e, f); 45 μm (g-t); 90 μm (u)
Initially a few basal bodies appear on the right of the old buccal cirri in the proter and the buccal cirri dedifferentiate simultaneously, which will become the frontoventral-transverse cirral anlagen (Figs. 3c, 5d). Then, the frontoventral-transverse cirral anlagen grows by increasing their numbers of basal bodies which become organized into numerous nascent oblique streaks as a ladder-like structure in the proter (Figs. 3c, d, 5e). Meanwhile, several nascent oblique streaks for the opisthe are formed to the right of the opisthe's oral primordium, with a few parental cirri from the midventral rows contributing to their formation (Figs. 3c, e, 5f).
In some middle to late stages, the frontoventral-transverse cirral anlagen for both daughter cells continue to develop and differentiate into many cirri (Figs. 3f, h, 4a, e, 5g, i, m-p, s). Anlage Ⅱ (the undulating membrane anlage is frontoventral-transverse cirral anlage Ⅰ) gives rise to the middle frontal cirrus and four to ten buccal cirri; anlage III produces the rightmost frontal cirrus and one or two parabuccal cirri. In the proter of Fig. 4a, anlagen IV-VII form a midventral pair each; anlagen Ⅷ-Ⅺ produce a midventral pair and a transverse cirrus each; the last anlage (the rightmost anlage) develops two frontoterminal cirri, one midventral row and one transverse cirrus; Other anlagen (anlagen XII-XV) form one midventral row and one transverse cirrus. Different specimens have different numbers of anlagen. Finally, the cell elongates and the new ciliary structures move further apart as they migrate towards their final positions. Note that the two anterior frontoterminal cirri from the last anlage migrate anteriorly and the parental structures are resorbed gradually (Figs. 4e, 5s). Meanwhile, the development of the cytostome is completed and the daughter cells begin to separate (Fig. 4e).
Within the right and innermost left parental marginal rows of both daughter cells, a few cirri near the anterior end, and a few others below the mid-body, differentiate to form two separate anlagen (Fig. 3f, h). These anlagen then increase in size by adding basal bodies and develop into cirri which gradually replace the parental structures (Fig. 4a, e). It is noteworthy that several small anlagen are formed de novo or intrakinetally to the left of the anlage to form the innermost left marginal rows, and then these short anlagen elongate and develop into the other short left marginal rows/segments of each daughter cell (Figs. 3f, h, 4a, e, 5h, j, k, r, t). The parental left marginal rows will eventually be resorbed (Figs. 4a, e, 5r, t).
Morphogenesis of the dorsal kineties is like most urostylids as they develop by intrakinetal basal body proliferation, i.e. two anlagen develop in each parental row (Figs. 3g, i, 5l). These anlagen subsequently elongate and the parental structures are either incorporated or resorbed (Fig. 4b, f). During the division process, neither surplus anlagen nor caudal cirri are formed.
The nuclear apparatus divides in the usual way for urostylids and hence needs no detailed comment. The most striking feature of the macronuclear division process is that all the macronuclear nodules fuse into a single mass (Figs. 3g, i, 4b, 5a, m, n, q). The micronuclei divide independently (Figs. 3i, 4b, f, 5q).
Only one physiological regeneration stage was observed for this species. According to the available data, the processes of reorganization during physiological regeneration and morphogenesis during binary fission are similar (Figs. 4c, d, 5u).
The SSU rDNA sequence of N. aenigmatica (Qingdao population) is deposited in the GenBank database with the accession number MN326296. The length and G + C content are 1656 bp and 44.26%. The sequences of the Qingdao and Korean populations are identical.