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The ultrastructure of ciliates carries important cytological, taxonomical, and evolutionary signals for these single-celled eukaryotic organisms. However, little ultrastructural data have been accumulated for most ciliate groups with systematic problems. In the present work, a well-known marine uronychiid, Diophrys appendiculata, was investigated using electron microscopy and a comparison with, and a discussion considering, phylogenetic analyses were made. The new findings primarily show that: (ⅰ) this species lacks the typical alveolar plate, bears cortical ampule-like extrusomes, and has microtubular triads in the dorsal pellicle, and thus exhibits some ultrastructural features in common with most of its previously studied congeners; (ⅱ) each adoral membranelle before the level of frontal cirrus Ⅱ/2 contains three rows of kinetosomes and each membranelle after the level of frontal cirrus Ⅱ/2 contains four rows, which might be related with morphogenesis and could be considered as a distinctive character of Diophrys; (ⅲ) some structural details of the buccal field, such as the extra-pellicular fibrils, pellicle, pharyngeal disks and microtubular sheet, were documented. In addition, based on the ultrastructural comparison of representatives, we discuss the differentiation between the subfamilies Diophryinae and Uronychiinae. A hypothetical systematic relationship of members in the order Euplotida based on a wide range of data is also provided.
Ciliated protists are a large assemblage of unicellular eukaryotes distributed in diverse habitats worldwide. They are widely used as model organisms in many research fields including molecular biology, evolution, cell biology, genetics and environmental biology (Adl et al. 2019; Bai et al. 2020; Corliss 1979; Hausmann and Bradbury 1996; Huang et al. 2018; Li et al. 2021; Sheng et al. 2020; Wang et al. 2022; Wu et al. 2020; Zhang et al. 2020b; Zhao et al. 2021, 2020; Zheng et al. 2021; Zhu et al. 2020). As one of the most highly developed groups, the euplotid ciliates have been the main subject of numerous recent investigations in various areas of research such as ecology, epigenetics, cytology, and systematics (Asghar et al. 2021; Chen et al. 2019; Di Giuseppe et al. 2014; Gao et al. 2020; Gong et al. 2020, 2022; Lian et al. 2020a, b; Serra et al. 2020). As research is developed and extended, detailed documentation revealed either by light or electron microscopy is recommended for describing new species and redescribing known species to improve knowledge and understanding of the biodiversity, taxonomy and systematics of euplotids (Gao et al. 2016; Hu et al. 2019; Song and Shao 2017; Vďačný and Foissner 2017).
For many years, within the order Euplotida, the systematics of Diophrys-like taxa (e.g., Diophrys Dujardin, 1841, Paradiophrys Jankowski, 1978, Diophryopsis Hill and Borror, 1992, Apodiophrys Jiang and Song, 2010, Heterodiophrys Jiang and Song, 2010, and Pseudodiophrys Jiang et al., 2011) and the closely related genus Uronychia Stein, 1859, have been in a state of flux (Jankowski 2007; Jiang and Song 2010; Jiang et al. 2011; Shao et al. 2010; Song et al. 2007). Originally, Diophrys and Uronychia were in the family Euplotidae, order Hypotrichida (Borror 1972; Corliss 1979; Tuffrau 1986). However, Jankowski (1979) considered that they should be separated into different families, so the families Diophryidae and Uronychiidae were established. Subsequently, Diophrys was reassigned to the family Euplotidae and Uronychia was retained within Uronychiidae (Shi et al. 1999; Small and Lynn 1985). In the systems of Jankowski (2007) and Lynn (2008), Diophrys and Uronychia are treated as members of the family Uronychiidae (Jankowski 2007; Lynn 2008) but further assigned into the subfamilies Diophryinae and Uronychiinae, respectively (Jankowski 2007). Jankowski (1978) established the genus Paradiophrys to distinguish six species with largely different characteristics from the remaining species in Diophrys at that time. Later, Hill and Borror (1992) added a new genus, Diophryopsis. Recently, Apodiophrys, Heterodiophrys and Pseudodiophrys have been established, and all five of the aforementioned Diophrys-like genera have been reassigned to the subfamily Diophryinae Jankowski, 1979 (Jankowski 2007; Jiang and Song 2010; Jiang et al. 2011; Shao et al. 2010). However, this sub-familial assignment has been challenged by molecular phylogenetic analyses since the Paradiophrys-Apodiophrys group always clusters with Uronychiinae rather than Diophrys (Huang et al. 2012; Zhang et al. 2020a). In addition, previous reports about Diophrys have mainly focused on its systematic position and on the taxonomy of its constituent species, while there is only minimal ultrastructural information about two species (D. oligothrix and D. scutum) (Gong et al. 2018; Rosati-Raffaelli 1970). In this paper, a detailed study of the ultrastructure of Diophrys appendiculata has been documented for the first time. Based on comprehensive morphological, ultrastructural and phylogenetic, information, the systematics of the genus Diophrys, the subfamilies Diophryinae and Uronychiinae and the order Euplotida are discussed.
Results
General morphology of the Qingdao population of Diophrys appendiculata
Detailed information revealed by optical microscopy about the general morphology of Diophrys appendiculata has been supplied by Song and Packroff (1997) and Kwon et al. (2008). The Qingdao population we studied matched well with these descriptions, so only a brief outline of its general morphology is supplied: the body shape was oval and the body size was 30−70 × 35−50 μm in vivo, about 55 × 40 μm after protargol staining and about 50 × 35 μm in SEM preparations. The buccal field occupied about half the body length. There were five frontals, two ventral, five transverse, two left marginal and three caudal cirri on the ventral side, and five dorsal kineties with fragmented rows on the dorsal side (Fig. 1A, B, E, H−L).
Figure
1.Diophrys appendiculata after protargol staining (A−D), from life (E, F) and following scanning electron microscopy (G–O). A, B Ventral (A) and dorsal (B) views of a representative individual. Blue arrows and arrowheads in (A) indicate caudal cirri and left marginal cirri, respectively. Blue arrows, arrowheads and double-arrowheads in (B) point to the macronucleus, dorsal kineties that are divided in two groups and dikinetids, respectively. C, D Ventral view of a representative individual (C) and buccal field (D). Red arrowheads point to the collar-lapel. Blue arrow marks the Ⅱ/2 frontal cirrus, dotted line represents the demarcation line of adoral membranelles differentiation: adoral membranelles above this line are each composed of three rows of cilia, those below this line are each composed of four rows of cilia. Inset shows the amplification of adoral membranelles near this demarcation line. E Dorsal kineties (red arrowheads). F Cortical ampule-like extrusomes grouped around the dorsal dikinetids. G Dorsal view of small intermembranellar ridges (cyan arrows). H−L Ventral (H), dorsal (I), right lateral (J), top (K) and bottom (L) views of representative individuals. Cyan arrows indicate the dorsal kineties that are divided into two groups, red arrowheads show the extra dorsal bristles that are between the anterior and posterior fragments in dorsal kinety 1. M Paroral membrane and endoral membrane. N Detail of adoral membranelles. Numbers (1−3) refer to the three ciliary rows that constitute the adoral membranelles in the anterior part. O Basal bodies are barren of cilia (cyan arrows) in transverse cirri. AZM adoral zone of membranelles, EM endoral membrane, PM paroral membrane. Scale bars: 30 μm (A, B, H−J); 20 μm (C, D, K, L); 10 μm (E−G, M); 5 μm (N); 2 μm (O)
In SEM preparations, a prominent buccal field extended over 50% of the ventral region. The adoral zone of membranelles (AZM) encircled the left side of the anterior portion of the cell (Fig. 1A, C, D, H). Two "separators" in the AZM were observed. One is a gap present between the "collar" and "lapel" regions of the AZM (Fig. 1C, D). The other is a potential boundary at about the level of the second frontal cirrus (cirrus Ⅱ/2) (Fig. 1C, D, N). Adoral membranelles situated before the level of the second frontal cirrus (cirrus Ⅱ/2) were each composed of three equal-length rows of kinetosomes and those after the level are each composed of four rows (Fig. 1C, D, N). Small intermembranellar ridges separated adjacent membranelles (Fig. 1G). The endoral and paroral membranes were located on the right side of the buccal field. The endoral membrane (EM) was slightly curved and comprised a single ciliary row (Fig. 1M). The paroral membrane (PM), which was formed by numerous short rows of cilia, posteriorly originated near the proximal end of the AZM and curved at the midpoint, crossing the entire peristome lengthwise (Fig. 1M). There were five large transverse cirri, some of which had about six kinetosomes barren of cilia (loss of cilia during preparation cannot be excluded) (Fig. 1H, L, O). There were five dorsal kineties, each composed of an anterior and a posterior fragment. Each fragment contained several closely arranged dorsal bristles and two or three extra dorsal bristles located between the anterior and posterior fragments in dorsal kinety 1 (Fig. 1I). A deep concavity with three caudal cirri was situated on the right posterior margin of the dorsal side (Fig. 1I, L).
Ultrastructure of the cortex
Pellicle
The pellicle included the plasmalemma and cortical alveoli (Fig. 2A). The plasmalemma covered the whole cell surface and the cortical alveoli were inconspicuous and surrounded by the outer and inner alveolar membranes (Fig. 2A). A series of differently oriented microtubules were present beneath the inner alveolar membrane (Fig. 2A−H). Longitudinal microtubules were arranged as a single layer on the ventral side and as two layers (appearing as triads in cross-section) on the dorsal side (Fig. 2B−D, F). At the junction of the dorsal and the ventral pellicle, where two types of microtubular arrangement met, the longitudinal microtubules were densely arranged in a single row (Figs. 2G, 7C). There was an additional layer of subpellicular microtubules that were oriented transversely beneath the longitudinal microtubules on both the ventral and dorsal sides (Fig. 2B, E, F, H). No clear perilemma, alveolar plates or epiplasm were observed.
Figure
2.
Transmission electron micrographs of cortex of Diophrys appendiculata. A Longitudinal section of the cell, showing the plasmalemma (red arrowhead), cortical alveoli (yellow arrow) and subpellicular microtubules (orange double-arrowheads). B−D, F Longitudinal microtubules (yellow arrows) arrange singularly on the ventral side (B, C) and as triads on the dorsal side (D, F), red arrowheads in B and F point to the transversal microtubules. E, H Section at different angles, showing crisscross subpellicular microtubules, yellow arrows indicate longitudinal microtubules, arrowheads show transversal microtubules. G Longitudinal microtubules densely arrange as a single layer (orange double-arrowheads) at the junction of dorsal and ventral pellicle where the two types of arrangement meet, yellow arrow shows the longitudinal microtubules arranged singularly, red arrowhead marks the longitudinal microtubules arranged as triads. Scale bar: 0.5 μm (A, C, D, F, G); 1 μm (B, E, H)
The cortical ampule-like extrusomes were colourless and densely grouped around the dorsal dikinetids (Fig. 1F), and no extrusion was observed in SEM preparations. In TEM preparations, these extrusomes were situated in the cortical cytoplasm and clustered near the bases of the ventral, transverse and left marginal cirri and adoral membranelles, and grouped around the kinetosomes of the dorsal bristles (Fig. 3A−C). They were usually ellipsoidal to cylindrical in shape, with a length of 0.2−0.7 μm and width of about 0.2 μm, and bounded by a unit membrane (Fig. 3D). Most of these extrusomes had an electron-dense content (Fig. 3E−I) but some had a more diffuse or partially less electron-dense content (Fig. 3E, G, H), possibly reflecting various stages of extrusion. Ellipsoidal vesicles that lacked any inclusion (possibly their contents had been extruded) were sometimes visible around the dorsal kinetosomes (Fig. 3J).
Figure
3.
Transmission electron micrographs showing the cortical ampule-like extrusomes of Diophrys appendiculata. A−C Sections near the base of ventral cirri (A), adoral zone of membranelles (B) and dorsal dikinetids (C), showing the arrangement of cortical ampule-like extrusomes (red arrows). D Mature cortical ampule-like extrusomes located beneath pellicle. E−G Various shapes of cortical ampule-like extrusomes. Red arrows show curved, long rod-shaped ones, orange arrowheads indicate lanceolate ones, yellow double-arrowheads mark the electron-lucent part. H, I Different electron density of cortical ampules-like extrusomes: red arrows show the cortical ampule-like extrusomes with electron-dense content, orange arrowheads indicate those with diffuse content. J Empty cortical ampule-like extrusomes in cross-section (red arrows) around dorsal dikinetids. Scale bars: 0.5 μm (A, E, H, I); 0.2 μm (D); 1 μm (B, C, F, G, J)
The buccal apparatus in Diophrys appendiculata was highly developed. It was composed of ciliary organelles (adoral membranelles and undulating membranes) and structures located in or associated with, the buccal cavity or peristome (Fig. 8A, B, D). The endoral membrane was constituted by a single row of kinetosomes (Fig. 4A−E, G, L). Extra-pellicular fibrils were located beneath the endoral membrane. These were highly electron-dense and arranged in parallel layers, up to approximately 0.5 μm in thickness at the middle part, becoming thinner or even bifurcated when extending to the brim (Fig. 4B−E, G, L). The pellicle of the dorsal wall of the oral cavity only contained a typical plasmalemma. It was difficult to distinguish between the pellicle and extra-pellicular fibrils in some sections because they were tightly connected (Fig. 4D). There was a single layer of subpellicular microtubules that might connect to other parts of the cell (Fig. 4C, E, J−L). The pharyngeal disks were electron-dense with multiple lamellar membranes, almost perpendicular to the dorsal wall, appearing as rods, strips or rings (Fig. 4A−I). A microtubular sheet commenced at the boundary area of the buccal field beneath the subpellicular microtubules and ran obliquely and posteriorly deep into the cytoplasm (Figs. 4A, F, J, K, 8A, B), possibly forming an unclosed circle because the cytoplasm was usually sparse in the area beneath the cytopharyngeal apparatus (Fig. 5A).
Figure
4.
Transmission electron micrographs showing structures in the buccal field of Diophrys appendiculata. A, B Longitudinal section of the buccal field, showing the arrangement and morphology of extra-pellicular fibrils (red arrow), pharyngeal disks (yellow arrowheads) and microtubular sheet (orange double-arrowheads). C, D Cross-section (C) and longitudinal section (D) of buccal field, showing endoral membrane, extra-pellicular fibrils (red arrows), pellicle (orange double-arrowheads), subpellicular microtubules, and pharyngeal disks (yellow arrowheads). E Detailed structures of buccal field, showing extra-pellicular fibrils (red arrow), pellicle (yellow arrowheads) containing plasmalemma and subpellicular microtubules, and pharyngeal disks (orange double-arrowheads). F Microtubular sheet (red arrow) beneath cytopharyngeal apparatus in the cytoplasm. G Extra-pellicular fibrils (yellow arrowheads) with thick middle and thin brims. H, I Variable shape of pharyngeal disks, red arrows point to ring-shaped, yellow arrowheads mark strip-shaped pharyngeal disks. J, K Longitudinal section of the boundary area of the buccal field, to show the microtubular sheet (red arrows) starting from the boundary area of the buccal field beneath the subpellicular microtubules and running obliquely and posteriorly deep into the cytoplasm. L Section of the boundary area of the buccal field, to show the separation (yellow arrowhead) of extra-pellicular fibrils (red arrows) from pellicle, and the bifurcation at the brim of extra-pellicular fibrils (orange double-arrowheads). AZM adoral zone of membranelles, EM endoral membrane. Scale bars: 5 μm (A, B); 2 μm (C, G, L); 1 μm (D, F, H, I); 0.5 μm (E, J, K)
Figure
5.
Transmission electron micrographs showing the cytoplasm and cytoplasmic organelles of Diophrys appendiculata. A, B Longitudinal section of the cell, showing cytoplasm and arrangement of cytoplasmic organelles. Red arrows mark food vacuoles, white double-arrowheads point to pharyngeal disks, orange arrowheads indicate macronuclear nodule. C Vesicles (red arrows) and small granules (yellow arrowheads, might be ribosomes) in the cytoplasm. D Mitochondria. E Food vacuoles containing bacteria. F Macronuclear nodule. Yellow arrowheads mark nucleoli, red arrow points to a chromatin body. AZM adoral zone of membranelles, CC caudal cirri, Mit Mitochondria. Scale bars: 10 μm (A, B); 2 μm (C); 1 μm (E); 0.5 μm (D, F)
Neighboring kinetosomes in the same row were linked by double linkages (containing anterior and posterior connections) that were parallel to the long rows (Fig. 6A, B, E). The diagonal connections splayed in a posterior direction interconnected the kinetosomes in different rows, and extended out to the electron-dense material called the radial ribbon of microtubules (Fig. 6A, B, E). Transverse connections interconnected the kinetosomes in different rows in the anterior direction (Fig. 6A, B, E). In longitudinal sections of the membranelles, there were numerous microtubules beneath the kinetosomes and parallel to the pellicle, which might have intertwined and turned posteriorly forming the submembranellar fibers (Fig. 6C, D).
Figure
6.
Transmission electron micrographs showing the ciliature of Diophrys appendiculata. A, B Cross-section of adoral membranelles, to show double linkages (red arrows), diagonal connections (white double-arrowheads), transverse connections (yellow arrowheads) and radial ribbon of microtubules. C, D Longitudinal section of the long (C) and short (D) axis of adoral membranelles. In (C), red arrow marks the postmembranellar connection, yellow arrowheads point to the intermembranellar fiber. In (D), red arrow marks the intermembranellar fiber forming bundles, yellow arrowheads point to the terminal fiber. E Cross-section of the adoral zone of membranelles, showing the arrangement of adoral membranelles. F Cross-section of paroral membrane. Red arrow marks the double linkages, yellow arrowheads point to the diagonal connections, white double arrowheads show the longitudinal linkages. G Cross-section of dorsal kineties. Red arrow shows inter-kinetosomal connection, white double-arrowheads mark a kinetosome-like structure which might be a kinetosome barren of cilia or a parakinetosomal body, yellow arrowheads points to the tangential transverse ribbon. H−K Cross-section of transverse cirri (H−J) and caudal cirri (K), to show double linkages (red arrows), diagonal connections (white double-arrowheads), transverse connections (yellow arrowheads), and kinetodesmal fiber. KD kinetodesmal fibril, RR radial ribbon of microtubules. Scale bars: 0.5 μm (A−D, F, H, I); 2 μm (E, K); 1 μm (G, I)
Multiple rows of kinetosomes constituted the paroral membrane, and each row contained four kinetosomes that formed four lines longitudinally. These kinetosomes were linked by electron-dense connections at five locations (Fig. 6F): (1) two of them obliquely interconnected with both neighboring kinetosomes, which were reminiscent of diagonal connections; (2) double linkages containing transverse and diagonal connections joined the basal bodies within the same row; (3) longitudinal linkages linked the kinetosomes in the same line longitudinally.
Ultrastructure of somatic ciliature
Dorsal kinetosomes
The dorsal bristle units were made up of one ciliated kinetosome and one unciliated kinetosome, and the two were connected by an electron-dense inter-kinetosomal connection. The tangential transverse ribbon was in front of the anterior kinetosome. There was a kinetosome-like structure behind the posterior kinetosome, which might be a parakinetosomal body (Fig. 6G).
Ventral and caudal cirri
A polykinetid pattern was observed in the caudal and transverse cirri (Fig. 6H−K). Double linkages, containing anterior and posterior connections, linked the kinetosomes in the same row in parallel. Neighboring kinetosomes in different rows were connected by diagonal connections (also called oblique fibrils/connections or diagonal fibrils) and transverse connections (also called transverse fibril or zig-zag fibril) forwards and backwards, respectively. The kinetodesmal fibrils were located behind and extended from the last kinetosome in each row (Fig. 6G).
Ultrastructure of cytoplasm, cytoplasmic organelles and nuclear apparatus
The cytoplasm was an electron-lucent colloidal material that contained numerous small granules, various electron-lucent vesicles, and cytoplasmic organelles (Fig. 5A, B). These cytoplasmic inclusions were characterized as follows (in ascending order of size). (1) The small granules were less than 50 nm in diameter, electron-dense, scattered in the cytoplasm, and might be ribosomes (Fig. 5C). (2) The vesicles had an electron-lucent membrane structure, mostly spherical or irregular in shape, with variable diameters (0.1−1 μm) (Fig. 5C). (3) The mitochondria were about 1 μm long, ellipsoidal, with tubular cristae, and mainly distributed beneath the pellicle (Fig. 5D). (4) The food vacuoles were 1−7 μm in diameter and contained copious amounts of food debris, possibly of bacterial origin (Fig. 5A, B, F). (5) Macronuclear nodules were surrounded by a karyotheca and contained irregular chromatin bodies that were more or less homogenous, and several spherical nucleoli that contained scattered, punctate, electron-dense particles (Fig. 5B, F).
SSU rDNA and phylogenetic analyses
The SSU rDNA sequence of the Qingdao population of Diophrys appendiculata has been deposited in the GenBank database. The accession number, sequence length, and GC content of this new sequence are as follows: OK039190, 1709 bp and 43.71%, respectively. The topologies of the ML and BI trees inferred from SSU rDNA sequence data were identical, therefore only the ML tree (with both support values) is shown (Fig. 7).
Figure
7.
Maximum likelihood (ML) tree inferred from SSU rDNA sequences of 107 taxa. Numbers followed by species name are accession numbers from GenBank database. Numbers near nodes are bootstrap values for ML and posterior probability values for Bayesian inference (BI) trees. Fully supported (100 ML/1.00 BI) branches are marked with solid circles. The scale bar corresponds to 0.1 expected substitutions per site. The newly sequenced population of Diophrys appendiculata is indicated in red (arrow)
In both analyses, the family Uronychiidae was monophyletic and was divided into two large clades: (1) Heterodiophrys, Diophryopsis and Diophrys formed the clade Diophryinae I (ML/BI, 95/1.00), with Heterodiophrys zhui (GU477635) deeply divergent from the remaining species at the base; (2) Paradiophrys, Apodiophrys and Uronychia formed a strongly supported clade (ML/BI, 97/1.00), i.e., Diophryinae Ⅱ-Uronychiinae, within which the close relationship of Paradiophrys and Apodiophrys was fully supported. Each of the families Gastrocirrhidae, Aspidiscidae, Certesiidae and Euplotidae was monophyletic and together formed a large clade that was sister to Uronychiidae.
The Qingdao population (OK039190) of Diophrys appendiculata clustered with three other populations (AY004773, EU267928 and JF694041) and sequences under the names of Diophrys oligothrix and D. blakeneyensis with high support (ML/BI, 95/1.00), forming a clade that was sister to a larger assemblage composed of D. scutum, D. apoligothrix, D. quadrinucleata, D. japonica and Diophrysis hytrix.
Details of the evolutionary relationship within the clade formed by four monophyletic families, Aspidiscidae, Certesiidae, Euplotidae and Gastrocirrihidae, are shown in Fig. 9 and Table 1.
Table
1.
Morphological and ultrastructural characteristics used for assessment of phylogenetic relationships among five families within the order Euplotida
Plesiomorph ○
Apomorph ●
1
Caudal cirri regular sized or absent
Caudal cirri hypertrophied
2
Caudal cirri located on dorsal side
Caudal cirri (if present) located on posterior pole
3
Cortical alveoli without platein
Cortical alveoli with platein
4
Buccal cavity without extra-pellicular fibrils
Buccal cavity with extra-pellicular fibrils
5
Pharyngeal disks: single-membrane-bound type
Pharyngeal disks: myeloid type
6
Ovoid or disc-like body shape with ventrally oriented oral cavity
Conoid body shape with anteriorly opened oral cavity
7
Four or five well-developed transverse cirri
Many conspicuous transverse cirri arranged in U-shape
8
Left marginal cirri present
Left marginal cirri absent
9
Adoral zone of membranelles continuous
Adoral zone of membranelles bipartite
10
With caudal cirri
Without caudal cirri
11
Fewer than three left marginal cirri
More than three left marginal cirri
12
Without condylopallium in the anterior end of cell
With condylopallium in the anterior end of cell
13
Significantly more than 10 extrusomes densely clustered around each ciliary basal body
Usually about 10 extrusomes around each ciliary basal body
14
Extrusomes anchored just beneath pellicle on dorsal side
Extrusomes and pellicle separated by a vesicle layer on the dorsal side
15
Caudal cirri originate from different dorsal kinety anlagen
Caudal cirri originate from right most dorsal kinety anlage
16
Frontoventral cirri dispersed
Frontoventral cirri grouped in anterior right of cell
The organization of a typical ciliate pellicle generally includes a plasmalemma, alveoli subtended by a membrane and an epiplasm (Lynn 2008; Pitelka 1965). In representatives of the order Euplotida, the alveoli can be present in one of two forms: (1) typical alveoli, with an electron-lucent inner space; (2) alveoli filled with alveolar plates (mainly composed of protein, thus also called platein) which consist of electron-dense materials (Kloetzel 1991). The latter form has been observed in Aspidisca (Rosati et al. 1987), Certesia (Wicklow 1983), Euplotes (Foissner 1978; Hausmann and Kaiser 1979; Ruffolo 1976) and Euplotidium (Lenzi and Rosati 1993). This study showed that the alveolar plates are not present in Diophrys appendiculata, which conforms to the previous description of its congeners, D. oligothrix and D. scutum, and in a representative of another uronychiid genus, namely Uronychia (Dong et al. 2020b; Gong et al. 2018; Rosati-Raffaelli 1970).
The detailed organization and orientation of longitudinal microtubules in Euplotida were first described based on Euplotes eurystomus as a series of closely associated longitudinal microtubules forming a triad configuration (called microtubular triads) beneath the dorsal pellicle, but appearing in a single configuration layer beneath the ventral pellicle (Grim 1967). This arrangement has been confirmed in other Euplotes species (Foissner 1978; Grim et al. 1980; Gu and Ji 1996; Ruffolo 1976) and other genera of Euplotida such as Uronychia (Dong et al. 2020b), Diophrys (Gong et al. 2018; Rosati-Raffaelli 1970), Certesia (Wicklow 1983), and Aspidisca (Rosati et al. 1987). The microtubular triads are similar to those of some cyrtophorids and are considered the main reason for cell rigidity (Hofmann and Bardele 1987; Kurth and Bardele 2001). A similar arrangement of dorsal longitudinal microtubules (showed as Fig. 8C) is also present beneath the dorsal pellicle of Diophrys appendiculata. Furthermore, it was shown that in the cortex where the dorsal and ventral microtubules meet, the microtubules appeared as a single-rowed layer with a dense arrangement, which differs from both the dorsal triads and the ventral pattern.
Figure
8.
Schematic reconstructions of the structures of buccal field (A, B, D) and longitudinal microtubules at the junction of the dorsal and ventral pellicle (C) in Diophrys appendiculata based on TEM investigation. A, B Longitudinal section (A) and cross-section (B) of the cell, to show the location and morphology of buccal structures. C Longitudinal microtubules arranged as triads on the dorsal side and as a single layer on the ventral side. Red arrows indicate the longitudinal microtubules arranged as two layers on the dorsal side, and red double-arrowheads show the longitudinal microtubules arranged as a single layer on the ventral side or densely arranged as a single layer at the junction of the dorsal and ventral pellicle. D Detailed scheme of the oral apparatus organization with the respective positions of endoral membrane, extra-pellicular fibrils, pellicle, subpellicular microtubules and pharyngeal disks. AZM adoral zone of membranelles
Cortical ampules are ovoidal to elongate membrane-bound extrusomes inserted under the cortex and associated with cirri, membranelles, and bristle kineties. They persist inside the cytoplasm and are not lost after discharge (Dallai and Luporini 1981; Görtz 1982; Lynn 2008; Ruffolo 1976). These organelles had a variety of names, such as ciliary vacuoles (Roth 1957), vacuoles or vesicles (Gliddon 1966), and secretory ampules (Fauré-Fremiet and André 1968), until they were defined by Ruffolo (1976). Cortical ampules have mostly been reported in Euplotes (Fauré-Fremiet and André 1968; Gliddon 1966; Görtz 1982; Rosati and Modeo 2003; Rosati et al. 1981; Roth 1957; Ruffolo 1976; Serra et al. 2020). In other genera, similar structures were sometimes referred to as "ampules", such as those in Diophrys (Gong et al. 2018), Certesia (named as muciferous-like bodies or vesicles; Wicklow, 1983) and Aspidisca (Rosati et al. 1987), due to having a similar shape and the inclusion of homogeneous material (Dong et al. 2020b; Gong et al. 2018). Rosati et al. (1987) demonstrated that the extrusomes of Aspidisca, Certesia, and Swedmarkia are similar to each other and differ in shape and arrangement from those structures associated with the ciliature of Diophrys and Euplotes. In the present study, we found that the extrusomes of Diophrys appendiculata are arranged more tightly around the dorsal bristles than those in Euplotes in vivo (Borror 1972; Chen et al. 2014; Curds 1975; Fotedar et al. 2016; Han et al. 2022; Lian et al. 2020, 2019; Song et al. 2009b; Tuffrau 1960). Based on the literature data, we speculate that extrusomes of euplotids might be more diverse than so far supposed. Since cytochemical studies of extrusomes are largely lacking, we suggest using the name 'cortical ampule-like extrusomes' for all these organelles in non-Euplotes species for the time being.
It must be emphasized that in the genus of Diophrys, the extrusomes in D. scutum are distinct from those in its congeners. Indeed, the extrusomes of D. scutum have an obvious "cap" in the distal portion which is lacking in the cortical ampule-like extrusomes of D. appendiculata and D. oligothrix, and can be considered as mucocysts or mucocyst-like extrusomes based on their shape, size and content appearance (Rosati-Raffaelli 1970). Furthermore, they seem to be densely arranged on the dorsal side rather than associated with the ciliature (Rosati-Raffaelli 1970).
Ultrastructural features in the buccal field
As shown in Fig. 8A, B, D, the detailed scheme of the buccal structure in Diophrys appendiculata is far more complicated than previously known. Extra-pellicular fibrils are located at the boundary between the external and internal environments of the oral cavity. Nobili and Rosati-Raffaelli (1971) defined the extra-pellicular fibrils as thin fibrils that run somewhat irregularly, parallel with and outside the pellicle. These structures were once misinterpreted as pharyngeal disks extending outside because they were both multi-layered and connected in some sections (Kloetzel 1974). However, there is no correlation in the electron density between these two structures, such as in Euplotes eurystomus (Kloetzel 1974). According to the limited ultrastructural information available for Diophrys, the extra-pellicular fibrils of D. appendiculata are extremely electron-dense and compact; the extra-pellicular fibrils in D. scutum have lower electron density and are loosely arranged (Nobili and Rosati-Raffaelli 1971; Rosati et al. 1987); and although not metioned in the description of D. oligothrix vegetative cells, some similar structures (named swollen membrane-packed discs) are observed between the endoral membrane and pellicle of the dorsal wall in cysts (Gong et al. 2018). Previous studies have shown that extra-pellicular fibrils are present in many euplotids, such as Aspidisca sp., Euplotes crassus, E. woodruffii, E. eurystomus, D. scutum and Swedmarkia arenicola (Kloetzel 1974; Luporini and Magagnini 1970; Nobili and Rosati-Raffaelli 1971; Rosati et al. 1987; Wicklow 1983), but are not present in Uronychia transfuga, U. binucleata and Euplotidium itoi (Dong et al. 2020b; Lenzi and Rosati 1993; Morelli et al. 1996).
The precursors that rapidly form food vacuole membranes (Kloetzel 1974; Lynn 2008), i.e., the pharyngeal disks, were classified into two morpho-types by Dong et al. (2019, 2020a). The first type, namely single-membrane-bound pharyngeal disks that are electron-lucent, are present in Climacostomum virens, Euplotidium itoi, Parabistichella variabilis, Uronychia transfuga and U. binucleata (de Puytorac et al. 1976; Dong et al. 2020b; Fischer-Defoy and Hausmann 1981; Hausmann and Hausmann 1981; Lenzi and Rosati 1993; Morelli et al. 1996; Nobili and Rosati-Raffaelli 1971). The other type, namely myeloid pharyngeal disks with high electron density, which are actually numerous compressed membranes, have been reported in Certesia quadrinucleata, Diophrys oligothrix, D. scutum, Euplotes eurystomus, E. crassus and E. woodruffii (Fauré-Fremiet and André 1968; Fischer-Defoy and Hausmann 1981; Gong et al. 2018; Kloetzel 1974; Lenzi and Rosati 1993; Morelli et al. 1996; Rosati et al. 1987; Wicklow 1983). According to the present study, the pharyngeal disks in D. appendiculata can be classed as the myeloid type, which is the same type as that in its congeners, Diophrys oligothrix and D. scutum (Gong et al. 2018; Nobili and Rosati-Raffaelli 1971; Rosati-Raffaelli 1970). The microtubular sheet was observed extending from the boundary area of the buccal field to the deep cytoplasm (Fig. 8A, B), which is also the case in Euplotes and Uronychia (Dong et al. 2020b; Kloetzel 1974; Luporini and Magagnini 1970; Nobili and Rosati-Raffaelli 1971; Rosati-Raffaelli 1970). This microtubular sheet is considered as forming the intracytoplasmic oral area and makes an actual barrier between the digestive and non-digestive cytoplasm (Luporini and Magagnini 1970; Nobili and Rosati-Raffaelli 1971; Rosati-Raffaelli 1970).
Two "separators" were present in the AZM in Diophrys appendiculata. The first "separator" is present between the "collar" and "lapel" regions of the AZM (Adl et al. 2012; Lynn 2008). Based on the present and previous studies, such a "separator" is usually present in all species of Diophrys (Chen and Song 2002; Fan et al. 2013; Hu et al. 2012; Kwon et al. 2008; Luo et al. 2014; Shao et al. 2010; Shen et al. 2011; Song et al. 2007, 2009a; Zhang et al. 2020a). Thus, we suggest that the genus definition of Diophrys should be updated to signify that a gap, which may be conspicuous or inconspicuous, is usually present between the "collar" and "lapel" regions of the AZM. Another "separator" is a potential boundary that is present at about the level of the second frontal cirrus (cirrus Ⅱ/2) on the left, separating the membranelles composed of three kinetosome rows from those composed of four kinetosome rows (Fig. 1C, D). This feature has not been used for identification so it was not mentioned in previous reports, however, many species of Diophrys (such as D. oligothrix, D. quadrinucleata and D. blakeneyensis) also present similar features according to published light micrographs (Hu et al. 2012; Luo et al. 2014; Zhang et al. 2020a). During morphogenesis in Diophrys, the parental AZM is partly retained by the proter and the marginal parts of the proximal membranelles dedifferentiate and are subsequently reorganized (Hill 1981; Hu 2008; Luo et al. 2014; Shao et al. 2010; Shen et al. 2011; Song and Packroff 1993; Song and Shao 2017; Song and Wilbert 1994; Song et al. 2009b; Yang et al. 2008). These reorganized proximal membranelles are coincidentally below the level of second frontal cirrus (cirrus Ⅱ/2) and contain four rows of kinetosomes. Thus, this unique "separator" at the level of the second frontal cirrus (cirrus Ⅱ/2) might have a relationship with morphogenesis and could be a taxonomically informative character for Diophrys. It is noteworthy that in D. parappendiculata, the only species within the genus Diophrys with an obviously bipartite AZM, there is a conspicuous gap (where adoral membranelles are lacking) in the AZM which starts at the collar-lapel separator and ends at the separator at the level of Ⅱ/2 cirrus (Shen et al. 2011).
Systematics of Euplotida based on phylogenetic and ultrastructural analyses
Based on a combination of the available microscopic, ultrastructural and phylogenetic information, we propose a hypothesis of the systematic relationships of the order Euplotida (Figs. 9, 10; Table 1). The monophyletic order Euplotida can be recognized by synapomorphies such as the rigid body, two-layered cyst wall and well-developed and sparse ventral cirri (Borror and Hill 1995; Huang et al. 2012; Jiang and Song 2010; Jiang et al. 2011; Shao et al. 2010; Yi et al. 2009; Zhang et al. 2020a). We speculate that the arrangement of longitudinal microtubules, which form a single layer on the ventral side and a double layer (triads) on the dorsal side, might possibly constitute another synapomorphy since it has been observed in numerous euplotids, whereas it is lacking in closely related orders such as Discocephalida (Wicklow 1982a, b).
Figure
9.
Hypothetical evolution of families and subfamilies in the order Euplotida according to the morphological features listed in Table 1 and molecular data. Members of the Euplotida share four synapomorphies (red asterisks at the tree base): rigid body, two-layered cyst wall, well-developed and sparse ventral cirri, and the highly special longitudinal microtubules, which are arranged as a single layer on the ventral side but as two layers (triad) on the dorsal side
Figure
10.
Review of the published ultrastructural data and some speculative structures of Euplotida in the framework of SSU rRNA gene phylogeny, showing representative structures of genera and families. The "?" indicates that there are no ultrastructural data. Fully supported (100 ML/1.00 BI) branches are marked with red circles. The blue dotted boxes indicate that these structures are speculative
Euplotida is composed of two major clades: the monophyletic family Uronychiidae and a clade that includes the remaining families of the order, i.e., Aspidiscidae, Certesiidae, Euplotidae and Gastrocirrihidae (Huang et al 2012; Jiang and Song, 2010; Jiang et al., 2011; Shao et al., 2010). The genera included in Uronychiidae share two notable features, i.e., three hypertrophied dorsally right-located caudal cirri and cortical alveoli without platein (Jankowski 2007; Shao et al. 2010). The family Uronychiidae has been further divided into two subfamilies, Uronychiinae and Diophryinae (Jankowski 2007; Shao et al. 2010). Uronychiinae used to be recognized by a highly developed undulating membrane that almost encloses the buccal field, and an invariable number of frontoventral cirri usually grouped in the anterior right of cell, whereas Diophryinae was recognized by undulating membranes located to the right of the buccal field and relatively dispersed frontoventral cirri of a variable number (Borror and Hill 1995; Jankowski 2007; Shao et al. 2010). However, according to phylogenetic analyses based on SSU rDNA sequence data, the Diophryinae is non-monophyletic and its two members, Paradiophrys and Apodiophrys, always cluster with Uronychiinae (Fan et al. 2013; Huang et al. 2012; Jiang and Song 2010; Jiang et al. 2011; Shao et al. 2010; Shen et al. 2010; Zhang et al. 2020a). In the present study, ultrastructural data revealed differences between the Diophryinae (represented by Diophrys) and Uronychiinae (represented by Uronychia). First, the extrusomes of Uronychiinae are located beneath the electron-lucent vesicles on the dorsal side, whereas those in Diophryinae (and other members of Euplotida) dock just beneath the pellicle on the dorsal side (Dong et al. 2020b; Morelli et al. 1996). Secondly, extra-pellicular fibrils are present in the Diophryinae but are absent in the Uronychiinae (Dong et al. 2020b; Gong et al. 2018; Morelli et al. 1996; Rosati-Raffaelli 1970). This difference in the presence of the extra-pellicular fibrils is also observed in the comparison of Aspidiscidae, Certesiidae and Euplotidae (A-C-E clade) versus Gastrocirrhidae (Kloetzel 1974; Luporini and Magagnini 1970; Nobili and Rosati-Raffaelli 1971; Rosati et al. 1987; Wicklow 1983). Unfortunately, ultrastructural information for Paradiophrys and Apodiophrys is still lacking. Thus, whether the characteristics of extrusomes and extra-pellicular fibrils can resolve the conflict between the molecular phylogeny and classical morphology in Uronychiinae requires further investigation. Additionally, there is also one more candidate difference between Diophryinae and Uronychiinae, i.e., the pharyngeal disks are myeloid in the former but single-membrane-bound in the latter (Dong et al. 2020b; Gong et al. 2018; Morelli et al. 1996; Rosati-Raffaelli 1970). Such a difference is also present between the A-C-E clade and Gastrocirrhidae (Fauré-Fremiet and André 1968; Fischer-Defoy and Hausmann 1981; Gong et al. 2018; Kloetzel 1974; Lenzi and Rosati 1993; Morelli et al. 1996; Rosati et al. 1987; Wicklow 1983), which might imply convergent evolution between the A-C-E clade and Diophryinae (Fig. 9).
As already mentioned, two different types of extrusomes arranged in two ways have been reported within the genus Diophrys: the cortical ampule-like extrusomes associated with ciliature in Diophrys oligothrix and D. appendiculata and the mucocysts arranged densely beneath the pellicle in D. scutum (Gong et al. 2018; Rosati-Raffaelli 1970). Coincidentally, in the molecular phylogenetic analyses, D. oligothrix and D. appendiculata cluster together whereas D. scutum is located in a separate clade. Therefore, the intrageneric differentiation of the extrusomes might suggest further separation of the genus Diophrys.
Materials and methods
Sampling, collection, and identification
A population of Diophrys appendiculata was collected from an indoor artificial seawater tank in the Laboratory of Protozoology, Ocean University of China, Qingdao, China. Details of the tank have been described in a previous publication (Dong et al. 2020b). Samples containing bottom sediment and water were collected from this tank and then examined with a stereomicroscope to isolate the target organisms. Raw cultures were established at room temperature (about 25 ℃) using artificial seawater (salinity: 30, prepared by dissolving Golden Trump sea salt (Shenzhen GCH. Industrial Co, Ltd) in Nongfu Spring mineral water) to which several wheat grains were added to promote the growth of bacteria as a food source for the ciliates. Specimens used for all subsequent studies were isolated from the raw cultures.
About 10 cells were observed in vivo using bright field and differential interference contrast microscopy (Olympus BX 51, Japan) to reveal the morphology of the cortical ampule-like extrusomes. Protargol staining (Wilbert 1975) was used to reveal the infraciliature and nuclear apparatus for species identification. The protargol powder was prepared according to Pan et al. (2013). Identification and general terminology are mainly according to Curds and Wu (1983) and Song et al. (2004).
Scanning electron microscopy (SEM)
The SEM method mainly followed the method described by Bardele et al. (1986), Gu and Ni (1993) and Dong et al. (2019). The specimens were fixed in a mixture of 1% OsO4 and a saturated solution of HgCl2 at room temperature (about 25℃) for 10 min. They were then dehydrated in a graded series of ethanol (30, 50, 70, 80, 90, 95 and 100%) for approximately 10 min at each concentration. A critical point dryer (Leica EM CPD300, Leica, Microsystems, Wetzlar, Germany) and a sputter coater (Leica EM ACE600, Leica, Microsystems, Wetzlar, Germany) were used to dry the specimens and coat them with platinum (about 2.46 nm thickness), respectively. Processed samples (about 60 cells) were studied in a scanning electron microscope (Hitachi S-4800, Tokyo, Japan) at an accelerating voltage of 10 kV.
Transmission electron microscopy (TEM)
The TEM procedure used was according to Gu and Ni (1995) and Dong et al. (2020a). The cells were fixed at 4℃ in a glutaraldehyde-OsO4 mixture for 10 min and in 2% OsO4 for one hour, successively. Cells were rinsed in 0.2 mol/L cacodylate buffer, dehydrated in a graded acetone series, and embedded in an epoxy resin (Eponate 12 resin). Ultrathin sections (approximately 70 nm thick) were stained with uranyl acetate and lead citrate and then observed in a transmission electron microscope (Hitachi 7700, Tokyo, Japan) at an accelerating voltage of 80 kV.
Genomic DNA extraction, high-throughput sequencing and phylogenetic analyses
The total genomic DNA (unpublished) was extracted using the Phenol–Chloroform method and digested with RNase A (Omega Bio-tek, Norcross, GA, USA; Lot No. L10RG) after sample collection (Zhang et al. 2021). Genomic DNA was submitted to the Novogene Company (Beijing, China) for Illumina sequencing (350 bp library, PE150) with a HiSeq4000 sequencer. The genome was assembled using SPADES version 3.13 (‐k 21, 33, 55, 77) (Bankevich et al. 2012; Chen et al. 2019; Nurk et al. 2013). The assembly was screened by BLAST + 2.2.28 (e-value = < 1 × 10−5) using two SSU rDNA sequences (Euplotes minuta FJ876977 and Moneuplotes crassus HQ413694) downloaded from the National Center for Biotechnology Information (NCBI) as references to detected rDNA sequences. Finally, the SSU rDNA was extracted from rDNA sequences with primers 18S-F (5′-AACCTGGTTGATCCTGCCAGT-3′) and 18S-R (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) (Elwood et al. 1985; Medlin et al. 1988).
The new sequence of Diophrys appendiculata was aligned with another 106 SSU rDNA sequences by BioEdit 7.0 with default parameters (Hall 1999). Five species (Paradiscocephalus elongatus EU684746, Leptoamphisiella vermis FJ865203, Pseudoamphisiella quadrinucleata EU518416, Discocephalus ehrenbergi FJ196397 and Prodiscocephalus borrori DQ646880) were chosen as outgroup taxa. Maximum likelihood (ML) analyses were conducted with RAxML-HPC2 on XSEDE (8.2.12) (Stamatakis et al. 2008) on the CIPRES Science Gateway (http://www.phylo.org/sub_sections/portal/) with the GTRGAMMA model (Miller et al. 2010). GTR + Ι + Γ was selected as the best model of nucleotide substitution with the Akaike Information Criterion (AIC) by the program MrModeltest v.2.0 (Nylander 2004). Bayesian inference (BI) analysis was performed with MrBayes on XSEDE 3.2.7a, with two sets of four chains for 10, 000, 000 generations (Ronquist and Huelsenbeck 2003). The first 25% of sampled trees were discarded as burn-in prior to constructing the majority rule consensus tree from the remaining trees. MEGA 6.0 was used to visualize tree topologies (Talavera and Castresana 2007).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (32170446, 32030015, 31961123002) and the National Natural Science Foundation of Shanghai (21ZR1419000). The authors would like to express their gratitude to Prof. Weibo Song (Ocean University of China) for his generous help and advice in preparing the manuscript and Dr. Bing Ni (East China Normal University) for his helpful guidance in SEM and TEM. We thank Dr. Tengyue Zhang (Ocean University of China) for providing original cells for this study, and Dr. Tengteng Zhang (Ocean University of China) for her help with the molecular data.
Author contributions
XP Fan conceptualized the project; JY Dong carried out the research; JY Dong, XP Fan, T Stoeck, YJ Liu, JY Ma and HG Ma helped with data interpretation, wrote, and revised the manuscript. All authors approved the final version of the manuscript.
Data availability
A voucher slide (registration number: DJY2018030701) with protargol-stained specimens of the Qingdao population of Diophrys appendiculata has been deposited into the collection of the Laboratory of Protozoology (OUC), Qingdao, China. The SSU rDNA sequence of Diophrys appendiculata Qingdao population has been deposited in the GenBank database with accession number OK039190.
Declarations
Conflict of interest
The authors declare that they have no conflicts of interest in this work.
Animal and Human Rights Statement
This article does not contain any studies with human participants or animals performed by any of the authors.
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Table
1.
Morphological and ultrastructural characteristics used for assessment of phylogenetic relationships among five families within the order Euplotida
Plesiomorph ○
Apomorph ●
1
Caudal cirri regular sized or absent
Caudal cirri hypertrophied
2
Caudal cirri located on dorsal side
Caudal cirri (if present) located on posterior pole
3
Cortical alveoli without platein
Cortical alveoli with platein
4
Buccal cavity without extra-pellicular fibrils
Buccal cavity with extra-pellicular fibrils
5
Pharyngeal disks: single-membrane-bound type
Pharyngeal disks: myeloid type
6
Ovoid or disc-like body shape with ventrally oriented oral cavity
Conoid body shape with anteriorly opened oral cavity
7
Four or five well-developed transverse cirri
Many conspicuous transverse cirri arranged in U-shape
8
Left marginal cirri present
Left marginal cirri absent
9
Adoral zone of membranelles continuous
Adoral zone of membranelles bipartite
10
With caudal cirri
Without caudal cirri
11
Fewer than three left marginal cirri
More than three left marginal cirri
12
Without condylopallium in the anterior end of cell
With condylopallium in the anterior end of cell
13
Significantly more than 10 extrusomes densely clustered around each ciliary basal body
Usually about 10 extrusomes around each ciliary basal body
14
Extrusomes anchored just beneath pellicle on dorsal side
Extrusomes and pellicle separated by a vesicle layer on the dorsal side
15
Caudal cirri originate from different dorsal kinety anlagen
Caudal cirri originate from right most dorsal kinety anlage
16
Frontoventral cirri dispersed
Frontoventral cirri grouped in anterior right of cell
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