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Nov.  2022
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Doublets of the unicellular organism Euplotes vannus (Alveolata, Ciliophora, Euplotida): the morphogenetic patterns of the ciliary and nuclear apparatuses associated with cell division

  • Corresponding author: Jiamei Jiang, jm-jiang@shou.edu.cn
  • Received Date: 2022-05-21
    Accepted Date: 2022-10-05
    Published online: 2022-11-24
  • Special topic: Ciliatology.
  • Edited by Jiamei Li.
  • Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
  • Ciliated protists are one of the most diverse and highly differentiated group among unicellular organisms. Doublets occur in ciliates when two cells fuse into a single individual. Doublets contain two major cellular components (either cell in a doublet) and have traditionally been considered as developmental anomalies. Nevertheless, doublets can divide or even conjugate effectively, which may represent dispersal forms of the life stages. In addition, morphogenesis, as an important process in the life cycle, will provide important insights into the complex differentiation mechanism and various physiological phenomena. However, morphogenetic studies focusing on doublets of ciliates are very limited, which has become an obstacle to understand their complete life history. Here we isolated a doublet strain from the marine species Euplotes vannus (Müller, 1786) Diesing, 1850 and investigated its morphogenetic events during asexual reproduction. Our results indicate that: (1) the opisthe's oral primordium develops de novo beneath the cortex; (2) the frontoventral and transverse cirral anlagen, cirrus Ⅰ/1, and marginal anlagen in both dividers develop de novo separately; (3) the dorsal kinety anlagen, the three rightmost ones of which produce three caudal cirri for the proter, occur within the parental structures in the mid-body region; (4) the opisthe acquires two caudal cirri, one from the end of each two rightmost kineties; and (5) there are two macronuclei and one micronucleus in the doublet and they divide amitotically and mitotically, respectively. Finally, we speculate that this special differentiation may be an adaptive form to adverse environments.
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Doublets of the unicellular organism Euplotes vannus (Alveolata, Ciliophora, Euplotida): the morphogenetic patterns of the ciliary and nuclear apparatuses associated with cell division

    Corresponding author: Jiamei Jiang, jm-jiang@shou.edu.cn
  • 1. Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China
  • 2. Department of Biology, Shenzhen MSU-BIT University, Shenzhen 518172, China
  • 3. Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
  • 4. Department of Biology, University of Pisa, 56126 Pisa, Italy
  • 5. Engineering Research Center of Environmental DNA and Ecological Water Health Assessment, Shanghai Ocean University, Shanghai 201306, China

Abstract: Ciliated protists are one of the most diverse and highly differentiated group among unicellular organisms. Doublets occur in ciliates when two cells fuse into a single individual. Doublets contain two major cellular components (either cell in a doublet) and have traditionally been considered as developmental anomalies. Nevertheless, doublets can divide or even conjugate effectively, which may represent dispersal forms of the life stages. In addition, morphogenesis, as an important process in the life cycle, will provide important insights into the complex differentiation mechanism and various physiological phenomena. However, morphogenetic studies focusing on doublets of ciliates are very limited, which has become an obstacle to understand their complete life history. Here we isolated a doublet strain from the marine species Euplotes vannus (Müller, 1786) Diesing, 1850 and investigated its morphogenetic events during asexual reproduction. Our results indicate that: (1) the opisthe's oral primordium develops de novo beneath the cortex; (2) the frontoventral and transverse cirral anlagen, cirrus Ⅰ/1, and marginal anlagen in both dividers develop de novo separately; (3) the dorsal kinety anlagen, the three rightmost ones of which produce three caudal cirri for the proter, occur within the parental structures in the mid-body region; (4) the opisthe acquires two caudal cirri, one from the end of each two rightmost kineties; and (5) there are two macronuclei and one micronucleus in the doublet and they divide amitotically and mitotically, respectively. Finally, we speculate that this special differentiation may be an adaptive form to adverse environments.

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Introduction
  • Ciliates are highly differentiated unicellular eukaryotes that can be found in diverse habitats. They are characterized by dimorphic nuclei (i.e., somatic macronucleus and germline micronucleus) and a unique sexual process called conjugation (Asghar et al. 2021; Chen et al. 2019; Lian et al. 2020; Liu et al. 2021). Some species, e.g., Euplotes vannus (the focus of this study), Tetrahymena thermophila and Paramecium tetraurelia, have been widely used as models in research on genetics, biochemistry, evolution, ecology, etc. (Abello et al. 2020; Chen et al. 2019; Duan et al. 2021; Munyenyembe et al. 2021; Wang et al. 2022; Zhang et al. 2022; Zhao et al. 2021; Zheng et al. 2021).

    A doublet is a deformed morph that occurs in ciliates when two ordinary cells fuse into one individual and survive in the form of double body (Dias et al. 2007). The two components of a doublet are fused in the cortex and their cytoplasm is interconnected. There are many fusion types of doublets, the most common of which is mirror-imaged, and a few are back-to-back fusion, right-side fusion, etc. (Bell et al. 2008; Jerka-Dziadosz 1983; Luporini and Giachetti 1980; Powers 1943; Shi and Frankel 1990). Doublets are rarely collected from natural environments (Ricci et al. 2000) and usually occur spontaneously during culture (deriving either from a conjugating pair or incomplete division) (Luporini et al. 1979; Miceli and Luporini 1982; Powers 1943) or induced by experiments (Bell et al. 2008; Jerka-Dziadosz 1983; Tchang and Pang 1965). The induction methods are as follows: (1) modifying culture conditions (Jerka-Dziadosz 1983); (2) microsurgery (Tchang and Pang 1965); (3) heat shock (Bell et al. 2008); (4) treating with colchicine (Kosaka 1990); and (5) translocation of cell parts during wound healing (Grimes 1982). Dawson (1920) observed dorsally joined homopolar doublets of Oxytricha hymenostoma in a conjugation-induced culture. More mirror-image doublets, which join side by side with the two sets of ventral ciliature on one plane, have been found in various taxa, such as euplotids, hypotrichs, spathidiids and synhymeniids (Jerka-Dziadosz 1983; Luporini et al. 1979; Vd'ačný and Tirjaková 2012; Xu and Foissner 2005). The morphogenesis of doublets is unclear except in the genera Paraurostyla and Stylonychia (He and Shi 1990; Li et al. 2004; Shi and Frankel 1990; Shi and Qiu 1989; Tchang and Pang 1965; Yano and Suhama 1991).

    The genus Euplotes Ehrenberg, 1830 is a species-rich and cosmopolitan group of ciliates. Among them, Euplotes vannus (Müller, 1786) Diesing, 1850 is a marine species that is easy to collect and cultivate in the laboratory, and thus has been widely used in cell biology, e.g., the structure and function of cilia (Tang et al. 2020), genomics (Chen et al. 2019), ecotoxicology (Pan et al. 2018), phylogenetics (Sheng et al. 2018), predator/prey relationships (Gruber et al. 2009), nuclear events during conjugation (Jiang et al. 2019) and morphogenesis (Gao et al. 2020).

    In the present study, we obtained a doublet strain of E. vannus in a conjugation-induced culture. The doublets divide by binary fission and thus could be maintained for generations in the laboratory. The morphogenetic events were investigated focusing on the aspects of the ciliature and nuclear apparatus during its binary fission.

Results

    Morphology of doublets

  • Two cells merged their pellicles on the right edge to form doublets that were about 80 × 70 μm in vivo (Fig. 1D, G, H). They joined side by side, with one component's ventral side and the other component's dorsal side on the same plane. In most doublets, the two components fused completely from anterior to posterior, although a few remained partially separated at their posterior end (Fig. 1D, H). Each component had similar morphological characters to those of singlets (Fig. 1AC) as described by Jiang et al. (2010): adoral zone prominent, composed of about 60 membranelles; paroral membrane short, located near to right of proximal portion of adoral zone; ten frontoventral, five transverse, two marginal and two or three caudal cirri; eight or nine dorsal kineties; macronucleus inverted C-shaped. There were two contractile vacuoles though one was difficult to recognize. It should be noted that the doublets possessed only one micronucleus (Fig. 1F). Since the morphogenetic process is synchronized in each component of the doublets, the following description of morphogenesis is based on one component.

    Figure 1.  Morphology of Euplotes vannus singlets (AC) and doublets (DH) in vivo (A, C–E, H), after protargol staining (B, G), and after Hoechst 33342 staining (F). A Schematic drawing of a representative individual. B Ventral view of a protargol-stained singlet, showing the ciliature and nuclear apparatus. C Ventral view of the representative singlet. D, H Different doublets, arrow marks the contractile vacuole. E Doublet in late stage of division. F A Hoechst 33342-stained specimen, showing the nuclear apparatus, arrowhead marks the micronucleus. G A protargol-stained specimen, arrows mark the AZM, arrowheads show the dikinetids of DK. Note: B and G are false-colored by inverting color in Photoshop. AZM adoral zone of membranelles, CC caudal cirri, DK dorsal kineties, FVC frontoventral cirri, Ma macronucleus, MC marginal cirri, Mi micronucleus, PM paroral membrane, TC transverse cirri. Scale bars: 50 μm

  • Stomatogenesis

  • The process of cortical morphogenesis commenced in the form of a small patch of kinetosomes (the opisthe's oral primordium), which appeared within a pouch beneath the cortex on the ventral side ahead of the marginal cirri (Figs. 2A, B, 3A, B). When this pouch started to expand, more kinetosomes assembled into new membranelles of the opisthe progressively backwards (Figs. 2C, D, 3CE). The row of newly constructed membranelles gradually extended forward and curved to form the opisthe's adoral zone (Figs. 2CE, 3CG). The parental adoral zone remained intact during the entire process.

    Figure 2.  Morphogenesis of Euplotes vannus doublets (AF) and singlets (G) after protargol staining. A A typical doublet, to show the ciliature, arrow marks the paroral membrane. B A specimen at early stage to show the oral primordium (arrow). C A specimen at slightly later stage, showing 5-FVT cirral streaks (arrowheads) and anlage of cirrus Ⅰ/1 for the proter (arrow). D A specimen showing the fragmentation of the cirri anlagen, the development of dorsal kineties anlagen (double-arrowheads), marginal cirri anlagen (arrows), the paroral membrane anlage for opisthe (blue arrowhead), and the cirrus Ⅰ/1 anlagen for proter and opisthe (red arrowheads). E A specimen at later middle stage, showing the differentiation of caudal cirri (double-arrowheads for proter's), the development of FVT cirral, the marginal cirri (arrows), the paroral membrane anlage for opisthe (blue arrowhead), and the cirrus Ⅰ/1 for opisthe (red arrowhead). F A specimen at late stage, arrowheads indicate new caudal cirri. G Morphogenesis of singlets after protargol staining, from Jiang et al. (2010). AZM adoral zone of membranelles, CC caudal cirri, DK dorsal kineties, MC marginal cirri. Scale bars: 40 μm

    Figure 3.  Photomicrographs of Euplotes vannus doublets during morphogenesis after protargol staining. A The ciliature of a typical doublet. B A very early divider to demonstrate the oral primordium (arrow). C, D Specimens at early stage, showing five FVT cirral streaks (arrowheads), the cirrus Ⅰ/1 anlagen (red arrows), and oral primordium (black arrows). E A slightly later divider, showing the anlagen of marginal cirri (blue arrows), the cirrus Ⅰ/1 anlage for proter (red arrow), and the FVT cirral streaks (arrowheads). F, G A divider at middle stage (F) and a divider at late stage (G), showing the development of dorsal kineties (red arrowheads), the paroral membrane anlage for opisthe (blue arrowhead), the marginal cirri (blue arrows), the cirrus Ⅰ/1 in proter and opisthe (red arrows), and the new caudal cirri for proter (double-arrowheads). H A late divider, showing the new caudal cirri (double-arrowheads), the paroral membrane for opisthe (arrowhead), and the marginal cirri for opisthe (arrow). AZM adoral zone of membranelles, CC caudal cirri, DK dorsal kineties, FVC frontoventral cirri, MC marginal cirri, PM paroral membrane, TC transverse cirri. Scale bars: 30 μm

    The primordium of the paroral membrane appeared later than the oral primordium and was located within a subcortical pouch near the posterior end of the oral primordium (Fig. 2D). It elongated, broadened, and eventually developed into the paroral membrane of the opisthe (Figs. 2E, F, 3G, H).

  • Development of cirral anlagen

  • As the oral primordium extended, two sets of basal bodies originated de novo to the right of the buccal cavity and anterior of the parental transverse cirri. These developed into two sets of five streaks which formed the frontoventral transverse (FVT) cirral anlagen (Figs. 2C, 3C). Subsequently, these cirral streaks extended in both directions with the proliferation of basal bodies, broadened, broke apart in a 3:3:3:3:2 pattern in each set (Figs. 2D, 3DF), and finally developed into nine frontoventral and five transverse cirri which eventually migrated to their final positions (Figs. 2E, F, 3G, H). No parental ciliary organelle was involved in the FVT-anlagen formation process.

    When the FVT cirral anlagen started to break apart, another anlage appeared de novo to the left of the FVT streaks, i.e., the leftmost frontal cirrus (cirrus Ⅰ/1) anlage for the proter (Figs. 2CF, 3DH). The cirrus Ⅰ/1 anlage of the opisthe appeared near the posterior of the new adoral zone and migrated to its final position (Figs. 2DF, 3D, FH). There is no evidence that the old cirri were involved in the formation of these anlagen.

    In the proter, the marginal cirral anlage originated de novo as a short row of kinetosomes near the left side of the bend of the parental adoral zone, while in the opisthe it commenced near the posterior part of the newly formed adoral zone (Figs. 2D, 3E, F). Each anlage subsequently divided and developed into two marginal cirri (Figs. 2E, F, 3G, H).

  • Development of dorsal ciliature

  • Several new basal bodies proliferated in the middle of each parental dorsal kinety (Figs. 2D, 3F). In the subsequent process, these loosely arranged anlagen extended towards both ends and finally replaced the old structures (Figs. 2E, F, 3G, H).

    During the proliferation of dorsal kineties in the proter, one caudal cirrus was formed at the posterior end of each of the rightmost three dorsal kinety anlagen. At the same time, in the opisthe, one caudal cirrus was formed at the posterior end of each of the rightmost two old dorsal kinety rows (Figs. 2E, F, 3G, H). Therefore, doublets with 3-3 or 2-2 caudal cirri were present in equal numbers in clonal cultures.

  • Development of nuclear apparatus

  • The doublets of Euplotes vannus had two C-shaped macronuclei but only one micronucleus which was located on one side (Fig. 4A). At the beginning of morphogenesis, replication bands formed at both ends of the two macronuclei, and these gradually moved from the ends to the middle, showing a significant increase in genetic material after replication (Fig. 4BF). Meanwhile, the micronucleus gradually expanded and stretched and divided mitotically to produce two micronuclei both of which were located close to the macronucleus on one side of the doublet (Fig. 4CF, H). When the replication of the macronuclei was completed, they changed from a C shape to a short rod shape (Fig. 4G). Then the short rod-shaped macronuclei gradually stretched into a dumbbell shape and the two micronuclei moved, one to each end of their associated macronucleus (Fig. 4I, J). During the late stage of cell division, the micronuclei were completely separated as one moved into the proter and the other into the opisthe, while the macronuclei continued to elongate and bend, and finally divided with the separation of daughter cells (Fig. 4K, L).

    Figure 4.  Photomicrographs of Euplotes vannus doublets during morphogenesis after acridine orange and Hoechst 33342 staining. Arrows indicate micronucleus. Arrowheads indicate macronuclear replication bands. A An undivided doublet, to show the general pattern of the nuclear apparatus. BD Showing the macronuclei beginning to replicate, with replication bands detected at both ends, and the single micronucleus which is undergoing mitosis in D. E, F Micronucleus divides into two and the replication bands of the macronuclei move towards the middle. G The replication of macronuclei is complete. H High magnification of detail in D showing the mitosis of micronucleus. IL Macronuclei gradually elongate and split during cytokinesis. Scale bars: 20 μm

Discussion

    Comparison of doublets among different species

  • With regard to doublets of euplotids, Luporini et al. (1979) reported three doublet strains and two singlet strains of Euplotes crassus. The doublet strain D-F1sH7 was found in the asexual reproductive system; some cells rearranged to form doublets due to the failure of separation at binary fission. The second doublet strain D-F2L was isolated from the conjugants of D-F1sH7 and a singlet strain, showing a different mating type from D-F1sH7. The third doublet strain, D-G14 was formed by the spontaneous fusion of mating pairs. The doublet of E. vannus is very similar to strain D-G14, that is, it was discovered during the conjugation process and was identified as a "combined deformed organism". After microscopic observation, we confirmed that the position of its adoral zone was completely different from that of the mating pairs. In the early stage of mating pairs, the adoral zones of the two cells are arranged back-to-back, whereas the adoral zone of each component in the doublets is located on the side of the cell opposite to the site of cellular fusion.

    The doublets of most reported ciliate species are mirror-imaged, having two sets of cortical structures juxtaposed on the same surface with the two components sharing a common ventral and dorsal surface (Bell et al. 2008; Dias et al. 2007; Jerka-Dziadosz 1983, 1985; Li et al. 2004; Shi and Frankel 1990; Shi and Qiu 1989; Vd'ačný and Tirjaková 2012). However, none of the doublets that have been reported in Euplotes are mirror-imaged. The doublet of E. crassus is formed by back-to-back fusion (Luporini and Giachetti 1980), while our doublet of E. vannus is another type which is similar to E. patella and E. woodruffi (Kimball 1941; Kosaka 1990; Powers 1943): the right edges of two ordinary individuals are fused, and finally the dorsal side of one cell is on the same horizontal plane as the ventral side of the other cell.

    In the reported morphogenesis of Paraurostyla weissei and Stylonychia mytilus, the ciliature of a doublet is not equivalent to the sum of two ciliary sets from two singlets, that is, both components lack some cirri (Jerka-Dziadosz 1983; Tchang and Pang 1980). During asexual division of doublets in P. weissei, the ciliary structures in both components develop synchronously in the same order of anterior to posterior sequence as their singlets (Jerka-Dziadosz 1983). According to Tchang and Pang (1980), the development of ciliature in each component of S. mytilus mirror-imaged doublets is not synchronous, that is, the ciliature of the right component, which is the same as the singlets, develops earlier than the left component, which is accompanied by disordered membranelles. Comparatively, the ciliature of each component in the Euplotes vannus doublet is identical to that of the singlets. Moreover, the morphogenetic process of both components in E. vannus doublets is synchronized and all structures in the offspring are consistent with those of their parent.

  • Comparison with asexual reproduction of normal cell

  • The morphogenetic process of Euplotes vannus singlets during cell division has been investigated in detail (Jiang et al. 2010). It is summarized as follows: (1) the oral primordium of opisthe develops de novo in a pouch beneath the cortex, while the proter inherits the parental adoral zone; (2) the paroral membrane of the opisthe generates independently within the same pouch; (3) two sets of FVT cirral anlagen originate de novo in both dividers and each set breaks into a 3:3:3:3:2 pattern; (4) the leftmost frontal cirrus and marginal cirri are also formed de novo; and (5) the dorsal kinety anlagen originate in the mid-region of the parental cell, while three (for proter) or two (for opisthe) caudal cirri are formed at the ends of the rightmost two or three dorsal kineties. The morphogenesis of doublets is consistent with that of the singlets.

  • Comparison with conjugative process

  • As discussed above, the doublets were isolated from a conjugation culture, thus they look very similar to mating pairs. However, they differ in some details such as (1) the fused position of two cells (right side in doublets vs. left side in mating pairs, i.e., the fusion of adoral zone); (2) the number of micronuclei (one in each doublet vs. two in each mating pair) (Gao et al. 2020; Jiang et al. 2019).

    The morphogenesis of Euplotes vannus during the conjugative process has also been studied in detail (Gao et al. 2020; Jiang et al. 2019). The two mating cells synchronously undergo two rounds of morphogenetic process, i.e., conjugational and postconjugational reorganizations. The postconjugational round of cortical morphogenesis is very similar to that of doublets in the following events: (1) the oral primordium is generated de novo within a subcortical pouch; (2) the paroral membrane anlage forms independently within the same pouch; (3) the anlagen for FVT cirri originate de novo and fragment in a 3:3:3:3:2 pattern; (4) the anlagen for the leftmost frontal and marginal cirri are generated de novo; (5) caudal cirri are formed at the ends of the rightmost dorsal kineties (Gao et al. 2020). The main difference is that the old dorsal kineties are unchanged during both rounds of morphogenesis in singlets (vs. replaced during division of doublets) (Gao et al. 2020).

    Doublets could be easily distinguished from mating pairs by the process of nuclear apparatus development. The nuclear events during conjugation are relatively complicated (Jiang et al. 2019) and can be summarized as follows: (1) three prezygotic divisions resulting in eight nuclei, two of which become pronuclei; (2) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; (3) two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely. In doublets, however, the micronucleus divides into two by mitosis, one for each of the two daughter cells. The macronucleus of each component becomes a short rod shape following the progress of replication bands. This then divides into two (one for each daughter), stretches, and then forms into a C shape.

  • The adaptive significance of doublets

  • As we have observed, Euplotes vannus singlets prefer to crawl or jerk incessantly on the substrate rather than swim. Only when they are disturbed do they swim briefly and then quickly return to the substrate, even when starved (Fenchel 2004). Doublets are formed by the fusion of the right edge of two cells in which the ventral side of one component is in the same plane as the dorsal side of the other component. This leads to a significant limitation on the crawling of doublets on the substrate since the cirri of only one component can be in contact the substrate. Nevertheless, these cirri need to drive the movement of the whole doublet when crawling, which is both unbalanced and expensive in terms of energy consumption. Therefore, locomotion in the vast majority of doublets is by swimming rather than crawling and, like the doublets of Oxytricha described by Ricci et al. (2000), they swim in straight lines with balanced rotation around their longitudinal axis, propelled by cilia on both sides.

    Our doublet strain was discovered in a conjugation system in which the cell density was exceptionally high and all cells were starved. Generally, cells under food-restricted or other adverse conditions will perform conjugation, which is a survival strategy (Miyake 1974). However, if the cells fail to conjugate, e.g., cannot find cells of complementary mating type, they may find another way to escape these adverse environments. The efficient swimming and subsequent dispersal of doublets may give them advantages over singlets in predator avoidance and feeding efficiency. Moreover, swimming is more conducive to alleviating the environmental pressure caused by high cell density, and swimming cells are also more easily dispersed by water flow than crawling cells (Ricci et al. 2000). After escaping from the harsh environment, the doublets can convert to singlets and back to the normal life, which was frequently seen in the doublet cultures. To some extent, this suggested that the appearance of doublets may be an adaptive form to escape adverse environments, and, as initially proposed by Ricci et al. (2000) for Oxytricha, they are likely to represent a differentiation state in ciliates similar to giants and cysts, rather than being the product of a mistake in a biological process (Ricci et al. 1989; Serra et al. 2021). Further studies are needed to determine the veracity of this speculation.

Materials and methods

    Ciliate sampling and identification

  • Euplotes vannus strains used in this study were the same strains as those used in previous studies (Gao et al. 2020; Jiang et al. 2019), which were isolated in July 2015 from Silver Sand Beach, Qingdao, China (35° 55′ 12″ N, 120° 11′ 48″ E, water temperature 24 ℃, salinity ~ 30). Species identification was based on morphological characters observed in vivo and after protargol staining, as previously described (Jiang et al. 2010; Song and Packroff 1997) (Fig. 1).

  • Cell isolation and cultivation

  • The doublet arose spontaneously in a conjugation system with mixed two strains of complementary mating types and was possibly produced by spontaneous fusion of a mating pair. Doublets were isolated and clonal cultures were established in sterilized seawater at 22 ℃ under a natural cycle of light and dark. The doublets were fed with sugar-free yogurt containing Lactobacillus bulgaricus and Streptococcus thermophilus. The E. vannus doublet strain has been cultured in the laboratory for more than one year, and they can still divide asexually in the same way as described in this paper.

  • Staining and observation

  • The protargol staining method according to Wilbert (1975) was used to reveal the ciliature and nuclei. The protargol powder was synthesized in-house according to Pan et al. (2013). Doublets were fixed with 50% formalin solution at room temperature for 1 min and then stained by Hoechst 33342 (100 ×) (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) and acridine orange (AO) (Shanghai Chemical Reagent Company, China, final concentration 0.1 μg/ml). The morphological studies were performed according to Wang et al. (2021) and Zhang et al. (2020). To show the changes during morphogenesis, the parental cirri and adoral membranelles are depicted in outline and the new cirri and adoral membranelles are shaded black. Systematics and terminology are mainly according to Jiang et al. (2010).

  • Acknowledgements

  • Our special thanks are given to Prof. Weibo Song (OUC) for his kind suggestions during drafting of the manuscript. This work was supported by the National Natural Science Foundation of China (32030015, 31922013 and 31961123002), the Natural Science Foundation of Shandong Province (ZR2020JQ13), the Fundamental Research Funds for the Central Universities (202141004), and the Researchers Supporting Project (RSP-2022R7) of the King Saud University, Saudi Arabia.

  • Author contributions

  • FG and JJ were responsible for the conceptualization and supervision of the study. JF performed cell culture and staining. YC performed live observations. JF, YC, GP and JJ contributed to the revision of the manuscript. All authors contributed to the article and approved the submitted version.

  • Data availability

  • The authors declare that all data supporting the findings of this study are available within the article.

Declarations

    Conflict of interest

  • The authors declare no conflicts of interest.

  • Animal and human rights statements

  • Not applicable.

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