1 Introduction

The ciliate order Euplotida Small & Lynn, 1985 is characterized by its sparse but well-developed ventral cirri arranged in frontal, ventral, and transverse groups (Fan et al., 2013; Jiang et al., 2013; Gao et al., 2017; Huang et al., 2011, 2012, 2018; Lian et al., 2018). Eupotids have been the subject of numerous recent investigations of their morphology, morphogenesis and systematics (Shao et al., 2010a, b; Yi et al., 2012; Liu et al., 2017; Luo et al., 2014, 2018; Wang et al., 2017a, b; Yan et al., 2018; Zhao et al., 2017, 2018). Uronychidae is a distinctive family in the Euplotida that is characterized by its highly developed paroral. Uronychia binucleata was first reported by Young (1922). It is characterized by its medium-sized, oval to almost rectangular body, two macronuclear nodules, high numbers of basal bodies in its dorsal kineties and characteristic buccal apparatus (Song and Wilbert, 1997).

Divisional morphogenesis of Uronychia has been studied for several species and appears to be highly conserved among all congeners for which data are available (Taylor, 1928; Wilbert and Kahan, 1981; Hill, 1990; Wilbert, 1995; Song, 1996; Song et al., 2004; Shen et al., 2009; Shi et al., 2017; Song and Shao, 2017). However, the development of the leftmost frontal cirrus and paroral in Uronychia has not been described in detail.

Uronychia binucleata is a rare species, and its morphogenesis was partially described by Hill (1990) (misidentified as U. transfuga) and Song (1996) (misidentified as U. uncinata). The rediscovery of U. binucleatain in coastal waters of northern China provided the opportunity to investigate its morphogenetic process in detail, including, for the first time in Uronychia, the formation of the leftmost frontal cirrus and the paroral.

2 Materials and Methods 2.1 Sampling, Culturing, and Isolation

Uronychia binucleata was collected from Yellow Sea coastal waters next to a sewage outfall at a beach near Zhanqiao Pier, (120˚32΄E, 36˚06΄N), Qingdao, China, on December 6, 2014. The water temperature was about 6.5⊥ and the salinity was 25%-28%. Individual U. binucleata cells were isolated in the laboratory using micropipettes. Cultures were established at 10-15⊥ using boiled sea water and adding small scuticociliates as the food source (Shao et al., 2007).

2.2 Morphogenesis

Protargol staining (Wilbert, 1975) was used to reveal the ciliature and nuclear apparatus. Counts and measurements of stained specimens were performed at a magnification of 1000℅. Drawings of protargol-stained cells were made with the help of a camera lucida (Yan et al., 2016, 2017). To illustrate the changes occurring during morphogenetic processes, old (parental) ciliary structures are depicted by contour whereas new structures are shaded black (Chen et al., 2018; Lu et al., 2018).

2.3 Terminology and Systematics

Terminology is mainly according to Song et al. (2004). Systematics is according to Lynn (2008), Gao et al. (2016) and Lyu et al. (2018).

3 Results and Discussion

The morphology of the present population matches almost exactly with that described by Song and Wilbert (1997), hence no further details are documented here (Figs. 1A-C; 4A, B; Table 1).

3.1 Morphogenesis During Binary Fission in Uronychia binucleata(Figs. 2-4)

Stomatogenesis Morphogenesis starts with the appearance of a small patch of kinetosomes, the opisthe* soral primordium (OOP) within a subsurface or subcortical pouch positioned between cytostome and left marginal cirri (Fig. 2A). At the same time, the proter*s oral primordium (POP) appears, also subcortically, slightly anterior of the parental adoral zone of membranelles (AZM2) (Figs. 2A, B). Both the OOP and POP develop by rapid proliferation of kinetosomes (Fig. 2D). Subsequently, two groups in the OOP, with eleven and five membranelles respectively, and two groups in the POP, each with five membranelles, differentiate and migrate to the surface of the cell (Figs. 2G, J; 3A, E, G, I).

Meanwhile, an additional anlage (anlage for leftmost frontal cirrus, LFCA) appears within the subcortical pouch adjacent to the posterior end of the oral primordium in each divider (Figs. 2D, E). The undulating membranes anlage (UM-anlage) then appears to the left of LFCA and locates between the oral primordium and LFCA in each divider (Figs. 2G, H; 5A). Because we cannot observe the earliest stages of morphogenesis, we were unable to determine whether the UM-anlage and LFCA develop from the same anlage. Later, the UM-anlage and the LFCA begin to lengthen and further develop along the right edge of the pouch opening (Figs. 2J, K; 5B). The anterior portion of the LFCA gradually widens (Figs. 3A, B; 5C-H). Finally the leftmost frontal cirrus develops from the LFCA and the paroral is formed from the UM-anlage (Figs. 3D, E, G; 5I, J).

During the late stages, the two groups of membranelles in both oral primordia separate. In the proter, the anterior group migrates anteriorly. As in its congeners, the anterior group will replace the posterior five membranelles of the adoral zone of the proter (AZM1), while the parental six membranelles of the adoral zone are retained (Fig. 3Ⅰ). In the opisthe, the anterior group migrates anteriorly and forms 11 membranelles in AZM1 (Fig. 3Ⅰ). In both daughter cells, the posterior groups form the membranelles in AZM2. In the posterior group of new membranelles, the most posterior one (which is also the smallest) moves apart from the other four and reaches its final position as the & buccal cirrus' (Figs. 3Ⅰ; 4L).

Development of the somatic ciliature in proter and opisthe At about the same time as the oral primordia are formed, basal bodies for FVT-cirral primordia (CA) develop on the cell surface firstly as four, and then five, streaks (anlagen I-V) anterior of the transverse cirri (Figs. 2A; 4C). No parental ciliary organelles are involved in the formation of these new primordia. Each of the five anlagen extends to its maximum length before they divide into two sets (Figs. 2A, D; 4D). Subsequently the streaks broaden, break apart and develop into distinct cirri as they migrate to their final positions (Figs. 2G, J; 3A, E; 4F). According to their segmentation, cirral anlagen I-V give rise to cirri in the pattern 3:3:2:2:3, respectively, while the leftmost cirrus is formed near the UM-anlage as described above (Figs. 3G, I; 4H).

The anlagen of the left marginal cirri are formed de novo and separately on the cell surface near the old AZM2 and the marginal cirri (Figs. 2A; 4E). These anlagen then enlarge and segment to form the new left marginal cirri (Figs. 2D, G, J; 3A, E, G, I). On the dorsal side, the proliferation of new basal bodies occurs at two levels within each of the six old kineties. The development of these anlagen seems to occur progressively from right to left (Figs. 2A, C, D, F, G, I; 4M). Two caudal cirri are formed at the posterior end of the rightmost anlage while the second primordium from the right gives rise to the third caudal cirrus (Figs. 2I, L; 3C, F, H, J; 4G, I).

3.2 Noteworthy Morphogenetic Features in Uronychia

Morphogenesis in five Uronychia species, including U. xinjiangensis, U. multicirrus, U. transfuga, U. binucleata, and U. setigera, has been investigated using silvers staining methods (Dembowska, 1926; Taylor, 1928; FaurëFaurfremiet 1964; Wilbert and Kahan, 1981; Hill, 1990; Wilbert, 1995; Song, 1996; Song et al., 2004; Shen et al., 2009; Chen et al., 2017; Shi et al., 2017). The pattern of morphogenesis is highly conserved among these congeners and the same general pattern was also observed for U. binucleata in the present study. The mode of development of leftmost frontal cirrus and paroral, however, was unknown in Uronychia and is described here for the first time. In U. binucleata, the anlage for the leftmost frontal cirrus is formed to the right of the UM-anlage and is earlier than the formation of the UM-anlage. This contrasts with the process in U. multicirrus, in which this anlage develops anterior of the UM-anlage after the formation of the latter (Shen et al., 2009).

Acknowledgements

This work is supported by the National Natural Science Foundation of China (Nos. 31872190, 31702009), the Key Laboratory of Mariculture (KLM), Ministry of Education, Ocean University of China (OUC) and the Innovation Team of Team of Higher Learning Insritutions of Tianjin (No. TD13-5089). We also thank Prof. Weibo Song (OUC) for his kind help and suggestions during drafting the manuscript.