Frontonia paramagna, Chen, Ying, Zhao, Yan, Pan, Xuming, Ding, Wenqiao, Al-Rasheid, Khaled A. S. & Qiu, Zijian, 2014

Chen, Ying, Zhao, Yan, Pan, Xuming, Ding, Wenqiao, Al-Rasheid, Khaled A. S. & Qiu, Zijian, 2014, Morphology and Phylogeny of a New Frontonia Ciliate, F. paramagna spec. nov. (Ciliophora, Peniculida) from Harbin, Northeast China, Zootaxa 3827 (3), pp. 375-386 : 377-378

Frontonia paramagna spec. nov.

Diagnosis. Freshwater Frontonia about 400–610 × 110–160 Μm in vivo; dorsoventrally slightly flattened with conspicuously small buccal cavity; about 179–201 somatic kineties; three vestibular and six to seven postoral kineties; peniculi 1–3 each with four kinetid rows; single ellipsoidal macronucleus; one contractile vacuole located equatorially on the right margin; extrusomes spindle-shaped.

Type slides. A silver carbonate-impregnated slide containing the holotype specimen is deposited in the Laboratory of Protozoology, Ocean University of China (CY 20111229 -1). Three silver nitrate slides containing paratype specimens are deposited in the Laboratory of Protozoology, Ocean University of China (CY 20111229 -2, 3, 4).

Type locality. A puddle in a freshwater wetland in Harbin (N45°52′ 02.67″, E126°32′ 56.23″), Heilongjiang Province, China.

Etymology. The prefix “para-” refers to the species being similar to Frontonia magna .

Morphological and Ultrastructural description. Table 1 and Figures 1–3 : Size huge and varied, in vivo about 400–610 × 110–160 Μm, but mostly 500–550 Μm in length; ratio of length: width about 3.5:1. Body shape constant, long ellipsoidal in outline; left margin straight while right margin slightly convex ( Figs. 1 A, 1B, 1C, 1D 2A,2B). Both ends rounded with the front end wider and slightly bent to the right. Dorsal-ventral side slightly flattened. Single ellipsoidal macronucleus, positioned at body centre ( Figs. 1 G, 2K). Single contractile vacuole, about 100 Μm in diameter, positioned in mid-body right of cell median; one contractile vacuole pore located on right dorsal surface ( Figs. 1 A, 1D, 1G, 2A, 2C). Most of the extrusomes spindle-shaped, about 5 Μm long, densely arranged beneath cortex ( Figs. 1 E, 2I, 2J, 3C, 3F). The other two types of extrusomes were observed for the first time ( Figs. 1 E, 3D, 3E). Cytoplasm is transparent, colourless to slightly grey. Cytoplasm filled by many yellow and grey food vacuoles, with different diameters, as well as dark granules and crystals containing bacteria, diatoms and other small ciliates ( Figs. 1 A, 2A, 2B). 180–199 somatic kineties. somatic cilia about 8 Μm long ( Figs. 1 F; 1G). Locomotion by revolving moderately rapidly on substrate or by swimming in water about long body axis. Buccal cavity small and shallow, approximately triangular in outline, occupying about 1/10 of body length ( Figs. 1 A, 1D, 1F; 1H, 2F, 2G, 2H, 2L, 3A, 3B). Three short vestibular kineties (VK1–3) on the right of the buccal cavity running from the anterior vertex to the postoral suture of the cavity, with dense kinetosomes ( Figs. 1 H; 2F, 2H). Three peniculi deeply located on the left wall of cavity: peniculi 1 and 2 (P1, 2) about equally long and close to each other, peniculus 3 (P3) slightly separated from P1, 2 and curved to right. Each peniculus composed of four rows of kinetosomes ( Figs. 1 F, 1H; 2H, 2L, 2M). Peniculus 3 composed of four shorter kineties, of which the right two are complete and the left two shortened ( Figs. 1 H; 2H, 2L). Single paroral membrane (PM) on right edge of the buccal cavity runs from anterior of the buccal overture to the posterior edge. Argentophilic line (AL) positioned parallel to VK ( Figs. 1 H; 2F, 2H).

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were combined to study the subpellicular ultrastructure of F. paramagna spec. nov. Three types of extrusomes were evident, including a spindle trichocyst, another new type of trichocyst and a mucocyst. A large number of spindle trichocysts with a cap-like structure were distributed densely in the specimens ( Figs. 3 C, 3F). The other two types of extrusomes, however, were rare and could be observed only by TEM. The mucocysts were an elongated oval-shape, about 2 Μm long and with transverse microfilaments on their surface, and can emit micro-mesh ( Fig. 3 D). The new type of trichocyst were about 5 Μm long, in the shape of a long rod with a dark, flat, front end, and appeared mainly around the oral apparatus ( Fig. 3 E).

This investigation also revealed a three layered fibre system beneath the pellicle. The first layer of fibre was close to the pellicle, with a diameter of about 0.12 Μm. The fibres of the second layer were densely wrapped around the extrusomes ( Figs. 3 F, 3G); while the fibres of the third layer were loose, covering most of the subpellicular structures and adjoined the cytoplasm.

Finally, the VK in the oral apparatus were observed by SEM. This showed that a membranous structure was wrapped around the root of each VK to form a coarse columnar structure, with interwoven fibres linking these columnar structures to the VK ( Fig. 3 H).

SSU rRNA gene sequence and phylogeny of Frontonia paramagna spec. nov. (Table 3 and Figure 4 ). The newly sequenced SSU rRNA gene of Frontonia paramagna spec. nov., with a length of 1,667 bp, has been deposited in the GenBank database with accession number JQ868786 . The genetic similarity and number of nucleotide differences between the new species and its congeners has been set out in Table 3, showing details of the molecular differences between this new species and other comparable species. Each of the SSU rRNA gene sequences of the species available from GenBank for genus Frontonia have similarities with this new species ranging from 91.1% to 98.6% with 22 to 147 nucleotides difference from other congeneric species (Table 3). The phylogenetic trees generated through both Bayesian Inference ( BI) and Maximum Likelihood (ML) methods had similar topologies, therefore, they were combined into a single consensus tree ( Fig. 4 ). The new species Frontonia spec. nov. clustered together with Frontonia sp. / vernalis with high support (100% ML, 1.00 BI) and then formed a clade with F. leucas (100% ML, 1.00 BI). The clade became the sister group with the clusters of F. tchibisovae / lynni / mengi / magna , also with high support (100% ML, 1.00 BI). But genus Frontonia did not form a monophyletic group since Frontonia sp. ( FN667825 ) and F. didieri were separated and clustered with Paramecium and Apofrontonia species instead of with their congeners (68% ML, 0.99 BI).

Figures

FIGURE 1. Frontonia paramagna spec. nov. from life (A, B, C, E) and after staining with silver carbonate (D, F – H). A. Ventral view of a typical individual, arrow refers to contractile vacuole. B. Lateral view. C. Different body shapes. D. Ventral views showing arrangement of extrusomes, sutures and contractile vacuole. E. Three types of extrusomes. F, G. Infraciliature of ventral and dorsal side. H. Structure of oral apparatus. Scale bars in (A, B, F, G) = 200 µm. AS, anterior suture; CV, contractile vacuole; CVP, contractile vacuole pore; Ma, macronucleus; P 1 – 3, peniculus 1, 2, 3; PK, postoral kineties; PM, paroral membrane; PS, postoral suture; VK, vestibular kineties.

FIGURE 2. Frontonia paramagna spec. nov. from life (A, B, F, G, I) and after staining with silver carbonate (D, E, H, J, K, M) and silver nitrate (C, L). A. Ventral view of a typical individual; arrow marks the contractile vacuole. B. Dorsal view of a typical individual. C. Arrow indicates the contractile vacuole pore. D. The postoral kineties. E. Arrows mark the somatic kineties. F-G. Photomicrographs of buccal field (arrows), showing vestibular kineties, paroral membrane and peniculi 1, 2, 3. H. To show the argentophilic line (the arrow below) and the paroral membrane (the arrow above). I, J. The spindle-shaped trichocyst. K. The macronucleus. L. Arrow indicates the peniculus 3. M. Photomicrograph of peniculi 1, 2, 3 (P 1, P 2, P 3). Scale bars in (B, C) = 200 µm; PM, paroral membrane; VK, vestibular kineties; PK, postoral kineties; Ma, macronucleus

FIGURE 3. Frontonia paramagna spec. nov. from scanning electron microscopy (SEM) (A, B, F, G, H) and transmission electron microscopy (TEM) (C – E). A. SEM photograph of a typical individual. B. Arrowhead shows the buccal field. C, F. Arrowhead shows the spindle-shaped trichocyst. D. Arrowhead shows the mucocyst. E. Arrowhead shows another type of trichocyst. G. Arrowheads show the fibre layers. H. Arrowheads show the vestibular kineties (VK) and the columnar structure. Scale bars in (A) = 20 µm; in (B) = 6 µm; in Fig (C, D) = 0.3 µm; in Fig. (E, F) = 1 µm; in Fig. G = 2 µm; in Fig. H = 4 µm.

FIGURE 4. ML and BI trees based on SSU rRNA gene sequences (Dataset II) computed with PHYML and MrBayes. The first and second values at the nodes represent bootstrap values for ML and BI analyses, respectively; a ‘ * ’ indicates a disagreement in topology that could not be represented on the consensus tree. The scale bar represents 5 changes per 100 positions. The sequences of Trimyema munutum and Plagiopyla frontata were used to root the tree. The position of the new species in the phylogenetic trees is marked with an arrow.

Tables

TABLE 1. Morphometric data of Frontonia paramagna spec. nov. Data based on randomly selected specimens with silver impregnation. Measurements in Μm. CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean; Min, minimum; SD, standard deviation.

Characters	Min	Max	Mean	SD	CV	n
Body length	318	362	340.8	13.0	3.7	15
Body width	96	124	113.0	7.6	6.8	15
Number of somatic kineties	180	200	189.7	6.3	3.3	15
Number of vestibular kineties	3	3	3.0	0	0	15
Number of postoral kineties	6	7	6.5	0	0	15
Number of ciliary row in peniculus 1	4	4	4.0	0	0	15
Number of ciliary row in peniculus 2	4	4	4.0	0	0	15
Number of ciliary row in peniculus 3	4	4	4.0	0	0	15