Light and transmission electron microscopy of Cepedea longa (Opalinidae) from Fejervarya limnocharis

Cepedea longa Bezzenberger, 1904, collected from Fejervarya limnocharis (Amphibia, Anura, Ranidae) from Honghu Lake, Hubei Province, China in May–July 2016, is described at both light and transmission electron microscope levels. This is the first electron microscopic study of this species. Cepedea longa possesses a developed fibrillar skeletal system, composed of longitudinal fibrillar bands and transversal fibrils as well as numerous thin microfibrils dispersed in the endoplasm, which may play an important role in morphogenesis and offer some resilience to deformations of the cell. Longitudinal microfibrils are polarizing elements of kineties, bordering the somatic kineties on the left side and possibly responsible for kinetosome alignment. Two types of vesicles exist in the somatic cortex: globular endocytotic vesicles and flattened exocytotic vesicles. As to the nuclei of C. longa, a thick microfibrillar layer was observed to attach to the cytoplasmic face of the nuclear envelope. This fact suggests no necessary connection between the presence of this microfibrillar layer and the number of nuclei. In addition, some unknown tightly-packed microtubular structures in the nucleoplasm were observed for the first time in opalinids; neither their nature nor physiological significance is known. A detailed list of all reported Cepedea species is included.


Introduction
Opalinids are commonly regarded as endocommensals in the guts of cold-blooded vertebrates, mostly amphibians, and have no known pathological effect on their hosts [13].
As to the phylogenetic affinities, opalinids were for a long time regarded as ''protociliates'' [4,29,31]. Then the hypothesis of opalinid-ciliate affinity was abandoned with the better understanding of ciliate biology. As a result, the opalines were transferred from the ciliates and placed with amebae and flagellates either as an isolated taxon in the phylum Zooflagellata or were treated as a separate phylum: Opalinata [5,6,54]. Recent works based on detailed ultrastructural study and convincing phylogenetic analyses suggest that opalinids belong to heterokonts as a sister group to Proteromonas Kunstler, 1883 within the order Slopalinida, with two families, the Proteromonadidae and the Opalinidae [8, 26-28, 35, 41, 43-46].
Cepedea is a common genus of the family Opalinidae that inhabits anuran amphibians. It was created by Metcalf in 1920 when he assigned the ''cylindrical multinucleated species'' to this genus and placed the ''flattened multinucleated species'' into the genus Opalina [30]. Metcalf also considered that Opalina arose from Cepedea by flattening the body [32]. Mohr stated that ''Cepedea is not a valid genus'' because ''flattened Opalina and cylindrical Cepedea have no discernible boundary'' [38]. The application of protargol (silver proteinate) impregnation revealed the arrangement of the kineties, which stem from the falx [9-12, 21, 55]. On the basis of the arrangement of the falx relative to the anteroposterior axis of the body, Delvinquier & Patterson proposed a more recent definition of Cepedea: Multinucleated, with a short, broad, axial falx almost parallel to the anteroposterior axis of the cell; kineties cover the body evenly [7].
To date, many new species of Cepedea have been found from anuran amphibians (Table 1). Cepedea longa was first discovered and named by Bezzenberger in 1904 from the intestines of Fejervarya limnocharis (= Rana limnocharis) [3]. Thereafter, the redescription and some revisions of this species were given by Metcalf [31] and Nie [40], respectively. However, the morphological data were still incomplete since no transmission electron microscopic study had been carried out. The present study therefore attempts to supplement the morphology-based descriptions at both light and transmission electronic microscopic levels, aiming to contribute to the knowledge of this genus and to provide useful information for its taxonomy.

Materials and methods
The frogs F. limnocharis used for this study were captured from Honghu Lake (29°40 0 -29°58 0 N; 113°12 0 -113°26 0 E), Hubei Province, China in May-July 2016. We obtained the permits allowing us to capture and sacrifice these specimens. The frogs were transported alive to the laboratory for further examination. All frog samples were dissected as soon as possible. The recta were collected into Petri dishes and examined with the aid of Stemi SV6/AxioCam MRc5 (Zeiss, Oberkochen, Germany). The opalinids were collected with Pasteur micropipettes and washed twice in 0.65% saline solution.
For identification, specimens were smeared on coverslips and stained with ammoniacal silver carbonate [20] or silver nitrate [53]. For measurements, we used freshly killed specimens (in 5% formalin) with no coverslips mounted (except for the nucleus, which was measured in the ammoniacal silver stained slides). The specimens were observed, measured at 200· or 400· magnification and photographed using Axioplan 2 imaging and Axiophot 2 (Zeiss, Oberkochen, Germany). All measurements are in micrometers. Slides 2016W001-004 of silver nitrate stained specimens and 2016W005-010 of ammoniacal silver stained specimens have been deposited at the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
For transmission electron microscopy (TEM), specimens were fixed directly in 2.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS, pH 7.4) for 2 h at 4°C, then postfixed in 1% (v/v) osmium tetroxide in PBS for 2 h at 4°C, followed by dehydration in a gradient acetone series and embedded in Araldite. Ultrathin sections were cut on a Leica Ultracut R ultramicrotome (Leica, Germany), stained with uranyl acetate and lead citrate before being observed in a JEM-1230 Transmission Electron Microscope (JEOL, Japan).

Results
Based on our survey, 76 (35.8%) of 212 examined F. limnocharis were found to be infected with Cepedea longa. Numerous opalinids were found mainly in the recta of frogs. The body is greatly elongated and cylindrical in form, slightly flattened and wedge-shaped at the anterior extremity, with the posterior end tapering or sharply pointed (Figs. 1A and 1C). Body length is 508.8-816.0 lm ( " X ¼ 727:7 lm; n = 20) and width 36.0-57.6 lm ( " X ¼ 46:9 lm; n = 20) in vivo. The animal is thickly flagellated and often coils when swimming (Fig. 1B), with its body surface twisting and giving a spiral appearance (Figs. 1C and 1D). The falx is quite short and thus difficult to observe, located at the margin of anterior extremity and parallel to the anteroposterior axis of the cell (Fig. 1E). All somatic kineties branch off from each side of the falx and follow a sigmoid course, numbering 64-87 (n = 8) in total (Figs. 1E and 1F). The organism possesses a large number of spherical or ellipsoidal nuclei (75-170; " X ¼ 129:6; n = 20), with a diameter ranging from 4.5 lm to 10.4 lm ( " X ¼ 7:5 lm; n = 40) (Fig. 1G). Data for measurements related to morphometric characteristics are given in Table 2.
With a transmission electron microscope, pellicular folds can be seen clearly, which are supported by ribbons of microtubules (Figs. 2A, 2B and 3A). Coated vesicles often  occur beneath the cortical folds, some of which are fused with the plasma membrane and seen as invaginations ( Fig. 2A). Pellicular folds vary between kineties, with their numbers varying at different intervals (Fig. 2B). Microfibrillar bands run through the cortex. In fact, a developed fibrillar skeletal system exists -it is made up of longitudinal fibrillar bands and fine transversal fibrils (Figs. 2C and 2D). Longitudinal microfibrils border the somatic kineties on the left side, with transversal branches running perpendicular to kinetal long axes and framing the ribs of the cortical vesicles (Figs. 2C and 2D). There are two types of cortical vesicles: globular endocytotic (endocytic/pinocytic) vesicles and elongated exocytotic (exocytic/membrane ''recycling'') vesicles. Endocytotic vesicles are often found in rows and alternate with these exocytotic vesicles (Fig. 2D). The somatic flagella emerge in cylindrical pits, around which there is also some skeletal material (Figs. 2B, 3A and 3D). The somatic kinetosomes are linked by desmoses, which have characteristic periodicity (Fig. 3B). Vacuoles are sometimes found between somatic kineties just beneath the cortical surface (Fig. 3C). Interkinetosomal desmoses are always composed of two parts: the trifurcated left branch and the right branch extend as one fibril to finally contact the left posterior of the next anterior kinetosome (Figs. 3B and 3C). The projecting part of a flagellum has a conventional (9 + 2) axonemal structure (Figs. 3A-3C). At a level slightly above the bases of the cortical folds, there is an electron-dense helix around the central pair of microtubules (Fig. 3D). The axosome is embedded in the proximal margin of the transitional plate (Fig. 3D). Each peripheral group of microtubules in the kinetosome gives rise to a curving arm (Fig. 3B) which extends out and up to make contact with the plasma membrane (Fig. 3D).
Bundles of microfilaments can be observed crossing the endoplasm between nuclei and mitochondria (Fig. 4A). As a multinucleate opalinid, of course, C. longa has many nuclei in the cell (Fig. 4B). Each nucleus has one nucleolus in the nucleoplasm and a thick microfibrillar layer attached to the cytoplasmic face of the nuclear envelope (Figs. 4B and 4C). It is noteworthy that some unknown tightly-packed microtubular structures distribute in the nucleoplasm (Fig. 4D). Mitochondria have tubular cristae at their periphery and a relatively large volume of matrix with an amorphic appearance (Fig. 4E).
As to the falcular area, we failed to observe its ultrastructure because of its quite limited length, although we attempted many times to prepare thin sections. Hence, there is no description presented here.

Discussion
As mentioned above, C. longa has been described from F. limnocharis by several authors. The average body size of opalinids examined in the present study (727.7 lm · 46.9 lm) bears the most resemblance to Bezzenberger's type specimens (680.0 lm · 52.0 lm) [3], and is smaller than that recorded by Metcalf (1000.0 lm · 75.0 lm) [31] and Nie (1162.0 lm · 42.5 lm) [40]. The longest specimen of C. longa recorded by Nie even reaches 1820 lm in length [40]. These data reveal that C. longa varies greatly in body dimensions. They also suggest that body dimension is not a reliable taxonomic parameter for opalinids. According to the aforementioned studies, C. longa shows strict host specificity to F. limnocharis [3,31,40]. However, the host species has now been recognized as a cryptic species complex [14,16]; thus, it is inappropriate to define C. longa as a host-specific endoparasite of F. limnocharis, since it shows at least some host variability. On the other hand, the body form and moving pattern of the living specimens, the arrangement of the falx and the nuclear features such as the number (mononucleated/binucleated/multinucleated), shape and position are most constant and important for specific identification [2,7,31].
The ultrastructural features of C. longa described herein closely resemble those of other opalinids: cortical folds  supported by ribbons of microtubules, coated vesicles (pinocytotic) at the base of the folds, a developed cortical fibrillar system, delicate kinetosomal architectures, etc. The multiplication of cortical folds and coated vesicles found in C. longa is similar to that described in C. dimidiata Stein, 1860 [42], C. sudafricana Fantham, 1923 [37], O. ranarum Ehrenberg, 1832 [34,43], P. polykineta Grim & Clements, 1996 [24] and P. pomacantha Grim et al., 2000 [25]. We think that the flattened exocytotic vesicles in rows under the cell surface may participate in the process of cell membrane reconstitution by which pinocytotic vesicles provide nutrients from the environment and then recycle back to the plasma membrane as the exocytotic, ''membrane reconstruction'' vesicles. This is a special adaptation strategy for these astomatous (no cytostome) opalinids.
According to our present study, C. longa possesses a developed fibrillar skeletal system, composed of longitudinal fibrillar bands and transversal fibrils as well as numerous thin microfibrils dispersed in the endoplasm. In fact, a network of microfibrils was also reported in some other opalines, such as C. dimidiata [42], C. sudafricana [37], O. ranarum [34,43], P. pseudonutti Sandon, 1976 [36] and P. pomacantha [25]. These previous studies showed that the existence of a microfibrillar skeleton may not be a unique characteristic of the genus Opalina but possibly a common feature to all opalines. The microfibrillar networks also recall some ciliate skeletal components, in particular the ecto-endoplasmic boundary layer in some rumen ciliates [22,23,[50][51][52]. As to their function, it is possible that they may play an important role in morphogenesis and offer some resilience to permanent deformations of the cell since the body is highly elastic and flexible. Moreover, these microfibrils, especially the longitudinal fibrillar bands, are polarizing elements of kineties and consequently may be responsible for kinetosome alignment. With respect to the nuclei of C. longa, a thick microfibrillar layer was observed here to attach to the cytoplasmic face of the nuclear envelope. According to the study of Mignot and Affa'a [36], there is a similar fibrillar structure in P. pseudonutti, while the cytoplasmic face of the nuclear envelope is bare in C. dimidiate, C. sudafricana and O. ranarum. Hence, they stated that in different species of Protoopalina (having two nuclei per cell), the cytoplasmic face of the nuclear envelope is always covered with a microfibrillar layer, while in the multinucleate opalinids it was lacking [36]. However, our aforementioned observation in C. longa contradicts their hypothesis and suggests no necessary connection between this microfibrillar layer and number of nuclei. In addition, it is noteworthy that some unknown tightly-packed microtubules distributed in the nucleoplasm were observed in our present study. Hence, this is the first report of such microtubules in opalinids. Neither their nature nor physiological significance is known.