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All issues Volume 24 (2017) Parasite, 24 (2017) 6 Full HTML
Open Access
Issue
Parasite
Volume 24, 2017
Article Number 6
Number of page(s) 10
DOI https://doi.org/10.1051/parasite/2017006
Published online 01 February 2017

© C. Li et al., published by EDP Sciences, 2017

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Materials and methods

The frogs F. limnocharis used for this study were captured from Honghu Lake (29°40′–29°58′ N; 113°12′–113°26′ 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 μm ($ \bar{X}=727.7\mathrm{\enspace \mu }\mathrm{m};$ n = 20) and width 36.0–57.6 μm ($ \bar{X}=46.9\mathrm{\enspace \mu }\mathrm{m}$; 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; $ \bar{X}=129.6$; n = 20), with a diameter ranging from 4.5 μm to 10.4 μm ($ \bar{X}=7.5\enspace \mathrm{\mu }\mathrm{m}$; n = 40) (Fig. 1G). Data for measurements related to morphometric characteristics are given in Table 2.

thumbnail Figure 1.

Light microscope images of Cepedea longa. (A) Overview of the living specimens, to show general form, greatly elongated and cylindrical, with the anterior extremity broader and the posterior end pointed. Scale bar = 100 μm. (B) Living specimens, to show C. longa thickly flagellated and often coils when swimming. Scale bar = 100 μm. (C)–(D) Living specimens, to show body surface twisting and giving a spiral appearance. Scale bar = 50 μm. (E) Specimens stained with ammoniacal silver, to show the falx (arrow) and somatic kineties branching off from each side. Scale bar = 25 μm. (F) Specimens stained with silver nitrate, to show somatic kineties follow a sigmoid course from anterior to posterior end of the cell. Scale bar = 25 μm. (G) Specimens stained with ammoniacal silver, to show the organism possessing a large amount of spherical or ellipsoidal nuclei (arrow). Scale bar = 25 μm.

Table 2.

Biometrical data (in μm) on Cepedea longa and comparison with former reports.

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).

thumbnail Figure 2.

Transmission electron microscope images of Cepedea longa, to show fine structures of the somatic cortex. (A) Section tangent to cell surface, to show pellicular folds (PF) supported by ribbons of microtubules (Mt). Some coated vesicles are fused with the plasma membrane and seen as invaginations (arrow). SK = somatic kinetosomes. Scale bar = 5 μm. (B) Section passing parallel to cell surface, to show pellicular folds (PF) interposing between somatic kineties (SK). FP = flagellar pit. Scale bar = 20 μm. (C)–(D) Selected enlargement of Figure 2A, to show a developed fibrillar skeletal system in the somatic cortex. Longitudinal microfibrils (LF) border the somatic kineties (SK) joined to each other by desmoses (Ds) on the left side, with transversal fibrils (TF) running perpendicular to kinetal long axes and framing the ribs of the cortical vesicles: globular endocytotic vesicles (EdV) and elongated exocytotic vesicles (ExV). Scale bar = 10 μm.

thumbnail Figure 3.

Transmission electron microscope images of Cepedea longa, to show fine structures of the somatic flagella. (A) Tangential section of a somatic kinety, to show fibrillar elements (arrow) between cortical microtubules (Mt) and around the membrane of each flagellar pit (FP). PF = pellicular folds. Scale bar = 2.5 μm. (B)–(C) Cross section through several kineties, to show somatic kinetosomes (SK) linked by desmoses (Ds) and sometimes interposed by vacuoles (V) just beneath the cell surface. A = kinetosomal arms. Scale bar = 2.5 μm. (D) Longitudinal section of kinetosomes, to show detailed fine structures. The axosome (Ax) is embedded in the proximal margin of transitional discs (TD), with curving arms (A) extending out and up. H = transitional helix, Mt = microtubules, SK = somatic kineties, PF = pellicular folds, FP = flagellar pit. Scale bar = 5 μm.

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).

thumbnail Figure 4.

Transmission electron microscope images of Cepedea longa, to show fine structures of the nuclei and mitochondria within the endoplasm. (A) Cross section observed at low magnification, to show numerous thin bundles of microfilaments (arrow) dispersed in the endoplasm between nuclei (N) and mitochondria (M). SK = somatic kinetosomes. Scale bar = 20 μm. (B)–(D) Cross section of the nuclei (N), to show the nuclear envelope (NE) covered by a thick layer of microfibrils (arrowhead) and some unknown microtubular structures (arrow) in the nucleoplasm. NL = nucleolus. Scale bar in B = 10 μm, in C and D = 5 μm. (E) Thin section shows mitochondria having tubular cristae at periphery with an amorphic appearance. Scale bar = 5 μm.

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 μm × 46.9 μm) bears the most resemblance to Bezzenberger’s type specimens (680.0 μm × 52.0 μm) [3], and is smaller than that recorded by Metcalf (1000.0 μm × 75.0 μm) [31] and Nie (1162.0 μm × 42.5 μm) [40]. The longest specimen of C. longa recorded by Nie even reaches 1820 μm 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, 50–52]. 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.

Acknowledgments

Financial support for this study was provided by the National Natural Science Foundation of China (No. 31471978), the earmarked fund for China Agriculture Research System (No. CARS-46-08) and the major scientific and technological innovation project of Hubei Province (No. 2015ABA045).

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Cite this article as: Li C, Jin X, Li M, Wang G, Zou H, Li W & Wu S: Light and transmission electron microscopy of Cepedea longa (Opalinidae) from Fejervarya limnocharis. Parasite, 2017, 24, 6.

All Tables

Table 1.

The first discoveries of Cepedea spp. from anuran amphibians.

In the text
Table 2.

Biometrical data (in μm) on Cepedea longa and comparison with former reports.

In the text

All Figures

thumbnail Figure 1.

Light microscope images of Cepedea longa. (A) Overview of the living specimens, to show general form, greatly elongated and cylindrical, with the anterior extremity broader and the posterior end pointed. Scale bar = 100 μm. (B) Living specimens, to show C. longa thickly flagellated and often coils when swimming. Scale bar = 100 μm. (C)–(D) Living specimens, to show body surface twisting and giving a spiral appearance. Scale bar = 50 μm. (E) Specimens stained with ammoniacal silver, to show the falx (arrow) and somatic kineties branching off from each side. Scale bar = 25 μm. (F) Specimens stained with silver nitrate, to show somatic kineties follow a sigmoid course from anterior to posterior end of the cell. Scale bar = 25 μm. (G) Specimens stained with ammoniacal silver, to show the organism possessing a large amount of spherical or ellipsoidal nuclei (arrow). Scale bar = 25 μm.
In the text
thumbnail Figure 2.

Transmission electron microscope images of Cepedea longa, to show fine structures of the somatic cortex. (A) Section tangent to cell surface, to show pellicular folds (PF) supported by ribbons of microtubules (Mt). Some coated vesicles are fused with the plasma membrane and seen as invaginations (arrow). SK = somatic kinetosomes. Scale bar = 5 μm. (B) Section passing parallel to cell surface, to show pellicular folds (PF) interposing between somatic kineties (SK). FP = flagellar pit. Scale bar = 20 μm. (C)–(D) Selected enlargement of Figure 2A, to show a developed fibrillar skeletal system in the somatic cortex. Longitudinal microfibrils (LF) border the somatic kineties (SK) joined to each other by desmoses (Ds) on the left side, with transversal fibrils (TF) running perpendicular to kinetal long axes and framing the ribs of the cortical vesicles: globular endocytotic vesicles (EdV) and elongated exocytotic vesicles (ExV). Scale bar = 10 μm.
In the text
thumbnail Figure 3.

Transmission electron microscope images of Cepedea longa, to show fine structures of the somatic flagella. (A) Tangential section of a somatic kinety, to show fibrillar elements (arrow) between cortical microtubules (Mt) and around the membrane of each flagellar pit (FP). PF = pellicular folds. Scale bar = 2.5 μm. (B)–(C) Cross section through several kineties, to show somatic kinetosomes (SK) linked by desmoses (Ds) and sometimes interposed by vacuoles (V) just beneath the cell surface. A = kinetosomal arms. Scale bar = 2.5 μm. (D) Longitudinal section of kinetosomes, to show detailed fine structures. The axosome (Ax) is embedded in the proximal margin of transitional discs (TD), with curving arms (A) extending out and up. H = transitional helix, Mt = microtubules, SK = somatic kineties, PF = pellicular folds, FP = flagellar pit. Scale bar = 5 μm.
In the text
thumbnail Figure 4.

Transmission electron microscope images of Cepedea longa, to show fine structures of the nuclei and mitochondria within the endoplasm. (A) Cross section observed at low magnification, to show numerous thin bundles of microfilaments (arrow) dispersed in the endoplasm between nuclei (N) and mitochondria (M). SK = somatic kinetosomes. Scale bar = 20 μm. (B)–(D) Cross section of the nuclei (N), to show the nuclear envelope (NE) covered by a thick layer of microfibrils (arrowhead) and some unknown microtubular structures (arrow) in the nucleoplasm. NL = nucleolus. Scale bar in B = 10 μm, in C and D = 5 μm. (E) Thin section shows mitochondria having tubular cristae at periphery with an amorphic appearance. Scale bar = 5 μm.
In the text

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