Redescription of Protoopalina pingi Nie, 1935 inhabiting the recta of Hylarana guentheri and Pelophylax nigromaculatus in China

A redescription of Protoopalina pingi Nie, 1935 is presented in this paper to complete Nie’s description at both light and scanning electron microscope levels. These organisms were collected from the recta of the frogs Hylarana guentheri Boulenger, 1882 and Pelophylax nigromaculatus Hallowell, 1861 from Jialing River, Sichuan Province and Honghu Lake, Hubei Province, respectively, in China. This is the first record of its occurrence in H. guentheri and P. nigromaculatus. The body of P. pingi is elongated and somewhat spindle-like in shape, slightly narrowed and bluntly rounded at the anterior extremity, while the posterior end is tapering or sharply pointed. The body surface is thickly flagellated, with the caudal tip being barren. The falx, located at the margin of the anterior end, is composed of a narrow band of kinetosomes. Four round or oval-shaped nuclei, usually arranged in a straight line, are situated in the middle region of the body. Comparisons are made between P. pingi and its congeners.


Introduction
Opalinids, originally discovered by Leeuwenhoek in 1683 [7], are multinuclear, mouthless, osmotrophic flagellated protozoa. They live as commensals in the digestive tracts of different poikilothermic vertebrates, especially anuran amphibians [15]. The opalinids were for a long time regarded as the astomatous (no cytostome) ciliates because of their superficial similarities with the ciliates and were given the status ''protociliates'' as opposed to ''euciliates'' since the monomorphic nuclei, in contrast to dimorphic nuclei, were suggested to be an ancestral state of ciliates [1,13,14,23]. Then the hypothesis of opalinidciliate affinity was abandoned since other characteristics, such as the structure of the nucleus, the mode of cell division and the reproductive cycle, differed remarkably from those of ciliates and these organisms were deemed to be either an isolated taxon in the phylum Zooflagellata or were treated as a separate phylum: Opalinata [3,4,8,24]. Now, it has been convincingly shown that opalinids belong to heterokonts as a sister group to Proteromonas within the order Slopalinida based on detailed ultrastructural study and believable phylogenetic analyses [2,6,10,11,16,17,19,20,22] The family Opalinidae can be separated into two subfamilies, Protoopalininae and Opalininae, based on the shape of the cell body and the number of nuclei. The subfamily Opalininae is comprised of the genera Cepedea Metcalf, 1920 and Opalina Duskinje and Valentin, 1835, while the subfamily Protoopalininae contains the genera Protoopalina Metcalf, 1918 andZelleriella Metcalf, 1920.
Protoopalina is the most common genus of opalinids inhabiting anuran amphibians [5,21]. It was established by Metcalf in 1918. Thereafter, many new species of Protoopalina have been found from the anuran amphibians. Protoopalina pingi was first discovered and named by Nie in 1935 from the intestines of Rana plancyi Lataste, 1880 [18]. Although discovered more than 70 years ago, many biological aspects of P. pingi are still unknown. After simple morphological information, no further data about this opalinid have been reported. The previous morphological data, however, are incomplete, and some descriptions of important taxonomic structures also need revision. This study adds to Nie's description and attempts to contribute to the knowledge of this genus.

Materials and methods
Host frogs, including 256 H. guentheri and 104 P. nigromaculatus, were captured from Jialing River in Pengan county (31°15 0 -31°29 0 N; 106°12 0 -106°25 0 E), Sichuan Province, China, in August 2011 and Honghu Lake (29°40 0 -29°58 0 N; 113°12 0 -113°26 0 E), Hubei Province, China, in June 2012, respectively. They were transported alive to the laboratory for further examination. We obtained the permits allowing us to capture and sacrifice these specimens. All frog samples were dissected, with the intestines and recta being opened and put into Petri dishes for examination. Then a 0.65% saline solution was added to the samples and we waited for a few minutes to allow P. pingi to swim out of the gut contents. The flagellates were collected with a Pasteur micropipette and washed twice in distilled water.
For light microscopy, individuals were observed, measured and photographed in vivo using both bright-field and differential interference contrast microscopy (Zeiss Axioplan 2 imaging and Axiophot 2, Oberkochen, Germany). The remaining specimens  were placed directly on coverslips, fixed in a saturated HgCl 2 solution and stained with Heidenhain's haematoxylin and a 1% Protargol solution. All measurements are in micrometres.
For scanning electron microscopy (SEM), the washed specimens were fixed in 2.5% glutaraldehyde in 0.2M phosphate buffered saline (PBS, pH 7.4) on a clean glass slide (1 cm · 1 cm), previously treated with 0.1% poly-L-Lysin and dried completely in air at room temperature (RT). After being washed with PBS three times, they were post-fixed in 1% osmium tetroxide at 4°C for 1 h, followed by serial dehydration in acetone and critical point drying using a HCP-2 critical point dryer (Hitachi Science Systems, Ibaraki, Japan). Then the glass slide was mounted on an aluminium stub using double-sided adhesive tape and sputter-coated with a thin layer of gold in an IB-3 ion coater (Eiko Engineering, Ibaraki, Japan) before observing and photographing with a Quanta 200 SEM (FEI, Amsterdam, Netherlands).

Results
One hundred and thirty-five of the 256 H. guentheri examined and 42 of the 104 P. nigromaculatus examined were found to be infected with P. pingi. Large numbers of P. pingi were found in the recta of all frog hosts that contained them.  round or oval-shaped nuclei are situated in the middle region of the body, usually with many nucleoli distributed within the karyoplasm (Figs. 2C, F and 3). Normally, the nuclei are arranged in a straight line running parallel to the longitudinal axis (Figs. 2C, F, and 3). The nuclei range in length from an average of 10.8 lm (7.0-15.0 lm, n = 15) and in width 8.0 lm (5.0-11.5 lm, n = 15) in Protargol specimens. Many apparent corpuscles of uneven size can be observed over the cytoplasm (Fig. 2F).
Data for measurements related to morphometric characteristics are given in Table 1.

Discussion
As mentioned above, P. pingi was first discovered and named by Nie from the intestines of Rana plancyi [18]. This is the first record of its occurrence in the recta of H. guentheri and P. nigromaculatus. The opalinids examined in the present study appear slightly bigger than Nie's type specimens, since he gave ranges of 55-160 lm by 12.5-57 lm in length and width. Also, the caudal tip of P. pingi is barren of flagella according to our SEM observation, which is different from that described by Nie [18]. He stated in his paper that ''the cilia covering the entire surface of the body are of moderate size and closely arranged in many oblique or longitudinal rows''. He was likely limited in his views of these flagellates due to the limits of staining techniques and observing equipment in his time. Due to the absence of other morphological data, it is impossible to compare our results with Nie's records.
In conclusion, based on general morphological characteristics, P. pingi is recorded and redescribed in detail from H. guentheri and P. nigromaculatus. Future collections will be made at different stages of the hosts' life cycles to determine if the trophonts always have four nuclei instead of the two usually found in Protoopalina, to determine if cysts are formed, to study its possible ''infection'' routes and further assess the host specificity.
Acknowledgements. Financial support for this study was provided by the National Natural Science Foundation of China (Grant Measurements in lm; Min = minimum, Max = maximum, Mean = arithmetic mean, SD = standard deviation, CV = coefficient of variation, N = number of individuals investigated.