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Ceratomyxa oplegnathus n. sp. from the gallbladder of cultured rock bream, Oplegnathus fasciatus
Jae Bum Cho, Eun Hye Lee, Se Ryun Kwon, Chun Soo Kim and Ki Hong Kim Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea
A new myxosporean species Ceratomyxa oplegnathus n. sp. was found from the gallbladder of cultured rock bream, Oplegnathus fasciatus. Mature spores were 44.14 2.41 (39.04-48.41) in length, 8.52 0.36 (7.9-9.26) in width in sutural view. Spores contained two spherical polar capsules measuring 3.04
0.2 (2.71-3.37) in diameter and a binucleated sporoplasm. Each polar capsule had a 5-6 coiled polar filament, and was situated near the sutural line. In ultrastructural observation, asynchronous divisions of generative cells without pansporoblast formation gave arise to 2 spores within a trophozoite. Sporoplasmic cell in the sporoblast was binucleated, and capsulogenic cells had large amounts of rough endoplasmic retic- ulum, external tubules, and capsular primordia.
Key words: Ceartomyxa oplegnathus n. sp., Myxosporea, Rock bream (Oplegnathus fasciatus), Gall blad- der
Introduction
More than 140 species of myxosporean parasites belonging to the genus Ceratomyxa have been described (Lom and Dyková, 1992), and most of them were coelozoic parasites of marine fish. In the present study, we found a new species Ceratomyxa oplegnathus n. sp. from the gallbladder of cultured rock bream (Oplegnathus fasciatus), a commercial- ly valuable marine fish in South Korea, and described morphological characteristics of develop- mental stages and spores using light- (LM) and transmission electron-microscopes (TEM).
Materials and Methods
A total of 15 rock bream, Oplegnathus fasciatus (15-20cm in body length) were obtained from a commercial fish farm at southern coast of South
Korea. Fish were killed by overexposure to MS-222 (Sigma Co.), excised all organs, and examined to determine the presence of parasites. As a result, trophozoites and spores of myxosporeans were found in the gallbladder.
Bile juice from gallbladder was collected with 1 syringe and deposited into an 1 effendorf tube. Tubes were centrifuged at 1500 rpm at room temperature. The resulted pellet was observed in a fresh material or fixed in 2% glutaraldehyde in phosphate buffer (pH 7.0, 4 ) for transmission electron microscopic study. Mature spores were described and measured by using an ocular micrometer and image analysis software (Image- Tool ver 3.0, UTHSCSA, USA) according to Lom and Arthur’s criteria (1989). Line drawings of spores were sketched with camera lucida. Mean and standard deviations of each spore characteristics were obtained from 80 fresh spores.
Corresponding Author : Ki Hong Kim, Tel : 051-620-6145, Fax : 051-628-7430, E-mail : [email protected]
19 2 (2006) J. Fish Pathol., 19(2) 109 ~ 117 (2006)
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For TEM study, the fixed pellet from gall bladder were plating in agar and then postfixed in same buffered OsO4, dehydrated and imbedded in resin.
Ultrathin-sectioned specimens were double-stained with uranyl acetate and lead citrate, and examined with a JEOL JEM1200 transmission electron microscope (JEOL LTD., Japan).
Results
Ceratomyxa oplegnathus n. sp.
Mature spores (Fig. 1-3) were elongate with flat- tened anterior and posterior ends, measured 44.14 2.41 (39.04-48.41) in length, 8.52 0.36 (7.9-9.26) in width in sutural view. Two valves were subequal and mostly straighten, and the tips of valves were little swollen. Two equal polar capsules
were situated near the sutural line, and almost spherical, 3.04 0.2 (2.71-3.37) in diameter and contained a 5-6 coiled polar filament (Fig. 14).
A sporoplasm with 2 nuclei was distributed in spore cavity (Fig. 15).
Trophozoites had numerous mitochondria, cyto- plasmic vesicles and endoplasmic reticulum in their cytoplasm (Fig. 4, 8). The earliest stage observed was a uninucleate primary cell (Fig. 4). As the cell developed, several secondary cells containing one or two tertiary cell(s) appeared within the cyto- plasm of the primary cells (Fig. 5, 6, 7, 8).
Generative cells (secondary and tertiary cell) gave arise to two sporoblasts in the trophozoite (Fig. 10). Each sporoblast was differentiated into compartmentalized constituents of a mature spore.
Two flattened valvogenic cells completely sur- rounded a binucleated sporoplasmic cell and two capsulogenic cells (Fig. 9, 10, 11), and were joined by junction (Fig. 9). Cytoplasm of capsulogenic cell contained lots of endoplasmic reticulum, mitochon- dria and numerous vacuoles (Fig. 9, 10, 11). In progress of maturation, the cytoplasm contained globular or long external tubule and capsular pri- mordia (Fig. 9, 10, 11).
In mature spores, valves tightly bound each other
Jae Bum Cho, Eun Hye Lee, Se Ryun Kwon, Chun Soo Kim and Ki Hong Kim
Fig. 1. Drawing of spore of Ceratomyxa oplegnathus from gallbladder of Oplegnathus fasciatus . Scale bar 20 .
Fig. 2-3. Mature spores of C. oplegnathus from gallbladder of Oplegnathus fasciatus. (arrow- suture line). Scale bars 10
111 Ceratomyxa oplegnathus n. sp. in rock bream
Fig. 4-7. Electron-micrographs of presporogonic stages of Ceratomyxa oplegnathus 4- earliest stage, primary cell (P) with a nucleus (n) and cytoplasmic vesicles (arrow). Mi- mitochondria; 5- three secondary cells (Sc) within the primary cell. Two closely associated cells contained an inner tertiary cells (Tc) each; 6- four secondary cells within the primary cell. Some sec- ondary cells contained one or two inner tertiary cells in the cytoplasm; 7- secondary cell harboring two tertiary cells. nii- nucleus of secondary cell. Scale bars 600 nm (4, 7); 1 (5,6)
Jae Bum Cho, Eun Hye Lee, Se Ryun Kwon, Chun Soo Kim and Ki Hong Kim 112
Fig. 8-11. Electron-micrographs of sporogonic stages of Ceratomyxa oplegnathus 8- five secondary cells (Sc) within prima- ry cell or plasmodium (P). n- vegetative nucleus, nii- nucleus of secondary cell, Tc - tertiary cell; 9- early sporoblast. Two valvogenic cells (Vc) enveloped a capsulogenic cell (Cc) with globular external tubule (Et). Each valvogenic cell joined together with junction (arrow). Er- endoplasmic reticulum; 10- two sporoblasts within plasmodium. Note the long or globu- lar external tubule within capsulogenic cell; 11- slightly matured sporoblast within primary cell. Valvogenic cells surround two capsulogenic cells and a binucleated sporoplasmic cell (S). nsi, nsii- nucleus of sporoplasmic cell. Each capsulogenic cells contained capsular primordia (Cp), external tubule and numerous rough endoplasmic reticulum. Scale bars 1 .
Ceratomyxa oplegnathus n. sp. in rock bream 113
Fig. 12-15. Electron-micrographs of mature spore of Ceratomyxa oplegnathus 12- longitudinal section of mature spore. Note dark lipid inclusion body (L) near polar capsule (Pc). Discharging channel (arrowhead) opened at the apical end of polar cap- sule. arrow - valvular suture; 13- magnification of valvular suture. Note tight junction (arrow) present between neighboring each valves (V); 14- polar capsule containing coiled polar filaments (Pf). Electron-dense zone (arrows) faced the apical end of polar capsule. Capsulogenic cells (Cc) still remained in fully matured spore; 15- binucleate sporoplasm containing numer- ous inclusion bodies (i), mitochondria (Mi) and membrane bound sporoplasmosome (Spo). arrow- valvular suture, nsi(nsii) - nucleus of sporoplasm; Scale bars 1 (12); 200 nm(13); 400 nm(14); 600 nm(15).
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by junction (Fig. 13). At the apical end of polar cap- sule, electron dense zone was seen in front of the discharging channel (Fig. 12). A sporoplasm con- tained 2 nuclei, sporoplasmosome and numerous various-sized inclusion bodies (Fig. 15). Electron- dense, osimiophilc granules near capsules were appeared in longitudinal section of mature spores (see “L” in Fig. 12).
Host: Rock bream, Oplegnathus fasciatus
Locality: Nam-Hae, Kyongsangnam-Do, South Korea.
Site of infection: Lumen and wall of the gallblad- der
Etymology: The species named after its host species name.
Materials deposited: 90% alcohol-fixed spores;
H&E stained histological sections. Laboratory of Fish and Shellfish Parasitology, Department of Aquatic Life Medicine, Pukyong National Univer- sity, South Korea. Accession number PKNU- ofpmy2-2003.
Discussion
Fish myxosporeans show their host, organ and tissue specificity, and these characteristics are avail- able in their taxonomy (Molnár, 1994). Although the present specimen was similar range in spore size with other 5 Ceratomyxa species - C. aggregata, C.
lunata, C. mesospora, C. orientalis and C. spectab- lis (Table 1), it was easily discriminated from them by different spore shapes. Spore of C. aggregata is strongly compressed parallel to longitudinal plane (Davis, 1917). C. lunata is distinctively curved toward postcapsular side (Davis, 1917). C.
mesospora have cone at the apex of spore (Davis, 1917). C. orientalis tapers to sharply pointed end (Shulman, 1988). C. spectabilis have highly
recurved horns at mid regions of spore and discov- ered from urinary bladder of the host fish (Shulman, 1988). Based on the differences in spore morpholo- gy, host specificity and geographical distribution, the present specimen was considered as a new species and named C. oplegnathus after its host name.
Fine structures of the trophozoites resembled those of other coelozoic myxosporeans from host gall bladder, such as Myxidium gadi (Feist, 1995), Ceratomyxa protopsettae (Cho et al., 2004) and Zschokkella mugilis (Sitjà-Bobadilla and Alvarez- Pellitero, 1993a). Numerous mitochondria, inclu- sion bodies and vesicles in peripheral layer are probably related with the pinocytosis as a mode of nutrition in the parasite (Lom and Dyková, 1996;
Uspenskaya, 1982).
Sitjà-Bobadilla et al. (1995) reported no pans- poroblast formation in the ultrastructure of Cerato- myxa sparusaurati. Their suggestion is evidenced by lack of a common membrane encircling two sporoblast and several secondary cells (possibly undifferentiated sporogonic cells) surrounded by a common membrane. It was also supported by the present C. oplegnathus that enveloping cell (peri- cyte) was not observed even at ultrastructural level.
Asynchronous sporogenesis and disporous spore formation without pansporoblast formation had been reported in other disporous myxosporeans including Ceratomyxa (Desportes and Théodoridès, 1982; Kalavati and MacKenzie, 1999; Yokoyama and Fukuda, 2001; Cho et al., 2004), Leptotheca (Desportes and Théodoridès, 1982; Tun et al., 2000) and Sphaerospora (Desser et al., 1983;
Dyková et al., 1990; Lom et al., 1982, 1985; Sitjà- Bobadilla and Alvarez-Pellitero, 1992, 1993b).
These patterns are common in the myxosporeans forming small disporic pseudoplasmodia (Lom and Dyková, 1992).
Jae Bum Cho, Eun Hye Lee, Se Ryun Kwon, Chun Soo Kim and Ki Hong Kim
Acknowledgements
This work was supported by the Brain Korea 21 Project in 2003, Republic of Korea.
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115 Ceratomyxa oplegnathus n. sp. in rock bream
Table 1. Comparison of spore characteristics of Ceratomyxa oplegnathus n. sp. with other closely related Ceratomyxa species
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Manuscript Received : July 12, 2006 Revision Accepted : August 1, 2006 Responsible Editorial Member : Myung-Joo Oh (Chonnam Univ.)
Ceratomyxa oplegnathus n. sp. in rock bream 117
