Parasitology International 63 (2014) 489–491

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Parasitology International

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Supplemental description of Thelohanellus wuhanensis Xiao & Chen, 1993 (Myxozoa: ) infecting the skin of Carassius auratus gibelio (Bloch): Ultrastructural and histological data

Yang Liu a,b,c, Junfa Yuan a,b,c,LuoJiaa,b,c, Mingjun Huang a,b,c, Zhigang Zhou d,ZemaoGua,b,c,⁎ a Department of Aquatic Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, People's Republic of China b Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, People's Republic of China c Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, People's Republic of China d Key Lab for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China article info abstract

Article history: Thelohanellus wuhanensis Xiao & Chen, 1993 infects allogynogenetic gibel carp Carassius auratus gibelio (Bloch) Received 3 July 2013 forming numerous plasmodia in the skin, resulting in high mortality of juvenile host fish. Within this paper we Received in revised form 7 January 2014 supplement the identification of this pathogenic with information on its ultrastructural and histological Accepted 13 January 2014 characteristics. Measurements of the spores corresponded to the original description, but asymmetry in the Available online 25 January 2014 spore shape was recorded. Scanning electron microscopy revealed that the spore surface was smooth and the sutural ridge of the anterior end was asymmetric as one side was thinner. In addition, the sutural line was either Keywords: “ ” Thelohanellus wuhanensis straight or S like. Transmission electron microscopy showed that most spores were surrounded by a Carassius auratus gibelio membranous sheath, while some sheaths were difficult to observe under light microscopy as the closeness of Redescription the sheath to the valves. The small electron-dense granules in the sporoplasm ranged from 0.66 to 1.14 μmin Ultrastructure diameter, but their function was unknown. Histology showed that the plasmodia of T. wuhanensis developed Histology in the stratum spongiosum of skin dermis. The presence of melanocytes around the plasmodia was responsible SSU rDNA for the black spots on T. wuhanensis cyst. The plasmodia were curved, perhaps to expand the surface area in contact with adjacent host tissue, facilitating the attainment of nutrients from the host. Crown Copyright © 2014 Published by Elsevier Ireland Ltd. All rights reserved.

Myxosporeans are an economically important group of microscopic microscopy and diagrammatic drawings [9]. However, spore morphology metazoan endoparasites with 2180 reported species infecting both cul- alone is insufficient to determine the validity of morphologically similar tured and wild fish [1]. The genus Thelohanellus Kudo, 1993 represents a myxosporeans. Here, we describe this species in detail and supplement small group of 75 nominal species, with at least 10 species described its ultrastructural and histological characteristics, which will aid our since 2006 [2,3], characterized by a tear-shaped, pyriform to broadly el- understanding of those features that may contribute to species lipsoidal spore and only one polar capsule discharging apically and axi- identification. ally[1]. Though most of the species do not cause severe disease, several In June 2010, 54 juvenile allogynogenetic gibel carp ranging from 6.4 Thelohanellus species have been reported as pathogens: Thelohanellus to 12.1 cm in length and from 9.0 to 18.0 g in weight were harvested hovorkai causes hemorrhagic thelohanellosis in Cyprinus carpio L. of up using a fine-mesh seine from a pond in Honghu City, Hubei Province, to two years of age [4]; Thelohanellus kitauei forms giant plasmodia in China. were then transported to the laboratory for myxosporean C. carpio intestine [5]; Thelohanellus nikolskii (syn. Thelohanellus cyprini) examination according to standard procedures [10].Plasmodia(Fig. 1) produces plasmodia up to 2 mm in the fins of C. carpio fingerlings [6]; containing spores morphologically consistent with those of the Thelohanellus zahrahae infects the gill of Barbonymus gonionotus (Bleeker) myxozoan genus Thelohanellus were found in the skin of 50 out of 54 [2]. Recently, an occurrence of thelohanellosis caused by T. wuhanensis allogynogenetic gibel carp. These plasmodia were whitish with black was reported in juvenile allogynogenetic gibel carp Carassius auratus spots, round or ellipsoidal measuring 2.7–6.3 mm in diameter. The gibelio (Bloch), an important commercial fish in China [7,8]. This parasite spore body (Fig. 2) was elongated elliptically in frontal view and forms numerous plasmodia in the skin and results in high mortality of ju- lemon-shaped in lateral view, measuring 22.9 ± 0.6 (21.8–24.0) μmin venile fish [7]. A previous study described T. wuhanensis using light length, 13.3 ± 0.5 (12.2–14.3) μm in width, and 10.6 ± 0.5 (9.9–11.6) μm in thickness. A single round polar capsule with an apophysis on its top end was close to the spore apex, measuring 10.8 ± 0.6 (9.6–11.9) ⁎ Corresponding author at: Department of Aquatic Animal Medicine, College of μm in length, 8.6 ± 0.5 (7.5–9.7) μm in width and contained coiled Fisheries, Huazhong Agricultural University, Wuhan 430070, People's Republic of China. fi Tel./fax: +86 27 87282114. polar laments with 8 to 10 turns. A small proportion of spores were E-mail address: [email protected] (Z. Gu). surrounded by a membranous sheath and numerous granules were

1383-5769/$ – see front matter. Crown Copyright © 2014 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2014.01.003 490 Y. Liu et al. / Parasitology International 63 (2014) 489–491

Fig. 1. Allogynogenetic gibel carp Carassius auratus gibelio infected with Thelohanellus wuhanensis in the skin. Arrows show the plasmodia under the scales. The inset shows the plasmodium marked by white arrow over the skin in a region without scales. Scale bar: 2 cm.

Fig. 4. T. wuhanensis spores observed by transmission electron microscopy. (a) Spores surrounded by the membranous sheath (arrow). Scale bar: 5 μm; (b) transverse section Fig. 2. Photomicrograph of fresh spores of T. wuhanensis. The big black arrow and the big of a spore showing the membranous sheath (arrow) around the spore valve and the whitearrowshowthemembranoussheathandthepitontheanteriorendofspore,respec- electron-dense pellets (asterisks) in the sporoplasm. Scale bar: 2 μm; (c) Higher tively. The small black arrows show the small granules in the sporoplasm. Scale bar: 20 μm. magnification of the membranous sheath (arrows). Scale bar: 0.5 μm.

present in the sporoplasm (Fig. 2). The morphology and morphometry of spores corresponded to the original description of T. wuhanensis [9], but the size of plasmodia in the present study differed from the original description (0.200–0.250 mm). This difference may be attributed to host size or strain. Myxosporean genomic DNA was extracted from a cyst fixed in 100% ethanol [11,12] and the SSU rDNA sequence was amplified using the primers MX5/MX3 [13]. The PCR products were separated using a 1.0% agarose gel and sequenced directly with an ABI PRISM® 3730XL DNA sequencer (Applied Biosystems Inc.). The 1525 base pair SSU rDNA se- quence was generated and deposited in GenBank (JQ968687). A BLAST search indicated that this sequence was identical to an existing GenBank entry for T. wuhanensis (JQ088179, HQ613410). Both the mor- phological and molecular data suggested that the species in the present study was T. wuhanensis. We examined spores with scanning electron microscopy (SEM) to further define ultrastructure. Fresh spores were fixed in a solution

of 3% glutaraldehyde, dehydrated with CO2 using the critical point method and sputter coated with gold. Samples were then examined with a JSM-6390 scanning electron microscope at 20 kV with a work- ing distance of 18 mm. The spore surface was smooth (Fig. 3a) and the sutural line was straight or “S” like (Fig. 3b, c), similar to Thelohanellus testudineus from the skin of allogynogenetic gibel carp [3]. SEM revealed that T. wuhanensis spores were lemon- shaped in lateral view (Fig. 3b).ThisisincontrasttoT. testudineus, Fig. 3. T. wuhanensis spores observed by scanning electron microscopy. (a) A spore in which were testudinate in lateral view with a flat and convex side frontal view. Scale bar: 10 μm; (b) A spore in lateral view with straight sutural line [3]. Interestingly, the sutural ridge of the anterior end of one side (arrow). Scale bar: 5 μm; (c) A spore in lateral view with “S” like sutural line (arrow). was much thinner in some T. wuhanensis spores (Fig. 3d, e), resulting Scale bar: 5 μm; (d) A spore in frontal view, showing the thinner sutural ridge (arrow). fi Scale bar: 10 μm; (e) Higher magnification of (d) showing the thinner sutural ridge in an asymmetric anterior end (Fig. 2)notidenti ed for other (big arrow) and the discharge pores of polar filaments (small arrows). Scale bar: 1 μm. myxosporean species [1]. Y. Liu et al. / Parasitology International 63 (2014) 489–491 491

were recorded, no severe inflammatory response was found, which was in accordance with other myxosporean infections [15,16]. In addition, we determined that the black spots observed in the skin around the plasmodia (Fig. 5b) on the cyst of T. wuhanensis were host melanocytes. We also observed that the membrane of the plasmodia curved to expand the surface area in contact with the adjacent host connective tissue (Fig. 5c), which might facilitate the nutrient acquisition from the host.

Acknowledgments

The authors would like to thank Dr. Jerri Bartholomew and Hurst Charlene from Oregon State University for critically reading the manu- script. This study was supported by the Nature Science Foundation of China (31172052), New Century Excellent Talents in University (NCET- 12-0866), the Fundamental Research Funds for the Central Universities (2011PY011, 2012PY004, 2012YB10, 2013PY023, 2013PY070), Science Fund for Distinguished Young Scholars of Hubei Province (2011CDA091).

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