Zhen Xu 1,2, Lu-Ping Zhang 1 and Liang Li 1,*

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1 DOI: /s W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(4), ; ISSN Morphological and molecular characterization of Cucullanus hainanensis sp. nov. (Ascaridida: Cucullanidae) from Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea Zhen Xu 1,2, Lu-Ping Zhang 1 and Liang Li 1,* 1 Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei Province, P. R. China; 2 Medical College of Hebei University of Engineering, Handan, Hebei Province, P. R. China Abstract Cucullanus hainanensis sp. nov., collected from Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea, was described using both light and scanning electron microscopy. The new species can be readily distinguished from its congeners by the large pseudobuccal capsule, the position of excretory pore and deirids, the length of spicules ( mm, % of body length) and gubernaculum ( mm), the number and arrangement of caudal papillae and the particular morphology of cloacal region in male. The new species was also characterized using molecular methods by sequencing and analysing the small subunit (18S) and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rdna). In addition, Cucullanus muraenesocis (Yin et Zhang, 1983) was regarded a homonym of C. muraenesocis Yamaguti, 1961, and a new name, Cucullanus wangi nom. nov. was given to it. Keywords Ascaridida, morphology, integrated taxonomy, Cucullanus, marine fishes, new species Introduction The genus Cucullanus Müller, 1777 is the largest group in Cucullanidae, currently including over 100 species parasitizing various freshwater, brackish and marine fishes or aquatic turtles (Petter 1974; Hasegawa et al. 1991; Moravec et al. 2005; Timi and Lanfranchi 2006). However, our present knowledge of Cucullanus nematodes in China is still fragmentary. As far as we are aware, only 10 species have been reported in China until now, C. anguillae Wang et Lin, 1975 from Anguilla japonica Temminck et Schlegel, C. cirratus Müller, 1777 from Pisodonophis cancrivorus (Richardson), C. muraenesocis (Yin et Zhang, 1983) from Muraenesox cinereus (Forsskål), C. heterochrous Rudolphi, 1802 from Psettodes erumei (Bloch & Schneider), C. hapalogengus (Luo, 2001) from Hapalogenys mucronatus (Eydoux et Souleyet) (now as Hapalogenys analis Richardson), C. decapteri Parukhin, 1966 from Decapterus sp., C. amadai Yamaguti, 1941 from tjanus erythopterus Bloch, C. spirocaudus Li, 1984 from Chelon haematocheilus (Temminck et Schlegel) [now as Liza haematocheila (Temminck & Schlegel)], C. carettas Baylis, 1923 from Pelochelys bibroni and C. fujianensis Wang, 1984 from Trionys sinensis (Parukhin 1966, 1967; Wang and Lin 1975; Yin and Zhang 1982, 1983; Wang 1984; Li 1984; Luo 2001). During a helminthological survey of the composition of the ascarididan nematode fauna of fishes in the Chinese waters, several cucullanid nematodes were collected from the intestine of Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea. Their examination using light and scanning electron microscopy revealed that these nematodes represented a hitherto unknown species of Cucullanus. Moreover, a specimen was also characterized using molecular methods by sequencing and analysing the small subunit (18S) and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rdna) in order to estimate the validity of the new species genetically. *Corresponding author: liangliangex369@126.com

2 Cucullanus hainanensis sp. nov. from China 711 Materials and Methods Light and scanning electron microscopy Fishes caught by commercial trawlers from the South China Sea (off Sanya, Hainan Island) were examined for parasites. Nematodes recovered from the intestine of Muraenichthys gymnopterus (Bleeker) were washed in physiological saline and then fixed and stored in 80% ethanol until studied. Light and scanning electron microscopical studies were prepared following the methods used by Li et al. (2013). Drawings were made with the aid of Nikon microscope drawing attachment. Measurements are given in micrometres unless otherwise stated. Voucher specimens are deposited in College of Life Science, Hebei Normal University, Hebei Province, China. Molecular procedures One female nematode was selected for the molecular analysis. Genomic DNA from this individual worm was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer s instructions. DNA was eluted in elution buffer and kept at 20 C until use. The partial 18S rdna was amplified by PCR using the primers 18SF (forward: 5 -CGCGAATRGCTCATTACAACAGC-3 ) and 18SR (reverse: 5 -GGGCGGTATCTGATCGCC-3 ) (Floyd et al. 2005). The ITS-2 region was amplified by PCR using the primers XZ1 (forward: 5 - ATTGCGCCATCGGGTTC ATTCC-3 ) and NC2 (reverse: 5 -TTAGTTTCTTTTCCTC- CGCT-3 ) (Zhu et al. 2002). The cycling conditions were described previously (Li et al. 2014). PCR products were checked on GoldView-stained 1.5% agarose gel and purified by the Column PCR Product Purification Kit (Shanghai Sangon, China). Sequencing was carried out using a DyeDeoxy Terminator Cycle Sequencing Kit (v.2, Applied Biosystems, California, USA) and an automated sequencer (ABI-PRISM 377). Sequencing for each sample was carried out for both strands. Sequences were aligned using ClustalW2 (Thompson et al. 1994) and adjusted manually. The sequences determined were compared (using the algorithm BLASTn) with those available in the National Center for Biotechnology Information (NCBI) database ( nih.gov). Results Cucullanus hainanensis sp. nov. (Figs. 1 3) General: Small-sized, whitish nematodes. Body elongate, cylindrical, widest at short distance posterior to oesophagus, tapering towards tail. Cuticle with fine transverse striations. Lateral alae absent. Oral aperture dorsoventrally elongate, slitlike, surrounded by distinct collarette, bearing row of small denticles on inner surface; 2 pairs of large, submedian cephalic papillae and 1 pair of small, lateral amphids present (Figs. 1A,B, 2A,C, 3C). Inner labial papillae not observed. Oesophagus muscular, remarkablely expanded at anterior end to form rather large pseudobuccal capsule (oesophastome); posterior part of oesophagus also expanded, slightly narrower than pseudobuccal capsule (Fig. 1A,B). Oesophagus opens posteriorly into intestine through valvular apparatus (Fig. 1A). Nerve-ring at about 40% of oesophageal length (Fig. 1A). Deirids situated laterally, hooked, at nearly middle of oesophagus (Fig. 2A,B). Post-deirids not observed. Excretory pore anterior to oesophago-intestinal junction (Figs. 1A, 3A,B). Intestinal caecum absent. Tail of both sexes conical, with subpointed tip (Figs. 1C E,G, 2D,E, 3E). Male (based on 2 mature specimens): Body mm long, maximum width Oesophagus long, representing % of body length, minimum width of middle part , maximum width of posterior part ; pseudobuccal capsule conspicuous, (Fig. 1A,B). Nerve-ring, excretory pore and deirids at , and from anterior extremity, respectively (Fig. 1A). Posterior end of body remarkably curved ventrally. Precloacal ventral pseudosucker well developed, distance from centre to cloaca (Fig. 1D). Ventral region of cloacal opening distinctly elevated (Figs. 1C,D,E 2D,E, 3D,E). Spicules alate, of almost equal length, , representing % of body length (Fig. 1D). Gubernaculum well sclerotized, slightly laterally expanded at about middle part, long (Fig. 1F). Caudal papillae 10 pairs in total. Precloacal papillae 3 pairs arranged as follows: first pair at level of ventral pseudosucker, second pair just posterior to ventral pseudosucker, third pair approximately mid-way between second pair and cloacal opening (Figs. 1D, 2E, 3E). Paracloacal papillae 4 pairs: first and second pairs very close to each other, subventral, slightly anterior to cloacal aperture; third pair subventral, almost at level of cloacal opening, fourth pair lateral, just posterior to cloacal opening (Figs. 1C,D,E, 2D,E, 3E). Three postcloacal pairs arranged as: first and third pair subventral, at nearly mid-length of tail and near tail tip, respectively; second pair subdorsal, situated between first and third subventral pairs (Figs. 1C,D,E, 2D,E, 3E). Single, medio-ventral precloacal papilla present on anterior cloacal lip (Fig. 1E). Phasmids lateral, very small, just anterior to first pair of postcloacal papillae (Figs. 1C,D, 2D,E, 3E). Tail long. Female (based on 2 gravid specimens): Body mm long, with maximum width Oesophagus long, wide at middle, representing % of body length; pseudobuccal capsule Nerve-ring, excretory pore and deirids , and from anterior extremity, respectively. Vulva slitlike, postequatorial, at mm from anterior extremity, % of body length (Fig. 1H). Ovijector directed anteriorly from vulva. Uteri amphidelphic. Eggs oval, thinshelled, with uncleaved contents, (n=20) (Fig. 1I). Tail long, with pair of lateral phasmids located at from tail tip (Fig. 1G).

3 712 Zhen Xu et al. Fig. 1. Cucullanus hainanensis sp. nov. from Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea. A anterior part of male, left lateral view; B cephalic end of male, right lateral view; C tail of male, left lateral view; D posterior end of male, left lateral view; E tail of male, ventral view; F Gubernaculum; G posterior end of female, left lateral view; H region of vulva; I eggs. Scale-bars: A,D,E,H = 250 µm; B,F = 120 µm; C = 150 µm; G,I = 130 µm

4 Cucullanus hainanensis sp. nov. from China 713 Taxonomic summary Type-host and type-locality: Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae); South China Sea, off Sanya, Hainan Island, China. Site of infection: Intestine. Prevalence and intensity of infection: 1 out of 2 fishes were infected with intensity 4 nematodes. Type specimens: Holotype: male (HBNU-F14012); allotype: female (HBNU-F14013); paratypes: 1 male, 1 female (HBNU-F14014). Etymology: The species name refers to the name of the type-locality, Hainan Island. Molecular characterization Partial 18S region. The length of the partial 18S sequence of Cucullanus hainanensis sp. nov. obtained herein was 884 bp. There are only four species of Cucullanus with the partial 18S sequences registered in GenBank, C. robustus (JF934726), C. baylisi (JF803935), Cucullanus sp. RAHAG-2013 (KF681520) and C. dodsworthi (HQ241923). Pairwise com- Fig. 2. Scanning electron micrographs of Cucullanus hainanensis sp. nov. from Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea, male. A anterior part of body, right lateral view (deirid arrowed); B magnified image of deirid; C cephalic extremity, apical view; D tail, left lateral view (phasmid arrowed); E posterior end of body, left lateral view (precloacal papillae and phasmid arrowed). Abbreviations: pp1-4, first to fourth pairs paracloacal papillae, ps1 3, first to third pairs postcloacal papillae

5 714 Zhen Xu et al. Fig. 3. Scanning electron micrographs of Cucullanus hainanensis sp. nov. from Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea, male. A anterior part of body, ventral view (excretory pore arrowed); B magnified image of excretory pore; C cephalic extremity, dorsal view; D magnified image of cloacal region; E posterior end of body, ventro-lateral view (precloacal papillae and phasmid arrowed). parison between the partial 18S sequence of C. hainanensis sp. nov. and the other four congeneric species showed % nucleotide differences. The partial 18S sequence of C. hainanensis sp. nov. is deposited in the GenBank database ( nih.gov) under accession number (KJ734676). Partial ITS-2 region. The length of the partial ITS-2 sequence Cucullanus hainanensis sp. nov. obtained herein was 465 bp. There is only C. dodsworthi with the ITS-2 sequence registered in GenBank (HQ241924). Pairwise comparison between the ITS sequence of C. hainanensis sp. nov. and C. dodsworthi showed 25.6% nucleotide differences. The ITS-2 sequence of C. hainanensis sp. nov. is deposited in the Gen- Bank database ( nih.gov) under accession number (KJ734675). Discussion The accurate identification of species of this extremely large group only based on the traditional approaches has never been easy; because the members of Cucullanus exhibit an extraordinary morphological similarity and some of them have not been completely described or illustrated. Consequently, when a new species is described, some authors commonly prefer to compare it to those from the same host group or the same zoogeographical region, as well as some particular traits (Moravec et al. 2005). According to Park and Moravec (2008), there have been ten species of Cucullanus recorded from anguilliform fishes. Of them, Cucullanus praecinctus (Dujardin, 1845) is considered a species inquirenda because of its original description only based on one female specimen (see Park

6 Cucullanus hainanensis sp. nov. from China 715 and Moravec, 2008). Cucullanus hainanensis sp. nov. differs from C. robustus Yamaguti, 1935, C. pedroi Timi et Lanfranchi, 2006 and C. hians (Dujardin, 1875) by the shorter spicules ( mm, representing % of body length in the former vs mm, representing % of body length in the latter three species) and the different arrangement of caudal papillae, especially the position of the first pair of precloacal papillae (situated well anterior to ventral sucker in the latter three species) (Yamaguti 1935; Park and Moravec 2008; Timi and Lanfranchi 2006; Rodrigues et al. 1975). The new species can be easily distinguished from C. oceaniensis Moravec, Sasal, Würtz et Taraschewski, 2005 by the different position of excretory pore and deirids (excretory pore at short distance below end of oesophagus and deirids just anterior to oesophago-intestinal junction in C. oceaniensis), longer gubernaculum ( mm in the new species vs mm in the latter) and the different arrangement of caudal papillae (Moravec et al. 2005). Cucullanus anguillae Wang et Lin, 1975 was collected from China and the position of the first pair of precloacal papillae is also at the level of ventral sucker (Wang and Lin 1975). However, the length of oesophagus and tail is much longer than that of C. hainanensis sp. nov. (oesophagus 1.6 mm, representing 16.3% of body length and tail 0.42 mm in male in C. anguillae vs oesophagus mm, representing % of body length and tail mm in male in the new species). Cucullanus hainanensis sp. nov. is different from C. filiformis Yamaguti, 1935 by the distinctly broader pseudobuccal capsule, the smaller body size ( mm in C. hainanensis sp. nov. vs 17.7 mm in the male in C. filiformis), the longer gubernaculum (vs mm in the latter) and the different position of excretory pore and the first pair of precloacal papillae (excretory pore posterior to oesophago-intestinal junction and the first pair of precloacal papillae situated well anterior to ventral sucker in C. filiformis) (Yamaguti 1935). From C. muraenesocis Yamaguti, 1961 and C. murenophidis Campana-Rouget, 1957, the new species differs by the longer spicules (vs mm in the latter two species) and broader pseudobuccal capsule (Yamaguti 1961; Campana- Rouget 1957). The new species can be readily differentiated from C. australiensis Baylis, 1927 by the distinctly smaller body size (vs mm in the male in C. australiensis), the shorter tail (vs mm in the male in the latter) and the different arrangement of caudal papillae (Baylis 1927; Morand and Rigby 1998). Among the species of Cucullanus reported from China, C. carettas and C. fujianensis are parasitic in turtles, which differ from the new species by possessing remarkable cuticular rings and the different arrangement of caudal papillae (Wang and Lin 1975; Wang 1984). Cucullanus hainanensis sp. nov. can be readily distinguished from C. hapalogengus by possessing well developed precloacal ventral pseudosucker (lacking of this structure in the latter) (Luo 2001). The new species is different from C. spirocaudus by the distinctly shorter spicules (vs mm, representing % of body length in C. spirocaudus). Cucullanus hainanensis sp. nov. can be readily differentiated from C. amadai, C. heterochrous, C. decapteri and C. cirratus by the particular morphology of the cloacal region (without the protruding cloacal region in the latter four species) and the different position of the first pair of precloacal papillae (Yamaguti 1941; Parukhin 1966; Vicente and Dos Santos 1973). Yin and Zhang (1983) described Indocucullanus muraenesocis Yin & Zhang, 1983 from Muraenesox cinereus (Forsskål) in the East China Sea (off Zhoushan Island, Zhejiang Province). Petter (1974) considered the genus Indocucullanus Ali, 1956 as a synonym of Cucullanus, and Peng et al. (2011) transferred it to Cucullanus, as C. muraenesocis (Yin & Zhang, 1983), unaware of the fact that this name had been given to C. muraenesocis Yamaguti, Therefore, C. muraenesocis (Yin et Zhang, 1983) is a homonym to C. muraenesocis Yamaguti, 1961 and a new name is required for the former according to the International Code of Zoological Nomenclature. We propose a replacement name C. wangi nom. nov. for C. muraenesocis (Yin et Zhang, 1983) to honour professor P.-Q. Wang (Fujian Normal University, Fujian Province, China) for his taxonomic contributions to helminth parasites in China. We also examined the type material of C. wangi nom. nov. (3 males, 2 females) deposited in Institute of Zoology, Chinese Academy of Sciences. From the new species, Cucullanus wangi nom. nov. differs by the larger body size (vs mm in male in C. wangi nom. nov.) and the absence of precloacal ventral pseudosucker. Genetic comparison of the partial 18S rdna of C. hainanensis sp. nov. and its congeneric species displayed % nucleotide differences, which is far lower than the level of the interspecific nucleotide variation in ITS-2 region (25.6% nucleotide differences between C. hainanensis sp. nov. and C. dodsworthi). Li et al. (2014) investigated the genetic diversity of Dichelyne pleuronectidis (Yamaguti, 1935) and found there is very low level of intraspecific nucleotide variability in the ITS region (0 0.58% nucleotide differences) among different individuals. However, there has been no record of the level of the intraspecific nucleotide variation in the partial 18S rdna in cucullanids so far. Li et al. (unpublished data) revealed that there are % nucleotide differences in the partial 18S rdna in different individuals of D. pleuronectidis. Thus, we could preliminarily consider that it is a practical method to utilize the partial 18S rdna and ITS-2 region as genetic markers for the separation and identification of species of Cucullanus. Acknowledgments. The authors wish to thank Dr. František Moravec (Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic) for sending us some literature on Cucullanus. This study was supported by the National Natural Science Foundation of China (No ), the Natural Science Foundation of Hebei Province (No. C ), the China Postdoctoral Science Foundation (Nos 2012M520593, 2014T70227), the Natural Science Foundation of Hebei Education Department (No. Y ), the Postdoctoral Research Projects Merit Subsidy of Human Resources

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