Parasitology International 65 (2016) 677–684 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint First report on nematode parasite infection in the yellowbar angelfish Pomacanthus maculosus (Perciformes: Pomacanthidae) from the Iraqi coral reef, with description of a new species of Cucullanus (Nematoda: Ascaridida) using the integrated approaches Liang Li a,⁎, Atheer H. Ali b,Wen-TingZhaoa, Liang Lü c,ZhenXud,⁎⁎ a Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Science, Hebei Normal University, 050024 Shijiazhuang, Hebei Province, PR China b Department of Fisheries and Marine Resources, College of Agriculture, Basrah University, Basrah, Iraq c Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Rd., Chaoyang District, 100101 Beijing, PR China d Medical College of Hebei University of Engineering, 056002 Handan, Hebei Province, PR China article info abstract Article history: The yellowbar angelfish Pomacanthus maculosus (Forsskål) (Perciformes: Pomacanthidae) is a beautiful, marine Received 16 April 2015 ornamental fish with very important commercial value. However, to date, no information is available on nema- Received in revised form 13 August 2016 tode parasite infection in P. maculosus. In the present study, the integrated approaches including light and scan- Accepted 27 August 2016 ning electron microscopy, and sequencing and analysing ribosomal [small ribosomal DNA (18S) and internal Available online 1 September 2016 transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions, respectively, were employed for the systematic evaluation of the nematode parasites firstly isolated from P. maculosus in the Keywords: Parasites Iraqi coral reef. The results revealed that these nematodes represent a new species of Cucullanus (Ascaridida: Ornamental fish Cucullanidae). The phylogenetic analyses based on 18S, ITS and cox1 sequences were constructed, respectively, Integrated taxonomy to assess the phylogenetic relationships between the new species and the other cucullanids, and the monophyly Phylogeny of Cucullanus, as well as some its related genus-level taxa. The results supported C. extraneus n. sp. appear to be Arabian gulf sister to C. hainanensis,andthegeneraCucullanus, Dichelyne and Truttaedacnitis may be not monophyletic assem- blages. This is the first report of the occurrence of nematode parasites in P. maculosus. © 2016 Elsevier Ireland Ltd. All rights reserved. 1. Introduction to exactly recognise of Cucullanus species purely based on morphologi- cal methods because of their considerably uniform morphology in the The yellowbar angelfish Pomacanthus maculosus (Forsskål) species. Consequently, in the present study, the integrated approaches, (Perciformes: Pomacanthidae) is a beautiful, omnivorous, coral reef including light and scanning electron microscopy, and sequencing and fish distributed throughout the Arabian Gulf, the northwestern Indian analysing the ribosomal [small ribosomal DNA (18S) and internal tran- Ocean and the Red Sea, which mainly feeds on algae, sponges and scribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit encrusting invertebrates [1–4]. Owing to its striking colour patterns, P. 1(cox1)] target regions, respectively, were used to exactly identify maculosus is also a marine ornamental fish with very important com- these parasites to the species level. In addition, in order to assess the mercial value [5]. However, to our knowledge, there have been no nem- phylogenetic relationships between the Cucullanus nematodes obtained atode parasites recorded from this fish. During a helminthological herein and the other cucullanids, and the monophyly of Cucullanus,as survey of the marine fishes in the Arabian Gulf, numbers of Cucullanus well as some its related genus-level taxa, the phylogenetic analyses nematodes were collected from P. maculosus in the Iraqi coral reef. based on different genetic markers were constructed, respectively. The accurate identification of parasites at any developmental stages is imperative to study parasite biology, resolve systematic problems and 2. Materials and methods diagnose and control of parasite disease [6–8]. However, it is not easy 2.1. Light and scanning electron microscopy ⁎ Correspondence to: L. Li, College of Life Science, Hebei Normal University, 20 East Road Ten P. maculosus with body length 18.5–31.0 cm caught in the Iraqi of 2nd South Ring, Yuhua District, 050024 Shijiazhuang, Hebei Province, PR China. ′ ′ ⁎⁎ Corresponding author. coral reef, Arabian Gulf (29°25 N, 48°48 E), were examined for para- E-mail addresses: [email protected] (L. Li), [email protected] (Z. Xu). sites. Nematodes recovered from the intestine of fish, were washed in http://dx.doi.org/10.1016/j.parint.2016.08.007 1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved. 678 L. Li et al. / Parasitology International 65 (2016) 677–684 physiological saline and fixed and stored in 80% ethanol until studied. For Medium-sized, whitish nematodes. Body elongate, cylindrical, maxi- light microscopical studies, nematodes were cleared in lactophenol. mum width at about region of middle body, tapering towards tail. Cuticle Drawings were made with the aid of a Nikon microscope drawing attach- with fine transverse striations. Lateral alae absent. Oral aperture dorso- ment. For scanning electron microscopy (SEM), specimens were fixed in ventrally elongate, slit-like, surrounded by distinct collarette, bearing 4% formaldehyde solution, post-fixed in 1% OsO4, dehydrated via an eth- row of small denticles on inner surface; 2 pairs of large, submedian ce- anol series and acetone and critical point dried. The specimens were coat- phalic papillae and 1 pair of prominent, lateral amphids present (Figs. ed with gold and examined using a Hitachi S-4800 scanning electron 1A,C, 2A). 2 pairs of small, submedian inner labial papillae observed microscope at an accelerating voltage of 20 kV. Measurements (the under SEM (Fig. 2A). Oesophagus muscular, slightly expanded at anterior range, followed by the mean in parentheses) are given in micrometres end to form pseudobuccal capsule (oesophastome); posterior part unless otherwise stated. The type-specimens are deposited in College of remarkably expanded, distinctly wider than pseudobuccal capsule Life Science, Hebei Normal University, Hebei Province, China. (Fig. 1A,B). Oesophagus opens posteriorly into intestine through valvular apparatus (Fig. 1A,B). Nerve-ring at about 2/5 of oesophageal length. In- 2.2. Molecular data testinal caecum absent (Fig. 1A,B). Deirids situated laterally, hooked, at anterior to oesophago-intestinal junction (Figs. 1B, 2B). Post-deirids not One female and two males were randomly selected for molecular observed. Excretory pore far posterior to oesophago-intestinal junction analysis. Genomic DNA from individual worms was extracted using a (Fig. 1A).Tailinbothsexesconical,withpointedtip(Figs. 1E,F, 2E,F). – Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according Male [Based on 6 mature specimens] Body 11.8 16.4 (14.4) mm – to the manufacturer's instructions. DNA was eluted in elution buffer and long, with maximum width 240 439 (366). Length of entire oesopha- – – kept at −20 °C until use. The partial 18S rDNA was amplified by poly- gus 828 966 (901), representing 5.65 7.02 (6.32)% of body length, – merase chain reaction (PCR) using the primers 18SF (forward: 5′-CGC minimum width of oesophagus 72 98 (83.0), maximum width of pos- – – – GAA TRG CTC ATT ACA ACA GC-3′) and 18SR (reverse: 5′-GGG CGG terior part 125 180 (148). Pseudobuccal capsule 169 250 × 70 82 – TAT CTG ATC GCC-3′) [9].Thecox1 was amplified by PCR using the (210 × 74.8). Nerve-ring, deirids and excretory pore at 280 375 – – primers CO1F (forward: 5′-TTT TTT GGT CAT CCT GAG GTT TAT-3′) (352), 670 887 (821) and 1.05 1.36 (1.22) mm from anterior extrem- and CO1R (reverse: 5′-ACA TAA TGA AAA TGA CTA ACA AC-3′) [10]. ity, respectively. Posterior end of body remarkably curved ventrally. – The ITS region was amplified by PCR using the primers A (forward: 5′- Precloacal ventral sucker, distance from centre to cloaca 814 985 GTC GAA TTC GTA GGT GAA CCT GCG GAA GGA TCA-3′)andB(reverse: (889) (Figs. 1G, 2C). Ventral region of cloacal opening distinctly elevated 5′-GCC GGA TCC GAA TCC TGG TTA GTT TCT TTT CCT-3′) [11]. The cy- (Figs. 1E, 2E). Spicules of almost equal length, alate, pointed at distal – – cling conditions were as described previously [12].PCRproductswere end, 1.23 1.90 (1.59) mm long, representing 10.3 11.6 (11.1)% of checked on GoldView-stained 1.5% agarose gels and purified with Col- body length (Fig. 1G). Gubernaculum well sclerotised, boat-like in later- – umn PCR Product Purification Kit (Shanghai Sangon, China). Sequencing al view, 130 188 (156) long (Fig. 1H). Caudal papillae 10 pairs in total. fi was carried out using a DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Precloacal papillae 3 pairs arranged as follows: rst pair at anterior to Applied Biosystems, California, USA) and an automated sequencer (ABI- precloacal ventral sucker, second pair just posterior to ventral sucker, PRISM 377). Sequencing for each sample was carried out for both third pair approximately mid-way between second pair and cloacal fi strands. Sequences were aligned using ClustalW2 [13] and adjusted opening (Figs. 1G, 2C). Paracloacal papillae 4 pairs: rst and