Myxosporea: Bivalvulida) Infecting the Gallbladder of the Orange-Spotted Grouper Epinephelus Coioides from the Arabian Gulf, Saudi Arabia
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The Journal of Published by the International Society of Eukaryotic Microbiology Protistologists Journal of Eukaryotic Microbiology ISSN 1066-5234 ORIGINAL ARTICLE Molecular and Morphometric Characteristics of Ceratomyxa hamour n. sp. (Myxosporea: Bivalvulida) Infecting the Gallbladder of the Orange-spotted Grouper Epinephelus coioides from the Arabian Gulf, Saudi Arabia Lamjed Mansoura,b, Hussain A. Al-Qahtania, Saleh Al-Quraishya & Abdel-Azeem S. Abdel-Bakia,c a Zoology Department, College of Science, King Saud University, Saudi Arabia, PO Box 2455, Riyadh, 11451, Saudi Arabia b Unite de Recherche de Biologie integrative et Ecologie evolutive et Fonctionnelle des Milieux Aquatiques, Departement de Biologie, Faculte des Sciences de Tunis, Universite De Tunis El Manar, Tunis, Tunisia c Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt Keywords ABSTRACT Bile; Myxozoa; new species; parasite; phylogeny. Ceratomyxa hamour n. sp. was found to infect the gallbladder of the orange- spotted grouper, Epinephelus coioides located off the Saudi Arabian coast of Correspondence the Arabian Gulf. The infection was reported as a free-floating spore in the A. S. Abdel-Baki, Zoology Department, Col- bile, and pseudoplasmodia were not observed. Mature spores were crescent- lege of Science, King Saud University, Saudi shaped and measured on average 7 lm in length and 16 lm in thickness. The Arabia, PO Box 2455, Riyadh 11451, Saudi polar capsule, meanwhile, had length to width measurements of 4 lm and Arabia 3 lm on average. A periodical survey was conducted throughout a sampling Telephone number: +9661 1 467 5754; period between December 2012 and December 2013, with the results show- FAX number: +9661 1 4678514; ing that the parasite was present throughout the year with a mean prevalence e-mail: [email protected] of 32.6%. The objective of this study was to characterize this new species based on its morphological and molecular differences from previously Received: 14 March 2014; revised 10 May described species. Molecular analysis based on the partial sequence of the 2014; accepted May 13, 2014. SSU rDNA gene, showed the highest similarity (97.8%) to Ceratomyxa buri, reported in the cultured yellow tail Seriola quinqueradiata in Japan. Indeed, doi:10.1111/jeu.12148 C. buri and the new species described here formed an individual cluster with a high degree of bootstrap support. This is the first reported species of genus Ceratomyxa from the Arabian Gulf fishes off Saudi Arabia. THE orange-spotted grouper, Epinephelus coioides, belongs parasites, with more than 280 species recounted globally to the family Serranidae (Randall 1997) and occurs in the (Gunter et al. 2010). Members of the genus Ceratomyxa western Indian Ocean from the southern Red Sea to Natal are mostly coelozoic, parasitizing the gallbladder of marine and east to the western Pacific, where it is distributed from fishes. Ceratomyxa spp. are distinguished by spores of the Ryukyu Islands to New South Wales. It ranges east into elongated shape with shell valves exceeding in extent the Oceania as far as Palau in the Northern Hemisphere and Fiji axial diameter of the spore (Lom and Dykova 2006). There in the Southern (Grandcourt et al. 2005; Russell and Hous- are two sub-spherical polar capsules near the sutural line ton 1989). Epinephelus coioides, common name hamour, is at the anterior pole of the spore (Sobecka et al. 2013). the commonest fish species in the Arabian Gulf and is one Binucleate sporoplasm does not fill the spore cavity com- of the most important commercially exploited species in pletely, and, in some species, two uninucleate sporo- Saudi Arabia (Grandcourt et al. 2005; Randall 1995). plasms have been reported (Lom and Dykova 2006). Myxosporean parasites have a worldwide distribution, Until recently, detection and identification of Cerat- mostly in fish hosts, although rarely in platyhelminths, rep- omyxa species are performed with microscopy-based tiles, and amphibians. Recently, a myxozoan-like parasite methods by examination of morphometrical features of was found in the brain of a mole Talpa europrea (Friedrich the mature spores. This could mask multiple distinct cryp- et al. 2000; Kent et al. 2001; Lom and Dykova 2006). tic species because of the plasticity on the spore mor- Ceratomyxa is the second largest genus of myxozoan phometry (Heiniger et al. 2008; Smothers et al. 1994). © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–7 1 New Ceratomyxa from the Gallbladder of Epinephelus coioides Mansour et al. Actually new species descriptions should include both 0.001 M EDTA pH 8.0), stained with ethidium bromide, and molecular and morphological data, and eventually ecobio- visualized on an UV transilluminator using a gel documenta- logical parameters of parasite (Fiala 2006; Smothers et al. tion system (BioRad Gel225 DocTMXR+, Ontario, Canada). 1994). Molecular analysis, mainly based on the SSU rDNA PCR products obtained from three different specimens sequence and sometimes completed with the LSU rDNA were sequenced by Macrogen Inc. (Seoul, South Korea), sequence, offer the possibility of more accurate and effi- using the two primers mentioned above and two additional cient methods for Ceratomyxa-specific characterization internal primers, MyxF1338 and MyxR1437 (Mansour et al. and may help on the elucidation of misclassified species 2013). (Gunter and Adlard 2010; Smothers et al. 1994). To our knowledge, no Ceratomyxa spp. have so far Phylogenetic analysis been described in fishes from the Arabian Gulf. Herein, we describe a new species of Ceratomyxa from the gall- Overlapping sequences were clipped and edited by visual bladder of an economically significant fish, the orange- inspection and assembled. The consensus sequence of spotted grouper, E. coioides. The identification was based 1,603 bp was used to extract the most similar sequence on spore morphometry and molecular phylogeny. from GenBank using a BLAST (Basic Local Alignment Search Tool) search at NCBI (National Center for Biotech- nology Information) (http://www.ncbi.nlm.nih.gov/). Thirty- MATERIALS AND METHODS three sequences of Ceratomyxa species and one Tetra- capsuloides bryosalmonae isolate were clipped (accession Host and parasite numbers and coordinates listed in Table 1) and aligned with Between December 2012 and December 2013, a total 85 ClustalX 2.1.0.12, applying the default parameters (Larkin orange-spotted grouper E. coioides were collected from et al. 2007). A phylogenetic tree, based on the obtained the boat landing site at Dammam (26°33020″N, 50°0025″E) alignment was generated using maximum likelihood and on the eastern coast of Saudi Arabia. The fish were exam- neighbor joining methods, using version 5 of the MEGA ined for myxosporean infection. Gallbladders were software (Tamura et al. 2011). The parameters for the max- extracted with caution and bile was collected by punctur- imum likelihood analyses were General Time Reversible ing the gall bladder with a delicately pointed glass pipette model, 2,000 bootstrap replications, Gamma distributed for light microscopic examination. Some positive gallblad- (G), number of discrete Gamma rates of 5, and complete ders with microscopic examination were preserved in deletion. The genetic distance matrix was estimated using 85% ethanol for molecular analysis. Fresh spores were the Kimura two-parameter model distance for transition photographed and measured using an Olympus micro- and transversion, for a total of 1,092 positions in the final scope equipped with a digital camera. Spore description dataset, and the rate variation among sites was modeled and measurements followed the guidelines of Lom and with a gamma distribution (shape parameter = 4). Arthur (1989). Measurements were based on 50 fresh spores using ocular micrometer and data were presented Statistical analysis as mean Æ SD (range). One-way ANOVA was carried out, and the statistical comparisons among the seasons were performed with SSU rDNA analysis Holm–Sidak method using a statistical package program DNA extraction was carried out from 50 ll of the infected (Sigma Plot version 11.0, Systat Software, San Jose, CA). bile preserved in ethanol using the QIAGEN DNeasy kit All p values are two-tailed, and p <0.001 was considered (QIAGEN Inc., Valencia, CA). SSU rDNA was amplified as significant for all statistical analysis in this study. using the PCR technique. The partial 18S sequence was amplified using the forward primer Myxospec F (50-TTC RESULTS TGCCGT ATC AAC TWG TTG) (Fiala 2006) and the reverse primer18R (50-CTACGG AAA CCT TGT TAC G) (Whipps The infection was observed in the form of free-floating et al. 2003). Amplifications were performed in a final vol- spores in the bile. Plasmodia were not observed. ume of 30 ll of PCR mixture containing 1X Taq DNA poly- Table 1. The relationship between the seasons and the prevalence merase buffer (MBI, Fermentas), 0.2 mmol of mixed dNTP, of Ceratomyxa hamour n. sp. infecting the gallbladder of Epinephelus 1.5 mmol of MgCl , 0.2 pmol of each primer, 1 U of Taq 2 coioides from the Arabian Gulf DNA polymerase and 50–100 ng of DNA, and ultra-pure water. The amplification was done in a thermocycler appa- No. examined No. infected Percent of ratus (Techne TC-Plus Satellites, Staffordshire, UK). The Seasons fish fish infection PCR cycling program used consisted of an initial step of denaturation at 94 °C for 4 min, followed by 35 cycles of Spring 36 12 33.3 Summer 36 11 30.5 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s, and a Autumn 36 7 19.4 final step of extension at 72 °C for 5 min. Subsequently, Winter 36 17 47.2 PCR products were separated in 1% agarose gel electro- Total 144 47 32.6 phoresis in a Tris-borate-EDTA buffer (0.045 M Tris-borate, © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 2 Journal of Eukaryotic Microbiology 2014, 0, 1–7 Mansour et al.