Myxobolus Buri, the Myxosporean Parasite Causing Scoliosis of Yellowtail, Is Synonymous with Myxobolus Acanthogobii Infecting the Brain of the Yellowfin Goby
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Blackwell Science, LtdOxford, UKFISFisheries Science0919 92682004 Blackwell Science Asia Pty LtdDecember 200470610361042Original ArticleRevised classification of Myxobolus buriH Yokoyama et al. FISHERIES SCIENCE 2004; 70: 1036–1042 Myxobolus buri, the myxosporean parasite causing scoliosis of yellowtail, is synonymous with Myxobolus acanthogobii infecting the brain of the yellowfin goby Hiroshi YOKOYAMA,* Mark Andrew FREEMAN, Tomoyoshi YOSHINAGA AND Kazuo OGAWA Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan ABSTRACT: Myxobolus buri Egusa, 1985, is a well-documented myxosporean parasite that causes the scoliosis of cultured yellowtail Seriola quinqueradiata. A similar parasite has been described as Myxobolus acanthogobii Hoshina, 1952, from the brain of the yellowfin goby Acanthogobius flavi- manus, although this parasite is not associated with skeletal abnormalities in host fish. The present study aimed to re-examine the identification of these two parasites by morphological and molecular analyses. Morphological characteristics (e.g. presence of the intercapsular processes and several folds on the sutural ridges) and spore dimensions were not distinguishable between the two species and were consistent with those in the original descriptions. Molecular analysis indicated that small subunit rRNA gene sequences shared 100% identity between the two parasites. Consequently, it can be concluded that M. buri is synonymous with M. acanthogobii, and thus this parasite can be reas- signed as M. acanthogobii. KEY WORDS: Acanthogobius flavimanus, Myxobolus, Myxozoa, parasite, scoliosis, Seriola quinqueradiata, yellowtail. INTRODUCTION the paper describing M. buri. Myxobolus acan- thogobii infects the cerebrospinal nervous system Myxosporean scoliosis is a significant cause of of the yellowfin goby Acanthogobius flavimanus, economic losses in cultured yellowtail Seriola but is not associated with spinal curvature of the quinqueradiata because of the unsightly appear- host fish. For this reason, little attention has ance of deformed fish and disease outbreaks in been paid to this parasite by fish pathologists marketable-sized fish. Since the 1970s, this dis- and fisheries scientists. Although M. acanthogobii ease has been observed in yellowtail farms in is obviously closely related to M. buri, it was not Japan, and several etiological factors, such as referred to in the original paper describing exposure to toxic substances, have been sus- M. buri as a new species.2 Therefore, the present pected as the cause of the disease. However, it study aimed to re-examine the identification of has been concluded that a brain infection by a these two parasites by morphological and molec- myxosporean is primarily responsible for the ular analyses. skeletal deformity,1,2 and this parasite was desig- nated as a new species, Myxobolus buri Egusa, 1985.2 Subsequently, this parasite has been found MATERIALS AND METHODS infecting the brain of other feral fish, such as Japanese bluefish Scombrops boops (Scombro- Parasite isolation pidae),3 red gurnard Chelidonichthys spinosus (Triglidae) and brown-lined puffer Canthigaster Myxosporean cysts and deformed yellowtail were rivulata (Tetraodontidae).4 transported to the University of Tokyo from fish The description of Myxobolus acanthogobii farms located in the Tokushima (January 1997) and Hoshina, 1952,5 far predates the publication of Nagasaki (October 2003) Prefectures of Japan. In November 2003, goby fishing was conducted at *Corresponding author: Tel: 81-35841-5285. Kanazawa Bay, Kanagawa Prefecture, which is the Fax: 81-35841-5283. Email: [email protected] type locality of M. acanthogobii. The first sampling Received 27 February 2004. Accepted 21 June 2004. (4 November 2003) yielded six yellowfin gobies Revised classification of Myxobolus buri FISHERIES SCIENCE 1037 (mahaze), one dusky tripletooth goby (chichibu) (Takara, Kyoto, Japan). Recombinant plasmids Tridentiger obscurus, one whitelimbed goby and PCR products were sequenced by the dideoxy (Ashishirohaze) Acanthogobius lactipes, one chain termination method8 using a BigDye Termi- chameleon goby (Akaobishimahaze) Tridentiger nator v3.1 Cycle Sequencing Ready Reaction Kit, trigonocephalus and one Richardson dragonet and a 310 capillary DNA sequencer (Applied (Nezumigochi) Repomucenus curvicornis (Cal- Biosystems, Foster City, CA, USA), according to lionymidae). In the second sampling (16 Novem- the manufacturer’s instructions. The consensus ber 2003), two yellowfin gobies, five streaked sequence obtained was compared to other gobies (Sujihaze) Acentrogobius pflaumii, five cha- sequences available in the databases; initial meleon gobies and one Richardson dragonet were sequence alignments were converted into captured. Following gross observation of the fishes, distances by the Kimura 2 parameter and a phylo- myxosporean cysts were collected to isolate spores, genetic tree was constructed using the neighbor- which were then preserved in the refrigerator (5∞C) joining (N-J) algorithm. or freezer (-80∞C) until required. The sequence data determined in the current study have been submitted to the GenBank data- base under accession number AY541585. The Gen- Morphological examination of spores Bank accession numbers for additional sequences used in the analysis are as follows: Ceratomyxa Spores were embedded in 1.5% melted agar on a shasta AF001579, Henneguya exilis AF021881, slide glass and observed by light microscopy. Digi- Henneguya ictaluri AF195510, Henneguya lesteri tal images were taken under an oil immersion AF306794, Kudoa amamiensis AF034638, Kudoa objective and measurements were made based on thyrsites AF031412, Myxidium truttae AF201374, 20 spores from multiple images. Potassium Myxidium lieberkuehni X76638, Myxobolus algon- hydroxide (0.2 mol/L) was applied to spores to quinensis AF378335, Myxobolus arcticus AF085176, induce extrusion of the polar filaments, which Myxobolus cerebralis U96492, Myxobolus hungari- were then measured. Descriptions and measure- cus AF448444, Myxobolus ichkeulensis AF378337, ments of spores were made according to Lom and Myxobolus insidiosus U96494, Myxobolus lentisu- Arthur.6 turalis AY119688, Myxobolus neurobius AF085180, Myxobolus pellicides AF378339, Myxobolus pen- dula AF378340, Myxobolus portucalensis Molecular analysis of spores AF085182, Myxobolus spinacurvatura AF378341, Myxobolus squamalis U96495, PKX organism Myxosporean cysts were homogenized in 0.4-mL U70623, Sphaerospora molnari AF378345, high concentration urea buffer containing 100 mg/ Sphaerospora oncorhynchi AF201373. mL proteinase K and digestion was allowed to occur overnight at 56∞C. DNA was subsequently extracted using a QIAamp DNA Mini Kit (Qiagen Detection of Myxobolus acanthogobii by Inc., Hilden, Germany) following the manufac- polymerase chain reaction assay turer’s tissue protocol; the purified DNA was used as template DNA for subsequent polymerase To enable accurate sequencing of the complete chain reactions (PCR). Small subunit (SSU) rDNA SSU rDNA, various internal oligonucleotide prim- was amplified using the universal primers 18e/ ers were designed for both sense and antisense 18g described by Hillis and Dixon.7 After an ini- DNA strands. The primer set: Ma-fwd 5¢-TGAG- tial denaturation at 95∞C for 4 min, samples were TAGTACACACGACACC-3¢, Ma-rev 5¢-CCACACA- subjected to 30 cycles of amplification (denatur- GACTCCACTGCA-3¢, utilizing the same PCR ation at 95∞C for 30 s, primer annealing at 55∞C conditions as above, were used to amplify an inter- for 30 s and extension at 72∞C for 30 s), followed nal amplicon of 750 base pairs (bp). These primers by a 7-min terminal extension at 72∞C. All ampli- were checked for cross-reactivity to other Myxobo- fications were performed on a Bio-Rad I-cycler lus spp. SSU rDNA sequences available in public (Bio-Rad Laboratories, Hercules, CA, USA). The databases and found to be specific for PCR products obtained were visualized in an M. acanthogobii are hence able to be used as a spe- ethidium bromide-stained 1% agarose gel. PCR cific PCR assay for the diagnosis and detection of amplicons were purified using a PCR purification M. acanthogobii. Although the sensitivity (detec- kit (Qiagen Inc.) and the resulting purified DNA tion threshold) of this PCR protocol has not been was used as template DNA in direct sequencing determined yet, a preliminary test of the PCR diag- reactions or cloned into a vector. PCR amplicons nosis was made on the fishes collected by the goby were cloned into the pt-7 Blue T-vector system fishing. 1038 FISHERIES SCIENCE H Yokoyama et al. RESULTS Gross observation of fishes collected by goby fishing No clinical signs of skeletal abnormalities were found in the fishes collected in the two samplings. However, heavily infected yellowfin gobies exhib- ited a slight exophthalmus and a swelling at the periphery of the eyes (Fig. 1a). After dissection of the yellowfin gobies, two of the six fish from the first sampling taken on 4 November and both fish from the second sampling taken on 14 November were visibly infected with M. acanthogobii. Numerous white-colored, bean-shaped cysts were found on the surface of the brain, olfactory nerve and vertebral column (Fig. 1b), whereas no cysts were observed in non-infected gobies (Fig. 1c). Morphological examination of spores Two myxosporean isolates from both yellowtail and yellowfin goby were morphologically indis- tinguishable. Spores were oval to ellipsoidal from the frontal view with