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 a slightly attenuated poste- rior end (Figs 2,3). The intercapsular process was prominent. Sutural ridges were conspicuous with 7–9 folds on the edge. The polar filament coil had three turns. The two pyriform polar capsules were slightly different in size; thus, they were sepa- rately measured. The spore dimensions of M. buri and M. acanthogobii are summarized in Table 1. The above data are consistent with those of the original descriptions of the two species. The neo- type specimens of the two myxosporean isolates from yellowfin goby and yellowtail were depos- ited in the collection at the National Science Museum, Tokyo, Japan, and assigned the acces- sion numbers NSMT-Pr180 and NSMT-Pr181, respectively.
Molecular analysis
Small subunit rDNA sequences (18e/18g) of 1877 bp were obtained from M. buri and
Fig. 1 Yellowfin goby Acanthogobius flavimanus. (a) External appearance of a goby heavily infected with Myx- obolus acanthogobii. Note the exophthalmus and an incision (arrow) as a result of the swelling at the periph- ery of the eyes. (b) Numerous cysts of M. acanthogobii formed in the cerebrospinal nervous system of an infected goby. (c) Brain tissue of an uninfected yellowfin goby. Revised classification of Myxobolus buri FISHERIES SCIENCE 1039
Fig. 2 Fresh spores of Myxobolus buri from yellowtail Seriola quin- queradiata. (a–d) Front views of spores; (e, f) side view of spores. Bar 10 mm.
Fig. 3 Fresh spores of Myxobolus acanthogobii from yellowfin goby. (a–d) Front views of spores; (e, f) side view of spores. Bar 10 mm.
M. acanthogobii, sharing 100% identity over the included in the analysis (Fig. 4), including spe- entire gene region. Phylogenetic analysis using cies such as M. spinacurvatura, which is associ- the N-J method revealed that the sequences ated with spinal deformities, and M. arcticus from M. buri and M. acanthogobii were grouped and M. neurobius, which infect host nervous distinctly from all other Myxobolus spp. tissues. 1040 FISHERIES SCIENCE H Yokoyama et al.
5 Detection of Myxobolus acanthogobii by 2 polymerase chain reaction assay esent esent study study Egusa Pr Pr Hoshina Using the specific PCR primers in a diagnostic assay, four of the six yellowfin gobies from the first sampling were shown to be positive for the myx- osporean infection. From the four positive fish, two fish were heavily infected (positive by visual exam- ination), whereas the other two were negative by microscopic observation. In the second sampling quinqueradiata quinqueradiata flavimanus flavimanus
Seriola Seriola Acanthogobius Acanthogobius (n = 2), both the yellowfin gobies were positive by PCR and by microscopic examination. The other gobiid fishes examined were all negative by PCR. However, two Richardson dragonets (one from each of the two samplings) showed a very weak ND band of the correct size by PCR, but the results (range) Host Reference erage polar erage were considered dubious. Av filament length 23.8 (19.7–28.2) 21.7 (16.0–31.2)
‡ § ¶ ‡ § ¶ DISCUSSION
† Light microscopic examination of the two parasites
(range) revealed that they were indistinguishable in their erage length erage
2.8 (2.6–3.2) 2.5 (2.0–2.9) 2.7 (2.0–3.2) 2.5 (2.2–2.6) 2.3 (2.0–2.5) 2.4 (2.0–2.6) morphology, even allowing for the minor variation Av in spore dimensions. Comparing the current study
‡ § ¶ ‡ § ¶ and the original description, the slight differences
Polar capsule Polar among spore size appear to lie within the range of intraspecific variation (Table 1). The relatively average. ¶ small sizes in the study by Hoshina could have (range) erage width erage been the result of the shrinkage of spores fixed in 4.3 (3.8–4.6) 4.5 (3.8–5.3) 3.8 (3.5–4.3) 4.0 (3.5–4.5) Av 5
small; formalin. The present study highlighted the size § differences of the two polar capsules, and hence
large; measurements were made separately. Egusa also ‡ mentioned that ‘the polar capsules were slightly
obolus buri and Myxobolus acanthogobii obolus buri and Myxobolus unequal in size in most cases’, although he mea-
(range) sured their size without distinguishing large and Myx 2
erage thickness erage small polar capsules. In any case, these minor dif-
Av ferences are likely to be insignificant. Until now, the taxonomy and classification of Myxozoa have been largely based on spore morphology and mor- phometrics.6 Even according to these traditional m) between m m) between pore
S criteria only, it is strongly suggested that the cre-
(range) ation of M. buri as a new species without compar- erage width erage
Av ison with M. acanthogobii is invalid, and that Egusa apparently overlooked the description of M. acanthogobii.2 Molecular analysis of SSU rRNA gene sequences from the parasites’ DNA adds further support to
(range) the synonymy of the two species. SSU rRNA gene erage length erage
9.8 (8.0–11.8) 8.1 (6.7–9.6) 6.2 (5.3–7.2) 3.5 (2.9–4.3)sequences 2.2 (1.9–2.9) of myxozoans can be variable between 10.3 (9.8–10.7) 8.5 (8.0–9.1) 6.6 (6.2–7.4) 4.2 (3.9–4.5) 10.6 (9.2–11.8) 9.2 (7.9–10.2) 6.6 (5.5–7.3) 4.5 (3.9–5.4) 2.8 (2.5–3.4) 30.6 (17.1–40.3) 10.6 (9.9–11.4) 9.1 (8.6–9.8) 6.9 (6.4–7.3) 4.8 (3.9–5.3) Av even closely related taxa, and intraspecific varia- tion of 2.6% between various isolates of Myxidium 9
omparison of spore dimensions ( omparison of spore lieberkuehni has been reported. Sequence data C alone might be inadequate to fully identify myx- osporean species, but it can be used in conjunction obolus obolus buri obolus obolus buri Large and small polar capsules were measured separately in this study; separately measured Large and small polar capsules were acanthogobii acanthogobii with morphological data for classification. ND, not determined because of formalin-fixed materials. ND, † able 1 Species T Myx Myx Myx Myx Recently, the need for a more accurate diagnostic Revised classification of Myxobolus buri FISHERIES SCIENCE 1041
PKX organism Ceratomyxa shasta 1000 Kudoa amamiensis 1000 Kudoa thyrsites Myxidium truttae 548 Sphaerospora oncorhynchi 1000 Myxidium lieberkuehni Myxobolus buri 1000 997 Myxobolus acanthogobii Henneguya lesteri 499 Myxobolus ichkeulensis 1000 1000 Myxobolus spinacurvatura 628 Henneguya ictaluri 1000 112 Henneguya exilis Myxobolus portucalensis 423 Myxobolus lentisuturalis Myxobolus squamalis 203 719 Myxobolus cerebralis 1000 Myxobolus insidiosus 992 Myxobolus neurobius 570 290 Myxobolus arcticus Myxobolus hungaricus 1000 Myxobolus pendula 1000 1000 Myxobolus pellicides Sphaerospora molnari 1000 Myxobolus algonquinensis
0.1
Fig. 4 Neighbor-joining phylogram of 26 myxozoan parasites, including Myxobolus acanthogobii and Myxobolus buri, based on small subunit rRNA gene sequences. Bootstrap confidence values shown at nodes; the tree is rooted to the related outgroup (PKX organism, the myxozoan causing proliferative kidney disease) and genetic distance is repre- sented by the scale bar. system for Myxozoa has increased because of tebrate hosts is presumed, as has been docu- insufficient morphological characteristics (e.g. mented for many fresh water myxozoans.10,11 many Myxobolus species). Thus, it is now recom- Recently, it has been demonstrated that the marine mended that SSU rRNA gene sequence data should myxozoan Ellipsomyxa gobii in the common goby be provided when describing new species.10 A com- Pomatoschistus microps uses polychaetes Nereis bination of the morphological and molecular evi- spp. as invertebrate hosts.12 The two-host life cycle dence in the present study leads to the conclusion complicates the analysis of the dynamics of the that M. buri and M. acanthogobii are the same par- infection cycle, but a molecular diagnostic tool will asitic organism and that M. buri is a synonym of aid such field studies. The epizootiology (e.g. geo- M. acanthogobii. Based on the International Code graphic distribution, host range) of this parasite in of Zoological Nomenclature, M. buri should be the natural environment and farm sites remains to suppressed and reassigned as M. acanthogobii. be clarified. The revised classification of M. buri as In the PCR tests on specimens collected by goby M. acanthogobii might provide a new perspective fishing, some fishes were found to be positive by for management strategies in yellowtail culture. If PCR but negative by microscopic observation (two yellowfin gobies act as a natural reservoir for this yellowfin gobies from the first sampling and one parasite in farm sites, control of scoliosis might be Richardson dragonet from each sampling). This achieved by reducing the population of goby by discrepancy could be explained by the difference enhanced fishing, or by avoidance of goby-pre- in the detection threshold of the two assays. Alter- ferred habitats, such as muddy estuaries. However, natively, it is possible that the PCR test detected the empirically, there appears to be no obvious rela- presporogonic stages of the myxosporean, which tionship between disease incidence and estuary could not be found by microscopic examination. sites. This suggests that the parasites from yellow- It should be noted that M. acanthogobii does not tail and yellowfin goby could be different ‘strains’ cause skeletal abnormalities in the yellowfin goby. or ‘populations’ of the same species. Only a little Maeno and Sorimachi indicated that the myx- information is available on the life cycles of marine osporean infection in the fourth ventricle of the myxozoans, but the involvement of alternate inver- brain mechanically affects the central nervous sys- 1042 FISHERIES SCIENCE H Yokoyama et al.
tem of yellowtail to induce motor disturbances.3 parasitic Myxobolus buri. Bull. Natl. Res. Inst. Aquacult. This parasite has also been found in other wild fish 1987; 12: 79–86. (e.g. Japanese bluefish, red gurnard and brown- 2. Egusa S. Myxobolus buri sp. n. (Myxosporea: Bivalvulida) lined puffer), which exhibit similar skeletal defor- parasitic in the brain of Seriola quinqueradiata Temminck mities (Y Maeno & M Sorimachi, unpubl. data, et Schlegel. Fish Pathol. 1985; 19: 239–244. 3. Maeno Y, Sorimachi M. Skeletal abnormalities of fishes 1991). Hoshina also noted that the brown-lined caused by parasitism of Myxosporea. NOAA Tech. Rep. puffer could be infected with a myxosporean sim- NMFS 1992; 111: 113–118. ilar to M. acanthogobii, but did not mention a pos- 4. Okada YK. Myxosporidie du cerveaux et de la moelle d’un 5 sible relation to spinal curvature in the host fish. Canthigaster (poisson tiliosteen). Bull. Soc. Zool. France Several myxosporean infections in the brains of 1932; 57: 39–44. fishes have been reported, but they are not always 5. Hoshina T. Notes on some myxosporidian parasites on associated with skeletal anomalies. Further studies fishes of Japan. J. Tokyo Univ. Fish 1952; 39: 69–89. are required to understand the varying pathologi- 6. Lom J, Arthur JR. A guideline for the preparation of species cal effects observed in different host fishes. descriptions in Myxosporea. J. Fish Dis. 1989; 12: 151–156. In conclusion, morphological and molecular 7. Hillis DM, Dixon MT. Ribosomal DNA: Molecular evolution and phylogenetic inference. Quart. Rev. Biol. 1991; 66: 411– analyses in the present study have demonstrated 453. that M. buri is synonymous with M. acanthogobii, 8. Sanger F, Nicklen S, Coulson AR. DNA sequencing with which was previously described in the brain of the chain terminating inhibitors. Proc. Natl Acad. Sci. 1977; 74: yellowfin goby. Consequently, this economically 5463–5547. important parasite should be reassigned as 9. Schlegel M, Lom J, Stechmann A, Bernhard D, Leipe D, Dyk- M. acanthogobii. ova I, Sogin ML. Phylogenetic analysis of complete small subunit ribosomal RNA coding region of Myxidium lie- berkuehni: evidence that Myxozoa are Metazoa and related ACKNOWLEDGMENTS to the Bilateria. Arch. Protistenk 1996; 147: 1–9. 10. Kent ML, Andree KB, Bartholomew JL, El-Matbouli M, We thank Dr Makoto Iwashita and other members Desser SS, Devlin RH, Feist SW, Hedrick RP, Hoffmann of our laboratory for collecting materials, and Mr RW, Khattra J, Hallet SL, Lester RJG, Longshaw M, Palenzuela O, Siddal ME, Xiao C. Recent advances in our Tetsuya Yanagida, The University of Tokyo, for tak- knowledge of the Myxozoa. J. Eukaryot. Microbiol. 2001; ing photographs of the spores and measuring their 48: 395–413. dimensions. 11. Yokoyama H. A review: Gaps in our knowledge on myxo- zoan parasites of fishes. Fish Pathol. 2003; 38: 125–136. 12. Køie M, Whipps CM, Kent ML. Ellipsomyxa gobii (Myxozoa: REFERENCES Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: 1. Sakaguchi S, Hara T, Matsusato T, Shibahara T, Yamagata Y, Polychaeta) as invertebrate hosts. Folia Parasitol. 2004; 51: Kawai H, Maeno Y. Scoliosis of cultured yellowtail caused by 14–18.