魚 病 研 究Fish Pathology,39(2),79-85,2004.6 2004 The Japanese Society of Fish Pathology

Kudoa thyrsites from Japanese Flounder and lateolabracis n. sp. from Chinese Sea Bass: Causative Myxozoans of Post-Mortem Myoliquefaction

Hiroshi Yokoyama1*, Christopher M. Whipps2, Michael L. Kent2, Kaori Mizuno3 and Hidemasa Kawakami3

1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan 2Center for Fish Disease Research , Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3404, USA 3 Ehime Prefectural Fish Disease Control Center , Uwajima, Ehime 798-0087, Japan

(Received November 17, 2003)

ABSTRACT--Post-mortem myoliquefaction caused by myxozoans of the Kudoa was observed in Japanese flounder Paralichthys olivaceus and Chinese sea bass Lateolabrax sp. cultured in Japan. Morphological and molecular analyses of the myxozoan from Japanese floun der identified it as , which has been described from various marine fishes in different oceans. The parasite from Chinese sea bass was similar to K. thyrsites, having stellate spores with one polar capsule larger than the other three. However, the spore size was smaller than that of K. thyrsites. The small subunit rDNA sequence from the Kudoa sp. of Chinese sea bass was distinct from that of K. thyrsites and phylogenetic analysis placed it as an outlier to K. thyrsites. Therefore, we describe the myxozoan from Chinese sea bass as Kudoa lateolabracis n.sp.

Key words: myxozoan, Kudoa thyrsites, Kudoa lateolabracis, Paralichthys olivaceus, Lateolabrax sp., Japanese flounder, Chinese sea bass, myoliquefaction

Post-mortem myoliquefaction, commonly referred to (Clupea harengus), K. cruciformum in as 'soft flesh','milky condition' or 'jellied meat' of wild Japanese sea bass (Lateolabrax japonicus), K. funduli in and cultured fishes is a well-known disease which is mummichog (Fundulus heteroclitus), K. histolytica in caused by myxozoan parasites, mostly belonging to the Atlantic mackerel (Scomber scombrus), K. mirabilis in genus Kudoa. Muscle degeneration is due to the pro ribbonfish (Trichiurus haumela), K. musculoliquefaciens teolytic enzymes possibly released from the pre in (Xiphias gladius), K. paniformis in Pacific sporogonic plasmodia of the parasite (Stehr and hake (Merluccius productus), K. peruvianus in Chilean Whitaker, 1986). Although there was no observable ef hake (Merluccius gayi), K. rosenbuschi in Argentine fect on physiology, behavior, or survival of the host fish, hake (Merluccius hubbsi) and K. thyrsites in farmed Kudoa infections have been of concern to (Salmo salar) and various marine fishes. and commercial fisheries due to their negative impact on Since the late 1980's, aquaculture of Japanese product quality (Moran et al., 1999a). Kudoa is com flounder (Paralichthys olivaceus) has expanded rapidly, prised of more than 40 identified (Moran et al., and its annual production has reached approximately a; Swearer and Robertson, 1999), and the following1999 7,000 metric tons in recent years. Japanese flounder is have been reported as causative agents of post-mortem usually raised in land-based tanks with running seawa myoliquefaction (Moran et al., 1999a): K. clupeidae in ter, and on occasion, several parasitic infections, e.g., ciliates and monogeneans, have become fatal problems * Corresponding author (Ogawa and Yokoyama, 1998). Accompanied by diver E-mail: [email protected] sification of cultured fish species, growing interest of 80 H. Yokoyama, C. M. Whipps, M. L. Kent, K. Mizuno and H. Kawakami

Chinese sea bass (Lateolabrax sp.) culture has led to cession numbers. Briefly, polymerase chain reaction the increased importation of seedlings to Japan from (PCR) primers and procedures of Whipps et al. (2003) China. As opposed to the Japanese sea bass were used to amplify overlapping fragments of SSU (Lateolabrax japonicus), Chinese sea bass has a greater rDNA. Fragments were sequenced in both directions tolerance to brackish or fresh water and a faster growth using PCR primers with AP Biotech(r) DYEnamic ET rate, suggesting that it is a promising species for marine Terminator cycle sequencing chemistry with Thermo finfish aquaculture. Sequenase II (Amersham Biosciences, USA) on an ABI Recently, muscle liquefaction was observed in fillets PRISM(r) 377 DNA Sequencer (Applied Biosystems, of cultured Japanese flounder and Chinese sea bass, USA). and stellate Kudoa spores were detected in these in Myxozoan small subunit (SSU) sequences for avail fected tissues. In the present study, we conducted able for members of the order of approxi morphological and molecular analyses of the parasites mately 1600 bp were aligned with ClustaIX (Thompson with the aim of identifying the causative organisms. et al., 1997) and examined by eye. A single variable sequence region of approximately 100 bp was realigned with Malign (Wheeler and Gladstein, 1994) with 10 Materials and Methods random sequence additions, an internal gap cost of 3, Fish sampling extra gaps cost 1, and tree bisection and reconnection Fillets of Japanese flounder (body weight, ca. 800 g) branch swapping. The following sequences (with showing the muscle degeneration were brought to our GenBank accession numbers) were used: Ceratomyxa laboratory from a processing company in Ehime Prefec shasta (AF001579), Kudoa amamiensis (AF034638), ture, western Japan, on January 10th and May 7th, Kudoa crumena (AF378347), Kudoa dianae 2003. The seedlings of Japanese flounder were im (AF414692), Kudoa lateolabracis n. sp. (AY382606), ported to Japan from Korea about one year before the Kudoa miniauriculata (AF034639), Kudoa minithyrsites harvest and then farmed in land-based tanks in Ehime (AY152749), Kudoa ovivora (AY152750), Kudoa Prefecture. Samples of liquefied Chinese sea bass paniformis (AF034640), Kudoa permulticapsula (body weight, ca. 2.3 kg) were transferred to our labora (AY078429), Kudoa quadricornis (AY078428), Kudoa tory on March 6th and June 30th, 2003. The seedlings shiomitsui (AY302724), Kudoa sp. from southern floun of Chinese sea bass were imported from China and der (Paralichthys lethostigma) (AY302723), K. thyrsites cultured in sea-cages in the Seto Inland Sea off Ehime from mahi mahi (Coryphaena hippurus) (AY152747), Prefecture. Incidence of •esoft flesh' (generalized K. thyrsites from Salmo salar (AF031412), K. thyrsites muscle liquefaction or even several pale foci) was exam from Thyrsites atun (AY078430), K. thyrsites from ined by visual observation and defined as the number of Japanese flounder (AY382607), Pentacapsula fish with liquefied area per the number of fish neurophila (AY172511), Hexacapsula sp. ex Gram examined. Prevalence of infection, the number of fish matorcynus bicarinatus (AY302723), Hexacapsula infected per number of fish examined, was determined sp. ex Scomberomorus commerson (AY302739), by microscopic observation for the presence of spores. Hexacapsula sp. ex Thalassoma lunare (AY302738), Infected muscle tissues were preserved in frozen for Septemcapsula yasunagai (AY302741) . morphological examination, in 10% formalin for histol Phylogenetic analyses of aligned sequences was ogy, and in 95% ethanol for DNA analysis. conducted using PAUP*4.0b1 (Swofford, 1998). Parsi mony analysis used the heuristic search algorithm with Morphological examination 50 random additions of sequences and tree bisection Thawed muscle tissues were examined macro reconnection (TBR) branch swapping. Bootstrap val scopically and followed by wet-mount preparations of ues were calculated with 1000 replicates using the heu muscle lesions using agar-coated slides for light micro ristic search algorithm with simple sequence addition scopical observations. Descriptions and measure and TBR branch swapping. Distance and maximum ments were made according to Lom and Arthur (1989) likelihood analyses were also performed. Distances and Langdon (1991). Formalin-fixed tissues from Chi were calculated using the HKY85 evolutionary model nese sea bass were processed by routine histology, and and a tree was constructed with the neighbor-joining

paraffin sections cut at 5 ƒÊm thick were stained with H & method. Maximum likelihood (ML) also employed the E or Diff-Quik and observed by light microscopy. HKY85 model and a heuristic search algorithm with 10 random sequence additions and TBR branch swapping. Molecular analysis Bootstrap values were calculated with 100 replicates for Small subunit rDNA sequence of approximately MI trees distance and 1680 bp was obtained for K. thyrsites from Japanese flounder and K. lateolabracis n. sp. from Chinese sea bass and deposited in GenBank with the following ac Kudoa causing myoliquefaction 81

able among the host species and the localities (Table Results 1) Spores from Chinese sea bass closely resemble to Diagnosis those of K thyrsites, having stellate spores with one po Based on visual observations of 'soft flesh' in har lar capsule larger than the other three (Figs 3 and vested fish, the incidence in Japanese flounder 4) However, they were clearly smaller than those of K appeared to be very low (less than 1%), whereas that in thyrsites and showed considerable differences in Chinese sea bass was 15% (3 out of 20) at one farm SSU rDNA sequence We, therefore, described the site No marked variation of disease incidence in differ myxozoan from Chinese sea bass as a new species ent localities was observed The somatic musculature Histological observations revealed that parasites of affected Japanese flounder and Chinese sea bass developed intracellulary in the muscle fibers (Fig exhibited a similar sign of the disease, a multifocal, pale 5A) No inflammatory response was observed directly blemish distributing sporadically in the trunk muscle, or generalized liquefaction in heavily infected fish (Fig 1) Microscopic examination revealed that spores from A B Japanese flounder were consistent with those of K thyrsites (Fig 2), though the spore dimensions are van

C D

Fig. 1. Liquefied somatic musculature of a fillet of cultured Fig. 2. Fresh spores of Kudoa thyrsites from Japanese Japanese flounder Paralichthys olivaceus Scale bar flounder A-C Top view, D Side view Scale bar = =5cm 5 ƒÊm

Table 1 Comparison of spore dimensions (ƒÊm) of Kudoa spp having stellate spores with unequal polar capsules (PC) 82 H Yokoyama, C M Whipps, M L Kent, K Mizuno and H Kawakami

A

Fig. 3. Schematic illustration of Kudoa lateolabracis n sp Left To view, Right Side view, Scale bar = 5 urn

A B B

C D

Fig. 5 Histological sections of Kudoa lateolabracis n sp from Chinese sea bass A Intracellular plasmodium in the muscle fiber, B Liquefaction of the musculature associated with dispersion of spores H & E stain Scale bar = 50 ƒÊm Fig. 4. Fresh spores of Kudoa lateolabracis n sp from Chi nese sea bass Lateolabrax sp A & B Top view, C & D Side view Scale bar = 5 ƒÊm length and 2 5 (1 5-3 0) ƒÊm in width Small polar cap sules were 2 8 (2 0-4 0) ƒÊm in length and 1 6 (1 5-2 5)

related to muscle fibers that contained the developing pm in width parasite Following maturation of the parasite, spores Remarks Small subunit rDNA sequence was most were liberated between disintegrated myofibers, sug similar to that from K thyrsites SSU sequence from Brit gesting that liquefaction of the musculature was ish Columbia, Canada, and showed 99 1% similarity. associated with spore dispersion (Fig 5B)

Kudoa lateolabracis Yokoyama, Whipps & Kent n sp

Description of parasites Type-host Chinese sea bass, Lateolabrax sp

Kudoa thyrsites (Gilchrist, 1924) (Percichthyidae, ) Host Japanese flounder, Paralichthys olivaceus Type-locality The Seto Inland Sea, Ehime Prefecture,

(Paralichtyidae Pleuronectiformes) Japan Locality Ehime Prefecture, Japan Prevalence of infection 33% (n = 6)

Prevalence of infection Not determined but probably Spores (Figs 3 and 4) Mature spores stellate in apical less than 1% view Four polar capsules pyriform in shape, one larger

Spores (Fig 2) Mature spores stellate in shape, with than the other three Polar capsules containing fila four unequal pyriform polar capsules Spore dimen ments with one to two loose coils Single bi-nucleate sions (n = 20, Table 1) were 14 7 (12 9-17 8) ƒÊm in sporoplasm at posterior Spore dimensions (n = 20, thickness, 10 0 (7 9-11 9) pm in maximum width, 7 6 Table 1), 11 5 (9 9-12 9) pm in thickness, 9 3 (8 4-9 9)

(6 9-7 9) ƒÊm in minimum width, 7 8 (6 9-8 9) ƒÊm in pm in maximum width, 6 5 (5 9-6 9) pm in minimum length Large polar capsules were 5 0 (4 0-5 9) ƒÊm in width, 6 4 (5 4-6 9) pm in length Large polar capsules Kudoa causing myoliquefaction 83

Fig. 6. Systematic tree of Kudoa myxozoans based on SSU rDNA sequences. Bootstrap confidence values shown at nodes.

were 5.2 (4.0-5.9) ƒÊm in length and 2.8 (2.5-3.5) ,ƒÊm in Discussion width. Small polar capsules were 3.6 (3.0-4.0) ,ƒÊm in

ength and 1.9 (1.5-2.0) ,ƒÊm in width. I Based on morphology and molecular analysis, the

Site of infection: Somatic muscle. Plasmodia observed myxozoan from Japanese flounder was identified as K.

within muscle fibers. thyrsites, one of the most noteworthy myxozoans of the

Type-material: Syntype specimens deposited in the order Multivalvulida, as it causes post-harvest

collection of the National Science Museum, Tokyo, myoliquefaction (Moran et al., 1999a). Kudoa thyrsites

Japan, accession no. NSMT-Pr 179. has been described from 35 fish species from several

Remarks: Kudoa lateolabracis n. sp. resembles K. families around the world, e.g., pen-cultured Atlantic

thyrsites, K. histolytica, K. cruciformum and K. salmon (Salmo salar) and wild-caught flatfish

minithyrsites, having stellate spores and unequal polar (Paralichthys adspersus) from Chile (Castro and Burgos,

capsules. However, the spores of K. lateolabracis n. 1996). In Japanese waters, three wild host species,

sp. (particulary, in thickness) are distinctly larger than Japanese (Engraulis japonicus), flying fish

those of K. minithyrsites, and smaller than those of the (Cypsilurus ago) and dolphin fish (Coryphaena hippurus)

other three species (Table 1; see perard (1928), have been reported to be parasitized by K. thyrsites

Matsumoto (1954), Kabata and Whitaker (1981), (Matsumoto, 1963; Langdon et al., 1992). The present

Langdon (1991), Shaw et al. (1997) and Whipps et al. study is the first report of K. thyrsites from farmed fish in

(2003)). Parsimony analysis of SSU rDNA sequence Japan, implying that K. thyrsites infection may be a consistently placed K. lateolabracis n. sp. as an outlier to potential threat not only to Japanese flounder culture but

a group of K. thyrsites representatives from British to other fish species culture in Japan, due to the

Columbia, England, Queensland, and Japan with strong parasite's broad host range.

bootstrap support (Fig. 6). Whether K. lateolabracis n. Morphological and molecular analyses demon

sp. was sister to K. minithyrsites or an outlier to K. strated that the myxozoan from Chinese sea bass was

minithyrsites and K. thyrsites was unresolved in our distinct from hitherto described species of Kudoa, and

analysis, reflected by poor bootstrap support at the K. assigned to a new species as K. lateolabracis n. sp.

minithyrsites branch. Distance and ML analysis yielded Parsimony analysis suggested that K. lateolabracis n.

similar trees. Based on genetic distance (uncorrected sp. was most closely related to K. minithyrsites (Fig. 6),

p-distance) K. lateolabracis n. sp. was 2.5% distant from and both have spores resembling K. thyrsites except that K. minithyrsites and 2.4% to 2.6% distant from K. they are smaller. We concluded that these myxozoan

thyrsites representatives. The possibility that K. were separate species based on differences in spore

lateolabracis n. sp. may represent a morphological vari size, hosts and geographic locations. In addition,

ant of K. thyrsites is refuted by our molecular analyses, analysis of SSU rDNA sequences indicates that they are

as it is distinct, even from the sympatric K. thyrsites from different; on average, K. lateolabracis n. sp. is 2.5% dis

Japanese flounder. tant from K. thyrsites, whereas the most distant sequences between K. thyrsites samples, based on 4 sequences in GenBank from representative hosts 84 H. Yokoyama, C. M. Whipps, M. L. Kent, K. Mizuno and H. Kawakami around the world, is 1.0%. the samples and a photograph of infected fish, and Dr. At first, we suspected that K. lateolabracis n. sp. Sung-Joung Lee for processing the histological sections. might be conspecific with K. cruciformum which was pre viously reported from Japanese sea bass. However, References the spore morphology distinctly separated them into the two different species (Table 1). Kudoa cruciformum Castro, R. R. and R. Burgos (1996): Kudoa thyrsites (, was originally described as Neochloromyxum Multivalvulida) causing "milky condition" in the musculature of Paralichthys adspersus (Neopterygii,Pleuronectiformes, cruciformum, having two spore valves (Matsumoto, Paralichthyidae) from Chile. Mem. Inst. Oswaldo Cruz, 1954). Later, however, Kudo (1966) and Shulman Rio de Janeiro, 91, 163-164. (1966) transferred the genus Neochloromyxum into Egusa, S. (1986): The order Multivalvulida Shulman, 1959 Kudoa, assuming that the description of Matsumoto (Myxozoa: ): a review. Fish Pathol, 21, 261- 274. (1954) was incorrect. If this parasite really belongs to Kabata, Z. and D. J. Whitaker (1981): Two species of Kudoa Kudoa, it might be synonymized to K. thyrsites, due to (Myxosporea: Multivalvulida) parasitic in the flesh of the close resemblance in morphology. However, the Merluccius productus (Ayres, 1855) (Pisces: Teleostei) in identification of this parasite has been deferred until the the Canadian Pacific. Can. J. Zool, 59, 2085-2091. presence of four, not two, valves is confirmed (Kabata Kudo, R. (1966): Protozoology, 5th ed. C. C. Thomas, and Whitaker, 1981; Egusa, 1986). Springueld, Illinois, 1174 pp. Langdon, J. S. 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