Heavy metacercarial infections of cyprinid fishes in Uji River

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1 Blackwell Science, LtdOxford, UK FISFisheries Science Blackwell Science Asia Pty Ltd 701February l infection in Uji River K Ogawa et al /j x Original Article132140BEES SGML FISHERIES SCIENCE 2004; 70: Heavy metacercarial infections of cyprinid fishes in Uji River Kazuo OGAWA, 1 * Toshio NAKATSUGAWA 2 AND Masayoshi YASUZAKI 1 1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo and 2 Kyoto Institute of Oceanic and Fishery Science, Miyazu, Kyoto , Japan ABSTRACT: Between January and April 2000, the cyprinid fishes Zacco platypus and Squalidus chankaensis in Uji River, Kyoto Prefecture showed inactive swimming and hemorrhages on the fins, skin and eyes. No bacterial or viral agents seemed involved in the disease outbreak; however, numerous metacercariae of unidentified trematodes of the family Bucephalidae were found encysted in the fins, skin, musculature and eyes of diseased fish, suggesting these parasitic infections were the etiological agents. The metacercariae comprised two species, designated here as A and B. It is assumed that the infection started with an accidental introduction of infected first intermediate hosts, the freshwater mussel, Limnoperna fortunei, into Uji River, and that Z. platypus and S. chankaensis served as the second intermediate hosts, and the sheatfish Silurus biwaensis as the final host to complete their life cycles. This is the first case of bucephalid infections of freshwater fishes in Japan. The Yodo River system, including Uji River, is the only area in Japan where the bucephalid infections have so far been confirmed. Comments are made on the possible danger of translocation of even a single infected first, second or final host from Uji River to Lake Biwa or other water systems. KEY WORDS: Bucephalidae, Digenea, Limnoperna fortunei, mass mortality, metacercaria, Zacco platypus. INTRODUCTION In Uji River, the cyprinid fish Zacco platypus (Japanese name: oikawa) is a popular species for angling, particularly in winter. In January 2000, large schools of Z. platypus with hemorrhages on the fins and skin were found swimming inactively in the shallow water near the banks of the river. Some fish became sufficiently weakened as to be easily caught by hand nets or apparently dead. Another cyprinid fish, Squalidus chankaensis (Japanese name: kohrai-moroko), was the only other fish species showing similar signs of disease. Based on the information collected from Uji River Fisherman s Cooperative Association and from anglers, moribund fish were noticed from the beginning of January until mid-april 2000 (water temperature: 6 12 C), and were most frequently observed in January and February The number of Z. platypus in the river had considerably decreased in March and April. Such a disease outbreak has *Corresponding author: Tel: Fax: aogawak@mail.ecc.u-tokyo.ac.jp Received 16 July Accepted 8 October never been noticed before during any season in this river. Surveys on the etiological agent revealed that diseased fish were heavily infected with metacercariae of trematodes of the family Bucephalidae in the fins, skin and other tissues near the body surface (Fig. 1). This is the first recorded case of infection of freshwater fish with bucephalid trematodes in Japan. The present study describes the observed features of the disease, the pathology condition of the fish and morphology and preliminary identification of the parasites. MATERIALS AND METHODS Study site Uji River is part of the Yodo River system, which originates from Lake Biwa, runs through Shiga, Kyoto and Osaka Prefectures and flows into Osaka Bay (Fig. 2). This water system consists of three continuous rivers, starting from Seta River in Shiga Prefecture, followed by Uji River in Kyoto Prefecture and finally Yodo River in Osaka Prefecture.

2 l infection in Uji River FISHERIES SCIENCE 133 Lake Biwa Seta River Yodo River Amagase Dam Uji River Osaka Bay 10 km Fig. 2 Map of the Yodo water system. other five fish with external lesions, the kidney was plated onto BHI agar (Oxoid Ltd, Basingstoke, UK) and sheep blood agar media (Nippon Becton Dickinson Co. Ltd, Tokyo, Japan). Plates were incubated at 18 C for 10 days. Fig. 1 Histological section of the caudal part of Squalidus chankaensis with numerous metacercariae collected in April Azan stain. Scale bar: 1 mm. Disease survey On 20 January 2000 when the outbreak of this disease was first investigated, fish with abnormal swimming behavior were only noticed in the area downstream from the Amagase Dam in Uji, Kyoto Prefecture. Moribund Z. platypus, with 10.7 cm in average fork length (FL) and 14.5 g in average body weight (BW) (n = 15), were collected by hand nets and examined grossly for external disease signs. External lesions were further observed under a dissecting microscope in five of these fish. Of the 15 diseased fish collected, 10 were examined for bacterial pathogens and the remaining five for isolation of viral agents, as described below. Bacteriological examination Bacterial isolation was attempted from the brain, using modified Cytophaga agar medium. For the Viral examination Viral isolation was attempted from the kidney of the above five fish. Kidneys were sampled, diluted (1 : 10) with sterile PBS( ), homogenized and centrifuged at 5 C at 2260 g for 15 min. The supernatant was passed through a 0.45 mm filter. Subsequently, 50 ml of the aliquot was inoculated onto CHSE-214 or FHM cell lines in 50 ml tissue culture flasks containing 5 ml E-RDF medium (Kyokuto Ltd, Tokyo, Japan) supplemented with 5% FBS (Bio Whittaker Inc., Walkersville, MD, USA) and incubated at 18 C for 1 month. Parasitological examination As all the fish examined in January were heavily infected with parasites of unknown taxonomic identity, parasitological examinations were conducted separately from the above survey for the description of the parasite and determination of the infection level. Fresh parasites were collected from Z. platypus and S. chankaensis in Uji River on 18 February and 6 April As they were encap-

3 134 FISHERIES SCIENCE K Ogawa et al. sulated within the host tissue, the parasites were observed using a compound microscope or with phase-contrast lighting after removal of the outer host connective tissue and an inner thin cyst wall of parasite origin. Fresh parasites in cysts were flattened between a coverslip and slide glass for measurement of the cysts. For a morphological description of the parasites, they were flattened as above and fixed in AFA (mixture of 70% ethanol 20 parts, 35% formaldehyde 1 part and acetic acid 1 part) after being released from the cysts in Z. platypus on 18 February and 6 April 2000 and in S. chankaensis on 6 April. Parasites were stained either with Heidenhein s iron hematoxylin or aceto-carmine, dehydrated, cleared in xylene and mounted in Canada balsam. Intensity of infection was determined by counting the number of parasites in the whole body of formalin-fixed Z. platypus (average FL 11.1 cm; average BW 14.7 g; n = 3) and Squalidus chankaensis (average FL 7.4 cm; average BW 3.8 g; n = 3) collected on 6 April. Histopathological examination Diseased Z. platypus sampled on 25 January, 18 February or 12 April 2000 (average FL 8.3 cm; average BW 7.2 g; n = 6) and S. chankaensis sampled on 12 April (average FL 7.0 cm; average BW 3.4 g; n = 6) were fixed in Bouin s solution or 10% formalin for histological examination. Paraffinsectioned materials were stained with hematoxylin and eosin (H&E) or Azan for microscopical observation. RESULTS Gross and microscopic observation of diseased fish Diseased fish had hemorrhagic lesions in the eyes, head, fin bases and erosions in the peduncle and at the base of the caudal and dorsal fins and, on occasion, pop-eye and pale gills. Stereomicroscopic observations of the eyes, caudal fin, peduncle and gills revealed parasites, mm in length, in the lesions. Parasites were numerous in the severe lesions of the caudal fin and peduncle, whereas only a small number were present in the gills. The parasites contained within the ellipsoid-shaped cysts could be seen actively stretching and contracting. Bacteriological and viral examinations From the external signs of the diseased fish and as outbreaks occurred at low temperatures, bacterial coldwater disease was suspected as the etiological agent. However, Flavobacterium psychrophilum was not isolated from the brains of the 10 fish examined and no bacteria were cultured from the kidneys of the five Z. platypus on BHI or sheep blood agar plates. No cytopathic effect (CPE) was detected in either the CHSE-214 or FHM cell lines during a 1- month culture period. These cell lines were further subcultured, but no subsequent CPE was apparent. Parasitological examination After the removal of host connective tissue, parasites were seen in either a thin- or rather thickwalled cyst (Fig. 3a,b). In April 2000, fresh parasites in a thin-walled cyst (Fig. 3a), elliptical or oval in shape, measured mm long by mm wide (n = 20) in Z. platypus and mm long by mm wide (n = 20) in S. chankaensis. They contained numerous, probably calcareous, bodies throughout the body, an elliptical structure in the center and a dark-colored tube originating from the body end. Observation of the parasites after being released from the cyst revealed that they had a mouth and pharynx in the central part of the body, and that the elliptical structure was the intestine. This indicates that they were metacercariae of the family Bucephalidae (Trematoda). Parasites within a thin-walled cyst are designated here as A (Fig. 3a) and those in a thick cyst wall as B (Fig. 3b). The latter species was rarely collected, but the central position of the mouth indicated that it was also a bucephalid metacercaria. The number of metacercariae (mostly A) from the whole body of Z. platypus and S. chankaensis collected on 6 April 2000 ranged from 650 to 9700 (average 5100) and from 1050 to 5110 (average 2890), respectively. Parasites were most abundant at the base of the fins, especially the caudal fin. Morphology of the metacercariae Stained metacercariae used for morphological descriptions also contained two species (Fig. 4), A constituting more than 90% of all the specimens examined. Measurements are shown in Table 1. Description of A (Fig. 4a c): Body is elongated and tapering toward both ends. The anterior adhesive apparatus is glandular, width being slightly larger than its length. Mouth opens at a level about two-thirds from the anterior end of the body. In most specimens, esophagus directs obliquely anteriorly and intestine obliquely poste-

4 l infection in Uji River FISHERIES SCIENCE 135 Fig. 3 Live parasites seen in a cyst wall of parasite origin. (a) A in a thin cyst wall (arrow), collected in February 2000, (b) B in a thick cyst wall (arrow), collected in April Scale bars: 0.1 mm. Fig. 4 Drawings of the two types of metacercariae. (a) A, ventral view, from Zacco platypus in February 2000, (b) A, ventral view, from Z. platypus in April 2000, (c) A, dorsal view, from Squalidus chankaensis in April 2000, (d) B, dorsal view, from S. chankaensis in April Scale bar: 300 mm. riorly, forming a V-shaped angle between esophagus and intestine. Excretory bladder is a narrow and straight tube, directing obliquely anteriorly, terminating before the level of the mouth. No morphological difference was found between specimens from Z. platypus and those from S. chankaensis. Remarks: The April specimens from Z. platypus were larger than the February specimens. Testes and ovary were poorly developed in February, but were more clearly visible in April, suggesting that invasion of the parasites into these fishes had ceased by April. In contrast, the relative position of the mouth opening on the body was quite stable among both specimens in February and April (DM/BL value in Table 1). There was no difference in the body length between the April specimens from Z. platypus and those from S. chankaensis (Wilcoxon test; P = 0.52). Description of B (Fig. 4d): Body is shorter than that of A. The anterior adhesive apparatus is a rounded sucker, width being as long as its length. Mouth opening is slightly muscular. Excretory bladder is a wide tube, directing obliquely anteriorly and terminating anterior to the mouth.

5 136 FISHERIES SCIENCE K Ogawa et al. Table 1 Host Parasite Measurements (in mm) of stained metacercariae from fishes in Uji River Date of sampling No. specimens examined Zacco platypus A 18 Feb Zacco platypus A 6 Apr Squalidus chankaensis A 6 Apr Zacco platypus B 18 Feb Squalidus chankaensis B 6 Apr Body: length (BL) width Oral sucker: length width Pharynx diameter Intestine: length width Length: anterior end to mouth (DM) Ratio: DM/BL % % % 73.1% % Excretory bladder: length (LEB) NO Ratio: LEB/BL % % % % Terminal genitalia: length NO Length in the horizontal level between the body end and anterior end of excretory bladder. Length in the horizontal level between the body end and anterior end of terminal genitalia. NO, Not observed. Remarks: This species is easily separated from A by the structure of the anterior adhesive apparatus and the excretory bladder. Histopathological observations From Z. platypus sampled in January and February 2000, numerous metacercairae, almost all of which were presumed to be A, were encysted near the surface over the whole body, especially at the basal part of the fins. The caudal fin was the most heavily infected site, harbouring parasites in the fin membrane and in the musculature surrounding the pterygiophore (Fig. 5a c). Some were also found in the cartilaginous tissue surrounding the pterygiophore. Heavy infection was sometimes accompanied by hemorrhages in the surrounding tissue (Fig. 5a,d,e). Host response including encapsulation of, and cell infiltration around, the parasite was observed in the connective tissue of the fins (Fig. 5b), but was generally weak or even negligible in the musculature (Fig. 5d). Rodlet cells were the main component of the infiltrating cells (Fig. 5c). e invading the eye were associated with hemorrhages around the worm (Fig. 5e). The two Z. platypus sampled in April 2000 were lightly infected, and cyst encapsulation was thicker than was observed in previous samples. Parasites in the fins were irregularly surrounded by up to several layers of fibroblasts (Fig. 5f). This thick encapsulation implies that cercarial invasion had subsided since February. In April, heavily infected S. chankaensis were also collected. Similarly, metacercarial cysts were observed in the cutaneous, muscle and cartilaginous tissues of the host (Fig. 6a). Heavy infection was often accompanied by hemorrhages and dissolution of muscle fibers around the cysts (Fig. 6a,b). Two types of cysts were recognized: cysts of A and B. The former were the same as those observed in Z. platypus, while the latter were encapsulated in collagenous tissues (Fig. 6c,d). A were always the dominant species, comprising more than 90% of metacercariae in histological sections. B were more frequently found in the musculature (about 12% [28 out of 235 metacercariae] examined histologically) than in the other tissues (only about 0.8% [3 out of 375 metacercariae]). No other potential pathogens such as bacteria or fungi were observed in histological sections of host tissues. DISCUSSION Bacteriological and histopathological examinations of Zacco platypus with erosion of the caudal fin and hemorrhages in the tail and eyes, collected

6 l infection in Uji River FISHERIES SCIENCE 137 a b Fig. 5 e encysted in Zacco platypus sampled on January, February or April (a) Many parasite cysts in the caudal fin, accompanied by local hemorrhage (arrow) (January), H&E. (b) Host encapsulation of parasites in the caudal fin (February), H&E. (c) Infiltration of rodlet cells (arrows) around cysts in the caudal fin (February), H&E. (d) Cysts associated with local hemorrhage (arrow) but no visible host encapsulation in the musculature surrounding the pterygiophore of the caudal fin (February), H&E. (e) Eye, showing hemorrhage (arrow) around the worm (February), H&E. (f) e encapsulated by several layers of host fibroblasts in the caudal fin (April), Azan. Scale bars: 400 mm for a, 200 mm for d and e, 100 mm for b and f and 40 mm for c. c e d f Fig. 6 e encysted in Squalidus chankaensis sampled in April (a) Thickened caudal fin membrane, caused by the presence of numerous parasites, with local hemorrhages (arrows), Azan. (b) Dissolution of muscle fibers (arrow) adjacent to a cyst in the musculature surrounding the pterygiophore of the caudal fin, Azan. (c) Cysts in the musculature at the basal part of the caudal fin represent those of A except one indicated by an arrow, representing B. A cyst in the center harbours two individuals of A. Azan. (d) Two cysts of B in the musculature, the left one harbouring two individuals, Azan. Scale bars: 400 mm for a, 200 mm for c and d, 100 mm for b. a c b d

7 138 FISHERIES SCIENCE K Ogawa et al. from Uji River in January 2000 revealed that neither bacteria nor fungi were involved in the disease. Furthermore, no CPE appeared on the cell lines inoculated with the kidney homogenates. Diseased fish with these gross signs were all heavily infected with metacercariae and it was confirmed histologically that parasites in the eye and fins were associated with local hemorrhages. Therefore, these external signs were thought to have been caused by the metacercarial infection, suggesting that this parasitic infection was the cause of the disease. Trematodes of the family Bucephalidae infect both marine and freshwater fish as the final hosts. 1 Their life cycle involves bivalves as the first intermediate hosts and fish both as the second intermediate hosts and as final hosts. In Japan, more than 20 species of bucephalid trematodes have been recorded from marine fish, 2 but none so far from freshwater fish. In the present study, Z. platypus and S. chankaensis serve as the second intermediate hosts for the trematode, harbouring metacercariae tentatively designated as A and B. Urabe et al. 3 surveyed molluscs from Uji River, and found that the freshwater mussel Limnoperna fortunei, an introduced bivalve from eastern Asia probably as a contaminant with imported hard clam, 4,5 was infected with sporocysts of a hitherto unknown bucephalid trematode. This strongly suggests that L. fortunei is the first intermediate host of either A or B, or both,although Urabe et al. did not confirm that their sporocysts represented one or two species. Furthermore, they found adult bucephalid trematodes from the intestine of the sheatfish Silurus biwaensis in the river. Identification of the adult worms remains for a future study. Thus, it is assumed that the life cycle of the trematodes described here started with an accidental introduction of infected L. fortunei into Uji River, where potential second and final hosts were distributed. Considering that more than 1 year is required for parasite numbers to build up to a sufficient infection level there to cause disease and that the parasites have an annual life cycle, the infection may have started in the late 1990s. The probable first intermediate host, L. fortunei, has been recorded in Japanese fresh waters since the early 1990s, first in Lake Biwa and later in several water systems including the Yodo River system. 5 Once introduced into a water system, this bivalve can easily propagate and is very difficult to eradicate. The Yodo River system, including Uji River, is the only area in Japan where the bucephalid infection has been confirmed. However, in Lake Biwa, also part of the Yodo River system, no such infection is known in either fish or bivalve hosts: L. fortunei, cyprinids or Silurus spp. (M. Urabe, unpublished data, 2002). This suggests that the infection originated in Uji River and that infected hosts are unable to move upstream to the lake. However, once even a single infected individual as the first, second or final host is introduced accidentally or intentionally to Lake Biwa or other water systems where L. fortunei is already established but no bucephalid infection confirmed, such an infected individual could be the source of possible disease outbreaks in these water bodies. This is because cyprinids and the sheatfish Silurus asotus, candidate second intermediate hosts and this final host, respectively, are commonly distributed there. As no control measures against the infection are available, translocation of such candidate animals from Uji River to other water systems should be strongly prohibited. Diseases of freshwater fish caused by bucephalid metacercarial infections have been reported in Europe. 6 8 Infections of Bucephalus polymorphus metacercariae were responsible for mass mortalities of cyprinid fishes. 6,8 Heavily infected fish showed abnormal swimming and hemorrhages in large areas including the head, eyes and fins. 8 Large masses of bacteria were demonstrated histologically in the damaged epidermis. 8 Host response included cell infiltration and fibroblast proliferation. 9 There are some differences between the B. polymorphus infections described above and the present case; metacercariae of B. polymorphus also invaded the gills 8 and visceral organs including the liver and kidney. 9 In the present case, encystment in the gills was rare and they never invaded deeper into the peritoneal cavity. Bacterial masses in the lesions were also not noticed in the present case. This may be because the outbreak occurred in August in Europe, whereas in Japan it was in winter, too cold for such bacterial proliferation. l infections of another bucephalid Rhipidocotyle illensis showed similar pathological effects on cyprinids. 9 e encysted mainly in the head region, including the eyes and gills, but sometimes penetrated deeper into the brain and peritoneal cavity, 9,10 with pathological changes including hemorrhages, necrosis, edema and inflammation. Zacco platypus fixed in April 2000 for histopathological examination were only moderately infected. However, Z. platypus fixed at the same time for other purposes included heavily infected fish similar to those sampled 2 months previously, suggesting that the apparent decrease in the infection level from February to April 2000 was not caused by the loss of parasites from the host during this period. It is possible that heavily infected fish had died or been carried downstream, leaving fewer heavily infected fish in the area by April.

8 l infection in Uji River FISHERIES SCIENCE 139 Two types of cysts, namely those of A and B, were recognized in S. chankaensis, while only cysts of A were found in Z. platypus histologically. This may be that S. chankaensis is more susceptible to infection by B than Z. platypus. However, B were also found in the parasitological examinations of Z. platypus (see Table 1), indicating that both fish can serve as a second intermediate host of B. B may have a greater site selectivity in fish, although it is not completely site specific; the musculature appears to be a more suitable habitat than the other tissues including the connective tissue of fin rays. However, with only six fish examined in the present study, further examination will be necessary to determine whether this parasite shows marked site selectivity. Two of the three specimens of B from S. chankaensis were obtained from a single cyst. Presence of more than one metacercaria in a single cyst has already been recorded in goldfish Carassius auratus experimentally infected with Parabucephalopsis prosthorchis. 11 Presence of two metacercariae in a single cyst is also demonstrated in a histological section of the caudal fin of S. chankaensis collected in April 2000 (Fig. 6d). From a year-round survey of L. fortunei in Uji River, Urabe et al. 3 indicated that the emergence of cercariae of the present bucephalids from the mollusc peaked in November January, when temperature ranged from 6 to 16 C. It is very unusual that the cercarial invasion into second intermediate hosts occurs at such low temperatures. In general, cercariae emerge from first intermediate hosts sometime from spring until autumn, the peaks of emergence being different from species to species, and cease to emerge as the water temperature decreases. 12,13 This is also the case for B. polymorphus and Rhipidocotyle illense (R. illensis), infecting freshwater fish, with the emergence of cericariae recorded from April to October and from May to October, respectively. 10 In Bucephalus varicus, a marine bucephalid, the cercarial emergence from the pearl oyster, Pinctada martensii, occurs from September to November. 14 Investigation into the pattern of cercarial release by L. fortunei will reveal the seasonality in the metacercarial infection of Z. platypus and S. chankakensis in Uji River in more detail. During the period of the disease outbreak (January April 2000), outflow of water from Lake Biwa to Uji River was limited because of low rainfall in the preceding months. Consequently, in Uji River, the water level was low and the current slow. This may have caused Z. platypus and S. chankaensis to become concentrated in shallower water and be subjected to increased cercarial exposure. The slow current may also have been favorable for the cercariae to invade these fishes. Zacco platypus examined the following winter, when the water level was normal, were all infected with the metacercariae, but the infection level was much lower. 3 It may be that the disease outbreak in early 2000 was largely affected by the low water level of the river. A long-term study in Uji River will reveal what factors contribute most to the infection levels among Z. platypus and S. chankaensis. ACKNOWLEDGMENTS We would like to thank the staff of Uji River Fisherman s Cooperative Association for collecting fish materials examined, and Dr Mark Freeman for critical reading of the manuscript. REFERENCES 1. Williams H, Jones A. Parasitic Worms of Fish. 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9 140 FISHERIES SCIENCE K Ogawa et al. ter fishes. Part II. Trematoda. Adv. Parasitol. 1979; 17: Paperna I. Digenea (Phylum Platyhelminthes). In: Woo PTK (ed). Fish Diseases and Disorders, Vol. 1. Protozoan and Metazoan Infections. CAB International, Wallingford. 1995; Sakaguchi S. Studies on a trematode parasite on the pearl oyster (Pinctada martensii). V. Development of its cercaria in the first intermediate host. Bull. Natl Pearl Res. Lab. 1965; 10: