Takifugu Rubripes

魚病研究 Fish Pathology,32(1),7-14,1997.3

Parasitesof Cultured Tiger Puffer (Takifugurubripes) and theirSeasonal Occurrences,with Descriptionsof Two New Speciesof Gyrodactylus

Kazuo Ogawa*1 and Kiyoshi Inouye*2

*1Departmentof AquaticBioscience , Faculty of Agriculture,The University of Tokyo,Bunkyo-ku, Tokyo 113, Japan *2NationalResearch Instituteof Aquaculture , Tamaki-cho,Watarai-gun, Mie519-04, Japan

(Received July 16,1996)

Parasitesof culturedtiger puffer, Takifugu rubripes, produced in a hatchery,introduced to a culturesite in Nagasaki Prefecturein summer and culturedfor one year and a halfin net cages,were examined bimonthly. Thirteenspecies of parasiteswere recorded:ciliates, Trichodina fuguand T. jadranicaon thegills, Cryptocaryon irritan s in the finsand body surface;myxosporeans, Kudoa shiomitsui inthe heartand Multivalvulida gen. sp. (syn.:Septemcapsula yasunagai Hsieh etChen,1984)in thebrain; monogeneans, Gyrodactylusrubripedis sp. n. on the finsand body surfaceand Heterobothriumokamotoi on the gillsand branchialcavity wall; trematodes, Tetrochetussp. and Maculiferpacificus in the rectum;acanthocephalan, Longicollum pagrosomi thein rectum; copepods,Caligus fugu on the buccalcavity wall, Pseudocaligus fugu on the body surfaceand Neobrachiella hugu(syn:Clavellopsis huguYamaguti,1939)on thebuccal cavity wall. Gyrodactylusrubripedis sp. n. isdifferent from themost closelyrelated G. pardalidissp. n. collected from the finsof pantherpuffer,Takifugu pardalis and finespottedpuffer, T. poecilonotusin smallersize of hamuli and shape of themarginal hook proper;thetwo new gyrodactylidsare similar to ones from freshwaterfish of thegenus Noemacheilusin havinginwardly bent rootof hamuli,but clearlyseparated from them by the shape of the ventral bar. In some selectedspecies of parasites,seasonal occurrences are describedand possiblesources of infection are discussed.

Key words: Takifugu rubripes,tiger puffer, parasite fauna, new species,Monogenea

Culture of tiger puffer, Takifugu rubripes, started in so far been reported from tiger puffer, but information western Japan in the 1950s (Okamoto, 1963), the annual about their pathogenicity is quite limited. As for ciliate production staying less than 100 metric tons during the 1950s-1970parasites, Cryptocaryon irritans was first reported from s. In those early years, feral puffers were tiger puffer as an unidentified ciliate by Sikama (1937) caught in spring and summer, and cultured until winter, and two species of Trichodina viz. T. fugu and T. when most of puffers were consumed. Since the 1980s, jadranica were described by Imai et al. (1997). As for however, the puffer culture industry has been rapidly myxosporeans, Kudoa shiomitsui was described by growing. The annual culture production of tiger puffer Egusa and Shiomitsu (1983) and Multivalvulida gen. sp. exceeded 4,000 metric tons in 1992, about 10 times as by Egusa (1986). The latter myxosporean was named much as that of 10 years before. This is mainly because as Septemcapsula yasunagai as a new genus and a new of a plentiful supply of artificially produced juveniles, species by Hsieh and Chen (1984), but there is some which has led to the recent expansion of tiger puffer discussion about its validity of the new species (Egusa, culture. With the change of the source of seedlings, 1986), and thus it is treated here as an unidentified culture methods have substantially changed. Typically, myxosporean as above. There are 13 species of hatchery-producedjuveniles are available to fish farmers metazoan parasites, of which are 3 species of mono in summer, and cultured for about 1 year and a half until geneans: Udonella caligorum reported by Yamaguti marketed in winter. (1958), Neobenedenia girellae by Ogawa et al. (1995) With regard to diseases associated with puffer culture, and Heterobothrium okamotoi first reported by Okamoto much remains to be studied, because of a rather short (1963) as H. tetrodonis and later described by Ogawa culture history. A total of 18 species of parasites have (1991) as a new species, 3 species of trematodes: 8 Kazuo Ogawa and Kiyoshi Inouye

Galactosomum sp. metacercaria by Yasunaga et al. Materials and Methods (1981), Pharyngora orientalis by Yamaguti (1934) and Psettarium japonicum by Yamaguti (1938), 3 species of Hatchery-reared juveniles of tiger puffer cultured at a

namatodes: Anisakis sp. larva by Yamaguti (1941), farm at Naru, Minami-Matsuura gun, Nagasaki

Contracaecum sp. larva by Fukuda et al. (1982) and Prefecture, Japan were used in this study. They were

Cuculanidae gen. sp. (originally? Spirruroidea cultured in an off-shore net pen and fed daily with

cuculanidae?) by Okamoto and Ogasawara (1965), and defrozen minced fish. Following the introduction of

4 species of copepods: Taeniacanthus yamagutii first the puffer seedlings to the farm early in July 1986, 20

reported by Okamoto and Ogasawara (1965) as Irodes individuals were randomly collected bimonthly from a yamagutii, Caligus fugu described by Yamaguti and single fish stock and examined for parasites from July Yamasu (1959a), Neobrachiella hugu, originally 1986, when they were 9.0•}0.8 cm in body length, 25.0

described by Yamaguti (1939) as Clavellopsis hugu, 7.4g in body weight, until November 1987, soon before•}

Argulus scutiformis by Yamaguti and Yamasu (1959b). harvested (28.8•}1.2cm long, 838•}145g in weight)

In this paper, an attempt is made to identify parasites (Fig. 1). Water temperature ranged from 13.5 to 28.0•Ž which appeared during a survey throughout the whole during the survey period. Preliminary parasitological period of culture and to describe their patterns of surverys of juvinile puffers were made on May 16, 1986,

occurrences,thus to provide basic information on parasite when the fish had still been reared in a hatchery in infections of cultured tiger puffer. Nagasaki Pref., and on July 4, a few days after arrival of

Fig. 1. Fluctuation of water temperature and growth of tiger puffer during the study period (July 1986-Nov 1987) at Naru, Nagasaki Prefecture. Parasites of cultured tiger puffer 9

the seedlings at the farm, 25 days before the first rugular in middle. Marginal hooks 19-21 in total length; handle sampling. 15-17 long; domus 8-10 long; hook proper 4 in length

Parasitological surveys were carried out for by 3.5-4 in distal width and 3-3.5 in proximal width. ectoparasites on the gills, fins (pelvic, dorsal and anal) Male copulatory organ 11-16 in diameter; cirrus 3.5- and body surface scrapings under a stereo-microscope, 5.5 long guarded by a single row of 4-7 minute spines. and the branchial and buccal cavity wall by the naked Remarks: This parasite is unique in having inwardly eye, and for endoparasites in the intestine, heart, brain bent root of hamuli. Similar structure was reported in under a stereo-microscope. Specifically, gill tricho Gyrodactylus species parasitic on Noemacheilus and its dinids were fixed in 5% formalin solution, and observed closely related genera of freshwater fish hosts (Ergens in wet mount under a compact microscope for and Kartunova, 1991). The present new species, identification. For practical reasons, trichodinids on the together with the following G. pardalidis sp. n., seems fins and body surface were not collected. Myxosporean to form an independent species group within the genus spores in the brain, heart and gills were smeared on a Gyrodactylus. slide glass and stained with May-Grunwald and Giemsa.

Gyrodactylids on the fins and body surface were fixed Gydodactylus pardalidis sp. n. in ammonium picrate-glycerin solution, and permanent (Figs. 3 and 4) mount preparations were made according to the method Habitat: fins of panther puffer, Takifugu pardalis of Ergens (1969). Other helminths were fixed in AFA, (Temminck et Schlegel).

stained with alum carmine or Heidenhein's iron Type locality and date: Hagi, Yamaguchi Prefecture, hematoxylin, dehydrated in alcohol series and mounted Japan (34•‹25' N, 131•‹23' E); Apr. 23, 1987. in Canada balsam. Parasitic copepods were fixed in Additional record: fins of finespotted puffer,

70% alcohol and observed in lactic acid. Takifugu poecilonotus (Temminck et Schlegel); Nomo,

Gyrodactylus from tiger puffer were compared with Nagasaki Prefecture; June 17, 1986. those from other puffer species of the genus Takifugu, Specimens deposited: Meguro, Pasasitological

namely T. pardalis at Hagi, Yamaguchi Prefecture in Museum, Tokyo, M. P. M. Coll. No. 19697-19698.

April 1987 and T. peocilonotus at Nomo, Nagasaki Pref. Description (based on 11 specimens): Body 0.33-

in June 1986. Drawings of Gyrodactylus species were 0.67 mm long. Hamuli 35-43 long excluding folded

made with the aid of a camera lucida. Measurements part of root; base 28-35 long, point 19-24 long, root are given in ƒÊm, unless otherwise stated. folded inwardly in middle. Ventral bar 17-24 long by

3.5-5.5 wide; ear-shaped processes 6.5-8.5 long;

membrane 9-12 long, with rounded margin. Dorsal bar Results 15-22 long by 1-2 wide, slightly swallen in middle.

Description of parasites Marginal hooks 18-21 in total length; handle 14-17 long;

Gydodactylus rubripedis sp. n. domus 8.5-10 long; hook proper 4-4.5 in length by 3.5

(Fig. 2) 4.5 in distal width and 3-3.5 in proximal width. Male- Habitats: fins and body surface of tiger puffer, copulatory organ 9-14 in diameter; cirrus 3-5.5 long

Takifugu rubripes (Temminck et Schlegel). guarded by a single row of 4-6 minute spines. Type locality and date: Naru, Nagasaki Prefecture, Remarks: G. pardalidis sp. n. is most similar to G.

Japan (32•‹52' N, 128•‹56' E); May 21, 1987. rubripedis sp. n., but different in smaller size of hamuli

Additional record: Naru, Nagasaki Prefecture, Nov. and shape of the marginal hook proper.

27, 1986, Mar. 16, 1987, July 23, 1987. Specimens deposited: Meguro Parasitological Occurrences of parasites Museum, Tokyo, M. P. M. Coll. No. 19696. In the two preliminary monitorings, no parasites were Description (based on 14 specimens): Body 0.39 found from fish in the hatchery in May (no. of fish 0.56 mm long. Hamuli 47-53 long excluding folded- examined=16), but 3 fish were already infected with part of root; base 40-46 long, point 27-29 long, root Longicollum pagrosomi in the rectum among 12 fish folded inwardly in middle. Ventral bar 20-25 long by examined in July, a few days after the arrival at the farm. 5-6 wide; well-developed ear-shaped processes, 9-15 A total of 13 species of parasites were found in or on long; membrane 11-15 long, with rounded margin. tiger puffer during the regular survey period from July Dorsal bar 16-25 long by 1.5-2 wide, slightly swallen 29, 1986 to Nov. 26, 1987 (Table 1). Among them, 10 Kazuo Ogawa and Kiyoshi Inouye

Figs. 2-4. Hard parts of Gyrodactylus species from the fins and body surface of puffers of the genus Takifugu . 2, Gyrodactylus rubripedis sp. n. from tiger puffer, Takifugu rubripes; 3 , Gyrodactylus pardalidis sp. n. from Takifugu pardalis; 4, Gyrodactylus pardalidis sp. n. from Takifugu poecilonotus. a, hamuli and bars; b, marginal hooks; c, male copulatory organ. Left scale for a and c, 20 ƒÊm; right scale for b, 10 ƒÊm. Parasites of cultured tiger puffer 11

Table 1. List of parasites recovered from cultured tiger puffer Takifugu rubripes in this study

Cryptocaryon irritans, Multivalvulida gen. sp., Tetrochetus sp., Maculifer pacificus, Caligus fugu and Pseudocaligus fugu were only occasionally found, and seasonality on the occurrence was not clearly demon strated. Thus, infections of the remaining 7 species of parasite were examined in detail (Fig. 5).

1. Trichodina fugu and Trichodina jadranica As is shown in Fig. 5, all the fish examined were infected with Trichodina spp. throughout the study period. Identification of the two trichodinids revealed that Trichodina fugu was always a dominant species on Fig. 5. Seasonal changes in prevalences of infection of tiger the gills (data not shown). puffer with some parasites during the survey period.

2. Kudoa shiomitsui The myxosporean was found all the time, with varying 3. Gyrodactylus rubripedis sp. n. prevalences of infection. Seasonality of infection was Although not all parts of fins and body surface were not clear. examined, the monogenean was sometimes found in large numbers, the highest number per fish being 50. The parasite tended to be abundant in winter. 12 Kazuo Ogawa and Kiyoshi Inouye

Fig. 6. Amphipod, Caprella acutifrons, found in the intestine of tiger puffer. a, major part of body of the amphipod with two cystacanth larvae (arrowheads) of Longicollum pagrosomi in the body cavity; b, enlargement of a, taken under dark-field illumination. Probosci are indicated by arrows. Scale, 1 mm. Fig. 7. Seasonal changes in frequency distribution of body length of Longicollum pagrosomi from the rectum of tiger puffer. White and black bars represent male 4. Heterobothrium okamotoi and female specimens, respectively. This monogenean was first recovered on the gills in Nov. 1986, about 5 months after introduction of the puffers to the farm, and found also on the branchial cavity host for the acathocephalan. wall in Jan, 1987. The prevalence of infection stayed Seasonal succession of the frequency distribution of high (75-100%) until the end of the survey. Parasites parasite body length (Fig. 7) suggests that the parasite on the gills were always immature, while those on the has a one-year life cycle and that invades the fish in branchial cavity wall were mostly adult. There was no summer. Largest worm measured 12.8 mm long, and correlation between the number of parasites on the two only a few fully formed eggs were observed in the female habitats of the host (data not shown). The highest body cavity. number of parasites per fish was 56. 6. Neobrachiella hugu 5. Longicollum pagrosomi This species was a very common parasite, and found In one of the three fish infected with the acanthoce- throughout the survey period. The mean intensity of phalan in the preliminary survey in July 1986, the infection stayed low (0.4-2.8) in the first year, but it amphipod, Caprella acutifrons, containing two rose to 5.6-6.7 from July to November in the second cystacanth stage larvae of L. pagrosomi in the body year. No clear seasonal change was noticed in the cavity, was found in the intestine (Fig. 6a, b). It was prevalence of adults with egg sacs or of immature forms. demonstrated that the amphipod acts as an intermediate These results suggest that copepodids, the infective stage Parasites of cultured tiger puffer 13 of the parasite, infect the fish all the year round with a (1996) experimentally demonstrated that amphipods peak period in warm water months. Carpella equilibra, C. californica and Jassa sp. aff. marmorata act as the intermediate hosts for L.

Discussion pagrosomi, though they did not specify any of the three amphipods harbored the infective stage of the parasite. Thirteen species of parasites were recorded from tiger The acanthocephalan has been reported from many puffer in this study. Among them, 5 species, including species of marine fishes, but mature worms were one new species, were first reported from tiger puffer; recovered only from red sea bream, Pagrosomus unicolor Gyrodactylus rubripedis sp. n., Tetrochetus sp., (=Pagrus major) (Yamaguti, 1935). This is the first Macurifer pacificus, Longicollum pagrosomi and record of the acanthocephalan from tiger puffer, but it Pseudocaligus fugu. seems that tiger puffer is not a suitable host for the Since no parasite was found from juvenile puffers acanthocephalan; specimens collected from puffers were reared in the hatchery, it was thought that tiger puffer considerably smaller than those from red sea bream had mostly been infected with the parasites after arriving (Yamaguti, 1935), and poorly matured. at the farm. However, Kudoa shiomitsui may have Cryptocaryon irritans, Gyrodactylus rubripedis sp. n., invaded the fish in the hatchery, for the myxosporean Heterobothrium okamotoi and Pseudocaligus fugu was already recovered from fish only about one month sometime heavily infect cultured tiger puffer, and are after the arrival. One-year old tiger puffer and several considered pathogens. In the present survey, however, other fish species, i. e. yellowtail,Seriola quinqueradiata, they appeared only occasionally, except for H. okamotoi. and red sea bream, Pagrus major, had also been cultured Trichodina spp. were ubiquitous parasites in this study. in the same area, when juvenile puffers were introduced Although no quantitative study of the infection was there. It is likely that the older puffers and other species conducted, fish infected with Trichodina did not show of fish contributed as the source of infection of the young any sign of disease. Longicollum pagrosomi and puffers with a possible exception for K. shiomitsui. Neobrachiella hugu provoked local hyperplasia of host Okamoto (1963) described the life cycle of tissue at the attachment site in the rectum and on the Heterobothrium tetrodonis (=H. okamotoi). A very buccal cavity wall, respectively, but the intensity of unusual observation in his description was that the infection with the two parasites never reached a state to oncomiracidium, after settling down on the gills or cause disease. On the other hand, H. okamotoi occurred branchial cavity wall of tiger puffer and shedding the almost on all tiger puffer examined in the second year. cillia, encysted for a while in the host tissue. We The pathogenicity of H. okamotoi will be described sometimes observed encapsulation of this parasite on the separately (Ogawa and Inouye, 1997b). branchial cavity wall. This was a host reponse to the parasite, and most probably he erroneously thought that Acknowledgements the encapsulation was part of a developmental stage of the parasite. We would like to thank Dr. R. Ergens, Institute of In the present study, adult parasites of H. okamotoi Parasitology, Czech Academy of Sciences, Ceske were found only on the branchial cavity wall, while Budejovice, Czech Republic for help in description of parasite on the gills were immature. This difference in Gyrodactylus species, and Dr. Z. Kabata, Pacific the habitat of adult and immature parasites on the host Biological Station, Nanaimo, B. C., Canada for strongly suggests that the monogenean migrates from identification of parasitic copepods, Dr. T. Shimazu, the gills to the branchial cavity wall in the life cycle. Nagano-ken Junior College, Nagano, Japan for The observation in this study will be described in more identification of the trematode Tetrochetus sp., Dr. I. detail separately (Ogawa and Inouye, 1997a; 1997b). Takeuchi, Otsuchi Marine Research Center, Ocean Acanthocephalan infections of vertebrates are closely Research Institute, the University of Tokyo, Otsuchi, related to the feeding habits of the hosts. It was Iwate, Japan for identification of the amphipod in the demonstrated that puffers not only were fed minced fish intestine of tiger puffer. Appreciation is also extended but also spontaneously ingested small invertebrates like to Mr. T. Kakimori, Naru, Nagasaki Pref., for providing amphipods propagated on the net meshing, and that the fish samples, and to the late Mr. S. Inouye, Daiichi amphipod, Caprella acutifrons, is an intermediate host Pharmaceutical Co., Ltd., Tokyo, for his help in the field. for Longicollumpagrosomi. Yasumono and Nagasawa 14 Kazuo Ogawa and Kiyoshi Inouye

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