魚 病 研 究 Fish Pathology,35(2),67-72,2000.6 2000 The Japanese Society of Fish Pathology

Tetrahymena Infection in Guppy, Poecilia reticulata

Soichi Imai1*, Sayaka Tsurimaki1, Eiko Goto1, Kunika Wakita2 and Kishio Hatai3 1Division of Veterinary Parasitology , Nippon Veterinary and Animal Science University, 1-7-1, Kyonan-cho, Musashino, Tokyo 180-8602, Japan 2Department of Pathology , Faculty of Veterinary Science, Azabu University, 1-17-71, Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan3 Division of Fish Diseases , Nippon Veterinary and Animal Science University, 1-7-1, Kyonan-cho, Musashino, Tokyo 180-8602, Japan

(Received January 19, 2000)

ABSTRACT--Out of 78 diseased guppies imported from Singapore to Japan, 43 fish were found infected with ciliates. Thus, pathological examination of guppies naturally infected with ciliates and identification of a ciliate sampled from guppies in Singapore were conducted. Most of the ciliates parasitized the inside of scales, muscles or internal organs, while some invaded into the eye socket and spinal cord. No damage was found in the intestinal epithelium. From these findings, it is considered that the main invasion route of these ciliates was from the scales of hosts to internal organs through muscles. As the results of morphological examination using silver impregnation, the examined ciliate was identified as Tetrahymena corlissi Thompson, 1955, which has been reported as a pathogen of guppies in North America.

Key words: ciliate, guppy, Tetrahymena, parasite, protozoa

Most of ciliate protozoa in fresh and marine water autopsy, all fish were fixed in 10% (v/v) phosphate-buffered are free living, but some, such as Trichodina and I formalin solution, embedded in paraffin, sectioned chthiophthirius, are parasitic to fishes (Bykhovskaya at 4 mm in thickness, and stained with haematoxylin and Pavlovskaya et al., 1964; Lom, 1995). Recently,- great eosin. damage of cultured guppies (Poecilia reticulata) by para Ciliate samples used for identification were col sitic ciliates has happened at farms in Southeast Asian lected from guppies cultured at farms A (Group A) and B countries, especially Singapore and Sri Lanka. (Group B), Singapore in July, 1998 and June, 1999, Although the most of guppies in Japan have been respectively. Protozoan specimens were fixed in imported as an aquarium fish from such countries, many Bouin's solution and washed in 70% (v/v) ethanol for of them have died by ciliate infection either during trans three times. The fixed specimens were air-dried and portation or soon after their arrival. So this damage impregnated with Protargol method (Foissner, 1992) for causes a serious problem for industries both in South the observation of adoral and somatic kineties. A part east Asian countries and in Japan. of the dried specimens was stained with Mayer's The present paper deals with the pathological find haematoxylin to observe the nuclei. Measurements of ings and species of this parasitic ciliate which is the respective parts of ciliates were performed on 30 cells. pathogen for guppy.

Results Materials and Methods Of 78 fish examined, invasion of ciliates was recog Seventy-eight guppies imported from Singapore to nized in 43 fish (55%). Obvious scale loss and ulcer Japan in September, 1998 to June, 1999, which showed were observed in the seriously parasitized fish (Fig. abnormal swimming or whitish appearance on the body 1). In histopathological examination, ciliates were surface, were used for pathological examination. After mainly found in the scale pockets (Fig. 2) and between muscle fibers (Fig. 3). The ciliates also invaded in the * Corresponding author abdominal cavity, internal organs, such as intestine (Fig. E-mail: [email protected] 4) and liver, eye socket (Fig. 5), cranial cavity and spinal 68 S. Imai, S. Tsurimaki, E. Goto , K. Wakita and K. Hatai

Fig. 1. Guppy seriously parasitized by ciliates. Scale loss, erosin and ulcer are observed. Fresh specimen. Bar = 2cm. Figs. 2-6. Histopathological sections of parasitized fish. All the sections are stained with haematoxylin and eosin. 2. Parasites found in the skin. Note many parasites in the scale pockets (arrows). Bar = 200 ƒÊm. 3. Parasites between the muscle fibers. Ciliates penetrate into the connective tissue of the muscle (arrows). Many cells, probably neutrophils, sorround the ciliates. Bar = 100 ƒÊm. 4. Parasites in the intestine. Many ciliates are observed in the loose connective tissue. Bar = 100 ƒÊm. 5. Ciliates gathering in the eye socket (arrows). Bar = 200 ƒÊm. 6. Ciliates invading into the spinal cord (arrows). Bar = 50 ƒÊm.

cord (Fig. 6). Distribution of the parasitized ciliates in body length was 47.0 ƒÊm (35.0-57.5 ƒÊm), and body

respective parts of the fish is shown in Table 1. width 17.6 ƒÊm (12.5-25.0 ƒÊm). The number of ciliary

In morphological examination, the ciliates of both A rows was 23-29, most frequently 27. The number of

and B groupes had pear-like body with somatic cilia on postoral ciliary rows was two in both A and B groups

the entire body surface (Fig. 7) and a caudal cilium (Fig. 7). A straight preoral suture was observed at the

which was longer than somatic cilia at the posterior end anterior part of the oral cavity (Fig. 9). In the oral cavity,

of the body (Fig. 8). In the ciliates of A group, body one undulating membrane at the right periphery of the

length was 61.9 ƒÊm on average (42.5-100.0 ƒÊm in cavity and three membranelles running obliquely at its

range), and body width 35.5 ƒÊm (20.0-62.5 ƒÊm). The left periphery were present (Fig. 9). Two pores of con-

number of ciliary rows running parallel to the body axis tractile vacuoles were observed at the posterior part of

was 17-25, most frequently 22. In the B group ciliates, the body (Fig. 10). Several ciliates in the group B had Tetrahymena infection in guppy 69

Figs. 7-12. Tetrahymena corlissi collected from guppies in Singapore. M: membranelle; Ma: macronucleus; Mi: micronucleus; OC: oral cavity; UM: undulating membrane. 7. Whole body of the ciliate. Two postoral ciliary rows are present

(arrows). Protargol staining. Bar = 10 ƒÊm. 8. Caudal cilium (arrow). The kinetosome of caudal cilium is larger than those of somatic cilia. Protargol staining. Bar = 5 ƒÊm. 9. Anterior part to the oral cavity. A straight suture is shown. Protargol staining. Bar = 5 ƒÊm. 10. Oral structure. Ciliary apparatus of the oral cavity consists of three membranelles (M1, M2 and M3) arranged obliquely at the left periphery of oral cavity and a bow-shaped undulating membrane at the right periphery of oral cavity. Protargol staining. Bar = 2 ƒÊm. 11. Pores of contractile vacuoles. Two pores situate back and forth (arrows). The ciliary rows on which the pores are present have no kineto- somes at the posterior to the pores. Protargol staining. Bar = 5 ƒÊm. 12. A macronucleus and a micronucleus. Both are spherical and the micronucleus is situated beside the macronucleus. Haematoxylin staining. Bar = 10 ƒÊm.

three pores. These pores opened at 8th and 9th ciliary row was situated anteriorly (Fig. 11). Macronucleus row both in groups A and B. The ciliary rows on which was spherical in shape and situated at the center of the the pores were present had no kinetosomes at the pos- body (Fig. 12). The size was 10.5 ƒÊm (8.0-16.0 ƒÊm) in terior to the pores (Fig. 11). The pores were situated group A, and 8.7 ƒÊm (7.5-10.0 ƒÊm) in group B. A small back and forth on the body surface, and it at 8th ciliary spherical micronucleus was situated beside the posterior 70 S. lmai, S. Tsurimaki, E. Goto , K. Wakita and K. Hatai

a b

Fig. 13. Schematic drawing of Tetrahymena corlissi. a. Ventral view; b. Right view. CC: caudal cilium; M: membranelle; Ma: ma- cronucleus; Mi: micronucleus; OC: oral cavity; PC: postoral ciliary row; PO: pore of conractile vacuole; PS: preoral suture; UM: undulating membrane. part of the macronucleus (Fig. 12). A schematic draw- seemed to occur, because the intestinal mucoepithelium ing of the ciliate is shown in Fig. 13. was not injured even in seriously parasitized fish. The ciliates appeared to destroy tissues of the hosts physi- cally. Discussion The findings that the ciliates of both A and B groups In pathological examinations, the ciliates were examined had a straight preoral suture, two postoral cili- detected from the most body parts of the guppies. ary rows and characteristic oral ciliary zone indicate that From the fact, it is assumed that no preferrable parasit- these ciliates are of the genus Tetrahymena (Corliss, ized sites of fish are present. However, the compara- 1970; Miyata, 1979). Although the measurements of tive examinations of parasitized parts of fish as shown in the ciliates of groups A and B were slightly different, it Table 1 suggest that the ciliates mainly penetrate from was concluded that these are the same species because scales to internal organs via muscle. Judging from the all the measurements were overlapped. stained sections of intestine (Fig. 4), no oral infection At least ten species into three complexes of this

Table 1. Parasitized locarities of ciliates in the guppies Tetrahymena infection in guppy 71

Table 2. Comparison of the present species to four known species

genus have been described up to this time (Corliss, fish at several aquariums and farms in North America 1970; Lynn, 1975). These ciliates have various life (Nigrelli et al., 1956; Hoffman et al., 1975). Hoffman et cycles, that is, free-living, parasitic or facultatively al. (1975) described the morphology of tetrahymenid parasitic. Of them, four species, T. corlissi, T. setifera, ciliated from guppies, which were identified as T. T. rostrata and T. paravorax, have been described as corlissi. Although their description was slightly different those with a caudal cilium (Holz and Corliss, 1956; from the original description by Thompson (1955) on the Corliss, 1970). Comparison of the data of the present points of the number of postoral ciliary row and the pres species to those of known species is shown in Table ence of caudal cilium, they stated that there is minor 2. Among them, T. corlissi was the most similar to the variation in this species. The morphology of the ciliates present species on the points of the numbers of the so observed in the present examination more agreed with matic ciliary rows, the pores of contractile vacuoles and the original description. the postoral ciliary rows as well as the shape and posi The present report demonstrated that this ciliate tion of micronucleus, although the number of the somatic species also plays a role as the causative pathogen of ciliary rows in T. corlissi is slightly more than that of the mass mortality of guppies in Southeast Asian countries, present species. Tetrahymena setifera also resembles especially in Singapore. the present species, but differs in the position of the pores of contractile vacuoles and the existence of kineto somes on the area posterior to the pores (Holz and References Corliss, 1956). In addition, this species is non-parasitic Bykhovskaya-Pavlovskaya, I. E., A. V.Gusev, M. N. Dubinina,, (Holz and Corliss, 1956). The present species clearly N. A. Izyumova, T. S. Smirnova, I. L. Sokolovskaya, G. A. differs from T. rostrata in the number of somatic ciliary Shtein, S. S. Shul'man and V. M. Epshtein, (1964): Key to rows, the shape of the micronucleus and the number and Parasites of Freshwater Fish of the U.S.S.R., Israel Prog. Sci. Trans., Jerusalem. 919 pp. positon of the pores of contractile vacuoles, and from T. Corliss, J. O. (1957): Tetrahymena paravorax n.sp., the first paravorax in the body size and the number and size of caudal-ciliated member of the genus referrable to the the micronucleus (Corliss, 1970). From the compari vorax-patula complex. J. Protozool., 4 (suppl.), 13. son, the present species was identified as Tetrahymena Corliss, J. O. (1970): The comparative systematics of species corlissi Thompson, 1955. Tetrahymena corlissi has composing the hymenostome ciliate genus Tetrahymena. J. Protozool., 17, 198-209. been known as a pathogen against amphibians and Foissner, W. (1992): Protargol methods. In "Protocols in pro freshwater fishes (Lom, 1995). When this ciliate spe tozoology". Lee, J.J. and Soldo, A. T. eds. Soc. Protozool., cies parasitizes guppy, it causes a high mortality of the Kansas, C6, pp. 1-8. 72S. Imai, S. Tsurimaki, E. Goto , K. Wakita and K. Hatai

Hoffman, G. L., M. Landolt, J. E. Camper, D. W. Coats, J. L. Lynn, D. H. (1975): The life cycle of the histophagous ciliate Stookey and J. D. Burek (1975): A desease of freshwater Tetrahymena corlissi Thompson, 1955. J. Protozool., 22, fishes caused by Tetrahymena corlissi Thompson, 1955 188-195. and a key for identification of holotrich ciliates of freshwater Miyata, A. (1979): Parasitic Protozoa. Their taxonomy, ecology fishes. J. Parasitol., 61, 217-223. and evolution. pp. 1498-1501, Kisei Genseidobutsu Holz Jr., G. G. and J. O. Corliss (1956): Tetrahymena setifera Kankokai, Nagasaki. (In Japanese.) n.sp., a member of the genus Tetrahymena with a caudal Nigrelli, R. F., S. Jakowska and M. Padnos (1956): Tetrahy cilium. J. Protozool., 3, 112-118. mena as pathogenic epibiont in fishes and urodeles. Lom, J. (1995): Trichodinidae and other ciliates (Phylum Cili J. Protozool., 3 (Suppl.), 10. phora). In "Fish diseases and disorders. vol. 1. Protozoano Thompson Jr., J. E. (1955): Morphology of a new species of and metazoan infections". Woo, P. T. K. ed. CAB Interna Tetrahymena. J. Protozool., 2 (Suppl.), 12. tional, Willingford, pp. 229-262.