DISEASES OF AQUATIC ORGANISMS Vol. 38: 219-224.1999 Published November 30 Dis Aquat Org

Experimental detection of the actinospores of pseudodispar (: ) in oligochaete alternate hosts

CS. Szekely*, K. Molnar, E. Eszterbauer, F. Baska

Veterinary Medical Research Institute, Hungarian Academy of Sciences, PO Box 18, 1581 Budapest, Hungary

ABSTRACT: The development of Myxobolus pseudodispar Gorbunova, 1936, an intracellular myx- osporean muscle parasite of the roach Rutilus rutilus L., was studied in experimentally infected oligochaetes. In one experiment, uninfected Tubifex tubifex Miiller and Lirnnodrilus hoffrneisteri (Cla- parede) were exposed to mature spores of M. pseudodispar. Triactinomyxon spores developed both in T. tubifex and L. hoffrneisteri specimens. Triactinospores were first released from the oligochaetes 76 d after initial exposure. At that time, pansporocysts containing 8 triactinospores were located in the gut epithelium of experimentally infected oligochaetes, but free actinosporean stages were also found in their gut lumen. Each triactinospore had 3 pyriform polar capsules and an elongated cylindrical sporo- plasm with 8 secondary cells. The spore body joined the 3 caudal projections with a relatively long style. One of the 3 caudal projections was shorter than the other two. The total length of the triacti- nospore was on average 206.5 pn~.

KEYWORDS: Myxobolus pseudodispar. Myxosporea . Rutilus rutilus . Development. Triactinomyxon stage . Tubifex tubifex . Lirnnodrilus hoffrneisteri

INTRODUCTION et al. 1993, 1995, Uspenskaya 1995, Trouillier et al. 1996, Yokoyama 1997) revealed that in each case vari- Myxobolus pseudodispar, a described by ous Oligochaeta spp. or Polychaeta sp. (Bartholomew Gorbunova (1936), is a myxosporean commonly occur- et al. 1997) acted as alternate hosts in the development ring in the skeletal muscles of the roach Rutilus rutilus of various myxosporean species. L. in Hungarian lakes, rivers and ponds (Baska 1986). Recently the life cycles of 7 species from the genera Some aspects of its location and intrapiscine develop- Myxobolus, Thelohanellus and Sphaerospora have ment were also studied by Baska (1986). Nothing was also been experimentally studied in our laboratory (El- known, however, about its extrapiscine developmental Mansy & Molnar 1997a,b, El-Mansy et al. 1998a, stage. Szekely et al. 1998, Molnar et al. 1999a,b). The extrapiscine phase of a myxosporean was first The work presented in this paper is part of continu- studied by Wolf & Markiw (1984), who revealed that ing experimental life cycle studies conducted on the the extrapiscine development of most common myxosporeans of Hungarian . In Hofer took place in an oligochaete alternate the experiments reported here, the development of (Tubifex tubifex Miiller). Following that pioneering Myxobolus pseudodispar was followed in Tubifex work, several other papers were published to support tubifex and Limnodrilus hoffmeisteri alternate hosts. its finding. These papers (El-Matbouli & Hoffmann 1989, 1993, Ruidisch et al. 1991, Styer et al. 1991, El- Matbouli et al. 1992, Grossheider & Korting 1992, MATERIALS AND METHODS Benajiba & Marques 1993, Kent et al. 1993, Yokoyama Myxospores of Myxobolus pseudodispar were col- lected from mature muscle cysts of 2 to 5 yr old roach Rutilus rutilus specimens seined in Lake Balaton, Hun-

O Inter-Research 1999 Resale of full article not permitted 220 Dis Aquat Org 38: 219-224, 1999

gary. Pieces of the muscle were cut, compressed after 1 d of incubation they were examined for the between 2 glass plates, and examined for the presence release of actinospores under a stereomicroscope. Tri- of myxosporean plasmodia. Plasmodia located intra- actinospores released by the oligochaetes were exam- cellularly within muscle cells were separated and ined under a coverslip. They were recorded on video- opened with a needle under a stereomicroscope. tapes and digitized pictures were taken with the help Mature spores obtained from plasmodia were identi- of the IMAGO@program as described by Szekely fied and counted. A total of 190 000 spores of M. pseu- (1997). Photographs were taken, drawings made and dodispar were collected. measurements of 47 actinospores recorded. In the Oligochaetes Tubifex tubifex Miiller and Limno- description, all measurements are given in pm. The drilus hoffmeisteri (Claparede), identified according to actinosporean stage of M. pseudodispar was described Brinkhurst (1963), were collected from the outlet of a using the terminology of Janiszewska (1957) as modi- duck farm in Szarvas, Hungary, where no fishes live. fied by Lom et al. (1997). After the first release of acti- This parasite-free oligochaete stock containing mem- nospores live T. tubifex and L. hoffmeisteri specimens bers of the 2 species in about the same number was were also examined for actinosporean stages under a transferred to sterilised mud, and propagated in the compound microscope. After finding the first released laboratory in aerated aquaria. The worms were fed on triactinospores 2 infected 7: tubifex and 1 L. hoffmeis- some drops of granulated food. teri specimen were fixed in 5% glutaraldehyde for The experiment started on 4 September 1998, when 1.5 h and post-fixed in 2% osmium tetroxide for 2 h, 100 specimens each of Tubifex tubifex and Limnodrilus washed several times with cacodylate buffer, dehy- hoffmeisteri were placed into a small plastic cup of drated and embedded in Durcupan ACM resin. Semi- 500 m1 volume, to which spores of Myxobolus pseudo- thin sections (0.5 to 1 p) were made and stained with dispar were added. The calculated spore count per 0.1 % toluidine blue solution. oligochaete was a total of 1000. The temperature of the room during the experiment varied between 20 and 23°C. All dishes were permanently aerated and regu- RESULTS larly supplied with fresh water to prevent evaporation and to refresh the water for the oligochaetes. Starting Light microscopy from 42 d post-exposure (p.e.),water from the dishes was filtered weekly through a fine mesh of 21 pm pore Actinospores of Myxobolus pseudodispar were size. The filtrates were taken up in a drop of water and released from both Tubifex tubifex and bmnodrilus examined for the presence of actinospores. On Day 76 hoffmeisteri, but no infection was found in the control p.e. 150 oligochaetes were placed into 2 m1 cell-well tanks of the same oligochaete species. Of 150 speci- plates as described by Yokoyama et al. (1991). and mens examined in cell-well plates, 4 T. tubifex (5.3 %)

Fig. 1. Cross-section of Tubifex tubifex infected by Myxobolus pseudodispar. The intestine of the oligochaete is heavily infected with pansporocysts (arrowheads) located in the epithelium. Some released pansporocysts are found also in the intestinal lumen (1). Semithin section. x250 Szekely et al. Experimental detection of A4yxobolus pseudodispar actinospores 221

Fig 2. Cross-section of Tubifex tubifex infected by Myxobolus pseu- dodispar. Enlarged part of Fig. 1 A spansporocyst containing 8 actino- spores is located in the gut epithe- lium. Inside the pansporocyst sporo- plasm, polar capsules (arrowhead) and folded membranes of the caudal processes (m) of triactinospores are seen. Semithin section. x2000 and 2 L. hoffmeisteri (2.7%) proved to be infected. processes (Fig. 4). Polar capsules, pynform in shape, During the experiment, only triactinomyxon-type acti- were 5.1 (4-6) pm in length and 3 pm in width. The nospores were obtained from all infected worms. The cylindrical sporoplasm, 45 (42-48) pm long and 15.8 release of triactinospores lasted for about 30 d. In (12-18) pm wide, contained 8 spherical secondary cells infected live oligochaete specimens placed under a 7.6 (6.4-8) pm in diameter (Fig. 3 inset). The style was coverslip, pansporocysts in the gut epithelium and free relatively long, 157.3 (145-173) pm in length. Its width spores in the gut lumen of the wornis were easily seen measured 15.8 (12.8-18) pm at the middle. Caudal under a compound microscope. No infection was found processes were unequal in length, one of them being in the control oligochaete stock examined in the same shorter. They were slightly bent, tapering toward the numbers. end and terminating in a sharply pointed end. The length of the 2 longer caudal processes was 196 (190-204) pm, that of the shorter one 127.2 (104-144) Histology pm. Their width at the middle was 13.6 (12-16) pm.

Pansporocysts of Myxobolus pseudodispar were mostly found in the gut epithelium, but pansporocysts Table 1. Myxobolus pseudodispar. Measurements of the tri- actinospores (on the basis of 47 triactinospores) in pm and free spores released into the intestinal lumen through the damaged gut epithelium were also seen in Measure Mean + SD Minimum Maximum semi-thin sections (Fig. 1). Eight triactinospores could pp - be counted in a pansporocyst. In horizontally sectioned Total length 206.5 + 15.6 179.2 244 triactinospores the spore body with the sporoplasm Polar capsules Length 5.1 + 0.5 4 6 and the dark-stained polar capsules could also be Width 3.0 + 0.0 3 3 recognised (Fig. 2) Inside the sporoplasm large, round Sporoplasm Length 45.0 + 2.2 42 4 8 secondary cells were distinguishable. The free part of Width 15.8 k 1.1 12.8 18 the spore body inside the pansporocyst was filled with Spore body Length 50.4 * 2.4 47 53 the folded styles and caudal processes of the acti- Width 15.8 k 1.1 12.8 18 nospores, which could easily be differentiated from the Style Length 157.3 ? 14.8 145 173 spore body by their pale colour. Width 15.8 ? 1.1 12.8 18 Description of triactinospores. Triactinospores Caudal processes (Table 1, Figs. 3 & 4) released from the tubificid body Longer Length Width and floating in the water were characterised by 3 pyri- Shorter Length form polar capsules, a sporoplasm, a style and 3 caudal 222 Dis Aquat Org 38: 219-224, 1999

Fig. 3. Myxoboluspseudodispar. Tri- actinospores floating in the water. x500. Inset: Spore body of the triacti- nospore with sporoplasm containing 8 sporoplasm cells and with polar capsules at the apical end. X 1200

The length from the apical point of polar capsules to cies. However, they are distinguishable by the low the end of sporoplasm (spore body) measured 50.4 number of secondary cells in the sporoplasm and by (47-53) pm. The total length from the polar capsules to the shorter size of 1 of the caudal processes. The sporo- the end of style (spore body with style) measured 206.5 plasm of this species has only 8 large sporoplasm cells (179-244) pm. and the third caudal process measures only two-thirds of the 2 others. The triactinomyxons obtained in this experiment are Differential diagnosis probably identical to one of the triactinomyxon types (T2) detected by El-Mansy et al. (1998b) during a sur- Both the myxospores and the actinospores of vey of actinospore infection of oligochaetes in a fish Myxoboluspseudodispar can readily be differentiated farm near Budapest. from those of other species by their asymmetric forms. The triactinomyxon stages of this species resemble other known triactinomyxospores of myxosporean spe- DISCUSSION

The species Myxobolus pseudodispar is a rather common parasite of the roach in Hungary (Molnar 1979, Baska 1986, Szekely & Molnar 1996-1997). The validity of the species was questioned by Lom & Dykova (1992), who regarded it as a synonym of M. cyprini. In the typical fish hosts (Cy-pfinus carpio and Rutilus rutilus)clear differences exist in the spore mor- phology (Molnar & Kovacs-Gayer 1985, Baska 1986), which supports the opinion represented by Gorbunova (1936) and Donec & Shulman (1984); therefore, we regard M. pseudodisparas a valid species. In spite of its common occurrence, little is known about its patho- genicity. No studies have been done on the intra- oligochaete development of this species. Data obtained in this study show that the intraoligochaete develop- ment of this parasite follows the same pattern as Fig. 4. Schematic drawing of the triactinospore of Myxobolus described by Wolf & Markiw (1984) for M. cerebralis. pseudodispar. Bar = 100 p In the present experiments the development of M. Szekely et al.: Experimental detection of Myxoboluspseudodispar actinospores 223

pseudodisparwas successfully completed in both Tubi- LITERATURE CITED fex tubifex and Limnodnlus hoffmeisten, in which tn- actinomyxon-type spores developed. In their shape Andree KB, Szekely CS, Molnar K,Gresoviac SJ, Hedrick RP (1999) Relationships among members of the Myxo- and size the spores resembled most of the triactino- bolus (: Bivalvidae) based on small subunit ribo- spores known from experimental studies on Myxobo- somal RNA sequences. J Parasitol8568-74 lus species (M. cerebralis, M. cotti, M. carassii, M. Bartholomew JL, Whipple MJ, Stevens DG, Fryer JL (1997) drjagini, M. portucalensis, M. hungancus), but due to The life cycle of shasta, a myxosporean para- site of salmonids, requires a freshwater as an the low number of secondary cells in the sporoplasm alternate host. J Parasitol83:859-868 and by the shorter length of one of the caudal pro- Baska F (1986) Histological studes on the development of cesses they distinctly differed from the known triacti- Myxobolus pseudodispar Gorbunova, 1936 in the roach nospores. (Rutilus ruuus). Acta Vet Hung 35:251-257 According to El-Matbouli et al. (1992), the intra- Benajiba MH, Marques A (1993) The alternation of actino- myxidan and myxosporidian sporal forms in the develop- oligochaete development of Myxobolus cerebralis, M. ment of giardi (parasite of Anguilla anguilla) cotti and M. carassii took 80 to 120 d, while for M. through oligochaetes. Bull Eur Assoc Fish Pathol 13: drjagini, M. hungancus and M. portucalensis El- 100-103 Mansy & Molnar (1997a,b) and El-Mansy et al. (1998a) Brinkhurst R0 (1963) A guide for the idenhfication of British aquatic Oligochaeta. Freshw Biol Assoc Sci Pub 22:l-55 reported development times of 91, 102 and 160 d, Donec S, Shulman SS (1984) Parasitic . Phylum respectively. The patent period of M. portucalensis Cnidosporidia. In: Bauer ON (ed) Key to determination of was somewhat shorter than that of the above-listed parasites of freshwater fishes of the USSR, Vol I. Pub1 species. House Nauka, Leningrad, p 88-251 (in Russian) The prevalence of infection (5.3 % in Tubifex tubifex El-Mansy A, Molnar K (1997a) Extrapiscine development of Myxobolus djagini Achmerov, 1954 (Myxosporea: and 2.7 % in Limnodrilus hoffmeisten')proved to be rel- Myxobolidae) in oligochaete alternative hosts. Acta Vet atively low compared to the high levels observed in Hung 45427-438 previous experiments on Myxobolus dgagini, M. por- El-Mansy A, Molnar K (1997b) Development of Myxobolus tucalensis, M. hungancus and M. dispar (El-Mansy & hungaricus Jaczo, 1940 (Myxosporea: Myxobolidae) in oligochaete alternate hosts. Dis Aquat Org 31:227-232 Molnar 1997a,b, El-Mansy et al. 1998a, Molnar et al. El-Mansy A, Molnar K, Szekely CS (1998a) Development of 1999a) in our laboratory. From a single experiment it is Myxobolus portucalensis Saraiva & Molnar, 1990 (Myx- impossible to draw conclusions as to whether the alter- osporea: Myxobolidae) in the oligochaete Tubifex tubifex nate hosts, the myxospores or some other factors are (Miiller). Syst Parasitol 41:95-103 responsible for this low prevalence. El-Mansy A, Szekely CS, Molnar K (199813) Studles on the occurrence of actinosporean stages of fish myxosporeans Histological studies prove that Myxobolus pseudo- in a fish farm of Hungary, with the description of triactino- dispar develops in the gut epithelium of the oligo- myxon, raabeia, aurantiactinomyxon and neoactinomyxon chaete alternate host, from where its pansporocysts types. Acta Vet Hung 46:259-284 and spores are released into the lumen and subse- El-Matbouh M, Hoffmann RW (1989) Experimental transmis- sion of two Myxobolus spp. developing by sporogeny via quently excreted with the faeces. tubificid worms. Parasitol Res 75:461-464 Up to now, the intraoligochaete Me cycle of about 20 El-Matbouli M, Hoffmann RW (1993) Myxobolus carassii species has been elucidated. However, a technique Klokacheva, 1914 also requires an aquatic oligochaete, enabling consistently successful laboratory infection is Tubifex tubifex as intermediate host in its life cycle. Bull known only in the case of Myxobolus cerebralis. The Eur Assoc Fish Pathol 13:189-192 El-Matbouli M, Fischer-Scherl T, Hoffmann RW (1992)Trans- species M. pseudodispar is not ideal for use in experi- mission of HofereLlus carassii Achmerov, 1960 to goldfish ments, as its relatively small, intracellular plasmoda Carassius auratus via an aquatic oligochaete. Bull Eur can be collected and identified only with great diffi- Assoc Fish Pathol 12:54-56 culty. Reinfection of the fish host with actinospores is Gorbunova M (1936) Changes in the parasitic fauna of pike and roach according to their age. Uchenye Zapiski also difficult to achieve, as roaches reared infection- Leningradskogo Ordena Lenina Gosudarstvennogo Uni- free are not yet available. At the same time, the col- versiteta, No. 7. (Biol Ser) Fasc 3, Problems of Economical lected myxospores and the obtained actinospores pro- Parasitology, Leningrad, p 5-30 (in Russian) vide excellent material for expanding the molecular Grossheider G, Korting W (1992) First evidence that Hoferel- biological studies that have already been done on lus cyprini (Doflein, 1898) is transmitted by Nais sp. Bull Eur Assoc Fish Pathol 12:17-20 other species (Andree et al. 1999). Janiszewska J (1957) Actinomyxidia 11. New systematics, sex- ual cycles, description of new genera and species. Zool Polon 8:3-34 Acknowledgements. The study was supported by the Hun- Kent ML, Whitaker DJ, Margolis L (1993) Transmission of garian Scientific Research Fund (OTKA, projects No. T Myxobolus arcticus Pugachev and Khokhlov, 1979, a myx- 020044 and T 029200). The authors thank MS Zsuzsa Kis for osporean parasite of Pacific , via a triactinomyxon her help in collecting and culturing tubificids, and are grate- from the aquatic oligochaete Stylodnlus heringianus ful to the Balaton Fisheries Co. for providing fish. (Lumbriculidae). Can J Zool 71:1207-1211 224 Dis Aquat Org 38: 219-224, 1999

Lom J, Dykova I (1992) Protozoan parasites of fishes. In: Series voir of the Kis-Balaton System. Parasit Hung 29-30:45-54 of developments in aquaculture and fishenes science, Vol Szekely CS, El-Mansy A, Molnar K,Baska F (1998) Develop- 26. Elsevier, Amsterdam, p 159-237 ment of Thelohanellus hovorkai and Thelohanellus nikol- Lom J, McGeorge J. Feist SW, Morris D. Adams A (1997) sku (Myxosporea: Myxozoa) in oligochaete alternate Guidelines for the uniform characterisation of the actino- hosts. Fish Pathol 33:107-114 sporean stages of parasites of the phylum Myxozoa. Dis Trouillier A, El-Matbouli M, Hoffmann R (1996) A new look at Aquat Org 30:l-9 the life-cycle of Hoferellus carassii in the goldfish (Caras- Molnar K (1979) Protozoan parasites of fish species indige- sius auratus auratus) and its relatlon to 'ludney enlarge- nous in Hungary. Parasit Hung 12:5-8 ment disease' (KED). Folia Parasitol43:173-187 Molnar K. Kovacs-Gayer g (1985) The pathogenicity and Uspenskaya AV (1995) Alternation of actinosporean and myx- development within the host fish of Myxobolus cyprini osporean phases in the life cycle of ZschokeUa nova (Myx- Doflein, 1898. Parasitology 90549-555 ozoa). J Euk Microbiol42:665-668 Molnar K. El-Mansy A. Szekely CS, Baska F (1999a) Develop- Wolf K, Marhw IME (1984) Biology contravenes in ment of Myxobolus dispar Thelohan, 1895 (Myxosporea: the myxozoa: new discoveries show alternation of inverte- Myxobolidae) in an oligochaete alternate host, Tubifex brate and hosts. Science 225:1449-1452 tubifex (Miiller). Folia Parasitol 46:15-21 Yokoyama H (1997) Transmission of Thelohanellus hovorkai Molnar K, El-Mansy A, Szekely CS, Baska F (1999b) Develop- Achmerov, 1960 (Myxosporea: Myxozoa) to common carp ment of Sphaerospora renicola Dykova and Lom. 1982 Cyprinus carpio through the alternate oligochaete host. (Myxosporea: Sphaerosporidae) in oligochaete alternate Syst Parasitol 3679-84 hosts. J Fish Dis 22:l-11 Yokoyama H, Ogawa K,Wakabayashi H (1991) A new collec- Ruidisch S, El-Matbouli M, Hoffmann RW (1991) The role of tion method of actinosporeans-a probable infective tubificid worms as an intermediate host m the life cycle of stage of myxosporeans to fishes-from tublficids and Myxobolus pavlovskii (Akhmerov, 1954). Parasitol Res experimental infection of goldfish with the actinosporean, 7:663-667 Raabeia sp. Fish Pathol 26:133-138 Styer EL, Harrison L, Burtle GJ (1991) Expenmental produc- Yokoyama H, Ogawa K, Wakabayashi H (1993) Involvement tion of proliferative gdl disease in channel catfish exposed of Branchiura sowerbyi (Oligochaeta: Annellda) in the to a myxozoan-infected oligochaete, Dero digitata. J Aquat transmission of Hoferellus carassii (Myxosporea: Myxo- Anim Health 3:288-291 zoa), the causative agent of kidney enlargement disease Szekely CS (1997) Possible applications of video technology (KED) of goldfish Carassius auratus. Fish Pathol 28: and hgital image processing in fish parasitology: morpho- 135-139 logical examination of the groups Apicomplexa and Myxo- Yokoyama H, Ogawa K, Wakabayashi H (1995) Myxobolus sporea-Actinosporea by video technology. Bull Eur Assoc cultus n. sp. (Myxosporea: Myxobolidae) in the goldfish Fish Pathol 17:81-82 Carassius auratus transformed from the actinosporean Szekely CS,Molnar K (1996-1997) Preliminary survey of the par- stage in the oligochaete Branchiura sowerbyi. J Parasitol asite fauna of some important fish species in the Upper Reser- 81:446-451

Editorial responsibility: Wolfgang Korting, Submitted: May 17, 1999; Accepted: September 3, 1999 Hannover, Germany Proofs received from author(s): November 16, 1999