Journal of Diseases 2001, 24, 461-468

Complete developmental cycle of pseudodispar (Gorbunova) (: Myxobolidae)

Cs Székely, K Molnár and O Rácz

Veterinary Medical ResearchInstitute, Hungarian Academy of Sciences,Budapest, Hungary

Hoffmann 1989, 1993; Styer, Harrison & Burde Abstract 1991; EI-Matbouli, Fischer-Scherl & Hoffmann Myxoboluspseudodispar (Gorbunova) is a common 1992; Grossheider & Körting 1992; Benajiba & parasite of the muscle of roach, Rutilus rutilus L, Marques 1993; Yokoyama, Ogawa & Wakabayashi whereas its actinosporean development occurs in 1993; Uspenskaya 1995; Trouillier, EI-Matbouli two oligochaete altemate hosts. This paper reports & Hoffmann 1996; Bartholomew, Whipple, the complete developmental cycle of this parasite Stevens & Fryer 1997; EI-Mansy & Molnár in the oligochaete altemate host Tubifex tubifex and 1997a,b; EI-Mansy, Molnár & Székely 1998; the roach. In laboratory experiments, parasite-free Székely,EI-Mansy, Molnár & Baska 1998; Székely, T tubifex specimenswere infected by myxospores Molná!, Eszterbauer & Baska 1999; Molnár, of M pseudodisparcollected from roach in Lake EI-Mansy, Székely & Baska 1999a,b; Eszter- Balaton. Parasite-free roach fingerlings were infec- bauer, Székely,Molnár & Baska2000). In most ted with Hoating triactinospores (TAMs) released of these studies, authors infected oligochaete or from oligochaeteson day 69 after challenge. Young altemate hosts with myxospores collec- plasmodia and spores in roach were first recorded ted from.fish and after development in theseworms on day 80 post-exposure (p.e.). Myxospores collec- they obtained sporesof actinosporean types such as ted from experimentally infected roach initiated a Triactinomyxon, Raabeia, Aurantiactinomyxon and new development in T tubiftx and the resulting Neoactinomyxum. Kent, Whitaker & Margolis TAMs infected roach. No infection of roach (1993) and Yokoyama, Ogawa & Wakabayashi resulted from feeding oligochaetes containing (1995) performed reverseexperiments, by infecting mature triactinospores. fish with actinospores obtained from oligochaetes. Kent et al. (1993) infected the sockeye salmon, Keywords: altemate hosts, experimental infection, Oncorhynchus nerka (WalbaUni), with triacti- life cycle, Myxobolus pseudodispar, Myxosporea, nospores releasedfrom the lumbriculid Stylodrilus Rutilus rutilus, Tubifex tubifex heringianus Claparéde, and these $tagesdevewped in the brain of salmon into Myxobolus arcticus Introduction Pugachev & Khokhlov, while Yokoyama et al. (1995) infected goldfish, Carassius auratus (L.), As Wolf & Markiw (1984) reported, the develop- with raabeia-type actinospores which transformed ment of Hofer was accom- in the altemate host Branchiura sowerbyiBeddard, plished through a salmonid fish and the oligochaete into a Myxobolus in the cartilage. This T tubifex (Müller), and a number of studies have species was described as M. cultus Yokoyama, demonstrated that other myxosporean speciesalso Ogawa & Wakabayashi. The complete develop- develop through oligochaete and, less frequen- mental cycle (myxosporean and actinosporean tly, polychaete alternate hosts (EI-Matbouli & phase) is known only for some species.EI-Matbouli

Correspondence Cs Székely, Veterinary Medical Research & Hoffmann (1998) successfullyrepeated Wolf & Institute, Hungarian Academy of Sciences, H-1581 Budapest, Markiw's experiment with M. cerebralis, while PO Box 18, Hungary Ruidisch, EI-Matbouli & Hoffmann (1991), (e-mail: [email protected])

@ 2001 BlackweUScience Ltd 461 Journal of Fish Diseases 2001, 24, 461-468 cs Székely et al. Compkte developmental')Ick of Myxobolus pseudodispar

Bartholomew et al. (1997) and Yokoyama (1997) In experiment 3, laboratory cultured parasite-free demonstrated the complete developmental cycle of oligochaetes were infected with M. pseudodispar M. pavlovskii Akhmerov, Ceratomyxashasta Noble myxospores.When floating T AMs appeared in the and Thelohanellushovorkai Akhmerov, respectively. water of the container the oligochaeteswere washed Myxobolus pseudodispar(Gorbunova), is a com- from the sediment and individually placed into mon myxosporean of the roach, Rutilus rutilus L. 2-mL cell-weIl plates (see Székely et al. 1999). The development in fish and pathogenic effects of Heavily infected specimens were selected under a this specieswere studied in detail by Baska (1987). stereomicroscope on the basis of released TAMs. Székely et al. (1999) studied the development of Infected oligochaetes were fed to SPF roach M. pseudodispar in' oligochaetes. These authors fingerlings. Each fingerling received one heavily collected myxospores from the muscles of roach TAM-infected oligochaete. The fish were killed and successfullyinfected T tubifex and Limnodrilus 90-215 days p.e. and the complete musculature hoffineisteri Claparéde. They reported that after a and kidneys were checked for the presence of 2.5-month prepatent period, triactinospores devel- M. pseudodisparplasmodia in smear preparations. oped in these oligochaetes. In experiment 4, the cycIe was repeated with This paper reports experimentsin which triactino- experimentally obtained myxospores. Myxobolus spores releasedfrQm r tubifexafterchallenge with pseudodisparmyxospores were collected from the M,p~dqdispar myxospores led to myxosporean muscle of fish from experiment 1. One hundred development resulting in M. pseudodisparinfection T tubifex were challenged with about 10 000 in the muscles of roach. myxospores of M. pseudodispar.When T AMs were releasedinto the water of the coritainers, 10 roach fingerlings were added for 24 h. Fish in this Materials and methods experiment were killed 77 days p.e. and examined Myxoboluspseudodispar myxospores were collected for the presence of plasmodia and myxospores as from mature intramuscular plasmodia in heavily described previously. infected roach from Lake Balaton and from the Roach cultured in a closed system under parasite Kis-Balaton water reservoir, Hungary. Muscles of free conditions served as controls. The musculatUre !oach were squashed between two glass plates. of five fish from this stock were examined by smear Intramuscular plasmodia were separatedfrom non- preparations in each experiment as a control. infected muscle cells and opened using a needle under a stereomicroscope to obtain myxospores. Laboratory-cultured parasite-free 7: tubifix were Histology challenged'in a 500-mL cup by the addition of When muscle cells on one side of the body of roach 50 000 myxospores. Water in the cups was regu- from experiments 1 and 2 showed relatively heavy larly checked for floating triactinomyxon spores infection with intramuscular plasmodia, the other (TAMs). The method used was that described by side of the bodywas fixed in Bouin's solution for Székdy ct:al. (1999). 3 h. The fish were embedded in paraffin wax and Fertilized eggsof roach were collected from Lake 4-6 J.1msections prepared and stained with hae- Balaton, hatched in aquaria and fed exclusively on matoxylin andeosin (H & E). Artemia nauplii and granulated food to obtain parasite free roach fingerlings. Results Four experiments were conducted. In experiments 1 and 2, 20 and 15 SPF roach In experiment 1 (T able 1) 7: tubifex specimens fingerlings (3-5 cm in body length), were placed infected with myxospores of M. pseudodisparfirst into containers containing approximately 3000 releasedTAMs on day 77 p.e. Releaseof TAMs freshly releasedT AMs for 24 and 20 h, respectively. increasedup to day 95 and then decreasedcontinu- Fish were killed 80-254 days post-exposure (p.e.) ously to day 120 poe.The fish were challengedwith and the complete musculature was checked for the TAMs on clay 90 p.e. Myxobolus pseudodíspar presence of M. pseudodisparplasmodia in smear developed into plasmodia in the muscle in 55% of preparations under a light microscope. Myxospores the exposed fish. Young plasmodia in the roach were counted by flattening plasmodia and estima- muscle harbouring developmental stageswere first ting the spüre numbers. found 80 clays p.e. (Fig. 1). Less frequently, these

@ 2001 Blackwell ScienceLtd 462 Journal of Fish Diseases 2001, 24, 461--468 Cs Székely et al. Compkte developmentalcyck of Myxobolus pseudodispar

Table 1 Challenge of roach fingerlings wirh Hoating TAMs of M. pseudodisparwirh an exposure time of 24 h (experiment 1)

Fishno. Fish length (cm) No. and development of plasmodia (spores) Necropsy(days p.e.)

1 3.4 2 3.6 Three young plasmodia 3 3.8 4 3.5 One young plasmodium with some spores 5 . 3.4 6 3.5 7 3.9 One plasmodium (20) 8 4.1 Three plasmodia (100) 9 4.2 Seven plasmodia (1500) 10 4.0 10plasmodia (3000) 11 4.1 12 4.5 Five plasmodia (400) 13 5.0 20 plasmodia (4000) 1~ 5.1 15 4.5 Nine plasmodia (3000) 16 5.0 17 4.5 - 18 7.0 19 5.2 11 plasmodia (30bO) 20 5.5 Five plasmodia (500)

Prevalence 11/20 (55%)

Figure 1 Experimental infection of a roach by Myxoboluspseudodispar triactinospores (TAMs) 80 days post-exposure (p.e.). Devdoping plasmodium (atrow) in one of the muscle fibtes (x 1000).

plasmodia also contained some young spores.More released spores on compression of the muscle developedplasmodia containing mostly sporeswere between glassplates (Fig. 3) and the spores showed found on clay 170 p.e. (Fig. 2). Most of the the characteristic asymmetric shape of M. pseudo- plasmodia were very small (50-120 X 20-40 !.lm), dispar (Fig. 4). Although plasmodia ruptUred easily containing not more than 20-400 myxospores.The on compression and spores were releasedinto the number of plasmodia and sporesincreased with time intermuscular space, no disseminated spores.were and some plasmodia reacheda size of 300 X 50 !.lm. found in the kidneys of the roach. In most infected fish only 1-11 plasmodia were In experiment 2 (Table 2) (which was performed found in the muscle; in one roach. however, 20 in a similar way as experiment 1) fish were developing plasmodia were counted. Infection in the examined only on day 254 p.e., at which time only challengedfingerlings was found up to day 239 p.e. mature plasmodia with spores were detected. The (8 mon ths). At that time the plasmodia easily prevalenceof infection was 62%.

@ 2001 BlackwellScience Ltd 463 Journal of Fish Diseases 2001, 24, 461-468

Figure 2 Experimenral infection of a roach by Myxoboluspseudodispar T AMs 170 days post-exposure (p.e.). A plasmodium containing developmental stagesand spores in the muscle /ibre (arrow) (x 500).

Figure 3 Myxospores of Myxobolus pseudodisparfrom a squashed musde plasmodium of an experimenta1ly infecred roach 8 monms post-exposure (p.e.) (x 500).

In experiment 3, none of the 10 roach fingerlings Discussion was found to be infected on days 90-215 p.e. by light-microscopic examination of fresh squash In the present study, roach and the oligochaete preparations made from the entire musculature 7: tubifex proved to be suitable altemate hosts for and kidneys of the fish. In experiment 4, on day 77 M. pseudodispar.Roach fingerlings reared in the p.e. plasmodia were seenin the muscle cells of 7 of laboratory under parasite-free conditions were suc- the 10 fingerlings challenged (Table 3). AlI control cessfully infected with experimentally produced fish were uninfected. floating T AMs of M. pseudodispar.After entering Histological examination of the experimentally the fish host, the infective cells of the sporoplasm of infected fish showed a typical M. pseudodispar the TAMs developed further and after 2.5 months infection with intracellular plasmodia in the muscle at 20 oC myxospores were formed. In this experi- fibres. Plasmodia fixed in the early stageof infection ment, two consecutive complete developmental contained vegetative developmental stages and cycles of M pseudodisparwere successfully repro- pansporoblasts (Fig. 5). duced by the use of oligochaete and fish altemate

@2001 Blackwell ScienceLtd 464 Figure 4 Characteristic asymmettic myxospores of Myxobolus pseudodispar releasednom a matute plasmodium in the muscle of an expetimentally infected toach 8 months post-exposure (p.e.) (x 1000).

Table 2 Challenge of roach fingerlings wirh floaringTAMs of M pseudodisparwirh an exposure time of20 hand necropsy at 245 days post-exposure (p.e.) (experiment 2)

No. fish Length ('Cm) Infection in muscle Intensi~ Development. of plasmodia

1 4.7 + b Large plasmodia 2 5..0 - 3 4.7 ,+ a Medium sized plasmodia . 4 5.Ö b Medium sized and large plasmodia 5 4.4 + a Medium sized plasmodia 6 5.7 + b Large plasmodia 7 4.6 +, a Medium sized plasmodia 8 4.5 + c Medium sized plasmodia .. 9 4.7 :;';1;, 10 4.7 ~ .. 11 4;9 ... .,. c'; 12 4.7 13 4.0 + a One small plasmodium Prevalence 8/13 (62%)

Infection in muscle: +: infected, -: non-infected. Intensity of infection: al-9 plasmodia, b 10-20 plasmodia, > 20 plasmodia.

Table 3 Challenge of roach fingerlings wirh floating TAMs of M pseudodisparwirh an exposure time of 20 h and necropsy 77 days post-exposure (p.e.) (experiment 4). TAMs derived from M. pseudodisparmyxospores obtained from experiment 1

Infection in muscle: +: ínfected. -: non-ínfeCted. Inrensítyof infecnon:. 1-9 plasmodía, b 10-20 plasmodia.

@ 2001 Blackwell ScienceLtd 465 Journal of Fish Diseases 2001, 24, 461-468 ú Székelyetal. Compkte developmentalcyc/e of Myxobolus pseudodispar

Figure 5 A relatively large Myxobolus pseudodispar plasmodium in me muscle fibre of an experimenrally infected roach 170 days post-exposure (p.e.) (H & E, X 500).

hosts, which suggeststhat M. pseudodisparinfection varying susceptibility to infection with M. cerebrali~ will be a suitable laboratory model in the future. as a result, in some biotopes high prevalence In our experiments, infection of the fish occurred M. cerebralis infections develop, while in others only as a result of invasion by floating actinospores the prevalence is low. The differences observed in and attempts to produce infection by feeding our experiments cannot apparently be e,xplainedby 7: tubifex specimensinfected with mature triactino- disparities in susceptibility, as both the oligochaete sporesfailed. This suggeststhat in the roach-oligo- and the fish stocks used were of identical origin and chaete M pseudodisparmodel the infective cells of presumably had very similar genetic propenies, and actinosporespresumably invade the fish through the the environmental factors, the temperature and the skin and gills, rather than through the gut wall, and age and number of the spores were also identical. reach the site of final development, the skeletal Whilethesuécessful infectionofoligochaetes and muscle cells, by a subsequent ~igration. These fish undoubtedly requires the presenceof a certain results are consistent with the accounts of several number of spores, in this study almost identical authors (Wolf & Markiw 1984; EI-Matbouli results were obtained by the use of a large number & Hoffmann 1989,1998; EI-Matbouli, McDowell, of spores collected from naturally infected fish and Mukkatira & Hedrick 1998), but are at vari- by the use of a relatively low number of spores ance with the observations of Yokoyama (1997), derived from experimentally infected fish. In the who successfullyproduced 7: hovorkai infection in latter, relatively small plasmodia containing as few common carp, Cyprinus carpio L., by feeding as 20-30 spores, a few hundred spores or ar most B. sowerbyi specimens containing aurantiactino- 1000 spores developed, which, however, very successfullyinfected the fish used in experiment 4. myxon spores. Although this study has brought us closer to A possible explánation is that the infectivity of attaining the objective of developing a model young spores not yet darnagedby the host reaction suitable for studying the development, ecology substantially exceededthat of sporescollected from and pathological füle of myxosporeans, many natural infectipns, which were probably aged and problems remain unsolved. It is unclear why it mostly releasedfrom a connective tissue capsule. was not possible to achieve 100% prevalence of No solitary spores were found in the melano- infection despite the use of very high numbers of macrophage centres of the kidney in these experi- myxospores and actinospores, and why substantial ments, although the occunence of such spores differences developed in the intensity of infection. proved to be common in naturally infected fish. Studying the susceptibility of different oligochaete The appearance of solitary or aggregated spores strains, EI-Matbouli et al. (1998) concluded that sunound~d and darnaged by macrophages in the within the species 7: tubifex there are strains of melanomacrophagecentres of the kidney was found

@ 2001 Blackwell ScienceLtd 466 Journal of Fish Diseases 2001, 24, 461-468 cs Székelyetal. Compkte developmental",ck of Myxobolus pseudodispar

to be common in severeMyxobolus cypnni Doflein, EI-Matbouli M. & Hoffmann R.W. (1998) Light and electron infection by Molnár & Kovács-Gayer(1985) and in microscopic studies on the chronological development of Myxobolus c~r~bralisto the actinosporean stage in Tubiftx M. pseudodisparinfection of the roach by Baska tubifex. International Journal for Parasitology28, 195-217. (1987). According to theseauthors, this was because EI-Matbouli M., Fischer-Scherl T. & Hoffmann R.W. (1992) spores released infO the intermuscular space after Transmission of Hoftr~llus carassiiAchmerov, 1960 to goldfish the disruprion of damaged muscle cells and mature Carassiusauratus via an aquatic oligochaete. Bulletin of th~ plasmodia were carried to the kidney via the blood Europ~an Association of Fish Pathologists12, 54-56. stream. The absenceof disseminated spores in the El-Matbouli M., McDowdl T., Mukkatira K. & Hedrick R.P. experimental material in this study can presumably (1998) Susceptibility of two different populations of tubificids be attributed to the relarively young age of the to Myxobolus cer~bralis.Proc~~dings of Whirling Dis~as~ Symposium, 19-.c21F~bruary, 1998. Fort Collins, CO, sporesand to the tower number of sporescontained USA, pp. 117-119. by the small-sized plasmodia. Eszterbauer E., Székely Cs., Molnár K. & Baska F. (2000) Development of Myxobolus bramM (Myxosporea: Myxoboli- dae) in an oligochaete altemate host, Tubifex tubifex. Journal óf Acknowledgements Fish Dis~as~s23, 19-25. The authors thank the Hungarian Scientific Grossheider G. & Körting W. (1992) First evidence that ResearchFund (OTKA), projects No. T 31755 and HOf"~llUS 'JIpnni (Doflein, 1898) is transmirted by Nais sp. T 29200, for financial support and Ms Zsuzsa Kis Bulletin of th~ Eur~p~anAssociation of Fish Pathologists12, 17-20. for preparing the histological sections. Thanks are Kent M.L., Whitaker D.J. & Margolis L. (1993) Transmission of due to the Balaton Fisheries Co. for providing Myxobolus arcticus Pugachev and Khokhlov, 1979, a myxosp- fertilized roach eggs. orean parasite of Pacific salmon, via a triactinomyxon from the aquatic oligochaete Stylodnlus heringianus (Lumbriculidae). Canadian Journal of Zoology 71, 1207-1211. References Molnár K. & Kovács-Gayer É. (1985) The pathogenicity and Bartholomew J.L., Whipple M.J., Stevens D.G. & Ftyet J.L. devdopment within the host fish of Myxobolus 'JIpnni Doflein, (1997) The life cycle of Ceratomyxashasta a myxosporean 1898. Parasitology90, 549-555. parasite of salmonids requires a freshwater polychaete as an Molnár K, EI-Mansy A., Székdy Cs. & Baska F. (1999a) altemate host. Journal of Parasitology83, 859-868. Development of Myxobolus dispar Thelohan, 1895 (Myxo- Baska F. (1987) Histological studies on the development of sporea: Myxobolidae) in an oligochaete altemate host Tubiftx Myxoboluspseudodispar Gorbunova, 1936 in the roach (Rutilus tubiftx (Müller). Folia Parasitologica46, 15-21. . TUtilus).Acta Veterinaria Hungarica 35,251-257. Molnár K., EI-Mansy A., Székely Cs. & Baska F. (1999b) Benajiba M.H. & Marques A. (1993) The altemation of acti- Development of Spha"ospora renico/a Dykova and Lom, 1982 nomyxidian and myxosporidian sporal forms in the devdop- (Myxosporea: Sphaerosporidae) in oligochaete alternate hosts. ment of Myxidium giardi (parasite of Anguil/4 anguil/4) Journal of Fish Dis~as~s22, 1-11. through oligochaetes. Bulletin of the European Associationof Ruidisch S., EI-Matbouli M. & Hoffmann R.W. (1991) The cole Fish Pathologists13, 100-103. of tubificid worms as an intermediate host in the life cycle of El-Mansy A. & Molnár K. (1997a) Extrapiscine development of Myxobolus pavlovskii (Akhmerov, 1954). ParasitologyRes~arch Myxobolus drjagini Akhmerov, 1954 (Myxosporea, Myxobol- 77, 663-667. idae) in oligoChaete alternative hosts. Acta Veterinaria Hun- Styer E.L., Harrison L.R. & Burtle G.J. (1991) Experimental garica 45, 427-438. production of proliferative gill diseasein channel cat/ish El-Mansy A. & Molnár K (1997b) Development of Myxobolus exposed to a myxowan-infected oligoChaete, D"o digitato. hungaricus (Myxosporea: Myxobolidae) in oligochaete alter- Journal of Aquatic Animai H~alth 3,288-291. nate hosts. Diseasesof Aquatic Organisms31, 227-232. Székely Cs., El-Mansy A, Molnár K & Baska F. (1998) El-Mansy A., Molnár K. & Székely Cs. (1998) Development of Devdopment ofTh~lohanellus hovorkai and Th~lohan~llus Myxobolusporeucalensis Saraiva & Molnár, 1990 (Myxosporea: nikolskii (Myxosporea: Myxowa) in oligochaete altemate Myxobolidae) in the oligochaete Tubifix tubifix (Müller). hosts. Fish Pathology 33, 107-114. SystematicParasitology 41, 95-103. Székely Cs., Molnár K., Eszterbauer E. & Baska F. (1999) El-Matbouli M. & Hoffmann R.W. (1989) Experimental Experimental detection of the acrinospores of Myxobolus transmission of tWo Myxobolus spp. developing by sporogeny pseudodispar(Myxosporea: Myxobolidae) in oligoChaete via tubificid worms. ParasitologyResearch 75, 461-464. altemate hosts. Dis~as~sof Aquatic Organisms38, 219-224. El-Matbouli M. & Hoffmann R.W. (1993) Myxobolus carassii Trouillier A., EI-Matbouli M. & Hoffmann R. (1996) A new Klokacheva, 1914 also requires an aquatic oligochaete, Tubifex IDok at the life-cycle of Hof"~llus carassiiin the goldfish tubifex as intermediate host in its life cycle. Bulletin of the (Carassiusauratus auratus) and its relation to 'kidney European Associationof Fish Pathologists13, 189-192. enlargement disease'(KED). Folia Parasitologica43. 173-187.

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Uspenskaya A.V. (1995) Alternation of actinosporean and transmission of Hoferellus carassii (Myxosporea: Myxozoa), the myxosporean phases in the life cyde of Zschokella nova causative agent of kidney enlatgement disease (KED) of (Myxowa). Journal of Eukaryotic Microbiology 42, 665-668. goldfish Carassius auratus. Fish Pathology 28, 135-139. WolfK. & Marklw M.E. (1984) Biology contravenestaxonomy Yokoyama H., Ogawa K. & Wakabayashi H. (1995) Myxobolus in the Myxowa: new discoveries show aItemation of cultus n. sp. (Myxospotea: Myxobolidae) in the goldfish invertebrate and vertebrate hosts. Science225, 1449-1452. Carassius auratus transformed from the actinospotean srage in Yokoyama H. (1997) Transmission of Thelohanellushovorkai the oligochaete Branchiura sowerbyi. Journal ofParasitology 81, Achmerov, 1960 (Myxosporea: Myxozoa) to common carp 446-451. Cyprinus carpio through the altemate oligochaete host. Received:23 Apri/200I SystematicParasitology 36, 79-84. Accepted:28 June 2001 Yokoyama H., Ogawa K. & Wakabayashi H. (1993) lnvolve- ment of Branchiura sowerbyi (Oligochaeta: Annelida) in the

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