Ahead of print online version FOLIA PARASITOLOGICA 58[2]: 157–163, 2011 © Institute of Parasitology, Biology Centre ASCR ISSN 0015-5683 (print), ISSN 1803-6465 (online) http://www.paru.cas.cz/folia/ The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore Szilvia Marton and Edit Eszterbauer Veterinary Medical Research Institute, Hungarian Academy of Sciences, P.O. Box 18, H-1581 Budapest, Hungary Abstract: Echinactinomyxon-type actinospores were found in a mixed-species oligochaete culture originating from the Temperate Water Fish Hatchery near Budapest, Hungary. On the basis of DNA sequence analysis, the actinospores were identified asMyxobolus pavlovskii (Akhmerov, 1954), the 18S rDNA sequence from myxospores of which is available in GenBank. Silver carp Hypoph- thalmichthys molitrix (Valenciennes) fry specimens were successfully infected by cohabitation with the echinactinomyxon-releasing oligochaetes, which confirmed the molecular data congruence. The echinactinomyxons and the myxospores that developed in the gills of exposed fish fry were analysed morphologically and on DNA basis. The infected gill tissue was examined histologically. As typical characters of M. pavlovskii, numerous small plasmodia were observed in the epithelia of gill lamellae. Plasmodia contained thousands of myxospores with polar capsules unequal in size and with large intercapsular processes. The 18S rDNA sequence from actinospores and those from myxospores originating from the experimentally infected fish were identical. The oligochaete species releasing actinospores was morphologically determined as Limnodrilus sp. This is the first record of an echinactinomyxon as an alternate stage within the genus Myxobolus. Keywords: Myxosporea, life cycle, silver carp, Hypophthalmichthys molitrix, 18S rDNA, histology, infection experiment Myxobolus pavlovskii (Akhmerov, 1954) is a myxo- a source of the infectious agent (Molnár 1979). Later, Ru- zoan parasite of the silver carp Hypophthalmichthys mo- idish et al. (1991) performed transmission experiments in litrix (Valenciennes) and the bighead carp Hypophthal- order to elucidate the life cycle of M. pavlovskii in greater michthys nobilis (Richardson). These two fish species detail. Myxospores originating from naturally infected were introduced to Hungary from Eastern Asia (China silver carp, identified as M. pavlovskii by morphological and the former USSR) in the early 1960s. The fish popu- measurements, were used for the infection of oligocha- lations in China already had been infected with M. pav- etes. Tubificid oligochaetes were added to the aquaria lovskii, and after the introduction to Hungary in the course containing myxospores, and after 93 days post exposure of a breeding programme of herbivorous fish, the parasite (p.e.), hexactinomyxon-type actinospores were detected was disseminated throughout the country (Molnár 1979). in the water. By cohabitation of infected oligochaetes and Myxobolus pavlovskii infects the gills and develops in silver carp, one-year-old fish specimens became infected. the stratified epithelium between gill lamellae, where After 120 days p.e., cysts containing myxospores were the developing plasmodia can fill the interlamellar space found between the lamellae of gills. In the study by these (Molnár 2002). In heavy infections, it may cause severe authors, only morphological characters were used to iden- pathological changes by hampering the host gas exchange tify the spores. and excretion. The present study was initiated after echinactino- Molnár (1979) was the first to conduct infection exper- myxon-type actinospores had been observed in a mixed iments with this parasite to obtain further information on oligochaete culture collected in the Temperate Water Fish its development. He examined several specimens of silver Hatchery (TEHAG) near Budapest, Hungary. The 18S carp and bighead carp from three fish farms in Hungary rDNA sequence of these actinospores had been found for the presence of M. pavlovskii, and performed both identical with that of M. pavlovskii myxospore isolates field and laboratory experiments. In the field, he could not previously studied by Eszterbauer (2004). This finding prevent uncontrolled infection of parasite-free fry despite contradicted the results obtained by Ruidish et al. (1991) a variety of pre-treatments (e.g., desiccation of the pond, and suggested that an echinactinomyxon instead of a hex- mud removal, quicklime treatment). Successful laborato- actinomyxon might be the actinospore stage of M. pav- ry infections of fish were only possible using mud/soil as lovskii. Address for correspondence: E. Eszterbauer, Veterinary Medical Research Institute, Hungarian Academy of Sciences, P.O. Box 18, H-1581 Budapest, Hungary. Phone: +36 1 467-4067; Fax: +36 1 467-4076; E-mail: [email protected] 157 Ahead of print online version Among other gill-parasitic Myxobolus species, Eszter- formalin, embedded in paraffin, cut approximately in 5 μm thin bauer et al. (2002) studied M. pavlovskii from silver carp sections, and stained with haematoxylin and eosin. and bighead carp on a molecular basis using PCR-RFLP Myxospores collected from the gills of five silver carp speci- (restriction fragment length polymorphism). They found mens infected experimentally in the present study were used to the same restriction pattern with three restriction enzymes infect oligochaetes. The M. pavlovskii cysts were scraped off and demonstrated that M. pavlovskii is able to infect both the gill filaments with a dissecting needle and ruptured. The ob- tained spore suspension was homogenized by vortexing and the fish hosts. In another study by Eszterbauer (2004), phylo- number of myxospores was estimated in a Bürker-chamber. The genetic analyses of gill-infecting Myxobolus species con- oligochaete culture used in the infection trial contained various firmed that the myxospores morphologically identified Tubifex tubifex lineages and Limnodrilus spp. The worm stock as M. pavlovskii are indeed identical in silver carp and originated from the Temperate Water Fish Hatchery (TEHAG) bighead carp. In the present study, transmission experi- located in Százhalombatta near Budapest, Hungary, as well as ments, morphological and histological methods as well from a small stream devoid of fish located near Aufseß, Ger- as molecular biological techniques were used to complete many. Prior to infection trial, the worm stock was kept under the characterisation of M. pavlovskii and clarify its con- laboratory conditions for over a year, during which time it was troversial life cycle. periodically checked for the presence of waterborne actino- spores, with negative results. This worm stock also served as MateRIALS AND METHODS a negative control in the infection trial. Approximately 10 g of oligochaetes were placed into a 5-litre aerated plastic tank con- Collection and examination of actinospores taining water and a 2-cm thick layer of mixed mud and sand. For Mud samples containing oligochaetes were collected from exposure, M. pavlovskii myxospores (>106) were added to the the Temperate Water Fish Hatchery (TEHAG) located at worm cultures. After one month p.e., the water from the exposed Százhalombatta near Budapest, Hungary. Mud and sediments cultures and the control was regularly checked for the presence were sifted in the laboratory to collect large oligochaetes (i.e. of actinospores by filtration for a period of 6 months. tubificids). The worms were kept in aerated 25-litre plastic tanks containing a layer of autoclaved mud with sand and were fed Molecular biological analysis a mixture of minced frozen lettuce, Spirulina powder (MaBitec), Myxospore and actinospore samples and one infected oligo- and frozen Artemia or bloodworms once a week. Water changes chaete specimen were brought to molecular identification. For were carried out weekly. To check for myxozoan infection, wa- DNA extraction, the samples were centrifuged at 7,000 × g for ter from the cultures was regularly filtered through 20 μm ny- 5 min, spores were suspended in 500 µl lysis buffer (100 mM lon mesh, and the presence of actinospores was checked with NaCl, 10 mM Tris, 10 mM EDTA, 0.2% sodium dodecyl sul- a Zeiss Axiostar Plus phase contrast microscope. In order to phate and 0.4 mg/ml proteinase K) and incubated at 55 °C for identify the infection of oligochaete specimens, approximately 3−4 hrs. DNA was purified using a Miniprep Express Matrix 300 worms were sampled from the culture and the individual (BIO 101, Qbiogene) as described by Eszterbauer (2004). worms were placed in cell-well plates as described by Yokoyama A nested PCR assay was applied for the amplification of the 18S et al. (1991). After 24 hrs, the water in the wells was examined rDNA from the myxospores and actinospores. In the first round, for the presence of released actinospores. Photomicrographs of primers 18e (5’-CTG GTT GAT TCT GCC AGT-3’) (Hillis and actinospores were taken using a Moticam 2000 (Motic) digital Dixon 1991) and 18r (5’-CTA CGG AAA CCT TGT TAC-3’) camera mounted on a Zeiss Axiostar Plus microscope. Measure- (Whipps et al. 2003) were used, followed by a second round ments were taken using Motic Images Plus 2.0 software after PCR with the primer pair SphF (5’-ACT CGT TGG TAA GGT calibration. Actinospores were collected for molecular biologi- AGT GGC T-3’) – SphR (5’-GTT ACC ATT GTA GCG CGC cal identification in 1.5-ml microtubes and stored at –20 °C until GT-3’) (Eszterbauer and Székely 2004). For both