New cell motility model observed in parasitic cnidarian Sphaerospora molnari (Myxozoa:Myxosporea) blood stages in fish.

Cellular motility is essential for microscopic parasites, it is used to reach the host, migrate through tissues, or evade host immune reactions. Many cells employ an evolutionary conserved motor protein- actin, to crawl or glide along a substrate. We describe the peculiar movement of Sphaerospora molnari, a myxozoan parasite with proliferating blood stages in its host, common carp. Myxozoa are highly adapted parasitic cnidarians alternately infecting vertebrates and invertebrates. S. molnari blood stages (SMBS) have developed a unique "dancing" behaviour, using the external membrane as a motility effector to rotate and move the cell. SMBS movement is exceptionally fast compared to other myxozoans, non-directional and constant. The movement is based on two cytoplasmic actins that are highly divergent from those of other metazoans. We produced a specific polyclonal actin antibody for the staining and immunolabelling of S. molnari's microfilaments since we found that neither commercial antibodies nor phalloidin recognised the protein or microfilaments. We show the in situ localization of this actin in the parasite and discuss the importance of this motility for evasion from the cellular host immune response in vitro. This new type of motility holds key insights into the evolution of cellular motility and associated proteins.

Myxozoans as biological tags for stock identification of the Argentine hake, Merluccius hubbsi (Gadiformes: Merlucciidae).

Myxozoans have been successfully used as tags for fish stock identification around the world. However, few studies using myxozoan tags have been carried out in the Southern Atlantic, a region with complex oceanography that constitutes a potentially suitable scenario for testing the utility of myxozoans as indicators. Its usefulness was tested using six samples of Merluccius hubbsi in two different regions of the Argentine Sea. Generalized linear models were performed to assess the effects of fish size and sex, and year and region of capture and selected using the Information Theoretic approach. Three myxozoan species were recorded: Kudoa rosenbuschi, Myxoproteus meridionalis and Fabespora sp. Results of modelling species individually showed differential capabilities for detecting geographical population structure at different spatial scales, with K. rosenbuschi and Fabespora sp. allowing the discrimination of northern and southern stocks, but Fabespora sp. also as a promissory indicator of intrapopulation sub-structure due to different migratory routes during non-reproductive periods. This work confirms that myxozoans offer a set of suitable markers at different spatial scales, which can be selected individually or in any combination, depending on the geographical extent of the study, constituting tools adaptable to the objectives of further research on fish population structure.

Molecular characterization of Sphaerospora molnari (Myxozoa), the agent of gill sphaerosporosis in common carp Cyprinus carpio carpio.

Sphaerospora molnari Lom, Dyková, Pavlásková and Grupcheva, 1983 often causes severe infections in the gills and skin of common carp fingerlings Cyprinus carpio carpio in Central Europe. Although most Sphaerospora spp. are coelozoic and affect the excretory system of fish, S. molnari develops mature spores in the epithelia of gill filaments, making it a rare representative of histozoic freshwater species within the genus. On the basis of a partial 18S rDNA sequence assigned as belonging to S. molnari, previous phylogenetic studies located the species within the Myxobolus clade. In the present study, S. molnari isolates from Hungary and the Czech Republic were characterized based on morphology, DNA sequence analysis and phylogenetic comparison. The obtained 3714 bp final consensus 18S rDNA sequence of the parasite showed several, sometimes extremely long inserts in the variable regions of the gene and differed considerably from the one published in GenBank in 2002. In situ hybridization confirmed the validity of the obtained DNA sequence and detected pre-sporogonic blood stages in the interstitium and blood vessels of the kidney. Phylogenetic analysis showed that S. molnari clusters within the Sphaerospora sensu stricto clade with a high support, revealing it as the first known histozoic member of the Sphaerospora subclade comprising parasites of freshwater fish.

In vitro treatments for the theront stage of the ciliate protozoan Cryptocaryon irritans.

The ciliate protozoan Cryptocaryon irritans Brown, 1951, the 'marine white spot', causes one of the most important parasitic fish diseases, with extensive losses every year in mariculture and in the ornamental fish industry. In the present study, we explore the in vitro use of 8 different compounds against the theront (infective) stage of C. irritans; these compounds include extracts of natural products (epigallocatechin gallate (EGCG), L-DOPA, papain), peracetic acid-based compounds (Proxitane 5:23 and 15% peracetic acid, PAA), quinine-based compounds (quinacrine hydrochloride and chloroquine diphosphate) and hydrogen peroxide. All of these compounds had an effect on theront survival; however, only EGCG caused significant theront mortality when applied in doses > or =50 mg l(-1) and over a period of 3 h; papain caused a maximum theront mortality of <50%. We discuss the type of application and potential utility of the compounds tested as part of a management control strategy for C. irritans infections in marine aquaculture and the ornamental fish industry.

Host-parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids.

Sparidae are economically important fishes to both, fisheries and aquaculture in the Mediterranean. Species diversification is an important strategy for the development of Mediterranean aquaculture. One of the species recently introduced is the sharpsnout seabream Diplodus puntazzo (Walbaum, 1792). During a parasitological study of fish from the Gulf of Valencia and the Mar Menor (Spain), myxozoan spores belonging to the genus Ceratomyxa were found in the gall bladder of D. puntazzo. A morphological description of the spores, which includes histology and transmission electron microscopy (TEM) as well as molecular (SSU ribosomal DNA) data resulted in the erection of a new species, Ceratomyxa puntazzi n. sp. A histopathological study of C. puntazzi n. sp. infection in D. puntazzo showed that the parasite causes necrosis and loss of epithelial cells in the gall bladder, and provokes a pericholangitis in the liver tissue surrounding the bile ducts. Furthermore, molecular data obtained from C. puntazzi n. sp. and three other ceratomyxids from the closely related fish species Diplodus annularis L. and Sparus aurata L. which share the same habitat suggest that the genus Ceratomyxa is host-specific in sparids, which agrees with data previously obtained from Serranidae, Labridae and Pomacentridae, and that ceratomyxid species from sparids in the Mediterranean originated from a common ancestor.

Infection dynamics of two renal myxozoans in hatchery reared fry and juvenile Atlantic cod Gadus morhua L.

In order to study the infection dynamics of 2 renal myxozoans, Zschokkella hildae Auerbach, 1910 and Gadimyxa atlantica Køie, Karlsbakk and Nylund, 2007 in cultured Atlantic cod, Gadus morhua L. aged 3-19 months, a specific single-round PCR assay and a double-label in situ hybridization protocol were developed. The results demonstrated that the 2 myxozoans show spatial separation of their development with regard to spore formation inside the renal tubules versus the collecting ducts and ureters, as well as temporal separation with Z. hildae proliferating and developing spores only once the G. atlantica infection decreases, despite the presence of both myxozoans in the smallest fry studied. These results strongly suggest within-host competition of the 2 myxozoans with potential suppression of Z. hildae by G. atlantica until G. morhua acquires immunity against G. atlantica. The quantification of the G. atlantica infection inside the renal tubules before and after a 29-day experimental growth performance study using fry from hatcheries with differing filtration systems showed that the intensity of infection with G. atlantica seems to be controlled if prolonged exposure to the myxozoan transmission stages takes place from hatching onwards. Surprisingly, growth rates in the trial were inversely affected suggesting that G. atlantica does not negatively influence cod fry growth performance.

Morphological and molecular redescription of the myxozoan Unicapsula pflugfelderi Schubert, Sprague & Reinboth 1975 from two teleost hosts in the Mediterranean. A review of the genus Unicapsula Davis 1924.

Elongate plasmodia with myxosporean spores belonging to the genus Unicapsula, Davis, 1924 were found in the skeletal muscle of the striped seabream, Lithognathus mormyrus (L.), a candidate for the mediterranean aquaculture. The only species of Unicapsula described from the Mediterranean is Unicapsula pflugfelderi Schubert et al. 1975, which occurs in the picarel, Spicara smaris (L.). For morphological and molecular comparison of U. pflugfelderi from S. smaris with Unicapsula sp. from L. mormyrus measurements of plasmodia and spores, ultrastructural details and 18S and 28S rDNA sequences were analysed. Whereas plasmodia were 2-3 times larger in S. smaris than in L. mormyrus (length 2.47-0.81 mm; width 0.22-0.09 mm; P = 0.000), spore morphology showed minor differences and both 18S and 28S rDNA sequences were 100% identical identifying the myxozoan as U. pflugfelderi. Scanning electron microscopy of the spores revealed a different shell valve distribution than the one used for the diagnosis of the genus Unicapsula. This resulted in a review of the genus Unicapsula dividing it into two morphological groups of different spore valve arrangement. TEM revealed the presence of a yet undescribed crystalline structure in the sporoplasm of the spores.

Cardicola aurata sp. n. (Digenea: Sanguinicolidae) from Mediterranean Sparus aurata L. (Teleostei: Sparidae) and its unexpected phylogenetic relationship with Paradeontacylix McIntosh, 1934.

A new sanguinicolid trematode, Cardicola aurata sp. n., is described from gilthead seabream Sparus aurata L., from off the Spanish Mediterranean coast. The morphology of C. aurata sp. n. generally agrees with the diagnosis of the genus, however, in contrast to all other reported Cardicola spp. the male pore is located sub-medially at the posterior end of the body instead of sinistrally before the posterior end of the body. Based on a comparison of the morphology as well as partial 28S and ITS2 rDNA sequence data from the present species with that from closely related species, it was decided to emend the diagnosis of Cardicola rather than create a new genus, as the aberrant position of the male pore is likely to be an autapomorphy. The phylogenetic analyses revealed a close relationship between Cardicola and Paradeontacylix, two genera with considerable morphological differences; C. aurata sp. n. occupies a position intermediate to these genera. Thus, a morphological comparison of Cardicola, Paradeontacylix and Braya, a genus which is morphologically similar to Cardicola but clusters basal to the Cardicola/Paradeontacylix clade, was conducted. The results of this comparison showed that despite large differences with regard to body shape, the organisation of the internal organs is very similar in species of Cardicola and Paradeontacylix. The synopsis of morphological data and molecular phylogeny allows for interpretations regarding the importance of different morphological features for the phylogenetic inference of the Sanguinicolidae.

Kudoa unicapsula n. sp. (Myxosporea: Kudoidae) a parasite of the Mediterranean mullets Liza ramada and L. aurata (Teleostei: Mugilidae).

A new multivalvulid myxozoan parasite, Kudoa unicapsula n. sp., is described from the intestinal mesentery, intestine and pyloric caeca of the thin-lipped grey mullet Liza ramada (Risso 1826) and the golden grey mullet L. aurata (Risso, 1810) from the Mediterranean coastal waters of Spain. It is characterized by the presence of elongated, rice corn-like white cysts of 0.47-0.56 x 0.18-0.38 mm, filled with tetracapsulate, slightly asymmetric spores, rectangular in apical view and tear-shaped in lateral view with four polar capsules of considerably different size and slightly unequal spore valves with rounded edges, overlapping each other on the apex of the spore. One large polar capsule includes a polar filament coiled in two to three turns, and the other three polar capsules, which are very small, posses only a rudimental filament. Both light and electron microscopy data showed that this species differs from all previously described Kudoa spp. with unequal polar capsules. The molecular analysis based on 18S and 28S ribosomal deoxyribonucleic acid DNA sequence data of K. unicapsula n. sp. indicates a close relationship and thus phylogenetic clustering together with K. trifolia, a myxozoan from the same host and the same geographical location.

Kudoa trifolia sp. n. - molecular phylogeny suggests a new spore morphology and unusual tissue location for a well-known genus.

A new species of myxozoan, Kudoa trifolia sp. n., was found in various organs of the golden grey mullet, Liza aurata (Risso), and the thinlip mullet, L. ramada (Risso), from the western Mediterranean. Spores developed in subspherical plasmodia of 0.28-1 mm diameter within connective tissue, predominantly in the spleen, the outer wall of the gall bladder and the gut, the mesenteries and occasionally also in the gills. The spores of K. trifolia differ from the commonly known shape of Kudoa by considerable enlargement of one of the four valve cells, thus forming a 'spore body', which contains the major part of the binucleate sporoplasm. Scanning electron microscopy of the spores revealed the presence of grape-like appendages, which occur in bundles terminally on the valve cells. Phylogenetic analysis based on the 18S rDNA sequence of K. trifolia showed that this species is deeply embedded in the genus Kudoa despite its aberrant morphology and host tissue location. This suggests important amendments to the morphological diagnosis of the genus Kudoa.

Molecular studies on the seasonal occurrence and development of five myxozoans in farmed Salmo trutta L.

Five myxozoan species, Tetracapsuloides bryosalmonae, Sphaerospora truttae, Chloromyxum schurovi, Chloromyxum truttae and a Myxobolus species were detected in farmed brown trout, Salmo trutta L. from Central Scotland. Using PCR and in situ hybridization, this study investigated the seasonal occurrence and tissue location of these species in young of the year brown trout. C. schurovi, C. truttae and Myxobolus sp. were first detected in brown trout in April, 2 months before T. bryosalmonae and S. truttae. T. bryosalmonae and S. truttae showed proliferation in the blood with intravascular stages of T. bryosalmonae accumulating in the heart. In contrast, only small amounts of PCR products of C. schurovi and C. truttae were obtained from the blood, suggesting that these species use the vascular system for transport but proliferate only in their target tissues from which large amounts of PCR product were obtained and where parasites were visible in histological sections. Large amounts of PCR product were obtained for T. bryosalmonae, S. truttae and both Chloromyxum species from the gills of brown trout, suggesting the gills as entry locus for these species. The neurotropic Myxobolus species formed plasmodia predominantly in the peripheral nerves, possibly indicating an entry route through the skin. Presporogonic stages of all other species had disappeared by September and mature spores were present from August onwards.

Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique.

Sphaerospora truttae is an important pathogen of Atlantic salmon parr in Scottish aquaculture. To trace the early development of S. truttae and to overcome the common problem of detecting low numbers of cryptic, early myxosporean stages, a DNA-based approach was applied in this study. Specific primers were designed for S. truttae from Atlantic salmon, based on 18S rDNA sequences, obtained from isolated myxosporean spores. These were 5' biotin-labelled and used in an optimized and rapid in situ hybridization (ISH) protocol, which provided a strong and specific signal of the parasite within host tissue sections and, at the same time, minimized structural damage to tissues due to processing. This methodology provided a reliable tool enabling the detection of S. truttae stages down to single cell level. Using ISH the epithelium of the gills was identified as the predominant entry locus of the parasite. By 3 days after infection S. truttae had penetrated the vascular epithelia and thereafter proliferated in the blood for at least 10 days before exiting the vascular system through capillary walls. From day 12 post-infection onwards, the kidney, as well as the spleen and the liver, were invaded. Numbers of S. truttae invading the kidney (37.3%) differed little from numbers found in the spleen (35.3%) and the liver (27.4%). The latter organs represented a dead end in the development of S. truttae as all stages in these organs degenerated and sporogony was found to take place exclusively inside the renal tubules. Early sporogonic stages were found from day 25 post-infection but mature spores only developed after at least 15 days of proliferation within the tubules.

Myxosporidia and macrophage centres in chub (Leuciscus cephalus)--quantitative interactions focus on Myxobolus cyprini.

Six myxosporidian species were found in chub (Leuciscus cephalus) originating from Lower Austrian rivers. The frequency of the parasites and their localization was recorded. In all chub, independent of size and origin, Myxobolus cyprini occurred predominantly in the macrophage centres (MCs) of the haematopoietic organs, spleen and kidney. Exclusively in the head kidney of young fish not yet described vermicular plasmodia containing spores of M. cyprini were found. In muscle tissue the prevalence of M. cyprini was comparatively low. Other species of Myxobolus characterized by plasmodial cysts frequently occurred in gills and swimbladder but were rarely detected, and only in small numbers, in the haematopoietic organs. The number of M. cyprini spores and the relative volume of MCs in the haematopoietic organs were estimated in order to examine possible correlations. Significant interrelated changes were found only in juvenile fish up to a size of 15 cm. In bigger fish, the number and size of macrophage aggregates were highly variable and independent of infection intensity and fish size, but the number of spores never exceeded that of the aggregated macrophages. The data suggest that due to an early date of infection M. cyprini is the only species which is closely associated with macrophage aggregation.