Myxosporean Stages Research Articles

Expression profiling and cellular localization of myxozoan minicollagens during nematocyst formation and sporogenesis

In free-living cnidarians, minicollagens are major structural components in the biogenesis of nematocysts. Recent sequence mining and proteomic analysis demonstrate that minicollagens are also expressed by myxozoans, a group of evolutionarily ancient cnidarian endoparasites. Nonetheless, the presence and abundance of nematocyst-associated genes/proteins in nematocyst morphogenesis have never been studied in Myxozoa. Here, we report the gene expression profiles of three myxozoan minicollagens, ncol-1, ncol-3, and the recently identified noncanonical ncol-5, during the intrapiscine development of Myxidium lieberkuehni, the myxozoan parasite of the northern pike, Esox lucius. Moreover, we localized the myxozoan-specific minicollagen Ncol-5 in the developing myxosporean stages by Western blotting, immunofluorescence, and immunogold electron microscopy. We found that expression of minicollagens was spatiotemporally restricted to developing nematocysts within the myxospores during sporogenesis. Intriguingly, Ncol-5 is localized in the walls of nematocysts and predominantly in nematocyst tubules. Overall, we demonstrate that despite being significantly reduced in morphology, myxozoans retain structural components associated with nematocyst development in free-living cnidarians. Furthermore, our findings have practical implications for future functional and comparative studies as minicollagens are useful markers of the developmental phase of myxozoan parasites.

Life cycles of three Myxobolus spp. from cyprinid fishes of Lake Balaton, Hungary involve triactinomyxon-type actinospores.

A study on the actinosporean fauna of oligochaetes of Lake Balaton was carried out from 2009 to 2011. The morphology of actinosporean stages of myxosporeans obtained from oligochaetes was studied, and their 18S rDNA structure was analyzed by molecular biological methods. Three triactinomyxon types were released from the oligochaete Isochaetides michaelseni (Tubificidae). The sequences of Triactinomyxon type 1 proved to be identical with those of Myxobolus fundamentalis. The sequences of Triactinomyxon type 2 showed 99.9% similarity to Myxobolus eryhtrophthalmi, while the sequences of Triactinomyxon type 3 showed a 99.9% similarity to those of Myxobolus shaharomae. The life cycles of the above species, just like those of other species with a known life cycle, suggest that most Myxobolus spp. develop through triactinomyxon-type actinosporean stages.

The marine myxosporean Sigmomyxa sphaerica (Thélohan, 1895) gen. n., comb. n. (syn. Myxidium sphaericum) from garfish (Belone belone (L.)) uses the polychaete Nereis pelagica L. as invertebrate host

Sigmomyxa sphaerica (Thélohan, 1892) gen. n. (Myxozoa, Myxosporea) with myxosporean stages in the gall bladder of Belone belone (L.) (Teleostei, Belonidae) uses the polychaete Nereis pelagica L. (Nereidae) from shallow water in the northern Øresund, Denmark, as invertebrate host. The nearly spherical tetractinomyxon-type actinospores of S. sphaerica differ from those of two species of Ellipsomyxa which also use Nereis spp. as invertebrate host. Pansporocysts of S. sphaerica were not seen. S. sphaerica is redescribed on the basis of myxospore stages from B. belone and actinospores from N. pelagica, and the phylogenetic affinities examined on the basis of ribosomal small subunit gene sequences. S. sphaerica is closest related to Ellipsomyxa spp., and is not congeneric with morphologically similar Myxidium spp. from gadids. This is the fifth elucidated two-host life cycle of a marine myxozoan.

Occurrence of actinosporean stages (Myxozoa) in the Nera River system (Umbria, central Italy)

We investigate the oligochaetes populations in the Nera River systems (central Italy) in order to highlight the occurrence of actinosporean stages in this natural environment. The survey was carried out during 2 years of monitoring 24 stations upstream and downstream the 12 trout farm along the rivers. In four of the 24 sites, oligochaetes releasing Echinactinomyxon and Aurantiactinomyxon types spore were collected. The actinospores observed were described morphologically, by transmission electron microscopy and by molecular analysis. Histological observations of the infected oligochaetes were carried out. A single morphotype of the collective group Echinactinomyxon were detected showing the presence of branches at the distal end of the caudal processes. The morphological and morphometrical characters showed that the Echinactinomyxon type herein described is a novel phenotype. This data has to be confirmed by the molecular comparison with sequences not yet available in GenBank. The molecular analysis of the Aurantiactinomyxon type, showed high genetic similarity (99.8%) with the two alternate stages of Chloromyxum truttae even if our type showed some differences in morphology stressing the importance of combining the two methods to avoid misidentifications. This is the first survey carried out in natural environment in Italy. Further analyses on fish population are required in order to detect myxosporean stage in the vertebrate hosts and confirm the molecular data.

Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification

The occurrence and morphology of actinosporean stages of myxosporeans were studied at a fish farm and in the River Tisza in Hungary. The 43 samples sequenced belonged to 10 'genotypes', from which six were determined as new actinosporean types. Based on DNA sequence analysis, the assumed actinosporean developmental stages of four myxozoan species were identified. The raabeia type collected from the worm Branchiura sowerbyi was identified as Myxobolus cultus Yokoyama, Ogawa & Wakabyashi, 1995. This was the first occurrence of this parasite in Hungary. Aurantiactinomyxon type 'A' was assigned as a developmental stage of Thelohanellus hovorkai Achmerov, 1964, triactinomyxon type 'D' was identified as Myxobolus sp. from the fins of roach Rutilus rutilus, while the DNA sequence of the guyenotia type actinosporean was 99.9-100% identical with Sphaerospora sp. from the kidney tubules of goldfish Carassius auratus auratus. Partial 18S rDNA sequences of the myxosporeans Thelohanellus hovorkai, T. nikolskii Achmerov, 1955 and Myxobolus sp. from Rutilus rutilus were new additions to GenBank. The DNA sequence analysis revealed that, in the case of actinosporeans, different 'morphotypes' can belong to the same 'genotype'. This study confirmed that actinospore classification based solely on traditional morphological features may lead to false conclusions, thus sequence analysis of the 18S rDNA and/or other genes is recommended in species and type descriptions.

Efficacy and toxicity of orally administrated anti-coccidial drug treatment on Enteromyxum leei infections in sharpsnout seabream (Diplodus puntazzo C.)

Three drugs effective against myxosporeans and commonly used to treat spore-forming para- sites were tested in sharpsnout sea bream (Diplodus puntazzo) infected by Enteromyxum leei. Two medicated diets were applied, one containing salinomycin and amprolium and the second containing fumagillin. Compared to untreated fish, both treatments resulted in significant reduc- tions in prevalence, intensity of all developmental myxosporean stages, and mortality. The effect was more prominent with the salinomycin and amprolium combination, where the significant reductions in intensity, prevalence, and mortality were unaccompanied by any histopathological evidence of toxic side effects or growth reduction. Sporoblasts and mature spores with distorted structures were observed in both drug treatments, but were more prevalent in the salinomycin and amprolium treatment than in the fumagillin treatment, indicating direct effectiveness on the parasite. Salinomycin with amprolium is a promising treatment for myxosporean infections in intensively cultured warmwater fish, leading to parasite elimination.

The life cycle of Henneguya nuesslini Schuberg & Schröder, 1905 (Myxozoa) involves a triactinomyxon‐type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.

Description of a new synactinomyxon type from the River Sousa, Portugal

Actinospore infection of oligochaetes collected from the mud of 2 freshwater biotopes in Portugal was studied. Using the 'cell-well plate method', a new synactinomyxon type was found in 2 specimens (1.3%) of the examined Tubifex tubifex oligochaetes from the River Sousa north of Porto, Portugal. In Criodrilus lacuum and Dero digitata specimens collected from the same river, no actinosporeans were released during the 12 wk observation period. Infected oligochaetes were only found immediately post-collection, and no further actinosporean release was recorded in Tubifex specimens kept alive for several weeks. Actinospore infection showed high intensity in oligochaetes in both positive cases. No actinosporean stages of myxosporeans have as yet been described from Portugal. On the basis of spore morphology and 18S rDNA sequence data, the synactinomyxon type presented in this paper differs from those already known and described in the literature.

Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts.

Nereis diversicolor O.F. Müller and N. succinea Frey et Leuckart (Polychaeta, Nereidae) living in brackish shallow areas in Denmark are naturally infected with tetractinomyxon actinospores. Infected Nereis spp. were experimentally fed to various potential fish hosts, and the actinosporean stages developed into myxosporean stages of Ellipsomyxa gobii Køie, 2003 (Ceratomyxidae) in the gallbladder of the common goby Pomatoschistus microps (Krøyer) (Gobiidae). The European eel Anguilla anguilla (L.), three-spined stickleback Gasterosteus aculeatus L., small sand eel Ammodytes tobianus L., flounder Platichthys flesus (L.), European plaice Pleuronectes platessa L. and common sole Solea solea (L.) did not become experimentally infected. In Danish shallow brackish areas P. microps is naturally infected with E. gobii, in some areas with a prevalence >90%. We compared small subunit ribosomal DNA sequences of the actinosporean with E. gobii from P. microps. Sequences were identical, which further verifies that both forms belong to the same organism. This is the first myxozoan two-host life cycle in the marine environment.

Description of a new actinosporean type from South African freshwaters

Actinospore infection of oligochaetes collected from the mud of 2 freshwater biotopes in South Africa was studied. Using the 'cell-well plate method', a new aurantiactinomyxon type was found in 1.1% of the examined Branchiura sowerbyi oligochaete specimens from the Rietvlei River, north of Johannesburg, Gauteng, South Africa. In 1.5% of B. sowerbyi collected in a pond (Padda Dam), near the Rand Afrikaans University, Johannesburg, the same aurantiactinomyxon type was found. Infected oligochaetes were found only after collection and no actinosporean release was recorded in Branchiura specimens kept alive for several weeks. Actinospore infection showed a high intensity in oligochaetes in both positive cases. Until now, no actinosporean stages of myxosporeans have been described from South Africa. The aurantiactinospore type presented in this communication differs from the known types already described in the literature.

Small subunit ribosomal DNA characterization of an unidentified aurantiactinomyxon form and its oligochaete host Tubifex ignotus.

In this study, the small subunit (18S) ribosomal DNA gene from an aurantiactinomyxon form of unknown taxonomic position (A1) and from its aquatic oligochaete host (Tubifex ignotus) were characterized. Molecular sequence information on A1 was obtained to allow comparisons of this gene with known sequences from known myxosporean forms, and therefore to investigate possible relationships between this organism and its alternate myxosporean stage. Sequence data for the oligochaete host, together with morphological features, will allow reliable identification of this species in the future. Sequence data derived from the 18S DNA gene and data from other related or non-related organisms were analyzed and used to construct a phylogenetic tree. Phylogenetic studies provided an insight into the taxonomic position of A1. Sequence similarities within the 18S rDNA A1 gene and compared organisms indicated that A1 was most closely related to members of the sub-order Variisporina (Myxidium lieberkuehni [Ml] and Sphaerospora oncorhynchi [So]). Clustering of the 3 organisms in the same branch was well supported by high bootstrap values (81%). A1 showed higher similarities with sequences of Ml (approximately 80%) than with So (approximately 79%). Myxosporean sequence analysis indicated that phylogenetic arrangements do not support traditional classification based on morphological criteria of the spores, but rather support arrangement by tissue location. Marine actinosporeans Triactinomyxon sp. and Tetraspora discoidea were found to be associated with Platysporinid myxosporeans, supporting previous findings. In this study, 18S rDNA sequence data are generated for first time for the aquatic oligochaete T. ignotus. Phylogenetic 18S rDNA gene analyses performed with T. ignotus support and confirm existing morphological and molecular phylogenetic studies. Paraphyly of the Tubificidae family was noticed.

Occurrence of actinosporean stages of myxosporeans in an inflow brook of a salmon hatchery in the Mena River System, Hokkaido, Japan.

Actinospore infection of oligochaetes living in the mud and on the roots of vegetation in an inflow brook of a Hokkaido salmon hatchery was studied within the framework of a Japanese-Hungarian research program. Two triactinomyxon types, 1 echinactinomyxon, and 1 neoactinomyxum type were isolated from the oligochaete Rhyacodrilus komarovi Timm, 1990 collected during the survey. The aurantiactinomyxons were recorded over a period of 3 mo starting from the day after oligochaete collection. The oligochaetes released actinospores for several weeks from the first day of the study. Spore excretion of individual oligochaetes was not synchronous. Of the oligochaetes examined, 0.7, 7, 3 and 3%, were infected with the echinactinomyxon, neoactinomyxum and the 2 types of triactinomyxon spores, respectively. Actinospore infection was intense in the positive oligochaetes in all 4 types. Of the 4 actinospore types presented here, 3 are described for the first time.

Cladistic analysis of myxozoan species with known alternating life-cycles.

The phylogenetic relationships of 15 myxozoan taxa with known alternating life-cycles were investigated in order to provide insight into the puzzling matches between myxosporeans and actinosporeans of the myxozoan life-cycle data. Phylogenetic analyses were performed using two partitioned data-sets of life-cycle stages, myxosporean stage from fish hosts versus actinosporean stage from annelid hosts, and a combined data-set of myxosporean and actinosporean stages. A cnidarian parasite of fish, Polypodium hydriforme Ussov, 1885, was used as the outgroup. The supraspecific level grouping in the conventional classification of actinosporeans was not supported in the analysis of the partitioned data from the actinosporean phase, which yielded two equally parsimonious trees. Analysis of the partitioned data from the myxosporean phase provided 24 equally parsimonious trees and did not support the current classification of myxosporeans. The analyses of the partitioned data of myxozoans by life-cycle stage revealed a lack of taxonomic congruence between the two life-stage partitions. Two equally parsimonious trees were obtained from analysis of the combined data. The suborder Variisporina of the Myxozoa was not supported by the total evidence trees, while the monophyly of the species of Myxobolus Butschli, 1882 and of the Myxidiidae were supported. The cladograms from the combined data revealed that these myxozoan species formed four major monophyletic groups. Among them, two were supported by the partitioned data of the actinosporean phase. The phylogenetic signals and the better resolution reflected by the trees of combined data suggest that the phylogenetic total evidence approach should be employed in future studies of the systematics of myxozoans.

Tetracapsula renicola n. sp. (Myxozoa : Saccosporidae); the PKX myxozoan--the cause of proliferative kidney disease of salmonid fishes.

Proliferative kidney disease (PKD) of salmonid fishes is caused by the extrasporogonic stage of an enigmatic myxozoan, referred to as PKX. Sporogenesis occurs in the renal tubules, resulting in monosporous pseudoplasmodia. The spores are ovoid with indistinguishable valves and measure 12 microm in length and 7 microm in width. Two spherical polar capsules (2 microm diameter) with 4 coils occur at the anterior end of the spore. Prominent capsulogenic cell nuclei posterior to the polar capsules are evident in histological sections stained with hematoxylin and eosin. Regardless of the true nature of the valves (indistinguishable or absent), this myxozoan is morphologically distinct from all other described members of the phylum Myxozoa. Comparisons of small subunit rDNA sequences of PKX with other myxozoans demonstrated that it branches from all other members of the myxosporeans from fish examined thus far, including representatives of the phenotypically most closely related genera, Sphaerospora and Parvicapsula. Recent reports, based on rDNA comparisons, indicate that the alternate stage of PKX occurs in bryozoans, and that PKX clusters in a clade with Tetracapsula bryozoides. Our analyses and those of others, along with phenotypic observations, indicate that salmonids are the primary myxosporean hosts for PKX, that the cryptic spores of PKX in salmonids are the fully formed myxospores as they occur in the fish host, and that PKX represents distinct species that we previously place in the genus Tetracapsula in the family Saccosporidae. The latter 2 taxa were described based on stages from bryozoans, and the myxosporean stage in fish of the type species, T. bryozoides, has not been identified (if it exists). Thus, more complete resolution of the life cycle of both PKX and T. bryozoides, as well as more genetic data, are required to determine the precise relationship of these organisms.

The life cycle of Sphaerospora truttae (Myxozoa: Myxosporea) and some features of the biology of both the actinosporean and myxosporean stages.

A previously undescribed echinactinomyxon type actinosporean was shown experimentally to be the alternate stage of Sphaerospora truttae. The echinactinomyxon type spores were found to be released from Lumbriculus variegatus and occasionally Tubifex tubifex. Overall infection prevalence of the echinactinomyxon was 0.14%. Peak release was in March-May each year. S. truttae spores developed in experimentally infected salmon 4.5 mo post-exposure to echinactinomyxon spores. Extrasporogonic stages of S. truttae first appeared in naturally infected salmon in early July and persisted for 8 to 10 wk. Correlation between peak release of echinactinomyxon spores and appearance of extrasporogonic stages of S. truttae is discussed.

First record of an actinosporean (Myxozoa) in a marine polychaete annelid.

The marine polychaete Nereis (Hediste) diversicolor (Annelida) from shallow water in the Oresund, Denmark, was found to be infected with an actinosporean stage of a myxozoan parasite. The body length of the pyriform actinospore is 12-16 microm and its maximum width is 10-12 microm. The spore is triangular in apical view, with the 3 spherical polar capsules distally. The spore is without caudal processes. Eight spores develop in each pansporocyst. Free spores and pansporocysts were found in the musculature and parapodia but not in the intestine. The myxosporean stage in fish is unknown. This is the first record of an actinosporean stage in a marine polychaete, but because marine oligochaetes are rare, compared with polychaetes, the latter are believed to play an important role as invertebrate (alternate) hosts in marine myxozoan life cycles.

Myxosporean infection causing intestinal disease in farmed turbot, Scophthalmus maximus (L.), (Teleostei: Scophthalmidae)

The present report describes an intestinal disease which causes important losses in farmed turbot. Mortality rates were higher in summer and reached 100% in all tanks where the disease was confirmed. Affected fish showed external signs consisting of anorexia, sunken eyes and a typical prominent bony ridge on the skull. These signs can be considered the pathognomonic signs of the disease, together with the gut lesions seen in the histological study. Pallor of the internal organs, intestinal haemorrhages and the presence of liquid in the intestine were also observed, with ascites in heavily infected fish. Histopathological damage was evident in the gut, with severe enteritis, detachment of epithelium, haemorrhages and inflammation of the subepithelial connective tissue. The myxosporean aetiology was demonstrated in all the fish showing the characteristic signs of disease. Myxosporean stages, including scarce spores, were found in the affected epithelium or free in the intestinal lumen together with epithelial debris. The present study demonstrates the importance of this myxosporean disease which represents a serious threat for turbot culture. This is also the first record of a member of the Myxosporea in turbot.

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) for the determination of viability of myxosporean and actinosporean spores

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) was used for determination of viability of myxosporean stage spores of Myxobolus artus and actinosporean stage spores of M. cultus. Viable spores stained green with FDA and non‐viable spores stained red with PI were clearly distinguishable in M. artus myxosporean spores released from live infected fish, while the spores collected from pseudocysts were not well stained with FDA. Immediately after release from the oligochaete, Branchiura sowerbyi, actinosporean spores of M. cultus, stained bright green in the sporoplasms and red in the spore processes. In vitro survivability tests revealed that the longevity of M. artus spores was 5 months at 25°C and 15 months at 18°C. Drying and ultraviolet irradiation (36 000 mW s cm–2 for M. artus myxosporean spores, 600 mW s cm–2 for M. cultus actinosporean spores) were most effective as sporicidal treatments.

Small subunit ribosomal RNA sequences unite alternate actinosporean and myxosporean stages of Myxobolus cerebralis the causative agent of whirling disease in salmonid fish.

The alternating myxosporean and actinosporean stages of the myxozoan parasite Myxobolus cerebralis (Hofer 1903) from its salmonid fish and aquatic oligochaete hosts, respectively, were compared for sequence homology of the small subunit (18S) ribosomal RNA genes. A 99.8% similarity between the sequences of these two stages was substantially greater than that of M. cerebralis compared to two other Myxobolus sp. from salmonid fish. Our results are the first molecular evidence confirming the alternating stages, initially described by Wolf and Markiw [25] for the life cycle of M. cerebralis but found in two different taxonomic classes (Myxosporea and Actinosporea) are indeed forms of the same organism. Sequencing of rRNA genes of the actinosporean stage followed by development of specific primers for DNA amplification of the myxosporean stage, as in our study, should be applied to solve other myxozoan life cycles. Additionally, these approaches will in the future provide useful diagnostic reagents for the detection and study of this important group of fish pathogens.

The demise of a class of protists: taxonomic and nomenclatural revisions proposed for the protest phylum Myxozoa Grassé, 1970

The phylum Myxozoa has been considered to comprise two classes, Myxosporea Bütschli, 1881 (primarily of fishes) and Actinosporea Noble in Levine et al., 1980 (primarily of aquatic oligochaetes). About 10 years ago it was demonstrated that the life cycle of Myxobolus cerebralis Hofer, 1903 (Myxobolidae: Platysporina) of salmonid fishes requires transformation of the myxosporean into an actinosporean stage in the oligochaete worm Tubifex tubifex (Tubificidae), and that the stage infective to fish is the actinosporean spore. This type of two-host life cycle has now been demonstrated or strongly implicated for 14 myxosporean species, belonging to 6 genera in 4 families. In light of these findings, the taxonomy of the Myxozoa is revised. We propose the following: suppression of the newer class Actinosporea and the order Actinomyxidia Štolc, 1899; and suppression of all families in the Actinosporea except Tetractinomyxidae. This family and its one genus, Tetractinomyxon Ikeda, 1912, are transferred to the order Multivalvulida Shulman, 1959 (Myxosporea). We also propose that actinosporean generic names be treated as collective-group names, thus they do not compete in priority with myxosporean generic names. Triactinomyxon dubium Granata, 1924 and Triactinomyxon gyrosalmo Wolf and Markiw, 1984 are suppressed as junior synonyms of Myxobolus cerebralis. The myxosporean stage of no other previously named actinosporean has been identified. Other actinosporean species are therefore retained as species inquirendae until their myxosporean stages are identified. A revised description of the phylum Myxozoa is provided that includes our proposed taxonomic and nomenclatural changes.