Sequential development of Buddenbrockia plumatellae (Myxozoa: Malacosporea) within Plumatella repens (Bryozoa: Phylactolaemata)

Colonies of the freshwater bryozoan Plumatella repens collected from a river in the UK were found to be infected with the myxozoan parasite Buddenbrockia plumatellae following laboratory maintenance. Optimisation of the bryozoan diet allowed maintenance of infected colonies for 90 d, permitting observation by light and electron microscopy of the sequential parasitic developmental cycle. Parasite stages were associated with host peritoneum, identifying the primary developmental phase. The association of B. plumatellae cells with peritoneal basal lamina and morphological similarities between parasite and host suggested that the parasite remodelled host tissue. Progressive expansion and elongation of individual parasites led to the release of freely floating vermiform stages within the host coelomic cavities. Within these 'worms', intraluminal masses developed, resulting in the formation of spores. Upon maturation, the 'worms' ruptured, releasing many spores within the host that were subsequently discharged. Although parasitism led to increased bryozoan fragmentation and lowered statoblast production, some colonies did survive, resulting in repeated waves of infection. Long-term laboratory maintenance of infected bryozoan colonies could provide a means of maintaining B. plumatellae for study until the full life cycle is ascertained.     

Ecology,development and pathogenicity of Buddenbrockia plumatellae Schröder, 1910 (Myxozoa,Malacosporea) (syn. Tetracapsula bryozoides) and establishment of Tetracapsuloides n. gen. for Tetracapsula bryosalmonae

Buddenbrockia plumatellae, an enigmatic worm-like myxozoan, was observed as continuously writhing free and attached 'worms' and as free mature spores in the coelom of the freshwater bryozoans Plumatella fungosa, Hyalinella punctata, and Fredericella sp. 'Worm' numbers could double every three days. 'Worms' and spores could be expelled from colonies by external pressure. Some mature 'worms' exited actively, entraining release of free spores, and gradually ceased movement outside the host. Bryozoans sealed off infected regions of the colony. Infected colonies grew slowly, produced no statoblasts, and eventually regressed and died. Transmission was not achieved and prevalence was low. Electron microscopy of 'worms' revealed a single layer of mural cells on a fibrous basal lamina overlying four longitudinal muscle blocks and an inner sheet of two types of proliferating cells, an organization indicative of the bilaterian ancestry of the Myxozoa. Primary type A cells were attached directly by striated tubules to mural cells at positions between muscle blocks. Secondary type A cells had a secretory function. Type B cells underwent meiosis and subsequently developed to typical malacosporean myxozoan spores filling the internal cavity of the 'worms'. External tubes were formed during capsulogenesis in 'worms' from Fredericella sp. Tetracapsula bryozoides is synonymised with Buddenbrockia plumatellae and a new genus is proposed for Tetracapsula bryosalmonae.     

A new model for myxosporean (Myxozoa) development explains the endogenous budding phenomenon, the nature of cell within cell life stages and evolution of parasitism from a cnidarian ancestor

The phylum Myxozoa is composed of endoparasitic species that have predominately been recorded within aquatic vertebrates. The simple body form of a trophic cell containing other cells within it, as observed within these hosts, has provided few clues to relationships with other organisms. In addition, the placement of the group using molecular phylogenies has proved very difficult, although the majority of analyses now suggest that they are cnidarians. There have been relatively few studies of myxozoan stages within invertebrate hosts, even though these exhibit multicellular and sexual stages that may provide clues to myxozoan evolution. Therefore an ultrastructural examination of a myxozoan infection of a freshwater oligochaete was conducted, to reassess and formulate a model for myxozoan development in these hosts. This deemed that meiosis occurs within the oligochaete, but that fertilisation is not immediate. Rather, the resultant haploid germ cell (oocyte) is engulfed by a diploid sporogonic cell (nurse cell) to form a sporoplasm. It is this sporoplasm that infects the fish, resulting in the multicellular stages observed. Fertilisation occurs after the parasites leave the fish and enter the oligochaete host. The nurse cell/oocyte model explains previously conflicting evidence in the literature regarding myxosporean biology, and aligns phenomena considered distinctive to the Myxozoa, such as endogenous budding and cell within cell development, with processes recorded in cnidarians. Finally, the evolutionary origin of the Myxozoa as cnidarian parasites of ova is hypothesised.     

3D Morphology, ultrastructure and development of Ceratomyxa puntazzi stages: first insights into the mechanisms of motility and budding in the Myxozoa

Free, amoeboid movement of organisms within media as well as substrate-dependent cellular crawling processes of cells and organisms require an actin cytoskeleton. This system is also involved in the cytokinetic processes of all eukaryotic cells. Myxozoan parasites are known for the disease they cause in economical important fishes. Usually, their pathology is related to rapid proliferation in the host. However, the sequences of their development are still poorly understood, especially with regard to pre-sporogonic proliferation mechanisms. The present work employs light microscopy (LM), electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) in combination with specific stains (Nile Red, DAPI, Phalloidin), to study the three-dimensional morphology, motility, ultrastructure and cellular composition of Ceratomyxa puntazzi, a myxozoan inhabiting the bile of the sharpsnout seabream.Our results demonstrate the occurrence of two C. puntazzi developmental cycles in the bile, i.e. pre-sporogonic proliferation including frequent budding as well as sporogony, resulting in the formation of durable spore stages and we provide unique details on the ultrastructure and the developmental sequence of bile inhabiting myxozoans. The present study describes, for the first time, the cellular components and mechanisms involved in the motility of myxozoan proliferative stages, and reveals how the same elements are implicated in the processes of budding and cytokinesis in the Myxozoa. We demonstrate that F-actin rich cytoskeletal elements polarize at one end of the parasites and in the filopodia which are rapidly de novo created and re-absorbed, thus facilitating unidirectional parasite motility in the bile. We furthermore discover the myxozoan mechanism of budding as an active, polarization process of cytokinesis, which is independent from a contractile ring and thus differs from the mechanism, generally observed in eurkaryotic cells. We hereby demonstrate that CLSM is a powerful tool for myxozoan research with a great potential for exploitation, and we strongly recommend its future use in combination with in vivo stains.     

Cytological aspects of similarity and difference of Myxozoa and Cnidaria

A comparative cytomorphological analysis of Myxozoa and parasitic Cnidaria Polypodium hydriforme has been carried out in view of the Weill (1938) hypothesis, which regards Myxozoa as a reduced Cnidaria. The question on the relation of Myxozoa and Cnidaria was arising several times with the application of some new methods during the Myxozoa studies. At present the idea on their phylogenetic relationships has appeared again in connection with an absolutely new understanding of the myxozoan life cycle (Wolf, Markiw, 1984), as well as with the application of molecular-biological methods for their phylogenetic studies. The latter, however, provided some diverse results. So far no comparative cytomorphological analysis of Myxozoa and Polypodium has been carried out. The present paper is to fill the gap on the basis of accumulated facts. According to Weill (1938), the features of similarity of parasitic Cnidaria and Myxozoa are the following: 1) the presence in both of extrusomes (nematocysts and polar capsules) whose structure and development are surprizingly similar; 2) the nuclear dimorphism and somato-generative segregation; 3) the presence of a somatic nutritional cell, surrounding the multiplying generative cells; at present it is known that polyploidy of somatic nuclei and the absence of parasitophorous vacuole are characteristic of trophamnion of Polypodium and trophozoite of Myxozoa; 4) the presence of radial symmetry in both groups; 5) the construction of a diblastic organism made of a cluster of endodermal cells and a few ectodermal cells; 6) the similarity of their cell contacts (Grassé, 1970). At present it is possible to add to Weill's (1938) list of features common for parasitic Cnidaria and Myxozoa the number of important similarities between Polypodium and Myxozoa, some of which being not characteristic of Cnidaria: 1) the "cell in cell" organization of all Polypodium parasitic stages and all Myxozoa life cycle stages; 2) the presence of gametophore supplied with extrusomes; 3) both organisms have haplophase in their life cycles preceded by two-step meiosis; 4) there are mitochondria with tubular cristae in both organisms; 5) the absence of spermatozoa and eggs in both organisms; 6) the similarity of Polypodium cnidocile structure and the cone-like formation situated at the anterior end of polar capsule of actinospore (Lom. Dykova, 1997); 7) the participation of MTOC in the formation of extrusomes in both animals. In spite of the obvious similarity between Myxozoa and parasitic Cnidaria (including Polypodium) it is, however, necessary to take into account differences between them, the main being as follows: the absence in Myxozoa of flagellated stages, centrioles, tissues and organs, true blastophylla, planula-like larvae, gastrulation; the presence of low cell integrations in Myxozoa; Cnidaria and Myxozoa have different types of mitosis, their life cycles and the discharge mechanism of their stinging apparatus being also different. We consider as quite valid a suggestion by Siddall et al. (1995) that parasitic Cnidaria could present an early separated branch of the cnidarian evolution. Further studies of Myxozoa life cycle may show their more definite relation to parasitic Cnidaria. The problem has not yet been solved completely since the available molecular-biological data are rather contradictory and moreover there is no distinct idea as to the Eumetazoa ancestor so far. A further thorough investigation is badly needed in the feelds of developmental cycle, cytomorphology and molecular biology of the variety of narcomedusae and representatives of Myxozoa. This may help to find some transitional forms and stages of the animals and to understand whether we deal with a regressive evolution of parasitic Cnidaria or with a parallel evolution of taxa originated from the common ancestor.     

Cell formation by myxozoan species is not explained by dogma

Eukaryotes form new cells through the replication of nuclei followed by cytokinesis. A notable exception is reported from the class Myxosporea of the phylum Myxozoa. This assemblage of approximately 2310 species is regarded as either basal bilaterian or cnidarian, depending on the phylogenetic analysis employed. For myxosporeans, cells have long been regarded as forming within other cells by a process referred to as endogenous budding. This would involve a nucleus forming endoplasmic reticulum around it, which transforms into a new plasma membrane, thus enclosing and separating it from the surrounding cell. This remarkable process, unique within the Metazoa, is accepted as occurring within stages found in vertebrate hosts, but has only been inferred from those stages observed within invertebrate hosts. Therefore, I conducted an ultrastructural study to examine how internal cells are formed by a myxosporean parasitizing an annelid. In this case, actinospore parasite stages clearly internalized existing cells; a process with analogies to the acquisition of endosymbiotic algae by cnidarian species. A subsequent examination of the myxozoan literature did not support endogenous budding, indicating that this process, which has been a central tenet of myxozoan developmental biology for over a century, is dogma.     

Diversity and systematics of the Malacosporea (Myxozoa)

Abstract. Myxozoans belonging to the recently described class Malacosporea parasitize freshwater bryozoans during at least part of their life cycle. There are at present only two species described in this class: Buddenbrockia plumatellae and Tetracapsuloides bryosalmonae . The former can exist as vermiform and sac-like stages in bryozoan hosts. The latter, in addition to forming sac-like stages in bryozoans, is the causative agent of salmonid proliferative kidney disease (PKD). We undertook molecular and ultrastructural investigations of new malacosporean material to further resolve malacosporean diversity and systematics. Phylogenetic analyses of 18S rDNA sequences provided evidence for two new putative species belonging to the genus Buddenbrockia , revealing a two-fold increase in the diversity of malacosporeans known to date. One new malacosporean is a vermiform parasite infecting the bryozoan Fredericella sultana and the other occurs as sac-like stages in the rare bryozoan, Lophopus crystallinus . Both bryozoans represent new hosts for the genus Buddenbrockia . Our results have established that the malacosporean which infected F. sultana was not a vermiform stage of T. bryosalmonae , although it was collected from a site endemic for PKD. Ultrastructural investigation of new material of B . plumatellae revealed the presence of numerous external tubes associated with developing polar capsules, confirming that the absence of external tubes should no longer be considered as a character of the class Malacosporea.     

The joint evolution of the Myxozoa and their alternate hosts: A cnidarian recipe for success and vast biodiversity

The relationships between parasites and their hosts are intimate, dynamic and complex; the evolution of one is inevitably linked to the other. Despite multiple origins of parasitism in the Cnidaria, only parasites belonging to the Myxozoa are characterized by a complex life cycle, alternating between fish and invertebrate hosts, as well as by high species diversity. This inspired us to examine the history of adaptive radiations in myxozoans and their hosts by determining the degree of congruence between their phylogenies and by timing the emergence of myxozoan lineages in relation to their hosts. Recent genomic analyses suggested a common origin of Polypodium hydriforme, a cnidarian parasite of acipenseriform fishes, and the Myxozoa, and proposed fish as original hosts for both sister lineages. We demonstrate that the Myxozoa emerged long before fish populated Earth and that phylogenetic congruence with their invertebrate hosts is evident down to the most basal branches of the tree, indicating bryozoans and annelids as original hosts and challenging previous evolutionary hypotheses. We provide evidence that, following invertebrate invasion, fish hosts were acquired multiple times, leading to parallel cospeciation patterns in all major phylogenetic lineages. We identify the acquisition of vertebrate hosts that facilitate alternative transmission and dispersion strategies as reason for the distinct success of the Myxozoa, and identify massive host specification‐linked parasite diversification events. The results of this study transform our understanding of the origins and evolution of parasitism in the most basal metazoan parasites known.     

Proliferative, presaccular stages of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) within the invertebrate host Fredericella sultana (Bryozoa: Phylactolaemata)

Proliferative kidney disease (PKD), caused by the malacosporean parasite, Tetracapsuloides bryosalmonae, is a major disease of salmonid culture both in western Europe and North America. The fish are infected from spores that develop within freshwater bryozoans and are released into the water column. Although sporogenesis has been studied in the bryozoan host and occurs within sacs, the formation of these sacs from presaccular stages has only been hypothesized. Examination of infected bryozoans by using a range of techniques identified proliferating, presaccular amoeboid stages of T. bryosalmonae on the body wall of the bryozoan Fredericella sultana. These stages possessed unique electron-dense bodies and were observed as aggregating within the bryozoan metacoel, differentiating to form spore sacs. Spore sac growth was associated with the assimilation of the presaccular parasites rather than through cryptomitosis of sac mural cells. This sac formation through aggregation and assimilation suggests an intriguing mechanism by which T. bryosalmonae can cross-fertilize.     

F-actin and beta-tubulin localization in the myxospore stinging apparatus of Myxobolus pseudodispar Gorbunova, 1936 (Myxozoa, Myxosporea)

To understand the discharge mechanism of Myxozoan polar capsule (cnida) it is necessary to verify the role of major cytoskeletal proteins in the process. With this aim F-actin and beta-tubulin localization in spores of myxosporean developmental phase (in myxospores) of Myxobolus pseudodispar Gorbunova, 1936 has been studied under confocal scanning laser microscope using phalloidin fluorescent staining of F-actin and indirect anti-beta-tubulin immunostaining. F-actin has been detected in walls of the stinging tube invaginated into the polar capsule of myxospore. The fact suggests the contractile proteins involvement in the process of myxozoan polar capsule extrusion. In addition, the cytoplasm of amoeboid sporoplasm inside the spore cavity is stained by phalloidin. A polar cap with strong beta-tubulin immunoreacton is observed at the front pole of fully mature myxospore above the outlets of the polar capsule discharge channels. The role of the beta-tubulin cap is supposed to be similar to that of the cnidarian cnidocil made of microtubules. The weaker beta-tubulin immunoreactivity has been found in stinging tubes, in polar capsule walls as well as in the suture line of spore walls and in the cytoplasm of amoeboid sporoplasm. The involvement of cytoskeletal proteins in the process of polar capsule extrusion is discussed. A hypothesis on the myxozoan polar capsule discharge mechanism is suggested. The mechanism of myxozoan cnida discharge is compared with that of cnidaria.     

Myxozoan parasitism in waterfowl

Myxozoans are spore-forming, metazoan parasites common in cold-blooded aquatic vertebrates, especially fishes, with alternate life cycle stages developing in invertebrates. We report nine cases of infection in free-flying native and captive exotic ducks (Anseriformes: Anatidae) from locations across the United States and describe the first myxozoan in birds, Myxidium anatidum n. sp. We found developmental stages and mature spores in the bile ducts of a Pekin duck (domesticated Anas platyrhynchos). Spores are lens-shaped in sutural view, slightly sigmoidal in valvular view, with two polar capsules, and each valve cell has 14-16 longitudinal surface ridges. Spore dimensions are 23.1 microm x 10.8 microm x 11.2 microm. Phylogenetic analysis of the ssrRNA gene revealed closest affinity with Myxidium species described from chelonids (tortoises). Our novel finding broadens the definition of the Myxozoa to include birds as hosts and has implications for understanding myxozoan evolution, and mechanisms of geographical and host range extension. The number of infection records indicates this is not an incidental occurrence, and the detection of such widely dispersed cases suggests more myxozoans in birds will be encountered with increased surveillance of these hosts for pathogens.     

Phylogeny of the multivalvulidae (Myxozoa: Myxosporea) based on comparative ribosomal DNA sequence analysis

Fish parasites of the Multivalvulida (Myxozoa, Myxosporea) are widespread and can be associated with mortality or poor flesh quality in their commercially important marine hosts. Traditional classifications divide members of this order into families based on spore valve and polar capsule numbers. Analyses of the small-subunit (SSU) ribosomal DNA (rDNA) sequences from all representative families in the order (Trilosporidae, Kudoidae, Pentacapsulidae, Hexacapsulidae, and Septemcapsulidae) indicate that a revision of the taxonomy and nomenclature is warranted. In our phylogenetic analysis of (SSU and large subunit) rDNA sequences, members of Pentacapsula, Hexacapsula, and Septemcapsula root within a clade of Kudoa species with Unicapsula (Trilosporidae) as an outlier to these genera. Therefore, we propose to synonymize Pentacapsulidae, Hexacapsulidae, and Septemcapsulidae with Kudoidae alter the diagnosis of Kudoidae and Kudoa to accommodate all marine myxozoan parasites having 4 or more shell valves and polar capsules.     

Development and molecular characterisation of the microsporidian Schroedera airthreyi n. sp. in a freshwater bryozoan Plumatella sp. (Bryozoa: Phylactolaemata)

The development of a new species of microsporidian, infecting a freshwater Plumatellid bryozoan, is described. The small-subunit rDNA, internal transcribed spacer region (ITS), and partial large-subunit rDNA genes were sequenced. Phylogenetic analysis demonstrated that the parasite clustered with Schroedera plumatellae. However, while there were morphological affinities with this species, significant differences were also observed. The infection initially appeared as a roughening of the peritoneum lining the metacoelom of the bryozoan. This roughening resolved into meront-infected syncytia, composed of interconnected cells of the body wall that detached to float in the coleomic cavity. Spores were observed to develop within these syncytia. All stages of development were diplokaryotic in contrast to S. plumatellae, which has a distinct monokaryotic merogony preceding sporogony. The infection was pathogenic to the host. Direct bryozoan-bryozoan transmission was not observed. We propose to name the microsporidian Schroedera aithreyi n. sp.     

Evaluation of a vector-control strategy of haemorrhagic thelohanellosis in carp,caused by Thelohanellus hovorkai (Myxozoa)

The life cycle of Thelohanellus hovorkai (Myxozoa), the causative agent of haemorrhagic thelohanellosis of carp Cyprinus carpio, involves the alternate oligochaete host Branchiura sowerbyi, which plays the role of vector in the parasite's transmission. Field investigations in carp farms suggested that oligochaete fauna were closely associated with the substrate type of the pond. The pond bottom in the enzootic farm consisted of clay soil and soft sediments comprised of organic mud, in which B. sowerbyi dominated in high densities, with a maximum of 5.6 ind. kg(-1) soil. In another case, in a carp farm with no previous history of the disease, the pond bottom was sandy soil, in which small-sized oligochaetes, composed mainly of Limnodrilus socialis, dominated. Laboratory studies on the substrate preference of oligochaetes proved that B. sowerbyi prefers mud to sand, whereas L. socialis has no tendency to substrate tropism. The delicate body surface of B. sowerbyi was subject to damage by rugged-edged sand particles, which inflicted severe injuries to the worms. Transmission experiments showed that L. socialis, which are non-susceptible to T. hovorkai, suppressed the production of T. hovorkai actinospores in B. sowerbyi in a mixed assemblage of oligochaetes. Field and experimental evidence in this study imply that substrate replacement in culture ponds might regulate the benthic oligochaete communities, resulting in minimization of the impact of haemorrhagic thelohanellosis. We propose that ecological control of oligochaete fauna by environmental management is a promising strategy against myxozoan diseases.     

Ultrastructure of Enteromyxum leei (Diamant, Lom, & Dyková, 1994) (Myxozoa), an Enteric Parasite Infecting Gilthead Sea Bream (Sparus aurata) and Sharpsnout Sea Bream (Diplodus puntazzo)

ABSTRACT. The ultrastructure of the developmental stages of the myxozoan Enteromyxum leei parasitizing gilthead seabream ( Sparus aurata ) intestine and sharpsnout sea bream ( Diplodus puntazzo ) intestine and gallbladder are described. The earliest stage observed was a small dense trophozoite located among enterocytes. Proliferative stages, observed intercellularly in the epithelium of the intestine and gallbladder as well as in the lumen, possessed the typical cell-in-cell configuration throughout their development. Secondary cells were seen undergoing division within a common vacuolar membrane that also encompassed pairs of tertiary cells. Cytochemical studies showed that primary cells stored mainly lipids whereas secondary cells stored abundant β-glycogen granules. Sporogonic development resembled that described for other disporous myxozoans. Within sporogonic stages, nonsporogonic secondary cells were observed accompanying two developing spores. Mature spores had a binucleated sporoplasm in which glycogen stores were abundant and no sporoplasmosomes were found. Our observations are discussed in relation to our knowledge on other myxozoans of the genus Enteromyxum .     

Synopsis of the species of Chloromyxum Mingazinni, 1890 (Myxozoa: Myxosporea: Chloromyxidae)

A synopsis of the species of Chloromyxum Mingazinni, 1890 (Myxozoa: Myxosporea: Chloromyxidae) is presented, including 140 nominal species. For each species the most relevant morphological and morphometric characteristics are indicated. Included are data on the site of infection within the host, the original host and the host locality, plus a full bibliography of the original records for these species. A diagrammatic illustration of a spore of each species is also provided.     

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.     

Synopsis of the species of Thelohanellus Kudo, 1933 (Myxozoa: Myxosporea: Bivalvulida)

A synopsis of the species of Thelohanellus Kudo, 1933 (Myxozoa: Myxosporea: Myxobolidae) is presented. It includes a total of 108 nominal species. For each species, the most relevant morphological and morphometric characteristics are provided, together with data on the type-host and locality, the site of infection within the host and the original references.     

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.     

Diagnostic polymerase chain reaction assay to detect Kudoa neurophila (Myxozoa: Multivalvulida) in a marine finfish hatchery

A single-round polymerase chain reaction (PCR) diagnostic assay was developed from a small subunit ribosomal DNA (SSU rDNA) gene sequence to detect the myxozoan parasite Kudoa neurophila, the causative agent of myxozoan disease in the hatchery reared marine finfish, striped trumpeter Latris lineata (Forster). The assay was developed for use as a disease control management tool in a hatchery system specifically designed to research and produce marine finfish such as striped trumpeter juveniles for aquaculture. The assay is sufficiently species specific and sensitive enough to detect a small fragment of the parasite's SSU rDNA. At the lower limits of detection, the test is consistently positive to an estimated 0.1 spore or 60 fg of parasite DNA per 25 microl PCR reaction in serial dilution and positive to an estimated 0.1 spore in 25 mg of infected fish CNS tissue (4 spores g(-1). Specifically, the test is capable of detecting early stages of the life cycle within the fish host and consequently diagnosing an infection not normally detected using traditional histological techniques. The test is also effective for screening water supplies and prey species cultures throughout the hatchery system to determine bio-security efficacy, to assist in identification of an alternate or other primary fish host, to indicate the location of potential disease reservoirs, and to enable a targetted approach to disease prevention.