Michael L. Kent

Recent advances in our knowledge of the Myxozoa

Abstract In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis, and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete, Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that:1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

The state of the art of the zebrafish model for toxicology and toxicologic pathology research--advantages and current limitations.

The zebrafish (Danio rerio ) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1—2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.

Biodiversity: Disease threat to European fish

The deliberate introduction of new species can have unexpected negative consequences and we show here how a recently introduced fish, the invasive Asian cyprinid Pseudorasbora parva, is causing increased mortality and totally inhibiting spawning in an already endangered native fish, the European cyprinid Leucaspius delineatus. This threat is caused by an infectious pathogen, a rosette-like intracellular eukaryotic parasite that is a deadly, non-specific agent. It is probably carried by healthy Asian fish, and could decrease fish biodiversity in Europe, as well as having implications for commercial aquaculture.

Disease threat to European fish.

The deliberate introduction of new species can have unexpected negative consequences and we show here how a recently introduced fish, the invasive Asian cyprinid Pseudorasbora parva, is causing increased mortality and totally inhibiting spawning in an already endangered native fish, the European cyprinid Leucaspius delineatus. This threat is caused by an infectious pathogen, a rosette-like intracellular eukaryotic parasite that is a deadly, non-specific agent. It is probably carried by healthy Asian fish, and could decrease fish biodiversity in Europe, as well as having implications for commercial aquaculture.

A review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial fisheries

Abstract The genus Kudoa Meglitsch, 1947 (Myxozoa: Myxosporea) is comprised of myxosporean parasites with four valves, each of which contains a polar capsule. Species within this genus are typically histozoic parasites of marine teleosts. However, since the establishment of the genus, a few coelozoic species have been described. Presently, there are 44 identified species within the genus. This genus is of concern to both aquaculture and commercial fisheries because several of its species either produce unsightly macroscopic cysts in the musculature or are associated with post-mortem myoliquefaction, and thus reduce the market value of the infected fish products. With the emergence of marine aquaculture, concerns regarding Kudoa infections have increased significantly in recent years. Based upon the literature gathered, a taxonomic review of the genus is warranted to verify the host range, geographical distribution, and validity of several of its species. The use of molecular systematics to answer these questions has been initiated. Furthermore, PCR tests have been developed using small-subunit rDNA sequence for a few Kudoa species, which will be useful for resolving the life cycles of these important marine parasites.

First Report of Three Kudoa Species from Eastern Australia: Kudoa thyrsites from Mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from Sweeper (Pempheris ypsilychnus)

Abstract Fish species around the world are parasitized by myxozoans of the genus Kudoa, several of which infect and cause damage of commercial importance. In particular, Kudoa thyrsites and Kudoa amamiensis infect certain cultured fish species causing damage to muscle tissue, making the fish unmarketable. Kudoa thyrsites has a broad host and geographic range infecting over 35 different fish species worldwide, while K. amamiensis has only been reported from a few species in Japanese waters. Through morphological and molecular analyses we have confirmed the presence of both of these parasites in eastern Australian waters. In addition, a novel Kudoa species was identified, having stellate spores, with one polar capsule larger than the other three. The SSU rDNA sequence of this parasite was 1.5% different from K. thyrsites and is an outlier from K. thyrsites representatives in a phylogenetic analysis. Furthermore, the spores of this parasite are distinctly smaller than those of K. thyrsites, and thus it is described as Kudoa minithyrsites n. sp. Although the potential effects of K. minithyrsites n. sp. on its fish hosts are unknown, both K. thyrsites and K. amamiensis are associated with flesh quality problems in some cultured species and may be potential threats to an expanding aquaculture industry in Australia.

Phylogeography of the Cosmopolitan Marine Parasite Kudoa thyrsites (Myxozoa: Myxosporea)

ABSTRACT. Kudoa thyrsites (Myxozoa: Multivalvulida) is a cosmopolitan marine parasite of fishes associated with post‐mortem tissue degradation. Financial losses incurred as a result of these infections are of concern to commercial fisheries. There is conflicting evidence whether K. thyrsites represents a cryptic species complex. Myxospore morphology is very similar for K. thyrsites across its range, but preliminary genetic analyses show some differences. Kudoa thyrsites and the morphologically similar Kudoa histolytica were examined from hosts in British Columbia, Canada, Oregon, USA, Chile, England, South Africa, Australia, and Japan. We compared myxospore morphology and DNA sequences of heat shock protein 70 and the small subunit, large subunit, and internal transcribed spacer 1 of the ribosomal DNA. There was some morphological variation between regional representatives, inconsistent with genetic analyses. Phylogenetically, major separations correlated to four broad geographic regions: Japan, Australia, eastern Pacific, and eastern Atlantic. Within these regions there was little additional genetic structure. These data are evidence for regional subdivision of K. thyrsites suggesting global transplantation of fishes has yet to homogenize these distinctions. Within regions, parasite gene flow appears to be high between host species, suggesting little host specificity and minimal cryptic speciation. Our data also indicate that K. histolytica is not a valid species, as it was morphologically and genetically indistinguishable from K. thyrsites.

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.

Survey of Salmonid Pathogens in Ocean-Caught Fishes in British Columbia, Canada

Abstract A survey of wild fishes captured around marine net-pen salmon farms and from open waters for certain salmonid pathogens was conducted in the coastal waters of British Columbia. Viral hemorrhagic septicemia virus was detected in Pacific herring Clupea pallasi, shiner perch Cymatogaster aggregata, and threespine sticklebacks Gasterosteus aculeatus. Infectious hematopoietic necrosis (IHN) virus was detected in one Pacific herring (collected well away from the farms) and in tube-snouts Aulorhynchus flavidus and shiner perch collected from a farm experiencing an IHN outbreak. Renibacterium salmoninarum was observed in moribund Pacific hakes Merluccius productus collected from within a net-pen and was also detected in several ocean-caught salmon. Aeromonas salmonicida subsp. salmonicida (typical strain) was isolated from a juvenile chinook salmon Oncorhynchus tshawytscha, whereas the atypical strain of this organism was isolated from a lingcod Ophiodon elongatus. Loma salmonae (Microsporea) was observe...

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 µm in length and 7 µm in width. Two spherical polar capsules (2 µm 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.