魚 病 研 究 Fish Pathology,38(4),125-136,2003.12 2003 The Japanese Society of Fish Pathology A Review:Gaps in Our Knowledge on Myxozoan Parasitesof Fishes* Hiroshi Yokoyama Department of Aquatic Bioscience, Graduate School of Agriculturaland LifeSciences, The Universityof Tokyo,Bunkyo, Tokyo 113-8657,Japan (Received June 23,2003) ABSTRACT-Since the epoch-making discovery of the life-cycleof Myxobolus cerebralisby Wolf and Markiw(1984), studies on myxozoan parasites of fishes have continued to progress rapidly. Molecular analyses of myxozoan SSUrDNA have been successful in clarifyingits system aticposition not as protozoans but as metazoans. However, there remain gaps in our understand ing of theirbiology and pathology. Alternate myxosporean and actinosporean stages in the life-cyclesof more than 25 species of freshwater myxozoan have been demonstrated, but many unresolved questions remain, e.g.,the relationshipbetween the parasiteand itsalternate inverte brate hosts and the mode of transmission in marine myxozoans. These unsolved questions have made itdifficult to design an effectivecontrol strategy for myxozoan diseases. Recent studies on countermeasures have been directedtowards the introductionof resistantfish strainsand control lingoligochaete populations. Key words: Myxozoa, myxosporean, actinosporean,parasite, taxonomy, biology,control strategy Myxozoans are multicellular,spore-forming para publicationsannually. Itis evident that two major find sitesof both marine and freshwater fishes. Out of more ings have been responsible for such a significant than 1,350 species described, most are not pathogenic advance in our knowledge in thisfield of study. One of to theirfish hosts. However, some have been docu these is the epoch-making discovery of the life-cycle of mented as serious pathogens, which have caused eco Myxobolus cerebralisby Wolf and Markiw(1984)and the nomic impacts to aquaculture and fisheriesindustries other is the elucidation of the taxonomic position of (Lom and Dykova,1992;Kent et al.,2001), e.g., Myxozoa using molecular systematics(see below). Myxobolus cerebralis,the cause of whirlingdisease in This review paper updates recent information on salmonid fish;Tetracapsuloidesbryosalmonae(=PKX), myxozoan studiesand highlightsnot only what we know the cause of proliferativekidney disease in salmonids; of these parasitesbut the gaps in our knowledge, with udoa thyrsites,the cause of post-mortem Kmyolique the aim of givinga directionfor future studies. factionin various marine fishes,particulary pen-cultured Atlantic salmon Salmo salar. While the above well Taxonomy of Myxozoa known species are widely epidemic throughout the world, many other species are endemic pathogens, only Myxozoa were long considered as an assemblage known as local diseases, such as Ceratomyxa shasta, of the protozoans, but this taxonomic placement has the cause of salmonid ceratomyxosis in the Pacific been challenged due to theirmulticellularity and cellular Northwest of the U.S.A. and Canada and Kudoa differentiation notfound in other protozoans. Recent amamiensis from cultured yellowtailSeriola quinquer molecular analyses using small-subunitribosomal DNA adiata on the Amami-Okinawa islandsof Japan(Fig.1). (SSUrDNA)sequences have been successful in clarify Recently, studies on myxozoan parasites have ing that myxozoans are not protozoans but metazoans made rapid progress, resultingin more than 60 related (Smothers et al.,1994;Siddal et al.,1995;Schlegel et *Invited paper given at the internationalconference"Impacts of al,1996). This view is now widely accepted, but con troversy remains as to whether theirphylogenetic origin myxozoan parasites in wild and farmed finfish",Nanaimo, BritishColumbia, Canada, July 31-August 2,2002. is diploblasticor triploblastic.Siddal et al.(1995)sug E-mail:[email protected] gested theirclose relationshipto the cnidarians,based 126 H. Yokoyama Fig. 1. Typical myxozoan diseases, myxospores and actinospores. a: yellowtail Seriola quinqueradiata infected with Kudoa amamiensis. Arrows show pseudocysts forming in the skeletal muscle. Scale bar = 5 cm. b: goldfish Carassius auratus infected with Hoferellus carassil. Arrow shows the enlarged kidney caused by the parasite. Scale bar = 1 cm. c: fresh myxospores of Myxobolus koi from the gills of common carp Cyprinus carpio. Scale bar = 10 ƒÊm. d: fresh actinospores of Myxobolus cultus from the intestine of oligochaete Branchiura sowerbyi. Scale bar = 100 ƒÊm. on the molecular evidence as well as morphological simi- Myxosporea and Malacosporea, the latter being recently larities between the polar capsules of Myxozoa and the created for uncommon myxozoans in bryozoan nematocysts of Cnidaria. In contrast, the triploblastic hosts. Following the clarification of the bi-phasic life- origin was suggested by the hox-gene analysis (Ander- cycle of many myxosporeans, the class Actinosporea son et al., 1998) and the morphological findings of was suppressed, and hitherto described actinosporeans Buddenbrockia, a possible ancestor of Myxozoa from were proposed to be downgraded to collective groups bryozoan hosts (Okamura et al., 2002). The ultrastruc- (Kent et al., 1994). However, it cannot be ruled out that tural study of Buddenbrockia revealed the triploblastic some actinosporean species, in particular marine spe- organization (the presence of an inner layer of cells and cies, have direct invertebrate-to-invertebrate transmis- 4 sets of longitudinal muscle blocks), thus demonstrating sion without a typical myxosporean phase (Lester et al., bilaterian affinities and a possible relation to nematodes 1999). Thus, it is still debated whether a newly found (Okamura et al., 2002). It also remains unclear which actinosporean, whose corresponding myxosporean came first, the myxosporean or the actinosporean stage is unknown, can be designated as a new species stages, in the evolutional scenario. Presence of sexual (Kent and Lom, 1999; Lester et al., 1998, 1999). reproduction in the actinosporean stage of Myxobolus Molecular phylogeny has revealed inconsistencies be- cerebralis (EI-Matbouli and Hoffmann, 1998) suggests tween the molecular and morphological systematics of that Myxozoa are originally parasites of invertebrates, the Myxozoa. In the order Multivalvulida, traditional although the sexual process has been poorly described classification dividing members of the order into families in other myxozoans. and genera were based on the number of polar capsules Intra-phylum classification has been largely revised. and spore valves. However, the molecular analysis of Presently, the Myxozoa is comprised of two classes; SSUrDNA of Pentacapsula, Hexacapsula, Septem- Review of Myxozoa 127 capsula and a newly found myxozoan having 13 polar (Feist et al., 2001; Canning et al., 2002), it is not well capsules (Whipps et al., 2003a) indicates that these understood whether the relationship between the two parasites cluster within the clade containing Kudoa spp., stages in bryozoan and salmonid hosts is biologically suggesting that the number of polar capsules is not a equivalent to that between myxo- and actinosporean life valid criterion for separating the multivalvulids into gen- stages. Further studies are required to elucidate the era and that the diagnosis of the genus Kudoa should be bi-phasic life-cycles of Myxozoa. amended to accommodate all myxozoans possessing 4 Much of what we know about biology of Myxozoa is or more polar capsules (Whipps et al., 2003b). based on studies on M. cerebralis (EI-Matbouli and Hoffmann, 1998; Bartholomew and Wilson, 2002), and it is necessary to determine similar information for other Biology of Myxozoa important myxozoans. The developmental cycle of M. Alternation of myxosporean and actinosporean cerebralis has been relatively well described, but many stages in the life-cycles of more than 25 species of fresh- questions still remain about various phases of the life- water myxozoans has been illustrated (Fig. 2). The cycle as described below. connection between the myxosporean genus, Myxobolus, and the actinosporean type (the former actinosporean Portals of entry of actinospores genus), triactinomyxon, is most common. Other corre- The findings of actinosporean stages have made it sponding patterns are not always predictable. Cladistic possible to experimentally determine the portals of entry analyses using the two different morphotypes showed for the parasite in the fish host. The skin, fins, gills and that the actinosporean morphology has a lack of taxo- buccal cavity of rainbow trout Oncorhynchus mykiss nomic congruity (Xiao and Desser, 2000a). Although have been demonstrated as the portals of entry for the PKX organism was identified as the malacosporean, triactinomyxon spores of M. cerebralis (Markiw, 1989; Tetracapsuloides bryosalmonae, infecting bryozoans El-Matbouli et al., 1995). Scanning electron micro- Fig. 2. Correspondence between myxosporean and actinosporean stages hitherto described. Breadth of connection lines repre- sents a relative frequency of the correspondence; 10 species for Myxobolus—triactinomyxon; 2 species each for Hoferellus aurantiactinomyxon, Myxobo/us—raabeia, Thelohanellus—aurantiactinomyxon, and Henneguya—aurantiactinomyxon; 1 species each for the other. 128 H.Yokoyama scopic studies revealed that as early as 1 min post expo (Yokoyama et al., 1997a). Myxobolus cerebralis devel sure to the fish, the polar filaments of the triactinomyxon ops in the cranial cartilage of rainbow trout, but localizes discharge and the