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Home , Ceratomyxa, Enteromyxum, Enteromyxum leei, Enteromyxum scophthalmi, Kudoa thyrsites, Multivalvulida

魚 病 研 究 Pathology,38(4),125-136,2003.12 2003 The Japanese Society of Fish Pathology

A Review:Gaps in Our Knowledge on Myxozoan Parasitesof *

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 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 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: , myxosporean, actinosporean,parasite, , biology,control strategy

Myxozoans are multicellular,-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 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). ,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 ,the cause of post-mortem Kmyolique the aim of givinga directionfor future studies. factionin various marine fishes,particulary pen-cultured Atlantic 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 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 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 . 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 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- and Malacosporea, the latter being recently larities between the polar capsules of Myxozoa and the created for uncommon myxozoans in bryozoan nematocysts of . In contrast, the triploblastic hosts. Following the clarification of the bi-phasic life- origin was suggested by the hox- 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 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 , 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- 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 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 . The skin, fins, gills and that the actinosporean morphology has a lack of taxo- buccal cavity of rainbow 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 of M. cerebralis (Markiw, 1989; 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 of , but localizes discharge and the sporoplasms penetrate through in the ribs or the gill arches of Salmo trutta, the opening of the mucous cells of the which are resistant to the pathogenic effects of whirling (El-Matbouli et al., 1999a). In contrast, the aurantiactin disease (Hedrick et al., 1999a). It is likely that infection omyxon spores of Thelohanellus hovorkai labeled with a in the head cartilage is more pathogenic than in the ribs fluorescent dye were observed to invade carp mainly via or gill arches, but the reason for the difference in tissue the gill filaments (Yokoyama and Urawa, 1997). distribution is unknown. Myxobolus buri develops in Aurantiactinomyxon spores of the PGD (= proliferative various parts of the brain of yellowtail but only cysts gill disease) organism primarily utilize the intestine of settled in the 4th ventricle have been closely associated channel lctalurus punctatus as the portal of entry with scoliosis of infected fish (Sakaguchi et al., 1987). (Bellem and Pote, 2001). This suggests a variety of In contrast, no consistent relationship between parasite invasion for which the stimulus is as yet unknown. Skin location and spinal curvature has been determined in the mucus of the host fish has been shown to induce the case of lordosis or lordo-scoliosis caused by Triangula polar filament extrusion of actinospores followed by the percae in redfin perch Perca fluviatilis (Langdon, 1987) release of sporoplasms, suggesting that actinospores and Myxobolus spinacurvatura in mullet Mugil cephalus find the host by certain chemical substances in fish (Maeno et al., 1990). Liyanage et al. (1998) showed mucus (Yokoyama et al., 1993; Uspenskaya, 1995; Xiao the causal relation between the tissue tropism of and Desser, 2000b; Ozer and Wootten, 2002). Thelohanellus hovorkai and the route of entry of the Yokoyama et al. (1995) demonstrated that low-molecular actinosporean. T. hovorkai infections that established mucin components stimulated the polar filament in the gill connective tissue were associated with water extrusion of raabeia actinospores of Myxobolus borne infection, whereas subcutaneous tissue infections cultus. A contradictory result was obtained by El occurred via an oral route. Subcutaneous infection by Matbouli et al. (1999a), where triactinomyxon spores -of T. hovorkai is likely to cause the cutaneous haemor M. cerebralis were not activated by the mucus of rainbow rhages clinically observed in diseased fish, implying that trout, perhaps indicating that both mechanical and oral infection, namely, the ingestion of oligochaetes, is chemical stimulations are required for host-finding in M. critical for disease outbreak. cerebralis. Regulation factors on developmental cycle Migration routes in fish hosts Environmental, physiological and immunological Bloodstream stages, extrasporogonic proliferation factors have been investigated as possible regulatory development, of Sphaerospora spp. have been well factors on the development of myxozoans in the fish documented in the fish host (Lom and Dykova, 1992). host. Temperature strongly influences the severity of Sphaerospora renicola also has swimmbladder stages, PKD in salmonids (Ferguson, 1981). Sexual matura often causing inflammation of the swimbladder in carp, tion of host fish may promote the development of Kudoa before sporogony develops in the kidney (Molnar and thyrsites (St-Hilaire et al., 1998). Entry to sea water of Kovacs-Gayer, 1986). El-Matbouli et al. (1995) demon salmonids, probably inducing physiological changes, strated that M. cerebralis migrates via the nervous sys results in the arrested development of salvelini tems prior to settling in the cartilage tissues of rainbow (Higgins et al., 1993). Dose of actinospores and fish trout. The presence of blood stages of age at the initial contact to actinospores influences the was suggested after the successful transmission of the severity of infection with Myxobolus cerebralis (Markiw, parasite to uninfected fish by intraperitoneal injection of a). However, it is unknown whether these 1992factors blood from infected fish (Moran et al., 1999), though the directly or indirectly influence the parasite's develop developmental form of K. thyrsites in blood has not yet ment. In other words, it is possible that temperature been found. For many other myxozoans, migration affects the development of the parasite directly, but it is routes remain to be solved as the missing link between also possible that an increase in temperature enhances the portals of entry and the target tissues. the host's immune responses, controlling the parasite's development. Also, this latter may either benefit the Settlement in the target tissues (associated with pathol fish, if it is a productive response, or increase the rate of ogy) the disease if there is an immunopathological Variable pathological effects associated with differ component. If in vitro culture techniques for myxozoans ent microhabitats of gill-infecting myxozoans have been are developed, the effects of factors on parasite devel reported (Molnar, 2002). For example, harmful largetyped opment can be analyzed individually. However, there plasmodia of Myxobolus koi develop in the primary are only a few convincing reports on the successful in lamellae of common carp Cyprinus carpio, whereas the vitro sporulation of myxozoans and this information is small-typed ones are situated in the secondary lamellae fragmentary (Wolf and Markiw, 1976; Siau, 1977). Review of Myxozoa 129

Release of myxospores from fish the environment with worm faeces. On the other hand, Most histozoic myxozoans were believed to only exit the developmental processes (the mechanisms of after host death and decomposition (Lom, 1987), but entry and release, etc.) of the actinosporean stage of there is also evidence that myxospores are released to Ceratomyxa shasta infecting the epidermis of a the environment while the fish host is still alive. In the freshwater (Bartholomew et al.,1997) and case of Myxobolus artus, which forms visible cysts in the malacosporeans developing in the body cavity of bryozo skeletal muscle of common carp, mature spores are ans (Canning et al., 2002) are still unknown. phagocytosed by host macrophages and transported to Actinospore releases from oligochaetes have been other organs such as the kidney, skin, intestine and gills, shown to have circadian cycles, occuring during the and are finally liberated from the fish (Ogawa et al., night or early morning(Yokoyama et al.,1993), and 1992). Associated with spore discharge of M. artus is appear to be temperature-dependent (El-Matbouli et al., the massive influx of spores into the gill capillaries which b; Blazer et al.,2003), resulting in seasonal cycles1999 may cause significant pathological changes in the gills (Gilbert and Granath, 2001; Allen and Bergersen, 2002; (Yokoyama et al., 1996). Spore discharge of M. artus Oumouna et al., 2003). In comparison to myxosporean has a seasonal pattern, possibly influenced by tempera stages, viability of actinospores in water is generally ture (Yokoyama et al., 1996). Other histozoic species short-lived, for several days (Markiw, 1992b; Yokoyama may rely on different routes and mechanisms of release. et al., 1993; Xiao and Desser, 2000b) or longer than 15 days

Regarding post-mortem myoliquefaction due to Kudoa (El-Matbouli et al., 1999b). Actinospores have spp., it is plausible that the parasite initiates the excre been shown to be susceptible to a wide range of chemi tion of proteolytic enzymes after host death in order to cals, ozone and UV treatments at practical levels facilitate spore release from fish (Langdon, 1991). (Yokoyama et al, 1997b; Hedrick et al., 2000; Wagner et al However, these enzymes appear not to be produced ., 2003). Development of triactinomyxon of M. from the myxospores but from the pre-sporogonic plas cerebralis in Tubitex is hampered by temperatures modia (Stehr and Whitaker, 1986). Compared to higher than 25℃, but recovered oligochaetes research on the route of entry, little attention has been remain susceptible to reinfection (Hedrick et al., 1998; paid on the route of exit. El-Matbouli et al,1999b). Collection methods for actinospores using multi-well Myxospore characteristics plates or simple filtration have been developed for stud Myxospores of M. cerebralis appear to remain ies on actinosporean biology (Yokoyama et al., 1991; viable for long periods of time, at least for several Thompson and Nehring, 2000). The former method months or for more than a year (Hoffman and Putz, might be useful for collection of a large quantity of

1969; El-Matbouli and Hoffmann,1991a). M. cerebralis actinospores at a time, while the latter is adapted for sur spores are resistant to freezing(-20℃ for 3 mo) and vey of actinospore emergence in a field. passage through the alimentary canal of predatory ani mals such as piscivorous birds (El-Matbouli and Biology of invertebrate hosts

Hoffmann,1991a), but are killed by heat(60℃ for 10min Oligochaetes, , sipunculids, bryozoans

)or chemical treatments with calcium hydroxide or and cephalopods have been described as invertebrate chlorine (Hoffman and Putz,1969). Polar filaments of hosts for myxozoans. Compared to our knowledge of Myxobolus koi, Myxobolus muelleriand Triangula percae fish biology, we have a very unsatisfactory understand were extruded by KOH (Nakajima and Egusa, 1974), ing of the biology of the alternate invertebrate hosts, e.g., urea (Lom, 1964) and fish tissue autolysis (Langdon, life-span, reproduction, feeding habits and defense 1987),respectively. But the in vivo mechanisms acti mechanisms. It is noteworthy that some oligochaetes vating the polar filament extrusion in the invertebrate have the ability to form cysts that allow their survival host have not been elucidated. through drought conditions for up to 5 months (Anlauf, 1990). It has been recently demonstrated that suscep Actinosporean characteristics tibility to M. cerebralis is variable among oligochaete Most actinosporeans have been reported to infect strains and genotypes (Stevens et al., 2001; Beauchamp the gut of oligochaetes (Kent et al,2001). et al., 2002). Environmental factors (e.g., habitat sub In case of M. cerebralis,myxospores released from the strates, nutritional potentials and dissolved oxygen lev fish host are ingested by the oligochaete hosts, the els) influencing the distribution of oligochaetes have valves open in the gut lumen, and then the sporoplasms been investigated in relation to the severity of myxozoan invade and develop in the intestinal epithelium. infections (Allen and Bergersen, 2002; Koprivnikar et al., Actinosporean stages undergo schizogony, gametogony 2002; Blazer et al., 2003; Liyanage et al., 2003). It is and sporogony, and finally produce usually 8 likely that anthropogenic activities in streams and rivers, actinospores in a pansporocyst. In the life-cyclesfully e.g. dam construction, agriculture and forestry practices described, the actinospores appear to be released into etc, affect oligochaete populations in a given watershed 130 H.Yokoyama

(Zendt and Bergersen, 2000). To explore a wide range of drugs more efficiently, either in vitro screening test or in vivo experimental infection Transmission of marine myxozoans model should be established. Failure to experimentally transmit Kudoa thyrsites infection by injection/ingestion of spores (Moran et al., Vaccination 1999) suggests the potential involvement of alternate Only a few reports have confirmed the presence of hosts in the life-cycles of marine myxozoans, as is well serum to myxozoans (Griffin and Davis, 1978; documented for their freshwater counterparts. Contrary Furuta et al., 1993; Hedrick et al., 1993). One explana to this belief, recent reports on the successful fish-to-fish tion of this inconsistent production of antibodies is that transmission of spp. give us new myxozoans may mimic antigens of host fish (Pauley, insight to the infection cycle of marine myxozoans from 1974). Acquired resistance to reinfection with PKD and the intestine of marine fish. Direct transmission whirling disease has been documented (Klontz et al., without a need of alternate hosts has been proven for 1986; Foott and Hedrick, 1987; Hedrick et al., 1998), (= Myxidium leei) from European though the mechanisms (specific and/or nonspecific seabream Sparus aurata (Diamant, 1997), E. defenses) have not been elucidated. Hedrick et al. scophthalmi from Scophthalmus maximus (1999a) suggested that innate such as cellular (Redondo et al., 2002) and Myxidium spp. from tiger immune responses by eosinophilic granular leukocytes puffer Takifugu rubripes (Yasuda et al., 2002). This might be involved in the resistance of brown trout to M. does not necessarily mean that invertebrate alternate cerebralis. No reports have determined protective anti hosts do not exist in their natural life-cycles. But, in an gens of the parasites responsible for the immunity. intensive culture system, it is highly likely that trophozoi tes released from the intestine are rapidly ingested by Introduction of the resistant strains other fish in the surrounding sea-cages, promoting a The genetic resistance of certain salmonid fish spe rapid multiplication of the parasite. Another uncommon cies or strains to C. shasta and M. cerebralis have been characteristic of E. leei is its wide host-range, which en reported (Bartholomew, 1998; Hedrick et al., 1999a, compasses at least 36 fish species belonging to 16 b; Thompson et al., 1999). However,1999 salmonid families (Padros et al., 2001). This implies that various strains resistant to C. shasta were found to be suscep fish species may act as a reservoir for this tible to M. cerebralis, suggesting that the mechanisms of parasite. Considering cannibalism and necrophagia resistance are different for the two myxozoans (Hedrick frequently occur in the natural environment, this method et al., 2001). Some evidence of the heritability of the of transmission may commonly exist among wild fish resistant trait suggests the potential application of selec too. tive breeding (Hemmingsen et al., 1986; Ibarra et al., 1992, 1994). If the application of recent molecular tech niques to develop gene markers identifying this trait is Potential Control Strategies successful, breeding programs may become effective in Chemotherapy the future (Arkush et al., 2002). Since the mid-1980s, many researchers have reported successful oral treatments with the fungal anti Management of aquaculture program biotic, (Molnar et al., 1987; Hedrick et al., Quarantine appears to be of primary importance in 1988; Szekely et al., 1988; Wishkovsky et al., 1990; preventing the spread of myxozoan parasites. Fish Yokoyama et al., 1990; El-Matbouli and Hoffmann, from an endemic area should not be transferred to non b; Sitja-Bobadilla and Alvarez-Pellitero, 1992;1991 Rhee endemic areas (Hoffman, 1990). However, a threat of et al., 1993; Yokoyama et al., 1999; Wang et al., 2001) parasite transmission via fish eggs is unlikely (Markiw, and its analog TNP-470 (Higgins and Kent, 1998) 1991). To enforce the quarantine policy rigorously, against many myxozoan diseases (Table 1), except for molecular-based detection methods will be useful for ceratomyxosis (Izbarra et al., 1990; Whipple et al., sensitive diagnosis of parasites even from live and 2002). However, the toxicities of these drugs have lim asymptomatic fish (Andree et al., 1998; Fox et al., ited their practical application for farmed fish 2000). Selection of disease-free sites (avoidance of (Wishkovsky et al., 1990). Toltrazuril, usually used for infected waters) is worthy of consideration, as some avian coccidian diseases, was successfully tested myxozoans have only a limited geographical distribution (Schmahl et al., 1989), but few researchers have studied (Hendrickson et al., 1989; Sugiyama et al., 1999). its efficacy. At present, there are no approved chemo Other factor that can be managed in aquaculture is therapeutants for myxozoans. In addition to anti-para the age at which fish are exposed to infection. For M. sitic drugs, efficacy of immunostimulants should be cerebralis, it has been well documented that delaying investigated, although Whipple et al. (2002) failed to exposure of rainbow trout to at least 9 weeks post hatch control ceratomyxosis by oral treatment with glucans. greatly decreases the incidence of whirling disease Review of Myxozoa 131

Table 1. Potentialcontrol strategies for myxozoan diseases

(Wagner,2002). For K. thyrsites,since sexually diation,chlorination, microfiltration and ozone was indi matured Atlanticsalmon Salmo salar are susceptible to cated to control whirling disease and ceratomyxosis infection,it has been recommended to remove recondi (Sanders et al.,1972;Hoffman,1974;Tipping,1988; tioned fish(grilse)before harvesting(St-Hilaireet al., Hedrick et al.,2000). Control of oligochaete popula 1998). Drying of infectedponds seems unlikelyto be tions may be achieved by reducing organic loads, effectivein mitigatingthe disease effects,because of the increasingflushing flows and limitingsiltation from water resistance of myxospores and encysted tubificids to shed activities(Brinkhurst,1996;Zendtand Bergersen, drought condition. Fallowing seems to be impractical 2000). Liyanage et al.(2003)showed that soilamend due to the complex life-cycleof myxozoans. However, ment(from organic mud to sand)of earthen ponds was assuming that the waterborne trophozoites of Enter effectiveat modifyingthe oligochaetecommunities, pos omyxum spp. are short-livedin sea water, fallowing sibly reducing the impact of Thelohanellus hovorkai strategiesmay successfully interruptthe fish-to-fish or infections. Furtherresearch on the habitatrequirement age-to-cage transmission. c of invertebratealternate hosts is required.

Environmental control *El-Matbouli Use of concrete tanks or raceways, possiblyhaving , M., M. Gay, T. S. McDowell, M. P. Georgiadis and R.P. Hedrick(1999):The potentialfor using biologicalcontrol some effectat reducing the habitatfor oligochaetes, was technologies in the management of whirlingdisease. Proc. a traditionalway to prevent whirlingdisease(Hoffman, 5th Annual Whirling Disease Symposium, , Whirling 1990). Disinfectionof rearingwater with ultravioletirra Disease Foundation, p.191-195. 132 H. Yokoyama

Biological control Anlauf, A. (1990): Cyst formation of (Muller)-an Some species or strains of oligochaetes, resistant to adaptation to survive food deficiency and drought. Hydrobiologia, 190, 79-82. Myxobolus cerebralis, have been shown to be capable of Arkush, K. D., A. R. Giese, H. L. Mendonca, A. M. McBride, G. ingesting and inactivating the myxospores of M. D. Marty and P. W. Hedrick (2002): Resistance to three cerebralis (Hedrick et al., 1998; El-Matbouli et al., 1999*: pathogens in the endangered winter-run see the footnote of the previous page). Benthos-eating (Oncorhynchus tshawytscha): effects of inbreeding and fishes may be useful for reducing the biomass of anne major histocompatibility complex genotypes. Can. J. Fish. lids in pond bottoms (Yokoyama et al., 2002). How Aquat Sci. , 59, 966-975. Bartholomew, J. L. (1998): Host resistance to infection by the ever, these approaches are still preliminary, and their myxosporean parasite Ceratomyxa shasta: a review. J. efficacies need to be evaluated quantitatively. Aquat. Anim. Health, 10, 112-120. Bartholomew, J. L. and J. C. Wilson (2002): Whirling disease: Post-harvest treatment reviews and current topics. Am. Fish. Soc., Bethesda, For the post-mortem myoliquefaction caused by pro 247 p. Bartholomew, J. L., M. J. Whipple, D. G. Stevens and J. L. Fryer teolytic enzymes from some marine myxozoans, post (1997): The life cycle of Ceratomyxa shasta, a harvest treatment might be adapted. Several protease- myxosporean parasite of salmonids, requires a freshwater inhibitors, e.g., potato extract and egg white, have been polychaete as an alternate host. J. Parasitol., 83, 859 reported to be effective against the proteolytic activities 868. - responsible for the muscle degeneration caused by Beauchamp, K. A., M. Gay, G. O. Kelly, M. El-Matbouli, R. D. Kathman, R. B. Nehring and R. P. Hedrick (2002): Preva marine myxozoans (Konagaya, 1984; Chang-Lee et al., lence and susceptibility of infection to Myxobolus 1989; Porter et al., 1993). However, this treatment is cerebralis, and genetic differences among populations of only applicable for the process of surimi. Microwave Tubifex tubifex. Dis. Aquat. Org., 51, 113-121. cooking may be another tactical option for inactivating Bellem, A. M. G. and L. M. Pote (2001): Portals of entry and the protease(s) in a whole fillet (Greene and Babbitt, systemic localization of proliferative gill disease organisms in channel catfish Ictalurus punctatus. Dis. Aquat. Org., 1990). To minimize diffusion of the histolytic enzymes 48, 37-42. into fish tissues, rapid cooking following minimal storage Blazer, V. S., T. B. Waldrop, W. B. Schill, C. L. Densmore and has been recommended (Lester, 1982; Patashnik et al., D. 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