Experimental evidence on prevention of infection by the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon using ultraviolet disinfection of rearing water.

In northern Japan, juvenile chum salmon Oncorhynchus keta (Walbaum) are released from hatcheries to enhance the fishery resource. Infections with ectoparasitic protozoans, particularly the flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae, occasionally cause severe mortality among hatchery-reared juveniles. This study examined the susceptibility of the two parasites to wide-ranging UV irradiation (experiment 1) and then investigated whether UV disinfection of the rearing water using a commercial device was useful for preventing infections among juveniles in a small-scale rearing system over a 28-day period (experiment 2). In experiment 1, parasite mortality reached 100% with UV irradiation doses of ≥9.60 × 105  µW s/cm2 for I. salmonis and ≥8.40 × 105  µW s/cm2 for T. truttae. In experiment 2, disinfection of the rearing water at a UV irradiation dose of 2.2 × 106  µW s/cm2 succeeded in complete prevention of both parasites in the juvenile salmon. These results elucidate the minimum dose of UV irradiation for inactivation of I. salmonis and T. truttae, and demonstrate the usefulness of water disinfection using a commercial UV irradiation device to prevent infections by these parasites in hatchery-reared juvenile chum salmon.

Effects of dietary supplementation with oregano essential oil on prevention of the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon Oncorhynchus keta.

The present study performed three experiments to establish a practical prevention strategy for the ectoparasitic flagellate Ichthyobodo salmonis and ciliate Trichodina truttae in hatchery-reared juvenile chum salmon Oncorhynchus keta using dietary supplementation with oregano essential oil. Experiment 1 showed that a diet supplemented for 3 weeks with 0.02% oregano essential oil significantly prevented infection with I. salmonis and T. truttae in juveniles reared in small tanks. Experiment 2, in outdoor hatchery ponds, demonstrated that the oregano treatment completely prevented I. salmonis infection for 52 days and T. truttae infection for 38 days. Oregano-treated juvenile mortality attributable to infection with these protozoans also decreased to 7.6% of control juvenile mortality, confirming the utility of this treatment in cultured O. keta. Physiological analyses of the oregano-treated juveniles elucidated the treatment's safety in relation to their metabolism, osmoregulation, natural immunity and olfactory responses and also detected carvacrol (a major component of oregano essential oil which shows antimicrobial activity) on the skin. In experiment 3, exposure of the two protozoans to oregano essential oil revealed a weak antiparasitic action on the body surface of the juvenile O. keta. The overall results demonstrate that dietary oregano supplementation is a practical prevention strategy for I. salmonis and T. truttae in hatchery-reared juvenile O. keta and suggest the possibility that its anti-parasitic action is attributable to a component of the oil that emerges onto the skin of the body of the fish.

Epizootiology of the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae on wild chum salmon Oncorhynchus keta.

Infestations of the ectoparasitic flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae have caused acute mortalities of hatchery-reared juvenile chum salmon Oncorhynchus keta in Hokkaido, northern Japan. This study examined the epizootiology of I. salmonis and T. truttae on wild chum salmon as a possible infection source of the 2 parasitic protozoans in hatcheries. Infestations by both ectoparasites were detected on freshwater-adapted adult and juvenile chum salmon in all 4 rivers examined. This is the first study of an anadromous Pacific salmonid to report infestation of I. salmonis and T. truttae in adults returning for spawning. Among the marine-inhabiting phase of chum salmon, infestation with I. salmonis, but not T. truttae, was observed on adults and juveniles. The 2 protozoans were experimentally transmitted at the same time from wild to hatchery-reared chum salmon juveniles, and caused a high rate of mortality in the hatchery fish. In freshwater, the proliferation rate of T. truttae was greater than that of I. salmonis. These observations show that the euryhaline ectoparasite I. salmonis can infest chum salmon throughout their life cycle, in both river and ocean habitats, whereas T. truttae is able to infest these salmonids only in freshwater. Furthermore, wild chum salmon were shown to be a potential infestation source for both T. truttae and I. salmonis in hatchery fish.

Henneguya cartilaginis n. sp. (Myxozoa: Myxosporea) in the head cartilage of masu salmon Oncorhynchus masou masou.

Henneguya cartilaginis n. sp. (Myxozoa: Myxosporea) is described from wild masu salmon Oncorhynchus masou masou in Hokkaido, Japan. H. cartilaginis n. sp. produced white cysts, up to 3mm in size, in the head of masu salmon. Infected fish exhibited cranial protrusion due to the cysts. Spores (11.4 × 8.6 µm) of H. cartilaginis n. sp. were egg-shaped with the posterior end more pointed and possessed two caudal appendages (34.2 µm average length). Histological observations revealed that large plasmodia possessing fine fibrous pseudopodia on the surface developed in the head cartilage. H. cartilaginis n. sp. resembles H. cerebralis, which was described from the cranial cartilage of Kosogol grayling Thymallus nigrescens in Mongolia. However, they were distinguishable by spore morphology. Molecular analysis of the 18S rDNA sequences indicated that H. cartilaginis n. sp. was most closely related to Henneguya zschokkei, H. nuesslini and H. salminicola of salmonid fish, with genetic similarities of 95.3%, 95.1% and 93.9%, respectively. Based on these differences in spore morphology, molecular data, the site of infection and geographical distribution, the present species is considered to be a new species.

Geographical variation in spore morphology, gene sequences, and host specificity of Myxobolus arcticus (Myxozoa) infecting salmonid nerve tissues.

Myxobolus arcticus Pugachev and Khokhlov, 1979 is a freshwater myxosporean parasite infecting the nerve tissues of salmonid fishes throughout the Pacific region of Far East Asia and North America. The principal fish host is sockeye salmon Oncorhynchus nerka in North America and masu salmon O. masou in Japan. Actinospores of M. arcticus were isolated from the lumbriculid oligochaetes Lumbriculus variegatus and Stylodrilus heringianus in Japan and Canada, respectively. Morphological comparisons indicated that Japanese actinospores from L. variegatus have significantly shorter caudal projections than Canadian isolates from S. heringianus, whereas the corresponding myxospores are indistinguishable. Transmission experiments showed that sockeye salmon were rarely susceptible to the Japanese actinospores, while masu salmon are highly susceptible to this parasite. Sequences of 4560 base pairs of the ribosomal RNA (rRNA) gene, including small subunit (SSU) and internal transcribed spacer (ITS) regions, from Japanese and Canadian isolates had a high similarity over 99.9%, suggesting that they may be conspecific. However, the biological data indicate that they are at least distinct strains. M. arcticus may be geographically isolated due to the specific homing migration of the anadromous fish hosts and has specialized its morphology and host selection for its local environment in the ongoing process of differentiation, potentially leading to speciation.

Population genetic structure and phylogeography of masu salmon (Oncorhynchus masou masou) inferred from mitochondrial and microsatellite DNA analyses.

The population genetic structure and phylogeography of masu salmon were investigated by using variation in the mitochondrial NADH dehydrogenase subunit 5 gene (ND5) and six polymorphic microsatellite loci among a total of 895 fish representing 18 populations collected from Japan (9), Russia (7), and Korea (2) from 2000 to 2008. An analysis of ND5 nucleotide sequences revealed 22 variable sites in about 560 bp in the 5' half of the gene, which defined 20 haplotypes, including some associated with geographical regions. Haplotype and nucleotide diversities were greater in the populations in Japan and Korea than in those in Russia, indicating greater genetic diversity in the Japanese and Korean populations than in the Russian populations. All the microsatellite loci examined showed a high level of variation, but the expected heterozygosity indicated a similar level of genetic diversity among the populations of the three regions, contrary to the results for ND5. However, AMOVA and pairwise population F (ST) estimates for both ND5 and the microsatellite markers indicated a similar pattern of moderate genetic differentiation among populations of the three regions, and large population groups on the coasts of the Sea of Japan, Sea of Okhotsk, and Pacific Ocean in the Far East. From a mismatch distribution analysis and neutrality test, the observed genetic structure appears to have been influenced primarily by bottlenecks during glacial periods and population expansions during interglacial periods in the late Pleistocene.

Genetic stock identification of chum salmon in the Bering Sea and North Pacific Ocean using mitochondrial DNA microarray.

A newly developed DNA microarray was applied to identify mitochondrial (mt) DNA haplotypes of more than 2200 chum salmon in the Bering Sea and North Pacific Ocean in September 2002 and also 2003, when the majority of maturing fish were migrating toward their natal river. The distribution of haplotypes occurring in Asian and North American fish in the surveyed area was similar in the 2 years. A conditional maximum likelihood method for estimation of stock compositions indicated that the Japanese stocks were distributed mainly in the north central Bering Sea, whereas the Russian stocks were mainly in the western Bering Sea. The North American stocks were abundant in the North Pacific Ocean around the Aleutian Islands. These results indicate that the Asian and North American stocks of chum salmon are nonrandomly distributed in the Bering Sea and the North Pacific Ocean, and further the oligonuleotide DNA microarray developed by us has a high potential for identification of stocks among mixed ocean aggregates of high-seas chum salmon.

Differences in host selection of actinospores of two myxosporeans, Myxobolus arcticus and Thelohanellus hovorkai.

We investigated the host selection mechanism of actinospore stages of 2 myxosporeans, Myxobolus arcticus and Thelohanellus hovorkai, infecting masu salmon (Oncorhynchus masou) and common carp (Cyprinus carpio), respectively. Discharge of the polar filaments and sporoplasm release by M. arcticus actinospores occurred within the first 5 min of exposure to skin mucus of masu salmon. The actinospores also reacted to the mucus of nonsusceptible fish, i.e., sockeye salmon (Oncorhynchus nerka) and goldfish (Carassius auratus), although the reactivity was comparatively lower. After exposure of masu, and sockeye and chum salmon (Oncorhynchus keta) to M. arcticus actinospores, the penetration of sporoplasms was observed in the fins and gills of masu and sockeye salmon to a similar extent and to a lesser extent in chum salmon. Thelohanellus hovorkai actinospores exhibited a slow response of sporoplasm release to common carp mucus as well as penetration into the gills of common carp. Neither chemoresponse to mucus of nonsusceptible fish (goldfish and sockeye salmon) nor sporoplasm invasion in goldfish was observed for T. hovorkai actinospores. These results indicate notable differences in the host selection at the time of entry between M. arcticus and T. hovorkai; the former responds quickly to fish mucus with low host specificity, whereas the latter was highly host specific in a dilatory reaction.

DNA microarray for rapid detection of mitochondrial DNA haplotypes of chum salmon.

For use in genetic stock identification, we developed an oligonucleotide (DNA) microarray hybridization method for rapid and accurate detection of nucleotide sequence variations in 20 previously identified variable nucleotide sites in about 500 bp within the 5' half of the control region of mitochondrial DNA of chum salmon (Oncorhynchus keta). The method includes immobilization of synthesized oligonucleotides containing respective polymorphic sites on a glass slide precoated with polycarbodiimide resin, a 2-hour hybridization with DNA microarray of biotinylated polymerase chain reaction fragments spanning the 5' variable portion followed by short washing, and visualization of hybridization signals by conventional ABC method and scanner-assisted computation of signal intensity on a computer. The entire process of hybridization and detection was completed within 4 hours. The resulting DNA microarray could detect all of the single nucleotide mutations and therefore could be used to identity the sequence variations defining 30 mtDNA haplotypes of chum salmon as revealed previously by nucleotide sequence analysis.

Ichthyobodo necator (Kinetoplastida)--a complex of sibling species.

Ichthyobodo necator is a parasitic flagellate that attacks fishes, causing disease problems in freshwater worldwide. Findings of similar flagellates in strictly marine fishes have indicated that ichthyobodiosis may be caused by more than 1 flagellate species. We obtained partial small subunit rDNA (ssu rDNA) sequences of 14 Ichthyobodo isolates originating from fishes in Norway, Japan, Singapore, South Africa and Brazil, and identified 8 strains or species, including 2 species infecting cultured salmon in Norway. An Ichthyobodo species isolated from the skin of Atlantic salmon parr in freshwater is suggested to represent L. necator sensu stricto, while another species, showing particular affinity for the gills, infects salmon in both fresh- and seawater. Atlantic cod is infected with a marine Ichthyobodo species unrelated to those infecting salmonids; 2 cyprinids originating from different parts of the world had related Ichthyobodo strains/species, and 2 isolates from unrelated North and South American fishes were also closely related. The phylogenetic relationships of the Ichthyobodo isolates is described, and the implications of the molecular findings on past and future morphological studies of Ichthyobodo spp. are discussed.

Description of two new actinosporean types from a brook of Fuji Mountain, Honshu, and from Chitose River, Hokkaido, Japan.

Actinospore infection of oligochaetes living in the mud of 3 freshwater biotopes in Japan was studied. Using the cell-well plate method, a new aurantiactinomyxon type was found in 0.77% of the examined Tubifex tubifex oligochaete specimens from a brook near Yamanashi Prefectural Fisheries Experimental Station on Fuji Mountain. In 0.14% of Lumbriculus variagetus collected from Chitose River, near Chitose Salmon Hatchery, a new siedleckiella type was found, while at the same time 8.1% of the Lumbriculus spp. oligochaetes released triactinomyxons of Myxobolus arcticus. Of the examined Rhyacodrilus komarovi oligochaetes collected from the Mena River system, Hokkaido, 0.2, 0.6, 0.5 and 0.8% were infected with echinactinomyxon, neoactinomyxum and 2 types of triactinomyxon spores, respectively, and described in our previous paper. The oligochaetes released actinospores for several weeks. Actinospore infection showed high intensity in positive oligochaetes in the case of all the actinosporean types. Two of the actinospore types (aurantiactinomyxon and siedleckiella) presented here have not been previously described.

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.