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.

Comparative transcriptome analyses of the third and fourth stage larvae of Anisakis simplex (Nematoda: Anisakidae).

We analyzed transcriptome profiles of Anisakis simplex (Nematoda: Anisakidae) 3rd (ASL3) and 4th larvae (ASL4) obtained by RNA-seq, to understand the molecular pathways linked to parasite survival and discover stage-enriched gene expressions. ASL3 were collected from chum salmon and ASL4 were obtained by in vitro culture. Whole transcriptome sequencing was conducted with Illumina sequencer, and de novo assembly was conducted. 47,179 and 41,934 genes were expressed in ASL3 and ASL4 transcriptomes. Of them, 17,633 were known and 29,546 were unmapped sequence for ASL3. 17,126 were known and 24,808 were unmapped sequence for ASL4. Polyubiquitins-related genes and collagen-related genes were the most abundantly expressed in ASL3 and ASL4. Mitochondrial enzyme-related genes were highly expressed both in ASL3 and ASL4. Among the transcripts, 675 were up-regulated in ASL3, while 1015 were up-regulated in ASL4. Several protease-related and protein biosynthesis-related genes were highly expressed in ASL3, all of which are thought to be crucial for invading host tissues. Collagen synthesis-related genes were highly expressed in ASL4, reflecting active biosynthesis of collagens during molting process. This information will extend our understanding of biology of the fish-borne zoonotic parasite A. simplex.

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.

Activation of an Aquareovirus, Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis.

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal-lysosomal system and released in a more infectious form. When allowed to swim in CSV-infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.

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.

Occurrence and identification of Anisakis spp. (Nematoda: Anisakidae) isolated from chum salmon (Oncorhynchus keta) in Korea.

The prevalence of infection and the identification of anisakid larvae in chum salmon (Oncorhynchus keta) from the Namdae River, the east coast of Korea, were investigated. In total, 8,358 larvae were collected from 120 fish samples (male = 58 fish, female = 62 fish) in 2008. Fish samples were collected during October and November 2008. All the chum salmon samples (120/120, 100%) caught were infected with anisakid larvae with a high intensity (69.65 ± 48.58 larvae/host). They were mostly found in muscles (98.00%). Based on the morphological and the molecular analysis of PCR-RFLP and sequencing of mitochondrial DNA cox2 gene markers, these nematodes were identified as Anisakis simplex (sensu stricto) third-stage larvae. This is the first report on the molecular identification of anisakid worms from salmonid fishes in Korea. The high occurrence of anisakid worms in chum salmon may pose considerable food safety problems if they were consumed as raw or undercooked, although their commercial value is relatively lower than other salmonid species.

Sea lice escape predation on their host.

Parasites seldom have predators but often fall victim to those of their hosts. How parasites respond to host predation can have important consequences for both hosts and parasites, though empirical investigations are rare. The exposure of wild juvenile salmon to sea lice (Lepeophtheirus salmonis) from salmon farms allowed us to study a novel ecological interaction: the response of sea lice to predation on their juvenile pink and chum salmon hosts by two salmonid predators-coho smolts and cut-throat trout. In approximately 70% of trials in which a predator consumed a parasitized prey, lice escaped predation by swimming or moving directly onto the predator. This trophic transmission is strongly male biased, probably because behaviour and morphology constrain female movement and transmission. These findings highlight the potential for sea lice to be transmitted up marine food webs in areas of intensive salmon aquaculture, with implications for louse population dynamics and predatory salmonid health.

Differential rejection of salmon lice by pink and chum salmon: disease consequences and expression of proinflammatory genes.

The consequences of high (735 copepodids fish-1) and low (243 copepodids fish-1) level exposures of size-matched juvenile pink and chum salmon to Lepeophtheirus salmonis copepodids were examined. At both levels of exposure the prevalence and abundance of L. salmonis was significantly higher on chum salmon. In addition, the weight of exposed chum salmon following the high exposure was significantly less than that of unexposed chum salmon. At both exposures, the haematocrit of exposed chum salmon was significantly less than that of unexposed chum. Neither weight nor haematocrit of pink salmon was affected by exposures at these levels. Despite the presence of microscopic inflammatory lesions associated with attachment of L. salmonis on the epithelium of gill and fin of both salmon species, there were no mortalities following either exposure. A transient cortisol response was observed in chum salmon 21 d after low exposure. An earlier and quantitatively higher expression of the proinflammatory genes interleukin-8 (IL-8), tumour necrosis factor alpha-1 (TNFalpha-1) and interleukin-1beta (IL-1beta) in fin and head kidney of pink salmon suggested a mechanism of more rapid louse rejection in this species. Together, these observations indicate a relatively enhanced innate resistance to L. salmonis in the juvenile pink salmon compared with the juvenile chum salmon.

The abundance and distribution of Lepeophtheirus salmonis (Copepoda: Caligidae) on pink (Oncorhynchus gorbuscha) and chum (O. keta) salmon in coastal British Columbia.

In total, 23,750 specimens of the salmon louse, Lepeophtheirus salmonis, were collected from 3,907 juvenile pink and 3,941 chum salmon caught within the Broughton Archipelago during a 2-yr survey. The prevalence on pink salmon was significantly higher than on chum salmon in 2004 (62.3% and 58.6%, respectively) and in 2005 (26.4% and 23.1%, respectively). The mean abundance on chum salmon was significantly higher than on pink salmon in 2004 (7.0 +/- 0.3 and 2.8 +/- 0.2, respectively), whereas in 2005 the mean abundance did not differ between species (0.6 +/- 0.1 and 0.5 +/- 0.0, respectively). The mean intensity on chum salmon was significantly higher than on pink salmon in 2004 (12.0 +/- 0.4 and 4.5 +/- 0.2, respectively) and in 2005 (2.5 +/- 0.2 and 1.7 +/- 0.1, respectively). The prevalence, intensity, and abundance of L. salmonis were significantly higher on salmon belonging to both host species in 2004 compared with 2005. In both years, a majority of pink and chum salmon had 2 or fewer lice. In general, a decline in abundance of L. salmonis over the 3 collection periods in each year coincided with an increased percentage of motile developmental stages. The abundance was lowest on fish collected from zones in which the seawater surface salinity was also lowest. Seawater surface temperature was higher and salinity was lower in 2004 compared with 2005. The spatial and temporal trends in the abundance of L. salmonis in relation to host size, infestation rates, and seawater salinity and temperature, evident in both years, must be considered in future studies assessing the role of farmed salmon in the epizootiology of this parasite on juvenile salmon in this area.

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.

Putative Diphyllobothrium nihonkaiense acquired from a Pacific salmon (Oncorhynchus keta) eaten in France; genomic identification and case report.

We report here a likely case of Diphyllobothrium nihonkaiense contracted in France through the consumption of a Pacific salmon imported from Canada. The species diagnosis was made by molecular analysis of two mitochondrial genes (COI & ND3). This case is rather unusual in that D. nihonkaiense has never been reported along the Pacific coast of North America.

[Peter-the-Great Gulf fishes are a source of human invasion of the nematode Anisakis simplex (the family Anisakidae)]. / Ryby zaliva Petra Velikogo--istochnik invazii cheloveka nematodoi Anisakis simplex (sem.Anisakidae).

The paper presents the results of the rate of infection with the nematode Anisakis simplex in Peter-the-Great gulf fishes that are a source of human invasion. Five of 19 fish types were found to be nematode-free, and the extensiveness and intensity of invasion were observed in Siberian salmons (Oncorhynchus keta) (61% and 2-700 worms, respectively), Alaska pollacks (Theragra chalcogramma) (75.7% and 1-31 worms), and brown rock trouts (Hexagrammos octogrammus) (100% and 1-60 worms). Autopsy showed that the distribution of larvae was ambiguous in different fish species. Data on the survival of Anisakis were also obtained under experimental conditions. The nematodes were ascertained to die at -8 degrees C at the moment of complete fish freezing. Anisakis may be alive in the unfrozen fish for more than 40 days. The death of worms in the salted fish occurs within 3-8 days only when 6-10/1000 concentrations of NaCl salts are achieved at the sites of worms.

[Infection of coastal catching fishes with a nematode of Anisakis simplex (Rud., 1809), L. in the Primor'e region]. / Zarazhennost' nematodoi Anisakis simplex (Rud., 1809), L. ryb pribrezhnogo lova v Primorskom krae.

The paper presents data on the infection of fishes from the Gulf of Peter the Great with an A. simplex nematode. A total of 308 fishes of 19 species were examined for parasites. Of them 5 species were found to be free from nematodes. The highest infection rates were noted in fishes of the species Oncorhynchus keta, Hexagrammos octogrammus, and Theragra chalcogramma. Differences in the infection rates of fishes of the gulf were explained by the specific features of their vital activity and by the saturation of the environment with an invasive agent. Based on the infection of secondary intermediate hosts with the nematoda, it may be suggested that there are the populations of fishes whose representatives accomplish their life cycle under the conditions of the internal waters of the gulf. The infection rate of these fishes with the parasite is low. The altered situation--an increase in the invasion rate--was associated with the seasonal migration of fishes of these species from the open marine spaces to the cost of the Gulf of Peter the Great. The opposite values of the infection rates of deep-sea (herrings) and bottom-dwelling (bases, greenlings) fishes make it possible to assume that bottom-dwelling invertebrates function as the first intermediate hosts in the life cycle of the nematode.