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.     

Comparative anatomy of the dorsal hump in mature Pacific salmon

Mature male Pacific salmon (Genus Oncorhynchus ) demonstrate prominent morphological changes, such as the development of a dorsal hump. The degree of dorsal hump formation depends on the species in Pacific salmon. It is generally accepted that mature males of sockeye (O. nerka ) and pink (O. gorbuscha ) salmon develop most pronounced dorsal humps. The internal structure of the dorsal hump in pink salmon has been confirmed in detail. In this study, the dorsal hump morphologies were analyzed in four Pacific salmon species inhabiting Japan, masu (O. masou ), sockeye, chum (O. keta ), and pink salmon. The internal structure of the dorsal humps also depended on the species; sockeye and pink salmon showed conspicuous development of connective tissue and growth of bone tissues in the dorsal tissues. Masu and chum salmon exhibited less‐pronounced increases in connective tissues and bone growth. Hyaluronic acid was clearly detected in dorsal hump connective tissue by histochemistry, except for in masu salmon. The lipid content in dorsal hump connective tissue was richer in masu and chum salmon than in sockeye and pink salmon. These results revealed that the patterns of dorsal hump formation differed among species, and especially sockeye and pink salmon develop pronounced dorsal humps through both increases in the amount of connective tissue and the growth of bone tissues. In contrast, masu and chum salmon develop their dorsal humps by the growth of bone tissues, rather than the development of connective tissue.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.     

Comparison of the spectral sensitivity of three species of juvenile salmonids

The spectral sensitivity of chum Oncorhynchus keta , pink Oncorhynchus gorbuscha and masu Oncorhynchus masou masou salmon was measured by the optomotor reaction index in monochromatic light of 400, 440, 480, 520, 560, 600 and 620 nm using an interference filter. The reaction rate of chum salmon was highest at 520 nm but the rates of pink and masu salmon were highest at 560 nm. In addition, a high reaction rate at 400 nm was also observed in masu salmon, suggesting that masu salmon are sensitive to ultraviolet light.     

Cloning and characterization of an odorant receptor in five Pacific salmon

The olfactory system of fish is extremely important as it is able to recognize and distinguish a vast of odorous molecules involved in wide ranges of behaviors including reproduction, homing, kin recognition, feeding and predator avoidance; all of which are paramount for their survival. We cloned and characterized one type olfactory receptors (ORs) from five congeneric salmonids: lacustrine sockeye salmon (Oncorhynchus nerka), pink salmon (O. gorbuscha), chum salmon (O. keta), masu salmon (O. masou) and rainbow trout (O. mykiss). Lacustrine sockeye salmon olfactory receptor 1 (LSSOR1) showed high sequence homology to the OR subfamily, and was expressed only in the olfactory epithelium (as indicated by PCR amplified genomic DNA and cDNA). OR genes from the five salmonids examined all showed strong homology (96-99%) to each other. Hypervariable regions, believed to be ligand-binding pockets, showed homologous completely matched amino acid sequences except for one amino acid in pink salmon olfactory receptor 1 (PSOR1), revealing that these ORs may be well conserved among salmon species. These results suggest that the isolated 5 salmonid ORs might play an important role in salmon life cycles.     

Transmission of Thelohanellus hovorkai Achmerov, 1960 (Myxosporea: Myxozoa) to common carp Cyprinus carpio through the alternate oligochaete host

Thelohanellus hovorkai (Myxosporea: Myxozoa) was transmitted to common carp Cyprinus carpio by exposing fish to Aurantiactinomyxon spores collected from the oligochaete Branchiura sowerbyi. The morphological characteristics of the actinosporean stage are described in detail. B. sowerbyi were exposed to T. hovorkai spores isolated from the experimentally infected carp, and after 3 and 4 months the worms exhibited prevalences of the actinosporean stage at 19.47% (7/36) and 14.6% (6/41), respectively. Control, unexposed worms were negative for the actinosporean infection. This is the first report of an Aurantiactinomyxon transforming into a myxosporean belonging to the suborder Platysporina.     

Evaluation of a vector-control strategy of haemorrhagic thelohanellosis in carp,caused by Thelohanellus hovorkai (Myxozoa)

The life cycle of Thelohanellus hovorkai (Myxozoa), the causative agent of haemorrhagic thelohanellosis of carp Cyprinus carpio, involves the alternate oligochaete host Branchiura sowerbyi, which plays the role of vector in the parasite's transmission. Field investigations in carp farms suggested that oligochaete fauna were closely associated with the substrate type of the pond. The pond bottom in the enzootic farm consisted of clay soil and soft sediments comprised of organic mud, in which B. sowerbyi dominated in high densities, with a maximum of 5.6 ind. kg(-1) soil. In another case, in a carp farm with no previous history of the disease, the pond bottom was sandy soil, in which small-sized oligochaetes, composed mainly of Limnodrilus socialis, dominated. Laboratory studies on the substrate preference of oligochaetes proved that B. sowerbyi prefers mud to sand, whereas L. socialis has no tendency to substrate tropism. The delicate body surface of B. sowerbyi was subject to damage by rugged-edged sand particles, which inflicted severe injuries to the worms. Transmission experiments showed that L. socialis, which are non-susceptible to T. hovorkai, suppressed the production of T. hovorkai actinospores in B. sowerbyi in a mixed assemblage of oligochaetes. Field and experimental evidence in this study imply that substrate replacement in culture ponds might regulate the benthic oligochaete communities, resulting in minimization of the impact of haemorrhagic thelohanellosis. We propose that ecological control of oligochaete fauna by environmental management is a promising strategy against myxozoan diseases.     

Evolutionary-physiological aspects of adaptation of the Pacific salmon fry of the Oncorhynchus genus to migration to the sea water

A complex of adaptive changes occurring in the Pacific salmon fry in the process of migration to the sea is described, including behavior, ion content in carcasses, and morphological changes in Stannius bodies, gill epithelium, and nephron tubular epithelium. Participating in experiments with transfer from fresh water into a two-layer aquarium (the lower layer - sea water, the upper layer - fresh water) were smolts of chum salmon and underyearlings of masu salmon as well as the trachurus and leiurus forms of the three-spined stickleback Casterosteus aculeatus. All fish, regardless of their salt preference, at once after placement into the two-layer aquarium, occupied the sea water zone, at the very bottom of the aquarium. After 1 h, there started brief excursions of masu salmon and chum salmon to the upper, fresh water layer; however, both forms of the three-spined stickleback did not participate in these excursions. After 12 h, the chum salmon settled down in the lower, sea water layer, while the masu salmon - in the upper, fresh water layer. Both forms of the three-spined stickleback never left the sea water layer and felt quite comfortably on the aquarium bottom. It seems that the high tolerance of the both stickleback forms to wide salinity limits allows them to choose the convenient position regardless of the water salt composition. By analyzing the material obtained for three years (2001-2003) on structure and functions of the gill epithelium chloride cells (CC), we have come to the conclusion that the fresh water fry of two salmon species, chum and masu salmons, caught at the same time and practically in the same water reservoirs can be divided into three groups. The underyearlings of the masu salmon as a rule are characterized by the thickened epithelium of secondary gill lamellae, but by a very small number of CC. In smolts of chum salmon, on the contrary, the epithelium is sufficiently thin, but enriched in the CC that demonstrate an active structure in the very beginning of migration to sea. However, with approaching the sea (and with an increase of terms of migration) the CC activity drops, but their amount does not change. And only after migration to the sea the CC activity rises again, although their amount seems to remain unchanged. The described peculiarities of behavior and of the ion composition regulation in the migrating salmon fry confirm the hypothesis that the salmons evolutionized in fresh water, that the Oncorhynchus genus appeared in large spaces of saltish waters, such as the Japan Sea at the period of the early Pleistocene, and that learning of fry of the Oncorhynchus genus (for instance, of O. gorbuscha and O. keta) is the most specialized in the salmons migrating to the sea, whereas the fresh water species of chars (Salvelinus) and of trouts (Salmo) are more primitive.     

Energy expenditures during spawning by chum salmon Oncorhynchus keta (Walbaum) in British Columbia

Physiological telemetry and proximate tissue analyses were used to assess energy expended by chum salmon Oncorhynchus keta on various behaviours during spawning in Kanaka Creek, British Columbia, Canada, and results were compared with published data on Fraser River sockeye salmon Oncorhynchus nerka , the only other species for which both types of measurements have been taken. Chum salmon arrived at the spawning grounds with body energy densities of 4·84 MJ kg⁻¹ in males and 4·62 MJ kg⁻¹ in females, lower than most sockeye salmon populations, and died with energy densities of c . 4 MJ kg⁻¹, similar to that observed in sockeye salmon and other salmonids. Moisture levels generally increased in body tissues over the spawning life, particularly in female gonads, and lipid levels decreased. Declines in protein observed over the spawning life of other Pacific salmon Oncorhynchus sp. were less evident in Kanaka Creek chum salmon. Holding behaviour constituted the dominant component of the activity schedule and energy budget of both sexes. After holding, the most expensive behaviours were nest digging in females and aggressive displays in males. Dominant males expended the most energy on behaviours each day, as indexed by oxygen consumption (3600 mgO₂ kg⁻¹), while satellite males expended nearly as much (3504 mgO₂ kg⁻¹) but females expended considerably less (2327 mgO₂ kg⁻¹). Kanaka chum salmon engaged more frequently in energetically expensive reproductive behaviours than Stuart River sockeye salmon.     

Physiological mechanisms of imprinting and homing migration in Pacific salmon Oncorhynchus spp

After several years of feeding at sea, salmonids have an amazing ability to migrate long distances from the open ocean to their natal stream to spawn. Three different research approaches from behavioural to molecular biological studies have been used to elucidate the physiological mechanisms underpinning salmonid imprinting and homing migration. The study was based on four anadromous Pacific salmon Oncorhynchus spp., pink salmon Oncorhynchus gorbuscha, chum salmon Oncorhynchus keta, sockeye salmon Oncorhynchus nerka and masu salmon Oncorhynchus masou, migrating from the North Pacific Ocean to the coast of Hokkaido, Japan, as well as lacustrine O. nerka and O. masou in Lake Toya, Hokkaido, where the lake serves as the model oceanic system. Behavioural studies using biotelemetry techniques showed swimming profiles from the Bering Sea to the coast of Hokkaido in O. keta as well as homing behaviours of lacustrine O. nerka and O. masou in Lake Toya. Endocrinological studies on hormone profiles in the brain-pituitary-gonad axis of O. keta, and lacustrine O. nerka identified the hormonal changes during homing migration. Neurophysiological studies revealed crucial roles of olfactory functions on imprinting and homing during downstream and upstream migration, respectively. These findings are discussed in relation to the physiological mechanisms of imprinting and homing migration in anadromous and lacustrine salmonids.     

Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.     

Straying of hatchery salmon in Prince William Sound,Alaska

The straying of hatchery salmon may harm wild salmon populations through a variety of ecological and genetic mechanisms. Surveys of pink (Oncorhynchus gorbuscha), chum (O. keta) and sockeye (O. nerka) salmon in wild salmon spawning locations in Prince William Sound (PWS), Alaska since 1997 show a wide range of hatchery straying. The analysis of thermally marked otoliths collected from carcasses indicate that 0–98% of pink salmon, 0–63% of chum salmon and 0–93% of sockeye salmon in spawning areas are hatchery fish, producing an unknown number of hatchery-wild hybrids. Most spawning locations sampled (77%) had hatchery pink salmon from three or more hatcheries, and 51% had annual escapements consisting of more than 10% hatchery pink salmon during at least one of the years surveyed. An exponential decay model of the percentage of hatchery pink salmon strays with distance from hatcheries indicated that streams throughout PWS contain more than 10% hatchery pink salmon. The prevalence of hatchery pink salmon strays in streams increased throughout the spawning season, while the prevalence of hatchery chum salmon decreased. The level of hatchery salmon strays in many areas of PWS are beyond all proposed thresholds (2–10%), which confounds wild salmon escapement goals and may harm the productivity, genetic diversity and fitness of wild salmon in this region     

Commercial blast-freezing of third-stage Anisakis simplex larvae encapsulated in salmon and rockfish

Sixty-four fish were blast-frozen to -35 C for 15 hr to determine the effects of commercial blast-freezing on the viability of third-stage larvae of Anisakis simplex encapsulated in the muscle and viscera of sockeye salmon (Oncorhynchus nerka) and canary rockfish (Sebastes pinniger). Parallel tests were conducted on larval nematodes in 16 whole (round) salmon, 16 dressed salmon (heads and viscera removed), and 32 whole (round) rockfish. After blast-freezing, 4 in-the-round salmon, 4 dressed salmon, and 8 in-the-round rockfish were examined at 1, 24, 48, and 72 hr. A total of 3,539 dead and 6 live larvae were collected from the fish tissues after standard enzymatic digestion. Salmon were infected with 1,245 of these larvae, and rockfish with 2,300. The 6 live worms, 2 from salmon and 4 from rockfish rounds, were recovered from muscle 1 hr after freezing; they were slightly motile and showed severe internal damage. No viable worms were found at or after 24 hr. The commercial blast-freezing process effectively killed larval nematodes in whole or dressed fish. Market-ready samples of previously blast-frozen silver salmon (O. kisutch) and chum salmon (O. keta) fillets and chum salmon steaks yielded no live worms, thereby confirming the efficacy of this process.     

Structure of spawning school of chum salmon Oncorhynchus keta from Olutorsky Bay of the Bering Sea (Northeastern Kamchatka)

Results of studies of spawning chum salmon Oncorhynchus keta (Walbaum) in Olutorsky Bay and the Apuka River—the largest river in northeast Kamchatka—inflowing Olutorsky Bay of the Bering Sea are presented. It was established that the first individuals of the chum salmon enter the river together with early sockeye salmon and chinook salmon in the first ten-day period of June, and mass-spawning run takes place in July–August. Analysis of biological characteristics of chum salmon caught in the Apuka River and Olutorsky Bay of the Bering Sea enabled us to reveal the inhomogeneity of its spawning school represented by two seasonal forms.     

The food web positioning and trophic niche of the non-indigenous round goby: a comparison between two Baltic Sea populations

Native species may show invasiveness toward a recipient ecosystem through increases in abundance as a result of artificial stocking events. Salmonid species are typical examples of native invaders whose abundance is increased after stocking with hatchery fish. This study evaluated the effects of hatchery chum salmon fry on sympatric wild masu salmon fry, benthic invertebrate prey, and algae, after a single stocking event in Mamachi stream, Hokkaido, northern Japan. The results suggested that the stocked hatchery chum salmon fry decreased the foraging efficiency and growth of the wild masu salmon fry through interspecific competition, and depressed the abundance of Ephemerellidae and total grazer invertebrates (Glossosomatidae, Heptageniidae, and Baetidae) through predation. Also, the hatchery chum salmon fry may increase algal biomass through depression of grazer abundance by predation (top-down effect). These results suggested that a single release of hatchery chum salmon fry into a stream may influence the recipient stream ecosystem.     

The distribution of juvenile Pacific salmon and associated fish species in Lake Sopochnoye,Iturup Island

A single survey using a minnow seine net showed that juvenile chum and sockeye salmon are distributed extremely unevenly along the shoreline of Lake Sopochnoye, on Iturup Island. Young-of-the-year chum salmon aggregate mainly near the estuaries of the rivers that flow into the lake; young-of-the-year and 2-year-old sockeye salmon aggregate closer to the central part of the lake. The distribution of associated fish species is also very uneven, but none of them dominates within the zone of catches; the abundance of each of these species is comparable to that of the juvenile Pacific salmon.     

No shot in the dark: myxozoans chemically detect fresh fish

This work reports the discovery of an hitherto unknown chemical recognition trait enabling a parasitic life cycle in aquatic habitats. We believe this is the first record of a natural, host-derived chemical molecule identified as a recognition cue for the phylum Myxozoa. The actinospores of these parasites attach to fish hosts via polar filaments that are extruded upon mechanical stimulation after preceding recognition of a chemical trigger contained in surface mucus. Our goal was to identify this signal. We separated compounds from a purified active fraction derived from trout mucus by a novel HPLC method. By subsequent nuclear magnetic resonance analysis of distinct components and testing in bioassays we elicited stimulation of polar filament discharge and sporoplasm emission in actinospores of three myxozoan spp., Myxobolus cerebralis, Myxobolus pseudodispar and Henneguya nuesslini, by the free nucleosides inosine, 2'-deoxyinosine and guanosine. These nucleosides also activated sporoplasm emission. Nucleosides appear to be appropriate cues for rapid host recognition by the waterborne parasite stages since they are continuously released into surface mucus. The recognition mechanism is not specific for susceptible host species, at least in the myxozoan spp. examined. In addition, a novel function of nucleobase derivatives as semiochemicals was uncovered and a wider impact of this molecule class in parasite recognition systems and aquatic chemical ecology is predicted. The relevance for disease prevention and cell culturing remains to be explored.     

Involvement of Manayunkia speciosa (Annelida: Polychaeta: Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon

A coelomic myxozoan infection was detected in freshwater polychaetes, Manayunkia speciosa from the Klamath River, Oregon/California, a site enzootic for the myxozoan parasites Ceratomyxa shasta and Parvicapsula minibicornis. The tetractinomyxon type actinospores had a near-spherical spore body 7.9 x 7.1 microm, with 3 spherical, protruding polar capsules, no valve cell processes, and a binucleate sporoplasm. Parvicapsula minibicornis-specific primers Parvi1f and Parvi2r amplified DNA from infected polychaetes in a polymerase chain reaction (PCR) assay. The small subunit 18S rRNA gene of the spores was sequenced (GenBank DQ231038) and was a 99.7% match with the sequence for P. minibicornis myxospore stage in GenBank (AF201375). Chinook salmon (Oncorhynchus tshawytscha) exposed to a dose of 1,000 actinospores per fish tested PCR positive for P. minibicornis at 14 wk postinfection and presporogonic stages were detected in the kidney tubules by histology at 20 wk. This life cycle is 1 of only about 30 known from more than 1,350 myxozoan species, and only the second known from a freshwater polychaete.     

Philomena oncorhynchi: Effect of hormones on maturation in anadromous sockeye, Oncorhynchus nerka

Adult sockeye salmon, Oncorhynchus nerka (Walbaum), treated with salmon pituitary extract in July survived for up to 47 days and developed nuptial colours in both male and female fish during this period, whereas no such characteristics appeared in untreated control fish. Furthermore, results showed that 20 of 50 adult female Philomena oncorhynchi recovered from the 15 treated fish contained fully developed tailed larvae in the uterus as compared with only one of 44 female P. oncorhynchi from 31 untreated control fish. None of 51 worms from 13 stilboestrol treated adult sockeye showed development in utero beyond an elongated embryo, nor did the host fish develop nuptial colours. P. oncorhynchi and its immature sockeye host (approx. 1 year prior to spawning) remained unaffected by any of three separate hormone treatment experiments using injection of salmon pituitary extract, injection of 17-β-estradiol, and stilboestrol mixed in the food.     

Age‐related thermal habitat use by Pacific salmon Oncorhynchus spp.

Age‐related thermal habitat use by sockeye Oncorhynchus nerka, chum Oncorhynchus keta and pink Oncorhynchus gorbuscha salmon was examined using trawl data obtained in spring in the North Pacific Ocean. Thermal habitat use differed by species and age. Larger and older fishes inhabited cooler areas, whereas smaller and younger fishes inhabited warmer areas.