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.     

Occurrence and genetic typing of infectious hematopoietic necrosis virus in Kamchatka, Russia

Infectious hematopoietic necrosis virus (IHNV) is a well known rhabdoviral pathogen of salmonid fish in North America that has become established in Asia and Europe. On the Pacific coast of Russia, IHNV was first detected in hatchery sockeye from the Kamchatka Peninsula in 2001. Results of virological examinations of over 10,000 wild and cultured salmonid fish from Kamchatka during 1996 to 2005 revealed IHNV in several sockeye salmon Oncorhynchus nerka populations. The virus was isolated from spawning adults and from juveniles undergoing epidemics in both hatchery and wild sockeye populations from the Bolshaya watershed. No virus was detected in 2 other watersheds, or in species other than sockeye salmon. Genetic typing of 8 virus isolates by sequence analysis of partial glycoprotein and nucleocapsid genes revealed that they were genetically homogeneous and fell within the U genogroup of IHNV. In phylogenetic analyses, the Russian IHNV sequences were indistinguishable from the sequences of North American U genogroup isolates that occur throughout Alaska, British Columbia, Washington, and Oregon. The high similarity, and in some cases identity, between Russian and North American IHNV isolates suggests virus transmission or exposure to a common viral reservoir in the North Pacific Ocean.     

Geographical and host distribution patterns of Parvicapsula minibicornis (Myxozoa) small subunit ribosomal RNA genetic types

Parvicapsula minibicornis is a myxozoan parasite implicated in mortalities of both juvenile and pre-spawning adult salmon in the Pacific Northwest of North America. Disease severity and presentation varies between salmon species and geographical localities. To better characterize population structure of the parasite, we sought genetic markers in the P. minibicornis ribosomal RNA gene. We compared samples from California with the type specimen from British Columbia, identified sequence variations, and then sequenced 197 samples from fish, river water and the parasite's polychaete worm host. Although DNA sequences of the parasite were >98·9% similar, there was enough variation to define 15 genotypes. All genotypes were detected in fish samples, although not in all species. A single genotype only was found in sockeye and pink salmon in the Fraser River Basin, but was not detected in sockeye from the adjacent Columbia River Basin. All coho salmon, irrespective of river basin, were infected with a unique mix of 2 genotypes. These data indicated that the P. minibicornis population exhibited strong signals of structuring by both geography and salmonid host species. Particular genotypes may correlate with disease differences seen in salmon populations in the Pacific Northwest.     

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.     

Comparison of small subunit ribosomal RNA gene and internal transcribed spacer sequences among isolates of the intranuclear microsporidian Nucleospora salmonis

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.     

The genetic population structure of lacustrine sockeye salmon, <Emphasis Type="Italic">Oncorhynchus nerka,</Emphasis> in Japan as the endangered species

Lacustrine sockeye salmon (Oncorhynchus nerka) are listed as an endangered species in Japan despite little genetic information on their population structure. In order to clarify the genetic diversity and structure of Japanese populations for evaluating on the bottleneck effect and an endangered species, we analyzed the ND5 region of mitochondrial DNA (mtDNA) and 45 single nucleotide polymorphisms (SNPs) in 640 lacustrine sockeye salmon in Japan and 80 anadromous sockeye salmon in Iliamna Lake of Alaska. The genetic diversity of the Japanese population in both mtDNA and SNPs was significantly less than that of the Iliamna Lake population. Moreover, all Japanese populations had SNP loci deviating from the HWE. In spite of low genetic diversity, the SNP analyses resulted that the Japanese population was significantly divided into three groups. These suggest that Japanese sockeye salmon populations should be protected as an endangered species and genetically disturbed by the hatchery program and transplantations.     

Molecular and morphological characterization of the willow rust fungus, Melampsora epitea, from arctic and temperate hosts in North America

Current taxonomy places all rust fungi that occur on willow (Salix spp.) in North America in one species complex, Melampsora epitea Thüm. Characteristics of M. epitea isolates from the Canadian arctic were compared to M. epitea isolates from temperate regions of North America. Sequences from internal transcribed spacer (ITS) regions of rDNA were obtained from urediniospores from rust-infected Salix leaves collected in the Canadian arctic and in Minnesota and compared. Phylogenetic analysis of nuclear ribosomal ITS regions indicated that arctic M. epitea samples were divergent from temperate M. epitea isolates, perhaps in part because all rusts examined diverged according to host species. Four urediniospore characteristics were examined: area, circularity (shape factor), major axis length and spine density. Statistically significant (P < 0.05) differences were observed for spine density among all host species except S. nigra and S. bebbiana. However major axis length differed between these species. These results represent the first evidence that arctic and temperate Melampsora species on Salix hosts in North America have evolved distinct molecular and morphological characters.     

Strategies for the conservation and management of isolated salmonid populations: lessons from Japanese streams

1. Endangered native populations of stream salmonids in Japan face three major threats: (i) negative interactions with introduced hatchery‐reared fish, (ii) fragmentation of habitat by impassable dams and (iii) recreational angling. 2. To prevent imminent extinction of many local populations, we evaluated these threats and possible conservation actions for red‐spotted masu salmon (Oncorhynchus masou ishikawae) and white‐spotted charr (Salvelinus leucomaenis japonicus) in the Fuji River system in central Japan. 3. Red‐spotted masu salmon and white‐spotted charr occupied only 0.73 and 2.4% of suitable thermal habitats, respectively, with masu salmon typically occupying habitats closer to human population centres. 4. Population viability analysis resulted in a 100‐year probability of extinction of 78.1% for masu salmon and 48.1% for charr. However, extinction risk of both species was predicted to be <5% if the carrying capacity increased from 141 to 303 for masu salmon and from 94 to 125 for charr, by allowing fish passage at the lower end of the habitat, and if annual adult survival rate increased by 0.04. Adult survival rate was the principal factor associated with population persistence. 5. To conserve isolated populations of stream‐dwelling salmonids, we recommend (i) assessing the distribution of remnant native and non‐native fish populations, (ii) that fishing regulations are modified to improve adult survival and population persistence and (iii) that fragmented reaches be reconnected to adjacent habitat, for example by removing or modifying artificial barriers to increase the carrying capacity of the isolated populations. Reconnection of fragmented reaches should, however, be avoided if it results in non‐native fish invading isolated populations.     

Effects of the brain parasite Myxobolus arcticus on sockeye salmon

Parasitism with Myxobolus arcticus did not affect smolt size of sockeye salmon or their osmocompetence, but had a deleterious effect ( P <0.001) on the swimming speed of naturally infected smolts. Parasitized fish had a mean swimming speed of 2.89 fork length s⁻¹ (L_F s⁻¹) compared with 4.37 L _F s⁻¹ for unparasitized fish. The parasite probably impairs swimming ability by affecting the central nervous system, but this effect does not appear severe enough to limit the parasite's usefulness in stock separation.     

Distribution,prevalence and severity of Parvicapsula minibicornis infections among anadromous salmonids in the Fraser River,British Columbia,Canada

Polymerase chain reaction (PCR) and microscopic examination of stained kidney sections were used to diagnose infections with the myxozoan parasite Parvicapsula minibicornis in maturing Fraser River salmon. In 2 series of collections, the parasite was detected in 109 of 406 migrating sockeye salmon Oncorhynchus nerka belonging to Early Stuart, Early Summer and Summer run-timing groups, mainly upper Fraser River stocks. However, the parasite was detected neither in fish at sea nor once they had migrated several 100 km upstream. Prevalence then increased to 95% or greater at the spawning grounds. Histological examination of kidney was less sensitive than PCR in detecting the parasite in salmon collected from the earliest sites in both collections found positive by PCR. Severity of infection was greatest at the spawning grounds. Development of infection in sockeye, measured by prevalence, severity or by the rate of false-negative histological diagnoses, appeared to be a useful estimate of in-river residence time. Prevalence and severity of infections in sequential samples of Harrison River and Weaver Creek sockeye stocks collected from the Harrison River indicated that more time had elapsed since parasite transmission than would be predicted based on migration distance alone. Pink salmon Oncorhynchus gorbuscha, coho salmon O. kisutch and chinook salmon O. tshawytscha were found to be infected with the parasite. Development of P. minibicornis in pink salmon was most similar to that in sockeye. Pink and coho salmon may be at risk to the pathological consequences of P. minibicornis infection.     

Isolation of Aeromonas salmonicida from masu salmon in the Republic of Korea

The isolation and identification of the Gram-negative fish pathogen, Aeromonas salmonicida salmonicida, from masu salmon (Oncorhynchus masou) at the Yang Yang Salmon Hatchery in the Republic of Korea is described. The bacterium possessed certain characteristics in common with isolates from fish in Japan. It is an important cause of mortality among cultured salmonids in the Republic of Korea. This is the first report of this fish pathogen from the Asian mainland.     

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.     

Age composition of masu salmon smolts in northern Japan

Smolts of anadromous masu salmon Oncorhynchus masou aged 3+ years were found in a northern Japanese river. This is the first recording of 3+ year smolts in Japan. These fish appeared to originate in the cold upper river where 2+ year parr were found during autumn.     

MOLECULAR GENETIC EVIDENCE FOR PARALLEL LIFE-HISTORY EVOLUTION WITHIN A PACIFIC SALMON (SOCKEYE SALMON AND KOKANEE,ONCORHYNCHUS NERKA)

The Pacific salmon Oncorhynchus nerka typically occurs as a sea-run form (sockeye salmon) or may reside permanently in lakes (kokanee) thoughout its native North Pacific. We tested whether such geographically extensive ecotypic variation resulted from parallel evolutionary divergence thoughout the North Pacific or whether the two forms are monophyletic groups by examining allelic variation between sockeye salmon and kokanee at two minisatellite DNA repeat loci and in mitochondrial DNA (mtDNA) Bgl II restriction sites. Our examination of over 750 fish from 24 populations, ranging from Kamchatka to the Columbia River, identified two major genetic groups of North Pacific O. nerka: a “northwestern” group consisting of fish from Kamchatka, western Alaska, and northwestern British Columbia, and a “southern” group consisting of sockeye salmon and kokanee populations from the Fraser and Columbia River systems. Maximum-likelihood analysis accompanied by bootstrapping provided strong support for these two genetic groups of O. nerka; the populations did not cluster by migratory form, but genetic affinities were organized more strongly by geographic proximity. The two major genetic groups resolved in our study probably stem from historical isolation and dispersal of O. nerka from two major Wisconsinan glacial refugia in the North Pacific. There were significant minisatellite DNA allele frequency differences between sockeye salmon and kokanee populations from different parts of the same watershed, between populations spawning in different tributaries of the same lake, and also between sympatric populations spawning in the same stream at the same time. MtDNA Bgl II restriction site variation was significant between sockeye salmon and kokanee spawning in different parts of the same major watershed but not between forms spawning in closer degrees of reproductive sympatry. Patterns of genetic affinity and allele sharing suggested that kokanee have arisen from sea-run sockeye salmon several times independently in the North Pacific. We conclude that sockeye salmon and kokanee are para- and polyphyletic, respectively, and that the present geographic distribution of the ecotypes results from parallel evolutionary origins of kokanee from sockeye (divergences between them) thoughout the North Pacific.     

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.     

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

Baltic salmon activates immune relevant genes in fin tissue when responding to Gyrodactylus salaris infection

Immune mechanisms in 2 strains of Salmo salar (Baltic salmon from River Ume Alv in Sweden and East Atlantic salmon from River Skjern? in Denmark) infected with the monogenean ectoparasite Gyrodactylus salaris were elucidated by molecular tools (real-time PCR). The gene expression in the fins (the preferred microhabitat of the parasite) of the susceptible but responding Swedish salmon was compared to the expression in the fins of the highly susceptible and nonresponding East Atlantic salmon. Experimental infections confirmed that both the Swedish and the Danish salmon allowed initial propagation of the parasite on the fins for a few weeks. Baltic salmon subsequently activated a response from Day 28 and limited the parasite population to a few parasites per host within the following weeks. In contrast, the Danish salmon did not respond and experienced a continuing increase in the parasite load during the same period, which reached several hundreds of parasites per host. RNA was isolated from fins of the 2 salmon strains during the course of infection and subsequent real-time PCR showed an increased expression of INFgamma, Mx and MHC I genes in Baltic salmon fins during large segments of the response phase. No upregulation of these genes could be detected in susceptible salmon. No increase in immunoglobulin genes was seen in any of the fish strains, which supports the notion that antibodies are not involved in the response. Further, the work suggests that cellular factors could at least partly contribute to the anti-parasitic response in Baltic salmon.     

Observations on saprolegniasis of adult sockeye salmon, Oncorhynchus nerka (Walbaum)

Some observations on the mycology and pathology of saprolegniasis of maturing sockeye salmon are reported. Saprolegnia spp. were isolated from all lesions examined. Some isolates did not produce oogonia and could not be identified further. The other isolates have affinities to the S. diclina-S. parasitica complex. Observations on the gross and histo-pathology of the lesions show the fungi can be active pathogens. It is hypothesized that increased levels of plasma corticosteroids and depletion of ascorbic acid reserves in the fish increase the probability that Saprolegnia spp. will initiate infections, either alone or concurrently with other opportunistic parasites.     

Fecundity and egg size variation in North American Pacific salmon (Oncorhynchus)

We examined regional and latitudinal variation in fecundity and egg weight for five species of Pacific salmon ( Oncorhynchus ) along the Pacific coast of North America. Data were examined for 24 chum salmon, 15 pink salmon, 34 sockeye salmon, 44 chinook salmon, and 40 coho salmon populations from published sources, unpublished Canadian hatchery records, our own laboratory investigations, and other unpublished sources. Substantial regional variation in fecundity and egg weight was observed, with salmon on the Queen Charlotte Islands and Vancouver Island in British Columbia generally having lower fecundity and larger egg size than nearby mainland populations. The relative distance of freshwater migration to the spawning grounds generally had a marked effect on both fecundity and egg size, with populations spawning in the upper portions in the drainages of large rivers like the Fraser River in British Columbia having reduced fecundity and egg size compared with coastal spawning populations. Fecundity was generally higher and egg size generally lower in more northern populations of sockeye, chinook, and coho salmon compared with southern ones. We suggest that egg size tends to be lower in northern populations of some species as a result of increased fecundity due to their older ages at maturity and a limited amount of energy that can be expended on egg production.