New polymorphic microsatellite markers for the masu salmon (Oncorhynchus masou masou) from Korea and their application to wild and hatchery populations

Masu salmon, Oncorhynchus masou masou, is an economically important fish species in the Far East and occurs in two life history forms: sea-run migratory (anadromous) and freshwater resident (non-anadromous). The non-anadromous form has recently become a popular freshwater food and game fish during a well-known Korean winter festival. However, the genetic background of this species remains largely unknown, partly due to a lack of molecular genetic markers. In this study, we developed new polymorphic microsatellite markers for masu salmon using next-generation sequencing technology. From 40 primer sets, 11 primer sets (27.5% of the primer sets selected) were successfully amplified with 106 alleles (range 2–9) in 64 individuals from different populations: two wild and one hatchery. Observed and expected heterozygosities ranged from 0.304 to 0.947 and 0.278 to 0.865, respectively. Significant departures from the Hardy–Weinberg equilibrium were detected for four markers (OMM11, OMM17, OMM28, and OMM33) in a single population. All pair-wise F_ST values were highly significant between the wild and hatchery populations (range 0.084–0.183, P < 0.0001). We identified a set of robust microsatellite markers that worked well even in formalin-fixed samples, which will be suitable for biogeographical and population structure analyses of the masu salmon.     

Carotenoids of hemipteran insects,from the perspective of chemo-systematic and chemical ecological studies

Carotenoids of 47 species of insects belonging to Hemiptera, including 16 species of Sternorrhyncha (aphids and a whitefly), 11 species of Auchenorrhyncha (planthoppers, leafhoppers, and cicadas), and 20 species of Heteroptera (stink bugs, assassin bugs, water striders, water scorpions, water bugs, and backswimmers), were investigated from the viewpoints of chemo-systematic and chemical ecology. In aphids, carotenoids belonging to the torulene biosynthetic pathway such as β-zeacarotene, β,ψ-carotene, and torulene, and carotenoids with a γ-end group such as β,γ-carotene and γ,γ-carotene were identified. Carotenoids belonging the torulene biosynthetic pathway and with a γ-end group were also present in water striders. On the other hand, β-carotene, β-cryptoxanthin, and lutein, which originated from dietary plants, were present in both stink bugs and leafhoppers. Assassin bugs also accumulated carotenoids from dietary insects. Trace amounts of carotenoids were detected in cicadas. Carotenoids of insects belonging to Hemiptera well-reflect their ecological life histories.     

Carotenoids of dragonflies,from the perspective of comparative biochemical and chemical ecological studies

Carotenoids of 20 species of dragonflies (including 14 species of Anisoptera and six species of Zygoptera) were investigated from the viewpoints of comparative biochemistry and chemical ecology. In larvae, β-carotene, β-cryptoxanthin, lutein, and fucoxanthin were found to be major carotenoids in both Anisoptera and Zygoptera. These carotenoids were assumed to have originated from aquatic insects, water fleas, tadpoles, and small fish, which dragonfly larvae feed on. Furthermore, β-caroten-2-ol and echinenone were also found in all species of larvae investigated. In adult dragonflies, β-carotene was found to be a major carotenoid along with lutein, zeaxanthin, β-caroten-2-ol, and echinenone in both Anisoptera and Zygoptera. On the other hand, unique carotenoids, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γ-carotene, were present in both Anisoptera and Zygoptera dragonflies. These carotenoids were not found in larvae. Food chain studies of dragonflies suggested that these carotenoids originated from aphids, and/or possibly from aphidophagous ladybird beetles and spiders, which dragonflies feed on. Lutein and zeaxanthin in adult dragonflies were also assumed to have originated from flying insects they feed on, such as flies, mosquitoes, butterflies, moths, and planthoppers, as well as spiders. β-Caroten-2-ol and echinenone were found in both dragonfly adults and larvae. They were assumed to be metabolites of β-carotene in dragonflies themselves. Carotenoids of dragonflies well reflect the food chain during their lifecycle.     

Latitudinal variation in sexual size dimorphism of sea-run masu salmon, Oncorhynchus masou

Sexual size dimorphism (SSD), a difference in body size between the sexes, occurs in many animal species. Although the larger sex is often considered invariable within species, patterns of selection may result in interpopulation variation or even reversal of SSD. We evaluated correlations between latitude and female body size, male body size, and relative body size (male body size/female body size) in 22 populations (ranging from 37 degrees N to 49 degrees N) of sea-run masu salmon (Oncorhynchus masou) that spawn in rivers along the Sea of Japan coast. Male size and the relative body size increased with latitude, but female size did not correlate with latitude. In addition, increase in male size with latitude was sufficient to result in a reversal of SSD, the switch-point being around 45 degrees N. We suggest that the positive correlation between latitude and male size is due to increasing operational sex ratios or sexual selection on sea-run male body size that result from sex-biased patterns of anadromy. In conclusion, our study provides the first example of predictable geographic variation in SSD shaped by apparent patterns of sexual selection.     

Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou

Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou was examined. Relatively greater variation in egg size occurred among rivers than among females within rivers or within females. Egg size was generally greater and egg number generally lower at more northerly latitudes.     

Genetic structure of masu salmon (Oncorhynchus masou) populations in Hokkaido, northernmost Japan, inferred from mitochondrial DNA variation

Genetic structure of masu salmon Oncorhynchus masou populations in Hokkaido was examined by analysing mtDNA NADH dehydrogenase subunit 5 gene (561 bp) of 382 individuals collected from 12 rivers, in which there were no records of artificial release. Analysis of molecular variance showed that between groups level and between populations within-group level explained each c. 10% of genetic variance. In neighbour-joining tree, four populations located in southern Hokkaido were clustered into a single group; however, other populations did not form any clear clusters. Fu's F _S, Tajima's D and a mismatch distribution test indicated a sudden expansion of population in the entire population of Hokkaido and the northernmost population of Chiraibetsu, which was genetically close to the southern Hokkaido group. The Sea of Japan and southern rivers, including those of southern Hokkaido, seem to have served as refugia for masu salmon during glacial periods, and their dispersal and straying in interglacial periods affected the genetic structure of masu salmon populations in Hokkaido.     

Functional analysis of thermo-sensitive TRPV1 in an aquatic vertebrate,masu salmon (Oncorhynchus masou ishikawae)

Transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in nociceptive primary sensory neurons and acts as a sensor for heat and capsaicin. The functional properties of TRPV1 have been reported to vary among species and, in some cases, the species difference in its thermal sensitivity is likely to be associated with thermal habitat conditions. To clarify the functional properties and physiological roles of TRPV1 in aquatic vertebrates, we examined the temperature and chemical sensitivities of TRPV1 in masu salmon (Oncorhynchus masou ishikawae, Om) belonging to a family of salmonids that generally prefer cool environments. First, behavioral experiments were conducted using a video tracking system. Application of capsaicin, a TRPV1 agonist, induced locomotor activities in juvenile Om. Increasing the ambient temperature also elicited locomotor activity potentiated by capsaicin. RT-PCR revealed TRPV1 expression in gills as well as spinal cord. Next, electrophysiological analyses of OmTRPV1 were performed using a two-electrode voltage-clamp technique with a Xenopus oocyte expression system. Heat stimulation evoked an inward current in heterologously expressed OmTRPV1. In addition, capsaicin produced current responses in OmTRPV1-expressing oocytes, but higher concentrations were needed for its activation compared to the mammalian orthologues. These results indicate that Om senses environmental stimuli (heat and capsaicin) through the activation of TRPV1, and this channel may play important roles in avoiding environments disadvantageous for survival in aquatic vertebrates.     

Differences of tributyltin accumulation in the masu salmon Oncorhynchus masou between sea‐run and freshwater‐resident types

In order to examine the accumulation pattern of tributyltin (TBT) in relation to the migratory pattern, TBT concentrations in muscles and strontium (Sr) concentrations in otoliths were determined in both sea‐run and freshwater‐resident masu salmon Oncorhynchus masou . Both TBT and Sr concentrations in sea‐run masu salmon were significantly higher than those of freshwater‐residents.     

Relationship between eyed-egg percentage and levels of cortisol and thyroid hormone in masu salmon Oncorhynchus masou

A possible cause of the low eyed-egg percentage that afflicts pond-reared masu salmon Oncorhynchus masou at a fish hatchery (Kumaishi Fish Hatchery, Hokkaido, Japan) was investigated. Serum cortisol levels of masu salmon during the spawning period were compared between individuals from Kumaishi and those from another station (Mori). Cortisol, thyroxine (T₄) and triiodothyronine (T₃) hormones were also measured in eyed eggs and their levels were compared to the eyed-egg percentage. Serum cortisol levels were significantly higher at Kumaishi than at Mori in May and July of the breeding season. At the Kumaishi station, there was a linear positive relationship between serum cortisol and fertilized-egg cortisol levels of female masu salmon. As cortisol levels in both ovulating females and eyed eggs increased, the eyed-egg percentage decreased, indicating a highly significant negative relationship between cortisol levels and eyed-egg percentage. In contrast, as T₃ and T₄ levels in eyed eggs increased, the eyed-egg percentage increased. The strong positive correlation between high cortisol levels in serum and in eyed eggs at Kumaishi indicates that the frequently higher cortisol levels may have caused the lowering of the eyed-egg percentage.     

Dispersal of wild and domestic masu salmon fry (Oncorhynchus masou) in an artificial channel

The day and night pattern of upstream and downstream dispersal of masu salmon fry of wild and domestic origin was compared in artificial channels (45 m long), for two ages of planting: unfed alevins and eyed eggs. Early dispersal was important for the wild stock (48–50%) compared with the domestic one (16–36%). More wild fry moved downstream than upstream, and more domestic fry dispersed upstream. Upstream movement in wild and domestic fry was more active by day than by night, except for wild fry planted as eyed eggs, where upstream migration was higher at night. In contrast, downstream movement in wild and domestic fry was more common by night than by day, but daylight catches were not negligible for the wild stock.     

Phylogenetic position and genetic diversity of masu salmon Oncorhynchus masou in the Mijiang River,China

Ichthyological Research -     

Comparison of the frequency of the growth hormone pseudogene between juvenile and adult female masu salmon Oncorhynchus masou

The proportions of individual masu salmon Oncorhynchus masou whose genotypic and phenotypic sex differed were compared among juvenile and adult fish in three rivers, and genotypically male but phenotypically female individuals were observed 6–16% more often among adults than among juveniles. This suggests that during the transition from juvenile to adult, survival rates of genotypically male but phenotypically female individuals are higher than those of normal females. In contrast, genotypically female but phenotypically male individuals were only found in the juvenile period, which suggests that they exhibit a decreased survival rate in comparison with normal males.     

Ascending speed and nocturnal activity of adult masu salmon (Oncorhynchus masou Brevoort) during upstream migration

We released five adult masu salmon (Oncorhynchus masou) tagged with external transmitters to track their ascending behaviour. The signals of all specimens were recorded in the upper area of the river system. Two patterns of ascending behaviour were recognized: ascending upward immediately after release and ascending during increased river discharge. The fastest ascending speed was about 1000 m h^–1. Active movements were detected at night. The signal recording duration at each receiver for each fish was generally brief. Most fish did not stay at the pools where the receivers were installed.     

Changes in melatonin binding sites under artificial light-dark, constant light and constant dark conditions in the masu salmon brain

To test whether the affinity (Kd) and total binding capacity (Bmax) of melatonin receptors exhibit daily and circadian changes in teleost fish whose melatonin secretion is not regulated by intra-pineal clocks, we examined the changes in melatonin binding sites in the brains of underyearling masu salmon Oncorhynchus masou under artificial light-dark (LD), constant light (LL) and constant dark (DD) conditions. In Experiment 1, fish were reared under a long (LD 16:8) or short (LD 8:16) photoperiod for 69 days. Blood and brains were sampled eight times at 3 h intervals. Plasma melatonin levels were high during the dark phase and low during the light phase in both photoperiodic groups. The Bmax exhibited no daily variations. Although the Kd slightly, but significantly, changed under LD 8:16, this may be of little physiological significance. In Experiment 2, fish reared under LD 12:12 for 27 days were exposed to LL or DD from the onset of the dark phase under LD 12:12. Blood and brains were sampled 13 times at 4 h intervals for two complete 24 h cycles. Plasma melatonin levels were constantly high in the DD group and low in the LL group. No significant differences were observed in the Kd and the Bmax between the two groups, and the Kd and the Bmax exhibited no circadian variation either in the LL or DD groups. These results indicate that light conditions have little effect on melatonin binding sites in the masu salmon brain.     

Molecular cloning and characterization of three distinct choriogenins in masu salmon, Oncorhynchus masou

Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg Halpha and Chg Hbeta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmHalpha and rmHbeta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high-molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg Halpha and Chg Hbeta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.     

Ultrastructure of the retinal pigment epithelium in the young masu salmon Oncorhynchus masou

The ultrastructure of retinal pigment epithelium (RPE) cells in the masu salmon Oncorhynchus masou fry was studied. The physiological state of the pigment was dependent on the level of light/dark adaptation. Similar morphological properties were observed in the response of the RPE cells to light and a magnetic field.     

Effects of stocking hatchery fish on the phenotype of indigenous populations in the amago salmon Oncorhynchus masou ishikawae in Japan

The expression of colour marks (parr marks, red and black spots) of the amago salmon Oncorhynchus masou ishikawae was compared with microsatellite information, to see the effects of stocking hatchery fish on the phenotype of indigenous populations, which face extinction through extensive stocking. A Bayesian-based assignment test suggested introgression of two exotic clusters into one indigenous cluster in the stocked area and its vicinity. The number of parr marks was significantly higher in one hatchery-origin population, which exclusively comprised one exotic cluster. An increased number of red spots in stocked hatchery fish was probably a consequence of hatchery feeding conditions. The number of black spots was correlated with body size in many populations, except for hatchery and heavily introgressed populations. Coefficients of correlation and regression of black spots with body size, which were largest in indigenous populations, decreased with an increase of introgression by hatchery fish. As indigenous populations have low genetic diversity with high relatedness, it was inferred that the height of correlation and regression coefficients in black spots is caused by high genetic homogeneity and fixation of alleles in loci related to the increase of black spots, both of which might have collapsed with introgression by hatchery fish. These results suggest the possibility that introgression by stocked fish causes a change of phenotype in indigenous populations.     

Spatial distribution of juvenile masu salmon (Oncorhynchus masou) with incongruent genotypic and phenotypic sex in Hokkaido, Japan

To clarify the spatial distribution of masu salmon Oncorhynchus masou with incongruent genotypic and phenotypic sex, we compared the phenotypic sex determined by gonad analysis with the genotypic sex identified by a male-specific genetic marker, Growth hormone pseudogene (GHp), in 584 individuals inhabiting 23 rivers in Hokkaido, Japan. We found that the genotypic sex is different from the phenotypic sex for 39 individuals in 10 rivers. Among them, 35 individuals (89.7?%) were GHp?+?females phenotypically. The opposite feature was noted in the remaining four individuals (10.3?%). This study also revealed that GHp?+?phenotypic females are distributed mainly in the Sea of Japan, whereas GHp- phenotypic male tend to appear in the Sea of Okhotsk. In addition, there was a significant negative correlation between the distance from the Bishabetsu River, where frequency of GHp?+?phenotypic females was the highest, and the frequency of those individuals in each river. These results suggest that the distribution of masu salmon with incongruent genotypic and phenotypic sex would be related to the movement of individuals among the populations and regions.     

Restricted movement of the fluvial form of red-spotted masu salmon,Oncorhynchus masou rhodurus,in a mountain stream,Central Japan

Movement of the fluvial form of red-spotted masu salmon (1⁺ and older),Oncorhynchus masou rhodurus, was studied using mark-recapture methods in a Japanese mountain stream. Most (63–91%) adult salmon were recaptured in the pool in which they were marked. The rest of the salmon moved upstream or downstream <20m during the non-breeding period. The proportion of the salmon moving increased slightly during the breeding period, but did not exceed 66%. The distance moved was also more variable during this period. The proportion of the smaller salmon which moved was larger than that of the larger fish during the non-breeding period. Conversely, during the breeding period, larger fish moved more frequently. Sedentary behaviour and local movements of adult salmon seem to be affected by their social relationships.