Masou Research Articles

Identification and characterization of pathogenic bacteria isolated from dead masu salmon Oncorhynchus masou masou and antibacterial activity of probiotic Lactococcus lactis subsp. lactis strain K-C2 against isolated pathogens

Identification and characterization of pathogenic bacteria isolated from dead masu salmon Oncorhynchus masou masou and antibacterial activity of probiotic Lactococcus lactis subsp. lactis strain K-C2 against isolated pathogens

Winter gifts for river ecosystems: A massive supply of earthworms in early winter.

Terrestrial resource pulses can significantly affect the community dynamics of freshwater ecosystems. Previously, its effect on the river community is considered to be stronger in summer, whereas weaker in winter when terrestrial invertebrates are less abundant. The movement of the terrestrial earthworms is triggered in winter, so they may be supplied to winter rivers as terrestrial resource pulse, but little is known about it. Here, we report that the massive numbers of the terrestrial earthworms were supplied intensively to an upstream of the small river in early winter. In particular, we found large numbers of megascolecid earthworms were supplied in an upstream of the small river in Northern Japan. Furthermore, we observed that supplied earthworms were consumed by salmonid fish species (masu salmon, white spotted char and rainbow trout) and aquatic invertebrates (gammarid amphipod, planarian flatworm, and stonefly larvae). These findings suggest that the terrestrial earthworms may play a key role in ecosystem functioning in winter when severe and other resources are scarce.

Cardiac hemodynamics and ventricular stiffness of sea-run cherry salmon (Oncorhynchus masou masou) differ critically from those of landlocked masu salmon.

Ventricular diastolic mechanical properties are important determinants of cardiac function and are optimized by changes in cardiac structure and physical properties. Oncorhynchus masou masou is an anadromous migratory fish of the Salmonidae family, and several ecological studies on it have been conducted; however, the cardiac functions of the fish are not well known. Therefore, we investigated ventricular diastolic function in landlocked (masu salmon) and sea-run (cherry salmon) types at 29-30 months post fertilization. Pulsed-wave Doppler echocardiography showed that the atrioventricular inflow waveforms of cherry salmon were biphasic with early diastolic filling and atrial contraction, whereas those of masu salmon were monophasic with atrial contraction. In addition, end-diastolic pressure-volume relationship analysis revealed that the dilatability per unit myocardial mass of the ventricle in cherry salmon was significantly suppressed compared to that in masu salmon, suggesting that the ventricle of the cherry salmon was relatively stiffer (relative ventricular stiffness index; p = 0.0263). Contrastingly, the extensibility of cardiomyocytes, characterized by the expression pattern of Connectin isoforms in their ventricles, was similar in both types. Histological analysis showed that the percentage of the collagen accumulation area in the compact layer of cherry salmon increased compared with that of the masu salmon, which may contribute to ventricle stiffness. Although the heart mass of cherry salmon was about 11-fold greater than that of masu salmon, there was no difference in the morphology of the isolated cardiomyocytes, suggesting that the heart of the cherry salmon grows by cardiomyocyte proliferation, but not cell hypertrophy. The cardiac physiological function of the teleosts varies with differences in their developmental processes and life history. Our multidimensional analysis of the O. masou heart may provide a clue to the process by which the heart acquires a biphasic blood-filling pattern, i.e., a ventricular diastolic suction.

A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences.

CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon (Oncorhynchus masou) elovl2 gene and rabbitfish (Siganus canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust target KI of elovl2 and Δ6 fad genes at multiple sites of LRS1 and LRS3, respectively, in the initial generation. This demonstrated that donor plasmid homology arms, which were nearly identical but not completely the same as the genome sequence, still led to on-target KI. Although the target KI efficiencies at LRS1, LRS2, and LRS3 sites were still relatively low in the current study, it is very promising that 100% KI efficiency in the future could be realized and perfected by selection of better LRSs and optimization of sgRNAs.

Recovery of white-spotted charr Salvelinus leucomaenis following the removal of stocked red-spotted masu salmon Oncorhynchus masou ishikawae in a small headwater tributary of Lake Biwa, central Japan

Recovery of white-spotted charr Salvelinus leucomaenis following the removal of stocked red-spotted masu salmon Oncorhynchus masou ishikawae in a small headwater tributary of Lake Biwa, central Japan

Domestication of captive-bred masu salmon Oncorhynchus masou masou (Salmonidae) leads to a significant decrease in numbers of lateral line organs

Because captive-bred animals gradually adapt to artificial rearing environments due to evolving life history traits, such individuals sometimes show lessened performance in natural environments. The lateral line system, one of the principal sensory organs of fishes, varies according to habitat environments, sometimes differing even within the same species. A reduction in lateral line elements may also occur in successive generations of captive-bred fish. Such a reduction, involving neuromasts over the entire body, was examined for the first time in captive-bred masu salmon Oncorhynchus masou masou. The total number of neuromasts in captive-bred fish was ca. 10% lower than in wild-caught and F1 fishes, suggesting that the system in captive-bred fish had reduced in number due to domestication. Furthermore, differences in total neuromast numbers between captive-bred and wild fish were greater than between anadromous and fluvial populations of the species. The lower number of neuromasts could be one of the reasons behind the lower survival of captive-bred fish in natural environments.

Feeding of mass fish species by natural and farmed juveniles of pacific salmons on example of the Obutonai River (Sakhalin Island)

Feeding of mass fish species caught in the Obutonai River (Sakhalin, Nevelsk district) was examined in May-July 2021, in the time of downstream migration of natural pink salmon Oncorhynchus gorbuscha juveniles and release of chum salmon O. keta juveniles from a fish hatchery. Three fish species capable to predate on salmon juveniles were presented in the catches in significant number, as whitespotted char Salvelinus leucomaenis, pacific redfin Tribolodon brandtii, and juveniles and dwarf males of masu salmon O. masou. These species occurred in different lower reaches of the river. Whitespotted char fed by both pink and chum juveniles, but was relatively few in number (single mass catch). Two-year-olds and dwarf males of masu salmon fed less intensively and pacific redfin fed exceptionally rarely on the migrating pink salmon juveniles of natural origin, but both these species consumed actively the chum juveniles released from a hatchery. The masu salmon juveniles had the maximum abundance in early June, then their number decreased. The pacific redfin abundance had complicated dynamics because of migrations both from the upper part of the river and from the sea; pacific redfin spawned in late June when ate exclusively eggs of the same species.

Juvenile semi-wild fish have a higher metabolic rate than farmed fish

Fish from commercially farmed stocks are often released into the natural environment to supplement wild populations. This practice is often applied to salmonid fish as they are an essential fishery resource and also used for recreational angling. However, farmed fish tend to show lower survival rates after release than wild fish. For this reason, the release of semi-wild fish is increasingly used in Japan; these fish are generated using female fish from domesticated stocks and male fish of wild origin. The survival rate of released semi-wild fish is higher than that of farmed fish, but the reason for this is unknown. This study compared the metabolism and swimming performance of semi-wild and farmed masu salmon (Oncorynchus masou). The analyses showed that resting metabolic rate (RMR), maximum metabolic rate (MMR) and swimming speeds that minimize energy costs of travel (optimal swimming speed) were higher in semi-wild fish than in farmed fish. Critical swimming speed did not differ significantly between the two groups of fish. Semi-wild fish with high RMR may have a social status advantage over farmed fish because a previous study reported that SMR, which is the value closest to basal metabolism significantly affects feeding motivation. This means that individuals with higher social status may be more motivated to feed. As RMR is proportional to food requirements, then release programs should be planned taking food resources at the release site into consideration.

Regional differences of temporal trends in masu salmon coastal catch

Regional differences of temporal trends in masu salmon coastal catch

DISTRIBUTION OF IODINE-127 IN MARINE ORGANISMS FROM COASTAL WATERS AROUND AOMORI, JAPAN.

We measured the concentrations and determined the distribution of the stable isotope 127I in the marine organisms. Marine fish were collected from the Pacific Ocean near Aomori, Japan. The mean 127I concentrations in the muscle of marine fish ranged from 0.67μgg-1-wet weight (ww) in cherry salmon to 0.84μgg-1-ww in fat greenling. Among the tissues measured, the muscle showed the lowest levels of 127I in all fish species. The highest 127I concentration in the tissues of marine fish was observed in the ovary of fat greenling. The mean 127I concentration in the of Japanese scallop ranged from 0.06μgg-1-ww in adductor muscle to 5.49μgg-1-ww in mid-gut gland. The 127I concentrations in seaweeds were 67μgg-1-dry weight (dw) in sea lettuce and 1783μgg-1-dw in kombu. Thus, the distribution of 127I concentration in marine organisms varied considerably depending on the tissue.

Pulsed supplies of small fish facilitate time-limited intraguild predation in salmon-stocked streams.

Pulsed supplies of prey generally increase predator food intake. However, it is unclear whether this holds true when predators and pulsed prey are in the same guild (i.e. intraguild (IG) predators and prey). IG prey may increase IG-predator food intake through predation, but they may decrease food intake through competition. To test these hypotheses, we compared the food intake of white-spotted charr (Salvelinus leucomaenis) (IG predator) in streams that were stocked or unstocked with masu salmon (Oncorhynchus masou) fry (IG prey) in streams in Hokkaido, Japan. One day after stocking, mean stomach content weight of charr was six times higher than in unstocked streams due to fry consumption. In particular, large charr showed intense piscivory. However, predation on fry was rare after about three weeks. Some factors that could explain this time-limited IG predation include the growth and decreasing abundance of fry over time and the acquisition of predator-avoidance behaviour. In days other than the first-day post-stocking, food intake by charr did not differ between stocked and unstocked streams. No effects of interspecific competition on charr food intake were observed.

Factors contributing to anglers’ satisfaction and their requests concerning recreational salmonid fisheries management: Insights from a questionnaire survey in strictly regulated, catch-and-release stream fishing areas

Regional fisheries managers need to consider anglers’ satisfaction and requests and balance them with resource conservation when making decisions to promote the sustainability of recreational fisheries. However, there is a marked paucity of information on inland recreational fisheries, particularly in non-Western countries. This study was designed to gain insight on the factors contributing to the anglers' satisfaction and their requests concerning salmonid recreational fisheries management. The approach used was a questionnaire survey of salmonid anglers in two strictly regulated, reservation-only, catch-and-release stream fishing areas in Japan. We evaluated the relationships among anglers’ characteristics and the fishing experience with their satisfaction degree. We also investigated the anglers’ requests concerning the use of fishing license proceeds. The catch numbers of the larger masu salmon available in the area (e.g., 20–40 cm standard length) were positively related to the satisfaction degree of anglers. This study confirms the findings on the determinants of anglers’ satisfaction in Western countries, especially concerning avid anglers. The highest priority for using the fishing license proceeds was for enhancing the number of fish and conserving the fish habitats. Surprisingly, enhancing the number of large fish received the lowest priority among the requests. These results suggest that anglers’ requests for recreational fisheries management do not necessarily reflect their preference to catch more and larger fish. Thus, reflecting only the anglers’ requests and discontinuing stocking the special areas with large fish could paradoxically reduce the anglers’ satisfaction. These results strongly point out that understanding the factors contributing to anglers' satisfaction is crucial to avoid misunderstandings and make sounder and more sustainable recreational fisheries management decisions.

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.

Effect of paternal life-history form and egg size on offspring life-history traits in masu salmon Oncorhynchus masou

Effect of paternal life-history form and egg size on offspring life-history traits in masu salmon Oncorhynchus masou

CRISPR/Cas9 - mediated knock-in method can improve the expression and effect of transgene in P1 generation of channel catfish (Ictalurus punctatus)

Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies.

Cryopreservation of masu salmon sperm using glucose-methanol extender and seminal plasma biomarkers related to post-thaw sperm motility

The aim of this study was to evaluate the cryopreservation of masu salmon (Oncorhynchus masou) semen, using 0.18 M glucose in 9% methanol as an extender, and to characterize semen parameters (sperm motility; sperm concentration; and seminal plasma parameters such as pH, electric conductivity, DNA concentration, and protein concentration), with respect to post-thaw sperm motility. Semen samples were collected from fishes in the middle of the spawning season, at Toya Lake station, Hokkaido University, and the end of the spawning season, at the Nanae freshwater station, Hokkaido University. The semen was diluted with the extender at a 1:5 ratio (semen:extender) and then equilibrated for 15 min. The final concentration was 0.15 M and 7.5% for glucose and methanol, respectively. Sperm motility was assessed for fresh, fresh-diluted, and post-thaw semen using computer-assisted sperm analysis. The post-thaw sperm showed lower motility, compared with those of fresh and fresh-diluted sperm, which ranged from 33.9% to 82.9% and 1.5% to 27.1% in the semen collected in the middle and end the spawning season, respectively. Sperm concentration, pH, and electric conductivity of the semen collected at the end of the spawning season were lower than those of semen collected in the middle of the spawning season. Moreover, remarkably high DNA concentrations were detected in the semen at the end of the spawning season, although no difference was observed in protein concentration. Finally, semen cryopreserved using the aforementioned method could be used for artificial fertilization to produce offspring. These results indicate that the glucose-methanol extender is useful for sperm cryopreservation in masu salmon and that the suitability of semen for cryopreservation is affected by the condition of the parental fish.

A novel ND1 mitochondrial DNA mutation is maternally inherited in growth hormone transgenesis in amago salmon (Oncorhynchus masou ishikawae)

Growth hormone (GH) transgenesis can be used to manipulate the growth performance of fish and mammals. In this study, homozygous and hemizygous GH-transgenic amago salmon (Oncorhynchus masou ishikawae) derived from a single female exhibited hypoglycemia. Proteomic and signal network analyses using iTRAQ indicated a decreased NAD+/NADH ratio in transgenic fish, indicative of reduced mitochondrial ND1 function and ROS levels. Mitochondrial DNA sequencing revealed that approximately 28% of the deletion mutations in the GH homozygous- and hemizygous-female-derived mitochondrial DNA occurred in ND1. These fish also displayed decreased ROS levels. Our results indicate that GH transgenesis in amago salmon may induce specific deletion mutations that are maternally inherited over generations and alter energy production.

Occurrence of a Skin Parasite <i>Argulus coregoni</i> (Branchiura: Argulidae) on Salmonids in Mountain Streams, Central Japan, with Discussion on Its Longitudinal Distribution and Host Utilization in Rivers

Argulus coregoni Thorell, 1864 was collected from white-spotted char, Salvelinus leucomaenis (Pallas, 1814), red-spotted masu salmon, Oncorhynchus masou ishikawae Jordan and McGregor, 1925, masu salmon, O. m. masou (Brevoort, 1856), and a hybrid between white-spotted char and masu salmon in mountain streams, Gifu Prefecture, central Japan. The host’s body surface under and near the pectoral fins was the most common infection site for A. coregoni. The infected white-spotted char were caught at high elevations (461–873 m) in the headwater streams. The infected individuals of the two masu salmon subspecies were mostly caught in the upper river reaches, but the elevations where they were caught were lower (237–733 m and 660–707 m, respectively) than those of the white-spotted char. Since A. coregoni is also known as a parasite of ayu, Plecoglossus altivelis altivelis (Temminck and Schlegel, 1846), in the middle and lower river reaches in the prefecture, this parasite utilizes different fish species as its hosts along a river course: white-spotted char and the two masu salmon subspecies serve as the hosts, respectively, in the headwater and the middle to lower sections of the upper reaches, but in the mid- and lower river reaches, ayu is the important host.

Length–weight relations of ten freshwater fish species (Actinopterygii) from Abashiri River basin, eastern Hokkaido, Japan

Length–weight relations (LWRs) were estimated for ten freshwater fish species such as gin-buna, Carassius langsdorfii Temminck et Schlegel, 1846; lake minnow, Rhynchocypris percnura (Pallas, 1814); Siberian loach, Barbatula toni (Dybowski, 1869); Japanese smelt, Hypomesus nipponensis McAllister, 1963; masu salmon, Oncorhynchus masou (Brevoort, 1856); rainbow trout, Oncorhynchus mykiss (Walbaum, 1792); whitespotted char, Salvelinus leucomaenis (Pallas, 1814); ninespine stickleback, Pungitius pungitius (Linnaeus, 1758); hana-kajika, Cottus nozawae Synder, 1911; and a species of goby Rhinogobius sp. Specimens were collected once a month except in the snow season from the Abashiri River basin, eastern Hokkaido, between June 2007 and November 2011. Fishes were captured by an electrofishing device (Smith–Root Inc., Model 12-b). The estimated allometric coefficient b values ranged from 2.790 (ninespine stickleback) to 3.294 (hana-kajika), and r2 values ranged from 0.772 (lake minnow) to 0.994 (goby). All the LWRs were highly significant, with P < 0.001. Besides, the study provides the first estimates of LWRs for the Siberian loach, Japanese smelt, masu salmon, whitespotted char, hana-kajika, and the goby.

To the question of the efficiency young pacific salmon at fish farms of the Sakhalin region

Effectiveness of the salmon juveniles feeding was examined during six cycles of fish breeding for pink salmon Oncorhynhus gorbuscha, chum salmon O. keta, cherry salmon O. masou, and coho salmon O. kisutsh at 12 fish hatcheries belonged to the Sakhalin branch of FBSI Glavrybvod with using of different starter feeds produced by Aller Aqua (Denmark) and Aquatech (Russia). All hatcheries worked in the coldwater regime of rearing. Both feeds were rather effective for all species that was evidenced by low feeding coefficients, usually 0.6–0.8. Anyway, average value of the feeding coefficient was lower for the starter feed Aquatech at all factories. Beyond a high quality of this feed, its availability in two variants of flotation was important, as the slowly sinking and floating forms. The floating food was found to be more suitable for the growing juvenile pink salmon at all farms. On the other hand, both starter feeds were suitable for chum salmon, their priority for this species depended on fish farm and obviously was determined by combination of many factors, such as temperature conditions, configuration of canals and ponds, their hydrodynamics, feed dispensers, etc.