Shinya Mizuno

Immunohistochemical study on changes in gill Na(+)/K(+)-ATPase alpha-subunit during smoltification in the wild masu salmon, Oncorhynchus masou

Changes in immunoreactivity of Na+/K+-ATPase α-subunit in gill sections of wild masu salmon (Oncorhynchus masou) during the parr-smolt transformation (smoltification) were compared with changes in gill Na+/K+-ATPase specific activity. Gill Na+/K+-ATPase specific activity increased from April and peaked in May. Immunohistochemical analysis, using an antiserum against a synthetic oligopeptide based on the conserved region of the Na+/K+-ATPase α-subunit, revealed that immunoreactivity was confined to chloride cells in the surface layer of primary lamellae and the proximal end of secondary lamellae. The size and number of these cells increased gradually from February to May; however, the number of chloride cells of the secondary lamellae decreased in May. These data suggest that the synthesis of Na+/K+-ATPase and the proliferation of chloride cells occur prior to the elevation of enzyme activity. Moreover, it is likely the proliferation and hypertrophy of chloride cells on primary lamellae prepare smolts for entry into seawater and migration in the ocean.

Changes in Transcript Levels of Gill Cortisol Receptor during Smoltification in Wild Masu Salmon, Oncorhynchus masou

Abstract We developed a quantitative PCR assay to investigate transcript levels of gill cortisol receptor (CR) in masu salmon (Oncorhynchus masou). Using this system, we examined changes in transcript levels of gill CR during smoltification of wild masu salmon while tracking serum cortisol and growth hormone (GH) concentrations patterns. The masu salmon were parr in January and February, and thereafter smoltified, migrating from the river to the sea in May. Gill CR transcript levels were very low in January and February, but thereafter increased and reached a maximum in April (5 fold increase over levels observed in January). In May, when smolt enter the sea, the gill CR transcript levels decreased. Serum cortisol concentrations were also low from January to March and increased to the peak in April. These changes correlated well with changes in CR transcript levels from March to April. In contrast, serum GH concentration began to increase in January, peaked in March and decreased from March to May. These results elucidated patterns in transcript levels of gill CR during smoltification of wild masu salmon and suggested that gill CR transcription was positively regulated by cortisol until serum cortisol level reached a peak, and negatively done after the peak in masu salmon.

Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou)

In the present study, the effects of starvation on kidney melano-macrophages (MM) in underyearling hatchery-reared masu salmon (Oncorhynchus masou) which were released into the river and underyearling wild masu salmon were examined histologically. One hundred and fifty fish were placed in a tank for each of the four study groups: wild-fed, wild-starved, hatchery-fed, and hatchery-starved. Starved groups were not fed during the 2-month course of the experiment, while fed groups were given krill. During the experiment, kidneys were collected and the mortality was calculated every 15 days in all groups. Moreover, hatchery-reared masu salmon released into the river were sampled on days 15 and 45 of the experiment. MMs with dark brown pigment were observed with random distribution in the kidneys in all groups during the experiment. The degree of MM deposition and mortality increased in parallel for both starved groups (wild and hatchery-reared), increasing rapidly after day 15, while deposition levels remained low throughout the experiment for both feeding groups as well as for the fish released into the river. There were no significant differences in the degree of MM deposition between hatchery-reared and wild fish for both starved and feeding groups. These results suggest that the degree of MM deposition in the kidney can be used as an indicator of starvation in both wild and hatchery-reared masu salmon.

Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou

We attempted establishment of quantitative system for dorsal fin pigmentation due to diffusion of melanin granules in melanophores during smoltification in masu salmon (Oncorhynchus masou) with image analysis and examined whether it is possible to determine the optimal time for smolt release of hatchery-reared masu salmon, which have many physiological characters in common with wild masu salmon, with the level of dorsal fin pigmentation. The effects of anesthesia with ethyl-m-aminobenzoate and fixation with paraformaldehyde on the pigmentation analysis were also investigated. There were no significant effects of 10-min full anesthesia or fixation on dorsal fin pigmentation. We were able to analyze dorsal fin pigmentation by observation of the fin within 10-min full anesthesia and in fixed fin tissue. Changes in dorsal fin pigmentation in hatchery-reared and two stocks of wild yearling masu salmon during smoltification from January or March to May in 2001 and 2002 were examined and compared with gill Na+,K+-ATPase activity. In both 2001 and 2002, the level of dorsal fin pigmentation and ATPase activity increased significantly from January or March to May during smoltification (P<0.05) in all fish. There was no significant difference in pigmentation level and ATPase activity in May among all fish in both 2001 and 2002. Means of the level of dorsal fin pigmentation in May for 2 years for all fish ranged between 83% and 84%. The earliest period, in which the pigmentation level began to increase significantly compared to initial period, was prior to the time, at which the ATPase activity increased, in all fish in 2001 and 2002. The levels of dorsal fin pigmentation and gill Na+,K+-ATPase activity exhibited a positive nonlinear correlation in wild and hatchery-reared fish. These observations suggest that the level of dorsal fin pigmentation could be used as an indicator of the suitable time for smolt release of our hatchery-reared masu salmon.

Quantitative Analysis of Body Silvering during Smoltification in Masu Salmon using Chromameter

Abstract In Japan, the phase distinction of masu salmon Oncorhynchus masou during smoltification is determined based on external appearance such as body silvering and black pigmentation of dorsal and caudal fin margin. This method is subjective, and it is difficult to quantify smoltification because body silvering and fin pigmentation level change continuously. Therefore, innovative and easily used criteria to establish smoltification are necessary. We therefore tested a chromameter (L-value) to measure body silvering in two brood years of hatchery-reared and natural (wild and hatchery-origin fish occurring in the wild) masu salmon and examined its effectiveness in determining the progress of smoltification as it relates to establishing smolt release time. Time required to measure body silvering using a chromameter was approximately 10 s per fish. For both brood years, the L-values of hatchery-reared and natural fish differed. With the progress of smoltification in both hatchery-reared and natural fish, t...

Relationships between gill Na⁺,K⁺-ATPase activity and endocrine and local insulin-like growth factor-I levels during smoltification of masu salmon (Oncorhynchus masou).

We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na(+),K(+)-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors.

Effects of cortisol and angiotensin II on the number and size of juxtaglomerular cells in masu salmon, Oncorhynchus masou

The authors previously reported that the number and size of juxtaglomerular cells (JGCs) in the kidney increased during smoltification in masu salmon, Oncorhynchus masou. In the present study, the effects of cortisol and/or angiotensin (Ang)xa0II ([Asn1, Val5]-Angxa0II) on the JGC number and size in masu salmon were examined to elucidate hormonal regulation of the changes in the JGC number and size during smoltification. These hormones were injected intraperitoneally every 2xa0days for a total of 6xa0injections. There was a significant increase in the JGC number and size with time following the start of the experiment in cortisol- and cortisol + Angxa0II-treated groups and no significant change in control and Angxa0II-treated groups. On both daysxa05 and 11, the JGC number and size in the cortisol-treated group were significantly large compared to those of control and Angxa0II-treated groups, respectively. The JGC number and size in the cortisol + Angxa0II-treated group were significantly large compared to those of control on both daysxa05 and 11, and those of the Angxa0II-treated group only on dayxa011, respectively. On the other hand, there was no significant difference in the JGC number and size between the Angxa0II-treated and control groups and between the cortisol- and cortisol + Angxa0II-treated groups during the experiment, respectively. The means of the JGC number and size in cortisol-treated group on dayxa011 were close to those previously reported in smolt. These results suggest that cortisol induces an increase in JGC number and size during smoltification in masu salmon.

Quantitative analysis of Ichthyobodo salmonis an ectoparasitic flagellate infecting juvenile chum salmon Oncorhynchus keta in hatcheries

Ichthyobodosis caused by the ectoparasitic flagellate Ichthyobodo salmonis is a significant cause of mortality in juvenile chum salmon Oncorhynchus keta reared in hatcheries of northern Japan. The present study established a real-time quantitative polymerase chain reaction assay (qPCR) of I. salmonis ribosomal DNA (rDNA) using SYBR Green. This assay allows monitoring of parasite infections for the epidemiological study and control of ichthyobodosis in hatcheries. qPCR showed high reproducibility for measurements between 1.0 and 1.0xa0×xa0108 rDNA copy/μl. There was a significant positive correlation between the number of parasites and the amount of I. salmonis rDNA. A survey using the qPCR assay indicated that infection by I. salmonis was present in 23 of 87 hatcheries; parasite loads were estimated to be between 50 and 750 parasites/g juvenile body weight. These results demonstrate that our qPCR assay enables the surveying of juvenile chum salmon reared in hatcheries for infection by I. salmonis.

Effects of dietary supplementation with oregano essential oil on prevention of the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon Oncorhynchus keta

The present study performed three experiments to establish a practical prevention strategy for the ectoparasitic flagellate Ichthyobodo salmonis and ciliate Trichodina truttae in hatchery-reared juvenile chum salmon Oncorhynchus keta using dietary supplementation with oregano essential oil. Experiment 1 showed that a diet supplemented for 3 weeks with 0.02% oregano essential oil significantly prevented infection with I. salmonis and T. truttae in juveniles reared in small tanks. Experiment 2, in outdoor hatchery ponds, demonstrated that the oregano treatment completely prevented I. salmonis infection for 52 days and T. truttae infection for 38 days. Oregano-treated juvenile mortality attributable to infection with these protozoans also decreased to 7.6% of control juvenile mortality, confirming the utility of this treatment in cultured O. keta. Physiological analyses of the oregano-treated juveniles elucidated the treatments safety in relation to their metabolism, osmoregulation, natural immunity and olfactory responses and also detected carvacrol (a major component of oregano essential oil which shows antimicrobial activity) on the skin. In experiment 3, exposure of the two protozoans to oregano essential oil revealed a weak antiparasitic action on the body surface of the juvenile O. keta. The overall results demonstrate that dietary oregano supplementation is a practical prevention strategy for I. salmonis and T. truttae in hatchery-reared juvenile O. keta and suggest the possibility that its anti-parasitic action is attributable to a component of the oil that emerges onto the skin of the body of the fish.

Epizootiology of the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae on wild chum salmon Oncorhynchus keta

Infestations of the ectoparasitic flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae have caused acute mortalities of hatchery-reared juvenile chum salmon Oncorhynchus keta in Hokkaido, northern Japan. This study examined the epizootiology of I. salmonis and T. truttae on wild chum salmon as a possible infection source of the 2 parasitic protozoans in hatcheries. Infestations by both ectoparasites were detected on freshwater-adapted adult and juvenile chum salmon in all 4 rivers examined. This is the first study of an anadromous Pacific salmonid to report infestation of I. salmonis and T. truttae in adults returning for spawning. Among the marine-inhabiting phase of chum salmon, infestation with I. salmonis, but not T. truttae, was observed on adults and juveniles. The 2 protozoans were experimentally transmitted at the same time from wild to hatchery-reared chum salmon juveniles, and caused a high rate of mortality in the hatchery fish. In freshwater, the proliferation rate of T. truttae was greater than that of I. salmonis. These observations show that the euryhaline ectoparasite I. salmonis can infest chum salmon throughout their life cycle, in both river and ocean habitats, whereas T. truttae is able to infest these salmonids only in freshwater. Furthermore, wild chum salmon were shown to be a potential infestation source for both T. truttae and I. salmonis in hatchery fish.