Enhanced osmoregulatory ability marks the smoltification period in developing chum salmon (Oncorhynchus keta).

The freshwater (FW) life of chum salmon is short, as they migrate to the ocean soon after emergence from the substrate gravel of natal waters. The alevins achieve seawater (SW) acclimating ability at an early developmental stage and the details of smoltification are not clear. We examined the stage-dependent SW acclimating ability in chum salmon alevins and found a sharp increase in SW tolerance during development that resembles the physiological parr-smolt transformation seen in other salmonids. Perturbation of plasma Na+ after SW exposure was prominent from the hatched embryo stage to emerged alevins, but the plasma Na+ became highly stable and more resistant to perturbation soon after complete absorption of yolk. Marker gene expression for SW-ionocytes including Na/K-ATPase (NKA α1b), Na-K-Cl cotransporter 1a (NKCC1a), Na/H exchanger 3a (NHE3a), cystic fibrosis transmembrane conductance regulators (CFTR I and CFTR II) were all upregulated profoundly at the same stage when the alevins were challenged by SW, suggesting that the stability of plasma Na+ concentration was partly a result of elevated osmoregulatory capability. FW-ionocyte markers including NKA α1a and NHE3b were consistently downregulated independent of stage by SW exposure, suggesting that embryos at all stages respond to salinity challenge, but the increase in SW osmoregulatory capability is restricted to the developmental stage after emergence. We propose that the "smoltification period" is condensed and integrated into the early development of chum salmon, and our results can be extrapolated to the future studies on hormonal controls and developmental triggers for smoltification in salmonids.

Evaluation of growth status using endocrine growth indices, insulin-like growth factor (IGF)-I and IGF-binding protein-1b, in out-migrating juvenile chum salmon.

This study aimed to utilize circulating insulin-like growth factor binding protein (IGFBP)-1b as a negative index of growth to evaluate the growth status of juvenile chum salmon (Oncorhynchus keta) in the ocean. First, rearing experiments using PIT-tagged juveniles were conducted to examine the relationship of circulating IGFBP-1b with growth rate of the fish in May and in June. The serum IGFBP-1b level negatively correlated with fish growth rate in both months, suggesting its utility as a negative index of growth. Next, the growth status of out-migrating juveniles in northeastern Hokkaido, Japan, was monitored for 3 years using the growth indices. Serum levels of IGF-I, a positive index of growth, in fish collected from the nearshore zone were low in May and high in June of all years. Levels of serum IGFBP-1b showed a trend opposite to that of serum IGF-I. However, the IGF-I/IGFBP-1b molar ratios well reflected the seasonal and regional trends. These findings suggest that the juveniles in June left the nearshore area under better growth conditions. The present study also suggests that the use of multiple growth indices would improve the sensitivity and accuracy to evaluate the current growth status of out-migrating juvenile chum salmon.

[Effects of salinity on haematological biochemistrical indices and liver tissue in juvenile Oncorhynchus keta].

In order to simulate the catadromous migration environment of the juvenile salmon (Oncorhynchus keta), five experimental groups containing different salinities (0, 5, 10, 15 and 20) were set up. The juvenile salmons with average body mass of (26.57 +/- 6.32) g and average full length of (14.44 +/- 1.05) cm were cultured for 130 days, and then the haematological biochemistrical indices was analyzed and the structure of liver tissue was observed to investigate the changes in physiological indices during the catadromous migration of juvenile salmon. The results showed that serum osmolality and the concentration of Na+, Cl- illustrated the same tendency with water salinity variation. Specifically, the serum Na+, C- and Mg2+ levels in the salinities of 15, 20 were significantly different from those in the salinity 5 and the freshwater and serum K+ in different salinities were all obviously lower than that in the freshwater. Also, the blood glucose level in salinity 10 was significantly higher than that in salinity 5 and 20, whereas total serum protein (TP), albumin (ALB) and globulin (GLB) levels all declined with the increase of salinity and the serum TP and GLB content in the freshwater group was remarkably higher than those in the salinity 15 and 20. There also existed an obvious discrepancy on total bile acids in freshwater group and the other salinity groups. The alanine aminotransferase (ALT) activity and aspartate aminotransferase (AST) activity in fresh water group were far different from those of the high salinity groups. Furthermore, in the low salinities (0 and 5), the liver cells of juvenile chum salmon partly broke down, and liver tissue was serious vacuolization. Collectively, the growth and survival rate had no significant difference in all groups and according to the physiological and biochemical indices, the appropriated salinity for juvenile chum salmon ranged from 10 to 20 during the catadromous migration.

Changes in the plasma levels of insulin-like growth factor-I from the onset of spawning migration through upstream migration in chum salmon.

An increase in activity of the pituitary-gonadal axis (PG-axis) and gonadal development are essential for the onset of spawning migration of chum salmon from the Bering Sea. In the Bering Sea, fish with larger body sizes initiated gonadal development and commenced spawning migration to the natal river by the end of summer. We thus hypothesized that insulin-like growth factor-I (IGF-I), a somatotropic signal that interacts with the PG-axis, can be one of such factors responsible for the onset of migration, and examined changes in plasma levels and hepatic expression of IGF-I gene in oceanic and homing chum salmon in 2001-2003. The plasma IGF-I levels and corresponding body sizes in maturing adults, which had developing gonads, were significantly higher than those in immature fish in all years examined. Such increase in the plasma IGF-I levels in maturing fish was observed even in the Gulf of Alaska during February 2006, while coincident increase was not observed in the hepatic amounts of IGF-I mRNA. In autumn, the plasma IGF-I levels in homing adults decreased during upstream migration in the Ishikari River-Ishikari bay water system in Hokkaido, Japan. In conclusion, the plasma IGF-I levels increased with gonadal development when chum salmon migrated from the winter Gulf of Alaska to the summer Bering Sea. Circulating IGF-I may interact with the PG-axis and promote gonadal development that is inseparable from the onset of spawning migration. Circulating IGF-I levels were thereafter lowered in accordance with final maturation during upstream migration in the breeding season.

Elevation of the plasma level of insulin-like growth factor-I with reproductive maturation prior to initiation of spawning migration of chum salmon.

When, where, and how oceanic chum salmon initiate spawning migration is unknown although gonadal development and elevation of the activity of the pituitary-gonadal axis (PG-axis) are essential. Insulin-like growth factor-I (IGF-I) is a somatotropic signal that interacts with the PG-axis for gametogenesis. We thus examined the plasma level of IGF-I in immature and maturing chum salmon in the Bering Sea and the Gulf of Alaska. The maturing adults which had maturing gonads left the Bering Sea for the natal river by the end of summer, because almost all fish were immature in September. The plasma level of IGF-I and corresponding body size in the maturing adults were two- to threefold that of immature fish. The plasma IGF-I level correlated positively with the pituitary contents of follicle-stimulating hormone and the plasma levels of 11-ketotestosterone and estradiol-17beta. Therefore, the plasma level of IGF-I increased with elevation of the PG-axis activity prior to the initiation of spawning migration from the Bering Sea. Circulatory IGF-I from visceral organs may inform the status of body growth to the PG-axis for gonadal development that is inseparable from decision of chum salmon whether to initiate homing behavior from the Bering Sea or not to initiate spawning migration by the coming spawning season.

Purification and characterization of a cysteine protease inhibitor from chum salmon (Oncorhynchus keta) plasma.

A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.

Year-to-year differences in plasma levels of steroid hormones in pre-spawning chum salmon.

Changes in plasma levels of steroid hormones in pre-spawning chum salmon (Oncorhynchus keta) were examined for 6 years in association with sexual maturation. Fish were sampled along their homing pathway from the coastal sea to the spawning ground from 1995 to 2000. Plasma levels of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), and cortisol were determined by enzyme immunoassays. Sexual maturity was comprehensively estimated by gonadosomatic indices, histology of gonads, nuptial color, spermiation or ovulation ratio. Since the plasma levels of steroid hormones and sexual maturation differed from year to year, they were compared with year-to-year variation of sea surface temperature (SST) of coastal sea to study influence of oceanographic environment on these physiological data. The SST of the migratory route varied among the years, so that we classified the 6 years into cool, intermediate, and warm years. Concerning maturity, the males that returned to the natal hatchery in the warm years were sexually more advanced than those in the cool years. Furthermore, histological data suggested that final oocyte maturation occurred before arrival at the hatchery in one of the warm years, i.e., 1999, while it occurred at the hatchery in one of the intermediate years, i.e., 2000. In the males, T and 11KT levels increased significantly on midway of the homing route in the warm years, whereas they did not show any noticeable changes in the cool years. Furthermore, the levels of T and 11KT on midway of the homing route in the warm years, i.e., 1998 and 1999, were significantly higher than those in one of the cool years, i.e., 1995, in both sexes. In the females, the levels of E2 decreased during upstream migration. Conversely, those of DHP considerably elevated at spawning ground in all years examined. The levels of cortisol were different from year to year regardless of the SST. The present results showed that there were year-to-year differences in plasma levels of steroid hormones and maturity, and some of them may be influenced by the year-to-year variation of SST.

Immune complex transfer two-site chemiluminescent immunoassay for serum growth hormone in alevin chum salmon.

An immune complex transfer two-site chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH) was developed to measure serum GH in alevin chum salmon (Oncorhynchus keta) using a chemiluminescent acridinium ester as a label. The immune complex transfer method dramatically reduced non-specifically bound of acridinium ester labelled antibody without a decrease in the specific binding. Consequently, we could detect lower levels of GH than achieved previously in a two-site CLIA for salmon GH. The detection limit of the assay was 7.8 fg/ml and the standard curve was linear up to 250 fg/ml. Coefficients of variation were 2.2-7.7% within-assay and 5.3-91% between-assay. We have developed a highly sensitive and reproducible GH method and applied it to measurement of GH in alevin chum salmon.

Development of an ELISA for chum salmon (Oncorhynchus keta) growth hormone.

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon (Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab' fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH's from several species of the genus Oncorhynchus.

Chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH).

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of growth hormone (GH) in salmonid species. The CLIA for salmon GH was performed using the sandwich method with anti-GH IgG as the first antibody and chemiluminescent acridinium ester-labelled specific anti-GH F(ab')2 as the second antibody. The measurable range of salmon GH in the CLIA was 39-1250 pg/mL using a short assay (1 day) protocol and 3.9-125 pg/mL in a longer (2-day) assay. The dilution curve in the CLIA of serum from masu salmon (Oncorhynchus masou) was parallel to the standard curve of recombinant chum salmon (Oncorhynchus keta) GH. Seasonal changes of serum GH levels were measured in 1 year-old masu salmon cultivated in a pond from March to November. Their serum GH levels increased during smoltification from March to April, achieved a maximum level of 21 ng/mL in August, and then declined gradually to 11 ng/mL in October.

Intracerebral expression of gonadotropin-releasing hormone and growth hormone-releasing hormone is delayed until smoltification in the salmon.

A developmental strategy was employed to investigate the functional assembly of neuropeptidergic systems in the migratory species of chum salmon Oncorhynchus keta. Using immunocytochemistry we have demonstrated that different groups of gonadotropin-releasing hormone- (GnRH)- and growth hormone-releasing hormone- (GHRH)-synthesizing neurons emerged according to very different developmental timetables. From the eye pigmentation stage (23 +/- 2 days after fertilisation (DAF)) through to the pre-smoltification stage (136 DAF), salmon-GnRH neurons originating from the olfactory placodes remained restricted to the extracerebral course of the terminal nerve. At the climax of smoltification (downstream migration 167 DAF), basal forebrain and midbrain GnRH neurons with elaborate neurite outgrowths in the brain and the pituitary became detectable. The GnRH neuroanatomical organization in the post-smoltification stage (197 DAF) was similar to that in the smoltification stage (167 DAF). In contrast to the case for other teleosts, chicken-GnRHII neurons were not found in the midbrain but were localized along the medial regions of the olfactory nerve. Growth hormone-releasing hormone immunoreactivity in the olfactory apparatus (21 DAF), and fibers along the basal telencephalon and hypothalamus and in the pituitary were observed during early embryogenesis (51 DAF) and in cells in the preoptic area on 167 DAF. The intracerebral expression of GnRH and GHRH was not detected until the peak of smoltification, which coincided with a peak in thyroid hormones, and precisely with downstream migratory behavior.

Immunocytochemical study of the somatolactin cells in the pituitary of Pacific salmon, Oncorhynchus nerka, and O keta at some stages of the reproductive cycle.

Cells of the intermediate lobe of salmonids, homologous to the PAS-positive cells of other teleost species, cannot be differentiated by normal staining techniques, but can be immunostained with an antiserum against cod somatolactin (SL). Immunocytochemical techniques were applied to pituitary sections of two Pacific salmon, Oncorhynchus nerka and O. keta. Samples of immature or early maturing fish were collected in the Pacific Ocean and from mature spawning fish from hatcheries near Seattle and Willard (Washington). SL cells were rather small and moderately immunoreactive in immature fish. They were slightly enlarged in the early stages of gonadal development and more often contacted the basal lamina through processes with terminal swellings, suggesting granule release into perivascular spaces. In spawning fish, cells were enlarged and frequently more granulated, showing a wide contact with the basal lamina and a proximodistal transport of granules. In addition, large and more or less degranulated cells were noted, also indicating an active release of SL granules. Spawning females tended to have more SL cells than equivalent males. The gradual stimulation of SL synthesis and release during sexual maturation suggests that SL may be involved in the control of some steps of reproduction as previously shown by the increase in SL plasma levels in maturing coho salmon.