Size-driven parr-smolt transformation in masu salmon (Oncorhynchus masou).

Anadromous salmonids exhibit partial migration, where some individuals within a population migrate down to the ocean through complex interactions between body size and photoperiod. This study aimed to integrate the ontogenetic and seasonal patterns of smoltification, a series of changes for future marine life, in a strain of masu salmon (Oncorhynchus masou). Spring smoltification, as evidenced by the activation of gill Na+,K+-ATPase (NKA), was induced during winter under an advanced photoperiod. In addition, juveniles showed an additional peak in gill NKA activity in August regardless of the photoperiod. When juvenile masu salmon were subjected to feeding manipulations during the first spring/summer, only fish exceeding a fork length of 12 cm exhibited an increased gill NKA activity. We tested whether size-driven smoltification required a long-day period by exposing juveniles to a constant short-day length (9-h light and 15-h dark) from January to November. Juveniles under short-day conditions exceeded 12 cm in June but showed no signs of smoltification. Thus, masu salmon undergo photoperiod-limited, size-driven smoltification during the first summer and size-limited, photoperiod-driven smoltification the following spring. The findings of the present study provide a framework for further elucidation of the physiological mechanisms underlying partial migration in salmonids.

Evaluation of binding capacity of circulating insulin-like growth factor binding protein-1b in salmonids using a ligand immunofunctional assay.

Insulin-like growth factor-binding proteins (IGFBPs) regulate the activity of insulin-like growth factor (IGF)-1. Among the three major circulating IGFBPs in salmonids, IGFBP-1b is an inhibitor of IGF activity induced under catabolic conditions. IGFBP-1b is considered to quickly sequester IGF-1 from the circulation. However, the level of circulating IGFBP-1b present in its unoccupied free form is unknown. Here, we aimed to develop a non-equilibrium ligand immunofunctional assay (LIFA) to evaluate IGF-binding capacity of circulating intact IGFBP-1b. Purified Chinook salmon IGFBP-1b, its antiserum, and europium-labeled salmon IGF-1 were used as the assay components. In the LIFA, IGFBP-1b was first captured by the antiserum, allowed to bind to the labeled IGF-1 for 22 h at 4 °C, and quantified its IGF-binding capacity. Serial dilutions of the standard and serum were prepared simultaneously within a certain concentration range (1.1-12.5 ng/ml). In underyearling masu salmon, IGF-binding capacity of intact IGFBP-1b was higher in fasted fish than in fed fish. Transferring Chinook salmon parr to seawater also increased IGF-binding capacity of IGFBP-1b, most likely due to osmotic stress. In addition, there was a strong relationship between total IGFBP-1b levels and its IGF-binding capacity. These results suggest that IGFBP-1b expressed under stress is mostly present in the free form. On the contrary, during smoltification of masu salmon, IGF-binding capacity of IGFBP-1b in the serum was relatively low and less related to the total IGFBP-1b level, suggesting its functional difference under certain physiological conditions. These results indicate that estimating both total IGFBP-1b level and its IGF-binding capacity is useful for evaluating the catabolic status and unraveling the regulation of IGF-1 activity by IGFBP-1b.

Development of a time-resolved fluoroimmunoassay for salmonid insulin-like growth factor binding protein-2b.

Insulin-like growth factor (IGF)-1 promotes the growth of vertebrates, and its binding proteins (IGFBPs) regulate the activity of circulating IGF-1. Three IGFBPs, IGFBP-2b, -1a, and -1b, were consistently detected in the circulatory system of salmonids. IGFBP-2b is thought to be the main carrier of IGFs and promoter of IGF-1-mediated growth in salmonids. Currently, there are no immunoassays for detecting IGFBP-2b. In this study, we developed a time-resolved fluoroimmunoassay (TR-FIA) for IGFBP-2b detection in salmonid fishes. To establish TR-FIA, we produced two recombinant trout (rt) IGFBP-2bs expressed, one with thioredoxin (Trx) and a histidine (His) tag, and the other with His-tag only. We labeled both recombinant proteins with europium (Eu). Only Eu-Trx.His.rtIGFBP-2b cross-reacted with anti-IGFBP-2b, and the addition of increasing amounts of Trx.His.rtIGFBP-2b replaced the binding, indicating its utility as a tracer and assay standard. The addition of unlabeled salmon IGF-1 did not affect the binding of the standard or sample. Serial dilution curves of sera from rainbow trout, Chinook salmon, and chum salmon were parallel to those of the standard. The assay range (ED80-ED20) of the TR-FIA was 60.4 to 251.3 ng/ml, and its minimum detection limit of this assay was 21 ng/ml. The intra- and inter-assay coefficients of variation were 5.68% and 5.65%, respectively. Circulating IGFBP-2b levels in fed rainbow trout were higher than those in fasted fish and were correlated with individual growth rates. This TR-FIA is useful for further exploring the physiological responses of circulating IGFBP-2b and evaluating the growth status of salmonids.

Changes in circulating insulin-like growth factor-1 and its binding proteins in yearling rainbow trout during spring under natural and manipulated photoperiods and their relationships with gill Na+, K+-ATPase and body size.

Smoltification in salmonids occurs during spring in response to increasing photoperiod to prepare for marine life. Smoltification is associated with increased hypo-osmoregulatory ability and enhanced growth potential, mediated by growth hormone and insulin-like growth factor (IGF)-1. Rainbow trout is uniquely insensitive to the induction of smoltification-associated changes by photoperiod, such as the activation of gill Na+,K+-ATPase (NKA). We measured the circulating IGF-1 and IGF-binding protein (IGFBP)-2b levels in yearling rainbow trout exposed to natural and manipulated photoperiods during spring and correlated these with gill NKA activity and body size. Although the effect of photoperiod manipulation on body size and circulating IGF-1 and IGFBP-2b was negligible, they were positively correlated with gill NKA activity in fish under simulated natural photoperiod. We next pit-tagged yearling rainbow trout and fed them a restricted ration or to satiation under a natural photoperiod. In April, gill NKA activity was higher in the satiation group than in the restricted group and positively correlated with body size and growth rate. In addition, circulating IGFBP-2b was positively correlated with gill NKA, size and growth, whereas circulating IGF-1 was correlated only with size and growth. The relationship between circulating IGF-1 and growth intensified from May to June, suggesting that the IGF-1-growth relationship was disrupted in April when gill NKA was activated. Two additional IGFBPs were related to growth parameters but not to gill NKA activity. The present study suggests that circulating IGFBP-2b and IGF-1 mediate the size-dependent activation of gill NKA in yearling rainbow trout during spring.

Evaluation of circulating insulin-like growth factor (IGF)-I and IGF-binding proteins as growth indices in rainbow trout (Oncorhynchus mykiss).

Circulating insulin-like growth factor (IGF)-I has been proposed as a growth index in several teleosts, including salmonids, and its level in circulation is stabilized by multiple IGF-binding proteins (IGFBPs). Three IGFBPs, IGFBP-2b, -1a, and -1b, are consistently detected in salmonid blood and are suggested to be indices of positive or negative growth, although their applicability to rainbow trout (Oncorhynchus mykiss) is unclear. The present study examined the usefulness of IGFBPs along with IGF-I as a physiological indicator of growth rate in rainbow trout through a rearing experiment. Two groups of underyearling rainbow trout were pit-tagged and either fed or fasted for 33 days. A third group was fasted for 22 days, followed by refeeding for 11 days. Serum IGF-I levels were reduced after fasting for 22 days, but refeeding did not retore its levels to those of the fed control. Nevertheless, there was a positive relationship between serum IGF-I levels and individual growth rates over 33 days of experimentation, confirming its validity as a growth index. Ligand blotting using labeled human IGF-I revealed two IGFBP bands at 43 and 32 kDa, which corresponded to IGFBP-2b and an unidentified form, respectively. In contrast, bands corresponding to IGFBP-1a and -1b, which usually increase after fasting, were hardly detected, even in the fasted fish. The responses of circulating IGFBP-2b to fasting and refeeding were similar to those of circulating IGF-I and positively correlated with growth rate and IGF-I levels. The intensity of the serum 32-kDa IGFBP band was higher in constantly fed fish than in the fasted fish; however, its correlation with growth rate was weaker than those of IGF-I and IGFBP-2b. The present study shows that IGF-I and IGFBP-2b can be used as growth indices for rainbow trout. In contrast, circulating IGFBP-1a and -1b may not serve as negative growth indices in rainbow trout under regular aquaculture conditions because they are rarely detected by ligand blotting or respond to fasting/refeeding.

Effects of Acclimation to Diluted Seawater on Metabolic and Growth Parameters of Underyearling Masu Salmon (Oncorhynchus masou).

We examined the effects of environmental salinity and feeding status on the growth and metabolic parameters of underyearling masu salmon. Fish were first acclimated to salinities of 0 (< 0.1), 11, or 22 psu for 10 days, after which time 50% of the fish in each group were fasted for 5 days followed by refeeding for 5 days. No effects on body length/weight were observed over the 20 days from the beginning of the experiment. Gill Na+, K+-ATPase (NKA) activity increased 20 and 10 days after transfer to water at 11 and 22 psu, respectively. Serum Na+ and Cl- levels were high in fish at 22 psu on day 20 but much lower than those in the environmental water, suggesting that fish at this salinity were able to hypo-osmoregulate. However, acclimation to 22 psu resulted in a reduction in feeding rate on day 20. Serum insulin-like growth factor (IGF)-I levels and liver glycogen content were reduced by fasting and restored after 5 days of refeeding, except in the fish at 22 psu. Intensities of serum IGFBP-1a and -1b bands were increased at higher salinities, whereas fasting/refeeding affected only IGFBP-1b. The present study suggests that acclimating masu salmon parr to 11 psu had no effect on metabolic and growth parameters, while 22 psu presumably suppressed their growth potential due to the possible energy cost or stress for osmoregulation. The disparate responses of circulating IGFBP-1a and -1b to higher salinity and fasting highlight their utility as indices of various catabolic statuses.

Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. RESULTS: No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. CONCLUSION: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

Effects of growth hormone and cortisol administration on plasma insulin-like growth factor binding proteins in juveniles of three subspecies of masu salmon (Oncorhynchus masou).

In this study, we examined the effects of porcine growth hormone (GH) and cortisol on plasma insulin-like growth factor binding proteins (IGFBPs) in juveniles of three subspecies of Oncorhynchus masou (masu, amago, and Biwa salmon). Ligand blotting using digoxigenin-labeled human IGF-I was used to detect and semi-quantify three major circulating IGFBP bands at 41, 28, and 22 kDa, corresponding to IGFBP-2b, -1a, and -1b, respectively. GH increased plasma IGFBP-2b concentration in masu and Biwa salmon but suppressed it in amago salmon. Plasma IGFBP-2b levels were increased by cortisol in the three subspecies. Cortisol induced plasma IGFBP-1a in the three subspecies, whereas GH had a suppressive effect in masu and Biwa salmon. Sham and cortisol injections increased plasma IGFBP-1b levels after 1 day in masu and amago salmon, suggesting that IGFBP-1b is induced following exposure to stressors via cortisol. Increased IGFBP-1b levels were restored to basal levels when co-injected with GH in Biwa salmon, and the same trend was seen in masu and amago salmon. However, the suppressive effect of GH disappeared 2 days after injection in the three subspecies. Despite some differences among subspecies, the findings suggest that cortisol is a primary inducer of plasma IGFBP-1b; however, GH counteracts it in the short term. Therefore, GH has the potential to modulate the degree of increase in circulating IGFBP-1b levels during acute stress.

Compensatory Response of the Somatotropic Axis from IGFBP-2b Gene Editing in Rainbow Trout (Oncorhynchus mykiss).

Rainbow trout with gene editing-induced reductions in serum insulin-like growth factor binding protein (IGFBP)-2b exhibit similar growth performance compared to fish without IGFBP-2b gene disruption. The objective of this study is to determine how the components of the insulin-like growth factor (IGF)/IGFBP system respond to a reduction in serum IGFBP-2b abundance. Editing the IGFBP-2b genes in rainbow trout resulted in an 83% decrease in serum IGFBP-2b in mutants. This resulted in a 35% reduction in serum IGF-I, which was offset by reduced expression of hepatic igfbp-1a2 and increased muscle igfr-1a; these responses suggest that an increased IGF-I signaling capacity offset reductions in serum IGF-I. During feed deprivation, the differential expression of igfbp genes supports the attenuation of the growth inhibitory response, likely due to the further reduction in serum IGF-I that alleviated the need for an IGF-inhibitory response. Unique igfbp expression patterns occurred during refeeding, suggesting an enhanced IGF-I signaling capacity in controls. Collectively, these findings support that the role of IGFBP-2b is to regulate serum IGF-I concentrations. The compensatory regulation of IGF/IGFBP system genes indicates that adjustments in other IGFBP, both circulating and at the local level, maintain IGF-I signaling at a level appropriate for the nutritional state of the fish.

Production of two recombinant insulin-like growth factor binding protein-1 subtypes specific to salmonids.

Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.

Hypothalamic agrp and pomc mRNA Responses to Gastrointestinal Fullness and Fasting in Atlantic Salmon (Salmo salar, L.).

The orexigenic agouti-related protein (AgRP) and the anorexigenic pro-opiomelanocortin (POMC) are crucial players in the control of feed intake in vertebrates, yet their role in teleosts has not been fully established. Triplicate groups of Atlantic salmon (Salmo salar) post smolts were subjected to (1) fasting for 3 days (fast) and (2) normal feeding (fed), resulting in a significant (p < 0.05) upregulation of hypothalamic agrp1 transcripts levels in the fast group. Moreover, the mRNA abundance of agrp1 was significantly (p < 0.05) correlated with the stomach dry weight content. Corresponding inverse patterns were observed for pomca2, albeit not statistically significant. No significant differences were found for the other paralogues, agrp2 and pomca1 and b, between fed and fast groups. The significant correlation between stomach fullness and agrp1 mRNA expression suggests a possible link between the stomach filling/distension and satiety signals. Our study indicates that hypothalamic agrp1 acts as an orexigenic signal in Atlantic salmon.

Intact rather than total circulating insulin-like growth factor binding protein-1a is a negative indicator of growth in masu salmon.

Insulin-like growth factor binding protein (IGFBP)-1a is one of three major circulating forms in salmon and induced under catabolic conditions. However, there is currently no immunoassay available for this form because of a lack of standard and specific antibodies. We developed a time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1a using recombinant protein for labeling, an assay standard, and production of antiserum. The TR-FIA had a low cross-reactivity (3.6%) with IGFBP-1b, another major form in the circulation. Fasting for 4 wk had no effect on serum immunoreactive (total) IGFBP-1a levels in yearling masu salmon, whereas 6-wk fasting significantly increased it. There was a significant, but weak, negative relationship between serum total IGFBP-1a level and individual growth rate (r2 = 0.12, P = 0.01). We next developed a ligand immuno-functional assay (LIFA) using europium-labeled IGF-I to quantify intact IGFBP-1a. In contrast to total IGFBP-1a, serum intact IGFBP-1a levels increased after 4 wk of fasting, and refeeding for 2 wk restored it to levels similar to those of the fed control. Serum intact IGFBP-1a levels showed a significant negative correlation with individual growth rate (r2 = 0.52, P < 0.001), which was as good as that of IGFBP-1b. Our findings using newly developed TR-FIA and LIFA suggest that regulation of intact IGFBP-1a levels has an important effect on growth in salmon and that intact IGFBP-1a is a negative index of salmon growth.

Utilization of an endocrine growth index, insulin-like growth factor binding protein (IGFBP)-1b, for postsmolt coho salmon in the Strait of Georgia, British Columbia, Canada.

Monitoring the growth of salmon during their early marine phase provides insights into prey availability, and growth rates may be linked to risks of size-dependent mortality. However, the measurement of growth rate is challenging for free-living salmon in the ocean. Insulin-like growth factor (IGF)-I is a growth-promoting hormone that is emerging as a useful index of growth in salmon. In addition, laboratory-based studies using coho salmon have shown that one of circulating IGF-binding proteins (IGFBPs), IGFBP-1b, is induced by fasting and thus could be used as an inverse index of growth and/or catabolic state in salmon. However, few studies have measured plasma levels of IGFBP-1b in salmon in the wild. We measured plasma IGFBP-1b levels for postsmolt coho salmon collected in the Strait of Georgia and surrounding waters, British Columbia, Canada, and compared regional differences in IGFBP-1b to ecological information such as seawater temperature and stomach fullness. Plasma IGFBP-1b levels were the highest in fish from Eastern Johnstone Strait and relatively high in Queen Charlotte Strait and Western Johnstone Strait, which was in good agreement with the poor ocean conditions for salmon hypothesized to occur in that region. The molar ratio of plasma IGF-I to IGFBP-1b, a theoretical parameter of IGF-I availability to the receptor, discriminated differences among regions better than IGF-I or IGFBP-1b alone. Our data suggest that plasma IGFBP-1b reflects catabolic status in postsmolt coho salmon, as highlighted in fish in Eastern Johnston Strait, and is a useful tool to monitor negative aspects of salmon growth in the ocean.

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.

Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).

In salmonids, the majority of circulating insulin-like growth factor-I (IGF-I) is bound to IGF binding proteins (IGFBP), with IGFBP-2b being the most abundant in circulation. We used CRISPR/Cas9 methodology to disrupt expression of a functional IGFBP-2b protein by co-targeting for gene editing IGFBP-2b1 and IGFBP-2b2 subtypes, which represent salmonid-specific gene duplicates. Twenty-four rainbow trout were produced with mutations in the IGFBP-2b1 and IGFBP-2b2 genes. Mutant fish exhibited between 8-100% and 2-83% gene disruption for IGFBP-2b1 and IGFBP-2b2, respectively, with a positive correlation (P < 0.001) in gene mutation rate between individual fish. Analysis of IGFBP-2b protein indicated reductions in plasma IGFBP-2b abundance to between 0.04-0.96-fold of control levels. Plasma IGF-I, body weight, and fork length were reduced in mutants at 8 and 10 months post-hatch, which supports that IGFBP-2b is significant for carrying IGF-I. Despite reduced plasma IGF-I and IGFBP-2b in mutants, growth retardation in mutants was less severe between 10 and 12 months post-hatch (P < 0.05), suggesting a compensatory growth response occurred. These findings indicate that gene editing using CRISPR/Cas9 and ligand blotting is a feasible approach for characterizing protein-level functions of duplicated IGFBP genes in salmonids and is useful to unravel IGF-related endocrine mechanisms.

Comparison between Atlantic salmon Salmo salar post-smolts reared in open sea cages and in the Preline raceway semi-closed containment aquaculture system.

The use of closed containment (CCS) or semi-closed containment systems (S-CCS) for Atlantic salmon Salmo salar aquaculture is under evaluation in Norway. One such system is the Preline S-CCS, a floating raceway system that pumps water from 35 m depth creating a constant current through the system. Exposing fish to moderate water currents is considered aerobic exercise and it is often perceived as positive for fish welfare, growth, food utilization, muscle development and cardiac health. The present study compared fish reared in the Preline S-CCS and in a reference open pen. Samples were taken in fresh water before being transferred to the seawater systems and after 1, 2 and 4 months in seawater and analysed for growth, mortality, muscle development and plasma insulin-like growth factor I (IGF-I) levels. Moreover, gene transcription were determined in the skeletal muscle [igf-I, insulin-like growth factor 1 receptor a (igf1ra) and insulin-like growth factor 1 binding protein 1a (igf1bp1a)] and cardiac transcription factors [myocyte-specific enhancer factor 2C (mef2c), gata4 and vascular endothelial growth factor (vegf)]. While the results suggest that post-smolts in Preline S-CCS were smaller than reference fish, fish from Preline S-CCS have less accumulated mortality at the end of the experiment and showed 2.44 times more small muscle fibres than the reference group fish after 4 months in seawater. These results confirmed what was previously observed in the second generation of Preline. Similar levels of big muscle fibres between Preline S-CCS and reference suggest a similar hypertrophy of muscle fibres even with lower IGF-I expression in the Preline S-CCS. Cardiac gene transcription suggests cardiac hypertrophy was observed after 4 months in seawater in the Preline S-CCS group. Altogether, Preline S-CCS is a promising technology able to produce more robust S. salar with a faster growth and lower mortality in the subsequent standard open cage system growth period.

Assessment of the timing and degree of smolt development in southern populations of masu salmon Oncorhynchus masou.

The present study assessed whether non-anadromous masu salmon Oncorhynchus masou in Miyazaki, southern Japan, smoltify, and if so, at what time of the year. Yearling O. masou of Miyazaki and an anadromous population from Hokkaido, northern Japan, were reared in hatcheries in their respective regions and sampled monthly from February to June to examine the spring smoltification period. The Hokkaido population showed a peak of gill Na+ -K+ -ATPase (NKA) activity in May, which was accompanied with an increase in mRNA levels of the seawater (SW)-type NKA alpha subunit, nkaα1b. Increases in gill NKA activity and nkaa1b levels were not seen in Miyazaki populations. Transferring yearling Miyazaki population to 70% SW (salinity of 23) in mid-April resulted in an increased serum osmolality over 4 days. These results suggest that they do not smoltify in their second spring. Next, profiles of gill NKA activity and its subunit mRNA levels in under-yearling Miyazaki population in the autumn were examined. Two phenotypes differing in body color during this period were categorized as parr and smolt-like fish. Smolt-like fish had higher gill NKA activity than parr in December while there was no significant difference in gill nkaα1b levels. Smolt-like fish acclimated to 70% SW better than parr as judged by lower serum osmolality. However, serum osmolality in smolt-like fish did not return to the basal level 7 days after transfer to 70% SW, suggesting that their hypo-osmoregulatory ability was not fully developed to a level comparable to anadromous populations of this species. The present study suggests that, if O. masou in Miyazaki go though a smoltification process, it occurs in its first autumn instead of the second spring and is less pronounced compared with anadromous populations.

Production of recombinant salmon insulin-like growth factor binding protein-1 subtypes.

Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development.

Circulating insulin-like growth factor binding proteins in fish: Their identities and physiological regulation.

Insulin-like growth factor binding proteins (IGFBPs) play crucial roles in regulating the availability of IGFs to receptors and prolong the half-lives of IGFs. There are six IGFBPs present in the mammalian circulation with IGFBP-3 being most abundant. In mammals IGFBP-3 is the major carrier of circulating IGFs, facilitated by forming a ternary complex with IGF and an acid-labile subunit (ALS). IGFBP-1 is generally inhibitory to IGF action by preventing it from interacting with its receptors. In teleosts, the third-round of vertebrate whole genome duplication created paralogs of each IGFBP, except IGFBP-4. In the fish circulation, three major IGFBPs are typically detected at molecular ranges of 20-25, 28-32 and 40-50kDa. However, their identities are not well established. Three major circulating IGFBPs in Chinook salmon have been identified through protein purification and cDNA cloning. Salmon 28- and 22-kDa IGFBPs are co-orthologs of IGFBP-1, termed IGFBP-1a and -1b, respectively. They are induced under catabolic conditions such as stress and fasting but their responses are somewhat different, with IGFBP-1b being the most sensitive of the two. Cortisol stimulates production and secretion of these IGFBP-1 subtypes while, unlike in mammals, insulin may not be a primary suppressor. Salmon 41-kDa IGFBP, a major carrier of IGF-I, is not IGFBP-3, as might be expected extrapolating from mammals, but is in fact IGFBP-2b. Salmon IGFBP-2b levels in plasma are high when fish are fed, and GH treatment increases its circulating levels similar to mammalian IGFBP-3. These findings suggest that salmon IGFBP-2b acquired the role and regulation similar to mammalian IGFBP-3. Multiple replications of fish IGFBPs offer a unique opportunity to investigate molecular evolution of IGFBPs.

Effects of seawater transfer and fasting on the endocrine and biochemical growth indices in juvenile chum salmon (Oncorhynchus keta).

Insulin-like growth factor (IGF)-I, IGF-binding protein (IGFBP)-1 and RNA/DNA ratio are endocrine and biochemical parameters used as growth indices in fish, however, they are subjected to environmental modulation. Chum salmon (Oncorhynchus keta) migrate from freshwater (FW) to seawater (SW) at fry/juvenile stage weighing around 1g and suffer growth-dependent mortality during the early phase of their marine life. In order to reveal environmental modulation of the IGF/IGFBP system and establish a reliable growth index for juvenile chum salmon, we examined effects of SW transfer and fasting on IGF-I, IGFBP-1 and RNA/DNA ratio, and correlated them to individual growth rate. Among serum IGF-I, liver and muscle igf-1, igfbp-1a, igfbp-1b and RNA/DNA ratio examined, muscle RNA/DNA ratio and muscle igfbp-1a responded to SW transfer. Serum IGF-I, liver igf-1 and liver RNA/DNA ratio were sensitive to nutritional change by being reduced in 1week in both FW and SW while muscle igf-1 was reduced 2weeks after fasting. In contrast, igfbp-1a in both tissues was increased by 2weeks of fasting and igfbp-1b in the liver of SW fish was increased in 1week. These results suggest that the sensitivity of igf-1 and igfbp-1s to fasting differs between tissues and subtypes, respectively. When chum salmon juveniles in SW were marked and subjected to feeding or fasting, serum IGF-I showed the highest correlation with individual growth rate. Liver igfbp-1a and -1b, and muscle igf-1 exhibited moderate correlation coefficients with growth rate. These results show that serum IGF-I is superior to the other parameters as a growth index in juvenile chum salmon in term of its stability to salinity change, high sensitivity to fasting and strong relationship with growth rate. On the one hand, when collecting blood from chum salmon fry/juveniles is not practical, measuring liver igfbp-1a and -1b, or/and muscle igf-1 is an alternative.