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.

Expression of Doublecortin, Glial Fibrillar Acidic Protein, and Vimentin in the Intact Subpallium and after Traumatic Injury to the Pallium in Juvenile Salmon, Oncorhynchus masou.

Fetalization associated with a delay in development and the preservation of the features of the embryonic structure of the brain dominates the ontogeny of salmonids. The aim of the present study was to comparatively analyze the distribution of the glial-type aNSC markers such as vimentin and glial fibrillar acidic protein (GFAP) and the migratory neuronal precursors such as doublecortin in the telencephalon subpallium of juvenile masu salmon, Oncorhynchus masou, in normal conditions and at 1 week after an injury to the dorsal pallium. Immunohistochemical labeling of vimentin, GFAP, and doublecortin in the pallium of intact juvenile masu salmon revealed single cells with similar morphologies corresponding to a persistent pool of neuronal and/or glial progenitors. The study of the posttraumatic process showed the presence of intensely GFAP-labeled cells of the neuroepithelial type that form reactive neurogenic zones in all areas of the subpallial zone of juvenile masu salmon. A comparative analysis of the distribution of radial glia in the dorsal, ventral, and lateral zones of the subpallium showed a maximum concentration of cells in the dorsal part of subpallium (VD) and a minimum concentration in the lateral part of subpallium VL. An essential feature of posttraumatic immunolabeling in the masu salmon subpallium is the GFAP distribution patterns that are granular intracellular in the apical periventricular zone (PVZ) and fibrillar extracellular in the subventricular (SVZ) and parenchymal zones (PZ). In contrast to those in intact animals, most of the GFAP+ granules and constitutive neurogenic niches in injured fish were localized in the basal part of the PVZ. With the traumatic injury to the subpallium, the number of Vim+ cells in the lateral and ventral regions significantly increased. At 1 week post-injury, the total immunolabeling of vimentin cells in the PVZ was replaced by the granular pattern of Vim immunodistribution spreading from the PVZ to the SVZ and deeper parenchymal layers of the brain in all areas of the subpallium. A significant increase in the number of DC+ cells was observed also in all areas of the subpallium. The number of cells increased both in the PVZ and in the SVZ, as well as in the deeper PZ. Thus, at 1 week after the injury to the dorsal pallium, the number of DC, Vim, and GFAP expressing cells of the neuroepithelial type in the subpallium of juvenile masu salmon increased, and additionally GFAP+ radial glia appeared in VD, which was absent from intact animals.

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.

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.

Parallel epigenetic modifications induced by hatchery rearing in a Pacific salmon.

Wild stocks of Pacific salmonids have experienced sharp declines in abundance over the past century. Consequently, billions of fish are released each year for enhancing abundance and sustaining fisheries. However, the beneficial role of this widely used management practice is highly debated since fitness decrease of hatchery-origin fish in the wild has been documented. Artificial selection in hatcheries has often been invoked as the most likely explanation for reduced fitness, and most studies to date have focused on finding signatures of hatchery-induced selection at the DNA level. We tested an alternative hypothesis, that captive rearing induces epigenetic reprogramming, by comparing genome-wide patterns of methylation and variation at the DNA level in hatchery-reared coho salmon (Oncorhynchus kisutch) with those of their wild counterparts in two geographically distant rivers. We found a highly significant proportion of epigenetic variation explained by the rearing environment that was as high as the one explained by the river of origin. The differentially methylated regions show enrichment for biological functions that may affect the capacity of hatchery-born smolts to migrate successfully in the ocean. Shared epigenetic variation between hatchery-reared salmon provides evidence for parallel epigenetic modifications induced by hatchery rearing in the absence of genetic differentiation between hatchery and natural-origin fish for each river. This study highlights epigenetic modifications induced by captive rearing as a potential explanatory mechanism for reduced fitness in hatchery-reared salmon.

Hydrogen Sulfide Modulates Adult and Reparative Neurogenesis in the Cerebellum of Juvenile Masu Salmon, Oncorhynchus masou.

Fish are a convenient model for the study of reparative and post-traumatic processes of central nervous system (CNS) recovery, because the formation of new cells in their CNS continues throughout life. After a traumatic injury to the cerebellum of juvenile masu salmon, Oncorhynchus masou, the cell composition of the neurogenic zones containing neural stem cells (NSCs)/neural progenitor cells (NPCs) in the acute period (two days post-injury) changes. The presence of neuroepithelial (NE) and radial glial (RG) neuronal precursors located in the dorsal, lateral, and basal zones of the cerebellar body was shown by the immunohistochemical (IHC) labeling of glutamine synthetase (GS). Progenitors of both types are sources of neurons in the cerebellum of juvenile O. masou during constitutive growth, thus, playing an important role in CNS homeostasis and neuronal plasticity during ontogenesis. Precursors with the RG phenotype were found in the same regions of the molecular layer as part of heterogeneous constitutive neurogenic niches. The presence of neuroepithelial and radial glia GS+ cells indicates a certain proportion of embryonic and adult progenitors and, obviously, different contributions of these cells to constitutive and reparative neurogenesis in the acute post-traumatic period. Expression of nestin and vimentin was revealed in neuroepithelial cerebellar progenitors of juvenile O. masou. Patterns of granular expression of these markers were found in neurogenic niches and adjacent areas, which probably indicates the neurotrophic and proneurogenic effects of vimentin and nestin in constitutive and post-traumatic neurogenesis and a high level of constructive metabolism. No expression of vimentin and nestin was detected in the cerebellar RG of juvenile O. masou. Thus, the molecular markers of NSCs/NPCs in the cerebellum of juvenile O. masou are as follows: vimentin, nestin, and glutamine synthetase label NE cells in intact animals and in the post-traumatic period, while GS expression is present in the RG of intact animals and decreases in the acute post-traumatic period. A study of distribution of cystathionine ß-synthase (CBS) in the cerebellum of intact young O. masou showed the expression of the marker mainly in type 1 cells, corresponding to NSCs/NCPs for other molecular markers. In the post-traumatic period, the number of CBS+ cells sharply increased, which indicates the involvement of H2S in the post-traumatic response. Induction of CBS in type 3 cells indicates the involvement of H2S in the metabolism of extracellular glutamate in the cerebellum, a decrease in the production of reactive oxygen species, and also arrest of the oxidative stress development, a weakening of the toxic effects of glutamate, and a reduction in excitotoxicity. The obtained results allow us to consider H2S as a biologically active substance, the numerous known effects of which can be supplemented by participation in the processes of constitutive neurogenesis and neuronal regeneration.

Differential Bioaccumulation of Mercury and Selenium in Stomach Contents and Tissues of Three Colorado, USA, Cutthroat Trout Populations.

Total mercury (THg) and selenium (TSe) levels were measured in stomach contents (SC) and twelve tissues of cutthroat trout (Oncorhynchus clarkii) occurring in three high-elevation lakes of Colorado, USA, inhabiting watersheds absent past and current mining activities. For 32 of 36 tissues, including muscle, mean THg wet weight (ww) concentrations were greater than in the diet (SC) for all sites, indicating biomagnification. Ranges of THg (µg/kg ww) for SC and stomach tissue (ST) were 1.23-73.54 and 14.55-61.35, respectively. Selenium concentrations in fish muscle were not greater than in the SC indicating a trophic transfer factor < 1.0. However, in several other tissues, mean Se dry weight (dw) levels were greater than in SC for all three lakes. Ranges of TSe for SC and ST were 166-7544 and 797-7523 (µg/kg dw), respectively. The muscle to egg/ovary ratio for Se averaged 2.30, 4.60, and 2.68 for the three populations. The variability of SC (planktonic vs. benthic) and differential distributions of THg and TSe in SC and organ-tissues generated questions focusing on the seasonal, physiological, and genetic drivers of these organometal(loid)s in subalpine trout.

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.

Olfactory gene expression in migrating adult sockeye salmon Oncorhynchus nerka.

Expression of 12 olfactory genes was analysed in adult sockeye salmon Oncorhynchus nerka nearing spawning grounds and O. nerka that had strayed from their natal migration route. Variation was found in six of these genes, all of which were olfc olfactory receptors and had lower expression levels in salmon nearing spawning grounds. The results may reflect decreased sensitivity to natal water olfactory cues as these fish are no longer seeking the correct migratory route. The expression of olfactory genes during the olfactory-mediated spawning migration of Pacific salmon Oncorhynchus spp. is largely unexplored and these findings demonstrate a link between migratory behaviours and olfactory plasticity that provides a basis for future molecular research on salmon homing.

Ovarian expression and localization of clathrin (Cltc) components in cutthroat trout, Oncorhynchus clarki: Evidence for Cltc involvement in endocytosis of vitellogenin during oocyte growth.

To evaluate potential involvement of clathrin in endocytosis of vitellogenin (Vtg) by teleost oocytes, cDNAs encoding clathrin heavy chain (cltc) were cloned from ovaries of cutthroat trout. Quantitative PCR revealed three types of cltc (cltc-a1, cltc-a2, cltc-b) to be expressed in 10 different tissues including the ovary. The cltc-a1 alone exhibited a significant decrease in ovarian expression during vitellogenesis; this was correlated with a corresponding decrease in transcripts encoding the major Vtg receptor (Vtgr). No development-related changes in ovarian cltc-a2 or cltc-b transcript levels were observed. In situ hybridization revealed a strong ctlc signal in pre-vitellogenic oocytes, but not in vitellogenic oocytes. Western blotting using a rabbit antiserum (a-Cltc) raised against a recombinant Cltc preparation detected a polypeptide band with an apparent mass of ~170kDa in vitellogenic ovary extracts. Immunohistochemistry using a-Cltc revealed Cltc to be uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, translocated to the periphery of lipid droplet stage oocytes, and localized to the oolemma during vitellogenesis. These patterns of cltc/Cltc distribution and abundance during oogenesis, which are identical to those previously reported for vtgr/Vtgr in this species, constitute the first empirical evidence that cltc-a1/Cltc-a1 is involved in Vtg endocytosis via the Vtgr in teleost fish.

Molecular cloning and partial characterization of a low-density lipoprotein receptor-related protein 13 (Lrp13) involved in vitellogenin uptake in the cutthroat trout (Oncorhynchus clarki).

Multiple ovarian membrane proteins that bind vitellogenin (Vtg) have been detected in teleosts. One of these Vtg receptors was recently identified as low-density lipoprotein receptor-related protein 13 (lrp13/Lrp13) in perciform species, but little is known about this Vtg receptor in salmonid fish. In this study, a cDNA encoding a putative Vtg receptor with 13+1 ligand binding repeats (lr13+1) was cloned from the ovary, and identified as an lrp13 ortholog for cutthroat trout (Oncorhynchus clarki). This lrp13 was predominantly expressed in the pre-vitellogenic stage ovary, and its expression decreased during vitellogenesis. Ovarian localization of Lrp13 was observed by immunohistochemistry using specific antiserum against recombinant Lrp13. Lrp13 immunoreactivity was observed at the oolemma, throughout the zona radiata, and within the perivitelline space between the zona radiata and granulosa cells in ovarian follicles at both the lipid-droplet and vitellogenic stages of growth-an expression pattern that mimics that of a lr8/LR8-type Vtg receptor in this species and of lrp13/Lrp13 in Morone species. Six discrete Vtg-binding proteins were detected in cutthroat trout ovarian membrane proteins when probing with a digoxygenin-labeled salmonid A-type Vtg (VtgAs) followed by chemiluminescent ligand detection. Western blotting using the anti-Lrp13 serum revealed a broad signal consisting of two proteins with masses ranging from ∼190 to ∼210 kDa, which corresponded with some of the VtgA-binding proteins. These findings suggest that, in addition to lr8/LR8, lrp13/Lrp13 acts as a VtgA receptor in trout.

Effects of dry brining, liquid smoking and high-pressure treatment on the physical properties of aquacultured King salmon (Oncorhynchus tshawytscha) during refrigerated storage.

BACKGROUND: Aquacultured King salmon (Oncorhynchus tshawytscha) pieces were dry brined with a salt/brown sugar mix, dipped in liquid smoke for 3 min, vacuum packed, high hydrostatic pressure (HHP) treated at 600 or 200 MPa for 5 min and stored at 4 °C for up to 40 days. RESULTS: The surface redness (average a*) of the samples increased after dry brining, then decreased after liquid smoke treatment. HHP did not change the outside color of liquid-smoked samples. However, the inside color changed depending on pressure. HHP-treated control samples without dry brining and liquid smoking changed to a pale pink color. HHP at 600 MPa resulted in a significant increase in hardness. Compared with fresh samples, dry-brined samples had reduced water activity, while samples dipped in liquid smoke had lower pH values. CONCLUSION: Dry brining and liquid smoking protect the outside color of salmon against changes caused by HHP. The increase in hardness may counteract the softening of the smoked salmon tissue over time.

Lack of hormonal stimulation prevents the landlocked Biwa salmon (Oncorhynchus masou subspecies) from adapting to seawater.

Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na(+),K(+)-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.

Molecular cloning and transcript expression of genes encoding two types of lipoprotein lipase in the ovary of cutthroat trout, Oncorhynchus clarki.

Large amounts of neutral lipids (NLs) are stored as lipid droplets in the ooplasm of fish oocytes, providing an essential energy resource for developing embryos and larvae. However, little is known about the origin of such lipids or about mechanisms underlying their uptake and accumulation in oocytes. We have proposed a model for this lipidation of teleost oocytes, as follows: very low density lipoprotein (Vldl) is metabolized by lipoprotein lipase (Lpl) outside and/or inside of the oocyte and the resulting fatty acids (FAs) are then utilized for de novo biosynthesis of NLs. As a first step toward verification of this model, cDNAs for genes encoding two types of Lpl, lpl and lpl2, were cloned from the ovary of cutthroat trout, Oncorhynchus clarki. Examination of Lpl polypeptide sequences deduced from the cDNAs revealed features similar to LPLs/Lpls in other species, including several conserved structural and functional domains. Both types of lpl mRNA were highly expressed in lipid storage tissues (e.g., adipose tissue, muscle, and ovary) and were predominantly expressed in the granulosa cells of ovarian follicles. Ovarian lpl1 mRNA levels showed a remarkable peak in April (early oocyte lipid droplet stage) and then decreased to low values sustained until November (mid-vitellogenesis), after which time a small peak in lpl1 gene expression was observed in December (late vitellogenesis). The mRNA levels of lpl2 also were elevated in April and were highest in June (late lipid droplet stage), but did not show other pronounced changes. These results suggest that, in the cutthroat trout, Vldl is metabolized by the action of Lpls in the granulosa cell layer to generate free FAs for uptake and biosynthesis of neutral lipids by growing oocytes.

Diurnal changes in salmon GnRH secretion in the brain of masu salmon (Oncorhynchus masou).

The day-night changes of salmon GnRH (sGnRH), which is secreted from various brain regions, were analyzed in maturing and matured masu salmon (Oncorhynchus masou). In maturing males, the levels of sGnRH secreted from the olfactory bulb (OB), terminal nerve (TN), and ventral telencephalon and preoptic area (VT+POA) were all significantly higher during midnight than daytime. However, the contents of sGnRH in the pituitary gland during midnight were not higher than those during daytime. In maturing females, the levels of sGnRH secreted from the VT+POA were higher during midnight than daytime, and the contents of sGnRH in the pituitary gland were also higher during midnight. In matured fish, the levels of sGnRH secreted from the OB, TN and VT+POA during midnight were significantly higher than those during daytime. There were also no significant differences in the contents of sGnRH in the pituitary gland. These results suggest that a short photoperiod may be involved in diurnal secretion rhythms of sGnRH in various brain regions and the pituitary gland.

Calibrating acoustic acceleration transmitters for estimating energy use by wild adult Pacific salmon.

This study is the first to calibrate acceleration transmitters with energy expenditure using a vertebrate model species. We quantified the relationship between acoustic accelerometer output and oxygen consumption across a range of swim speeds and water temperatures for Harrison River adult sockeye salmon (Oncorhynchus nerka). First, we verified that acceleration transmitters with a sampling frequency of 10 Hz could be used as a proxy for movement in sockeye salmon. Using a mixed effects model, we determined that tailbeat frequency and acceleration were positively correlated (p<0.0001), independent of tag ID. Acceleration (p<0.0001) was positively related to swim speed while fork length (p=0.051) was negatively related to swim speed. Oxygen consumption and accelerometer output (p<0.0001) had a positive linear relationship and were temperature dependent (p<0.0001). There were no differences in swim performance (F(2,12)=1.023, p=0.820) or oxygen consumption (F(1,12)=0.054, p=0.332) between tagged and untagged individuals. Five tagged fish were released into the Fraser River estuary and manually tracked. Of the five fish, three were successfully tracked for 1h. The above relationships were used to determine that the average swim speed was 1.25±0.03 body lengths s(-1) and cost of transport was 3.39±0.17 mg O(2) kg(-1)min(-1), averaged across the three detected fish. Acceleration transmitters can be effectively used to remotely evaluate fine-scale behavior and estimate energy consumption of adult Pacific salmon throughout their homeward spawning migration.

Responses of insulin-like growth factor (IGF)-I and two IGF-binding protein-1 subtypes to fasting and re-feeding, and their relationships with individual growth rates in yearling masu salmon (Oncorhynchus masou).

Two subtypes of insulin-like growth factor binding protein (IGFBP)-1 are present in salmon blood and they are both up-regulated under catabolic conditions such as stress. The present study examined effects of fasting and re-feeding on IGFBP-1a (28-kDa form) and IGFBP-1b (22-kDa form) both at mRNA and protein levels along with IGF-I and RNA/DNA ratio in yearling masu salmon. Fish were individually tagged and assigned to one of three treatments: Fed, Fasted or Re-fed. Circulating IGF-I levels significantly decreased after fasting for 5 weeks and were positively correlated with individual growth rates. Liver igf-1 mRNA levels were not affected by the treatment. Muscle RNA/DNA ratio did not respond to fasting nor showed correlations with growth rates. Circulating IGFBP-1a and IGFBP-1b increased during fasting and decreased after re-feeding. Both serum levels were inversely correlated with growth rates, while IGFBP-1b had consistent negative relationships with growth rates. Fasting/re-feeding also affected their mRNA levels in the liver. These results suggest that circulating IGF-I and IGFBP-1b could serve as positive and negative indices of growth, respectively, in masu salmon. Different sensitivities of IGBP-1a and IGFBP-1b may be useful to assess a broad range of catabolic conditions when they are combined.

Combined cytotoxicity of ZnO nanoparticles and chlorpyrifos in the rainbow trout, Oncorhynchus mikyss, gonadal cell line RTG-2.

The toxicity of ZnO nanoparticles (ZnO NPs) in aquatic organisms has been extensively studied, but little information is available on the effects associated with their interaction with other contaminants. In this context, the in vitro effects of co-exposure of chlorpyrifos (CPF) and ZnO NPs on fish-derived cells were investigated. A selection of concentrations was tested in single and binary exposures: CPF (0.312 - 75 mg/L) and ZnO NPs (10 - 100 mg/L). Cytotoxicity was measured using commonly used cellular endpoints: Alamar Blue/CFDA-AM for viability and plasma membrane integrity, NRU for lysosomal disruption and MTT for mitochondrial function. In addition, specific mechanisms of toxicity for CPF and ZnO NPs were tested: acetylcholinesterase (AChE) activity and ROS generation, respectively. AChE was by far the most sensitive assay for single exposure to CPF. There was no concentration-response relationship for ROS after single exposure to ZnO NPs, but 10 mg/L produced significant effects only for this cellular endpoint. Co-exposure of CPF with 10 m/L of ZnO NPs produced significant effects in almost all endpoints tested, which were enhanced by co-exposure with 100 mg/L of ZnO NPs. AChE testing of additional co-exposures with bulk ZnO, together with the application of the Independent Action (IA) prediction model, which allowed us to draw more in-depth conclusions on the toxicological behavior of the mixture. Synergism was observed at 0.625 mg/L CPF concentration and antagonism at 5 mg/L CPF in mixtures containing 100 mg/L of both ZnO NPs and bulk ZnO. However, more cases of synergism between CPF and ZnO NPs occurred at intermediate CPF concentrations, demonstrating that nano-sized particles have a more toxic interaction with CPF than bulk ZnO. Therefore it can be argued that in vitro assays allow the identification of interaction profiles of NP-containing mixtures by achieving multiple endpoints with a large number of concentration combinations.

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.

[Gaseous intermediates in the brain of the salmon Oncorhynchus masou].

Distribution of nitroxidergic and H2S-producing neurons in the brain of the salmon Oncorhynchus masou was studied by methods of histochemical markering of NADPH-diaphorase and by immunohistochemical markering of the neuronal nitric oxide synthase and cystathionin beta-synthase (CBS). The established distribution of CBS and nNOS/NADPH-d of neurons and fibers in the salmon telencephalon, optic tectum, and cerebellum allows suggesting that the NO- and H2S-producing systems, represent individual, non-overlapping neuronal complexes performing specialized functions in the activity of local neuronal networks. In the brainstem part, the nNOS-ir and NADPH-d-positive neurons were detected in the composition of viscerosensor (V, VII, and IX-X) and visceromotor (III, IV, and VI) nuclei of craniocerebral nerves, octavolateral afferent complex, reticulospinal neurons, and medial reticular formation. CBS in the salmon medulla was revealed in neurons of the X nerve nucleus, reticulospinal neurons, and ventrolateral reticular formation. Distribution of NO-ergical and H2S-producing neurons in the salmon medulla nuclei indicates that NO in salmon is the predominant neuromodulator of medulla viscerosensory systems, while H2S seems to modulate only the descending motor systems. The results of the performed study allow suggesting that NI in the descending motor systems. The results of the performed study allow suggesting that NO in the salmon medulla periventricular area can act as a regulator of postnatal ontogenesis.