Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.     

Isolation of apolipoproteins from carotenoid-carrying lipoprotein in the serum of chum salmon, Oncorhynchus keta

Carotenoid-carrying lipoprotein (CCL) was rapidly isolated from the high density lipoprotein (HDL) fraction of the upstream migrating male chum salmon (Oncorhynchus keta) by a single-step density gradient ultracentrifugation. The two apolipoproteins (Mr = 24,000 and 12,000; designated apo-I and apo-II, respectively) were readily dissociated and separated in 0.1% SDS by gel filtration chromatography. Prominent features of the amino acid composition in the CCL included the relative high levels of glutamic acid, alanine, leucine, and lysine, and the low cysteine content. Apo-I, as well as the CCL, was rich in glutamic acid, alanine, leucine, and lysine. Compared to the amino acid composition of apo-I, apo-II included relatively high levels of glycine and tyrosine, and low threonine, serine, and arginine contents. When the intact CCL particle was treated with trypsin, apo-I was rapidly proteolyzed, while apo-II was resistant. However, both apo-I and apo-II isolated from the CCL particle were readily digested with trypsin. This suggested that a different structural arrangement rather than the amino acid compositions of the apolipoproteins was associated with the limited trypsin digestion of the CCL particle. Apo-II may be sheltered from the aqueous environment and lie partly within the CCL particle. The properties of both the HDL fraction and apolipoproteins from pink salmon (Oncorhynchus gorbuscha) were similar to those of the CCL from chum salmon.     

Effects of varying salinities on Lepeophtheirus salmonis survival on juvenile pink and chum salmon

Survival of the sea louse Lepeophtheirus salmonis on juvenile Pacific salmon Oncorhynchus gorbuscha and Oncorhynchus keta was examined with respect to salinity (0, 7, 14, 21 and 28). Rapid mortality was observed in fresh water (0) but motile stage sea lice tolerated higher salinities (7, 14, 21 and 28) for up to 7 days. These findings suggest that salinities juvenile Pacific salmon typically encounter during early marine residence have little affect on motile sea louse survival.     

Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of alpha and beta subunits with N-terminal amino acid residues of Tyr, Gly (alpha, beta of GTH I) and Tyr,Ser (alpha, beta of GTH II). Coho salmon GTH I-beta and GTH II-beta differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-beta and GTH II-beta, respectively. Specific rabbit antisera to the beta subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.     

Transmission dynamics of parasitic sea lice from farm to wild salmon

Marine salmon farming has been correlated with parasitic sea lice infestations and concurrent declines of wild salmonids. Here, we report a quantitative analysis of how a single salmon farm altered the natural transmission dynamics of sea lice to juvenile Pacific salmon. We studied infections of sea lice (Lepeophtheirus salmonis and Caligus clemensi) on juvenile pink salmon (Oncorhynchus gorbuscha) and chum salmon (Oncorhynchus keta) as they passed an isolated salmon farm during their seaward migration down two long and narrow corridors. Our calculations suggest the infection pressure imposed by the farm was four orders of magnitude greater than ambient levels, resulting in a maximum infection pressure near the farm that was 73 times greater than ambient levels and exceeded ambient levels for 30 km along the two wild salmon migration corridors. The farm-produced cohort of lice parasitizing the wild juvenile hosts reached reproductive maturity and produced a second generation of lice that re-infected the juvenile salmon. This raises the infection pressure from the farm by an additional order of magnitude, with a composite infection pressure that exceeds ambient levels for 75 km of the two migration routes. Amplified sea lice infestations due to salmon farms are a potential limiting factor to wild salmonid conservation.     

First report of growth rate of juvenile chum salmon Oncorhynchus keta captured in the Sea of Okhotsk offshore

Ichthyological Research - The growth rate of 16 juvenile chum salmon Oncorhynchus keta (180–286 mm fork length) captured in the Sea of Okhotsk offshore during autumn 2002 was...     

Proliferation and Neuro- and Gliogenesis in Normal and Mechanically Damaged Mesencephalic Tegmentum in Juvenile Chum Salmon,Oncorhynchus keta

Russian Journal of Developmental Biology - Processes of proliferation and constitutive neuro- and gliogenesis in the mesencephalic tegmentum of intact juvenile chum salmon, Oncorhynchus keta, and...     

Isolation, primary structure, and biological and immunological properties of pink and chum salmon insulins

1. Insulins have been isolated from islet tissue of pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon. The primary structure of chum and pink salmon insulins was found to be identical. Compared to the amino acid sequence of human insulin, the salmon insulins under study differed at 14 positions. 2. Biological activity of pink salmon insulin was 83% of that of standard porcine insulin. 3. The immunological properties of fish insulins were investigated in specific radioimmunoassay (RIA) systems, based on porcine and pink salmon insulins. 4. A significant difference in the antigenic determinants of these fish and mammalian hormones was revealed.     

Functional response of juvenile pink and chum salmon: effects of consumer size and two types of zooplankton prey

Feeding rate experiments were conducted for pink salmon Oncorhynchus gorbuscha fry [mean fork length ( L _F) 39 mm], juveniles (103–104 mm L _F) and juvenile chum salmon Oncorhynchus keta (106–107 mm L _F). Fishes were presented with small copepod ( Tisbi sp.) or larger mysid shrimp ( Mysidopsis bahia ) prey at varying densities ranging from 1 to 235 prey l⁻¹ in feeding rate experiments conducted at water temperatures ranging from 10·5 to 12·0° C under high light levels and low turbidity conditions. Juvenile pink and chum salmon demonstrated a type II functional response to mysid and copepod prey. Mysid prey was readily selected by both species whereas the smaller bodied copepod prey was not. When offered copepods, pink salmon fry fed at a higher maximum consumption rate (2·5 copepods min⁻¹) than larger juvenile pink salmon (0·4 copepods min⁻¹), whereas larger juvenile chum salmon exhibited the highest feeding rate (3·8 copepods min⁻¹). When feeding on mysids, the maximum feeding rate for larger juvenile pink (12·3 mysids min⁻¹) and chum (11·5 mysids min⁻¹) salmon were similar in magnitude, and higher than feeding rates on copepods. Functional response models parameterized for specific sizes of juvenile salmon and zooplankton prey provide an important tool for linking feeding rates to ambient foraging conditions in marine environments, and can enable mechanistic predictions for how feeding and growth should respond to spatial-temporal variability in biological and physical conditions during early marine life stages.     

Gastric Pepsin in an Anuran Larva

Larval stomach development was studied in the obligate carnivorous larva of the frog Lepidobatrachus laevis . Pepsin producing cells of the larval stomach were identified using rabbit anti-porcine pepsin and immunohistochemical techniques. Pepsin production was detected at a very early stage of development (stage 24: during opercular development) when the larvae were first competent for feeding. Peptic activity in isolated larval stomachs was demonstrated in a microassay using acid denatured hemoglobin at pH 1.7. The total activity per stomach increased 5,400 fold through the beginning of metamorphosis and the specific activity increased 345 fold through the same period. Electrophoretic analysis of the larval pepsinogens, using a caseinolytic assay revealed the presence of one major pepsinogen at stage 24; two additional isozymes were observed during later larval development. The molecular weight of the isopepsinogens was 34,800.     

Fish gonadotropin(s). II. Isolation of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography.

A highly purified gonadotropic hormone preparation has been obtained from chum salmon (Oncorhynchus keta) pituitaries by extraction with ethanolic or aqueous buffer, affinity chromatography on Con A Sepharose and gel filtration on Sephadex G-75 superfine. A purified fraction from Sephadex G-75 averaged 448 mug NIH-LH-S18/mg glycoprotein as measured by the uptake of radiophosphate into chick testes. A total of 1.1 g of salmon gonadotropin (s) (SG)/kg fresh tissue was recovered when the isolation began with an aqueous extraction. Analytical polyacrylamide gel electrophoresis (P.A.G.E.) of the purified fraction from Sephadex G-75 displayed a single broad zone in non-dissociating conditions and two bands in 8 M urea. Polyacrylamide gel electrofocusing yielded six sharp bands with an isoelectric point range of 4.38 to 5.05, and four bands with an isoelectric point range of 4.31 to 4.95 in 8 M urea. A molecular weight of 41,000 was determined by gel filtration. A subunit molecular weight of 17,800 +/- 10% was found by P.A.G.E. in 0.1% sodium dodecyl sulphate (SDS), suggesting that native SG consists of two subunits. Purified preparations were highly stable in Tris-Cl buffers and retained their activity for several months when stored at -73 degrees C.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Physiological mechanisms of imprinting and homing migration in Pacific salmon Oncorhynchus spp

After several years of feeding at sea, salmonids have an amazing ability to migrate long distances from the open ocean to their natal stream to spawn. Three different research approaches from behavioural to molecular biological studies have been used to elucidate the physiological mechanisms underpinning salmonid imprinting and homing migration. The study was based on four anadromous Pacific salmon Oncorhynchus spp., pink salmon Oncorhynchus gorbuscha, chum salmon Oncorhynchus keta, sockeye salmon Oncorhynchus nerka and masu salmon Oncorhynchus masou, migrating from the North Pacific Ocean to the coast of Hokkaido, Japan, as well as lacustrine O. nerka and O. masou in Lake Toya, Hokkaido, where the lake serves as the model oceanic system. Behavioural studies using biotelemetry techniques showed swimming profiles from the Bering Sea to the coast of Hokkaido in O. keta as well as homing behaviours of lacustrine O. nerka and O. masou in Lake Toya. Endocrinological studies on hormone profiles in the brain-pituitary-gonad axis of O. keta, and lacustrine O. nerka identified the hormonal changes during homing migration. Neurophysiological studies revealed crucial roles of olfactory functions on imprinting and homing during downstream and upstream migration, respectively. These findings are discussed in relation to the physiological mechanisms of imprinting and homing migration in anadromous and lacustrine salmonids.     

Primary structure of chum salmon prolactins: occurrence of highly conserved regions

The complete amino acid sequence of prolactin from the pituitaries of salmon (Oncorhynchus keta) has been determined. Salmon prolactin comprised two variants, I and II, which were separated by reverse-phase high-performance liquid chromatography. Each variant was reduced, S-carboxymethylated, and then cleaved with cyanogen bromide and enzymes. The resulting fragments were separated by reverse-phase high-performance liquid chromatography, as well as gel filtration, and subjected to sequence analysis by the dansyl-Edman method. Both variants contain 187 amino acid residues with two disulfide linkages at residues 46-160 and 177-187, lack a linkage in the N-terminal portion of mammalian prolactins, and differ from each other by the replacement of only four amino acid residues. Salmon prolactin (sPRL) shows 31% sequence identify with ovine prolactin. Moreover, four restricted regions, i.e., sPRL (3-21), (46-60), (68-83), and (160-178), encompass this significant conservatism between the teleost and the mammalian hormone, with identities of 47, 87, 62, and 68%, respectively. Such considerable identity between these distant phylogenic species strongly suggests that these regions may be responsible for the biological activity of prolactin.     

Detection of Renibacterium salmoninarum antigen in migrating adult chum salmon (Oncorhynchus keta) in Japan.

Renibacterium salmoninarum antigen was detected in the kidney of migrating chum salmon (Oncorhynchus keta) using the indirect dot blot assay and indirect fluorescent antibody test. The adult chum salmon had migrated into a bay in which cultured coho salmon infected with R. salmoninarum were present. Antigen was detected in 5% of the chum salmon although they did not have clinical signs of bacterial kidney disease (BKD). This report describes the first case of R. salmoninarum antigen detection among wild chum salmon populations in eastern Asia.     

Carp maturational-ovulatory gonadotropin but not carp vitellogenic gonadotropin or salmon maturational-ovulatory gonadotropin stimulates testosterone production by rat Leydig cells in vitro

Carp (Cyprinus carpio) maturational-ovulatory gonadotropin, prepared from the fraction of pituitary extract adsorbed on Con A-Sepharose (Con A II) and subsequently adsorbed on CM-cellulose (Whatman CM-52), stimulated testosterone production by isolated rat Leydig cells. The fraction of carp pituitary extract unadsorbed on the immobilized lectin (Con A I) with a mol. wt of 30,000, which had previously been shown to contain vitellogenic gonadotropin, was devoid of steroidogenic activity. Salmon (Oncorhynchus keta) pituitary Con A I and Con A II fractions containing vitellogenic and maturational-ovulatory gonadotropin respectively did not enhance steroidogenesis in the same assay system. The results indicated that carp maturational-ovulatory gonadotropin resembled mammalian luteinizing hormone (LH) in its chromatographic behavior on Con A-Sepharose and CM-cellulose and also in its steroidogenic activity in rat Leydig cells. However, not all teleost maturational-ovulatory gonadotropins are LH-like: the salmon hormone is a notable exception. The data further supports the distinctiveness of carp vitellogenic gonadotropin and maturational-ovulatory gonadotropin.     

Comparison of the spectral sensitivity of three species of juvenile salmonids

The spectral sensitivity of chum Oncorhynchus keta , pink Oncorhynchus gorbuscha and masu Oncorhynchus masou masou salmon was measured by the optomotor reaction index in monochromatic light of 400, 440, 480, 520, 560, 600 and 620 nm using an interference filter. The reaction rate of chum salmon was highest at 520 nm but the rates of pink and masu salmon were highest at 560 nm. In addition, a high reaction rate at 400 nm was also observed in masu salmon, suggesting that masu salmon are sensitive to ultraviolet light.     

Isolation of collagen from the skins of Ehlers-Danlos syndrome-affected dogs by acetic acid extraction and pepsin digestion

Collagen was isolated by acetic acid extraction in the presence of protease inhibitors and also by pepsin digestion from the skins of dogs affected with the Ehlers-Danlos syndrome and the skins on non-affected dogs. The collagen preparations isolated by acetic acid extraction from the Ehlers-Danlos syndrome-affected dog skin contained a greater proportion of alpha-chains than the collagen preparations from the normal dog skin. When the collagen from the Ehlers-Danlos syndrome-affected dog skin was reduced with NaBH4 before heat denaturation, and electrophoresis, there was a greater proportion of beta-chains present. The collagen isolated from the normal dog skin was not affected by the NaBH4 reduction. Collagen preparations isolated by pepsin digestion from both the Ehlers-Danlos syndrome-affected dog skin and the non-affected dog skin contained the same quantity of alpha- and beta-chains. In addition, collagen from both affected and non-affected dog skins isolated by pepsin digestion contained 10-11% type III collagen as determined by the interrupted sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Pepsin digestion of the collagens isolated by acetic acid extraction in the presence of protease inhibitors from the skins of affected and non-affected dogs eliminated the differences between the alpha:beta ratios of the affected and non-affected collagen preparations.     

Sequence divergence of the mitochondrial DNA of Pacific Ocean salmon

Restriction assay of mtDNA has been made in 6 salmon species form the genus Oncorhynchus and one species from the related genus, i.e. Salvelinus malma. The size of the mitochondrial genome was found to be identical and equal to 16.7 kilobases. The digestion patterns of mtDNA cleaved by 5 restriction endonucleases (Eco RI, Bgl I, Bgl II, Hind III, and Pst I) were used for analysis of the levels of interspecific variation and for estimation the matrix of mtDNA sequence differentiation. It was found that the level of nucleotide sequence divergence (p) in the genus Oncorhynchus varies within 1.7-6.7%. Minimum p value was observed in a pair O. keta--O. gorbuscha, maximum one--between O. masu and other species. With respect to similarity in their mtDNA, three groups may be distinguished: 1) O. gorbuscha--O. keta; 2) O. nerka--O. kisutch, O. tschawytscha; 3) O. masu. Mean value of intergeneric level of sequence divergence between Oncorhynchus and Salvelinus was found to be equal to 8%. On the basis of mtDNA analysis, the dendrogram of similarity of the species was plotted which is consistent in principle with current viewpoints on phylogenetic relations among the Pacific salmon.     

cDNA cloning and sequence analysis of preproendothelin-1 (PPET-1) from salmon, Oncorhynchus keta

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).