Cultured Rainbow Trout Research Articles

Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes

The suitability of cultured rainbow trout hepatocytes as a model system for the assessment of xenobiotic effects on hepatic biotransformation enzymes in fish was examined. Two model water pollutants, the imidazole fungicide prochloraz and the alkylphenolic compound nonylphenol diethoxylate (NP2EO), were investigated in a comparative in vitro/in vivo approach. Biotransformation enzymes were measured in cultured rainbow trout hepatocytes following exposure to xenobiotics in vitro, or in the liver of juvenile rainbow trout ( Oncorhynchus mykiss) exposed in vivo. The patterns of biochemical responses to the model pollutants were generally similar between in vitro and in vivo investigations. Levels of cytochrome P4501A (CYP1A) protein and the catalytic activity of the CYP1A-dependent enzyme 7-ethoxyresorufin- O-deethylase (EROD) were induced in vitro after 24 h of exposure to 1.0 μM prochloraz. In vitro, higher prochloraz concentrations induced only the levels of CYP1A above control levels, but not EROD activity. In vivo exposure of juvenile trout to 0.27 μM prochloraz resulted in an induction of CYP1A and EROD after 7 and 14 days, while 0.027 μM prochloraz had no effects. In vitro, the 6β- and 16β-hydroxylation of testosterone was significantly decreased by 1.0–3.0 μM prochloraz, while in vivo these variables were significantly inhibited after exposure to 0.27 μM prochloraz for 7 and 14 days. NP2EO did not affect EROD activity in vitro. In vivo, EROD activity and CYP1A remained unchanged following 7 days of exposure to 0.32 or 1.30 μM NP2EO. NP2EO (15–50 μM) inhibited the 16β-hydroxylation and glucuronidation of testosterone in vitro. In vivo, 7 days of exposure to 0.32 or 1.30 μM NP2EO resulted in a significant inhibition of the 6β- and 16β-hydroxylation of testosterone. The good qualitative correspondence between in vitro and in vivo results indicates that studies using trout hepatocytes allow the identification of biochemical targets of xenobiotic effects in fish liver. However, more research is needed before quantitative predictions, e.g. of effective concentrations, can be made from in vitro investigations.

Mesophilic Aeromonads in Wild and Aquacultured Freshwater Fish

Numbers and species of motile Aeromonas were determined in freshly caught freshwater fish, in the surrounding environment, and also during iced chilled storage of fish specimens. Although no significant differences were observed in water samples, initial levels for skin, gill, and intestines were significantly lower in farmed rainbow trout (Oncorhynchus mykiss) than in wild brown trout (Salmo trutta) and pike (Esox lucius). During storage of wild specimens, naturally occurring aeromonads grew fairly well on the surfaces of skin and body cavity. Of 171 strains assigned to the genus Aeromonas, 88% were identified to phenospecies and putative genospecies level by using comprehensive biochemical schemes. The isolates were allocated to putative hybridization groups (HGs) 1 and 3 Aeromonas hydrophila (29%); putative HG 8 Aeromonas veronii biovar sobria (19%); putative HG 2 Aeromonas bestiarum (18%); putative HG 9 Aeromonas jandaei (16%); putative HGs 4 and 5a Aeromonas caviae (2%); putative HG 12 Aeromonas schubertii (2%); and putative HG 11 (unnamed, 0.6%). The remaining 20 isolates (12%) resembled A. schubertii but could not be allocated to currently recognized phenospecies or to putative HGs. Although cultured rainbow trout yielded strains of putative HGs 1, 4, and 8, which appear to be of major clinical importance, most isolates assigned to putative HGs 1 and 8 were recovered from pike. Differences among HGs found in wild animals could be related to their origin (unpolluted rivers for brown trout and urban rivers for pike). The recovery of these aeromonads species was not related to sampling site. The initial levels of motile aeromonads, their behavior during storage, and the strong potential spoilage activity of most isolates confirm that these bacteria can contribute to deterioration of iced wild freshwater fish. Although adequate cooking would inactivate motile aeromonads, the high incidence of isolates belonging to gastroenteritis-associated HGs should be regarded as a potential health concern, particularly for susceptible populations when there is a possibility of cross-contamination.

Cytochrome P450 1A- and stress protein-induction in early life stages of medaka (Oryzias latipes) exposed to trichloroethylene (TCE) soot and different fractions

It has previously been shown that trichloroethylene (TCE) soot extracts cause dioxinlike toxic effects in medaka fish (Oryzias latipes) and primary liver cell culture of rainbow trout (Oncorhynchus mykiss). This study examines embryonic and larval induction of cytochrome P450 1A and stress proteins after exposure of medaka embryos to extracts and fractions of TCE combustien-generated aerosols. Embryos were exposed to three concentrations of whole soot extract (WE; 2.7, 7.2 and 18 μg l -1 incomplete combustion by-products), TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, 3 ng l -1 ) and four TCE fractions with different polarity (Fr 1-4; 18 μg l -1 ) for 8 days. Approximately 50% of the embryos were then transferred to control water and allowed to hatch. EROD activity in embryos was significantly higher than in controls after the 8 day-exposure to TCE soot extract (WE), with activity being highest at 2.7 μg l -1 WE (5.6× control). Of TCE fractions, only fraction 1 (Fr1, non-polar compounds) caused a significant increase in EROD activity. In larvae, significantly induced EROD activity was detected following the 7.2 μg l -1 WE treatment (3.30 pmol min -1 mg prot. -1 ). Dioxin treatment did not result in increased embryonal or larval EROD activity. Larval CYP 1A was localized mainly in liver, gut, kidney, cornea and chondrocytes of cranium and tail. Hsp70 was induced in larvae but not in embryos. Statistically significant induction over controls was observed in two WE groups (2.7, 7.2 μg l -1 ) and in the group exposed to dioxin (WE 18 μg l -1 not analysed). Mean hsp60 levels were not significantly higher than controls. Apparent bacterial contamination may have induced hsp70 in one control group including embryos and larvae (C/Fr3).

Heat shock protein 70 levels in rainbow trout primary epidermal cultures in response to 2,4-dichloroaniline exposure: A novelin vitro aquatic toxicity marker

The aim of this work was to investigate the use of the heat shock protein, HSP 70, as a sublethal measurement of ecotoxicity and to identify if the amount of HSP 70 synthesized is proportional to the chemical stress applied. This was achieved by quantifying the HSP 70 levels in primary cultured rainbow trout, Oncorhynchus mykiss (R.), skin epidermal cells in response to 2,4-dichloroaniline (2,4-DCA) exposure. The cellular stress response protects organisms from damage resulting from exposure to a wide variety of stressors including xenobiotics. The use of a HSP 70 polyclonal antibody on rainbow trout primary epidermal skin cultures exposed to 2,4-DCA was investigated as a possible biomarker for environmental stress using an immunocytochemical approach. The epidermis is highly susceptible, as it is the interface between the fish and its aquatic environment. In this study we have developed a simple in vitro system for aquatic-toxicity risk assessment. A method for the quantification of heat shock (stress) protein levels by immunocytochemistry is described. The antibody dilution range enabled the detection and quantification of only the inducible HSP 70 fraction. A 1:2000 dilution was decided upon. This assay was effective in detecting and quantifying the induced HSP 70. There was a direct toxicant concentration-dependent increase in the levels of the cellular stress protein in the primary epidermal cultures. Enhanced localization of HSP 70 in the nuclei of the epidermal cells was observed following exposure to 2,4-DCA. This work indicated the possibility of using heat shock protein induction and subsequent quantification as a sensitive system for aquatic toxicity risk assessment.

Genetic analyses reveal unusually high diversity of infectious haematopoietic necrosis virus in rainbow trout aquaculture.

Infectious haematopoietic necrosis virus (IHNV) is the most significant virus pathogen of salmon and trout in North America. Previous studies have shown relatively low genetic diversity of IHNV within large geographical regions. In this study, the genetic heterogeneity of 84 IHNV isolates sampled from rainbow trout (Oncorhynchus mykiss) over a 20 year period at four aquaculture facilities within a 12 mile stretch of the Snake River in Idaho, USA was investigated. The virus isolates were characterized using an RNase protection assay (RPA) and nucleotide sequence analyses. Among the 84 isolates analysed, 46 RPA haplotypes were found and analyses revealed a high level of genetic heterogeneity relative to that detected in other regions. Sequence analyses revealed up to 7.6% nucleotide divergence, which is the highest level of diversity reported for IHNV to date. Phylogenetic analyses identified four distinct monophyletic clades representing four virus lineages. These lineages were distributed across facilities, and individual facilities contained multiple lineages. These results suggest that co-circulating IHNV lineages of relatively high genetic diversity are present in the IHNV populations in this rainbow trout culture study site. Three of the four lineages exhibited temporal trends consistent with rapid evolution.

Antiestrogenicity of β-naphthoflavone and PAHs in cultured rainbow trout hepatocytes: evidence for a role of the arylhydrocarbon receptor

The aims of the present study were to assess, (1) if polyaromatic hydrocarbons (PAHs) are able to inhibit estradiol-regulated vitellogenin synthesis in fish; and (2) if this antiestrogenic activity is mediated through the binding of PAHs to the arylhydrocarbon receptor (AhR). Cultured liver cells of rainbow trout, Oncorhynchus mykiss, were co-exposed to PAHs and 17β-estradiol (E2), and the resulting effects on induction of AhR-regulated 7-ethoxyresorufin- O-deethylase (EROD) activity and on E2-regulated vitellogenesis were investigated. The following test compounds were compared: the PAH 3-methylcholanthrene (3MC), which is a strong EROD inducer, the PAH anthracene (ANT), which is not an inducer of EROD activity, and the model EROD inducer, β -naphthoflavone (βNF). 3MC and βNF led to significant decreases of E2-triggered hepatocellular VTG synthesis, whereas ANT exerted no antiestrogenic activity. The rank order of the antiestrogenic activity of the test substances agreed with their EROD-inducing potency suggesting that their antiestrogenicity might be mediated through the AhR. Further evidence for this assumption comes from the observation that inhibitors such as α-naphthoflavone which interferes with ligand–AhR binding, and 8-methoxypsoralen (8MP), which prevents binding of the occupied AhR to responsive DNA elements, clearly reduced the antiestrogenic effects of the xenobiotics. Furthermore, from the comparison of estradiol concentrations in media of liver cells exposed to the CYP 1A-inducing agents and in media of control cells it is unlikely that the observed antiestrogenic effects were caused by an enhanced E2 catabolism. In conclusion, the results from this study indicate that, (1) AhR-binding PAHs possess an antiestrogenic activity; and (2) that the antiestrogenic activity is mediated through the AhR.

Enhanced susceptibility of seawater cultured rainbow trout, Oncorhynchus mykiss (Walbaum), to the microsporidian Loma salmonae during a primary infection with the sea louse, Lepeophtheirus salmonis

Enhanced susceptibility of seawater cultured rainbow trout, <i>Oncorhynchus mykiss</i> (Walbaum), to the microsporidian <i>Loma salmonae</i> during a primary infection with the sea louse, <i>Lepeophtheirus salmonis</i>

An in vitro assessment of the toxicity of 2,4‐dichloroaniline using rainbow trout primary epidermal cell cultures

Abstract2,4‐Dichloroaniline (2,4‐DCA) is a common pollutant of freshwaters. Information on the effects of this chemical on the aquatic environment is lacking. In this work, we present a technique using primary cell culture of rainbow trout (Oncorhynchus mykiss) epidermal cells as a model for the evaluation of the potential ecotoxicologic hazards of 2,4‐DCA. The effects of a wide dose range of the chemical on survival and function of the cells were studied. 2,4‐Dichloroaniline was found to show an increasingly toxic effect over the dose range of 100 to 1,000 μM. The toxicity threshold was observed to occur at approximately 400 μM in serum‐containing media and 200 μM in serum‐free media. The LC50 in serum‐containing media at 24, 48, 72, and 96 h of exposure ranged from 650 to 740 μM. In serum‐free media the LC50 ranged from 340 to 500 μM over a similar time period. Therefore, the toxicity of 2,4‐DCA to the primary cells increased in the absence of serum in the culture medium. As the 2,4‐DCA concentration increased the number of goblet (mucus) cells present in the cultures decreased, as did the normal healthy epidermal cells. Cell death was predominantly necrotic as opposed to apoptotic. This study is the first demonstration of an in vitro technique using fish primary epidermal cultures as a toxicology assessment tool. The major advantage of using primary cultures as opposed to immortalized cell lines is in the ability of these cultures to maintain their in vivo characteristics for approximately 10 d postexposure, allowing the study of the acute effects of aquatic contaminants. The characteristics of established cells deviate substantially from those of normal cells.

Effects of Selected Chemotherapeutants on Nitrification in Fluidized-Sand Bioftlters for Coldwater Fish Production

Four fish chemotherapeutants, formalin, benzalkonium chloride, chloramine-T, and hydrogen peroxide were evaluated for their effect on the nitrification efficiency of fluidized-sand biofilters. The chemotherapeutants were added at conventional concentrations to two small-scale (2,200 L) coldwater recirculating rainbow trout (Oncorhynchus mykiss) culture systems each containing six fluidized-sand biofilters operating in parallel. Nitrification efficiency of biofilters was calculated before and after chemotherapeutant treatments by determining ammonia removal efficiency at ambient conditions, and also when challenged with a sudden increase of ammonium chloride at a concentration four times that of the ambient total ammonia-nitrogen (TAN). Two formalin treatments in recycle bath mode at 167 and 300 ppm were conducted with only the 300 ppm treatment having a significant negative effect on biofilter nitrification efficiency. Four single benzalkonium chloride treatments of one and 2 ppm were conducted; two static bath treatments and two recycle bath treatments. Of these four tests, only the recycle bath treatments caused biofilter nitrification efficiency to be significantly impaired. Two multiple treatments with benzalkonium chloride were conducted: one static bath treatment and one recycle.bath treatment. These treatments caused ammonia removal efficiency to decrease by 18% in the static bath treatment and by 63% in the recycle bath treatment. Of these two tests, only the recycle bath treatment caused a significant impairment of nitrification. Single static bath and recycle bath treatments with 9 ppm of chloramine-T both resulted in significant impairment of nitrification, as did a 12 ppm multiple static bath treatment. A single static bath treatment with 100 ppm of hydrogen peroxide caused almost total failure of nitrification within 24 h of treatment but biofilters were able to remove 23% of TAN within 48 h of treatment.

Factors influencing the expression of interleukin-1β in cultured rainbow trout ( Oncorhynchus mykiss) leucocytes

The dose dependency and kinetics of lipopolysaccharide (LPS) induced interleukin-1β (IL-1β) mRNA expression in rainbow trout leucocytes has been studied. Northern blot analysis revealed weak hybridisation of a trout IL-1β probe to RNA from head kidney leucocytes stimulated with 0.1 μg LPS/ml, with a large increase in IL-1β transcript level seen between 0.1 and 1.5 μg LPS/ml. Using 5 μg LPS/ml expression was first detectable 1–2 h post-stimulation. By 4 h post-stimulation maximal induction was seen but by 24–48 h the level had fallen and by 72 h no transcript was detectable. Culture temperature had a marked effect on IL-1β expression, with low temperatures inhibiting transcription. Indeed, an eight-fold increase in IL-1β transcript level was seen between cells cultured at 4°C and 22°C. Preincubation with cortisol was also shown to inhibit LPS-induced IL-1β expression.

Aquaculture–agriculture systems integration: an Australian prospective

The concept of integrating aquaculture with irrigated farming systems was evaluated in three projects in the Goulburn–Murray Irrigation District (GMID) in south‐eastern Australia. (1) Semi‐intensive cage culture trials using silver perch, Bidyanus bidyanus (Mitchell), were undertaken in a range of irrigated farming systems: irrigation supply channels; groundwater supplies; and on‐farm storage dams. Survival and growth of fish under these conditions were comparable to fish reared in conventional aquaculture ponds. However, poor water quality at some sites reduced survival and growth. (2) A study was undertaken to assess the feasibility of the cage culture of silver perch and rainbow trout, Oncorhynchus mykiss (Walbaum), in three irrigation water storage reservoirs. Conditions within each reservoir, particularly water quality, wind and wave action, and cage fouling, varied considerably, and influenced fish growth and survival. (3) Mariculture trials were undertaken in two saline groundwater evaporation basins, which were part of an integrated agriforestry and salt reclamation system. The species tested included two oyster, two prawn and nine fish species. Some species exhibited exceptional survival and growth rates under trial conditions. These projects indicate that the concept of integrating aquaculture with existing irrigated farming systems has the potential to enhance productivity, water use efficiency and overall environmental sustainability in the GMID.

AN IN VITRO ASSESSMENT OF THE TOXICITY OF 2,4-DICHLOROANILINE USING RAINBOW TROUT PRIMARY EPIDERMAL CELL CULTURES

2,4-Dichloroaniline (2,4-DCA) is a common pollutant of freshwaters. Information on the effects of this chemical on the aquatic environment is lacking. In this work, we present a technique using primary cell culture of rainbow trout (Oncorhynchus mykiss) epidermal cells as a model for the evaluation of the potential ecotoxicologic hazards of 2,4-DCA. The effects of a wide dose range of the chemical on survival and function of the cells were studied. 2,4-Dichloroaniline was found to show an increasingly toxic effect over the dose range of 100 to 1,000 μM. The toxicity threshold was observed to occur at approximately 400 μM in serum-containing media and 200 μM in serum-free media. The LC50 in serum-containing media at 24, 48, 72, and 96 h of exposure ranged from 650 to 740 μM. In serum-free media the LC50 ranged from 340 to 500 μM over a similar time period. Therefore, the toxicity of 2,4-DCA to the primary cells increased in the absence of serum in the culture medium. As the 2,4-DCA concentration increased the number of goblet (mucus) cells present in the cultures decreased, as did the normal healthy epidermal cells. Cell death was predominantly necrotic as opposed to apoptotic. This study is the first demonstration of an in vitro technique using fish primary epidermal cultures as a toxicology assessment tool. The major advantage of using primary cultures as opposed to immortalized cell lines is in the ability of these cultures to maintain their in vivo characteristics for approximately 10 d postexposure, allowing the study of the acute effects of aquatic contaminants. The characteristics of established cells deviate substantially from those of normal cells.

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.

Use of rainbow trout primary epidermal cell cultures as an alternative to immortalized cell lines in toxicity assessment: A study with nonoxynol

AbstractIncreased concern has centered around environmental contaminants that exert estrogenic effects. A class of nonionic surfactants, the nonylphenol ethoxylates (such as the compound nonoxynol), are an example of one such group of contaminants. We studied the effects of nonoxynol (α‐(4‐nonylphenyl)‐ω‐hydroxypoly‐(oxy‐1‐ethanediyl)) on an epithelial primary culture. The culture system that was employed is a development of an in vitro technique for the culture of rainbow trout (Onchorynchus mykiss) epithelium, one that allows for the culture of both epithelial cells and goblet cells. The LC50 after 24 h was found to be 16.6 μmol/L. The LC50 after 48 h was 12.9 μmol/L. Nonoxynol was found to exhibit an acute lethal dose of 75 μmol/L. A decrease in the number of goblet cells present in the system was observed as exposure to nonoxynol increased. Nonoxynol was found to kill epithelial cells mainly by necrosis. A comparative study of the effects of nonoxynol on the epithelium papulosum cyprini cell line was also carried out. The LC50 of nonoxynol on this cell type was 4.1 μmol/L, with an acute lethal dose of 10 μmol/L.

Genetic vaccination of rainbow trout against viral haemorrhagic septicaemia virus: small amounts of plasmid DNA protect against a heterologous serotype

Viral haemorrhagic septicaemia (VHS) is known as one of the most important diseases in cultured rainbow trout in Europe. An efficient vaccine is highly desirable, but so far only limited success has been obtained with traditional products based on killed or attenuated virus. Genetic immunization with a plasmid vector containing the VHS virus glycoprotein gene under the control of a cytomegalovirus promoter has recently been shown to induce high levels of protection against the homologous virus isolate. Expressed glycoprotein could be detected immunohistochemically in fish muscle and about 70% of the vaccinated animals had neutralizing antibodies in their serum. To further evaluate the potential of the DNA vaccine technology for prophylaxis of VHS, a vaccination trial including lower doses of DNA and different virus isolates was performed. Eight weeks after injection, rainbow trout were challenged by immersion with the homologous virus isolate or with a serologically different isolate. Cumulative mortalities demonstrated that even the lowest dose of DNA tested (0.1 μg per fish) induced protective immunity against both virus isolates. Virus neutralization tests in cell culture indicated that trout sera neutralized VHS virus isolates independently of serotypes defined with mammalian mono- and polyclonal antibodies. No protecion was observed following vaccination with a plasmid construct carrying the VHS virus nucleocapsid–protein gene.

Differential effect of insulin-like growth factor I on in vitro gonadotropin (I and II) and growth hormone secretions in rainbow trout (Oncorhynchus mykiss) at different stages of the reproductive cycle.

The short-term effect of insulin-like growth factor I (IGF-I) on GTH I (FSH-like), GTH II (LH-like), and GH production by cultured rainbow trout pituitary cells was studied in immature fish of both sexes, at early gametogenesis and in spermiating and periovulatory animals. IGF-I had no effect on basal GTH I and GTH II release, whereas it always inhibited basal GH, showing decreasing intensity with the gonad maturation. In absence of IGF-I, GTH I and GTH II cells were always responsive to GnRH, whereas no response was observed for GH cells whatever the sexual stage. The action of IGF-I on the sensitivity to GnRH differs between GTH and GH cells. The former requires a coincubation with IGF-I (10(-6) M)/GnRH to show an increase in sensitivity, independent of the sexual stage. To be responsive to GnRH, the GH cells require longer exposure to IGF-I, the efficiency of which decreases with gonad maturation. The action of IGF-I (10(-6) M) on GTH cell sensitivity to GnRH does not seem to be related to a mitogenic effect or to an improvement in cell survival. It seems to be IGF-I specific, not passing via the insulin receptor. Certain hypotheses on the putative role of IGF-I and GnRH as a link between growth and puberty are suggested.

Differential Effect of Insulin-Like Growth Factor I on in Vitro Gonadotropin (I and II) and Growth Hormone Secretions in Rainbow Trout (Oncorhynchus mykiss) at Different Stages of the Reproductive Cycle

The short-term effect of insulin-like growth factor I (IGF-I) on GTH I (FSH-like), GTH II (LH-like), and GH production by cultured rainbow trout pituitary cells was studied in immature fish of both sexes, at early gametogenesis and in spermiating and periovulatory animals. IGF-I had no effect on basal GTH I and GTH II release, whereas it always inhibited basal GH, showing decreasing intensity with the gonad maturation. In absence of IGF-I, GTH I and GTH II cells were always responsive to GnRH, whereas no response was observed for GH cells whatever the sexual stage. The action of IGF-I on the sensitivity to GnRH differs between GTH and GH cells. The former requires a coincubation with IGF−I (10−6m)/GnRH to show an increase in sensitivity, independent of the sexual stage. To be responsive to GnRH, the GH cells require longer exposure to IGF-I, the efficiency of which decreases with gonad maturation. The action of IGF-I (10−6m) on GTH cell sensitivity to GnRH does not seem to be related to a mitogenic effect or to an improvement in cell survival. It seems to be IGF-I specific, not passing via the insulin receptor. Certain hypotheses on the putative role of IGF-I and GnRH as a link between growth and puberty are suggested.

The immune response of rainbow trout (Oncorhynchus mykiss) againstAphanomyces invadans

Aphanomyces invadans, also referred toA. invaderisorA. piscicida, is the aetiological agent of epizootic ulcerative syndrome (EUS). Immune responses of rainbow trout (Oncorhynchus mykiss) against this fungus were examined, including both antibody and macrophage responses. The fish antibodies to the fungus, assessed by an enzyme-linked immunosorbent assay (ELISA) were generally non-specific, but sera from fish vaccinated withA. invadansreacted with bands at 55 and 40kDa by Western blot analysis. Macrophage activity was examinedin vitroby culturing rainbow trout head kidney macrophages with fungal spores. The macrophages were able to phagocytose sporesin vitroand, in some cases, hyphae were observed growing from germinated spores within the macrophage. When low concentrations of spores (1×102and 1×103spore ml−1) were cultured with macrophage monolayers, no germination of spores or growth of hyphae was observed. At concentrations of 1×104spore ml−1or higher, macrophages were unable to kill all the spores and fungal mycelium eventually overgrew the culture. Transmission electron microscopy (TEM) was used to examine the ultrastructural interaction between macrophages and the fungus. The possible role of rainbow trout as a model forA. invadansinfection in salmonids is also discussed.

Characterization of intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus.

Viral hemorrhagic septicemia virus (VHSV) infections cause high losses in cultured rainbow trout in Europe. Attempts to produce a recombinant vaccine based on the transmembrane glycoprotein (G protein) have indicated that proper folding is important for the antigenicity and immunogenicity of the protein. The present study was initiated to identify the disulfide bonds and other structural aspects relevant to vaccine design. The N-terminal amino acid residue was identified as being a pyroglutamic acid, corresponding to Gln21 of the primary transcript. Peptides from endoproteinase-degraded G protein were analyzed by mass spectrometry before and after chemical reduction, and six disulfide bonds were identified: Cys29-Cys339, Cys44-Cys295, Cys90-Cys132, Cys172-Cys177, Cys195-Cys265, and Cys231-Cys236. Mass spectrometric analysis in combination with glycosidases allowed characterization of the glycan structure of the G protein. Three of four predicted N-linked oligosaccharides were found to be predominantly biantennary complex-type structures. Furthermore, an O-linked glycan near the N terminus was identified. Alignment of the VHSV G protein with five other rhabdovirus G proteins indicates that eight cysteine residues are situated at conserved positions. This finding suggests that there might be some common disulfide bonding pattern among the six rhabdoviruses.

Short Communication Economic dimension of gas bubble disease effects on rainbow trout culture

Short Communication Economic dimension of gas bubble disease effects on rainbow trout culture