The Effects of Immunostimulation Through Dietary Manipulation in the Rainbow Trout; Evaluation of Mucosal Immunity

Immunostimulant-containing diets are commonly used in aquaculture to enhance the resistance of cultured fish to disease and stress. Although widespread in use, there have been conflicting results published, and surprisingly little is known about the regulation of immune response-related genes in tissues key to mucosal immunity induced by immunostimulant dietary feeding. Using a salmonid-specific microarray platform enriched with immune-related genes and in situ hybridization, we investigated dietary acclimation in two organs relevant to mucosal immunity, the gills and the intestine, in the rainbow trout (Oncorhynchus mykiss). Immunostimulant diets significantly changed gene expression profiles and gene distribution in a tissue-specific manner: genes and functional Gene Ontology categories involved in immunity were differently expressed at portals of entry where significant changes in genes and functional groups related to remodeling processes and antigen presentation were observed. Furthermore, genes involved in chemotaxis, cell differentiation, antigen-presenting capacity and tissue remodeling were localized in both organs.     

Effects of thermal acclimation on transcriptional responses to acute heat stress in the eurythermal fish Gillichthys mirabilis (Cooper)

The capacities of eurythermal ectotherms to withstand wide ranges of temperature are based, in part, on abilities to modulate gene expression as body temperature changes, notably genes encoding proteins of the cellular stress response. Here, using a complementary DNA microarray, we investigated the sequence in which cellular stress response-linked genes are expressed during acute heat stress, to elucidate how severity of stress affects the categories of genes changing expression. We also studied how prior acclimation history affected gene expression in response to acute heat stress. Eurythermal goby fish (Gillichthys mirabilis) were acclimated to 9 ± 0.5, 19 ± 0.5, and 28 ± 0.5°C for 1 mo. Then fish were given an acute heat ramp (4°C/h), and gill tissues were sampled every +4°C to monitor gene expression. The average onset temperature for a significant change in expression during acute stress increased by ～2°C for each ～10°C increase in acclimation temperature. For some genes, warm acclimation appeared to obviate the need for expression change until the most extreme temperatures were reached. Sequential expression of different categories of genes reflected severity of stress. Regardless of acclimation temperature, the gene encoding heat shock protein 70 (HSP70) was upregulated strongly during mild stress; the gene encoding the proteolytic protein ubiquitin (UBIQ) was upregulated at slightly higher temperatures; and a gene encoding a protein involved in cell cycle arrest and apoptosis, cyclin-dependent kinase inhibitor 1B (CDKN1B), was upregulated only under extreme stress. The tiered, stress level-related expression patterns and the effects of acclimation on induction temperature yield new insights into the fundamental mechanisms of eurythermy.     

Influence of temperature on silver accumulation and depuration in rainbow trout

To assess the influence of water temperature on silver uptake, rainbow trout Oncorhynchus mykiss ( c . 50 g; held at 13° C) were exposed to 0·1 μM AgNO₃ in ion‐poor water for 1 week at 4 and 16° C without previous temperature acclimation. To assess the influence of temperature on elimination of previously accumulated Ag, rainbow trout were exposed to 0·1 μM AgNO₃ in ion‐poor water for 1 week at 12° C, then were randomly divided amongst two Ag‐free water containers, differing only in temperature (3 and 16° C), for 2 months. In the uptake study greater accumulation of Ag was seen in the gills, plasma and especially the livers and bile of 'warm' rainbow trout (16° C) compared to 'cold' rainbow trout (4° C), which can be explained by the higher metabolic rates of the warmer fish. In the depuration study there was no net elimination of Ag from the livers and bile but there was biphasic elimination of Ag from the gills and plasma of 'warm' and 'cold' fish, but with few differences between them. This indicated that temperature‐dependent processes were less important in Ag elimination than in Ag uptake. Toxicokinetic modelling of Ag uptake by livers indicated four‐fold greater uptake of Ag by 'warm' rainbow trout compared to 'cold' rainbow trout (one compartment uptake model). Elimination of previously accumulated Ag from the plasma was best fitted by a two compartment rate‐constant based model, with approximately half the plasma Ag load eliminated within 24 h, followed by slower elimination of Ag over 2 months.     

Peptidylarginine deiminase gene is differentially expressed in freshwater and brackish water rainbow trout

Peptidylarginine deiminase (PADI)-like cDNA sequence was isolated from rainbow trout (Oncorhynchus mykiss). It consists of a 111-bp 5′-untranslated region, a 731-bp 3′-UTR, and a 2,010-bp open reading frame encoding a protein of 669 amino acids. In the presence of calcium ions, PADI enzymes catalyze the post-translational modification reaction generating citrulline residues. Mammalian PADI enzymes are involved in a number of regulatory processes during cell differentiation and development such as skin keratinization, myelin maturation, and histone deimination. Though five PADI isotypes have been isolated from mammals, in bony fish only one PADI enzyme is present, which contains conserved amino acid residues responsible for catalysis and calcium ion-binding. Sequence identity of piscine PADI protein sequences available at gene databases exceeds 67%. Phylogenetic analyses revealed that not only piscine, but also amphibian and avian PADI-like proteins share most identical amino acid residues with mammalian PADI2. mRNA level of trout PADI-like gene is high in skin, fin, gills, brain, and spleen of rainbow trout. Quantitative Real-Time RT-PCR revealed that PADI gene is differentially expressed in liver, trunk kidney, and spleen of two trout strains, the freshwater-cultured STEELHEAD trout and the brackish water strain BORN.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.     

Beta-adrenergic stimulation enhances the heat-shock response in fish

We have taken advantage of the unique properties of nucleated rainbow trout (Oncorhynchus mykiss) red blood cells (rbcs) to demonstrate that beta-adrenergic stimulation with the agonist, isoproterenol, significantly enhanced the heat-induced induction of heat-shock proteins (Hsps) in trout rbcs without affecting hsp expression on its own. Furthermore, this beta-adrenergic potentiation of hsp expression occurred only at physiologically relevant concentrations of adrenergic stimulation. In further experiments, we found that adrenaline increased 100-fold and noradrenaline increased 50-fold in trout after a 1-h heat shock at 25 degrees C, approximately 12 degrees C above acclimation temperature. This is the first time the adrenergic heat-shock response has been described for a temperate fish species. We conclude that beta-adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish.     

Crowding causes prolonged leucopenia in salmonid fish, despite interrenal acclimation

Crowding for 3 weeks significantly reduced the coefficient of condition of both brown trout and rainbow trout. However, acclimation of the hypothalamic-pituitary-interrenal (HPI) axis, as assessed by changes in plasma cortisol levels, occurred within 6 days for brown trout and within 10 days for rainbow trout. Blood lactate levels were significantly reduced in the crowded fish of both species throughout the experiment. Sexual maturation of the male fish significantly elevated the number of circulating red blood cells in both species, reduced the lactate levels in brown trout and elevated cortisol levels in the rainbow trout. Despite the relatively rapid interrenal acclimation, the numbers of thrombocytes and lymphocytes in the blood of both species were significantly reduced during the period of crowding and it is concluded that changes in the composition of circulating blood cells are more reliable indicators of chronic crowding stress than are plasma cortisol levels. These findings are discussed in relation to the role of the HPI axis in suppressing the defence systems of salmonid fish during periods of chronic stress.     

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss)

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.     

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.     

Influence of dissolved organic matter on copper binding, and calcium on cadmium binding, by gills of rainbow trout

Complexation of Cu by 5 mg Cl⁻¹ dissolved organic matter (DOM) from a marsh kept Cu from binding to gills of small rainbow trout Oncorhynchus mykiss in 9-day exposures to 0.5 μM Cu in soft water. The protective effect of DOM occurs because the formation of Cu-DOM complexes reduces the amount of free Cu in the water, so the disruptive effects of Cu on ionoregulation, such as inhibited Na uptake, cannot develop. The Cu-DOM complexes themselves do not bind to the gills. Calcium (1100 μm) reduced the accumulation of Cd by trout gills in short, 2-h exposures through competition for gill binding sites but not over longer, 7-day exposures to 0–14 μM Cd. However, the protective effect of Ca against Cd toxicity persisted throughout the longer experiment, likely due to the decrease in the electrochemical gradient for diffusive loss of Ca from the fish to the water. Rainbow trout and fathead minnows Pimephales promelas accumulated Cu and Cd on their gills in a similar manner; thus, binding constants for metal-gill interactions determined for one species of fish can be generalized to other fish species. When literature binding constants determined for fathead minnows were applied to our studies with rainbow trout, computer modelling of Cu-gill and Cu-DOM interactions simulated our results well. In contrast Cd-gill and Ca-gill modelling predicted the initial competitive effect of Ca against Cd accumulation by trout gills, but did not predict the longer-term accumulation of Cd by trout gills.     

Critical thermal minima and maxima of three freshwater game-fish species acclimated to constant temperatures

A total of 120 critical thermal maxima (CT maxima) and 120 critical thermal minima (CT minima) were determined for channel catfish, largemouth bass and rainbow trout acclimated to three constant temperatures: 20, 25 and 30 °C in catfish and bass, and 10, 15 and 20 °C in trout. Highest mean CT maximum and lowest mean CT minimum measured over these acclimation temperatures were 40.3 and 2.7 °C (catfish), 38.5 and 3.2 °C (bass) and 29.8 and ∼ 0.0 °C (trout). Temperature tolerance data were precise with standard deviations generally less than 0.5 °C. Channel catfish had the largest thermal tolerance scope of the three species while rainbow trout had the lowest tolerance of high temperatures and the highest tolerance of low temperatures. In all species CT minima and CT maxima were highly significantly linearly related to acclimation temperature. Within each species, slopes relating CT maxima to acclimation temperature were approximately half as large as those relating CT minima to acclimation temperature, suggesting that acclimation temperature has a greater influence on tolerance to low rather than high temperatures. Slopes relating both CT minima and CT maxima to acclimation temperature for the two warm-water species were similar and approximately twice those for the rainbow trout. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

Thermoacclimatory changes in blood oxygen binding properties and gill secondary lamellar structure ofSalmo gairdneri

Summary The blood oxygen binding properties and gill secondary lamellar structure of rainbow trout acclimated to several temperatures were studied. The blood oxygen carrying capacity decreased as acclimation temperature increased from 2 to 15 °C; the decrease was probably caused by an increase in plasma volume. Also the blood oxygen affinity decreased as the acclimation temperature increased from 2 to 15 °C. This change had no effect on the oxygen loading in gills, since the efferent arterial oxygen tension was adequate for approximately 100% erythrocytic O₂ saturation at all acclimation temperatures, but facilitated the oxygen unloading in tissues. At the highest acclimation temperature (18 °C) the oxygen loading in gills was facilitated by the changes in the secondary lamellar structure; the proportion of erythrocytes in the secondary lamellar capillaries was higher than at the other acclimation temperatures (2 and 10 °C).     

Increase of Metallothionein-immunopositive Chloride Cells in the Gills of Brown Trout and Rainbow Trout After Exposure to Sewage Treatment Plant Effluents

Metallothionein, a biomarker of exposure and toxicity of heavy metals, has been detected in the gills of brown trout (Salmo trutta fario L.) and rainbow trout (Oncorhynchus mykiss Richardson) by means of immunohistochemistry. A very prominent labelling of chloride cells was found after exposure to diluted sewage plant effluents. No significant increase was observed in either the number of labelled cells or their labelling intensity after exposure to water of a polluted river compared to fish kept in tap water. These results do not correlate with findings of a histopathological study, suggesting that the metal levels at the sewage treatment plant were too low to produce gross histopathology. A comparison between the species indicated that the rainbow trout showed a generally higher metallothionein expression than the brown trout.