Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss)

AIMS: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. METHODS AND RESULTS: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. CONCLUSIONS: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.     

Characterization of 38 polymorphic microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Thirty‐eight new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage of heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Five markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

Diagnostic single nucleotide polymorphisms for identifying westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss)

We describe 12 diagnostic single nucleotide polymorphism (SNP) assays for use in species identification among rainbow and cutthroat trout: five of these loci have alleles unique to rainbow trout (Oncorhynchus mykiss), three unique to westslope cutthroat trout (O. clarkii lewisi) and four unique to Yellowstone cutthroat trout (O. clarkii bouvieri). These diagnostic assays were identified using a total of 489 individuals from 26 populations and five fish hatchery strains.     

Mitochondrial and nuclear DNA sequence variability among populations of rainbow trout (Oncorhynchus mykiss)

Mitochondrial and nuclear DNA variability was examined to assess population genetic structure and phylogeographic relationships in rainbow trout. Single-strand conformation polymorphisms and restriction site differences within 1055 bp of the mitochondrial D-loop region and 1566 bp of nuclear DNA in six single-copy nuclear DNA regions identified 31 mitochondrial genotypes and 50 nuclear alleles. Gene trees were constructed by sequencing each variant allele or mitochondrial genotype identified. Examination of 30 populations in 10 native rainbow trout groups using an analysis of molecular variance ( AMOVA ) indicated that 65% of mitochondrial variability and 35% of nuclear variability was explained by differences among the 10 groups. Phylogenetic patterns evident in mitochondrial and nuclear DNA were not always concordant. Differences in the evolutionary patterns detected by mitochondrial and nuclear DNA may reflect the differential impact of past introgression events on variability in the two genomes.     

Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine

Aims:  To characterize two probiotic carnobacterial isolates, Carnobacterium maltaromaticum (B26) and C. divergens (B33), derived from rainbow trout ( Oncorhynchus mykiss ) intestine.
Methods and Results:  Both cultures, which were able to colonize the fish gut mucosal layer, comprised nonsporogenous, nonmotile, Gram-positive, catalase and oxidase-negative rods. The growth of both carnobacteria occurred between 0 and 37°C, in 0–10% (w/v) NaCl and at pH 5–10. Specifically, strain B26 grew in nutrient broth supplemented with 15% (w/v) NaCl. The most abundant cellular fatty acid of both cultures was 9-octadecenoic acid (18 : 1 n -9) (B26 = 52·6%; B33 = 40·6%), which was characteristic of Carnobacterium . Both cultures were inhibitory to Aeromonas salmonicida , Aer. hydrophila , Streptococcus iniae and Vibrio anguillarum , and strain B33 inhibited Listeria monocytogenes . Both carnobacteria, which did not contain plasmids, produced inhibitory compounds against Gram-positive and Gram-negative bacteria.
Conclusions:  Both probiotic cultures, B26 and B33, had unique phenotypic characteristics and showed a broad spectrum of antibiotic resistance against varying pathogenic bacteria.
Significance and Impact of the Study:  The results of this study contribute to new information and significance of carnobacterial species.     

Characterization of twenty‐four microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Twenty‐four new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Seven markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

Brain distribution of myosin Va in rainbow trout Oncorhynchus mykiss

This study presents data on myosin Va localization in the central nervous system of rainbow trout. We demonstrate, via immunoblots and immunocytochemistry, the expression of myosin Va in several neuronal populations of forebrain, midbrain, hindbrain and spinal cord. The neuronal populations that express myosin Va in trout constitute a very diverse group that do not seem to have many specific similarities such as neurotransmitters used, cellular size or length of their processes. The intensity of the immunoreactivity and the number of immunoreactive cells differ from region to region. Although there is a broad distribution of myosin Va, it is not present in all neuronal populations. This result is in agreement with a previous report, which indicated that myosin Va is approximately as abundant as conventional myosin II and kinesin, and it is broadly involved in neuronal motility events such as axoplasmatic transport. Furthermore, this distribution pattern is in accordance with what was shown in rats and mice; it indicates phylogenetic maintenance of the myosin Va main functions.     

Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

A suite of 26 PCR‐based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species‐specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.     

Metal-ion-mediated oxidative stress in the gill homogenate of rainbow trout (Oncorhynchus mykiss)

Human activities play a major role in toxic and carcinogenic metal pollution of the environment. This study was undertaken to evaluate the effects of copper and mercury at the 400-to 1000-μM concentration range on some biochemical markers of oxidative stress, such as lipid peroxidation (LPO), glutathione-S-transferase (GST) activity, and reduced glutathione (GSH) content in the rainbow trout gill homogenates with or without supplementation of manganese, selenium, and bovine serum albumin (BSA). The integrity of DNA was also measured to assess metal ion toxicity. The results showed that the LPO and specific activity of GST were elevated. This indicated that cell-protecting antioxidant mechanisms were overtaxed and could not prevent membrane peroxidation. Following the addition of metals, the GSH content was also significantly reduced in a concentration-dependent manner. Mercury was found to be more effective than copper. The application of antioxidants proved beneficial in inhibiting LPO, reducing GST activity, and elevating the GSH levels in the gill samples. Manganese was more effective than selenium and BSA. Surprisingly, when BSA (1.0%) was added to the gill homogenates treated with a 1000-μM concentration of metal ions, instead of alleviating malondialdehyde (MDA) generation, a drastic elevation in the MDA levels, alleviation in GST activity, and a further decrease in glutathione (GSH) levels were observed, which were most likely the result of pro-oxidant activity of BSA. The results also indicated that mercury and copper functioned as genotoxic pollutants, which altered the DNA integrity by inducing the single and double-stranded DNA breaks in the gill cell nuclei. Collectively, toxicity of metal ions is related to the depletion of GSH content and inhibition of antioxidant enzyme GST, resulting in the propagation of LPO and DNA damage.     

Analysis of chorion hardening of eggs of rainbow trout, Oncorhynchus mykiss

We estimated changes of chorion hardness of rainbow trout (Oncorhynchus mykiss) egg by the use of three parameters, namely increase of resistance of an egg to rupture by extraneously applied pressure, decrease of solubility of chorion proteins in 8 mol/L urea and a change in the content of γ-glutamyl-ε-lysine crosslink. Unfertilized egg chorions became hardened after egg activation. During chorion hardening, 49, 56 and 65 kDa protein components of the chorion gradually disappeared, high molecular weight intermediates (113,160–170 and higher than 250 kDa) were newly formed and, finally, all components became undetectable by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The content of γ-glutamyl-ε-lysine (γ-Glu-ε-Lys) crosslink in the chorion increased after hardening. Chorion hardening was inhibited by the incorporation of monodansyl-cadaverine, a competitive inhibitor for transglutaminase (TGase), into the chorions. TGase activity was detected in unfertilized eggs and localized in the chorion fraction rather than in the ooplasmic fraction. The findings suggest that chorion hardening depends upon polymerization of the chorion components by TGase-dependent γ-Glu-ε-Lys crosslink formation.     

Amitosis‐like nuclear division in erythrocytes of triploid rainbow trout Oncorhynchus mykiss

This work shows that the atypical erythrocytes in triploid rainbow trout Oncorhynchus mykiss were morphologically similar to those of toads. The nuclei of the cells can be bell‐shaped, constricted or irregular. It is presumed that such nuclear division is probably amitosis.     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

溶解氧对虹鳟呼吸机能及红细胞特性的影响

目的：初步研究溶解氧对虹鳟呼吸机能及红细胞特性的影响。方法：设四组溶氧水平,测定呼吸频率、血液中PO2、PCO2和pH值,并以RBC、Hb、脆性、膜粘滞性及丙二醛含量等指标观察溶氧对红细胞的影响。结果：溶解氧升高使鱼呼吸频率降低,血液PO2升高（P〈0.05）;高溶氧导致红细胞数、丙二醛含量降低。红细胞膜粘滞系数在试验第10d时,高氧组明显高于其它组,但30d时各溶氧组差异不显著。结论：在一定范围内溶解氧升高可以改善机体组织氧含量,提高呼吸功能,红细胞对溶氧变化有其生理适应性。     

Cortisol and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for high or low tolerance to stress

The influence of exposure to stressors on cortisol and the non-specific immune traits lysozyme and serum haemolytic activity were examined in second generation rainbow trout ( Oncorhynchus mykiss ) selected for either high or low serum cortisol level following a confinement stress. Lysozyme and serum haemolytic activity were also assessed, together with levels of specific antibodies against Aeromonas salmonicida A-layer, Vibrio salmonicida O-antigen and Vibrio anguillarum O-antigen, following injection of vaccines against these pathogens.
Significant differences in mean cortisol levels between the two selection lines were observed, but in only one of two stress experiments was the 'high-stress' line found to have the higher cortisol level; in the other experiment the 'high-stress' line had significantly lower cortisol levels than the 'low-stress' line. Lysozyme levels were in four of four assessments higher in the high-stress line than in the low-stress line, whereas components of serum haemolytic activity tended to be lower in the high-stress line than in the low-stress line. Levels of specific antibodies against all three bacterial pathogens were elevated following the injection of the vaccines. Only antibody production against A. salmonicida A-layer was significantly different between the two lines, the higher production of antibody being in the high-stress line.     

Histology and carbohydrate histochemistry of the alimentary canal in the rainbow trout Oncorhynchus mykiss

A histological and histochemical analyses were carried out on the entire alimentary canal of the rainbow trout Oncorhynchus mykiss . In particular the oesophageal region showed presence of terminal β‐D‐galactose(1–3)‐N‐acetylgalactosamine and α‐N‐acetylgalactosamine. In the anterior and posterior regions of the stomach, lining epithelium and gastric pits exhibited the presence of β‐gal and α‐GalNAc. In addition sialoglycoconjugates having sialic acid–β–galactose(1–3)‐N‐acetylgalactosamine and sialic acid‐N‐acetylgalactosamine as terminal tri‐ and di‐saccharides, were demonstrated. In proximal and distal intestine goblet cells showed the presence of sialoglyconjugates, having sialic acid‐β‐gal(1–3)‐GalNAc and sialic acid‐GalNAc as terminal sequences, belonging to N‐linked chains. In the enterocytes of the entire intestine, terminal GlcNAc, α‐Gal, α‐fucose were found.     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re‐arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.