Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.     

Effects of ambient colour on colour preference and growth of juvenile rainbow trout Oncorhynchus mykiss (Walbaum)

Colour preference of individual juvenile rainbow trout Oncorhynchus mykiss was tested at 1 and 12° C, and also at 12° C after a 42 day growth experiment under white, blue, green, yellow or red ambient colour. All experiments were carried out under controlled laboratory conditions and the preference was assessed by the location of the fish in a preference tank with four chambers. Rainbow trout showed a preference for blue and green at 1° C and for green at 12° C. After the growth experiment the fish reared in blue tanks preferred blue and green but green was the most preferred colour for the fish reared in green, yellow and red tanks. Yellow and especially red chambers were avoided, irrespective of the ambient colour during the growth trial. The final mass of fish reared in the red aquaria was significantly smaller than that of the fish in green tanks. In addition, when the data of the preference tests were correlated with the data of the growth experiment using mean values of the four tested colours, a very good linear relationship was observed between the preference ( i.e. visit frequency in coloured compartments) and growth rate as well as food intake. When considering the results both from the preference and growth trials it is suggested that green is the best environmental colour for rearing juvenile rainbow trout while rearing in a red environment cannot be recommended.     

Dietary vitamin E and the response of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Yersinia ruckeri

Rainbow trout fed diets containing 7, 86 or 806 mg vitamin E kg⁻¹ for 22 weeks were exposed to virulent Yersinia ruckeri by bath or injection. Mortalities were always least among those fed the highest concentration of vitamin E but serum antibody production was not affected by vitamin E levels.     

The ontogeny of drinking in the rainbow trout, Oncorhynchus mykiss (Walbaum)

Drinking was measured by the rate of uptake of tritiated dextran in fresh water by the alevins and fry of rainbow trout at various stages of development during a 40-day post-hatch period. Drinking increased almost S-fold during the initial 16 day yolk-sac stage. Drinking rate increased most between 16 and 23 days, the transitional period between yolk-sac absorption and first feeding. The maximum weight-specific drinkingrateof 3.24μlg⁻¹ h⁻¹ recorded for 40-day-old fry was higher than previously recorded for adults. Abrupt transfer from an adaptation temperature of 10 to 19° C increased drinking significantly (Q₁₀= 1.2), but sudden transfer to 1 1‰ salinity sea water caused a substantial fall in drinking rate in 23-day fry. A 24-h period of adaptation to 1 1‰ and 15‰ salinity restored drinking to a rate similar to that in fresh water. The water drunk by 31-day fry fed to satiation was initially higher than by unfed fry, but drinking rates subsequently fell below the control level. The results are discussed in terms of osmoregulation and the uptake of dissolved or suspended substances by the intestinal tract for the purpose of immunization.     

Environmental influences on cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two groups of wild (lakedwelling and anadromous), and a group of hatchery-reared Oncorhynchus mykiss (Walbaum) (rainbow trout) were sampled in order to measure cardiac morphometrics, haemoglobin concentration, and the DNA and protein concentration in cardiac muscle. A combination of these variables was used to distinguish wild fish from domestic ones.
The wild fish had significantly higher levels of haemoglobin (for male fish, 10.10 and 10.07 g 100 ml⁻¹ vs. 7.69 g d⁻¹) and larger relative ventricle mass (females, 0.091 and 0.099% ofbody mass vs. 0.073%; males, 0.108 and 0.134% vs. 0.102%; immatures, 0.086 and 0.094% vs. 0, 072%, respectively) than the domestic fish. The anadromous and domestic fish had significantly higher amounts of compact tissue when compared with lake fish (females, 43 and 47% of ventricle mass vs. 34%, respectively). Ventricle size distinguished wild fish from domestic fish, except male anadromous and male domestic fish which were distinguished only by haemoglobin and compact tissue values. Immature fishes from all groups had lower total protein levels in the ventricle, lower compact tissue levels, and less haemoglobin. Points regarding the potential environmental influences in determining these cardiovascular trends are discussed.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution

Blood lysozyme activity was determined in rainbow trout that were stressed by handling and transport, or suffered from acute ammonia poisoning. To optimize the lysoplate assay, the pH dependence of the lysozyme activity was studied. For serum and kidney lysozyme, the pH optimum was 5.5 to 6.0. Ventilation frequency and plasma glucoseconcentration of the fish were used as stress indicators. Less severely stressful situations, such as 30 min of handling, brought about either a stimulation or a suppression of the lysozyme activity. Strong stressors, such as transport lasting 2 h or acute water pollution, reduced the lysozyme level significantly. The decrease of lysozyme activity due to handling and transport persisted for 24 h, but the activity returned to normal within 2 weeks following confrontation with the stressor.     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Testing the relationship between domestication and developmental instability in rainbow trout,Oncorhynchus mykiss (Teleostei,Salmonidae)

Fish domestication is an evolutionary process arising in captivity through genetic and developmental mechanisms, producing organisms performing more poorly than wild conspecifics in the natural environment. Culture conditions could be suboptimal for fish at particular life cycle stages, presenting environmental disturbances leading to developmental instability. The limited size of captive lots, moreover, can result in the loss of genetic variation, and the resulting homozygosity (as well as hybridization and mutation) could have strong harmful effects on developmental stability. Rainbow trout are the most widely‐cultured species in Europe and North America, having been in culture for more than a century. Prolonged artificial selection for desired traits and incidental effects of domestication has led to the development of a ‘farmed type’. Fluctuating asymmetry, variations in meristic counts, and skeletal anomalies were examined in several rainbow trout captive and wild clonal lines as indicators of developmental instability. Differences in developmental stability were identified among lines and correlated with different degrees of exposure to captivity. Some relationship between meristic counts and domestication level was found in the present study, with the number of vertebrae and of dorsal pterygiophores and rays being the strongest predictors of the domestication level. However, the occurrence of skeletal anomalies and fluctuating asymmetry were apparently not related to the level of exposure to captivity. The findings of the present study will facilitate the selection of clonal lines with divergent phenotypes for subsequent quantitative trait loci analyses aimed at identifying genome regions linked with morpho‐anatomical and physiological adaptive responses to captivity. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 608–628.     

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.     

Mechanisms of aluminium extraction and accumulation at the gills of rainbow trout, Oncorhynchus mykiss (Walbaum), in acidic soft water

Rainbow trout were fitted with latex masks for the measurement of ventilatory flow rate (V?_w) and with opercular catheters for sampling expired water from close to the branchial surface. Fish were exposed for 6 h to pH 5.1, 4.7 or 4.1 in the presence (138 μg l ¹) or nominal absence (1 μg 1^–1) of Al. Inspired and expired Al concentrations and water pH were measured via the opercular catheters. Gills were sampled for accumulated Al at the end of the experiments. V?_w increased during Al exposures at all three pHs. However, in the absence of Al, V?_w increased only at pH 4.1. Aluminium extraction from the water and Al accumulation on the gills were highest at inspired pH 5.1 and lowest at pH 4.1, and correlated well with expired pH, which was 0.2–0.7 pH units higher than inspired pH. Gill Al accumulations amounted to only about one-tenth of deposition calculated from Al extraction from the water and V?_w, and gill Al was tightly bound to the branchial surface. Calculations of Al solubility, oversaturation, and species composition were made using measured expired pH values, and were compared with Al extraction from the water and measured gill Al concentrations. In general, these analyses indicated that reduced Al solubility near the gills is a reasonable explanation of Al extraction from the water, and that Al(OH)₂⁺ and Al(OH)₃° are the Al species most likely to interact initially at the gills. It is suggested that mucus sloughing removes most precipitated Al, and that only the charged form persists, bound to structural elements on the gill surface. A model incorporating these results, and pH changes in the fish gill micro-environment in general, is presented to explain previously-reported ionoregulatory and respiratory effects of Al.     

The respiratory burst of rainbow trout, Oncorhynchus mykiss (Walbaum), phagocytes is modulated by sympathetic neurotransmitters and the'neuro'peptide ACTH

The phagocytic leukocytes of teleosts, like those of many animal groups, are capable of converting molecular oxygen to a series of highly reactive oxygen intermediates. These constitute part of the cytotoxic defences of the animal. Reactive oxygen intermediates produced in vitro by rainbow trout, Oncorhynchus mykiss (Walbaum), pronephric myeloid cells have been detected by means of luminol-enhanced chemiluminescence, and by the reduction of cytochrome c in the presence and absence of exogenous superoxide dismutase and catalase. A panel of potential biological response modifiers, when added individually to cell suspensions, showed that the catecholamine receptor agonists phenylephrine and isoproterenol differentially modulate the respiratory burst. Receptor antagonists yohimbine, phentolamine and prazosin showed that the effects were receptor-mediated. Adrenocorticotropin (ACTH) enhanced the apparent respiratory burst in both assays. Pathways of neuroimmunological regulation, therefore, include non-lymphoid cells as targets of classical neurotransmitters and a neurohormone.     

Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy

Fixation of mucus for the assessment of biofilms and surface associated pathogens often involves complex and expensive techniques. Rainbow trout killed by an overdose of MS 222 had their gills removed and immersion-fixed gently in buffered glutaraldehyde containing 2% Alcian blue. Control tissues consisted of gills fixed in Alcian blue-free fixative. Trout were also killed and directly immersed in liquid nitrogen and the gills freeze-dried then vapour fixed with osmium tetroxide at −50° C. Following fixation gill tissue was processed for light and electron microscopy. A continuous and intact mucous coat was not detected on tissue fixed by conventional methods but the addition of Alcian blue to the fixative preserved an extensive mucous coat trapped between the lamellae and overlying the epithelia. Electron microscopic examination revealed that mucus preservation with the conventional fixative was poor and intermittent whereas the addition of Alcian blue to the fixative greatly enhanced the preservation of the branchial mucous coat. Mucus appeared as interdispersed flocculant material between the epithelial microridges and formed extensive superficial sheets over the epithelium. Freeze-dried/vapour-fixed gill tissue also provides excellent preservation of the integrity of branchial mucous coats, the mucus appearing as a continuous sheet over the filament and secondary lamellae. However, freeze-dried tissue fails to preserve sufficient cellular integrity for this technique to be useful for light or transmission electron microscopy. The potential for use of glutaraldehyde-Alcian blue fixed-gill tissue diagnostically and in research are discussed.