Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

The generic term \"Gill disease\" refers to a wide range of disorders that affect the gills and severely impact salmonid aquaculture systems worldwide. In rainbow trout freshwater aquaculture, various etiological agents causing gill diseases have been described, particularly Flavobacterium and Amoeba species, but research studies suggest a more complex and multifactorial aetiology. Here, a cohort of rainbow trout affected by gill disease is monitored both through standard laboratory techniques and 16S rRNA Next-Generation Sequencing (NGS) analysis during a natural disease outbreak and subsequent antibiotic treatment with Oxytetracycline. NGS results show a clear clustering of the samples between pre- and post-treatment based on the microbial community of the gills. Interestingly, the three main pathogenic bacteria species in rainbow trout (Yersinia ruckeri, Flavobacterium psychrophilum, and Flavobacterium branchiophilum) appear to be weak descriptors of the diversity between pre-treatment and post-treatment groups. In this study, the dynamics of the gill microbiome during the outbreak and subsequent treatment are far more complex than previously reported in the literature, and environmental factors seem of the utmost importance in determining gill disease. These findings present a potential novel perspective on the diagnosis and management of gill diseases, showing the limitations of conventional laboratory methodologies in elucidating the complexity of this disease in rainbow trout. To the authors\' knowledge, this work is the first to describe the microbiome of rainbow trout gills during a natural outbreak and subsequent antibiotic treatment. The results of this study suggest that NGS can play a critical role in the analysis and comprehension of gill pathology. Using NGS in future research is highly recommended to gain deeper insights into such diseases correlating gill\'s microbiome with other possible cofactors and establish strong prevention guidelines.

UNASSIGNED: Pharmaceutical residues are widely detected in aquatic environment and can be taken up by nontarget species such as fish. The cytochromes P450 (CYP) represent an important detoxification mechanism in fish, like in humans. In the present study, we assessed the correlation of the substrate selectivities of rainbow trout CYP1A and CYP3A homologues with those of human, through determination of the half-maximal inhibitory concentrations (IC50) of a total sixteen human pharmaceuticals toward CYP1A-like ethoxyresorufin O-deethylase (EROD) and CYP3A-like 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (RT-S9).
UNASSIGNED: The inhibitory impacts (IC50) of atomoxetine, atorvastatin, azelastine, bimatoprost, clomethiazole, clozapine, desloratadine, disulfiram, esomeprazole, felbinac, flecainide, orphenadrine, prazosin, quetiapine, sulpiride, and zolmitriptan toward the EROD and BFCOD activities in RT-S9 were determined using the IC50 shift assay, capable of identifying time-dependent inhibitors (TDI). Additionally, the nonspecific binding of the test pharmaceuticals to RT-S9 was assessed using equilibrium dialysis.
UNASSIGNED: Most test pharmaceuticals were moderate to weak inhibitors of both EROD and BFCOD activity in RT-S9, even if most are noninhibitors of human CYP1A or CYP3A. Only bimatoprost, clomethiazole, felbinac, sulpiride, and zolmitriptan did not inhibit either activity in RT-S9. EROD inhibition was generally stronger than that of BFCOD and some substances (atomoxetine, flecainide, and prazosin) inhibited selectively only EROD activity. The strongest EROD inhibition was detected with azelastine and esomeprazole (unbound IC50 of 3.8 ± 0.5 µM and 3.0 ± 0.8 µM, respectively). None of the test substances were TDIs of BFCOD, but esomeprazole was a TDI of EROD. Apart from clomethiazole and disulfiram, the nonspecific binding of the test pharmaceuticals to the RT-S9 was extensive (unbound fractions <0.5) and correlated well (R 2 = 0.7135) with their water-octanol distribution coefficients.
UNASSIGNED: The results indicate that the P450 interactions in RT-S9 cannot be explicitly predicted based on human data, but the in vitro data reported herein can shed light on the substrate selectivity of rainbow trout CYP1A1 and CYP3A27 in comparison to their human homologues. The IC50 concentrations are however many orders of magnitude higher than average environmental concentrations of pharmaceuticals. The time-dependent EROD inhibition by esomeprazole could warrant further research to evaluate its possible interlinkages with hepatotoxic impacts on fish.

Fish in their natural environments possess elaborate mechanisms that regulate physiological function to mitigate the adverse effects of multiple environmental stressors such as temperature, metals, and hypoxia. We investigated how warm acclimation affects mitochondrial responses to Cd, hypoxia, and acute temperature shifts (heat shock and cold snap) in rainbow trout. We observed that state 3 respiration driven by complex I (CI) was resistant to the stressors while warm acclimation and Cd reduced complex I +II (CI + II) driven state 3 respiration. In contrast, state 4 (leak) respirations for both CI and CI + II were consistently stimulated by warm acclimation resulting in reduced mitochondrial coupling efficiency (respiratory control ratio, RCR). Warm acclimation and Cd exacerbated their individual effect on leak respiration to further reduce the RCR. Moreover, the effect of warm acclimation on mitochondrial bioenergetics aligned with its inhibitory effect on activities of citrate synthase and both CI and CII. Unlike the Cd and warm acclimation combined exposure, hypoxia alone and in combination with warm acclimation and/or Cd abolished the stimulation of CI and CI + II powered leak respirations resulting in partial recovery of RCR. The response to acute temperature shifts indicated that while state 3 respiration returned to pre-acclimation level, the leak respiration did not. Overall, our findings suggest a complex in vivo interaction of multiple stressors on mitochondrial function that are not adequately predicted by their individual effects.

With the rapid and significant cost reduction of next-generation sequencing, low-coverage whole-genome sequencing (lcWGS), followed by genotype imputation, is becoming a cost-effective alternative to single-nucleotide polymorphism (SNP)-array genotyping. The objectives of this study were 2-fold: (1) construct a haplotype reference panel for genotype imputation from lcWGS data in rainbow trout (Oncorhynchus mykiss); and (2) evaluate the concordance between imputed genotypes and SNP-array genotypes in 2 breeding populations. Medium-coverage (12×) whole-genome sequences were obtained from a total of 410 fish representing 5 breeding populations with various spawning dates. The short-read sequences were mapped to the rainbow trout reference genome, and genetic variants were identified using GATK. After data filtering, 20,434,612 biallelic SNPs were retained. The reference panel was phased with SHAPEIT5 and was used as a reference to impute genotypes from lcWGS data employing GLIMPSE2. A total of 90 fish from the Troutlodge November breeding population were sequenced with an average coverage of 1.3×, and these fish were also genotyped with the Axiom 57K rainbow trout SNP array. The concordance between array-based genotypes and imputed genotypes was 99.1%. After downsampling the coverage to 0.5×, 0.2×, and 0.1×, the concordance between array-based genotypes and imputed genotypes was 98.7, 97.8, and 96.7%, respectively. In the USDA odd-year breeding population, the concordance between array-based genotypes and imputed genotypes was 97.8% for 109 fish downsampled to 0.5× coverage. Therefore, the reference haplotype panel reported in this study can be used to accurately impute genotypes from lcWGS data in rainbow trout breeding populations.

The rapid increase in aquaculture over the last several decades has led to concerns about the environmental impact of fish feeds relying on marine resources for fishmeal (FM). We aim to assess Nannochloropsis sp. QH25 co-product as a viable and sustainable replacement for FM in juvenile rainbow trout, Oncorhynchus mykiss, feeds. We formulated four experimental diets: a reference (FM based), 33N, 66N, and 100N diet (33%, 66%, and 100% co-product replacement). Rainbow trout were randomly assigned to one of 16 tanks and randomly assigned an experimental diet to consume throughout the experiment (64 days total), with four replicate tanks per diet. We compared the phosphorus (P) and nitrogen (N) digestibility, emissions, and growth between diets and, compared six environmental impacts (biotic resource use (BRU), global warming potential (GWP), water use, land use, marine eutrophication potential (MEP), and freshwater eutrophication potential (FEP)) of each diet. Our results indicate that replacing FM with co-product did not significantly alter growth. P digestibility of the experimental and reference diets was comparable. BRU conversion ratio was significantly lower in the experimental diets. However, there were significantly higher water and land use conversion ratios but insignificantly higher results in GWP, MEP, and FEP between the reference and 100N diet.

The aim of this study was to evaluate a new Bacillus amyloliquefaciens B-1895 probiotic as a feed additive for farmed trout. Final weight, absolute and average daily gain of fish, and average daily growth rate were higher in the group that received the probiotic than in the control group (p<0.05). Moreover, the probiotic-fed trout had more intense growth rates than the control group (higher by 15.7%; p<0.05). A decrease in feed ratio was also observed in the group that received probiotic (25% decrease; p<0.05), indicating more efficient digestion and assimilation of feed. In general, the introduction of probiotic in the feed did not adversely affect the functional status of the fish. In young trout of the control group, when assessing the general chemical composition of the organism in the muscle tissue revealed significantly (p≤0.001) higher level of moisture content by 5.1% and lower by 11.0% dry matter content. In muscle, the protein content was higher by 1.33% (p≤0.001) and fat content by 2.1% (p≤0.001) in experimental fish. Generally, Lactobacilli, Enterococcus, Vibrio, Bacillus, and coliform bacteria were found in the intestinal samples of rainbow trout. Significant reliable difference (p≤0.05) between the samples of experimental and control groups was noted in the content of Bacillus bacteria. In the control group, 5.0±0.4×103 CFU/g was detected, while in the experimental group 8.4±0.8×104 CFU/g. Overall, the data indicate that probiotic bacteria B. amyloliquefaciens B-1895 has no adverse effect on selected microorganisms in the study fish.

BACKGROUND: Although research on the mechanism and control of pain and inflammation in fish has increased in recent years, the use of analgesic drugs is limited due to the lack of pharmacological information about analgesic drugs. Tolfenamic acid is a non-steroidal anti-inflammatory drug and can be used in fish due to its low side effect profile and superior pharmacokinetic properties.
OBJECTIVE: The pharmacokinetics, bioavailability and plasma protein binding of tolfenamic acid were investigated following single intravascular (IV), intramuscular (IM) and oral administration of 2 mg/kg in rainbow trout at 13 ± 0.5°C.
METHODS: The experiment was carried out on a total of 234 rainbow trout (Oncorhynchus mykiss). Tolfenamic acid was administered to fish via IV, IM and oral route at a dose of 2 mg/kg. Blood samples were taken at 13 different sampling times until the 72 h after drug administration. The plasma concentrations of tolfenamic acid were quantified using high pressure liquid chromatography-ultraviolet (UV) and pharmacokinetic parameters were assessed using non-compartmental analysis.
RESULTS: The elimination half-life (t1/2ʎz) of tolfenamic acid for IV, IM and oral routes was 3.47, 6.75 and 9.19 h, respectively. For the IV route, the volume of distribution at a steady state and total body clearance of tolfenamic acid were 0.09 L/kg and 0.03 L/h/kg, respectively. The peak plasma concentration and bioavailability for IM and oral administration were 8.82 and 1.24 µg/mL, and 78.45% and 21.48%, respectively. The mean plasma protein binding ratio of tolfenamic acid in rainbow trout was 99.48% and was not concentration dependent.
CONCLUSIONS: While IM route, which exhibits both the high plasma concentration and bioavailability, can be used in rainbow trout, oral route is not recommended due to low plasma concentration and bioavailability. However, there is a need to demonstrate the pharmacodynamic activity of tolfenamic acid in rainbow trout.

Recent work has revealed the importance of contemporary evolution in shaping ecological outcomes. In particular, rapid evolutionary divergence between populations has been shown to impact the ecology of populations, communities, and ecosystems. While studies have focused largely on the role of adaptive divergence in generating ecologically important variation among populations, much less is known about the role of gene flow in shaping ecological outcomes. After divergence, populations may continue to interact through gene flow, which may influence evolutionary and ecological processes. Here, we investigate the role of gene flow in shaping the contemporary evolution and ecology of recently diverged populations of anadromous steelhead and resident rainbow trout (Oncorhynchus mykiss). Results show that resident rainbow trout introduced above waterfalls have diverged evolutionarily from downstream anadromous steelhead, which were the source of introductions. However, the movement of fish from above to below the waterfalls has facilitated gene flow, which has reshaped genetic and phenotypic variation in the anadromous source population. In particular, gene flow has led to an increased frequency of residency, which in turn has altered population density, size structure, and sex ratio. This result establishes gene flow as a contemporary evolutionary process that can have important ecological outcomes. From a management perspective, anadromous steelhead are generally regarded as a higher conservation priority than resident rainbow trout, even when found within the same watershed. Our results show that anadromous and resident O. mykiss populations may be connected via gene flow, with important ecological consequences. Such eco-evolutionary processes should be considered when managing recently diverged populations connected by gene flow.

Astaxanthin (AST) is a natural compound derived from shellfish, microorganisms, and algae, with several healthy properties. For this reason, it is widely used in the diet of humans and animals, such as pigs, broilers, and fish, where its addition is related to its pigmenting properties. Moreover, AST\'s ability to reduce free radicals and protect cells from oxidative damage finds application during the weaning period, when piglets are exposed to several stressors. To better elucidate the mechanisms involved, here we generate ad hoc pig and rainbow trout in vitro platforms able to mimic the intestinal mucosa. The morphology is validated through histological and molecular analysis, while functional properties of the newly generated intestinal barriers, both in porcine and rainbow trout models, are demonstrated by measuring trans-epithelial electrical resistance and analyzing permeability with fluorescein isothiocyanate-dextran. Exposure to AST induced a significant upregulation of antioxidative stress markers and a reduction in the transcription of inflammation-related interleukins. Altogether, the present findings demonstrate AST\'s ability to interact with the molecular pathways controlling oxidative stress and inflammation both in the porcine and rainbow trout species and suggest AST\'s positive role in prevention and health.