Ichthyophonosis is a disease caused by the mesomycetozoean parasite Ichthyophonus hoferi that affects a variety of fish species, including rainbow trout Oncorhynchus mykiss Walbaum. This disease is characterized by granulomatous lesions and necrosis in various organs, which can have severe impacts on the health and welfare of the fish. Ichthyophonosis has been found in several parts of the world, including Europe, and is a significant concern in the aquaculture industry and for populations of wild marine fishes. The rainbow trout is a widely cultured salmonid species in many countries, including Serbia. Although the presence of I. hoferi in rainbow trout has been reported in several countries, it has never been documented in Serbia. In this article, we report the first case of ichthyophonosis in rainbow trout in Serbia.

Current prophylactic and disease control measures in aquaculture highlight the need of alternative strategies to prevent disease and reduce antibiotic use. Mucus covered mucosal surfaces are the first barriers pathogens encounter. Mucus, which is mainly composed of highly glycosylated mucins, has the potential to contribute to disease prevention if we can strengthen this barrier. Therefore, aim of this study was to develop and characterize fish in vitro mucosal surface models based on commercially available cell lines that are functionally relevant for studies on mucin regulation and host-pathogen interactions. The rainbow trout (Oncorhynchus mykiss) gill epithelial cell line RTgill-W1 and the embryonic cell line from Chinook salmon (Oncorhynchus tshawytscha) CHSE-214 were grown on polycarbonate membrane inserts and chemically treated to differentiate the cells into mucus producing cells. RTGill-W1 and CHSE-214 formed an adherent layer at two weeks post-confluence, which further responded to treatment with the γ-secretase inhibitor DAPT and prolonged culture by increasing the mucin production. Mucins were metabolically labelled with N-azidoacetylgalactosamine 6 h post addition to the in vitro membranes. The level of incorporated label was relatively similar between membranes based on RTgill-W1, while larger interindividual variation was observed among the CHSE in vitro membranes. Furthermore, O-glycomics of RTgill-W1 cell lysates identified three sialylated O-glycans, namely Galβ1-3(NeuAcα2-6)GalNAcol, NeuAcα-Galβ1-3GalNAcol and NeuAcα-Galβ1-3(NeuAcα2-6)GalNAcol, resembling the glycosylation present in rainbow trout gill mucin. These glycans were also present in CHSE-214. Additionally, we demonstrated binding of the fish pathogen A. salmonicida to RTgill-W1 and CHSE-214 cell lysates. Thus, these models have similarities to in vivo mucosal surfaces and can be used to investigate the effect of pathogens and modulatory components on mucin production.

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.

Fish are currently used models for the toxicity assessment of chemicals, including polycyclic aromatic hydrocarbons (PAHs). Alternative methods including fish cell lines are currently used to provide fast and reliable results on the toxic properties of chemicals while respecting ethical concerns about animal testing. The Rainbow trout liver cell line RTLW1 was used to analyze the effects of two water-accommodated fractions from two crude oils: Arabian Light crude oil (LO) and refined oil from Erika (HO). Several toxicity endpoints were assessed in this study, including cytotoxicity, EROD activity, DNA damage (comet and micronucleus assays), and ROS production. RTL-W1 cells were exposed for 24 h at two or three dilutions of WAF at 1000 µg/L (0.1% (1 μg/L), 1% (10 μg/L), and 10% (100 μg/L)) for cytotoxicity and EROD activity and 1% and 10% for ROS production and genotoxicity). Exposure of RTL-W1 cells to LO WAF induced a significant increase of EROD activity and ROS production and altered DNA integrity as revealed by both the comet assay and the micronucleus test for 10 µg/L of LO. On the other hand, HO WAF exhibited limited toxic effects except for an EROD induction for 1% WAF dilution. These results confirmed the usefulness of RTL-W1 cells for in vitro toxicological assessment of chemical mixtures.

The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.

Activin signaling is essential for proper embryonic, skeletal muscle, and reproductive development. Duplication of the pathway in teleost fish has enabled diversification of gene function across the pathway but how gene duplication influences the function of activin signaling in non-mammalian species is poorly understood. Full characterization of activin receptor signaling pathway expression was performed across embryonic development and during early skeletal muscle growth in rainbow trout (RBT, Oncorhynchus mykiss). Rainbow trout are a model salmonid species that have undergone two additional rounds of whole genome duplication. A small number of genes were expressed early in development and most genes increased expression throughout development. There was limited expression of activin Ab in RBT embryos despite these genes exhibiting significantly elevated expression in post-hatch skeletal muscle. CRISPR editing of the activin Aa1 ohnolog and subsequent production of meiotic gynogenetic offspring revealed that biallelic disruption of activin Aa1 did not result in developmental defects, as occurs with knockout of activin A in mammals. The majority of gynogenetic offspring exhibited homozygous activin Aa1 genotypes (wild type, in-frame, or frameshift) derived from the mosaic founder female. The research identifies mechanisms of specialization among the duplicated activin ohnologs across embryonic development and during periods of high muscle growth in larval and juvenile fish. The knowledge gained provides insights into potential viable gene-targeting approaches for engineering the activin receptor signaling pathway and establishes the feasibility of employing meiotic gynogenesis as a tool for producing homozygous F1 genome-edited fish for species with long-generation times, such as salmonids.

OBJECTIVE: To test the use of rainbow trout skin as a surgical mesh in abdominal hernioplasties in rats.
METHODS: The experiment involved 20 Wistar rats receiving implants of trout skin processed for disinfection in 0.5% glutaraldehyde and preserved in 100% glycerin. The animals were divided into four groups, divided at 7, 15, 30, and 90 days postoperatively. Clinical and infrared thermography evaluations were performed, and after euthanasia, assessments of adhesion formations and sample collection for histological evaluation were conducted.
RESULTS: The implant was observed to be intact, ensuring the integrity of the abdominal wall, support for the viscera, and normal mobility for the rats for up to 90 days. Low rates of clinical alterations were observed, with an intense inflammatory reaction up to day 7, chronic inflammation and the onset of angiogenesis at day 15, and a low inflammatory reaction with collagenous infiltrate and fibrosis at day 30. At day 90, the implants showed a collagenous and fibrotic infiltrate with a minimal inflammatory infiltrate.
CONCLUSIONS: The surgical mesh of trout skin performed well, making it a potential alternative for surgical procedures in muscle aponeurotic corrections in the abdominal wall.

Recirculating aquaculture systems (RAS) have become more attractive due to reduced water consumption and effluent discharge. However, intensification of production increases the risk of introducing pathogens at farming sites. The emergence of uncultivable pathogens and RAS pathobiome diversity shifts the traditional disease paradigm from \"one pathogen, one disease\" to complex multiple-pathogen disease cases. Piscine orthoreovirus genotype 3 (PRV-3) is an excellent example, as it is capable of inducing anemia and heart pathology resembling heart and skeletal muscle inflammation under experimental conditions, and is associated with increased mortality in association with other pathogens in the field. The aim of this study was to develop a method for detection of multiple pathogens and putative pathogens, as co-infections are common in aquaculture. To do this, in the pilot study, we mapped the pathobiome of RAS-farmed rainbow trout (Oncorhynchus mykiss) (commercial RAS, farm A) using both standard diagnostic methods and metabarcording (16S rRNA) to investigate the gill microbiome. During this study, we observed infections with multiple pathogens, and detected two putative gill pathogens Candidatus Branchiomonas cysticola and Candidatus Piscichlamydia salmonis, both of which have been linked with complex gill disease in Atlantic salmon (Salmo salar). Based on the pilot study, we developed and tested a high throughput qPCR (HT-qPCR) chip targeting 22 viral and bacterial pathogens and putative pathogens, followed by a surveillance of a fish cohort in a commercial RAS farm during production (farm B). Co-infection with PRV-3 and Ca. B. cysticola combined with stress inducing management practices may explain the severe disease outbreak observed (37% mortality). The time course study sets the base for a future screening scheme for disease prediction and addresses limitations of the method when testing environmental DNA/RNA.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.