Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

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.

Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms.

BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Hyperoxia disproportionally benefits the aerobic performance of large fish at elevated temperature.

Increasing evidence shows that larger fish are more vulnerable to acute warming than smaller individuals of the same species. This size-dependency of thermal tolerance has been ascribed to differences in aerobic performance, largely owing to a decline in oxygen supply relative to demand. To shed light on these ideas, we examined metabolic allometry in 130 rainbow trout ranging from 12 to 358 g under control conditions (17°C) and in response to acute heating (to 25°C), with and without supplemental oxygen (100% versus 150% air saturation). Under normoxia, high temperature caused an average 17% reduction in aerobic scope compared with 17°C. Aerobic performance disproportionally deteriorated in bigger fish as the scaling exponent (b) for aerobic scope declined from b=0.87 at 17°C to b=0.74 at 25°C. Hyperoxia increased maximum metabolic rate and aerobic scope at both temperatures and disproportionally benefited larger fish at 25°C as the scaling exponent for aerobic scope was reestablished to the same level as at 17°C (b=0.86). This suggests that hyperoxia may provide metabolic refuge for larger individuals, allowing them to sustain aerobic activities when facing acute warming. Notably, the elevated aerobic capacity afforded by hyperoxia did not appear to improve thermal resilience, as mortality in 25°C hyperoxia (13.8%, n=4) was similar to that in normoxia (12.1%, n=4), although we caution that this topic warrants more targeted research. We highlight the need for mechanistic investigations of the oxygen transport system to determine the consequences of differential metabolic scaling across temperature in a climate warming context.

Flavobacterium acetivorans sp. nov. and Flavobacterium galactosidilyticum sp. nov., psychrotolerant flavobacteria isolated from cultivated fish.

Two yellow-coloured strains, F-29T and F-340T, were isolated from fish farms in Antalya and Mugla in 2015 and 2017 during surveillance studies. The 16S rRNA gene sequence analysis showed that both strains belong to the genus Flavobacterium. A polyphasic approach involving a comprehensive genome analysis was employed to ascertain the taxonomic provenance of the strains. The overall genome-relatedness indices of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) between the strains and the other members of the genus Flavobacterium were found to be well below the established thresholds of 70 and 95 %, respectively. The whole-genome-based phylogenetic analysis revealed that strain F-29T is closely related to Flavobacterium granuli (dDDH 39.3 % and ANI 89.4 %), while strain F-340T has a close relationship with the type strain of Flavobacterium pygoscelis (dDDH 25.6 % and ANI 81.5 %). Both strains were psychrotolerant with an optimum growth temperature of 25 °C. The chemotaxonomic characteristics of the strains were typical of the genus Flavobacterium. Both strains had phosphatidylethanolamine, aminolipids and unidentified lipids in their polar lipid profile and MK-6 as the isoprenoid quinone. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The genome size of the strains was 3.5 Mb, while G+C contents were 35.3 mol% for strain F-29T and 33.4 mol% for strain F-340T. Overall, the characterizations confirmed that both strains are representatives of two novel species within the genus Flavobacterium, for which the names Flavobacterium acetivorans sp. nov. and Flavobacterium galactosidilyticum sp. nov. are proposed, with F-29T (JCM 34193T=KCTC 82253T) and F-340T (JCM 34203T=KCTC 82263T) as the type strains, respectively.

Host specificity and virulence of Flavobacterium psychrophilum: a comparative study in ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss) hosts.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species.

A cell line derived from heart of rainbow trout is refractory to Tilapia lake virus.

Cell lines are important in vitro models to answer biological mechanisms with less genetic variations. The present study was attempted to develop a cell line from rainbow trout, where we obtained a cell line from the heart, named "RBT-H." The cell line was authenticated using karyotyping and cytochrome c oxidase subunit I (COI) gene sequencing. The karyotype demonstrated diploid chromosome number (2n) as 62 and the sequence of partial COI gene was 99.84% similar to rainbow trout COI data set, both suggesting the origin of RBT-H from the rainbow trout. The heart cell line was mycoplasma-free and found to be refractory to infection with the Tilapia lake virus. The RBT-H cell line is deposited in the National Repository of Fish Cell Line (NRFC) at ICAR-NBFGR, Lucknow, India, with Accession no. NRFC0075 for maintenance and distribution to researchers on request for R&D.

Comparative safety and efficacy of autogenous vaccine administrated by different routes against furunculosis caused by Aeromonas salmonicida sub. salmonicida in large Rainbow trout (Oncorhynchus mykiss).

The development and growth of fish farming are hindered by viral and bacterial infectious diseases, which necessitate effective disease control measures. Furunculosis, primarily caused by Aeromonas salmonicida, stands out as a significant bacterial disease affecting salmonid fish farms, particularly rainbow trout. Vaccination has emerged as a crucial tool in combating this disease. The objective of this experiment was to assess and compare the efficacy and duration of different vaccine protocols against furunculosis in large trout under controlled rearing conditions, utilizing single and booster administrations via intraperitoneal, oral, and immersion routes. Among the various vaccination protocols tested, only those involving intraperitoneal injection, administered at least once, proved truly effective in preventing the expression of clinical signs of furunculosis and reducing mortality rates. A single intraperitoneal administration provided protection for up to 2352°-days, equivalent to approximately 5 months in water at 16 °C. However, intraperitoneal vaccination may lead to reduced growth in the fish due to resultant intraperitoneal adhesions. Additionally, protocols incorporating booster doses via intraperitoneal injection demonstrated efficacy regardless of the administration route of the primary vaccination. Nevertheless, the use of booster vaccinations via the intraperitoneal route did not confer any significant advantage over a single intraperitoneal injection in terms of efficacy.

Antiviral effect of miR-206 in rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus infection.

Infectious hematopoietic necrosis (IHN), caused by IHN virus, is a highly contagious and lethal disease that seriously hampers the development of rainbow trout (Oncorhynchus mykiss) aquaculture. However, the immune response mechanism of rainbow trout underlying IHNV infection remains largely unknown. MicroRNAs act as post-transcriptional regulators of gene expression and perform a crucial role in fish immune response. Herein, the regulatory mechanism and function of miR-206 in rainbow trout resistance to IHNV were investigated by overexpression and silencing. The expression analysis showed that miR-206 and its potential target receptor-interacting serine/threonine-protein kinase 2 (RIP2) exhibited significant time-dependent changes in headkidney, spleen and rainbow trout primary liver cells infected with IHNV and their expression displayed a negative correlation. In vitro, the interaction between miR-206 and RIP2 was verified by luciferase reporter assay, and miR-206 silencing in rainbow trout primary liver cells markedly increased RIP2 and interferon (IFN) expression but significantly decreased IHNV copies, and opposite results were obtained after miR-206 overexpression or RIP2 knockdown. In vivo, overexpressed miR-206 with agomiR resulted in a decrease in the expression of RIP2 and IFN in liver, headkidney and spleen. This study revealed the key role of miR-206 in anti-IHNV, which provided potential for anti-viral drug screening in rainbow trout.

MK-0608 inhibits in vitro and in vivo RNA replication of infectious pancreatic necrosis virus.

Infectious pancreatic necrosis virus (IPNV) is an important pathogen that is threatening the worldwide salmon and trout industry. But there is no therapeutic drug available for now. In this study, we demonstrate that MK-0608 is highly efficient against IPNV and low cytotoxic, with a 50 % effective concentration (EC50) of 0.20 µM and selectivity index (SI) of about 268. Time of addition assay illustrated that MK-0608 targeted the early stage of IPNV life cycle. Furthermore, we found that MK-0608 blocked IPNV attachment on the premise of sufficient pre-incubation time but MK-0608 did not influence viral internalization and release. MK-0608 could inhibit IPNV genome synthesis, and combination with ribavirin enhanced the inhibition effect, which might be functional via binding to IPNV RNA dependent RNA polymerase (RdRp), which was predicted by using molecular docking methods. In vivo test showed that IPNV was extremely suppressed in the rainbow trout (Oncorhynchus mykiss) with one single dose of MK-0608, and the higher dosage of 50 mg/kg could cause 3 log decrease of IPNV loads in fish tissues.

Rainbow trout DUBA inhibits type I interferon signaling by deubiquitinating TRAF3.

Deubiquitinating enzyme A (DUBA), a member of the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs), is recognized for its negative regulatory role in type I interferon (IFN) expression downstream of Toll-like receptor 3 (TLR3). However, its involvement in the TLR3 signaling pathway in fish remains largely unexplored. In this study, we investigated the regulatory role of DUBA (OmDUBA) in the TLR3 response in rainbow trout (Oncorhynchus mykiss). OmDUBA features a conserved OTU domain, and its expression increased in RTH-149 cells following stimulation with the TLR3 agonist poly(I:C). Gain- and loss-of-function experiments demonstrated that OmDUBA attenuated the activation of TANK-binding kinase 1 (TBK1), resulting in a subsequent reduction in type I IFN expression and IFN-stimulated response element (ISRE) activation in poly(I:C)-stimulated cells. OmDUBA interacted with TRAF3, a crucial mediator in TLR3-mediated type I IFN production. Under poly(I:C) stimulation, there was an augmentation in the K63-linked polyubiquitination of TRAF3, a process significantly inhibited upon OmDUBA overexpression. These findings suggest that OmDUBA may function similarly to its mammalian counterparts in downregulating the poly(I:C)-induced type I IFN response in rainbow trout by removing the K63-linked ubiquitin chain on TRAF3. Our study provides novel insights into the role of fish DUBA in antiviral immunity.

Evaluation of a DNA vaccine with self-designed CpG sequences against J genotype IHNV infection in rainbow trout (Oncorhynchus mykiss).

Rainbow trout suffer from infectious hematopoietic necrosis virus (IHNV) outbreaks, which lead to massive mortality and huge economic loss worldwide. The approved commercial vaccine is used for the prevention of IHN in Canada. Given that Chinese domestic J-genotype isolates are different from North American IHNV isolates, the development of an effective DNA vaccine against Chinese J-genotype isolates is urgent. In this study, we developed a DNA vaccine encoding glycoprotein based on our previously isolated IHNV GS21 strain and self-designed CpG sequences were supplemented as molecular adjuvants. The vaccinated rainbow trout were significantly protected against IHNV with approximately a relative percent survival (RPS) of 94.74% compared to the unvaccinated group. Moreover, the specific antibody of IgM and neutralizing antibody (NAb) was significantly provoked after the vaccination. Particularly, the antiviral immune response was rapidly evoked in the early stage of vaccination including the up-regulation of Mx-1, IFN-Ⅰ, and IFN-γ. The IHNV load in vaccinated fish was apparently lower than that in the unvaccinated group. Furthermore, the integration of exogenous genes into the host chromosome and the spread of antimicrobial-resistant genes were not found. These results suggested that our vaccine enhances robust immune responses and evokes considerable protection against IHNV with limited genetically modified risk, which is an effective and promising vaccine candidate for further prevention of IHNV outbreaks.

Effect of ß-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells.

ß-glucans are carbohydrates present in the cell wall of many fungi, which are often used as immunostimulants in feeds for farmed species. Their capacity to activate innate immune responses directly acting on innate cell populations has been widely documented in fish. However, whether they can affect the functionality of adaptive immune cells has been scarcely explored. In this context, in the current work, we have determined the effects of ß-glucans on rainbow trout blood IgM+ B cells in the presence or absence of 2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS), a model antigen. For this, rainbow trout peripheral blood leukocytes were incubated with different doses of ß-glucans or media alone in the presence or absence of TNP-LPS for 48 h. The size, levels of expression of surface MHC II, antigen processing and phagocytic capacities and proliferation of IgM+ B cells were then studied by flow cytometry. The number of IgM-secreting cells in the cultures was also estimated by ELISpot. ß-glucans significantly decreased the levels of surface MHC II expression and the antigen processing capacities of these cells, especially in the presence of TNP-LPS, while they increased their phagocytic activity. On their own, ß-glucans slightly activated the proliferation of IgM+ B cells but reduced that induced by TNP-LPS. In contrast, ß-glucans significantly increased the number of cells secreting IgM in the cultures. This effect of ß-glucans on the IgM-secreting capacity of B cells was also confirmed through a feeding experiment, in which the IgM-secreting capacity of blood leukocytes obtained from fish fed a ß-glucan-supplemented diet for one month was compared to that of leukocytes obtained from fish fed a control diet. Altogether, these findings contribute to increase our knowledge regarding the effects of ß-glucans on fish adaptive responses.

Systemic immune response of rainbow trout exposed to Flavobacterium psychrophilum infection.

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is one of the most serious bacterial diseases leading to significant economic loss for rainbow trout (Oncorhynchus mykiss) aquaculture. However, little is known about the systemic immune response of rainbow trout against F. psychrophilum infection. This study investigated the immune response of rainbow trout to F. psychrophilum infection using multiple experiments, including bacterial load detection, phagocyte activity assessment, enzyme activity evaluation, and gene expression profiling. Results showed that the spleen index and intestinal pathogen load reached a peak at 3 days post-infection, with strong pro-inflammatory gene expression observed in rainbow trout. Leukocytes RBA and PKA were significantly elevated in the spleen, blood and intestine at 7 days post-infection. Heat map analysis demonstrated that the spleen had a more substantial pro-inflammatory response compared to the intestine post-infection and exhibited higher expression levels of immune-related genes, including IgM, il1ß, il6, cd4, cd8a, cd8b, c1q, chathelicidin, inos, and lysozyme. Both Th1 and Th2 polarized responses in the spleen were activated, with Th2 (il4/13a, gata3) (FC > 4) being more intense than Th1 (tnfα, t-bet) (FC > 2). Tight junction proteins exhibited down-regulation followed by up-regulation post-infection. Collectively, the results of this study expand our current understanding of the immune response of rainbow trout post F. psychrophilum infection but also provide new avenues for investigation in salmonid aquaculture.

Interaction of Per- and Polyfluoroalkyl Substances with Estrogen Receptors in Rainbow Trout (Oncorhynchus mykiss): An In Silico Investigation.

Fresh water sources, including lakes, such as the Great Lakes, are some of the most important ecosystems in the world. Despite the importance of these lakes, there is increasing concern about the presence of per- and polyfluoroalkyl substances (PFAS)─among the most prevalent contaminants of our time─due to the ability of PFAS to bioaccumulate and persist in the environment, as well as to its linkages to detrimental human and animal health effects. In this study, PFAS exposure on rainbow trout (Oncorhynchus mykiss) is examined at the molecular level, focusing on the impact of PFAS binding on the alpha (α) and beta (ß) estrogen receptors (ERs) using molecular dynamics simulations, binding free energy calculations, and structural analysis. ERs are involved in fundamental physiological processes, including reproductive system development, muscle regeneration, and immunity. This study shows that PFAS binds to both the estrogen α and estrogen ß receptors, albeit via different binding modes, due to a modification of an amino acid in the binding site as a result of a reorientation of residues in the binding pocket. As ER overactivation can occur through environmental toxins and pollutants, this study provides insights into the influence of different types of PFAS on protein function.

Bioconcentration Assessment of Three Cationic Surfactants in Permanent Fish Cell Lines.

Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid-water partitioning coefficient, DMLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

Purification and characterization of glutamate dehydrogenase from rainbow trout (Oncorhynchus mykiss) liver and molecular docking studies.

Glutamate dehydrogenase (GDH) participates in the energy metabolism of proteins and the synthesis of metabolites important for the organism. In this study, GDH enzyme was purified from the liver of rainbow trout (Oncorhynchus mykiss) by 2',5'-ADP Sepharose 4B affinity chromatography in one step. As a result of this purification process, GDH enzyme was purified 171-fold with 5.83 U/mg protein-specific activity. The characterization experiments presented that the storage stability of the purified GDH enzyme was determined as -80°C; optimum temperature 40°C; it was determined that the optimum ionic strength was 100 mM phosphate buffer and the optimum pH was 8.00. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PAGE studies showed that the natural molar mass of the purified GDH enzyme was 346.74 kDa, and the molar mass of its subunits was 53.71 kDa. Km and Vmax values for substrates and coenzymes of GDH enzyme purified from rainbow trout liver were calculated, and the lowest Km value was found in NAD+ (1.86 mM) and the highest Vmax value in NH4 + (1.79 U/mL). The effects of some metal ions, vitamins, and solvents on the activity of the purified GDH enzyme were investigated and also IC50 values and inhibition types. The metal ion with the lowest IC50 value is Ag+ (8.65 ± 1.68 µM), and the vitamin is B6 (0.77 ± 0.04 mM). The binding affinities of inhibitors were investigated with molecular docking, based on the conformational state of GDH.

Applying a heat transfer mathematical model for the cryopreservation of rainbow trout (Oncorhynchus mykiss) sperm: How straw location over liquid nitrogen level affects freezing rate and fertilization yield.

Cryopreservation of rainbow trout semen under field conditions was analyzed. Straw location over liquid nitrogen level is a crucial variable that affects freezing rate and fertilization yield due to changes in nitrogen vapor external temperature. The objectives were: to analyze cryopreservation protocols by experimentally measuring the cooling rates and fertilization yield of 0.5 ml plastic straws located in nitrogen vapor at different heights corresponding to different external temperatures; to numerically simulate the freezing process, by solving the heat transfer partial differential equations with the corresponding thermo-physical properties of the biological system and the plastic straw; to evaluate and analyze the surface heat transfer coefficient (h) during the freezing process of the straws; to introduce a new variable, the characteristic freezing time (tc), that enables comparison between protocols; this variable was defined as the elapsed period between the initial freezing temperature and a final reference temperature of -40 °C (temperature in which more than 80 % of the water is in a frozen state). The mathematical model predicted the temperature distribution inside the straw, showing a low effect of straw plastic materials (polyethylene-terephthalate glycol, polyvinyl-chloride, and polypropylene) on freezing rates. The average h value obtained from numerical simulations was 25.5 W/m2 K, close to that obtained from the analytical Nusselt correlation for natural convection. An improvement on fertilization trials was observed when the average external nitrogen temperature was -129.6 °C (temperature range: -94 to -171 °C) with an average tc of 56.8 s (ranging between 47 and 72 s). These results corresponded to a height above the level of liquid nitrogen of 2 cm. Comparison with literature reported data showed satisfactory results. Applying mathematical models in the cryobiology field achieved results that are relevant for cryopreservation activities.

Genetic diversity patterns in farmed rainbow trout (Oncorhynchus mykiss) populations using genome-wide SNP and haplotype data.

Rainbow trout is one of the most popular aquaculture species worldwide, with a long history of domestication. However, limited information exists about the genetic diversity of farmed rainbow trout populations globally, with most available reports relying on low-throughput genotyping technologies. Notably, no information exists about the genetic diversity status of farmed rainbow trout in Sweden. Double-digest restriction-site-associated DNA sequencing was performed on more than 500 broodfish from two leading producers in Sweden and from the country's national breeding program. Following the detection of single nucleotide polymorphisms (SNPs), genetic diversity was studied by using either individual SNPs (n = 8680; one SNP retained per 300 bp sequence reads) or through SNP haplotypes (n = 20 558; all SNPs retained in 300 bp sequence reads). Similar amounts of genetic diversity were found amongst the three populations when individual SNPs were used. Furthermore, principal component analysis and discriminant analysis of principal components suggested two genetic clusters with the two industry populations grouped together. Genetic differentiation based on the FST fixation index was ~0.01 between the industry populations and ~0.05 when those were compared with the breeding program. Preliminary estimates of effective population size (Ne ) and inbreeding (based on runs of homozygosity; FROH ) were similar amongst the three populations (Ne ≈ 50-80; median FROH ≈ 0.11). Finally, the haplotype-based analysis suggested that animals from the breeding program had higher shared coancestry levels than those from the other two populations. Overall, our study provides novel insights into the genetic diversity and structure of Sweden's three main farmed rainbow trout populations, which could guide their future management.

First case of ichthyophonosis in farmed rainbow trout Oncorhynchus mykiss in Serbia.

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.

Antimicrobial susceptibility of Peruvian Yersinia ruckeri isolates to oxytetracycline and florfenicol using MIC testing.

Yersiniosis, caused by Yersinia ruckeri, has become the most common disease in farmed rainbow trout Oncorhynchus mykiss in Peru, affecting Puno and Junín Regions, important aquaculture areas in the country. Florfenicol (FLO) and oxytetracycline (OXY) are the antimicrobials most frequently used to mitigate losses attributed to this pathogen. This study presents an analysis of the susceptibility patterns of 60 Y. ruckeri isolates (30 isolates each from Puno and Junín), including the type strain CECT 4319T and the strains CECT 955 and CECT 956, against FLO and OXY. Minimum inhibitory concentrations (MICs) were determined following the guideline for standard broth dilution method published by the Clinical and Laboratory Standards Institute. MIC results ranged from 4.0 to 8.0 µg ml-1 for FLO and 0.5 to 4.0 µg ml-1 for OXY. Normalized resistance interpretation (NRI) analysis identified epidemiological cut-off values of ≤16.0 µg ml-1 for FLO and ≤4.0 µg ml-1 for OXY. All Peruvian isolates, including the collection strains, were categorized as wild-type for both antimicrobials. Even though the number of Y. ruckeri isolates with MIC values of 8 µg ml-1 for FLO is more than double in Puno than in Junín (15 vs. 7 isolates), the NRI analysis showed the same epidemiological cutoff of 16 µg ml-1; while for OXY, it was 4.0 µg ml-1 for Puno and 2.0 µg ml-1 for Junín. This study establishes the basis for monitoring susceptibility to FLO and OXY in new Y. ruckeri isolates in Peruvian rainbow trout farming.