Rainbow Trout Cell Line Research Articles

PACAP binds conserved receptors and modulates cytokine gene expression and protein secretion in trout cell lines

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1β and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1β protein from previously accumulated transcripts or the cleavage of accumulated IL-1β precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

Non-microcystin extracellular metabolites of Microcystis aeruginosa impair viability and reproductive gene expression in rainbow trout cell lines

Microcystis aeruginosa is a ubiquitous freshwater cyanobacterium best known for producing hepatotoxic microcystins; however, this common bloom-forming species also produces myriad biologically active and potentially deleterious other metabolites. Our understanding of the effects of these non-microcystin metabolites on fish is limited. In this study, we evaluated cytotoxicity of extracellular metabolites harvested from both microcystin-producing (MC+) and non-producing (MC-) strains of M. aeruginosa on rainbow trout (Oncorhynchus mykiss) cell lines derived from tissues of the brain, pituitary, heart, gonads, gills, skin, liver, and milt. We also examined the influence of M. aeruginosa exudates (MaE) on the expression of critical reproduction-related genes using the same cell lines. We found that exudates of the MC- M. aeruginosa strain significantly reduced viability in RTBrain, RTgill-W1, and RT-milt5 cell lines and induced significant cellular stress and/or injury in six of the eight cell lines—highlighting potential target tissues of cyanobacterial cytotoxic effects. Observed sublethal consequences of Microcystis bloom exposure occurred with both MC+ and MC- strains' exudates and significantly altered expression of developmental and sex steroidogenic genes. Collectively, our results emphasize the contributions of non-MC metabolites to toxicity of Microcystis-dominated algal blooms and the need to integrate the full diversity of M. aeruginosa compounds—beyond microcystins—into ecotoxicological risk assessments.

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.

Evaluation of tire tread particle toxicity to fish using rainbow trout cell lines

Tire and road wear particles (TRWP) resulting from tire abrasion while driving raise concerns due to their potential contribution to aquatic toxicity. Our study aimed to assess cryogenically milled tire tread (CMTT) particle toxicity, used as a proxy for TRWP, and associated chemicals to fish using two Rainbow Trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgutGC) epithelium. CMTT toxicity was evaluated through several exposure pathways, including direct contact, leaching, and digestion, while also assessing the impact of particle aging. Following OECD TG249, cell viability was assessed after 24 h acute exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. In vitro EC50 values for the fish cell lines exceeded river TRWP concentrations (2.02 g/L and 4.65 g/L for RTgill-W1 and RTgutGC cell lines, respectively), and were similar to in vivo LC50 values estimated at 6 g/L. Although toxicity was mainly driven by the leaching of tire-associated chemicals, the presence of the particles contributed to the overall toxicity by inducing a continuous leaching, highlighting the importance of considering combined exposure scenarios. Aging and digestion conditions were also found to mediate CMTT toxicity. Thermooxidation resulted in a decreased chemical leaching and toxicity, while in vitro digestion under mimicked gastrointestinal conditions increased leaching and toxicity. Specific chemicals, especially Zn, 2-mercaptobenzothiazole, 1,3-diphenylguanidine, and N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) were identified as contributors to the overall toxicity. Although 6PPD-quinone was detected in CMTT digestate, cytotoxicity assays with RTgill-W1 and RTgutGC cell lines showed no toxicity up to 6 mg/L, supporting the notion of a specific mode of action of this chemical. This study provides insights into the toxicological mechanisms induced by tire particles and their associated chemicals and can help in the evaluation of potential risks to aquatic life associated with TRWP.

The Role of Glutathione and Sulfhydryl Groups in Cadmium Uptake by Cultures of the Rainbow Trout RTG-2 Cell Line.

The aim of this study is to investigate the role of cellular sulfhydryl and glutathione (GSH) status in cellular cadmium (Cd) accumulation using cultures of the rainbow trout cell line RTG-2. In a first set of experiments, the time course of Cd accumulation in RTG-2 cells exposed to a non-cytotoxic CdCl2 concentration (25 μM) was determined, as were the associated changes in the cellular sulfhydryl status. The cellular levels of total GSH, oxidized glutathione (GSSG), and cysteine were determined with fluorometric high-performance liquid chromatography (HPLC), and the intracellular Cd concentrations were determined with inductively coupled plasma mass spectrometry (ICP-MS). The Cd uptake during the first 24 h of exposure was linear before it approached a plateau at 48 h. The metal accumulation did not cause an alteration in cellular GSH, GSSG, or cysteine levels. In a second set of experiments, we examined whether the cellular sulfhydryl status modulates Cd accumulation. To this end, the following approaches were used: (a) untreated RTG-2 cells as controls, and (b) RTG-2 cells that were either depleted of GSH through pre-exposure to 1 mM L-buthionine-SR-sulfoximine (BSO), an inhibitor of glutathione synthesis, or the cellular sulfhydryl groups were blocked through treatment with 2.5 μM N-ethylmaleimide (NEM). Compared to the control cells, the cells depleted of intracellular GSH showed a 25% reduction in Cd accumulation. Likewise, the Cd accumulation was reduced by 25% in the RTG-2 cells with blocked sulfhydryl groups. However, the 25% decrease in cellular Cd accumulation in the sulfhydryl-manipulated cells was statistically not significantly different from the Cd accumulation in the control cells. The findings of this study suggest that the intracellular sulfhydryl and GSH status, in contrast to their importance for Cd toxicodynamics, is of limited importance for the toxicokinetics of Cd in fish cells.

Isolation and characterization of the morphology, size and particle number of rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) cell line derived large and small extracellular vesicles

Extracellular vesicles (EVs) are 50–1,000 nm lipid bilayer-bound vesicles, released into the extracellular environment by various cell types for intercellular communication purposes. The quantitative and qualitative characteristics of EVs can be affected by stress and pathological conditions. The majority of extracellular vesicle (EV) studies have been performed on mammalian cell lines or bodily fluids. EVs have been previously described from bodily fluids like plasma, serum or mucus in different fish species, however the available knowledge of fish cell line derived EVs is limited and in the vast majority of studies, the overall focus is on small EVs (< 200 nm). We isolated large and small extracellular vesicles from zebrafish (Danio rerio) liver (ZFL), rainbow trout (Oncorhynchus mykiss) liver (RTL-W1), gill (RTgill-W1) and intestinal epithelial (RTgutGC) cell lines using stepwise centrifugation and characterized the size and morphology of EVs. Here we demonstrated that large and small extracellular vesicles can be successfully isolated using stepwise centrifugation from the serum-free medium of the selected piscine cell lines after a 24-h incubation period. The size distribution of large and small EVs isolated from the piscine cell lines suggest that large and small EV groups show high diversity in size ranges, containing heterogenous subpopulations in sizes, and the results highly depend on the applied method and whether filtration steps were included following the isolation. The spherical morphology of EVs was verified by transmission electron microscopy.

The Potentiating Effect of Graphene Oxide on the Arylhydrocarbon Receptor (AhR)-Cytochrome P4501A (Cyp1A) System Activated by Benzo(k)fluoranthene (BkF) in Rainbow Trout Cell Line.

The increasing use of graphene oxide (GO) will result in its release into the environment; therefore, it is essential to determine its final fate and possible metabolism by organisms. The objective of this study was to assess the possible role of the aryl hydrocarbon receptor (AhR)-dependent cytochrome P4501A (Cyp1A) detoxification activities on the catabolism of GO. Our hypothesis is that GO cannot initially interact with the AhR, but that after an initial degradation caused by other mechanisms, small fractions of GO could activate the AhR, inducing Cyp1A. The environmental pollutant benzo(k)fluoranthene (BkF) was used for the initial activation of the AhR in the rainbow trout (Oncorhynchus mykiss) cell line RTL-W1. Pre-, co-, and post-exposure experiments with GO were performed and Cyp1A induction was monitored. The strong stimulation of Cyp1A observed in cells after exposure to GO, when BkF levels were not detected in the system, suggests a direct action of GO. The role of the AhR was confirmed by a blockage of the observed effects in co-treatment experiments with αNF (an AhR antagonist). These results suggest a possible role for the AhR and Cyp1A system in the cellular metabolism of GO and that GO could modulate the toxicity of environmental pollutants.

Novel insights into the cytokine network of rainbow trout Oncorhynchus mykiss using cell lines and primary leukocyte populations

Cytokines are small proteins that regulate innate and adaptive immune responses and are released by both immune and non-immune cell types. In the current study, the constitutive and induced gene expression profiles of a suite of proinflammatory and regulatory cytokines was examined comparatively in eight rainbow trout (Oncorhynchus mykiss) cell lines, in order to establish the cytokine repertoires of these different cell types, especially the understudied non-immune cells. They included three epithelial cell lines (RTgut, RTgill, and RTL), one endothelial cell line (RTH), one fibroblast cell line (RTG-2), two stromal cell lines (TSS and TPS-2) and one monocyte/macrophage-like cell line (RTS-11). Three types of primary leukocytes (derived from blood, spleen and head kidney) of trout were also included in the analysis, to allow comparison to the repertoires expressed in T cells, as a major source of cytokines in immune responses. The major findings are: 1) IL-2A, IL-2B, IL-4/13B1, IL-4/13B2, IL-10b, P40B1, P28B, IL-17A/F1b, TNF-α3, TNF-α4, IFNγ1, CCL20L2b and CCL20L3a are expressed mainly in leukocytes but IL-17 N, IL-17D, IL-20 and CCL20L1b2 are not expressed in these cells. Hence future studies in these cell lines will help establish their function in fish; 2) Some of the cytokines were differentially expressed in the cell lines, revealing the potential role of these cell types in aspects of trout mucosal and inflammatory immune responses, 3) Similar cell types grouped together in the cell cluster analysis, including the leukocyte cluster, stromal cell cluster, and epithelial and endothelial cell cluster. Taken together, this investigation of these trout cell lines forms a good database for studying the function of cytokines not expressed in isolated leukocytes or that are preferentially expressed in the cell lines. Furthermore, the cytokine expression analysis undertaken confirmed the phenotypic relationship of these cell types at the molecular level.

Investigating the bioaccumulation potential of anionic organic compounds using a permanent rainbow trout liver cell line

Permanent rainbow trout (Oncorhynchus mykiss) cell lines represent potential in vitro alternatives to experiments with fish. We here developed a method to assess the bioaccumulation potential of anionic organic compounds in fish, using the rainbow trout liver-derived RTL-W1 cell line. Based on the availability of high quality in vivo bioconcentration (BCF) and biomagnification (BMF) data and the substances’ charge state at physiological pH, four anionic compounds were selected: pentachlorophenol (PCP), diclofenac (DCF), tecloftalam (TT) and benzotriazol-tert-butyl-hydroxyl-phenyl propanoic acid (BHPP). The fish cell line acute toxicity assay (OECD TG249) was used to derive effective concentrations 50 % and non-toxic exposure concentrations to determine exposure concentrations for bioaccumulation experiments. Bioaccumulation experiments were performed over 48 h with a total of six time points, at which cell, medium and plastic fractions were sampled and measured using high resolution tandem mass spectrometry after online solid phase extraction. Observed cell internal concentrations were over-predicted by KOW-derived predictions while pH-dependent octanol–water partitioning (DOW) and membrane lipid-water partitioning (DMLW) gave better predictions of cell internal concentrations. Measured medium and cell internal concentrations at steady state were used to calculate RTL-W1-based BCF, which were compared to DOW- or DMLW-based model approaches and in vivo data. With the exception of PCP, the cell-derived BCF best compared to DOW-based model predictions, which were higher than predictions based on DMLW. All methods predicted the in vivo BCF for diclofenac well. For PCP, the cell-derived BCF was lowest although all BCF predictions underestimated the in vivo BCF by ≥ 1 order of magnitude. The RTL-W1 cells, and all other prediction methods, largely overestimated in vivo BMF, which were available for PCP, TT and BHPP. We conclude that the RTL-W1 cell line can supplement BCF predictions for anionic compounds. For BMF estimations, however, in vitro-in vivo extrapolations need adaptation or a multiple cell line approach.

RTG-TOF, a rainbow trout (Oncorhynchus mykiss) cell line with an inducible gene expression system.

RTG-TOF, a rainbow trout (Oncorhynchus mykiss) cell line with an inducible gene expression system.

Gut Associated Lymphoid Tissue (GALT) primary cells and stable cell lines as predictive models for intestinal health in rainbow trout (Oncorhynchus mykiss).

The use of functional feeds for farmed fish is now regarded as a key factor in improving fish health and performance against infectious disease. However, the mechanisms by which these nutritional components modulate the immune response are not fully understood. The present study was undertaken to identify the suitability of both primary gut-associated lymphoid tissue (GALT) leucocyte cells and established rainbow trout cell lines as potential alternative methods to test functional feed ingredients prior to full fish feeding trials that can take months to complete. In addition to the primary GALT culture cells, the two rainbow cell lines RTS11 and RTgutGC which are from macrophage and gut epithelial cells, respectively. The cells were stimulated with a variety of pathogen associated molecular patterns (PAMPs) (PHA and Poly I:C) and recombinant rainbow trout IL-1β (rIL-1β), a proinflammatory cytokine, additionally two forms of β-glucan, a prebiotic commonly used aquafeeds were used as stimulants. From this, the suitability of cell models as a health screen for functional feeds was assessed. GALT leucocytes were deemed most effective to act as a health screen over the 4hr time point demonstrating responses to Poly I:C, PHA, and rIL-1β. RTS11 and RTgutGC also responded to the stimulants but did not give a strong T-cell response, most likely reflecting the nature of the cell type as opposed to the mixed cell populations from the primary GALT cell cultures. When stimulated with both forms of β-glucan, GALT leucocytes demonstrated a strong proinflammatory and T-cell response.

Short and long-term effects of nanobiomaterials in fish cell lines. Applicability of RTgill-W1

Nanobiomaterials (NBMs) are nanostructured materials for biomedical applications that can reach aquatic organisms. The short and long-term effects of these emerging contaminants are unknown in fish. The RTgill-W1 cell line has been proposed as a model to predict the acute toxicity of chemicals to fish (OECD Test Guideline nº 249). We assessed the applicability of this cell line to study the short and long-term toxicity of 15 NBMs based on hydroxyapatites (HA), lipid (LSNP/LNP), gold, iron oxide, carbon, poly l-Lactide acid (PLLA) fibers with Ag and poly (lactide-co-glycolide) acid. Two more rainbow trout cell lines (RTL-W1, from liver, and RTS-11, from spleen) were exposed, to identify possible sensitivity differences among cells. Exposures to a range of concentrations (0.78–100 μg/mL) lasted for 24 h. Additionally, the RTgill-W1 was used to perform long-term (28 d exposure) and recovery (14 d exposure/14 d recovery) assays. Cells were exposed to the 24 h-IC20 and/or to 100 μg/mL. A triple cytotoxicity assay was conducted. After 24 h, only PLLA Fibers-Ag showed cytotoxicity (IC50 < 100 μg/mL). However, the NBMs in general provoked concentration-dependent effects after long-term exposures, except the LSNPs. A recovery of viability was only observed for AuNPs, AuNRods, Fe3O4PEG-PLGA, MgHA-Collag_Scaffolds, Ti-HA and TiHA-Alg NPs.These results evidenced the need to test the long-term toxicity of NBMs and showed differences in cytotoxicity probably associated to different mechanisms of toxic action. The RTgill-W1 was useful to screen short and long-term toxicities of NBMs and appears as a promiseful model to assess possible toxicity of NBMs in fish.

Immunostimulant properties of full-length and truncated Marinobacter algicola flagellins, and their effects against viral hemorrhagic septicemia virus (VHSV) in trout

Adjuvants that would help optimize fish vaccines against bacterial and viral pathogens are highly demanded by the aquaculture sector. Flagellin has been proposed as an immunostimulant and an adjuvant for more than a decade. However, the adjuvant ability of flagellins with hypervariable region deleted is still unclear in fish. In this study, we evaluated the immune-stimulating capacity of two recombinant flagellins, the wild-type flagellin F from Marinobacter algicola and a version with the hypervariable region deleted (FredV2), to induce the transcription of a wide range of immune genes using two rainbow trout cell lines: a monocyte/macrophage-cell line (RTS-11) and an epithelial cell line from intestine (RTgutGC). Additionally, we studied the capacity of both flagellins to limit the replication of viral hemorrhagic septicemia virus (VHSV) on the RTgutGC cell line. Our results demonstrated that both recombinant flagellins can significantly increase the transcription of IL-1β1, IL-6, and IL-8 in both cell lines. However, other cytokines such as IFNγ1, and TNFα or antimicrobial peptides such as hepcidin were induced by both flagellins in RTgutGC but not in RTS-11 cells. Furthermore, both flagellins were capable of reducing the replication of VHSV in RTgutGC cells. Although the immunostimulatory and the antiviral capacities exerted by F were slightly more potent than those obtained with FredV2, the effects were retained after losing the hypervariable region. Our results provide new information on the immunostimulating and antiviral capacities of flagellins that point to their potential as suitable adjuvants for the future optimization of vaccines in aquaculture.

Integrity and wound healing of rainbow trout intestinal epithelial cell sheets at hypo-, normo-, and hyper-thermic temperatures

How temperature influences fish physiological systems, such as the intestinal barrier, is important for understanding and alleviating the impact of global warming on fish and aquaculture. Monolayers of the rainbow trout cell line, RTgutGC, with or without linear 500μm wide gaps (wounds) were the in vitro models used to study the integrity and healing of intestinal epithelial sheets at different temperatures. Cultures at hypothermic (4°C) or hyperthermic (≥ 26°C) temperatures were compared to normothermic control cultures (18-22°C). Monolayers remained intact for at least a week at temperatures from 4 to 28°C, but had lost their integrity after 3h at 32°C as the cells pulled away from one another and from the plastic surface. F-actin appeared as prominent stress fibers in cells at 28°C and as blobs in cells at 32°C. At normothermia and at 26°C, cells migrated as sheets into the gaps and closed (healed) the gaps within 5-6 days. By contrast, wounds took 14 days to heal at 4°C. At 28°C some cells migrated into the gap in the first few days but mainly as single cells rather than collectively and wounds never healed. When monolayers with wounds were challenged at 32°C for 3h and returned to 18-22°C, cells lost their shape and actin organization and over the next 6 days detached and died. When monolayers were subjected to 26°C for 24h and challenged at 32°C for 3h prior to being placed at 18-22°C, cell shape and actin cytoskeleton were maintained, and wounds were healed over 6 days. Thus, intestinal epithelial cells become thermostabilized for shape, cytoskeleton and migration by a prior heat exposure.

Metabolism and cytotoxicity of diphenylarsinic acid, a degradation product of sea-dumped chemical warfare agents, in a rainbow trout liver cell line RTL-W1.

Recent studies have found primary degradation products of phenylarsenic chemical warfare agents (CWAs) accumulating in fish tissues, while the potential effects of these dumped phenylarsenic CWAs, such as Clark I and II, in the Baltic Sea biota are poorly understood. In this study, the metabolism and cytotoxicity of diphenylarsinic acid (DPA), a primary degradation product of phenylarsenic CWA, was studied by incubating rainbow trout cell line RTL-W1 cells in media with 100mg/L DPA. Previously undescribed metabolites were identified by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPHLCHRMS). Moreover, the cytotoxicity of diphenylarsine glutathione conjugate (DPA-SG), the major metabolite of DPA, was studied. Cytotoxicity of the compounds was evaluated using the Neutral Red retention test (NRR), showing an IC50 value of 278mg/L for DPA and 1.30mg/L for DPA-SG, indicating that the glutathione (GSH) conjugate of DPA is more than two orders of magnitude toxic than DPA itself, suggesting that toxic properties of DPA are increased after conjugation with intracellular GSH leading enhanced toxicity after uptake. Results gained in this study give more detailed information for elucidating biological effects of dumped chemical munitions in marine environment. Moreover, the results help in assessing the environmental and health risks posed by marine munition continued presence and deterioration in the sea bottom.

Effect of PET and PVC microplastics on rainbow trout cell lines RTgill-W1, RTG-2 and RTL-W1

Effect of PET and PVC microplastics on rainbow trout cell lines RTgill-W1, RTG-2 and RTL-W1

Evidence of Transcriptional Shutoff by Pathogenic Viral Haemorrhagic Septicaemia Virus in Rainbow Trout.

The basis of pathogenicity of viral haemorrhagic septicaemia virus (VHSV) was analysed in the transcriptome of a rainbow trout cell line inoculated with pathogenic and non-pathogenic VHSV isolates. Although both VHSV isolates showed similar viral replication patterns, the number of differentially expressed genes was 42-fold higher in cells inoculated with the non-pathogenic VHSV at 3 h post inoculation (hpi). Infection with the non-pathogenic isolate resulted in Gene Ontologies (GO) enrichment of terms such as immune response, cytokine-mediated signalling pathway, regulation of translational initiation, unfolded protein binding, and protein folding, and induced an over-representation of the p53, PPAR, and TGF-β signalling pathways. Inoculation with the pathogenic isolate resulted in the GO enrichment of terms related to lipid metabolism and the salmonella infection KEGG pathway involved in the rearrangement of the cytoskeleton. Antiviral response was evident at 12hpi in cells infected with the pathogenic isolate. Overall, the data showed a delay in the response of genes involved in immune responses and viral sensing in cells inoculated with the pathogenic isolate and suggest transcriptional shutoff and immune avoidance as a critical mechanism of pathogenicity in VHSV. These pathways offer opportunities to further understand and manage VHSV pathogenicity in rainbow trout.

A ribonucleoprotein transfection strategy for CRISPR/Cas9\u2010mediated gene editing and single cell cloning in rainbow trout cells

BackgroundThe advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example.ResultsRibonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively.ConclusionsThe designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

Analysis of immunostimulatory responses and immune tolerance to β-glucans in rainbow trout cell lines

β-glucans are widely used in fish diets as immunomodulators to enhance the defensive capacities of farmed animals and improve their resistance to pathogens. Despite their extensive use, the precise effects that β-glucans provoke on immune and intestinal cells in teleost fish have not yet been fully elucidated. In the current work, we have used the RTS11 monocyte-macrophage cell line and the RTgutGC intestinal epithelial cell line to further investigate the effects of β-glucans on rainbow trout (Oncorhynchus mykiss), determining the levels of transcription of a wide range of immune genes that code for pro-inflammatory cytokines/molecules, antimicrobial peptides or adhesion molecules. Our results show that RTS11 monocytes/macrophages are more reactive to β-glucans than intestinal epithelial cells. Despite this, β-glucans were able to enhance some of the transcriptional effects of Aeromonas salmonicida on RTgutGC cells as they did on RTS11 cells, evidencing some immunostimulatory effects on gut epithelial cells. Finally, we have also used these two cell lines to establish whether β-glucans induce tolerance, comparing the effects to those exerted by LPS. Pre-exposure of both cell lines to LPS strongly inhibited a secondary response to LPS that affected the transcription of many of the genes studied, confirming the potential of these cell lines to study mechanisms of immune tolerance. However, when both cell lines were stimulated with β-glucans, only interleukin 6 (IL6) transcription was significantly lower in cells that had been previously exposed to β-glucans in comparison to cells that had not been previously stimulated. These results provide valuable information concerning the immunostimulatory and potential tolerogenic effects exerted by β-glucans in both macrophages and gut epithelial cells.

The sensitivity of multiple ecotoxicological assays for evaluating Microcystis aeruginosa cellular algal organic matter and contribution of cyanotoxins to the toxicity

Secondary metabolites of cyanobacteria and algae released during algal blooms often exhibit toxic effects, but only a small number of the metabolites are the subject of routine analytical screenings. Alternatively, ecotoxicological assays offer a better representation of the overall negative effects. The aim of this work was to compare multiple assays in their sensitivity towards cellular algal organic matter (COM) of the toxin-producing cyanobacterium Microcystis aeruginosa. Multiple endpoints were investigated: mortality, growth inhibition, bioluminescence inhibition, genotoxicity, endocrine-disrupting effects, oxidative stress, and the induction of ethoxyresorufin-O-deethylase (EROD). Three rainbow trout (Oncorhynchus mykiss) cell lines as well as representatives of bacteria, yeasts, algae, vascular plants, and crustaceans were employed, and the results were expressed per mg of dissolved organic carbon (DOC) in the COM. M. aeruginosa COM was toxic to the RTgill-W1, RTG-2, and RTL-W1 cell lines (EC50 values ranging from 0.48 ± 0.02 to 1.9 ± 0.1 mgDOC/L), to the crustacean Thamnocephalus platyurus (LC50 = 20 ± 1 mgDOC/L), and to Lepidium sativum (IC50 = 241 ± 13 mgDOC/L). In contrast, no effect was observed for bacteria and yeasts, and the growth of the alga Desmodesmus subspicatus was even stimulated. No genotoxicity, endocrine-disrupting effects or increase in oxidative stress or EROD activity was detected. The content of six microcystins (MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF), anatoxin-a, cylindrospermopsin, and nodularin in the M. aeruginosa COM was determined by liquid chromatography–tandem mass spectrometry. An artificially prepared mixture of the detected cyanotoxins in the corresponding concentrations did not induce response in the O. mykiss cell lines and T. platyurus, suggesting that other cyanobacterial metabolites are responsible for the toxicity of M. aeruginosa.