Rainbow Trout Gill Research Articles

Environmental Implications of Ionic Liquid and Deep Eutectic Solvent in Geothermal Application: Comparing Traditional and New Approach Methods.

The significant surge of ionic liquids (ILs) research over the past decade has led to the formation of various novel ionic liquid compounds and their diverse applications. Enhanced geothermal systems (EGS) for geothermal power generation are an emerging IL application as a heat extraction fluid. The once widely held belief in the environmentally friendly characteristics of ionic liquids, mainly due to their insignificant vapor pressure, is now being scrutinized. It has become apparent that while ILs do not readily evaporate into the atmosphere, they are not guaranteed to remain entirely isolated from the environment. Recent attention has been directed toward toxicological studies, including ecotoxicity impacts, with the long-accepted assumption of ILs having low toxicity being invalid. This paper aims to shed light on the toxicity of hexylepyradinium bromide (HPyBr) IL and a deep eutectic solvent (DES) comprising choline chloride with magnesium chloride hexahydrate (ChCl:MgCl2·6H2O) to five test species, an algal species (Raphidocelis subcapitata), the water flea (Ceriodaphnia dubia and Daphina magna), the fathead minnow (Pimephales promelas), and the earthworm (Eisenia fetida), to measure acute and chronic toxicity. Additionally, new approach methods (NAMs) are presented using the fathead minnow embryo and the rainbow trout (Oncorhynchus mykiss) gill cell line and the RTgill-W1 assay to compare sensitivity across species. Overall, ChCl:MgCl2·6H2O displayed lower toxicity, while HPyBr demonstrated higher toxicity, highlighting the need for caution in handling it to prevent harm to aquatic ecosystems. Comparative analysis underscored the potential threat of ChCl:MgCl2·6H2O to aquatic life, highlighting the cumulative effects of the environmental components.

In Vitro Predictions of Acute Fish Toxicity for Development of Sustainable Crop Protection Products.

New agrochemicals must demonstrate safety to numerous ecological systems, including aquatic systems, and aquatic vertebrate toxicity is typically evaluated by using the in vivo acute fish toxicity (AFT) test. Here, we investigated two alternative in vitro assays using a cell line isolated from rainbow trout (Onchorhynchus mykiss) gill tissue: (i) adenosine triphosphate (ATP) luminescence and (ii) cell painting. The former assay measures cytotoxicity, while the latter measures changes in cellular morphology in response to chemical exposure. We assessed how well end points in these two assays predicted acute lethality (i.e., LC50 values) in independent in vivo AFT tests. When compared to results from OECD TG 249 (in vitro), we found that the ATP assay was not as predictive (R2 = 0.53) as the cell painting assay. Similarly, when compared to results from OECD TG 203 (in vivo), the cell painting was much more predictive (R2 = 0.67). Our results show that such in vitro assays are useful for fast and efficient screening alternatives to in vivo fish testing that can aid in the agrochemical discovery phase, where thousands of potential new actives are tested each year.

Single and combined effects of titanium (TiO2) and zinc (ZnO) oxide nanoparticles in the rainbow trout gill cell line RTgill-W1.

Understanding the environmental impact of nanoparticle (NP) mixtures is essential to accurately assess the risk they represent for aquatic ecosystems. However, although the toxicity of individual NPs has been extensively studied, information regarding the toxicity of combined NPs is still comparatively rather scarce. Hence, this research aimed to investigate the individual and combined toxicity mechanisms of two widely consumed nanoparticles, zinc oxide (ZnO NPs) and titanium dioxide (TiO2 NPs), using an in vitro model, the RTgill-W1 rainbow trout gill epithelial cell line. Sublethal concentrations of ZnO NPs (0.1µgmL-1) and TiO2 (30µgmL-1) and a lethal concentration of ZnO NPs causing 10% mortality (EC10, 3µgmL-1) were selected based on cytotoxicity assays. Cells were then exposed to the NPs at the selected concentrations alone and to their combination. Cytotoxicity assays, oxidative stress markers, and targeted gene expression analyses were employed to assess the NP cellular toxicity mechanisms and their effects on the gill cells. The cytotoxicity of the mixture was identical to the one of ZnO NPs alone. Enzymatic and gene expression (nrf2, gpx, sod) analyses suggest that none of the tested conditions induced a strong redox imbalance. Metal detoxification mechanisms (mtb) and zinc transportation (znt1) were affected only in cells exposed to ZnO NPs, while tight junction proteins (zo1 and cldn1), and apoptosis protein p53 were overexpressed only in cells exposed to the mixture. Osmoregulation (Na + /K + ATPase gene expression) was not affected by the tested conditions. The overall results suggest that the toxic effects of ZnO and TiO2 NPs in the mixture were significantly enhanced and could result in the disruption of the gill epithelium integrity. This study provides new insights into the combined effects of commonly used nanoparticles, emphasizing the importance of further investigating how their toxicity may be influenced in mixtures.

Novel insights on microbiome dynamics during a gill disease outbreak in farmed rainbow trout (Oncorhynchus mykiss)

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.

In vitro cultivation of adherent cells on microscope slides for downstream applications

In vitro studies with cultured cells are often conducted as an important part of basic research. Adherent cells are typically cultivated in flasks or trays, for which cell staining and subsequent visualization become impractical. We here present a simple step-by-step method for growing adherent cells directly on glass microscope slides, using low-cost equipment readily available in most laboratories. Most parameters such as type of microscope slide (e.g. surface coating), cell seeding concentrations and incubation times can be adjusted according to cell line characteristics and experimental aims, reflecting the methods’ flexibility. Through our experiments, microscope slides proved to provide an acceptable surface for cell adhesion and growth of the tested cell lines, as well as being robust and functional with respect to downstream procedures. The method can potentially be combined with different techniques for visualization of experimental effects, such as histological staining methods, fluorescent staining, and immunochemistry. In our method development we have successfully cultivated three different cell lines directly on microscope slides – Atlantic salmon kidney cells (ASK), rainbow trout gill cells (RTgill-W1), and human cancerous lung cells (A549) – and subjected them to various experimental treatments. Finally, as proof-of-concept we provide examples of successful histological staining of the fixed cells.Experimental design in short:•Cultivate cells and calculate cell concentration•Seed a small volume of growth medium with an appropriate number of cells on microscope slide in an area confined by hydrophobic marker•Let cells adhere over night before adding more growth medium or directly conducting experiments and fixing cells for downstream applications

Characterization and inter-strain variability in ichthyotoxicity of Heterocapsa ovata (Peridiniales, Dinophyceae) from temperate waters of South Australia

IntroductionHarmful algal bloom (HAB)-forming species and populations exhibit substantial intraspecific functional trait variation, which can confer eco-evolutionary advantages. Phenotypic variability among populations can buffer the immediate detrimental effects of environmental fluctuations, with more diverse populations expected to survive changing conditions more efficiently than their uniform counterparts.MethodsIn February 2014, a mixed fish-killing dinoflagellate bloom occurred in the temperate waters of Coffin Bay, South Australia, causing the death of oysters and fish in the area. The bloom was dominated by Karenia mikimotoi and a cryptic species of Heterocapsa. Twenty-one monoclonal Heterocapsa isolates were established from the site and identified as H. ovata using microscopy and universal ribosomal markers (ITS/5.8S and LSU D1/D3 rDNA regions; SSU and cob were used for amplicon sequencing). These isolates were tested for ichthyotoxicity using a bioassay based on cells from the gills of rainbow trout (Oncorhynchus mykiss). Culture fraction preparations (whole cells, supernatant, and lysed cells) were analyzed to determine ichthyotoxicity levels.ResultsThe highest ichthyotoxicity was observed in lysed cells, with surprisingly high inter-strain variability. This suggests that different strains of H. ovata have varying levels of toxicity.DiscussionResults from this study expand our understanding of the adaptive strategies of HAB species and enable predictions of future population dynamics under changing climatic conditions. The substantial phenotypic variability among H. ovatastrains highlights the potential for diverse responses to environmental stressors, underscoring the importance of considering intraspecific variation in ecological and evolutionary studies of HABs.

Mixtures of legacy and replacement perfluorosulphonic acids (PFSAs) demonstrate ratio-, concentration- and endpoint-dependent synergistic interactions in vitro

The extensive use of poly- and per-fluoroalkyl substances (PFASs) has les to their widespread presence in the environment, raising concerns about potential toxicity. While certain PFASs of concern have been phased-out or banned, new PFASs continue to be produced. Two such substances are perfluoroethylcyclohexane sulphonate (PFECHS) and perfluorobutane sulphamide (FBSA), replacements of perfluoroctanesulphonic acid (PFOS) that have recently been detected in multiple environmental media around the globe. Despite PFASs generally occurring in the environment as mixtures, few data are available outlining the effects of PFAS mixtures. Therefore, this research investigated the interaction potential of binary and ternary mixtures of emerging and legacy PFASs. The immortalized rainbow trout gill cell line (RTgill-W1) was chosen as the experimental model to investigate two apical endpoints: cytotoxicity and phospholipidosis. RTgill-W1 cells were exposed for 24 h to each compound to obtain endpoint-specific effect concentrations (LCx; ECx). These values were then applied to formulate mixture predictions following the Loewes Additivity and Steel and Peckham methods. Based on cytotoxicity, relative potencies of individual compounds were: PFOS > PFECHS > FSBA. PFOS and PFECHS had nearly identical effects on phospholipidosis, while FSBA did not have any effects. Most mixtures had a synergistic effect on cytotoxicity, but the effect was both dose- and ratio-dependent. PFOS and PFECHS were additive at lower concentrations (LC10) and synergistic at higher concentrations (LC50; 3:1, 1:1, and 1:3). PFECHS and FSBA mixtures were synergistic at all doses and ratios (3:1, 1:1, 1:3), while FBSA and PFOS were mainly synergistic at higher concentrations and at ratios favouring PFOS (1:1, 1:3). Tertiary combinations were mainly synergistic. For phospholipidosis, mixtures were strictly additive. These results are strongly suggestive of synergism between emerging PFAS replacements and highlight that independent apical mechanisms of different PFASs could combine to induce unexpected toxicity. Considering that emerging replacements are continuing to increase in concentration in the environment, such mixture scenarios are also likely to continue to increase in probability.

The myxozoans Myxobolus cerebralis and Tetracapsuloides bryosalmonae modulate rainbow trout immune responses: quantitative shotgun proteomics at the portals of entry after single and co-infections.

Little is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae. One group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection. In the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated. The results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Morphometric and Enzymatic Changes in Gills of Rainbow Trout after Exposure to Elevated Temperature-Indications for Gill Remodeling.

Seven-month-old rainbow trout acclimated to 9 °C were used. The fish were gradually adapted to a water temperature of 20 °C over a period of seven days and then exposed to this temperature for 32 days. Changes in gill morphometry and histology and in enzyme activities in comparison to fish kept at 9 °C were investigated. No histopathological abnormalities were discerned at the heightened temperature. The gill epithelium thickened by approximately 40%, suggesting an increase in the branchial diffusion barrier for ions, water, and gases. Concurrently, there was a significant decrease in the activities of gill H+-ATPase and Na+/K+-ATPase, indicative of a reduction in osmoregulation under elevated temperatures. Carbonic anhydrase activity exhibited an increase following the 32-day exposure to 20 °C, potentially mitigating the adverse effects of increased gill epithelium thickness on gaseous exchange. There were no indications of gill surface enlargement as the measurements of the length of the primary and secondary lamellae, as well as of the distances between them, were similar at 9 and 20 °C. The activities of the gill enzymes associated with glycolysis and the citric acid cycle displayed a varied response following the 32-day exposure of rainbow trout to 20 °C. Pyruvate kinase decreased, while lactate dehydrogenase increased, and malate dehydrogenase remained constant. This might suggest a decrease in the glycolytic rate, a greater reliance on anaerobic pathways at 20 °C compared to 9 °C, and the consistent efficiency of the citric acid cycle in the gills of rainbow trout in response to elevated temperatures. In summation, the data suggest a remodeling of rainbow trout gills in response to elevated temperatures, affecting both morphometric and metabolic aspects.

The flame retardant triphenyl phosphate alters the epigenome of embryonic cells in an aquatic in vitro model.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant and plasticizer that is added to a wide variety of consumer and industrial products. It is also a ubiquitous environmental pollutant. Exposure to TPhP has been shown to alter gene expression in metabolic and estrogenic signaling pathways in in vitro and in vivo models of a variety of species, and as such, is considered to be an endocrine disrupting chemical. Exposure to endocrine disrupting chemicals is increasingly being associated with changes to the epigenome, especially during embryonic development. The aim of this study was to evaluate whether TPhP exposure in aquatic ecosystems has the ability to alter the epigenome in two immortal cell lines derived from trout (Oncorhynchus mykiss). This study assessed whether 24 h exposure to TPhP resulted in changes to histone modification and DNA methylation profiles in steelhead trout embryonic cells and rainbow trout gill epithelial cells. Results show that several epigenetic modifications on histone H3 and DNA methylation are altered in the embryonic cells following TPhP exposure, but not in the gill epithelial cells. Specifically, histone H3 acetylation, histone H3 mono-methylation and global DNA methylation were found to be reduced. The alterations of these epigenetic modification profiles in the embryonic cells suggest that exposure to TPhP during fetal development may alter gene expression in the developing embryo, likely in metabolic and estrogenic pathways. The impacts to the epigenome determined in this study may even carry multigenerational detrimental effects on human and ecosystem health, which requires further investigation.

Cytotoxicity of 19 Pesticides in Rainbow Trout Gill, Liver, and Intestinal Cell Lines.

The rainbow trout gill cell line (RTgill-W1), via test guideline 249 of the Organisation for Economic Co-operation and Development, has been established as a promising New Approach Methodology, although to advance confidence in the method more case studies are needed that: 1) expand our understanding of applicability domains (chemicals with diverse properties); 2) increase methodological throughput (96-well format); and 3) demonstrate biological relevance (in vitro to in vivo comparisons; gill vs. other cells). Accordingly, the objective of our study was to characterize the cytotoxicity of 19 pesticides against RTgill-W1 cells, and also liver (RTL-W1) and gut epithelial (RTgutGC) cell lines, and then to compare the in vitro and in vivo data. Of the 19 pesticides tested, 11, 9, and 8 were cytotoxic to the RTgill-W1, RTL-W1, and RTgutGC cells, respectively. Six pesticides (carbaryl, chlorothalonil, chlorpyrifos, dimethenamid-P, metolachlor, and S-metolachlor) were cytotoxic to all three cell lines. Aminomethylphosphonic acid, chlorantraniliprole, dicamba, diquat, imazethapyr, and permethrin exhibited cell-line-specific toxicity. No cytotoxic responses were observed for three herbicides (atrazine, glyphosate, and metribuzin) and four insecticides (clothianidin, diazinon, imidacloprid, and thiamethoxam). When cytotoxicity was measured, there was a strong correlation (rs = 0.9, p < 0.0001) between in vitro median effect concentration (EC50) values (based on predicted concentrations using the In Vitro Mass Balance Model Equilibrium Partitioning (IV-MBM EQP) Ver. 2.1) derived from RTgill-W1 and RTL-W1 cells with in vivo median lethal concentration (LC50) values from 96-h acute toxicity studies with trout. In all 28 cases, the in vitro EC50 was within 18-fold of the in vivo LC50. These data help increase our understanding of the ecotoxicological domains of applicability for in vitro studies using cultured rainbow trout cells, while also demonstrating that these assays performed well in a 96-well format and have promise to yield data of biological relevance. Environ Toxicol Chem 2024;00:1-13. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Toxicological Effects of Synthesized Pyrazoline Derivative 4-(3-(4-Bromophenyl))- 5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl) Benzenesulfonamide, Compound B8 on Antioxidant Enzymes and Hematological Parameters of Rainbow Trout Alevins (Oncorhynchus mykiss)

The toxicological effects of a synthesized pyrazoline derivative 4-(3-(4-bromophenyl))- 5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl) benzene sulfonamide, compound B8, on rainbow trout (Oncorhynchus mykiss) were evaluated by following the activity of the antioxidant enzymes and the hematological parameters. Fish were exposed to variant doses (0.5, 1, and 2.5 mg L-1) of the compound B8 for 48 h. Antioxidant enzymes and activities were measured, and the obtained data were statistically analyzed. Our results showed that concentration at 1 mg L-1 significantly increased the malondialdehyde (MDA) levels (p &lt; 0.05) in the liver and gills of rainbow trout (Oncorhynchus mykiss), while glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were significantly decreased. The increase in MDA levels in the liver and gill could be related to tissue damage induced by oxidative stress after exposure to compound B8. Significant increases in white blood cell count (WBCs), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentrations (MCHC) were obtained (p &lt; 0.05). Moreover, significant decreases were observed in erythrocyte numbers, hemoglobin concentrations, hematocrit levels, and platelet numbers (p &lt; 0.05). The results showed that exposure to high doses (2.5 mg L-1) of the compound B8 caused oxidative stress in the liver and gills of fish and may lead to acute anemia.

In vitro fish mucosal surfaces producing mucin as a model for studying host-pathogen interactions.

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.

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.

Mucosal immunity and microbiota change in the rainbow trout (Oncorhynchus mykiss) gills after being challenged with infectious hematopoietic necrosis virus

Respiratory structures are crucial for vertebrate survival, as they serve not only to perform gas-exchange processes but also as entry points for opportunistic pathogens. Previous studies have demonstrated that fish contain gill mucosal-associated lymphoid tissue, and harbor a large number of commensal bacteria on their surface and contribute to maintaining fish health. However, by far, very limited information is known regarding the effects of viral infection on gill mucosal immunity and microbiota homeostasis. In this study, we conducted an infection model by bath with infectious hematopoietic necrosis virus (IHNV) and revealed a 27 % mortality rate among rainbow trout in the first two weeks after infection. Moreover, we found that diseased fish with the highest IHNV loads in gills exhibiting severe damage, as well as increased goblet cell counts in both primary lamellae (PL) and secondary lamellae (SL). Additionally, RT-qPCR and RNA-seq analyses revealed that IHNV infection induced a strong innate and adaptive antiviral immune responses. Interestingly, an antibacterial immune response was also observed, suggesting that a secondary bacterial infection occurred in trout gills after viral infection. Furthermore, 16S rRNA analysis of trout gills revealed a profound dysbiosis marked by a loss of beneficial taxa and expansion of pathobionts following IHNV infection. Overall, our finding demonstrates that IHNV infection induces significant changes of the microbial community in the fish respiratory surface, thus triggering local antiviral and bacterial mucosal immunity.

Yersinia ruckeri infection activates local skin and gill B cell responses in rainbow trout.

Teleost fish lack organized structures in mucosal tissues such as those of mammals, but instead contain dispersed B and T cells with the capacity to respond to external stimuli. Nonetheless, there is still a great lack of knowledge regarding how B cells differentiate to plasmablasts/plasma cells in these mucosal surfaces. To contribute to a further understanding of the mechanisms through which fish mucosal B cells are activated, in the current study, we have studied the B cell responses in the skin and gills of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri. We have first analyzed the transcription levels of genes related to B cell function in both mucosal surfaces, and in spleen and kidney for comparative purposes. In a second experiment, we have evaluated how the infection affects the presence and size of B cells in both skin and gills, as well as the presence of plasmablasts secreting total or specific IgMs. The results obtained in both experiments support the local differentiation of B cells to plasmablasts/plasma cells in the skin and gills of rainbow trout in response to Y. ruckeri. Interestingly, these plasmablasts/plasma cells were shown to secrete specific IgMs as soon as 5 days after the exposure. These findings contribute to a further understanding of how B cells in the periphery respond to immune stimulation in teleost fish.

The Effect of Malathion Concentration and Exposure Time on Histopathological Changes in the Liver and Gill of Rainbow Trout

Exposure of aquatic organisms to organophosphorus pollutants is a subject of keen interest to biologists and environmental scientists. Examining histopathological changes in the tissues of exposed animals can provide great insights to understand the health condition of the organisms. This study examined the effects of malathion concentration and exposure time on the liver and gill tissues of the rainbow trout (Oncorhynchus mykiss) in a laboratory condition and tried to provide a quantitative assessment for the analysis of these effects. The experiment was conducted in three treatments including 0.025, 0.05, and 0.075 mg/L of malathion for 1, 5, and 9 days with a nonexposed group as control, in three replicates. The liver and gill samples were fixed in buffered formalin. About 5 µ tissue sections were prepared using the conventional histological methods and stained using the hematoxylin–eosin method. Histopathological changes in the liver and gill tissues were quantified by grading and the resulting data were analyzed by rank-based estimation. The results showed that histopathological changes in the liver and gill tissues are more affected by the malathion concentration than by the duration of the exposure. However, longer exposure had an intensifying effect on the tissue damage caused by the malathion at higher concentrations. The presence of melanomacrophages as an indicator of malathion toxicity was determined. The fish exposed to 0.075 mg/L malathion for 9 days showed atrophy in the liver and gill tissues, indicating cell death and functional inactivation. Histopathological changes in the liver and gills confirmed the dose-dependent effect of malathion on the rainbow trout.

Teleost IgD+IgM− B cells in gills and skin have a plasmablast profile, but functionally and phenotypically differ from IgM+IgD− B cells in these sites

Although most B cells in teleost systemic compartments co-express IgM and IgD on the surface, cells exclusively expressing either of the two Igs are common in fish mucosal tissues, providing us with a unique opportunity to further characterize IgD+IgM- B cells, an intriguing B cell subset. Hence, we compared the phenotype of IgD+IgM- cells to that of IgM+IgD- B cells in rainbow trout gills and skin, also establishing the response of these subsets to immune stimulation. The transcriptional profile and secreting capacity of IgD+IgM- B cells corresponded to that of cells that have started a differentiation program toward plasmablasts, similarly to IgM+IgD- B cells. Yet, IgM+IgD- B cells retained high levels of surface MHC II and antigen-processing abilities, while these were much lower in IgD+IgM- cells, suggesting important differences in their antigen-presenting capacities. Our findings contribute to a deeper understanding of the enigmatic role of IgD in mucosal surfaces.

Amoeba species colonizing the gills of rainbow trout (Oncorhynchus mykiss) in Swiss aquaculture.

Nodular gill disease (NGD) is an infectious condition characterized by proliferative gill lesions leading to respiratory problems, oxygen deficiency and mortality in fish. Globally, NGD primarily impacts freshwater salmonids in intensive aquaculture systems. In recent years, numerous outbreaks of severe gill disease have affected more than half of the larger rainbow trout (Oncorhynchus mykiss) farms in Switzerland, mainly during spring and early summer. Mortality has reached up to 50% in cases where no treatment was administered. Freshwater amoeba are the presumed aetiologic agent of NGD. The gross gill score (GS) categorising severity of gill pathology is a valuable first-line diagnostic tool aiding fish farmers in identifying and quantifying amoebic gill disease (AGD) in farmed marine salmonids. In this study, the GS was adapted to the NGD outbreak in farmed trout in Switzerland. In addition to scoring disease severity, gill swabs from NGD-affected rainbow trout were sampled and amoeba were cultured from these swabs. Morphologic and molecular methods identified six amoeba strains: Cochliopodium sp., Naegleria sp., Vannella sp., Ripella sp., Saccamoeba sp. and Mycamoeba sp. However, the importance of the different amoeba species for the onset and progression of NGD still has to be evaluated. This paper presents the first description of NGD with associated amoeba infection in farmed rainbow trout in Switzerland.

Accumulation of Thallium in Rainbow Trout (Oncorhynchus mykiss) Following Acute and Subchronic Waterborne Exposure.

The accumulation and tissue distribution of toxicants in aquatic biota can be determinative of their toxic impact to both exposed organisms and their potential human consumers. In the present study, accumulation of the trace metal thallium (Tl) in gill, muscle, plasma, and otoliths of rainbow trout (Oncorhynchus mykiss) following acute (96-h) and subchronic (28-day) waterborne exposures was investigated. Owing to known interactions between Tl and potassium ions (K+ ), plasma and muscle K+ concentrations were also determined. Branchial Tl accumulated in a dose-dependent manner in both acute and subchronic exposures, while plasma Tl was rapidly mobilized to tissues and accumulated only at exposure concentrations of 141 µg L-1 or higher. For muscle tissue, Tl concentrations at 28 days were markedly lower than those at 96 h at comparable exposure concentrations (0.9 µg L-1 ), indicating the presence of mechanisms that act to reduce Tl accumulation over time. However, after acute exposure, muscle Tl reached concentrations that, if consumed, would exceed acceptable daily intake values for this element, indicating some risk to human health from the consumption of fish from waters heavily contaminated with Tl. Otoliths showed Tl concentrations that reflected exposure concentration and length, confirming their capacity to provide insight into fish exposure history. No changes in tissue K+ concentrations were observed, suggesting that accumulation of Tl in rainbow trout plasma and muscle does not occur at the expense of K+ homeostasis. In addition to highlighting the capacity of rainbow trout to accumulate Tl to levels that exceed recommended dietary doses to human consumers, the present study provides the first data of tissue-specific Tl accumulation in an important regulatory species. Environ Toxicol Chem 2023;42:1553-1563. © 2023 SETAC.