Fish Skin and Gill Mucus: A Source of Metabolites for Non-Invasive Health Monitoring and Research

Mucous membranes such as the gill and skin mucosa in fish protect them against a multitude of environmental factors. At the same time, changes in the molecular composition of mucus may provide valuable information about the interaction of the fish with their environment, as well as their health and welfare. In this study, the metabolite profiles of the plasma, skin and gill mucus of freshwater Atlantic salmon (Salmo salar) were compared using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Several normalization procedures aimed to reduce unwanted variation in the untargeted data were tested. In addition, the basal metabolism of skin and gills, and the impact of the anesthetic benzocaine for euthanisation were studied. For targeted metabolomics, the commercial AbsoluteIDQ p400 HR kit was used to evaluate the potential differences in metabolic composition in epidermal mucus as compared to the plasma. The targeted metabolomics data showed a high level of correlation between different types of biological fluids from the same individual, indicating that mucus metabolite composition could be used for fish health monitoring and research

Protective Immunization of Atlantic Salmon (Salmo salar L.) against Salmon Lice (Lepeophtheirus salmonis) Infestation

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Mucosal and Systemic Immune Responses to Salmon Gill Poxvirus Infection in Atlantic Salmon Are Modulated Upon Hydrocortisone Injection

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD

Protective Immunization of Atlantic Salmon (Salmo salar L.) against Salmon Lice (Lepeophtheirus salmonis) Infestation

Vaccination against salmon lice (Lepeophtheirus salmonis) is a means of control that averts the negative effects of chemical approaches. Here, we studied the immunogenicity and protective effect of a vaccine formulation (based on a salmon lice-gut recombinant protein [P33]) against Lepeophtheirus salmonis infestation in Atlantic salmon in a laboratory-based trial. Our findings revealed that P33 vaccine can provide a measure of protection against immature and adult salmon lice infestation. This protection seemed to be vaccine dose-dependent, where higher doses resulted in lower parasitic infestation rates. We also provide immunological evidence confirming that P33-specific immune response can be triggered in Atlantic salmon after P33 vaccination, and that production of P33-specific antibodies in blood can be detected in vaccinated fish. The negative correlation between P33-specific IgM in salmon plasma and salmon lice numbers on vaccinated fish suggests that protection against lice can be mediated by the specific antibody in salmon plasma. The success of P33 vaccination in protecting salmon against lice confirms the possibility of employing the hematophagous nature of the parasite to deliver salmon-specific antibodies against lice-gut proteins

Workflow for the Targeted and Untargeted Detection of Small Metabolites in Fish Skin Mucus

The skin mucus of fish is in permanent contact with the aquatic environment. Data from the analysis of the chemical composition of skin mucus could potentially be used for monitoring the health status of the fish. Knowledge about mucus composition or change in composition over time could also contribute to understanding the aetiology of certain diseases. The objective of the present study was the development of a workflow for non-invasive sampling of skin mucus from farmed salmon (Salmo salar) for the targeted and untargeted detection of small metabolites. Skin mucus was either scraped off, wiped off using medical wipes, or the mucus’ water phase was absorbed using the same type of medical wipes that was used for the wiping method. Following a simple filtration step, the obtained mucus samples were subjected to hydrophilic interaction chromatography coupled to high-resolution mass spectrometry. Post-acquisition processing included the targeted analysis of 86 small metabolites, of which up to 60 were detected in absorbed mucus. Untargeted analysis of the mucus samples from equally treated salmon revealed that the total variation of the metabolome was lowest in absorbed mucus and highest in the scraped mucus. Thus, future studies including small-molecule metabolomics of skin mucus in fish would benefit from a sampling regime employing absorption of the water phase in order to minimize the bias related to the sampling step. Furthermore, the absorption method is also a less invasive approach allowing for repetitive sampling within short time intervals

Identification of Novel Glycans in the Mucus Layer of Shark and Skate Skin

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage. While the mucus layers of various bony fish species have been investigated, the composition and glycan profiles of shark skin mucus remain relatively unexplored. In this pilot study, we aimed to explore the structure and composition of shark skin mucus through histological analysis and glycan profiling. Histological examination of skin samples from Atlantic spiny dogfish (Squalus acanthias) sharks and chain catsharks (Scyliorhinus retifer) revealed distinct mucin-producing cells and a mucus layer, indicating the presence of a functional mucus layer similar to bony fish mucus albeit thinner. Glycan profiling using liquid chromatography–electrospray ionization tandem mass spectrometry unveiled a diverse repertoire of mostly O-glycans in the mucus of the two sharks as well as little skate (Leucoraja erinacea). Elasmobranch glycans differ significantly from bony fish, especially in being more sulfated, and some bear resemblance to human glycans, such as gastric mucin O-glycans and H blood group-type glycans. This study contributes to the concept of shark skin having unique properties and provides a foundation for further research into the functional roles and potential biomedical implications of shark skin mucus glycans

First record of experimentally induced salmon gill poxvirus disease (SGPVD) in Atlantic salmon (Salmo salar L.)

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Vaccination with ectoparasite proteins involved in midgut function and blood digestion reduces salmon louse infestations

Infestation with the salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) affects Atlantic salmon (Salmo salar L.) production in European aquaculture. Furthermore, high levels of salmon lice in farms significantly increase challenge pressure against wild salmon populations. Currently, available control methods for salmon louse have limitations, and vaccination appears as an attractive, environmentally sound strategy. In this study, we addressed one of the main limitations for vaccine development, the identification of candidate protective antigens. Based on recent advances in tick vaccine research, herein, we targeted the salmon louse midgut function and blood digestion for the identification of candidate target proteins for the control of ectoparasite infestations. The results of this translational approach resulted in the identification and subsequent evaluation of the new candidate protective antigens, putative Toll-like receptor 6 (P30), and potassium chloride, and amino acid transporter (P33). Vaccination with these antigens provided protection in Atlantic salmon by reducing adult female (P33) or chalimus II (P30) sea lice infestations. These results support the development of vaccines for the control of sea lice infestations.This research was funded by PHARMAQ AS, grant number Agreement PHARMAQ-CSIC.Peer reviewe

Protective Immunization of Atlantic Salmon (Salmo salar L.) against Salmon Lice (Lepeophtheirus salmonis) Infestation

Vaccination against salmon lice (Lepeophtheirus salmonis) is a means of control that averts the negative effects of chemical approaches. Here, we studied the immunogenicity and protective effect of a vaccine formulation (based on a salmon lice-gut recombinant protein [P33]) against Lepeophtheirus salmonis infestation in Atlantic salmon in a laboratory-based trial. Our findings revealed that P33 vaccine can provide a measure of protection against immature and adult salmon lice infestation. This protection seemed to be vaccine dose-dependent, where higher doses resulted in lower parasitic infestation rates. We also provide immunological evidence confirming that P33-specific immune response can be triggered in Atlantic salmon after P33 vaccination, and that production of P33-specific antibodies in blood can be detected in vaccinated fish. The negative correlation between P33-specific IgM in salmon plasma and salmon lice numbers on vaccinated fish suggests that protection against lice can be mediated by the specific antibody in salmon plasma. The success of P33 vaccination in protecting salmon against lice confirms the possibility of employing the hematophagous nature of the parasite to deliver salmon-specific antibodies against lice-gut proteins

Long-Term Feeding with Curcumin Affects the Growth, Antioxidant Capacity, Immune Status, Tissue Histoarchitecture, Immune Expression of Proinflammatory Cytokines, and Apoptosis Indicators in Nile Tilapia, <i>Oreochromis niloticus</i>

The impact of dietary curcumin (CUR) on the growth, antioxidant activity, histomorphology of certain organs, proinflammatory cytokine production, and immune status of Oreochromis niloticus was evaluated. The fingerlings (n = 225, 41.60 ± 0.09 g/fish) were randomly allotted into five experimental groups in triplicate. Fish were fed basal diets complemented with 0, 200, 400, 600, or 800 mg curcumin/kg diet (CUR0, CUR200, CUR400, CUR600, and CUR800, respectively) for 10 weeks. An increase in fish growth was reported in the CUR200 and CUR400 groups. The feed conversion ratio was enhanced by 15% in the CUR400 group. Fish body protein content was increased in the CUR600 group (p ≤ 0.01). Body fat was decreased, and ash content was increased by CUR supplementation in a level-related way (p O. niloticus challenged with Aeromonas hydrophila was highest in the CUR200-CUR600 groups (100%) and decreased in the CUR800 group (80%). This study concluded that CUR could be added to Nile tilapia diets up to 400 mg·kg−1 to achieve better growth, antioxidant capacity, immune response, and intestinal histology. Long feeding periods on high levels of CUR (600 and 800 mg·kg−1) stimulate inflammatory reactions in fish tissues