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.

Quantification of Defensive Proteins in Skin Mucus of Atlantic salmon Using Minimally Invasive Sampling and High-Sensitivity ELISA.

Protocols used to collect fish skin mucus may inadvertently compromise the sampled fish or the resulting sample. Here, we used three methods (wiping, scraping, and absorption) to collect skin mucus from Atlantic salmon and compared their invasiveness on fish skin epithelium. We found that the absorption method was the least invasive. We also compared the abundance of antigen-specific immunoglobulin M subtype A antibodies (IgM-A Ab) and complement component 5 (C5) in mucus samples collected from vaccinated fish by the three methods. An enzyme-cascade-amplification strategy colorimetric immune assay was optimized and used to analyze IgM-A, and ELISA was used to analyze C5. The abundance of antigen-specific IgM-A in skin mucus was comparable between the three methods, but C5 was significantly lower in absorbed mucus in comparison to in the wiped or scraped mucus samples. Absorbed skin mucus samples collected from various body regions of salmon, levels of C5 were comparable, while specific IgM-A amounts varied between the regions. By comparing three mucus-absorbing materials (medical wipe, gauze, and cotton) for their ability to absorb and release IgM-A and C5, medical wipes proved to be ideal for IgM-A analysis, whereas gauze was the best for C5 analysis.

The predominant role of mucosal immunoglobulin IgT in the gills of rainbow trout (Oncorhynchus mykiss) after infection with Flavobacterium columnare.

Columnaris disease, induced by Flavobacterium columnare, seriously affects the health of freshwater fish species and damages the mucosal tissues, such as the fins, skin, and gills. Teleosts represent the first bony vertebrate to contain both innate and adaptive immune responses against pathogens. So far, three immunoglobulin isotypes (IgM, IgD, and IgT/IgZ) have been identified in teleost fish, and IgT in mucosal tissues of teleost fish was reported to perform a similar function to IgA in mammals during parasitic infection. However, very limited information is known about the function of IgT in gill mucosal tissues during bacterial infection. In the present study, rainbow trout (Oncorhynchus mykiss) was infected with F. columnare (Fc) via immersion. After Fc infection, the gill structure of rainbow trout showed serious hyperplasia symptoms on the secondary lamellae at 12 h post infection (hpi). Moreover, the mRNA expression levels of NOS2 and cathelicidin-1 were significantly upregulated immediately at 12 hpi and showed high expression throughout the experiment. IgT and IgM showed much higher mRNA expression levels at 28 days post infection (dpi) and 75 dpi, while IgD only showed high mRNA expression levels at 28 dpi. Importantly, the accumulation of IgT+ B cells and strong bacteria-specific IgT responses were detected in the gill lamellae of both infected fish (28 dpi) and survivor fish (75 dpi). Overall, our results suggest that IgT and IgT+ B cells play a central role in the adaptive immune responses of fish gill mucosa against bacterial infection.

Local and systemic humoral immune response in farmed Atlantic salmon (Salmo salar L.) under a natural amoebic gill disease outbreak.

Amoebic gill disease (AGD), caused by the protozoan parasite Neoparamoeba perurans, is one of the most significant infectious diseases for Atlantic salmon (Salmo salar L.) mariculture. The present study investigated the humoral immune response (both local in gill mucus and systemic in serum) of farmed Atlantic salmon naturally infected with N. perurans in commercial sea pens, at two different stages of the disease and after freshwater treatment. Parameters analysed included activity of immune related enzymes (i.e. lysozyme, peroxidase, protease, anti-protease, esterase, alkaline phosphatase), IgM levels, and the terminal carbohydrate profile in the gill mucus. Overall, greater variations between groups were noted in the immune parameters determined in gill mucus than the equivalent in the serum. In gill mucus, IgM levels and peroxidase, lysozyme, esterase and protease activities were decreased in fish showing longer exposure time to the infection and higher disease severity, then showed a sequential increase after treatment. Results obtained highlight the capacity of gills to elicit a local response to the infection, indicate an impaired immune response at the later stages of the disease, and show partial reestablishment of the host immune status after freshwater treatment. In addition to providing data on the humoral response to AGD, this study increases knowledge on gill mucosal humoral immunity, since some of the parameters were analysed for the first time in gill mucus.

Interaction of TiO2 nanoparticles with proteins from aquatic organisms: the case of gill mucus from blue mussel.

To better understand the mechanisms of TiO2 nanoparticle (NP) uptake and toxicity in aquatic organisms, we investigated the interaction of NPs with the proteins found in gill mucus from blue mussels. Mucus is secreted by many aquatic organisms and is often their first line of defense against pathogens, xenobiotics, and other sources of environmental stress. Here, five TiO2 NPs and one SiO2 NP were incubated with gill mucus and run out on a one-dimensional polyacrylamide gel for a comparative qualitative analysis of the free proteins in the mucosal solution and the proteins bound to NPs. We then used nanoscale liquid chromatography coupled with tandem mass spectrometry to identify proteins of interest. Our data demonstrated dissimilar protein profiles between the crude mucosal solution and proteins adsorbed on NPs. In particular, extrapallial protein (EP), one of the most abundant mucus proteins, was absent from the adsorbed proteins. After thermal denaturation experiments, this absence was attributed to the EP content in aromatic amino acids that prevents protein unfolding and thus adsorption on the NP. Moreover, although the majority of the protein corona was qualitatively similar across the NPs tested here (SiO2 and TiO2), a few proteins in the corona showed a specific recruitment pattern according to the NP oxide (TiO2 vs SiO2) or crystal structure (anatase TiO2 vs rutile TiO2). Therefore, protein adsorption may vary with the type of NP. Graphical abstract Proteins with adsorption selectivity as identified from isolated bands.

Characterisation of the gill mucosal bacterial communities of four butterflyfish species: a reservoir of bacterial diversity in coral reef ecosystems.

While recent studies have suggested that fish mucus microbiota play an important role in homeostasis and prevention of infections, very few studies have investigated the bacterial communities of gill mucus. We characterised the gill mucus bacterial communities of four butterflyfish species and although the bacterial diversity of gill mucus varied significantly between species, Shannon diversities were high (H = 3.7-5.7) in all species. Microbiota composition differed between butterflyfishes, with Chaetodon lunulatus and C. ornatissimus having the most similar bacterial communities, which differed significantly from C. vagabundus and C. reticulatus. The core bacterial community of all species consisted of mainly Proteobacteria followed by Actinobacteria and Firmicutes. Chaetodonlunulatus and C. ornatissimus bacterial communities were mostly dominated by Gammaproteobacteria with Vibrio as the most abundant genus. Chaetodonvagabundus and C. reticulatus presented similar abundances of Gammaproteobacteria and Alphaproteobacteria, which were well represented by Acinetobacter and Paracoccus, respectively. In conclusion, our results indicate that different fish species present specific bacterial assemblages. Finally, as mucus layers are nutrient hotspots for heterotrophic bacteria living in oligotrophic environments, such as coral reef waters, the high bacterial diversity found in butterflyfish gill mucus might indicate external fish mucus surfaces act as a reservoir of coral reef bacterial diversity.

Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.