Microbial contributions to the Atlantic salmon (Salmo salar) skin mucosal barrier

Fish live in close contact with bacteria; in seawater, there are around one million per millilitre. The fish secret mucus, which is covering the skin. Together with the skin, this mucus layer constitutes a barrier which protects the fish against bacteria and other substances in the water. However, at the same time, this mucus is inhabited by bacteria, which appear to be a natural part of the skin mucosal surface. It is not known whether this skin microbiota has a functional role in this mucosal barrier. The mucosal surfaces of fish skin have similarities to the mucosal surfaces covering the intestines of vertebrates and humans. Bacteria also colonize these surfaces, and recent research has demonstrated that paradoxically, these bacteria are required for obtaining protection against other bacteria. We hypothesize that natural microbiota inhabiting fish skin mucus is essential for the skin mucus to function as a barrier. In this project, we used an interdisciplinary approach to examine how bacteria affect the skin mucosa of yolk sac fry of Atlantic salmon. An important milestone was to establish a protocol for germfree rearing of fry throughout the yolk sac stage. Comparative analyses of germfree and conventionally raised fry provide information about the microbial contributions to this mucosal barrier. This model system also allows for examination of the mucus properties on an intact, natural mucosal surface. By using rheological methods, we found that the skin mucus of the yolk sac fry is a layered and heterogenous structure. While one layer was firm, dense, and closely attached to the skin, the other was more viscous and only loosely associated with the skin. We found that this loose mucus was rapidly secreted from the fish, probably representing a mechanism for the fish to remove detrimental particles from the skin mucosa. The firm mucus layer became thicker, and the loose mucus stronger, throughout the yolk sac stage. We further examined how this mucosal surface was influenced by the presence of bacteria. By using rheology, reflected light microscopy, and AFM (Atomic Force Microscopy) we found that the germfree fry had a thinner dense mucus layer, while the loose mucus layer has altered viscoelastic properties, and was softer, compared to that of conventionally raised fish. Thus, the commensal fish microbiota appears to influence the barrier properties of the skin mucosa. The results also suggest that the composition of the fish microbiota to some extent is relevant for these effects. We found indications that some microbial communities also might affect the thickness of the skin (epidermis). The presence of bacteria did not influence of the total number of cells producing the mucus (goblet cells), but the number of actively secreting goblet cells appeared to increase under some microbial conditions. Moreover, we found that the expression of some genes was altered in the presence of bacteria. In particular, the expression of two genes encoding antimicrobial peptides increased significantly. Furthermore, two genes encoding the main skin mucins seemed to by higher for conventionally reared fry. By using AFM, we found that the two fish pathogens Aeromonas salmonicida and Yersinia ruckeri bound to mucins that had been purified from salmon skin and gut, and that A. salmonicida bound more strongly to skin than to gut mucins. However, we observed no binding of the pathogens to the intact skin mucosa on fry. A possible explanation is that the loose mucus on the skin mucosa has liquid-like properties and that bacteria trapped in this mucus is rapidly removed together with the secreted mucus. We characterised the skin microbiota for yolk sac fry that had been colonized by distinct bacterial sources. The skin microbiota differed from the surrounding water microbiota but was still influenced by the microbial environment of the newly hatched fry. The composition of the skin microbiota varied among individuals and changed over time. In this early life stage, prior to the onset of active feeding, the skin and gut microbiota was relatively similar. The skin microbiota was resilient towards manipulations, and the fry resistant towards bacterial disease: Exposure to high doses of the pathogen Yersinia ruckeri and a commensal Janthinobacterium strain did not result in notable changes in the microbiota composition, and the fish showed no signs of bacterial disease. In this project, we have demonstrated that the mucus covering the skin of salmon yolk sac fry is heterogenous and complex. Bacteria are needed to obtain full barrier properties and affect this mucosal surface at several levels: the thickness and viscoelasticity of the mucus, the activity of the goblet cells in epidermis, and the expression of genes encoding antimicrobial peptides. The skin microbiota was diverse, varied among individuals, changed with age, and was resilient towards exposure to high doses of both pathogenic and commensal bacterial strains.

The project brought new, valuable knowledge about the skin mucosa of Atlantic salmon fry, the skin microbiota, and the microbial influence on this mucosal barrier. This contributed to move forward the knowledge status in the field of host-microbe interactions on mucosal surfaces. The development of a new model system based on germfree salmon fry, and the implementation of analytical methods rarely used to study host-microbe interactions, will be important for developing new basic and applied research projects. On a longer term, the results obtained in this project will may contribute to promote mucosal health and microbial management in the aquaculture industry. A total of 14 master students did their research projects directly in relation to this project. A PhD candidate funded by the project is expected to complete her thesis in near future. The project contributed to interdisciplinary collaboration and developed and expanded the competence of all researchers involved in the project.

In the proposed project, we will investigate the role of fish skin microbiota in the mucosal barrier function for Atlantic salmon. Fish live in close contact with bacteria, and their skin mucosa is colonized by a distinctive microbiota. Fish skin represents an ancient vertebrate mucosal surface sharing functional and structural characteristics with the intestinal mucosa. While the importance of the gut microbiota to the mucosal barrier function is increasingly recognized, the role of the fish skin microbiota is not known. We hypothesize that the commensal microbiota colonizing fish skin mucosa is essential for obtaining full barrier function. In this project we will examine microbial effects on various aspects of the skin mucus barrier function in salmon fry. To achieve this, we will use a highly multidisciplinary approach, including national and international experts from divergent research fields. The project involves establishment of a germ-free model for salmon fry, allowing for examining well-defined, controlled microbial conditions. The potential effects of different microbial conditions on the mucosal barrier will be addressed at several levels: A) At the molecular level, using ultra-sensitive force probe (Atomic Force Microscopy; AFM) to study adhesion properties and bacteria-mucins interactions. B) Mucus barrier properties (mechanical, interactive, immunological) using micro-rheology (Multiple Particle Tracking), AFM, and gene expression analysis of immune factors. C) Finally, the barrier function will be tested by introducing fish pathogens to fry and mucus (Obj.4). A new approach here is to study intact natural mucosal surfaces by these high-resolution techniques. The project is expected to provide new basic insight in mucus-microbe interactions on mucosal surfaces, about barrier properties of fish skin mucosa and the role of the commensal microbiota. Improved understanding the skin barrier function is highly relevant to aquaculture of Atlantic salmon.