In vitro modelling of the immunological interactions between the salmon louse, Lepeophtheirus salmonis (Kroyer, 1837), and the Atlantic salmon, Salmo salar (L., 1758)
Atlantic salmon, SaI1M salar. L., have been shown to be more susceptible to infections by the caligid copepod Lepeophtheirus salmonis (Kreyer, 1837) than other salmonid species. Atlantic salmon exhibit a reduced cellular immune response to the attached parasite which has led to the hypothesis of the presence of sea louse associated compounds that depressed the fish's normally efficient inflammatory mechanisms. The aim of the current study was to test this hypothesis. A biochemically defmed in vitro culture system was developed that would allow collection of the secreted/excreted products of the copepodid larvae of L. salmonis, and avoid their contamination by metabolites of the host from their collection, in vivo. Available tissue culture methods proved inadequate in supporting copepodid culture because of the louse requirement for a seawater maintenance medium that was osmotically unsuitable for cultured cells. Tissue engineering technologies developed in the construction of human living skin equivalents were successful in the development of an Atlantic Salmon Skin Equivalent (ASSE). ASSE is a novel organotypic tissue culture substrate that was constructed from Atlantic salmon fibroblasts (AS-6) and primary cultures of Atlantic salmon epithelial cells. Cells were supported in a matrix of collagen fibres, acid extracted from the tails of rats, and combined using a layering technique to create a substitute salmon skin. ASSE has a fibroblastic dermal equivalent overlaid by an epidermis-like layer and a layer of collagen, and was maintainable in a seawater media. During its development, the cells within ASSE showed signs of differentiation that included stratification, increased fibronectin production by cultured fibroblasts, and the formation of a basement membrane-like layer at the junction of the dermis and epidermis. In this environment, ASSE allowed the survival of copepodid larvae for an average of 12 days, a period approximately 5 days longer than that of their free-swimming counterparts. Furthermore, cultured copepodids were observed feeding, exhibiting the normal range of settlement behaviours, and also showed increases in their length. However, metamorphosis to the chalimus I stage did not occur and was not stimulated by the supplementation of ASSE with salmon mucus, salmon peptone or DL methionine. Nevertheless, copepodid maintenance on ASSE allowed the collection of the compounds they released into the culture media. These were assayed for their effects on the immunological functioning of salmon macrophages. These assays demonstrated that the chemotaxic ability of macrophages was significantly reduced following treatment with louse culture supernatants . (LCS), as was their phagocytic ability. In both assays, the activity of each function was approximately 50% lower than that recorded in untreated cells. Intracellular respiratory burst and the phagocytic index of active phagocytes, however, was not affected. The biological activity of LCS was inhibited following heating, dilution, and treatment with proteinase K, indicating that the active immunosuppressive compounds were either themselves proteins, or required protein to be effective. The active substance was designated Louse Immunomodulatory Factor (LIF). The enzyme profiles of culture supernatants were investigated using API ZYM test strips. The profiles of LCS supernatants were significantly different to those of control supernatants from the early stages of louse incubation with ASSE. LCS supernatants showed elevated levels of leucine aminopeptidase, C4 and C8 esterases, alkaline phosphatase, P-glucuronidase, and Nacetyl- j-glucosaminidase. The involvement of these enzymes in the digestive processes of arthropods is well documented. However, the presence of the chitin hydrolysing enzyme Nacetyl- Beglucosaminidase, may also be suggestive of the preparation of copepodids for moulting. This, and the involvement of these enzymes in digestion in copepodids, and as possible immunomodulatory compounds is discussed. Gel filtration chromatography identified 14 proteins in the LCS that were not present in the control supernatants. These proteins were in the molecular weight range <1 kOa to 2665 kOa. No biological activity was attributed to these proteins when isolated by chromatography and assayed for their effects on macrophage chemotaxis. This lack of activity may be associated with their dilution during the chromatography process in which the supernatants were diluted approximately 5000 times. Experimental evidence showed that the activity of LIF was absent when supernatants were diluted to 1:1000, and so methods of concentration may be required in order to establish the immunological activity of these proteins. This study has developed an Atlantic salmon skin substitute that could have a great number of applications in the study of salmonid metabolism, cellular communication, immunology, and drug and chemical testing. Here, it supported the extended maintenance of sea lice larvae in vitro and allowed the collection of the products of their culture. The findings have shown that the copepodid stage of L. salmonis produces substances, LIF, that depress the chemotaxic and phagocytic activity of salmon macrophages, in vitro. These active substances may also be responsible for the depression of the inflammatory responses of sea lice infected Atlantic salmon, in vivo.