Molecular characterisation of key components of the mucosal immune system in Atlantic salmon (Salmo salar L) and transcriptome analysis of responses against the salmon louse (Lepeophtheirus salmonis)

Abstract

Mucosal immunity in mammals is mediated mainly by secretory immunoglobulin A (SIgA), which is produced by IgA plasma cells commonly located in the lamina propria, and a transport system involving the polymeric Ig receptor (pIgR). In teleost fish, IgM plays some roles associated with mucosal defence. Very recent findings indicate that IgT, an antibody exclusive to teleosts, might have a special role in mucosal immune responses, and a possible pIgR counterpart has been identified. The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod targeting the skin (and the gill to a lesser extent), has been a major challenge to the aquaculture industry. While the first line of defence against this parasite is crucial, equally important, in the context of vaccine development is the generation of information on the adaptive immune system. Based on this line of reasoning, Atlantic salmon IgT and pIgR were selected as targets for further characterization in the present study. Three distinct IgT heavy chain (����) sub-variants, with an identity index of 76-80%, were described. The identity index between ����1 and ����1 (the first constant domains of the IgT and IgM heavy chains, respectively) in Atlantic salmon is 52%. It is plausible to assume that this relatively high similarity is a result of interactions with common light chains. The relative abundance of ������������������������and�������� transcripts in a series of tissues revealed an overall expression pattern of IgM >> IgT > IgD. Interestingly, challenge experiments with salmon louse showed 10 fold increase of IgM and IgT mRNA in skin samples, supporting the assumption that these antibodies are involved in mucosal immune responses. The search for pIgR homologues in Atlantic salmon resulted in two pIgR-like candidates: Salsal pIgR and Salsal pIgRL. Meanwhile, a comparative evaluation was made to a series of CD300-like molecules (CMRF-35 like molecules, CLM) reported to the databanks. Salsal pIgR and Salsal pIgRL were identified on the basis of similarity to homologous genes, and like the counterparts in other teleosts they are composed of two Ig superfamily (IgSF) V-like domains, a transmembrane region, a connecting peptide, and a cytoplasmic tail. Two CD300-like molecules in salmon (CLM1 and CLM7) also have the same domain structure, but their cytoplasmic region is predicted to contain putative immunoreceptor tyrosine based inhibition motifs (ITIM), which is a typical feature of CD300A and CD300F in humans. While Salsal pIgR and Salsal pIgRL were expressed in tissues of skin and gill respectively, their expression pattern is not restricted to mucosal tissues, but notably, their expression increased during infection with salmon louse. Further studies are needed to elucidate the transport mechanisms of mucosal antibodies in salmon. To investigate how the salmon louse evades the immune system of the host, temporal immune gene expression changes in skin, spleen, and head kidney of Atlantic salmon were analysed using microarray and quantitative real-time PCR (qPCR) during the first 15 days post infection (dpi). This window represents the copepodid and chalimus stages of lice development. Transcriptomic responses, recorded already at 1 dpi, were highly complex and large by scale. Many genes showed bi-phasic expression profiles with abrupt changes taking place between 5 and 10 dpi (the copepodid-chalimus transitions). Large group of secretory splenic proteases with unknown roles showed the greatest fluctuations: up-regulated 1-5 dpi and markedly down regulated afterwards. T cell related transcripts showed a short term (1-5 dpi) increase. After 5 dpi, the magnitude of transcriptomic responses decreased markedly in skin. The findings provided an insight into the time windows in the development of the parasite which are critical to the host and where modulation of the host immune system might occur.