Norwegian aquaculture shares a large proportion (more than half) of global production, where Atlantic salmon ( Salmo salar) is one of the most intensified farmed fish species . However, there is a significant number of fish mortality in the production line resulting in huge economic losses due to various infectious agents, selective breeding practice and high rearing temperature causing impaired immune functions . Moreover, higher temperature can be detrimental for fish health and can pose a threat for developing deformities during early life stages in Atlantic salmon . Continuous attempts have been undertaken to produce robust fish and rearing circumstances, particularly by adjusting water temperature during early life stages, which have been shown to be effective in regulating adult salmon phenotypes. In this study, we aim to assess if low rearing temperature during embryogenesis of Atlantic salmon can manipulate innate immunity which may improve disease resistance potential against potent pathogens in later life stages such as Yersinia ruckeri which is predominant in salmonids. Y. ruckeri causes enteric redmouth disease (ERM) and is more prevalent in rainbow trout in countries such as Scotland, Australia and Chile. However, the number of outbreaks in farmed Atlantic salmon has increased in Norway in recent years. Y . ruckeri is o ften a problem during the freshwater stage of production . Therefore, it is crucial to find sustainable options on how to improve disease resistance in Atlantic salmon. Manipulating embryonic rearing temperature may help producing robust phenotypes, as it is a strong regulator of immunity and changes occurring during early life stages may have a pervasive influence on the fish .
Materials and methods
F ertilized eggs of Atlantic salmon were reared under different temperature regimes of 4, 6 and 8℃ until the eyed-egg stage (~320 day degrees), and thereafter reared under similar conditions as the 8℃ group. L arvae (before start-feeding) were exposed for 2h to a Y. ruckeri bath challenge in a multi-well plate and followed for 72h. Bacterial localization was assessed by immunohistochemical (IHC) detection and followed up by histological examination in the gills and skin. Multi- gene expression analysis was performed on the BioMark HD platform to assess early immune competence (ImCom) . Multiplex fluorescent in situ hybridization (FISH) was established, using RNAscope technology, to detect monocyte and lymphoid progenitor cells in whole larvae.
Larvae from the 6℃ group showed a better survival probability (up to 16.69 %) than in the 8℃ at 72h post challenge . IHC staining showed localization of the bacterial antigen mostly in the secondary lamellae of gills, and the epithelial and basal layers of skin. However, very mild histopathological changes such as sparse epithelial lifting of gills secondary lamellae and epithelial disruption of skin tissue were noticed after 24 and 72h . ImCom results showed that the l arvae of the 4℃ group had a significant ly differential expressio n pattern of innate immune genes compared to the 8℃ group. For example, toll-like receptor 13 (tlr13) involved in pathogen recognition and stress proteins such as heat shock protein 70 (hsp1a1) in the gills, whereas gelsolin (gsn) , collagen 1 (col1a) and claudin 4 (cldn4), which are important extra cellular matrix (ECM) and tight junction proteins for their regulatory roles during inflammatory and antimicrobial response, were differentially expressed in the skin after 24 and 72h of challenge . However, gsn , hsp1a1 and cldn4 were also upregulated in 8℃ , but immune response was overall more pronounced in the gills than in the skin. Moreover, 6℃ group had a relatively suppressed immune genes expression in both gills and skin compared to 8℃ group. Other pro-inflammatory genes also showed higher tendencies in the low temperature groups. The FISH method demonstrated the detection of monocyte lineage cell population in the head kidney region and lymphoid progenitor cells, which were more abundant in the thymus tissue in all temperature group. However, 4℃ group larvae showed a relatively low number of these long living immune cells which could relate to thermal sensitivity of progenitor cell differentiation during embryogenesis.
Altogether, our results depict that low rearing temperature during early life stage may have an influence on developmental plasticity of innate immunity and better survival in later life.
The research was supported by the Research Council of Norway (COOLFISH; No. 29685)