Unpredictable Repeated Stress in Rainbow Trout (Oncorhynchus mykiss) Shifted the Immune Response against a Fish Parasite

Abstract

Farmed fish are regularly subjected to various stressors due to farming practices, and their effect in the context of a disease outbreak is uncertain. This research evaluated the effects of unpredictable repeated stress in rainbow trout challenged with the ciliate Ichthyophthirius multifiliis, known to cause white spot disease in freshwater fish. Before and after the pathogen exposure, fish were handled with a random rotation of three procedures. At 7 days post-infection (dpi), the parasite burden was evaluated in fish and in the tank's water, and the local and systemic immune responses were investigated in the gill and spleen, respectively. The fish mortality was recorded until 12 dpi, when all the fish from the infected groups died. There was no statistical difference in parasite burden (fish and tank's water) and infection severity between the two infected fish groups. The immune gene expression analysis suggested a differential immune response between the gill and the spleen. In gills, a T helper cell type 2 immune response was initiated, whereas in spleen, a T helper cell type 1 immune response was observed. The stress has induced mainly upregulations of immune genes in the gill (cat-1, hep, il-10) and downregulations in the spleen (il-2, il-4/13a, il-8). Our results suggested that the unpredictable repeated stress protocol employed did not impair the fish immune system.

Keywords: Ichthyophthirius multifiliis; fish handling; immune gene expression; immune signaling; parasite infection.

Figure 1

Recorded cumulative mortality in percentage of rainbow trout after exposure to Ich theronts (500 per fish). Each treatment was composed of 60 fish (n = 30 fish per tank). The tank duplicates of each fish treatment are represented separately dpi: days post infection. ****: p < 0.0001.

Figure 2

Evaluation of the Ich burden in fish (n = 5 fish per tank; 10 fish per treatment) and water at 7 dpi. (A) Ich spot count in rainbow trout skin and gill. (B) iag52a Ct value from water sampling (n = 1 per tank) and fish gill. ns: no statistical significance. No statistical analysis was performed for eDNA results due to limited sampling size.

Figure 3

Gene expression of immune relevant gene (A–K) in rainbow trout gill at 7 dpi (n = 5 fish per tank; 10 fish per treatment). Data are represented as geometric mean with geometric standard deviation (n = 10 fish per group). Only significant results were represented. Ctrl: control. Inf: infected. S: stress. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Figure 4

Gene expression of immune relevant gene (A–I) in rainbow trout spleen at 7 dpi (n = 5 fish per tank; 10 fish per treatment). Data are represented as geometric mean with geometric standard deviation (n = 10 fish per group). Only significant results were represented. Ctrl: control. Inf: infected. S: stress. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Figure 5

Correlation analysis of immune gene expression (gill and spleen) and infection severity (gill) measured with Ct value of iag52a (A–G) from Ich at 7 dpi (n = 5 fish per tank; 10 fish per treatment). Dots represent results from a single fish. Dotted lines represent simple linear regressions and colored areas represent 95% confidence intervals associated with each regression. Only significant results (p < 0.05) were presented.