The objective of the present study is to identify and evaluate informative indicators for the welfare of rainbow trout exposed to (A) a water temperature of 27 °C and (B) a stocking density of 100 kg/m3 combined with a temperature of 27 °C. The spleen-somatic and condition index, haematocrit and the concentrations of haemoglobin, plasma cortisol and glucose revealed non-significant differences between the two stress groups and the reference group 8 days after the onset of the experiments. The transcript abundance of almost 1,500 genes was modulated at least twofold in in the spleen of rainbow trout exposed to a critical temperature alone or a critical temperature combined with crowding as compared to the reference fish. The number of differentially expressed genes was four times higher in trout that were simultaneously challenged with high temperature and crowding, compared to trout challenged with high temperature alone. Based on these sets of differentially expressed genes, we identified unique and common tissue- and stress type-specific pathways. Furthermore, our subsequent immunologic analyses revealed reduced bactericidal and inflammatory activity and a significantly altered blood-cell composition in challenged versus non-challenged rainbow trout. Altogether, our data demonstrate that heat and overstocking exert synergistic effects on the rainbow trout's physiology, especially on the immune system.

Faculty of Agriculture and Environmental Sciences University of Rostock 18059 Rostock Germany

Institute of Aquaculture and Protection of Waters Faculty of Fisheries and Protection of Waters University of South Bohemia Husova tř 458 102 370 05 České Budějovice Czech Republic

Institute of Fisheries State Research Centre of Agriculture and Fisheries Mecklenburg Vorpommern Südstraße 8 18375 Born Germany

Institute of Genome Biology Leibniz Institute for Farm Animal Biology Wilhelm Stahl Allee 2 18196 Dummerstorf Germany

Institute of Immunology Federal Research Institute for Animal Health Friedrich Loeffler Institute 17493 Greifswald Insel Riems Germany

Sea Lice Research Centre Department of Biology University of Bergen Thormøhlensgate 55 5008 Bergen Norway

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