Experimental design of the multi-stressor approach to investigate environmental stress responses. The experiments were performed in an ectothermic vertebrate, the coral reef fish Pomacentrus moluccensis. Text within each circle names the stress and the duration it was applied, being three hours (3 h) or five days (5 d). Numbers above arrows indicate the number of microarray hybridisations used in the analysis of gene expression changes. Each microarray hybridisation compared relative expression levels in two samples (two-colour microarray analysis). Each sample in the microarray hybridisations represented one individual fish (no sample pooling) and a total of 118 individual fish were analysed in this study. Arrowheads indicate use of Alexa Fluor® 555 dye (green), which was swapped between samples to account for potential dye bias. The dotted lines within the ambient 3 h group indicate five groups of fish exposed to ambient conditions on separate days in order to provide time-matched controls for each of the stress treatments. Ambient conditions were 28°C in summer and 26°C in winter.