## 4 Discussion  
Farmed fish are commonly exposed to many pathogens in intensive systems. Bacterial diseases, such as furunculosis, are a major concern for aquaculture due to heavy economic losses [52]. Fish skin is a multifunctional organ that protects the organism from the environment and is the first barrier against infection. One of the first symptoms of furunculosis is the darkening of the skin and the emergence of ulcers, which lead to sepsis and fish death. Despite massive vaccination, this disease is still a threat for fish, which can worsen because temperature increases due to climate change will promote _A. salmonicida_ infection [18]. In the present study, we applied a 4 \(\times\) 44 oligonucleotide microarray to investigate the gene expression in the skin with adhering skeletal muscles of the rainbow trout subjected to _A. salmonicida_ infection. Further, the gene expression results obtained using the microarray method were validated via a RT-qPCR analysis of the selected genes. The results of the mRNA quantification using these two methods were consistent. Our studies showed that in both the moribund and survivor fish (2 and 6 dpi), a bacterial infection caused the down-regulation of several genes involved in the glycolysis/gluconeogenesis pathway, such as aldolase and fructose-bisphosphate (_aldoc_ and _aldo_), bisophosphoglycerate mutase (_bpgm_), phosphofractokinase, muscle (_pfkm_) and phos  
Figure 8: (**a**) RT-qPCR results for moribund (blue) and survivor (orange) fish; (**b**) Pearson correlation between microarray and RT-qPCR expression levels for moribund fish (2 dpi); (**c**) Pearson correlation between microarray and RT-qPCR expression levels for survivor fish (6 dpi). Red line represents a best fit line (with confidence intervals around the slope). phoglycerate kinase 1 (_pgk1_). In both groups, the expression of the leptin (_lep_) gene was raised, which is involved in the regulation of food intake and body fat and exerts powerful peripheral modulations on immune cells; thus, it may participate in the interaction between the immune response and metabolism [23; 53]. Since a bacterial infection affects the glycolysis/gluconeogenesis, leptin changes the fuel source to fatty acids (lipid) [54], and this shift might protect them from sepsis [55]. Up-regulated phospholipase A2 (_pla2g12a_ and _pla2g4a_) and 5-lipoxygenase activating protein (_alox5 ap_) are involved in biosynthesis eicosanoids, which are lipid-derived mediators of inflammation. Other genes involved in eicosanoids metabolism and lipid peroxidation such as prostaglandin-endoperoxide synthase 2 (_ptgs2_), 15-hydroxyprostaglandin dehydrogenase (_hpgd_) and prostaglandin reductase 1 (_ptgr1_) were differentially expressed between the moribund and survivor fish. The expression of these genes was higher in the first 2 days post infection and decreased in time to the control level (_p_-value < 0.05). Previously, the role of _ptgs2_ has been studied in the immune response against _Aeromonas hydrophila_ infection in common carp [56]. In summary, in accordance with previous studies in vertebrates [57; 58], these results indicate metabolic reprogramming in conjunction with the immune response in rainbow trout infected by _A. salmonicida_. Lysozyme is an important mucosal antibacterial enzyme that lyses pathogens. In this study, two types of lysozymes (c-type and g-type) were overexpressed in the skin of rainbow trout after 2 days of infection and then decreased in the next days; however, only _lysozyme c_ showed a statistically significant difference. Further, the lysozyme activity in the blood also increased at 2 dpi compared to the uninfected fish, and then it decreased at 6 dpi to the control level, which additionally confirms the activation of the innate humoral system up to 2 days post infection. Lysozyme was decreased in the head kidney of rainbow trout stimulated with _A. salmonicida_[23].