Function of Akt in immunity in Chinese mitten crab (Eriocheir sinensis)

Eriocheir sinensis is one of the most important aquaculture species in China. With intensification of high-intensity feeding and degradation of the environment, the increasing diseases caused by bacteria infections have resulted in enormous economic loss in crab aquaculture. Akt is a core molecule of PI3K-AKT signaling pathway, which is highly conserved from invertebrate to vertebrate that plays essential roles in various physiological and pathological processes, such as cell proliferation, metabolism, development and survival. Although several Akts among invertebrates have been explored, the identities of Akt and their downstream effectors, especially in pathogen infection of crustaceans, remains poorly understood. In the present study, an Akt gene (designated as EsAkt) was identified in Chinese mitten crab E. sinensis, and the mRNA expression patterns of EsAkt in response to immune stimulations were investigated. The specific primers and PCR technology were used to clone and obtain the E. sinensis Akt gene (EsAkt). The expression level EsAkt in different tissues and after different immune stimulations were detected by real-time quantitative PCR (qRT-PCR). The cellular localization of EsAkt protein was detected by immunofluorescence assay. The changes of apoptotic gene after EsAkt RNA interference were analyzed by double stranded RNA interference. The results showed that EsAkt molecule contained the PH domain, the central catalytic domain (S_TKc) and the carboxy-terminal hydrophobic domain (S_TK_X).EsAkt was constitutively expressed in all tested tissues, and its expression in immunity related tissues (such as hepatopancreas, gills and blood lymphocytes) was significantly higher than that in stomach and intestine (P<0.01). Immunofluorescence assay results indicated that the oval hemocytes were observed in the bright field, and the cell nuclei were stained by DAPI in blue. The EsAkt-reactive areas were stained in green, and the positive green signals were mainly diffuse localization in the cytoplasm. After injecting lipopolysaccharide (LPS) andAeromonas hydrophila into E. sinensis, the expression of EsAkt was significantly induced. After LPS immune stimulation for 12 h, the response of EsAkt reached the peak, which was 4.35 times that of the control group (P<0.01). After stimulation for 48 h, the mRNA expression level ofEsAkt decreased, but it was still 2.62 times that of the control group (P<0.01); After 6 h ofA. hydrophila immune stimulation, the response of EsAkt reached the peak, which was 9.05 times higher than that of the control group (P<0.01). After 48 h of stimulation, the mRNA expression level ofEsAkt fell back to normal. Upon double stranded RNA interference with EsAkt, the mRNA expression level of EsAkt was 0.38 times higher than that of the control group (P<0.05), while the expression level of apoptosis related geneEscaspase-3-like was significantly up-regulated, which was 2.69 times higher than that of the control group (P<0.05). These results imply thatEsAkt plays an important role in pathogen challenge by regulating the apoptosis related gene expression in E. sinensis, which enriches the basic data of the PI3K-AKT signaling pathway in E. sinensis and lays a foundation for further research on the immune defense mechanism of crustaceans.