Characterization of PmSpӓtzle and SPIPm5 from black tiger shrimp Penaeus monodon

Humoral response proteins in shrimp, antimicrobial proteins (AMPs) and proteinase inhibitors, are implicated in host defenses against the pathogen infection because their expression is changed in response to microbial infection. After the pathogen infection in shrimp, the Spätzle protein is synthesized and activated the pathogenic signaling via Toll pathway by its active protein for the synthesis of AMPs. In this study, three PmSpätzle isoforms, PmSpz1, 2 and 3 genes, were identified in Penaeus monodon. The PmSpz1 gene was chosen for studied in details. Its gene was expressed in all shrimp tissues tested and in response to WSSV infection. Western blot analysis of hemolymph showed that the PmSpz1 mostly existed as an active form awaiting to activate Toll pathway. Injection of a recombinant PmSpz1 rendered the shrimp less susceptible to the WSSV challenge. The recombinant of active Spätzle activated the synthesis of AMP genes: crustinPm1, crustinPm7, ALFPm3, penaeidin3 but not penaeidin5. It is indicating that the expression of all antimicrobial proteins but not penaeidin5 was expressed under the regulation of Toll pathway. For SPIPm5 protein, the Kazal-type serine proteinase inhibitor from P. monodon was also studied. Its transcripts were also expressed in all tissues tested and its gene expression was up-regulated by heat stress, WSSV and YHV. Injection of rSPIPm5 into shrimp to mimic heat stress condition had more or less no effect on the crustinPm1, penaeidin3, penaeidin5, Hsp70, SPIPm2 and SPIPm5. The effect of rSPIPm5 protein inhibited the hemolymph proPO activity. In survival experiments, the rSPIPm5 could also prolong the shrimp from pathogen infection, WSSV, YHV, Vibrio harveyi and Vibrio parahaemolyticus in shrimp. The increased endurance against the microbial infection was due to the inhibitory effects presumably activated by rSPIPm5 on viral replication and bacterial growth but not the expression of AMPs gene. The microbial inhibitory effects may be activated by rSPIPm5. Therefore, the SPIPm5 plays an important role in shrimp innate immunity against the viral and bacterial infection besides its function as a proteinase inhibitor.