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Spätzle is a signaling ligand in innate immune response that signals pathogenic infection via Toll receptor and Toll pathway into the cells for the synthesis of antimicrobial proteins. Herein, three PmSpätzle isoforms were identified in Penaeus monodon, namely PmSpz1, 2 and 3. The PmSpz1 was chosen for detailed study. The PmSpz1 gene was expressed in all nine tissues tested including the hemocytes, stomach, hepatopancreas, gill, lymphoid tissue, eyestalk, muscle, intestine and heart. Its expression was up-regulated upon white spot syndrome virus (WSSV) infection. Western blot analysis of hemolymph showed that the PmSpz1 mostly existed as a cleaved active form awaiting to activate the Toll pathway. Injection of a recombinant PmSpz1 rendered the shrimp less susceptible to the WSSV infection. Injection of a recombinant active form of PmSpz1 into a normal shrimp activated the synthesis of crustinPm1, crustinPm7, ALFPm3, penaeidin3 but not penaeidin5 indicating that the expression of all antimicrobial proteins but not penaeidin5 was under the regulation of Toll pathway.Three PmSpätzle isoforms are identified in Penaeus monodon.The PmSpz1 gene is up-regulated in response to WSSV infection.The PmSpz1 mostly exists as an active form awaiting to activate the Toll pathway.The recombinant PmSpz1 renders the shrimp less susceptible to the WSSV infection.The recombinant active form of PmSpz1 activates the synthesis of AMPs.