Cloning and characterization of a melanization inhibition protein (PmMIP) of the black tiger shrimp, Penaeus monodon

Melanization is an important component of the innate immune responses in invertebrates and it is essential for defense against invading microorganism. Melanin formation, which is a result of activation of the so called prophenoloxidase activating system, needs to be controlled due to the dangerous effects of quinones and melanin which are produced during the process of melanization. Here, a cDNA for a melanization inhibition protein (MIP), named PmMIP, was identified from the black tiger shrimp, Penaeus monodon by RT-PCR using degenerated oligonucleotide primers and RACE-PCR. The complete sequence significantly matched MIP of the freshwater crayfish Pacifastacus leniusculus (PlMIP). PmMIP contains an N-terminal signal peptide and a fibrinogen related domain (FReD). RT-PCR was applied to examine the expression profiles of PmMIP in various tissues of juvenile P. monodon. PmMIP was expressed in all examined tissues except hemocytes and at very low levels in hepatopancreas and ovaries. The expression of this gene was very low during the larval stages and hardly present in egg and at the nauplius stage. A time-course expression analysis of PmMIP upon Vibrio harveyi challenge at protein levels in plasma was determined. The result shows that MIP protein in plasma was induced at 6 h and disappeared at 12 and 24 h and then the protein reappeared at 48 and 72 h post injection. These results suggest that upon bacterial infection the PmMIP protein is first released from tissues into hemolymph and then degraded to allow melanization to occur for fighting against bacteria.