Two cDNA libraries were prepared from hemocytes of normal and Vibrio harveyi-challenged black tiger shrimp Penaeus monodon. A total of 1062 expressed sequence tag (EST) clones were sequenced unidirectionally. ESTs representing the antimicrobial peptide (AMP) homologues, antilipopolysaccharide factors (ALF), penaeidins and crustins were discovered. They predominated among immune-related genes, representing 29.2% and 64.0% of the normal and challenged libraries, respectively. Several types of each AMP homologue were found. Sequence alignments of ALF in P. monodon (ALFPm 1 to 5) implied possible alternative splicing of different exons at both NH2 and COOH-termini. Only one major type of penaeidin (penPm3) was found in P. monodon. In addition, crustin homologues (crusPms1 to 4) and a newly identified glycine-rich antibacterial peptide (GAMPPm1) were also isolated and characterized. Using RT-PCR analysis, expression of ALF, penaeidin and crustin transcripts was detected in various tissues but the main expression site was in hemocytes. Expression of these antimicrobial peptides in P. monodon subjected to V. harveyi challenge revealed a significant increase in expression of ALFPms (p < 0.05) but a decrease in expression of crustins and penaeidins.

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to β-1,3-glucan and LPS with a dissociation constant of 6.86 × 10⁻⁷ m and 3.55 × 10⁻⁷ m, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or β-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and β-1,3-glucan in the shrimp proPO activating system.