Cloning and expression analysis of an anti-lipopolysaccharide factor from giant freshwater prawn, Macrobrachium rosenbergii.

Anti-lipopolysaccharide factors (ALFs) are a group of effector molecules that are classified as antimicrobial peptides (AMPs). They are found in limulids and crustaceans and show a broad range of antimicrobial activity. In the current study, an ALF gene from Macrobrachium rosenbergii (MrALF) was identified. Its full length was 690 bp and it encoded a 124 amino acid protein. A signal peptide and a conserved LPS-binding domain with two conserved cysteine residues that comprise a cluster of positive charged residues within a disulfide loop were predicted in MrALF. The M. rosenbergii ALF clusters with the Macrobrachium olfersii ALF and further clusters with most crustacean ALFs, suggesting that they should originate from one common ancestor. Positive selections should have sharpen the evolution of M. rosenbergii and M. olfersii ALF genes. Reverse transcriptase polymerase chain reaction analysis showed that MrALF was expressed in all detected tissues. In the epidermis, MrALF was obviously upregulated 24 h after the LPS challenge. In the stomach and gills, MrALF was upregulated upon LPS challenge. The results show that MrALF might have important roles in the immune defense against invading bacteria. The positive selections that occur in the ALFs of crustaceans might have resulted from a Red Queen's race with its pathogens. We found evidence of positive selection acting to drive functional divergence during the evolution crustacean ALF genes, especially in the M. rosenbergii ALF gene. The evolutionary changes might correspond to the challenges induced by pathogens that infect crustaceans.