The abaecin family comprises a class of proline-rich antimicrobial peptides (AMPs) with restricted distribution in hymenopteran insects. Intriguingly, in the parasitic wasp Nasonia vitripennis its members (termed nabaecin-1 to -3) have gained a carboxyl terminal glycine-rich antimicrobial unit through exon-shuffling. Here, we describe cDNA cloning of nabaecin-3 and the donor gene (navitripenicin) of the shuffling, and structural and functional features of nabaecin-3 and its two domains (respectively called amino-terminal abaecin unit (NtAU) and carboxyl-terminal navitripenicin unit (CtNU)). Nabaecin-3 and navitripenicin were found to be transcriptionally Up-regulated in response to bacterial challenge. By using recombinant expression and chemical synthesis techniques, we produced nabaecin-3, NtAU and CtNU. Circular dichroism (CD) analyses show that these peptides remarkably differ in their structures. Functionally, nabaecin-3 displayed a wide spectrum of antimicrobial activity against an array of bacteria, yeasts and fungi at micromolar concentrations, while CtNU only had a weak antibacterial activity and NtAU completely lacked activity. Our results indicate that in Nasonia the antimicrobial function of abaecin depends on the combination of NtAU with CtNU and thus suggest a new role of exon-shuffling in buffering loss-of-function mutation of a gene. (C) 2012 Elsevier Inc. All rights reserved.