Identification and characterization of the parasitic wasp Nasonia defensins: positive selection targeting the functional region?

Abstract

Defensin is a crucial component of innate immunity highly conserved across different insect orders. Here, we report identification and characterization of defensins in the parasitic wasp Nasonia (Hymenoptera: Pteromalidae). In comparison with those in the non-parasitic insect Apis mellifera, two different subtypes of defensins (defensin1 and defensin2) have undergone independent gene duplication to create a mutigene family of five members (named 1-1, 1-2, 2-1, 2-2 and 2-3) in the Nasonia lineage. Such duplication occurred before the divergence of three sibling species (N. vitripennis, N. giraulti and N. longicornis) and the duplicated genes was subsequently subjected to positive selection at the amino-terminal loop and the gamma-core region. RT-PCR identified that only the subtype 1 of defensins were constitutively expressed in the N. vitripennis adult stage and none of the five defensins was expressed in other developmental stages (i.e. the infected Musca domestica pupae). A functional form of 2-2 in N. vitripennis (named navidefensin2-2) was produced in Escherichia coli by an on-column refolding approach. The recombinant peptide presented a typical defensin structure, as identified by CD analysis, and selectively inhibited the growth of two Gram(+) bacteria at low micromolar concentrations. The bioactive surface responsible for antibacterial activity of navidefensin2-2 was identified in the gamma-core region of this molecule. Positive selection targeting the antibacterial region of defensins could be a consequence of evolutionary arms race between Nasonia and its pathogens.