Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 Page 11 of 19 Figure 8 Semi-quantitative RT-PCR detecting the inducible expression of AMP genes in N. vitripennis before and after bacterial infection. M: DNA marker. -: non-challenged; +: challenged. [17-21], N. vitripennis still have evolved more AMP genes both in kinds and numbers (Figure 9A). Based on the phylogenetic relationship of holometabolous insects [85], we elucidate several evolutionary events (e.g. gene expansion, terminal extension and tandem repeat) which might have occurred along the parasitoid branch (Figure 9B) to result in the lineage-specific complexity increase in N. vitripennis AMPs for synergetic defense in parasitic condition [86]. Evolution of the N. Vitripennis AMPs Three different strategies have been recognized to shape the complex antimicrobial immune system in N. vitripennis: Gene duplication Adjacent chromosome location together with gene structure conservation strongly supports tandem gene duplication occurring in at least three AMPs in the N. vitripennis genome, including navidefensins, nabae- cins and nasonins (Figure 10A). An emerging trend in the study of AMPs is that their evolution is often driven by Darwin positive selection following gene duplication [87]. However, we only detected weak positive selection signal in the amino-terminal abaecin unit (Table 2). No such signal was found in the gene families of defensins and nasonins, which is likely due to the sparse sam- plings of species used in this analysis because when more defensin sequences are included, accelerated amino acid substitutions were detected in the Nasonia lineage [25]. An additional change after gene duplication is structural diversification of N. vitripennis AMPs (Figure 10B). These structural variations may result in evolutionary novelty by changing structural basis related to function. For example, n-loop has been proven to be functionally important in mosquito defensin [50] and its change in size will probably influence the activity and antibacterial spectra of these defensins, as observed in navidefensin2-2 [25] and nasonin-1. There is also increasing awareness that alteration of the highly exposed wrapper disulfide bridge linkage pattern can lead to functional switch of scorpion sodium channel toxins via adjusting the conformation of key residues associated with toxin function [84]. In this regard, navi- tricins with such a disulfide bridge are examples for