Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 Page 9 of 19 Figure 6 Hymenoptaecin-like peptides. A. Multiple sequence alignment. Red arrows label the position of phase 0 introns. A red “^” indicates a site in droyataecin that is corrected from TAG to GAG codon. Proline residues in amino-termini of nahymenoptaecins are bolded and shown in brown. Putative proprotein cleavage sites are boxed in black; B. Phylogenetic tree constructed with the NJ method of MEGA 4.0. Solid circle represents gene duplication; C. Comparison of pronavicin with known Pro-rich AMPs. Sequence identity with pronavicin and proline contents are shown here. Dse: D. sechellia, Dsi: D. simulans, Dpe: D. persimilis, Dps: D. pseudoobscura, Dvi: D. virilis, Dya: D. yakuba, Der: D. erecta, Dan: D. ananassae, Dgr: D. grimshawi, Dwi: D. willistoni, Mg: Myrmecia gulosa, Pa: Pyrrhocoris apterus, Bp: Bombus pascuorum. Table 1 Antibacterial activity of pronavicin. Microorganism B. megaterium M. luteus Bacillus sp. S. typhimurium E. coli Stenotrophomonus sp. YC-1 Stenotrophomonus sp. LZ-1 Lethal conc. (CL) (μM) 3.11 (y = 1.0867x + 1.019, R2 = 0.998) 42.4 (y = 0.65x + 0.0175, R2 = 0.9956) 19.5 (y = 0.7125x - 0.2313, R2 = 0.995) 15.8 (y = 0.3458x + 0.2525, R2 = 0.9993) 61.8 (y = 0.2375x + 0.0712, R2 = 0.9774) 78.0 (y = 0.3x + 0.0292, R2 = 0.9959) 12.0 (y = 0.3625x + 0.3, R2 = 0.9542)* * indicates the semi-inhibition of pronavicin against Stenotrophomonus. sp. LZ-1. Inducible expression profiles of N. Vitripennis AMP-like genes The up-regulation of AMP expression after infection is a common defense strategy by hosts to rapidly destroy invaders [5]. To evaluate whether N. vitripennis AMPs have such expression feature, we chose twelve genes that belong to 4 different categories (i.e. nabaecins, nahymenoptaecins, navidefensins, and nasonins) as representatives. By using RT-PCR and DNA sequencing, we found that six genes are constitutively expressed because their cDNAs can be amplified from both infected and non-infected N. vitripennis adults. These genes are respectively nasonin-1, nasonin-3, nasonin-4, nabaecin-2, together with navidefensin1-1 and 1-2, two recently cloned genes from the non-infected N. vitripen- nis adults [25]. Subsequently, we studied the expression profiles of all twelve genes after bacterial challenge by semi-quantitative RT-PCR. Results showed that only two genes (nasonin-3 and nasonin-4) whose expression was not induced by the bacteria. Four genes (navidefen- sin1-1, 1-2, nasonin-1 and nabaecin-2) weakly expressed in the non-infected wasp were significantly up-regulated by bacterial challenge. Transcripts of nabaecin-1, nahy- menoptaecin-1 and nahymenoptaecin-2 were only detected in the infected wasp, indicating that their expression is initiated by infection (Figure 8). Differen- tial expression patterns among different AMPs may represent a combinational strategy to form a defense network against diverse microbial pathogens. Although three navidefensin2 genes were not detected to be expressed in the non-infected and infected adults, their defense role in different developmental stages of Naso- nia is possible [25]. Discussion AMPs in the parasitic wasp With more and more genome sequences released, iden- tifying new bioactive peptides/proteins by computational