Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 melanogaster, there are 20 AMPs characterized [16]. In the past ten years, a number of genome sequencing pro- jects have promoted application of computational approaches for the discovery of components involved in innate immunity of a given species. Some examples include D. melanogaster, Anopheles gambiae, A. melli- fera, Tribolium castaneum and Bombyx mori [17-21]. This will undoubtedly extend our knowledge in basic biology of insect innate immunity system. Furthermore, these data also provide new clues for elucidation of immunological adaptation of insects to environmental changes. For example, analysis of the complete immune system of A. gambiae results in the discovery of a marked deficit of orthologues due to gene loss and excessive expansion of some specific genes, which is sig- nificantly different from that of D. melanogaster. This possibly reflects differential selective pressures from dif- ferent pathogens between these two species. These results also facilitate further in-depth analysis of the mosquito immune system’s impact on the malaria para- site [17,22]. Parasitoids (Hymenoptera, Insecta) are a group of parasitic insects. Some of them attach significant vectors of human disease, such as house flies, roaches and ticks, and some of them are extremely important regulators of agricultural pests [23]. To study molecular mechanism of the innate immunity of parasitic wasps, the basic biol- ogy of their AMPs is needed to be established. As the first parasitic hymenopteran insect to have its gen- ome sequenced [24], N. vitripennis provides a new resource for the identification of AMP genes in this class of insects in a genomic scale [25,26]. Here, we report systemic characterization of the AMP repertoire from the N. vitripennis genome, which provide a per- spective for the understanding of a possible relationship between immunity and parasitism. In comparison with A. mellifera and several non-Hymenopteran insects, N. vitripennis has developed a more complex antimicrobial immune system through genetic duplication and exon- shuffling. Results Computational identification of AMPs in the N. Vitripennis genome To identify N. vitripennis AMP repertoire, we employed three complementary approaches to database search of the N. vitripennis genome (see additional file 1). Firstly, we chose A. mellifera AMPs (e.g. mellitin, apidaecin, apisimin, abaecin, hymenoptaecin, defensins) as queries to recognize their orthologues in N. vitripennis by BLASTP and TBLASTN. Secondly, we performed pat- tern search by the Scanprosite program based on the cysteine arrangement pattern of CSab-type defensins [8]. This strategy allowed identifying a large family of Page 2 of 19 DLPs with a typical CXXXC and CXC motif and an amino-terminal signal peptide, which can be divided into three distinct subfamilies, including the known navidefensins [25] and two new subfamilies named nasonins and navitricins, respectively. Thirdly, we screened putative secreted peptides of < 150 amino acids from the N. vitripennis proteome by recognizing an amino-terminal signal sequence, from which AMP- like peptides rich in specific amino acids were identified. All AMPs found by the above approaches were again used as query to carry out BLASTP and TBLASTN iteratively until no new hits appeared. As a result, we identified a total of 44 AMPs in the genome of N. vitri- pennis, as shown in additional file 2, in which only nahymenoptaecin-1 and five defensins are recently reported [25,26]. All these newly discovered peptides display remarkable AMP characteristics, as reflected by their small size and net positive charges at pH 7.0. Most peptides described here are smaller than 150 amino acids in length, except for nahymenoptaecin-1, nahymenoptaecin-2, nasonin-2 and nasonin-6, which all have undergone internal dupli- cation (see additional file 2). N. vitripennis AMPs exhibit extensive sequence and structural diversity and can be distinguished into multiple molecular types including insect and fungal DLPs with the CSab fold; Pro- or Gly- rich abaecins and hymenoptaecins; horseshoe crab tachystatin-type AMPs with the ICK fold; and a linear a-helical peptide. To confirm the reliability of our computational predic- tion, we undertook the N. vitripennis EST database search and found more than 60% peptides have corre- sponding transcripts, suggesting the genes encoding these peptides are expressed at the transcriptional level. For remaining 40% peptides whose cDNA sequences were not found in the EST database, one possibility is that their expression depends upon suitable microbial challenges [27] or is associated with different develop- mental stages of N. vitripennis, as observed in the D. melanogaster antifungal drosomycin [28], although pseudogene or wrong prediction resulted from computa- tional methods cannot be completely excluded. Classification of N. Vitripennis AMPs Defensin-like peptides Classical insect-type defensins (CITDs) Defensins with the CSab structure are crucial effectors of innate immu- nity [7] which have been found in many insect species, the majority of them coming from different orders of the subclass Neoptera (Diptera, Coleoptera, Lepidopera, Hemiptera and Hymenoptera). These AMPs are primar- ily active against Gram-positive bacteria likely by form- ing voltage-dependent channels in bacterial membrane leading to a loss of cytoplasmic potassium [29,30]. Their