Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 out in 0.1 M Tris-HCl (pH = 8.0) with 2 mM GSH and 0.2 mM GSSG. Refolded peptide was purified to be homogeneity by C18-RP-HPLC. Its MW was determined by MALDI-TOF MS on a Kratos PC Axima CFR plus (Shimazu Co. LTD, Kyoto). Antimicrobial assays Inhibition zone assays were carried out to evaluate anti- microbial activities of peptides [28,52,117]. Briefly, 50 μl bacteria or fungal spores with OD600 = 0.5 were inocu- lated to pre-heated 6 ml Luria-Bertaini’s medium (1% bactotryptone, 0.5% bactoyeast extract and 0.5% NaCl) for bacteria or MEA medium (1% Malt Extract, 0.1% Peptone, 2% Glucose) for fungi containing 0.8% agar. The mixture was then spread on a 9-cm Petri dish, giv- ing a depth of 1 mm. After settling, 2-mm wells were punched in the plate and then 2 μl of peptide under dif- ferent concentrations was added to each well. Diameters of the inhibition zones were measured 16 hours after incubation at 30°C. CL was calculated by the Hultmark’s method [52]. The microorganisms used in this assay were listed in additional file 3. Establishment of the infection model N. vitripennis was maintained as a laboratory culture on pupae of the housefly Musca domestica. They were reared under a 14:10 light-dark cycle at 25°C in glass containers fed on 20% (v/v) honey solution. A standard method was applied to establish an infection model [118] of N. vitri- pennis. Briefly, a bacterial mixture of E. coli ATCC 25922 and M. luteus which had been separately incubated over- night was injected into abdomen of N. vitripennis adults with a micro-injector. About 50 wasps were challenged in total. Non-injected wasps were used as a control. Preparation of total RNA For isolating N. vitripennis total RNA, 50 wasps chal- lenged for 16 hours by bacteria were grounded into fine powder in liquid nitrogen. The Trizol reagent (SBS Gen- etech, Beijing) was used to prepare total RNA according to the supplier’s instructions. This method was also applied to prepare non-challenged total RNA [25]. cDNA Cloning Reverse transcription of total RNAs prepared from non- infected or infected N. vitripennis was performed using RT PreMix kit (TransGen, Beijing) and a universal oligo (dT) - containing adaptor primer dT3AP. The first- strand cDNA amplification in both non-infected and infected wasps was performed by using an AMP-like gene-specific primer and the universal primer 3AP according to the method described [119]. PCR product was cloned into pGEM-T (TIANGEN, Beijing), and transformed into E. coli DH5a competent cells Page 16 of 19 (TransGen, Beijing). Their sequences were confirmed by DNA sequencing. Primers used in this study are all listed in additional file 4. Semi-quantitative RT-PCR For semi-quantitative RT-PCR, an AMP-like gene were amplified from non-infected or infected cDNAs by using a gene-specific primer and 3AP and PCR products obtained from different cycles (i.e. 25, 30, 35, 40, 45) were taken for comparison of the amounts of these pro- ducts by electrophoresis on 1.5% agarose gels. N. vitri- pennis ribosome protein RP49 was chosen as an internal control which was amplified by using primers NvRp49- F2/3AP and the same cDNA templates. Additional file 1: Strategy of database searches of putative Nasonia vitripennis antimicrobial peptides. A diagram for the computational identification of putative AMPs. Additional file 2: Summary of N. vitripennis antimicrobial peptides. A table for the summary of N. vitripennis antimicrobial peptides. Additional file 3: Microorganism sources used in this work. A table for the microorganisms used here. Additional file 4: The PCR primers used in this work. A table for primers used here. Acknowledgements We thank Human Genome Sequencing Center, Baylor College of Medicine http://www.hgsc.bcm.tmc.edu/ for providing N. vitripennis sequence data for public use and Prof. Chuanling Qiao (Institute of Zoology, Chinese Academy of Sciences, China), Prof. Fengyan Bai (Institute of Microbiology, Chinese Academy of Sciences, China) and Dr. Jianguo Zhou (Oak Ridge National Laboratory, USA) for providing microbial strains used in this study. We also thank Prof. Fengqin He (Institute of Zoology, Chinese Academy of Sciences, China) for providing M. domestica. This work was supported by grants from the National Natural Science Foundation of China (30730015, 30621003 and 90608009) and the 973 Program from the Ministry of Science and Technology of China (2010CB945304). Author details 1Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China. 2State Key Laboratory of Rice Biology, Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, PR China. Authors’ contributions SZ designed the experiments. CT, SZ and GY wrote the paper. CT conducted database search. CT, BG and QF performed the experiments. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 25 August 2009 Accepted: 19 March 2010 Published: 19 March 2010 References 1. Ezekowitz RA, Hoffmann JA: Innate immunity Totowa, New Jersey: Human press 2003. 2. Hale JD, Hancock RE: Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther 2007, 5:951-959.