## 5 Materials and Methods
### Insets, Rearing Conditions and Bioassay  
Adults of _H. oblita_ and _H. parallelism_ were collected from Baoding and Cangzhou, Hebei province, China. These adults were reared in plastic boxes filled with 5 cm thick sieved soil and fed with elm (_llmus pumila_ L.) leaves, at 25 \(\pm\) 0.5 \({}^{\circ}\)C, 16L: 8D photoperiod condition and 60% humidity. The eggs were collected and hatched in the same conditions. For bioassay activity against adults, we used the leaf-dipping method. Before feeding, the cleaned elm leaves were soaked in Cry protoxin protein solutions, and then the surface liquid was dried at room temperature. In each treatment, ten adults were feed with the dipped leaves, and then, we replaced the protein dipped elm leaves and check insect mortality every day. These assays were done in triplicate for each protein concentration, using a two-fold dilution protein concentration series (0.03125 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL, and 2.0 mg/mL). For bioassays against larvae, we used a procedure modified from [PERSON] (2009) [20]. Five-day larvae were feed with a mixture of potato with spores/crystal suspension. Firstly, the potato was shred, washed, air driedand then mixed with Bt spore/crystal water suspension; then, the mixture was further mixed with sieved soil and divide evenly into four six-well culture plates. In each treatment, 24 larvae were feed separately in each well performed in triplicate and mortality was determined after 14 day. The adults of _A. glabripennis_ were collected from Beijing, China, and reared in plastic boxes filled with pieces of willow branches (_Salix babylonica_), at \(25\pm 0.5\)\({}^{\circ}\)C, 16L: 8D photoperiod condition, 60% humidity. The adults feed on the bark and lay their eggs in the bark. The eggs hatch and larvae bioassay were performed on artificial diet. To prepare the artificial diet, we made willow bark powder and willow xylem powder. Branches with a diameter of 5-6 cm from the growing willow were used to separate the willow bark and xylem. Then, we washed, dried, ground, and sieved (80 meshes) the bark and xylem, respectively. The resulted powders were ready for use. The artificial diet was produced by mixing 60 g willow bark powder, 30 g willow xylem powder, 22.5 g agar, 10 g yeast powder, 1.5 g sorbic acid, 1.5 g methyl 4-hydroxybenzoate, 1.5 g ascorbic acid, and 520 mL sterile water, and sterilized at 121 \({}^{\circ}\)C for 20 min. To perform bioassays, the Cry proteins were mixed with the artificial diet and divided into a 24-well culture plates, with final protein concentrations of 100 ug/g, and 300 ug/g; then, 24 two-day larvae were feed separately in each well in triplicate in a dark condition, and mortality was determined after 10 days. The _C. bowringi_ Baly population was reared in the lab using rape leaves. The bioassays were performed in triplicate against larvae by using the leaf dipping method. Before feeding, the cleaned rape leaves were soaked in Cry protein solutions with two-fold dilution series of protein concentrations of 0.125 ug/mL, 0.0625 ug/mL, 0.03125 ug/mL, 0.01563 ug/mL, and 0.007815 ug/mL, and then the surface liquid was dried at room temperature. In each treatment, 20 larvae were feed with the dipped leaves, at \(25\pm 0.5\)\({}^{\circ}\)C, 16L: 8D photoperiod, 60% humidity, the mortality was determined after two days. As mentioned above three replicates were performed for each treatment for all bioassays. The 50% lethal concentrations (LC\({}_{50}\)) were determined by Probit analysis. For all leaf dipping method, 0.005% Triton X-100 was added to increase fluid ductility and allowing uniform protein dip. The following are available online at [[http://www.mdpi.com/2072-6651/12/10/642/s1](http://www.mdpi.com/2072-6651/12/10/642/s1)]([http://www.mdpi.com/2072-6651/12/10/642/s1](http://www.mdpi.com/2072-6651/12/10/642/s1)). Figure S1. UPGMA phylogenetic analysis of the amino acid sequences from domain I of Cry proteins, Figure S2.