## 2 Results
### Cloning and Sequence Analysis of crys8 Ha1 and crys8 Ha1 from BtSU4
Figure 2 shows that BtSU4 had typical Bt spores and relatively small irregular crystals (indicated by arrow). The ICPs accumulated in BtSU4 crystals were composed of 135 kDa protein, as revealed by SDS-PAGE electrophoresis (Figure 2E). Figure 1: Phylogenetic analysis of the protoxin or toxin core amino acid sequences from different Cry proteins. These analyses involved 52 amino acid sequences. Evolutionary analyses were conducted in Clustal Omega and Neighbour-joining Phylogenetic analysis ([PERSON] et al. 2019). The Cry8 Ha1 and Cry8 Ia1 proteins were labeled in red letters. The SEM images of Figure 2 showed that both Cry8 Ha1 and Cry8 Ia1 crystals inclusions accumulated forming large irregular crystals (Figure 2B,C, indicated by arrows). SDS-PAGE analysis showed that the expressed Cry8 Ha1 and Cry8 Ia1 protoxins were resolved also as 135 kDa bands (Figure 2E), similar to the ICPs molecular weight band observed for BtSU4 strain. To compare the toxicity of protoxins with that of activated toxins of both Cry8 Ha1 and Cry8 Ia1 proteins, the protoxins of Cry8 Ha1 and Cry8 Ia1 were activated by chymotrypsin digestion. We decided to activate Cry8 toxins with chymotrypsin since it was previously shown that this protease was highly efficient for activation of Cry3 Aa toxin that is toxic to coleopteran larvae in contrast to trypsin protease [10]. After digestion with chymotrypsin, the activated toxins were again analyzed by SDS-PAGE electrophoresis, showing that the Cry8 Ha1 is activated into a 70 kDa protein fragment after 2 h of treatment with the protease. The 70 kDa protein fragment was relative stable since it remained after 24 h of treatment, although some of the 70 kDa fragment was further degraded into a smaller fragment with a molecular weight about 55 kDa (Figure 3). In contrast, the protoxin of Cry8 Ia1 after protease treatment showed two main fragments of 97 and 66 kDa (Figure 3). The toxicity of the two Cry8 Ha1 and Cry8 Ia1 protoxins was first analyzed against different insects (Table 1, Figure 4). The data showed that the Cry8 Ha1 protoxin was toxic to both _H. parallels_ larvae and adults. This protoxin was also toxic against _H. oblita_ adults, but not to _H. oblita_ larvae neither to _C. bovringi_ larvae (Table 1). Analysis against _A. glabripennis_ larvae showed that Cry8 Ha1 protoxin was also toxic to the larvae of this insect (Figure 4). The Cry8 Ha1 treated larvae showed a severe  
Figure 3: SDS-PAGE protein profile of Cry8 Ha1 and Cry8 Ia1 proteins, after digestion with chymotrypsin protease. “M” means the marker, and the molecular weight of the bands are 212, 116, 97, 66 and 45 kDa, respectively. Figure 2: SEM observation and Electrophoretic analysis of Bt spore and crystal mixtures. Panels (**A**–D), SEM images of the spore and crystal mixtures of Bt strain BtSU4, HD8H, HD8I and crystal negative strain HD-73’. The scale bar in panels A, B, C and D was 2 \(\upmu\)m. Panel (**E**), Sodium dodecyl sulfate-polyacrylamideamide gel electrophoresis (SDS-PAGE) profiles of these spore and crystal mixtures. M, Bio-Rad high-molecular-mass protein marker. The other lanes were total protein samples from Bt strain BtSU4, HD8H, HD8I and crystal negative strain HD-73’, respectively. effect in their growth, they remain small, lack mobility and looks severely damaged, after few days of treatment they died. The image of Figure 4B shows these effects. In contrast, the Cry8 Ia1 protonix, showed toxicity only against _H.__parallela_ larvae and did not show toxicity to any the other insect tested in this study (Table 1). We then analyzed toxicity of the activated toxin samples, chymotrypsin activation caused the complete loss of toxicity of Cry8 Ha1 to _H.__oblita_ adults and seems to reduce the activity against _H.__parallela_ adults when compared to the Cry8 Ha1 protonix, although these data are not significant since their fiducial limits overlapped. The activation of Cry8 Ha1 toxin with chymotrypsin resulted in enhanced toxicity against _C.__bowringi_ larvae (Table 1).