## 2 Results  
### Cloning and Sequence Analysis of crys8 Ha1 and crys8 Ha1 from BtSU4  
After BtSU4 genome sequencing and insecticidal gene annotation, two genes coding for proteins with similarity to Cry8 family were discovered. The two Cry8 proteins consisted of 1199 and 1194 amino acids, respectively. The two novel proteins were subsequently named as Cry8 Ha1 and Cry8 la1 ([[https://www.bpprc.org](https://www.bpprc.org)]([https://www.bpprc.org](https://www.bpprc.org))). After CDD search, the core region of the two novel insecticidal proteins were identified. The domains I, II, and III of Cry8 Ha1 protein were located from amino acids 94 to 290, 300 to 510 and 512 to 657, respectively. While for Cry8 Ia1 protein these domain regions were located from amino acids 106 to 288, 296 to 516, and 517 to 665, respectively. BLAST analysis results showed that some domains of the two Cry8H proteins have high similarity to other proteins from the Cry family such as Cry3, Cry1B, or Cry1I, rather than to Cry8 members. Phylogenetic analysis were performed with the complete protoxin or the toxin core sequences of these two Cry8 proteins and compared with different Cry proteins (Figure 1). These analyses showed that protoxin sequences of Cry8 Ha1 and Cry8 Ia1 clustered in a single branch with the other Cry8 protoxins as expected. In contrast the sequence of the toxin cores of these proteins were found in different branches. The toxin core of Cry8 Ha1 toxin was found clustered in the same branch with Cry3 proteins together with Cry8 Ba, Cry8 Pa, Cry8 Ca, and Cry8 Ia, while Cry8 Ia1 was found grouped with Cry8G, Cry8K, and Cry7G toxins (Figure 1). Suggesting that these two novel proteins Cry8 Ha1 and Cry8 Ia1 may have differential specificity. We also performed phylogenetic analyses of the different domains. Analysis of domain I sequences from several Cry proteins showed that this region is distributed in three main clusters (Figure S1). Cluster A includes 11 different Cry8 proteins clustered with domain I sequences from Cry1B and Cry1I (Figure S1, Cluster A); the second group (Figure S1, Cluster B), includes domain I sequences from Cry8 Ha1 and Cry8 Ia1 that were found to be grouped with other nine Cry8 and Cry3 proteins; the third group (Figure S1, Cluster C) includes six members, forming a cluster including more distant Cry8 proteins (Figure S1). The sequences from domains II and III showed larger variation, where the Cry8 Ha1 and Cry8 Ia1 were located in different clusters. For domain II sequences six different clusters were identified, the Cry8 Ha1 was located in Cluster A (Figure S2, Cluster A), which included sequences of domain II from Cry1B and Cry1I proteins, while Cry8 Ia1 is found in cluster E with other Cry8 members such as Cry8 Ib1, Cry8 Ga1, Cry8 Ka1, and Cry8 Kb1 (Figure S2, Cluster E). Finally, for domain III sequences, they were classified in seven clusters, where the domain III of Cry8 Ha1 is found in Cluster A, which includes sequences from Cry1I, Cry1B and Cry3 proteins (Figure S3, Cluster A), while Cry8 Ia1 is found in Cluster E together with other Cry8 proteins, such as Cry8 Ilb1, Cry8 Sa1, Cry8 Ta1 and Cry8 La1 (Figure S3, Cluster E). These analyses show that Cry8 Ha1 toxin is more closely related with Cry3, Cry1B, and Cry1I. Differential Toxicity of Cry8 Ha1 and Cry8 Ia1 Protaxins and Activated Toxins Against Different Coleopteran Insets  
To express the _cry8 Ha1_ and _cry8 la1_ genes, primers were designed according to their gene sequences, amplified from the total DNA of BtSU4 strain and cloned into pTSK shuttle vector as described in Materials and Methods. The acrylosulfierous HD-73' strain was transformed with these constructions by electroporation, and the resulted recombinant strains HD8H and HD8I were used to express these proteins and determine their toxicity against different coleopteran insects as described below. The morphology of the crystals produced by BtSU4, HD8H, and HD8I strains was analyzed in spore/crystal suspensions observed under scanning electron microscopy (SEM).