## 3 Discussion  
The Cry8 toxin family has been reported to have toxicity against coleopteran insects and until now 60 different Cry8 proteins (including 2 Cry8-like proteins) that belong to 28 holotypes by the new Bacterial Pesticidal Protein Resource Center (BPPRC) ([[https://www.bpprc.org](https://www.bpprc.org)]([https://www.bpprc.org](https://www.bpprc.org))) [6]. Cry8 proteins have been shown to be mainly toxic to Scarabs, but some patents claimed that certain Cry8 proteins also show toxicity to Chrysomeloidea insects [11]. The larvae and adults of both Scarabs and Chrysomeloidea insects feed on plants, thus it is important to find effective strategies to control both stages of these insects. Until recently, as shown in Figure 5, the Cry8 Ab, Cry8 Ca, Cry8 Da, Cry8 DB, Cry8 EA, Cry8 Ga, Cry8 Na, and a Cry8-like protein have been confirmed toxic to six main harmful scarabs. Among these proteins, the Cry8 Da, Cry8 DB and Cry8 Ga proteins have shown to be effective against both adults and larvae stages of the insects [12; 13]. In this study, we show that the protoxin from the novel Cry8 Ha1 showed toxicity against both adults and larvae of _H.__parallela_. In contrast, this Cry8 Ha1 protonix was only toxic to the adults of _H.__oblita_, but not to the larvae. In addition, Cry8 Ha1 was toxic to the larvae of _A.__glabripennis_. The protoxin of the new Cry8 Ia1 was only toxic to the larval stages of _H.__parallela_. The reason for the differential toxicities of these protooxins observed in adults or larvae stages of certain species is not known, but one alternative could be related to a differential expression of Cry8 midgut receptors in both developmental stages. This hypothesis remains to be analyzed. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multicolumn{2}{c}{**H. _oblita_} & \multicolumn{2}{c}{**H. _parallela_**} & _C. _bowringi_ \\ \cline{2-5}  & **Adult** & & **Adult** & **Lavae** & **Lavae** \\  & **LC50 Values in** & **Lavae** & **LC50 Values in** & **LC50 Values in** & **LC50 Values in** \\  & **mg/mL** & **10\({}^{6}\) CFU/g** & **mg/mL** & **CFU/g** & **mg/mL** \\  & **(Confidence Limits)** & **(Confidence Limits)** & **(Confidence Limits)** & **(Confidence Limits)** \\ \hline Cry8 Ha1-Pro & 0.99 (0.62–2.05) & NA & 0.16 (0.00–0.57) & 218.90 (68.21–661.10) & NA \\ Cry8 Ia1-Pro & NA & NA & NA & 85.03 (03.59–176.08) & NA \\ Cry8 Ha1-Act & NA & NT & 0.66 (0.20–12.36) & NT & 0.02 (0.01–0.02) \\ Cry8 Ia1-Act & NA & NT & NA & NT & NA \\ \hline \multicolumn{5}{l}{Note: the NA = no activity; NT = not tested; “-Pro” = protonix; “-Act” = activated toxin; “CFU” = the colony-forming unit of crystal-spore mixture.} \\ \end{tabular}
\end{table} 
Table 1: The LC50 Cry8 Ha1 and Cry8 Ia1 against _Holotrichia oblita_, _Holotrichia_ _parallela_, and _Colaphellus__bowringi_. Figure 4: The bioassay of Cry8 Ha1 protonix against _Anoplophora glabripennis_. (**A**): the mortality of _A.__glabripennis_ treated by different dose of Cry8 Ha1 protonix. This experimented was performed in triplicate. For each repeat, 24 larvae were tested; (**B**): Morphology of toxin-treated _A.__glabripennis_ larvae. The phylogenetic analysis of Cry8 Ha toxin core sequence showed that this proteins is more related to Cry3 proteins and the analysis of the different domains of Cry8 Ha1 and Cry8 Ia1 proteins confirmed that Cry8 Ha1 is more related to the coleopteran active proteins Cry3, Cry1B, and Cry1I that have been shown to be specifically toxic to Chrysomeloidea insects [5]. Our data suggest that the novel Cry8 Ha1 and Cry8 Ha1 proteins have evolved from different origins, since these Cry8 Ha1 and Cry8 Ia1 proteins do not cluster together. Only Domain I of both Cry8 Ha1 and Cry8 Ia1 proteins clustered together with domain I from Cry3 proteins. Domain II of Cry8 Ha1 is more related to domain II from Cry1B and Cry1I proteins; while Domain III of this protein is closer to domain III from the Cry1B, Cry1I, and Cry3 coleopteran active proteins.