## 3 Discussion
In contrast Domains II and III of Cry8 Ia1 are not related to the corresponding domains of other coleopteran active toxins, only to other Cry8 proteins, such as Cry8 Hb and Cry8 Ga. Based in these close phylogenetic relationships with other Cry toxins active against Chrysomeloidea insects we decided to determine the toxicity of the two novel Cry8 Ha1 and Cry8 Ia1 proteins against some Chrysomeloidea pests, such as _C. bowringi_ and _A. glabripennis_. For _C. bowringi_, Cry8 Ha1 was effective, but the toxicity was revealed only with the Cry8 Ha1 activated toxin after in vitro activation. Previously, a similar observation was made with the Cry7 Aa1 protein against Colorado potato beetle where only the in vitro activated Cry7 Aa1 protein showed to be toxic [9]. It has been shown that the content of midgut proteases may have great variations between different insects orders, for example, the main digestive proteases of Lepidoptera and Diptera are serine proteases, whereas those of Coleoptera are mainly cysteine and aspartic proteases [14]. Therefore, it is possible that the Cry8 Ha1 is not processed correctly by the _C. bowringi_ midgut proteases but the in vitro processing could activate the toxin properly revealing its toxicity against _C. bowringi_ larvae. In the case of _A. glabripennis_, the Cry8 Ha1 protoxin was effective against _A. glabripennis_ larvae, which is a Cerambycidae insect that harms many forests plants. Until recently, only Cry3 Aa protein was shown to has toxicity to some Cerambycidae species, including _Phytoecia rufuentris_ [PERSON], _Apriona germari_, as well as _A. glabripennis_[15; 16; 17]. The discovering of new insecticidal proteins with different amino acid sequences that may have different modes of action is important to have additional tools for the control of Cerambycidae and Chrysomeloidea insects. The toxicity of Cry8 Ha1 to two different Cerambycidae and Chrysomeloidea species suggests that other Cry8 proteins could also potentially show toxicity to different coleopteran species. In addition, we also find that the in vitro solubilization and activation was adverse for Cry8 Ha1 activity to scarab adults and also for its toxicity against the larval stages of _H. parallels_, since a reduction in toxicity was observed with the activated toxin. These data could indicate that the treatment with chymotrypsin inactivates the Cry8 Ha1 protoxin or that two different mechanisms exist for protoxin or activated toxin, as previously suggested [15]. The fact that Cry8 Ha1 activated toxins increases is toxic effect against _C. bowringi_ larvae indicates that chymotrypsin treatment did not inactivate Cry8 Ha1. Rather, the data indicate that both Cry8 Ha1 protoxin and activated toxin may have independent toxicity pathways in the different insect species analyzed. In the case of Cry1 Ab and Cry1 Ac toxins, it has been shown that protoxins and activated toxins exert toxicity by independent pathways, where two different oligomer prepores with different pore formation activity are formed depending if protoxin or activated toxin bind to cadherin receptor [18]. Furthermore, it has been shown that the C-terminal region of Cry1 Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) receptors providing a higher binding affinity of the protoxin to the gut membrane which contributes to the higher toxicity of Cry1 Ab protoxin compared to the activated toxin [19]. It remains to identify the different protoxin or activated Cry8 Ha1 binding proteins in the different insects analyzed and in the different developmental stages. However, an alternative explanation could be that the differential toxicity observed between Cry8 Ha1 protoxin or activated toxin is related to the differential proteases that are present in the different insects analyzed. In any case, our data point out that the differential toxicities of protoxin or activated toxin should be taken into account to decide which form of the toxin should be expressed in transgenic plants, depending on the target pest.