Identifying Insect Protein Receptors Using an Insecticidal Spider Toxin
The insecticidal spider toxin PaluIT1 was used to identify potential protein receptors in lepidopteran larvae. PaluIT1 was reacted with both biotin-N-hydroxy-succinimide (BHS) and fluorescein isothiocyanate (FITC) to obtain biotinylated and fluorescent probes, respectively. BHS and FITC reacted either to the N-terminal of the residue Ala1 or to the ε-amine of the Lys8 residue of PaluIT1; therefore, mono- and di-labeled products were obtained. The mono-labeled fluorescent probes were lethal to pest larvae species such as Galleria mellonella, Spodoptera frugiperda, Spodoptera litura, and Diatraea magnifactella with LD50 values from 10 to 33 μ g/g of larvae. In addition, rabbit primary antibodies against PaluIT1 were made for histochemical and immunochemical assays in order to identify protein receptors of PaluIT1 in lepidopteran larvae. Western blot assays using PaluIT1, PaluIT1-biotin, PaluIT1-FITC, and antibodies against PaluIT1 helped to identify insect protein receptors from ganglia cord homogenates. Protein bands of 250–260 kDa in S. frugiperda, G. mellonella, and D. magnifactella and above 207 kDa in S. litura were observed suggesting a Nav α-subunit protein receptor in these lepidopteran species. In addition, protein bands of 80 kDa in S. frugiperda and D. magnifactella and of 75 and 80 kDa in G. mellonella were also identified. A proteomic analysis of those protein bands suggested that PaluIT1 interacts with the cutworm larvae voltage-gated sodium channel, hexamerin and arylphorin.
KeywordsInsecticidal Receptors Nav PaluIT1 Hexamerin Arylphorin
This work was financed by grants from Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) number IN204415 and from SEP-CONACyT number 240616 to GC and CONACYT CB 106949 to EV.
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