Schneider, K.D., Tourtois, J., Bertrand, D., Kambara, K., Hulbert, D., Haase, A., Kanniainen, S.R., DeVree, B., Kennedy, R.M., Bao, L. (2026). Mechanistic characterization of insecticidal Shiva toxin interactions with Drosophila melanogaster nicotinic acetylcholine receptors.  Pestic. Biochem. Physiol. 221(): 107136.

FBrf0265621

Research paper

Shiva family toxins are naturally occurring insecticidal peptides that target insect nicotinic acetylcholine receptors (nAChRs) and show strong utility as bioinsecticides. However, the molecular basis of their interaction with insect nAChRs remains poorly understood. In this study, we demonstrate that Shiva toxins exhibit no cross-resistance with spinosad, indicating that they do not interact with the Drosophila nAChR α6 subunit. Using CRISPR-generated Drosophila melanogaster lines carrying homozygous knockout alleles of individual nAChR subunit, insect bioassays revealed that the nAChR β1 subunit is critical for Shiva toxin activity. To further define the interaction site, Drosophila nAChR α4 and β1 subunits (Dmα4-β1) were heterologously co-expressed in Xenopus oocytes and examined by two-electrode voltage clamp (TEVC). Shiva toxins directly activated Dmα4-β1 receptors and also acted as positive allosteric modulators (PAM), enhancing acetylcholine-evoked currents with EC50 values in the nanomolar range. Consistent with the bioassay results, electrophysiological analysis confirmed the essential role of the β1 subunit in Shiva toxin interaction. Site-directed mutagenesis identified two residues, E206 and I231, within the Drosophila nAChR β1 subunit that are required for Shiva toxin activity. Together, these results further clarify the molecular mechanism underlying Shiva toxin interaction with insect nAChRs and provide insights into their mode of action as bioinsecticides.