Title

Developing the next generation of chemical and bioinsecticides from insecticidal neurotoxins of Hadronyche versuta

Date of Completion

January 2004

Keywords

Biology, Molecular|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Due to increased resistance and human health concerns associated with current insecticides there is an urgent need for novel, more specific methods of arthropod pest control. The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory neurotoxins that we isolated from the venom of Australian funnel-web spiders. In order to shed light on the mechanism of action of these toxins, and to enhance their utility as lead compounds for insecticide development, we developed an efficient Escherichia coli expression system for production of recombinant J-ACTX-Hv1c so that structure-function relationships could be elucidated using site-directed mutagenesis. A comprehensive alanine-scanning mutagenesis generated the first complete map of the key functional residues of a spider toxin. Additional mutagenesis of specific functional moieties on the critical side chains provided a detailed mechanism of toxin-target interaction. This data, along with NMR refinement of the 3D toxin structure, has supplied necessary details for development of a small molecule mimetic to employ as a novel chemical insecticide. ^ The activity and phenotype of J-ACTX-Hv1c across several arthropod Orders and the spatial orientation of critical side chains supplied valuable clues as to the molecular target of the J-ACTXs. Ultimately, electrophysiology in cockroach dorsal unpaired median (DUM) neurons revealed that J-ACTX-Hv1c blocks potassium channels. Thus the renamed kappa (κ)-atracotoxins represent the first known insect-specific K+ channel blockers. ^ As a method to individually target a pest species, we also determined the applicability of κ-ACTX-Hv1c in an insect release program and in biopesticide development. For validation of insect release programs, we engineered transgenic Drosophila that express κ-ACTX-Hv1c under control of a heat shock promoter (Hsp70). Mating transgenic flies with wild-type flies results in heterozygous offspring that exhibit 100% death within 24 hrs following induction of the toxin gene. For biopesticide development, we generated recombinant baculoviruses (Autographa californica) containing κ-ACTX-Hv1c or an insect-selective Ca2+ channel blocker, ω-ACTX-Hv1a. Assessment of these recombinant viruses in pest lepidopteran species revealed a significant improvement in time to paralysis/death over wild type virus. ^