The characterisation of GNA based fusion proteins and their potential as a novel class of bio-insecticides.

The human population is predicted to surpass 9 billion by 2050, and producing enough food remains a global challenge. Food security is paramount; with 16% of all crop losses due to insect pests and current chemical control agents for these species largely non-specific, new classes of novel insecticides are needed. ω-Hv1a-ACTX (Hv1a), a peptide from the Blue Mountain funnel web spider (Hadronyche versuta) has been fused to the plant lectin Galanthus nivalis agglutinin (GNA). GNA acts as a ‘carrier’ molecule allowing venom derived peptides to be delivered to the insect haemolymph and reach their site of action. The resulting fusion protein Hv1a-GNA is toxic orally towards Lepidopteran, Coleopteran and Hemipteran pests but displays no toxicity towards honeybees (Apis mellifera), a beneficial pollinator species. In this study the expression profiles of four Hv1a/GNA based fusion proteins produced in the yeast Pichia pastoris were assessed. The oral toxicity of the Hv1a/GNA fusion proteins to Acyrthosiphon pisum (pea aphids) was established and LC50 values calculated. Proof of concept was provided for transiently expressing a GNA/Hv1a fusion protein in tobacco (Nicotina benthamiana) and future work to produce of stable expressing Arabidopsis thaliana lines is recommended. The first ever double toxin-GNA fusion protein (DT1) containing the spider toxins Hv1a and ω-Hv1a-Hv1g (Hv1g) was purified from fermented culture supernatant. It was shown that DT1 exhibits toxicity by injection to Mamestra brassicae and LD50 values were calculated. This work proved that both toxins within the fusion protein are active. Results in this thesis demonstrate the importance of developing fusion protein based biopesticides, which will be essential for protecting crops future