| Summary: | Farmers are required to increase their food productivity to meet the demand from a continually enlarging population. A major constraint on meeting this requirement is the damage created by insect pests. Agricultural pests and associated diseases destroy 30%-40% of the world’s crop produce. The withdrawal of broad-spectrum chemical insecticides, the threat of the development of resistance in insect pests to remaining compounds and the possibility of new pest species spreading into the UK as a result of environmental change have prompted research into alternative pesticides. Protein-based insecticides offer the possibility of producing compounds that are specific to pest species and environmentally benign. The production of orally active insecticidal fusion proteins, containing toxins from Segestria florentina (tube-web spider) or Mesobuthus tamulus (Indian red scorpion) fused to a ‘carrier’ protein (snowdrop lectin; Galanthus nivalis agglutinin; GNA) which transports them across the insect gut epithelium, has shown that recombinant protein expression systems and protein engineering techniques can be used to produce novel insecticidal proteins. The main aim of the work described in this thesis is to extend this technology. Avidin, a biotin-binding protein known to be insecticidal, was evaluated as a possible ‘carrier’ protein. Recombinant avidin was produced in high yields using Pichia pastoris and was compared to the native egg white protein. Recombinant avidin has insecticidal activity towards hemipteran plant pests. It was highly toxic to Acyrthosiphon pisum (pea aphid) when fed in liquid artificial diet, causing 100% mortality after four days when present at concentrations ≥0.25mg/ml (250ppm). The toxicity towards A. pisum was prevented by biotin supplementation of the diet. In contrast, recombinant avidin had no significant effects on the survival of Sitobion avenae (cereal aphid) at concentrations up to 2mg/ml (2000ppm) in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to sub-optimal levels of biotin and possess a more effective system for scavenging biotin from recombinant avidin in the diet. Avidin is readily transported to the haemolymph of lepidopteran larvae after feeding, which suggested that it might replace GNA in synthetic insecticidal fusion proteins. Numerous attempts were made to produce a fully functional insecticidal avidin-based fusion protein containing scorpion or spider toxins by expression as recombinant proteins in P. pastoris but, following tests against Mamestra brassicae (cabbage moth) larvae, the fusion proteins were found to be non-toxic. The lack of toxicity was most likely due to incorrect folding of the toxin component of the fusion, since the avidin component was functional. Avidin fed to A. pisum was found to bind to the stomach region of the gut after ingestion and was retained for at least 72 hours. Feeding conjugates of avidin with fluorescently labelled biotin, or a fluorescently labelled, biotinylated peptide, showed that the conjugated compound was also retained in the aphid gut after feeding. A conjugate between avidin and biotinylated leucomyosuppressin (LMS), a myoinhibitory peptide hormone that affects gut contractions in insects, was prepared and fed to aphids. The avidin : biotin-LMS conjugate had insecticidal activity towards A. pisum when fed in diet at levels which neither of the components (avidin or biotin-LMS) caused significant mortality. It was hypothesised that binding to the gut, through the avidin moiety, was responsible for the observed oral toxicity of the avidin : biotin-LMS conjugate. The same principle was applied to lepidopteran larvae. A conjugate between avidin and biotinylated allatostatin was prepared and fed to M. brassicae larvae. The avidin : biotin-allatostatin was non-toxic, most likely due to cleavage between the biotin molecule and allatostatin as a result of the high levels of gut proteolysis in lepidoptera. Avidin conjugates of peptides that have little or no oral toxicity to insects, as a result of restricted access to sites of action, could have the potential to form a novel class of insecticidal compounds.
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