Isolation and characterization of orally active insecticidal peptides from spider venoms

Hardy, Margaret Carter (2011). Isolation and characterization of orally active insecticidal peptides from spider venoms PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Hardy, Margaret Carter
Thesis Title Isolation and characterization of orally active insecticidal peptides from spider venoms
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-07
Thesis type PhD Thesis
Supervisor Glenn F. King
Total pages 112
Total colour pages 29
Total black and white pages 83
Language eng
Subjects 06 Biological Sciences
Formatted abstract
The goal of the work presented in this thesis is to develop novel, sustainable approaches to manage arthropod pests that cause economic damage. Several species of introduced and native Australian insects cause significant economic damage to buildings, agricultural areas, and public property (e.g., telephone poles) in Queensland. Arthropod pests have traditionally been controlled with chemical insecticides. However, the evolution of insecticide resistance in target insects, combined with the revocation of key insecticides due to their adverse environmental effects, has created an unmet demand for a new generation of environmentally friendly insecticides. To date no one has directly examined whether spider venom is toxic to termites. In the course of this thesis it has been determined that the venom of the Australian tarantula Selenotypus plumipes Pocock (Araneae: Opisthothelae: Myglaomorphae: Theraphosidae) is lethal to the most economically important species of termite in Australia, Coptotermes acinaciformis (Froggatt) (Isoptera: Rhinotermitidae). I have explored the hypothesis that individual peptide toxins from tarantula venom are orally active in insects. These peptides have been isolated and sequenced, and the three dimensional structure of one of these peptides has been determined. The aim of this project is to determine the extent to which tarantula-venom components can be developed as environmentally friendly bioinsecticides.

The primary aims of these studies were to screen and analyze venom toxicity from different spiders through assays using a diverse range of insects, including mealworms, crickets, and Australian termites; to isolate active peptidic components from the venom using chromatographic fractionation; to obtain the amino acid sequence of these peptides using a combination of mass spectrometry, Edman degradation, and rapid amplification of complementary ends (RACE); and to determine the structure of the active peptides using nuclear magnetic resonance (NMR) spectroscopy. Using this combination of techniques, it was anticipated that a novel insecticide, perhaps with a novel mode of action, could be isolated, purified, and produced using recombinant methods.

By screening for oral toxicity, five novel, orally active insecticidal peptides were discovered in the venom of an Australian tarantula, S. plumipes. Active insecticidal peptides were purified from crude venom via a combination of reverse-phase high performance liquid chromatography and cation exchange chromatography.

Partial amino acid sequences obtained using N-terminal sequencing were used to design primers for obtaining the sequence of full-length toxin transcripts from a venom gland transcriptome obtained using 454 next-generation sequencing. One peptide toxin was produced via chemical synthesis, and its three-dimensional structure was determined using NMR spectroscopy. Structural homologies with other spider venom toxins have provided clues to its likely molecular target in the insect nervous system.

In summary, the insecticidal toxins discovered during the course of this thesis could be deployed as bioinsecticides in the form of baits or sprays, or by engineering transgenes encoding the peptide toxins into plants or entomopathogens. The activity and mechanism of action of these novel insecticidal toxins will be explored in future work and may lead to a novel commercial product to meet current market needs.
Keyword Australian funnel-web spider
Inhibitor Cystine Knot
Insect pests
Insecticidal toxin
Nuclear Magnetic Resonance (NMR)
Spider-venom peptide
Venom gland
Venom peptidome
Venom transcriptome

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Created: Mon, 14 Nov 2011, 10:48:59 EST by Mrs Margaret Hardy on behalf of Scholarly Communication and Digitisation Service