title: The use of spider venom neurotoxin-based fusion proteins as species-selective pesticides.
Creators: name: Wahlers, J
Publisher: name: Liverpool John Moores University
Year: 2020

description: The current generation of synthetic pesticides and anti-parasitic compounds have been linked to numerous ecological issues, such as pollinator population decline and human health risks. There is also a growing level of resistance amongst the pests and parasites these compounds are used to control. However, without their use agricultural output will suffer tremendously and thus alternative solutions must be investigated. One such solution is the use of fusion proteins based on neurotoxic peptides derived from spider venom. Spider venoms have been shown to contain peptides that have been optimised over hundreds of millions of years to show extreme selectivity towards their target receptors in prey species, a trait lacking in the current generation of synthetic pesticides. Generally, however these peptides lack oral toxicity, severely restricting their use as bio-pesticides (bio-pesticides can be defined as a form of pesticide based on natural products such as venom). By combining the genes for these peptides with the gene for an orally toxic protein known as Galanthus nivalis agglutinin an orally toxic fusion protein that shows a high level of specificity is produced. Such fusion proteins have been previously shown to be selective and effective pesticides, with exposure through oral and injection routes being deleterious to lethal for several pest species yet without harming beneficial hymenopterans. This thesis explores the efficacy of some of three such neurotoxic fusion proteins in the potential control of helminthic parasites using the model nematode species Caenorhabditis elegans. Survival bioassays and time budget studies were undertaken to determine any potential negative effects of oral, contact and injection exposure to field-realistic levels of Hv1a/GNA on the survival and behaviour of the British bumble bee species Bombus terrestris. Preliminary electrophysiology experiments were also conducted, using affordable extra-cellular recording equipment to determine Hv1a/GNA’s effects on the nervous activity of B. terrestris and the cricket species Gryllus assimilis. Finally, the interactions between a Hv1a/GNA and the CaV1s of B. terrestris and C. elegans were modelled. Manually observed thrashing assays were used to explore the potential effects of ButaIT/GNA, Hv1a/GNA and Pl1a/GNA on C. elegans. Worms were suspended in 100 µl of M9 media containing concentrations of 0, 50 ppm, 125 ppm, 250 ppm or 500 ppm of each fusion protein, with thrash counts taken after five minutes, an hour and four hours. In all three fusion proteins, a rise in thrashing rate was observed after 5 minutes (although only with 500 ppm in the case of Pl1a/GNA), followed by a decrease after both one and four hours. Interestingly, in all cases non-linear dose-response curves were observed, with the peak effect on mobility observed at sub-maximal concentrations in all three fusion proteins. Of further interest is the observations of spastic paralysis in both Hv1a/GNA and Pl1a/GNA treated nematodes. Spastic paralysis runs counter to Hv1a’s proposed mode of action as a calcium channel blocker. Spastic paralysis matches the mode of action of Pl1a, but Pl1a is active on voltage-gated sodium channels which are absent in C. elegans. Potential reasons for these surprising results are discussed, along with the conclusion that all three fusion proteins showed the potential to be anthelmintic treatments. In B. terrestris, oral exposure to field-realistic concentrations of Hv1a/GNA did not cause significant mortality after 48 hours in any of the tested exposure routes. Beyond this point, significant mortality was observed with oral exposure, and no significant differences were observed between Hv1a/GNA and pure GNA. In addition, exposure to Hv1a/GNA and GNA led to increased stationary behaviour and decreased feeding (consistent with the observed effects of GNA exposure in other hymenopterans). This suggests the toxicity of Hv1a/GNA is caused by GNA, rather than the effects of Hv1a. Evidence to support this theory came from the extracellular recording experiments, where no significant effects on the neural coding (as measured by spike rate) were observed following injections with Hv1a/GNA. Significant decreases in spike rate were, however, observed following Hv1a/GNA injections in Gryllus assimilis. The docking studies illuminated an interesting problem in Hv1a (and thus Hv1a/GNA) being too large to fit into the pore of the CaV. Thus, two other binding sites were investigated based on the binding poses of Dc1a and ProTxII on their respective NaV targets. In both binding sites presented by this research binding was stronger in C. elegans than in B. terrestris, potentially explaining the mechanism behind Hv1a’s selectivity against hymenopteran CaVs.