The mechanisms of action of insecticidal lectins from snowdrop (GNA) and jackbean (concanavalin a) ontomato moth larvae

Artificial diet bioassays were carried out to investigate the impact of GNA and Con A upon the development of L. oleracea larvae. GNA, at 2 % of total dietary protein, exerted a significantly detrimental effect upon larval development, growth and consumption, with little effect upon survival. Con A was shown to be the more toxic of the 2 lectins. When tested at concentrations of 2.0 %, 0.2 % and 0.02 %, Con A caused a significant decrease in survival and larval development, and caused greater reductions in larval growth and consumption compared to GNA. The potential for GNA and Con A to exert insecticidal effects via binding to the brush border membrane (BBM) and peritrophic membrane (PM) of L. oleracea larvae was investigated. Con A, which specifically binds a-D-mannopyranoside and a-D- glucopyranoside residues, was shown to bind in vitro to the majority of BBM and PM proteins. In contrast GNA, which exhibits strict specificity for a(l,3) and a(l,6)-linked D-mannose residues, bound to only 5 BBMV and 2 PM proteins. In agreement, higher levels of Con A, compared to GNA, were shown to accumulate in larval gut tissue after feeding the proteins in vivo. Despite this both lectins were shown to have a similar ability to disrupt the digestive capacity of the larval midgut. GNA and Con A stimulated similar short term elevations in BBM enzyme and soluble trypsin activities and a long-term reduction in a-glucosidase activity. Increases in levels of trypsin activity in faecal material collected from lectin-fed larvae suggested that the proteins may act by disrupting mechanisms of enzyme recycling. Aminopeptidase, an abundant and avidly binding BBM protein (120 kDa), was identified as a major Con A binding species in L. oleracea. A 98 kDa GNA-binding BBM protein was purified and amino acid sequence data was obtained from digest polypeptides allowing oligonucleotide primers to be designed. Subsequent attempts to amplify (by PCR and RT-PCR) fragments containing coding sequence corresponding to the 98 kDa protein were unsuccessful. This was attributed to oligonucleotide degeneracy together with the low abundance and relatively large size of the protein. The potential for GNA and Con A to exert systemic effects upon I. oleracea was demonstrated by the detection of both lectins in the haemolymph of larvae exposed to experimental diets. GNA was detected in haemolymph of larvae exposed to experimental diet for just 2 hours. In contrast, no Con A was detectable in haemolymph extracted from larvae fed for 24 hours, although it was present in the haemolymph after 5 days of exposure to the diet. That GNA and Con A may act directly upon organs other than the insect gut was indicated by the detection of lectins in vivo in malpighian tubule and fat body tissue extracts. A significant reduction in haemocyte abundance in haemolymph samples extracted from lectin-fed larvae suggested that both GNA and Con A may also act by disrupting the immune system of L. oleracea

Mathematical modelling of blood glucose regulation

Exercise is beneficial for all individuals; it lowers blood pressure, keeps the heart healthy and increases insulin sensitivity. Recent studies have shown the power that regular exercise has to improve metabolic health, which in turn works to prevent and to reverse the onset of the widespread epidemics of type 2 diabetes (T2DM). However, diabetics taking insulin are required to meticulously plan exercise around meals and intake of insulin as they face an increased risk of hypoglycaemia from physical activity, which can discourage them from taking part. This thesis describes the use of systems of ordinary differential equations to model the effects of exercise on the glucose regulatory system, for both healthy and diabetic individuals. A particular focus is given to the role of glucagon, whose role is often neglected in glucoregulatory models, and its ability to enhance hepatic glucose production and so to prevent hypoglycaemia. Models of glucose-insulin-glucagon dynamics are first developed to describe an Intravenous glucose tolerance test (IVGTT), as the processes involved are simpler than in exercise and already widely modelled for glucose and insulin, thus is a good basis for validating the incorporation of glucagon. Mathematical models are used as tools within biological applications as they allow for an investigation into the dynamics that are involved in complex regulatory processes. The mathematical models in this thesis serve as accurate tools to predict blood glucose levels during exercise for both a non-diabetic and type 1 diabetic individual (T1DM) and emphasise exercise as a key element in the prevention of T2DM. By mathematically modelling the system and the mechanisms that occur to maintain glucose homeostasis an insight is gained into what the principal factors are for the greatest increase in insulin sensitivity and for the reduction in the likelihood of either hypoglycaemic or hyperglycaemic episodes. This may lead to recommendations for exercise plans which not only provide the greatest benefits for everyday health ant to assist with preventing the onset of diabetes but also to offer safer regimes for individuals with T1DM

The solvolysis of some primary alkyl bromides

Recent evidence suggests that the solvolysis of organic halides is associated with Arrhenius activation energies which have negative temperature coefficients. It is considered that this is most probably due to the increased solvation of the transition state of the reaction relative to the initial state of the reactants. Bensley and Kohnstam have suggested that the heat capacity of activation (ΔCP * = dE/dT - R) is a measure of this increase in solvation and also that in S(_N)1 reactions,the entropy of activation, Δ S * , is a measure of the same phenomenon; whereas in S(_N)2 reactions an additional negative contribution to Δ S * due to the covalent binding of the nucleophilic reagent must be considered. The solvolysis of R-Hal i n a given solvent and at a given temperature should, therefore, have a constant value for the ΔCp */ΔS * ratio for SNl reactions, independent of the nature of the group R and the ratio should have a lower value for S(_N)2 reactions (since ΔCp* and ΔS*are both negative in the type of reaction being considered]] At the commencement of this study these tentative suggestions required experimental verification. A parallel study has shown the constancy of ΔCp*/ΔS* for S(_N)l reactions and this thesis describes the determination of this ratio, for the solvolysis of some n-alkyl bromides in 50% aq.acetone when the extreme form of the S(_N)2 mechanism is operative. These compounds all showed negative temperature coefficients of activation energy and the ΔCp*/ΔS* ratio was always lower than for S(_N)l reactions, in agreement with the earlier predictions. It can be concluded that the determination of this ratio provides an additional test of solvolytic mechanism which seems likely to be applicable to border-line reactions in which there is a transition from mechanism S(_N)2 to mechanism S(_N)1. Here, the 'classical' mechanistic tests do not always yield unambiguous results

Which Insect Species and Why?

This chapter describes the common features determining the suitability of insects for small- and industrial-scale farming, the main insect species currently being produced on a large scale for feed production and other potential candidate species. Natural consumption of insects by animals and which insects are suitable for which animal feed is also briefly discussed

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects

Background Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake. Results A gene encoding a toxin, ButaIT, from the red scorpion (Mesobuthus tamulus) was synthesised and assembled into expression constructs. One construct contained ButaIT alone, whereas the other contained ButaIT fused N-terminally to a GNA polypeptide (ButaIT/GNA). Both recombinant proteins were produced using the yeast Pichia pastoris as an expression host, and purified. Recombinant ButaIT and ButaIT/GNA were acutely toxic when injected into larvae of tomato moth (Lacanobia oleracea), causing slow paralysis, leading to mortality or decreased growth. ButaIT/GNA was chronically toxic when fed to L. oleracea larvae, causing decreased survival and weight gain under conditions where GNA alone was effectively non-toxic. Intact ButaIT/GNA was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. Proteolysis of the fusion protein was also observed. ButaIT/GNA was significantly more toxic that GNA alone when fed to the homopteran Nilaparvata lugens (rice brown planthopper) in liquid artificial diet. Conclusion The ButaIT/GNA recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of GNA as a carrier to transport potentially toxic peptides and proteins across the insect gut. Although ButaIT has been claimed to be lepidopteran-specific, the fusion protein has more wide-ranging insecticidal activity. Fusion proteins based on plant lectins have potential applications in crop protection, both as exogenously applied treatments and as endogenous products in transgenic plants

Analysis of unpolarised π +π − photoproduction with the GlueX Experiment

This thesis presents measurements of spin-density matrix elements in unpolarised π+π − photoproduction on a proton target. The dominant resonance contribution to the dipion system is the ρ(770) meson. Due to the large production cross section for this resonance, a valid comparison can be made between the obtained final results and ρ(770) spin-density matrix elements measured previously with other experiments. The measurement was performed over the 3.0 – 11.6 GeV beam energy regime, which is a more extensive energy range than has ever been studied previously for the ρ(770). Results were obtained by analysing data from the GlueX experiment based at Jefferson Lab. Extended maximum likelihood fits were applied to extract three spin-density matrix elements using Markov chain Monte Carlo based parameter estimations. This was performed using various binning configurations to probe the energy, mass, and four-momentum transfer dependence of the determined physics observables. Spin-density matrix elements are shown to be consistent with the model of s-channel helicity conservation at low −t. The effects of pomeron and f2 exchanges are clearly visible in the energy dependence of the measured observables. These observations provide valuable insights into the relative strengths of both processes as a function of the photon energy, and may enable theorists to disentangle the f2/P coupling ratio. Spin-density matrix elements are seen to be highly dependent on the reconstructed resonance mass. This observation is likely to be a result of non-resonant S-wave background processes, and emphasises the need for a more detailed model of the π +π − angular distribution that considers all of the competing angular momentum components that contribute to the measured final state. The statistical precision of measurements performed for this thesis surpass what was achievable in previous studies of the ρ(770) by several orders of magnitude. Studies of the energy and four-momentum transfer dependence, and insights into the effects of non ρ(770) background contributions provide valuable input for production models. This will help inform the choice of wave-sets used for partial wave analyses, supporting GlueX in its search for exotic hybrid meson states

Fusion to snowdrop lectin magnifies the oral activity of insecticidal omega-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system

Background: The spider-venom peptide v-hexatoxin-Hv1a (Hv1a) targets insect voltage-gated calcium channels, acting directly at sites within the central nervous system. It is potently insecticidal when injected into a wide variety of insect pests, but it has limited oral toxicity. We examined the ability of snowdrop lectin (GNA), which is capable of traversing the insect gut epithelium, to act as a ‘‘carrier’’ in order to enhance the oral activity of Hv1a. Methodology/Principal Findings: A synthetic Hv1a/GNA fusion protein was produced by recombinant expression in the yeast Pichia pastoris. When injected into Mamestra brassicae larvae, the insecticidal activity of the Hv1a/GNA fusion protein was similar to that of recombinant Hv1a. However, when proteins were delivered orally via droplet feeding assays, Hv1a/ GNA, but not Hv1a alone, caused a significant reduction in growth and survival of fifth stadium Mamestra brassicae (cabbage moth) larvae. Feeding second stadium larvae on leaf discs coated with Hv1a/GNA (0.1–0.2% w/v) caused $80% larval mortality within 10 days, whereas leaf discs coated with GNA (0.2% w/v) showed no acute effects. Intact Hv1a/GNA fusion protein was delivered to insect haemolymph following ingestion, as shown by Western blotting. Immunoblotting of nerve chords dissected from larvae following injection of GNA or Hv1a/GNA showed high levels of bound proteins. When insects were injected with, or fed on, fluorescently labelled GNA or HV1a/GNA, fluorescence was detected specifically associated with the central nerve chord. Conclusions/Significance: In addition to mediating transport of Hv1a across the gut epithelium in lepidopteran larvae, GNA is also capable of delivering Hv1a to sites of action within the insect central nervous system. We propose that fusion to GNA provides a general mechanism for dramatically enhancing the oral activity of insecticidal peptides and proteins

Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae)

BACKGROUND: The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918. RESULTS: Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL−1 when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2–6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance. CONCLUSION: Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand–channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies