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Inducible Tolerance to Bacillus Thuringiensis (Bt) Endotoxins Based on Cell-Free Immune Reactions

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Inducible Tolerance to Bacillus Thuringiensis (Bt) Endotoxins Based on Cell-Free Immune Reactions Inducible tolerance to Bacillus thuringiensis (Bt) endotoxins based on cell-free immune reactions by Mohammad Mahbubur Rahman B. Sc (Hons.) & M. Sc in Zoology (Dhaka University, Bangladesh) A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Science at the University of Adelaide Discipline of Plant and Pest School of Agriculture, Food and Wine Waite Campus, Glen Osmond SA 5064, Australia November 2OO6 To my father Late M. Abdur Rahman TneL¡ or CoNTENTS Abstract I Statement iv Acknowledgements V Chapter One 1 Overview of the study Ghapter Two 12 Literature Review 1. General introduction 14 2. Literature review 15 2.1 Introduction 15 2.2 Bt-endotoxins (Cry-toxins) 16 2.2.1 Abriefhistory as biopesticídes 2.2.2 Host range and commercial products 2.2.3 Classification of Cry-toxins 2.2.4 Structure of Cry-toxins 2.2.5 Mode of action of Cry-toxins in insect midgut 2.2.6 Cry-toxin receptors in insect midgut 2.2.7 Pests resistant to Cry-toxins 2.2.8 Mechanisms of Cryloxins resistance in insects 2.3 Inducible tolerance to Bt Cry-toxins in insect pests 33 2.3.1 Introductíon 2.3.2 Inducibletolerancemechanisms 2.3.3 Insect immunity 2.3.4 Parasitoid-derivedimmunesuppressors 2.3.5 Mode of action of Cryloxíns in insect midgut 2.3.6 Cry-toxin receptors ín insect midgut 2.3.7 Pests resistant to Cry-toxins 2.3.8 Mechanisms of Cry-toxins resistance in insects 3. The aim of the study 41 4. Reference cited 43 Ghapter Three 60 Paper l: Cell-free immune reactions in insects Abstract 62 1. lntroduction 62 2. Materials and methods 62 2.1 Low-density gradient centrifugation 2.2 LPS bioassay 2.3 Lectin bioassays 3. Result 63 3.1 Lipophorin particles contain immune proteins 3.2 Lip ophorin particles in immune-induced ins ects 3..1 LPS-induced melønisation is associated with lipophorin particles 3.4 Lectin-induced aggregation of líp ophorin particles 3.5 PNA -m e di at e d s e lf- as s emb ly of lip op h orin p ar t i c I es 3.6 Self-as s embly into cage-like aggregates 4. Discussion 66 5. References 69 Ghapter Four 71 Paper ll: Mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides :Hypothesis (Review) Abstract 73 1. lntroduction 73 2. Membrane trafficking an achilles heel? 74 3. Pore-forming toxins 74 4. Lipid exchange 78 5. Configurational specificity 79 6. References 79 Ghapter Five 82 Paper lll: lnduction and transmission of Bacillus thuringiensls tolerance in the flour moth Ephestia kuehniella Abstract 84 1. lntroduction 84 2. Experimental procedures 84 2.1 Brformulation 2.2 Insects 2.3 Selection 2.4 Melanizationassays 2.5 Tolerance bioassay 2.6 Induction bioassay 2.7 Reciprocal crosses 2.8 Relationship betvveen Bt-tolerance and the rate of the melanization 2.9 Statisticalanalysis 3. Result 85 3.1 Relationship between Bt-tolerance and immune response 3.2 Effects of pretreatmentwith a low concentration of Btformulation 3.3 Transmission of Bt tolerance and immune status by a maternal effect 3.4 Genetic dispositionfor Bt tolerance and immune induction 4. Discussion 86 5. References 87 Ghapter Six 88 Paper lV: The development of the endoparasiloid Venturia canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella Abstract 90 1. lntroduction 90 2. Materials and Methods 91 3. Result 91 4. Discussion 92 5. References 93 Chapter Seven 93 Paper V: Tolerance to Bacillus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella Abstract 96 1. lntroduction 97 2. Materials and methods 99 2.1 Insect 2.2 Parasitism 2.3 Melanizationassays 2.4 Bt-toxin bioassøy of parasitised larvae 2.5 Low-density gradient centrifugation of gut content 2.6 Protein separation and Western blots 2.7 Statisticalanalysis 3. Results 103 3.1 Plasma melanization in parasitised larvae 3.2 Plasma melanization in tropolone-treated larvae 3.3 Tropolone-treatment and parasitism success 3.4 Bt-tolerance in parasitised larvae 3.5 Btlolerance in tropolone-treated larvae 3.6 Bt-toxin interacts with lipophorin particles in the gut 3.7 Separation by low-density gradient centrifugation 4. Discussion 107 5. References 111 6. Llst of tables 116 5. Figure legends 117 Chapter Eight 124 Paper Vl: Sequestration of mature Bacillus thuringiensis endotoxin by lipophorin particles Abstract 127 1. lntroduction 128 2. Materials and methods 130 2.1 Bt-toxin 2.2 Low-density gradient centrifugation 2.3 Western blots 2.4 Insect gut staining 2.5 Bt-toxin aggregation assay 3. Results 132 3.1 CrylAc cquses aggregation of lipophorin particles 3.2 Lipophorin-staining in the gut 3.3 Lipophorin particles form aggregates in the gut 4. Discussion 135 5. References 137 6. Figure legends 139 Ghapter Nine 144 Summary and conclusion Appendix A) Arsenic interactions with lipid particles containing iron (Manuscript under review in Environmental Pollution) 159 B) Factors affecting growth in the koinobiont endoparasitoid Venturia canescens in the flour moth Ephestia kuehniella (Manuscript under review in Journal of lnsect Physiology) 178 C) Bt{olerance by an elevated immune response in the flour moth Ephestia kuehniella (Poster presented in the XXll lnternational Congress of Entomology) 195 D) Cell-free immune reactions in insect (Poster presented in the School Research Day) 197 E) Cell-free sequestration of mature Bt-toxin CrylAc in the gut lumen of Ephestia kuehniella larvae (Poster presented in the School Research Day) 199 F) lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins based on cell-free immune reactions (PhD Final Seminar) 201 Abstract The use of Bacillus thuringiensrs (Bt) endotoxins to control insect vectors of human diseases and agricultural pests is threatened by the possible evolution of resistance in major pest species. Despite the use of Bt-endotoxins in transgenic crops covering about B0 million hectares, the precise details of how endotoxins bind to gut cells to kill insects are poorly understood. This limitation impedes our understanding of potential mechanisms of insect resistance to Bt- endotoxins other than the loss or modification of receptors. We explored a novel mechanism, where tolerance to Bt-endotoxins is correlated with an elevated immune status involving cell-free immune reactions in the gut lumen. The thesis project is based on investigations of a laboratory culture of the flour moth Ephestia kuehniella, which showed induction of hemolymph melanization a sign of immune induction, after feeding sub-lethal concentrations of a Bt-formulation. Since the elevated immune status was transmitted to subsequent generations by a maternal effect, an increase of toxin in the food by increments was possible every generation. Investigations of strains exposed to various toxin levels revealed a correlation between systemic immune induction and Bt- tolerance. Molecular analysis revealed a possible mechanism of immune mediated inactivation of Bt-endotoxins in the gut lumen. To gain more specific information about the effector pathways involved in the protection against the toxin, we studied the effects of Bt-toxin formulations in susceptible (non-induced) and tolerant (immune-induced) larvae after natural (parasitism-mediated) and chemical (tropolone-mediated) suppression of defence reactions. Although melanization in hemolymph was significantly reduced, there was no significant effect on susceptibility to the toxin in parasitised or tropolone-treated larvae. This suggests that melanization of hemolymph is correlated with an elevated immune status but not responsible for the observed tolerance to Bt-toxin. This leaves coagulation as a likely mechanism for Bt-tolerance in the gut lumen. To examine whether hemolymph proteins exist in the gut lumen were they could function as pro-coagulants to inactivate the toxin; we compared gut and plasma proteins of immune-induced larvae with those of non-induced larvae. This analysis revealed that the lipid carrier lipophorin represents a major component in the gut lumen and interacts with mature Bt-toxin like an oligomeric lectin that may inactivate the toxin in a cell free coagulation reaction in the gut lumen before it can reach the brush border membrane. Further analysis showed that lipophorin particles are the regulatory and effector components in innate immune defence reactions, which are involved in the recognition and inactivation of lipopolysaccharides (LPS) and bacteria even in the absence of hemocytes. Examination of proteins from lipophorin particles separated by low-density gradient centrifugation have shown that in immune- induced insects sub-populations of lipophorin particles are associated with pattern recognition proteins, phenoloxidase and regulatory proteins that activate prophenoloxidase. Moreover, interactions with lectins resulted in the assembly of lipophorin particles into cage-like coagulation products, effectively protecting the surrounding tissues and cells from the potentially damaging effects of pathogens and phenoloxidase products. This cell'free immune reaction mediated by lipophorin particles may potentially involve in detoxification of pore- forming toxins (Bt-endotoxins) in the gut lumen. This thesis is based on the following publications and manuscripts: Rahman, M.M., Ma, G., Roberts, H.L.S. and Schmidt, O. (2006). Cell-free immune reactions in insects. Journal of Insect Physiology 52:754-762 Schmidt, O., Rahman, M.M., Ma, G., Theopold, U., Sun, Y., Sarjan, M., Fabbri, M. and Roberts, H.L.S. (2005). Mode of action of antimicrobial proteins, pore- forming toxins and biologically active peptides (Hypothesis). Invertebrate Survival Journal. 2: 82-90. Rahman, M.M., Roberts, H.L.S., Sarjan, M., Asgari, S. and Schmidt, O. (2004). lnduction and transmission of Bacillus thuringiensrs tolerance in the flour moth Ephestia kuehniella. Proceedings of the National Academy of Sciences of the United Sfafes of America 101(9): 2696-2699. il Rahman, M.M., Roberts, H.L.S. and Schmidt, O. (2004).
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