Inducible tolerance to Bacillus. on cell-free immune reactions. thuringiensis (Bt) endotoxins based

Transcription

1 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) 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 2006

2 TABLE OF CONTENTS Statement Acknowledgements iv v Chapter One 1 Overview of the study Chapter Two 12 Literature Review 1. General introduction 2. Literature review 2.1 Introduction 2.2 Bt-endotoxins (Cry-toxins) A brie/history as biopesticides Host range and commercial products Classification o/cry-toxins Structure o/cry-toxins Mode 0/ action 0/ Cry-toxins in insect midgut Cry-toxin receptors in insect midgut Pests resistant to Cry-toxins Mechanisms o/cry-toxins resistance in insects 2.3 Inducible tolerance to Bt Cry-toxins in insect pests Introduction Inducible tolerance mechanisms Insect immunity Parasitoid-derived immune suppressors Mode 0/ action o/cry-toxins in insect midgut Cry-toxin receptors in insect midgut Pests resistant to Cry-toxins Mechanisms o/cry-toxins resistance in insects 3. The aim of the study Reference cited Chapter Three 60 Paper I: Cell-free immune reactions in insects Introduction Materials and methods Low-density gradient centrifugation 2.2 LPS bioassay 2.3 Lectin bioassays

3 3. Result Lipophorin particles contain immune proteins Lipophorin particles in immune-induced insects 3.3 LPS-induced melanisation is associated with lipophorin particles 3.4 Lectin-induced aggregation of lipophorin particles PNA-mediated self-assembly of lipophorin particles 3.6 Self-assembly into cage-like aggregates 4. Discussion References 69 Chapter Four Paper II: Mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides :Hypothesis (Review) Introduction Membrane trafficking an achilles heel? Pore-forming toxins Lipid exchange Configurational specificity 6. References Chapter Five 82 Paper III: Induction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella 1. Introduction Experimental procedures Bt-formulation 2.2 Insects 2.3 Selection 2.4 Melanization assays 2.5 Tolerance bioassay 2.6 Induction bioassay 2.7 Reciprocal crosses 2.8 Relationship between Bt-tolerance and the rate of the melanization 2.9 Statistical analysis Result 3.1 Relationship between Bt-tolerance and immune response 3.2 Efects 3.3 Transmission of Bt tolerance and immune status by a maternal effect 3.4 Genetic disposition for Bt tolerance and immune induction 85

4 4. 5. Discussion References Chapter Six 88 Paper IV: The development of the endoparasitoid Venturia canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella 1. Introduction Materials and Methods Result Discussion References Chapter Seven 93 Paper V: Tolerance to Bacillus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella A rnct Introduction 2. Materials and methods Insect 2.2 Parasitism 2.3 Melanization assays 2.4 Bt-toxin bioassay of parasitised larvae 2.5 Low-density gradient centrifugation of gut content 2.6 Protein separation and Western blots 2.7 Statistical analysis 3. Results 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 Bt-tolerance in tropolone-treated larvae 3.6 Bt-toxin interacts with /ipophorin particles in the gut 3.7 Separation by low-density gradient centrifugation 4. Discussion References List of tables Figure legends

5 Chapter Eight 124 Paper VI: Sequestration of mature Bacillus thuringiensis endotoxin by lipophorin particles Introduction Materials and methods 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 Cry1Ac causes aggregation of/ipophorin particles 3.2 Lipophorin-staining in the gut 3.3 Lipophorin particles form aggregates in the gut 4. Discussion References Figure legends 139 Chapter 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 Insect Physiology) 178 C) Bt-tolerance by an elevated immune response in the flour moth Ephestia kuehniella (Poster presented in the XXII International 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 Cry1Ac in the gut lumen of Ephestia kuehniella larvae (Poster presented in the School Research Day) 199 F) Inducible tolerance to Bacillus thuringiensis (Bt) endotoxins based on cell-free immune reactions (PhD Final Seminar) 201

6 The use of Bacillus thuringiensis (St) 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 St-endotoxins in transgenic crops covering about 80 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 Stendotoxins other than the loss or modification of receptors. We explored a novel mechanism, where tolerance to St-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 St-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 Sttolerance. Molecular analysis revealed a possible mechanism of immune mediated inactivation of St-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 St-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 St-toxin. This leaves coagulation as a likely mechanism for St-tolerance in the gut lumen. To examine whether hemolymph proteins exist in the gut lumen were they could function as pro-coagulants to

7 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 /ipopolysaccharides (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 immuneinduced 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 poreforming 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. immune reactions in insects. Journal of Insect Physiology 52: (2006). Cell-free Schmidt, 0., 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, poreforming toxins and biologically active peptides (Hypothesis). Invertebrate Survival Journal. 2: Rahman, M.M., Roberts, H.L.S., Sarjan, M., Asgari, S. and Schmidt, O. (2004). Induction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella. Proceedings of the National Academy of Sciences of the United States of America 101(9):

8 Rahman, M.M., Roberts, H.L.S. and Schmidt, O. (2004). The development of the endoparasitoid Venturia canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella. Journal of Invertebrate Pathology 87 (2-3): Rahman, M.M., Roberts, H.L.S. and Schmidt, O. Tolerance to Bacillus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella. Applied and Environmental Microbiology (under review). Rahman, M.M. and Schmidt, O. Cell-free sequestration of mature Bacillus thuringiensis endotoxin by lipophorin particles. Applied and Environmental Microbiology (under review).