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). The development of the endoparasitoid Venturia canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella. Journal of lnvertebrate Pathology 87 (2'31: 129-131
Rahman, M.M., Roberts, H.L.S. and Schmidt, O. Tolerance to Bacillus thuringiensrs 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 thuringiensrs endotoxin by lipophorin particles. Applied and Environmental
M i crobi ology ( u nd er review).
ilt This thesis contains.no material, whicJr has beon accepted for the award of anlt other degree or díploma ín any universìty or tertÍary institution. To the bes,t of the my knowledge and beliel this thesis oontains no materials previously publîshed
I in text. I or written by another person, exeept, where due reference is made the I
I
I I I give consent to this cCIpy of my theeis, when deposited in the University of . Adelaide library, being available for photocopying and loan.
Mohammad Mahbubur Rahman November 2O06
IV Acknowledgement
First I like to take the opportunity to thank my principal supervisor Professor Otto Schmidt and co-supervisor Assoc. Professor Michael Keller for their willingness to accept me as PhD research fellow and their countless support through the years. I sincerely believe that their novel and creative ideas and discussions, which in turn stimulated my vision on my project in more advanced level, are of crucial importance for the successful completion of my PhD project within two-year time frame. Professor Schmidt, in particular brought me to the world of molecular biology over medical entomology as a visiting research student, which certainly enriched my scientific background and broadened my vision of such a fascinating science. I am very grateful as one of his students having the opportunity to share his compassion and enthusiasm, professional as well as mental courage whenever I encountered difficulties, and being able to use the freedom to pursue what I thought was important during the course of my study.
I do acknowledge Dr Harry Roberts for his advice on experimental design, constructive criticism on results, help in analysis and interpretation of data, and manuscript preparation. Thanks to Professor Ravi Naidu and his PhD student Ms. Farzana (CRC Care, University of South Australia) providing me blood samples and instrumental support for ICP-MS analysis.
Then I would like to thanks my fellow colleagues Drs. Muhammad Sarjan, Mehdi Sohani, Sassan Asgari, Richard Glatz, Dongmei Li, Guangmei Zhang, Gang Ma, Ms. Nicki Featherstone, Mr. Sohbat Bahraminejad, Mr. Reza Hosseini, Ms. Judy Bellati and work experience students Caspar Jonker, NatashaMclnnes, Jana Bradley and Kathrin Janssen, who at various stages assisted with my research. I would also like to thank all those whom I am supposed to thank but can't mention them all individually by name. Thanks to Adelaide University for financial support without which successful completion of my PhD study would not be possible. At last but not least, I like to acknowledge my family members for their encouragement, support, understanding, patience, and love. lt was impossible for me to start my PhD research project without the mental and emotional support from my wife Ms. Farzana, given that she was also quite busy as a full- time PhD research fellow at the same time. Farzana, thanks to you for your help either assisting directly to my work leaving aside of your own or allowing me to work long hours even overnight whenever I needed. Master Fardeen Rahman is still too little to understand why his dad has been leaving him in the childcare for a long time. I am grateful to you Fardeen-my lovely son for your cooperation, love and wonderful smile. Last not least my late father Mr. M. Abdur Rahman, we do miss you dad now and then. I am doubt without the inspiration from you all; life would be very different and much less have been achieved.
VI IndødÞIe fiole¡snce. fo Baaf/l¡¡s f/r¿¡r?frgrlensiË fiBfJ ondoloxins M M.Rahman based on cell-free immune reaci¡iana
Overuiew of the Thesis
1: Overview of the Thesis
Endotoxins from spore-forming soil bacterium Bacillus thuringiensis (Bt), are the most valuable biopesticides used worldwide in commercial agriculture, forest management, and control of vector insects (Ferre and Van Rie, 2002). But the development of Bt-resistance among target populations is a threat to the success story of Bt-toxins, and its long-term use in the face of enormous selective pressure generated by widespread use of Bt Cry toxins in crops and organic farming (Ferre and Van Rie, 2002; Griffitts et al., 2005). Although only few insect species have developed resistance to Bt-endotoxins in the field (Ferre and Van Rie, 2002; Morin et al., 2003) despite the use of Bt-Cry proteins in transgenic crops covering about 80 million hectors (Bates et al., 2005), many species have the potential to become resistant, and genetic resistance has been selected in several species in the laboratory (Ferre and Van Rie, 2002; McGaughey, 1985). lt is important, however, to keep in mind thatconditions in the laboratory are very different from selection pressures that occur in the field. Moreover, laboratory colonies might have a considerably lower level of genetic diversity than field populations (Schnepf et al., 1998).
The ability to detect resistance in the field, a precondition for developing and implementing resistance management strategies, relies entirely on molecular and genetic knowledge of the genes and pathways that give rise to resistance (Griffitts et al., 2005). lt is therefore surprising that the precise details of how toxins bind to insect gut cells to kill insects are still poorly understood (Chilcutt and Tabashnik, 2004; Ferre and Van Rie, 2002; Gould, 1998). While known cases of receptor inactivation generate high levels of resistance, lethality is often restricted to neonates or early larval stages (Gilliland et al., 2002). One explanation is that older larvae stop feeding after encountering contaminated food and with enough fat reserves available may survive the bioassay. However, some insects are known to resume feeding after several days overcoming othenruise lethal doses of the toxin. This could suggest that other
2 modes of resistance exist, some based on inducible mechanisms (Griffitts and Aroian, 2005).
The project has uncovered novel tolerance mechanisms against low to medium levels of Bt-formulation in the flour moth Ephestia kuehniella. The observed tolerance is dependent on the induction of immune proteins, which occurs with the ingestion of sub-lethal doses of Bt-formulations. To understand the tolerance mechanisms we therefore studied innate immunity and its reactions in the gut lumen. ln innate immunity how microbes and other potentially damaging objects are recognised and inactivated in the absence of cells are not known (Hall et al., 1999, Kanost et al., 2004; Karlsson et al., 2004). lt has been suggested that proteolytic coagulation cascades and antimicrobial peptides (Boman and Hultmark, 1987) are known to inactivate potentially damaging organisms and toxins in invertebrates (Hoffmann et al., 1999). Although lipophorin and phenoloxidase are involved in coagulation (Li et al., 2002) and that the addition of LPS to purified lipophorin particles causes aggregation and inactivation of the toxin (Ma et al., 2006), it is unclear how is the binding of LPS to pattern recognition proteins translated into the inactivation of pathogens and toxins? lndirect evidence suggests that invertebrates with an open circulatory system sequester damaging microorganisms and toxins by a combination of coagulation (Nagai and Kawabata, 2000) and melanization reactions (Kanost et al., 2004), involving adhesive (Lee et al., 1998) and covalent cross-linking of plasma components (Jiang et al., 2003), melanin synthesis and reactive oxygen production (Nappi and Ottaviani, 2000). Since the mechanisms underlying these processes are not known, we asked whether lipophorin particles are the regulatory and effector components for cell-free immune reactions. lt is also known that pore-forming toxins have lectin-like properties, so we asked whether the mature Bt-toxin can interact with lipophorin particles like an oligomeric lectin and become inactivated in a cell-free coagulation reaction in the gut lumen
3 The main objective of the research undertaken during my candidature was to provide answer to these questions. To achieve these objectives, a number of specific processes within these broad areas were investigated.
Paper l. The concept of cell-free defence reactions was introduced after it was discovered in Ephestia kuehniella, that lipophorin particles were found to be associated with immune proteins, such as pattern recognition proteins, prophenoloxidase and its activating proteases. The study demonstrates that the recognition and inactivation of lipopolysaccharides (LPS) and bacteria is mediated by lipophorin particles, which are the lipid carrier in insects. ln immune-induced insects sub-populations of lipophorin particles are associated with pattern recognition proteins 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 (Rahman et al., 2006).
Paper ll. A hypothetical mode of action of antimicrobial proteins, pore- forming toxins and biologically active peptides was proposed with special emphasis on Bt-toxins. Antimicrobial proteins and pore-forming toxins are important effectors in innate immune defence reactions with their unique structural properties mediating mainly lysis activities in cellular membrane (Boman, 2000). But their mode of action, comprising the insertion of channels into cholesterol-containing membranes is not known. The study demonstrates the mechanical implications of pore-formation by extracellular protein assemblies that drive cellular uptake reactions by leverage-mediated (LM) processes, where oligomeric adhesion molecules bent membranes-receptors around 'hinge'-like lipophorin particles (Schmidt et al., 2005). Since many pore- forming toxins, such as Bt-toxins are lectins, the insertion into the membrane may be mediated by an LM-uptake reaction (Schmidt and Theopold, 2004). ln the LM-scenario, the ring-shaped pore complex is formed before or during the assembly of receptors. This is in contrast to the current assumption that Bt- toxins are inserted into the membrane as monomers by a receptor-mediated reaction and only assembled into pore-forming oligomeric complexes once
4 inside the membrane bilayer (de Maagd et al., 2001). However, it has been shown that some mature Bt-toxins form tetrameric complexes when processed in vitro (ltla et al., 2005), which would enable multiple interactions with receptors and lipoproteins before membrane insertion. Since many pore- forming toxins are lectins, which recognise mucin-like glycoprotein-receptors (Armstrong et al., 1996) and glycolipids (Griffitts et al., 2005), such ring-shaped adhesion complexes could potentially be internalised by an LM-mechanism (Schmidt and Theopold, 2004), where the structural features of the complex predict a disruption of the lipid bilayer during uptake reactions.
Paper ttl. The process of immune induction by feeding of a sublethal dose of Bt- formulation to lepidopteran larvae generates an elevated immune status in the hemolymph, probably by localised cellular damage to the gut lining, exposing hemolymph to elicitors from the gut lumen. The elevated immune status, which is correlated with the degree of plasma melanization, provides larvae with a limited but nevertheless significant protection against subsequent application of lethal doses of the toxin (Rahman et a1.,2004a). Moreover, the elevated immune status and with it the tolerance to Bt-formulations, is transmitted to subsequent generations by a maternal effect. Given that the level of melanization is correlated with immune-induction and the elevated immune status with Bt-tolerance, the question is whether melanization is responsible for the observed Bt-tolerance in immune-induced larvae? Several observations indicate that this is not the case:
Paper IV. lmmune suppression and a series of control investigations, show that an elevated immune status does not cross-protect against parasitiods that oviposit inside the hemocoel (Rahman et al., 2004b), while it does protect against some gut-derived pathogens (Reeson et al., 1998). This raises two questions: Firstly, how does immune suppression by parasitoids affect melanization levels and Bt-toxicity?
Hymenopteran parasitoids that lay their eggs inside the hemocoel of another insect, suppress the host's immune system thereby precluding the immune- mediated inactivation of the egg and emerging larvae. lt is also possible that
5 the growing larvae, which have to digest and endocytose up hemolymph components inside the gut lumen, would be negatively affected by the melanization and coagulation of immune-active host plasma. The suppression of the host immune system, which occurs by maternal protein secretions of the female parasitoid, involving serpins (Beck et al., 2000; Nappi et al., 2005) and other immune suppressors (Asgari et a1.,2003; Lavine and Beckage, 1995; Shelby et al., 2000) is apparently strong enough to reduce the immune status to a level, where it allows the development of the parasitoid inside the host larvae. The question is, whether parasitoid survival is dependent on low levels of melanization in the host. Since immune suppression by parasitoids can increase the virulence of pathogens (Washburn et al., 2000), we also asked the question whether Bt-toxicity is increased in parasitised larvae.
Paper V. Tolerance to Bt-toxin in the gut is not affected by parasitism- mediated immune suppression in the hemolymph (Rahman et al., under review). This raises the question whether melanization, which is a measure of immune induction, constitutes the cause of Bt{olerance or represents an independent pathway that is irrelevant to Bt-toxicity and Bt-tolerance. Results from an in vivo suppression of gut melanization involving the specific inhibition of phenoloxidase by a metal ion-chelating agent tropolone (Morita et al., 2003) suggests that reduction in melanization has no effect on Bt-toxicity (Rahman et al., under review), which implies that plasma melanization is correlated with the immune status but is not responsible for the observed tolerance to the Bt-toxin in the gut lumen. We did not observe any signs of melanization in the gut tissue or the gut lumen of immune-induced E. kuehniella lawae, which is in contrast to the mid-gut of Bt-tolerant Helicoverpa armigera laryae, where the peritrophic membrane was darkened by melanization reactions (Ma et al., 2005). Although melanization is correlated with immune-induction, the reduction of melanization has no effect on Bt-toxicity. This leaves coagulation as a likely mechanism for Bt-tolerance in the gut lumen.
Paper Vl. The lipid carrier lipophorin, which is a pro-coagulant in hemolymph, is present in the gut lumen and interacts with Bt-toxins by an aggregation
6 reaction, sequestering the toxins into coagulation products (Rahman and Schmidt, under review). lsolation of lipophorin particles by low-density gradient centrifugation revealed that these particles possess the ability to interact with lipopolysaccharide (LPS), forming aggregates (Rahman et al., 2006). The aggregation appears to require at least two steps, one where the LPS molecule is incorporated into the lipid moiety of the lipophorin particle and a subsequent activation of putative lectin-like adhesion molecules that cross-link sugar determinants across lipid particles. While the first step occurs in the absence of calcium, the second step requires calcium but can be simulated by the addition of oligomeric adhesion molecules, such as lectins binding to galactosamine residues. For example, when Gal-specific peanut agglutinin (PNA) was added to isolated lipophorin particles in the absence of calcium, the outcome was aggregation of lipid particles into globular structures with F|TC-conjugated PNA trapped inside a lipid layer effectively shielding the lectin from further interactions. Since pore-forming toxins have lectin-like properties, we asked whether the mature toxin can interact with lipophorin particles and become inactivated by aggregation reactions. We have shown that purified lipid particles from hemolymph plasma and the gut lumen are able to interact with mature Cry1Ac (Bt-Cry toxin) like oligomeric lectins causing adhesive aggregation (Rahman and Schmidt, under review).
Conclusion; The inducible tolerance mechanisms are quite different from other Bt-resistance mechanisms (Gonzalez-Cabrera et al., 2001), where the observed reductions in Bt-toxicity are exclusively explained in terms of alterations to receptor properties in the gut epithelium (Darboux et al., 2002; Gahan et al., 2001). The observed immune mediated inactivation of Bt Cry- toxins in the gut lumen of Bt-tolerant larvae may have profound implications for Bt-resistant management in the field. Although it may be effective only against low to medium levels of toxin and of no immediate threat to most transgenic crops expressing the Bt Cry-proteins, it may pose long-term threat through genotype selection, given that the toxin levels in transgenic crops may not be sufficient to kill tolerant insect populations in conjunction with semi-dominant receptor mutations. The results in E. kuehniella are consistent with the similar
7 observation of a field-collected strain of cotton bollworm Helicoverpa armigera that has developed genetic resistance in the laboratory (Akhurst et al., 2003), comprising a complex mixture of putative receptor inactivation and elevated immune status (Ma et al., 2005) and P. xylostella (M. Sarjan, unpublished data), suggesting that the initial development of low-level Bt-tolerance through immune-related processes may be a common phenomenon.
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I Lee, S., Cho, M., Hyun, J., Lee, K., Hommâ, K., Natori, S., Kawabata, S', lwanaga, S., and Lee, B. (1998): Molecular cloning of cDNA for pro- phenol-oxidase-activating factor l, a serine protease is induced by lipopolysaccharide or 1,3-beta-glucan in coleopteran insect, Holotrichia diomphalia larvae. European Journal Biochemistry 257 , 615-621. Li, D., Scherfer, C., Korayem, A. M.,Zhao,2., Schmidt, O., and Theopold, U. (2002): lnsect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. lnsect Biochemistry and Molecular Biology 32, 919-928. Ma, G., D. Hay, et al. (2006). Recognition and inactivation of LPS by lipophorin particles. Developmental & Comparative lmmunology 30, 619-626. Ma, G., Roberts, H., Sarjan, M., Featherstone, N., Lahnstein, J', Akhurst, R', and Schmidt, O. (2005): ls the mature endotoxin Cry1Ac from Bacillus thuringiensrs inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae? lnsect Biochemistry and Molecular Biology 35,729-739. McGaughey, W. H. (1985): lnsect resistance to the biological insecticide Bacillus thuringiensis. Science 229, 1 93-1 95. Morin, S., Biggs, R. W., Sisterson, M. S., Shriver, L., Ellers-Kirk, C', Higginson, D., Holley, D., Gahan, L. J., Heckel, D. G., Carriere, Y., Dennehy, T. J', Brown, J. K., and Tabashnik, B. E. (2003): Three cadherin alleles associated with resistance to Bacillus thuringiensrs in pink bollworm. Proceeding of the Nationat Academy of Sciences of the United Sfafes of America 1 00, 5004-5009. Morita, Y., Matsumura, E., Okabe, T., Shibata, M., Sugiura, M., Ohe, T., Tsujibo, H., lshida, N., and lnamori, Y. (2003): Biological activity of tropolone. Biological and Pharmaceutical Bulletin 26, 1 487-1490. Nagai, T., and Kawabata, S.-i. (2000): A link between blood coagulation and prophenoloxidase activation in arthropod host defense Journal of Biological Chemi stry 27 5, 29264-29267 . Nappi, A. J., Frey, F., and Carton, Y. (2005): Drosophla serpin 27Ais a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response. Journal of Insect Physiology 51,197-205. Nappi, A. J., and Ottaviani, E. (2000): Cytotoxicity and cytotoxic molecules in invertebrates. BioEs says 22, 469-480. Rahman, M. M., G. Ma, et al. (2006). Cell-free immune reactions in insects. Journal of Insect Physiology 52:754-762. Rahman, M. M., Robefts, H. L. S., Sarjan, M., Asgari, S., and Schmidt, O' (2OO4a): lnduction and transmission of Bacillus thuringiensrs tolerance in the flour moth Ephestia kuehniella. Proceeding of the National Academy of Sciences of the United Sfafes of America 101, 2696-2699. Rahman, M. M., Roberts, H. L. S., and Schmidt, O. (2004b): The development of the endoparasitoid Venf uria canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella. Journal of lnvertebrate Pathology 87, 129-131.
10 Rahman, M. M., Roberts, H. L. S., and Schm¡dt, O. (under review): Tolerance to Bacittus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella. Applied and Environmental Microbiology. Rahman, M. M., and Schmidt, O. (under review): Cell-free sequestration of mature Bacillus thuringiensis endotoxin by lipophorin particles. Applied and Environmental Microbiology. Reeson, A. F., Wilson, K., Gunn, A., Hails, R. S., and Goulson, D. (1998): Baculovirus resistance in the noctuid Spodoptera exempta is phenotypically plastic and responds to population density. Proceedings of the Royal Society of London Series B, Biological Sciences 265, 1787- 1791. Schmidt, O., Rahman, M. M., Ma, M., Theopold, U., Sun, Y., Sarjan, M., Fabbri, M., and Roberts, H. (2005): Mode of action of antimicrobial proteins, pore- forming toxins and biologically active peptides (Hypothesis). lnvertebrate Survival Journal 2, 82-90. Schmidt, O., and Theopold, U. (2004): An extracellular driving force of endocytosis and cell-shape changes (Hypothesis). BioEssays 26, 1344- 1 350. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R., and Dean, D. H.(1998): Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews 62,775-806. Shelby, K. S., Adeyeye, O. A., OkoþKotber, B. M., and Webb, B. A. (2000): Parasitism-linked block of host plasma melanization. Journal of I nvertebrate Pathology 7 5, 21 8-225. Washburn, J. O., Haas-stapleton, E. J., Tan, F. F., Beckage, N. E', and Volkman, L. E. (2000): Co-infection of Manduca sexfa larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa californica M Nucleopolyhedrovirus. Journal of lnsect Physiology 46, 179-190.
11 lnducible lalerianee fs Baei/¡is th urtngitt ns¡s (B1) e n d otoxin s M M Rakman based on cell-free immune reactions
I ,! L
Literature Review
il
12 1, General introduction 14
2. Literature review 15 2.1 lntroduction 15 2.2 Bt-endotoxins (Cry-toxins) 16 2.2.1 A brìef hìstary øs biopestícides 16 2.2.2 Hast range and cornttereial products 18 2.2.3 Classífieaiíon of Cryloxins 18 2.2.4 Strucfi'tre of Cry'toxins 19 2.2,5 lúod,e of ac,tìan of Cry-toxins ínínsectrnídgut 20 2.2,6 C4t toxìn receptors in ínseet midgltt 24 2.2,7 Pesß resìstant to Cry-taxiw 26 2.2.8 Meehanismt afBt Cry+oxÍns resßtsnce ín í:nsects 27 2.3 lnducible tolerance to Bt'toxin in insect pests 33 2.3,1 Introduetian 33 2.3.2 Inducibletolerancemechønisms 34 2.3.3 Insect ìmmunity 36 2.3.4 Parasìtoid-derived imnune ruppressors 38 3. The aims of the study 41
4. Reference 43
13 1. General introduction
Soil bacterium Bacillus thuringiensis (Bt) produces insecticidal crystal toxins that are used widely as an alternative or supplement to synthetic chemical pesticide application in commercial agriculture, forest management, and control of vector insects (Ferre and Van Rie, 2002), since they are specific to their target pests, are safe to other animals and plants, and are biodegradable (Schnepf et al., 1998). Bt-toxins are also a key source of genes for transgenic expression to provide pest resistance in plants and thereby reduce reliance on conventional pesticides, providing economic, health and environmental benefits (Shelton et al., 2002). Therefore, the adoption of Bt transgenic crops by the farmers has exceeded all expectations, covering more than 80 million hectares since 1996 (Bates et al., 2005). ln some respects, a Bt-transgenic crop plant is an important technological and practical development, because it ensures that only those insects that attack the crop will be exposed to Bt toxins - there is no risk to other types of insects. lt also ensures that the range of uses for Bt is extended to insects that feed on roots or tunnel into plant tissues - for example, the European corn borer - because such insects cannot be controlled by Bt suspensions sprayed onto plant surfaces.
The success story of Bt-toxins has been overshadowed by at least 12 documented cases of Bt resistance in laboratory selected populations of various insect species (Ferre and Van Rie, 2002) and nematodes (Griffitts et al., 2005). Moreover, the emergence of resistance in field populations of the diamondback moth Plutella xylostella (Baxter et al., 2005; Ferre et al., 1991;Sayyed et al., 2004; Tabashnik, 1994) and cabbage loopers Trichoplusia ni (Janmaat and Myers, 2003) have highlighted the inherent danger of the evolution of resistance against these environmentally friendly insecticides. Although the ability to detect resistance in the field, which is important for developing and implementing strategies to delay and monitor resistance before it becomes a widespread problem, entirely relies on molecular and genetic knowledge of the genes and pathways that give rise to
14 resistance, it is surprising that the precise details of how toxins bind to insect gut cells to kill insects are poorly understood (Ferre and Van Rie, 2002; Griffitts and Aroian,2005).
However, it is known that when susceptible insects ingest Bt-toxins with their diet, the toxins are proteolytically activated by insect midgut proteases and bind to membrane gut receptors, leading to pore formation, cell lysis, and eventually insect death (de Maagd et al., 2001). ln principle, the mechanism of insect resistance to Bt-toxins could be localised to each of the various steps known to constitute the mode of action (Ferre and Van Rie, 2002). The crucial issue is to identify the major resistance genes to monitor the extent of variation for the genetic basis of resistance to Bt in the field. This limitation impedes our understanding of potential mechanisms of insect resistance to Bt endotoxins other than the loss or modification of receptors (Darboux et al., 2002; Gahan et al., 2001; Morin et al., 2003), altered proteolysis of protoxin and/or toxin (Forcada et al., 1996; Oppert et al., 1994) and repair and/or replacement of damaged cells (Loeb et al., 2001; Martinez-Ramirez et al., 1999).
The thesis project describes an investigation of a laboratory culture of the flour moth Ephestia kuehniella for a possible correlation between systemic immune induction and Bt-tolerance. The observed tolerance is dependent on the induction of immune proteins, which occurs with the ingestion of sub-lethal doses of Bt- formulation and is based on cell-free immune reactions in the gut lumen removing the mature toxin before it can reach the brush border membrane.
2. Literature rev¡ew 2.L Introduction
ln this review, a brief description of B. thuringiensrs endotoxins is provided and their mode of action will be discussed. Secondly, Bt-resistance mechanisms in
15 different insects will be summarized with special emphasis in inducible tolerance mechanism based on cell-free immune reactions.
2.2 Bt-endotox¡ns (Cry-toxins)
B. thuringiensls, commonly known as Bt, is a naturally occurring, spore-forming soil bacterium which produces large quantities of insecticidal crystal proteins or õ- endotoxins or Cry-toxins, during the stationary phase as part of its natural life cycle (Agaisse and Lereclus, 1995). B. thuringiensls produces a large quantities of Cry- toxins, each having defined host specificities and have gained large popularity for their ability to control a wide range of insect pests and nematodes in a natural, environmentally friendly manner (Ferre and Van Rie, 2002; Gould, 1998). Changes in a single amino acid in Crytoxins have been shown to have dramatic effects on the range and specificity of activity against insect species; (Chandra et al., 1999; Rajamohan et al., 1996). With modern biotechnology, the genes coding for Bt Cry- toxins are readily isolated and introduced into plants and become a stably inherited traits of new cultivars (Babu et al., 2003; Deacon, 2003).
Fig 1. 8. thuringiensrs viewed bY phase contrast microscopy. The vegetative cells contain endospores (phase bright) and crystals of an insecticidal toxin (õ-endotoxin), Most cells have lysed and released the spores and toxin crystals (Deacon, 2003)
2.2.1 A brief history as biopesficldes
B. thuringiensrs was first identified by lshawata in grain storage facilities in Japan in
1901 (lshw ala, 1901). Then, in 191 1 , Berliner rediscovered it as a pathogen in flour moths in the province of Thuringia, Germany (Berliner, 1911). Bt Cry-toxins was first used as a commercial insecticide in France in 1938 (Worthington, 1991), and
16 then in the USA in the 1950s (Ghassem¡, 1981). However, more effective Bt formulations were introduced in the 1960s with the discovery of various highly pathogenic strains, each of which produces its own unique insecticidal crystal protein, with particular activity against different types of insect (McGaughey and Johnson, 1994). Bt Cry-toxin gene was transferred and expressed in E. coli in 1981 (Schnepf and Whiteley, 1981) and in vegetable plants in 1987 (Vaeck et al., 1987). First Bt corn was registered by US EPA in 1995 and commercially introduced in USA in 1996 (Shelton et a1.,2002).
Fig.2. Global area planted to Bf crops (Bates et al., 2005)
Three Bt crops are now commercially available: corn, cotton, and potato, and several other are in various stages of development including potato, rice, maize, wheat, canola, soybean, tobacco, tomato, apple, peanuts and broccoli (Babu et al., 2003; Shelton et al., 2002). Bt crops are currently grown in 17 countries and the adoption rates are rising rapidly with the global area of Bt crops growned increasing 24Yobelween2002 and 2003 (Bates et al., 2005). As of now, cotton is the most popular of the Bt crops and has led to a 3o/o-27o/o increase in cotton yield in countries where it is grown since it was commercialized in Australia and USA in 1996 (Jarman, 2006). The adoption of Bt crops is predicted to continue its steady
17 growth, influenced by an increasing confidence in the benefits of Bt crops and the availability of new Bt varieties that confer protection against additional crop pests (Bates et al., 2005).
2.2.2 Host range and commercial products
For many years, Bt Cry{oxins was available only for the control of Lepidoptera, using a highly potent strain (8. thuringiensis var kurstaki). Toxin from this strain still forms the basis of many Bt formulations (Meadows et al., 1992). Further screening of a large number of toxins from other Bt strains revealed some that are active against larvae of Coleoptera or Diptera. Most of these toxins have the same basic toxin structure, but differ in insect host range, perhaps because of different binding affinities to the toxin receptors in the insect gut (Deacon, 2003). Many commercial products from the toxin of B. thuringiensis var kurstaki for the control of Lepidoptera are marketed under various trade names such as Biobit@, Dipel@, and Javelin@.
The toxins produced by strains of B. thuringiensis var israelensrs are highly active against simuliid blackfly vectors of some tropical diseases, and also against fungus gnat larvae. This strain is also a key source of toxin for larval control of vector mosquito (Abdullah et al., 2003). The commercial products of this strain are Skeetal@, Vectobac@ and Mosquito Attack@. Toxins from B. thuringiensrs var san diego or B. thuringiensis var tenebrionis are marketed for the control of some Coleoptera - especially for the control of the important Colorado potato beetle.
2.2.3 Classification of Cry-toxins
Toxins from B. thuringiensls are classified according to their size, crystal shape and specificity: the major group is insect-specific Cry toxins and the other is cytolytic Cyt toxins (Schnepf et al., 1998). The Cry toxins are encoded by different genes and have distinct three-domain structure with amphipathic c¡-helix (Li et al., 1991), where Cyt-toxins have only one p-domain that can enhance the effectiveness of Cry-toxins for insect control (Li et al., 1996). As of now, about 311
18 different genes that encode the Cry-toxins have been identified and sequenced (Crickmore et a|.,2006) and this enables the toxins to be assigned to more than 15 groups on the basis of sequence similarities (Deacon, 2003).
Table 1. Bt-endotoxins and their host specificity (adopted from Deacon, 2003)
Protein size Bt cry-toxins lnsect activity
Cry1 [several subgroups: a), A(b), A(c), B, c, D, E, F, Gl 1 30-1 38 Lepidoptera Cry2 [subgroups A, B, C] 69-71 Lepidoptera and Diptera Cry3 [subgroups A, B, C] 73-74 Coleoptera Cry4 [subgroups A, B, C, D] 73-134 Diptera Cry 5-9 35-129 Nematodes and others
2.2.4 Structure of Cry-toxins
The three dimensional structures of trypsin activated Lepidoptera specific Cry1Aa (Gnochutski et al., 1995), Coleoptera specific Cry3A and Cyt2A (Li et al., 1991) have been proposed. Cry3A and Cry1Aa possess three domains and 36% amino acid sequence identity (Li et al., 1991). But C\i2A consists of a single domain and 2Oo/o amino acid sequence identity with Cry3A and CrylAa (Hofte and Whiteley, 1989; Li et al., 1996).
¡ I I \ ^t { ¡ r'- t' I , I f f
,J I
t, l\
ìrflI ;
+
Fig. 3. Three dimensional structures of trypsin activated Bt Cry-toxin (Li et al. 1991)
19 Domain I in Cry3A and Cry1Aa consists of seven antiparallel cx-helices in which the
Sth hel¡x is encircled by the remaining helices and some or all of the helices are thought to have pore forming activity (Grochulski et al., 1995); Domain ll consists of three antiparallel B-sheets, similar to the antigen-binding regions of immunoglobulins and believed to bind to receptors in the gut lumen (Lee et al.,
2000b); Domain lll consists of two twisted, antiparallel B-sheets forming a B- sandwich, which is thought to responsible for the toxicity (de Maagd et al., 1996) and to protect the exposed C{erminal region of the active toxin, preventing further cleavage by gut proteases (Aronson and Shai, 2001). The ability to enhance toxicity by domain lll is due for its lectin-like fold in B-sheets, which can facilitate the toxin binding to receptors with specific glycodeterminants (Akao et al., 1999; Burton et al., 1999) and trigger the conformational change in Doman lfor insertion into the membrane (Li et a1.,2001).
2.2.5 Mode of action of Cry-toxins rn rnsecf midgut
The Bt-crystals are p-sheet aggregates of a ca. 130 KDa protein that is actually a protoxin and must be activated by certain midgut proteases to become a 60 to 65 KDa active toxin (Hofte and Whiteley, 1989). The crystal protein is highly insoluble in normal and acid pH conditions, so it is entirely safe to humans, higher animals and most insects (Schnepf et al., 1998). However, it is solubilised under reducing conditions of high pH (above about pH 9.5) - the conditions commonly found in the mid-gut of lepidopteran larvae (Knowles and Dow, 1993).
ln vitro studies of the mode of action of Bt Cry-toxins by using a variety of systems including insect cell lines, whole insect gut tissues, and brush border membrane vesicle preparations of insect midgut revealed that elucidation of the toxicity mechanism is made complicated by the number of subspecies of B. thuringiensis studied by different investigators, the production of more than one toxin by the same bacterium, a wide number of susceptible hosts, the interaction between crystals and spores in toxicity in some laryae, and multiple receptors for the same
20 toxin in individual host. The molecular mechanisms of Cry-toxins have been discussed intensively, with several hypothesis based cascades of multi-step activitities (de Maagd et al., 2001; Knowles and Dow, 1993).
.'...... 4þl -.------+
Solubilization process lrol Binding with ."."pür,
Pore fcirmatî6ñ-
rrîil æmffiæ
Fig. 4. Mode of action of Cry toxins (adapted from de Maagd et al, 2001) (a) After ingestion by the insect, crystals dissolve in the gut juice. (b) Gut proteases subsequently clip off the C-terminal region of Cry proteins (purple) as well as a small N- terminal fragment (yellow). (c) The resulting 'activated' toxin binds to receptors on the epithelial cell membrane, a process in which both domain ll and domain lll are involved. (d) Structural rearrangement of domain I might follow allowing a two-helix hairpin to insert into the membrane. (e) lnserted toxins form pores probably as oligomers, but the architecture of the pore is still unknown.
A basic model for the mechanism of action of Bt Cry{oxins has been widely accepted for many years and involves solubilization of the crystal in the insect midgut, proteolytic processing of the protoxin by midgut proteases, binding of the Cry toxin to midgut receptors, and insertion of the monomeric toxin into the apical membrane to create tetrameric ion channels or pores (de Maagd et a|.,2001). A susceptible insect digests Cry-toxins with its diet. Once digested, the protoxin is solubilized under midgut alkaline conditions and the protoxin is cleaved by a gut protease such as trypsin or chymotrypsin to produce an active toxin (Carroll and Ellar, 1993), where chymotrypsin has been shown to be necessary for
21 solubilisation at least for Cry3A (Carroll et al., 1997). During solubilisation, a large segment of the protoxin is removed and leaves a protein-resistant active core toxin (Knowles, 1994). Genetic deletion studies showed that Cterminal half of the 130 kDa Cry1 toxins is removed in this process, indicating that the first 600 amino acid residues are the insecticidal determinants in this group of toxins, while Cry2 and Cry3 toxins could be truncated versions of Cry1 where proteiolytic activation occurs primarily at the N-terminus (Bravo et al., 2002\. Differences in the extent of solubilisation may affect the degree of toxicity among Cry-toxins (Bora et al., 1994). For example, a reduction in solubility is thought to be involved for some insect resistance to Cry toxins (Crickmore et al., 1998).
After proteolytic activation, the active toxin passes through the peritrophic membrane (PM) and binds to specific receptors in the brush border membrane vesicles (BBMVs) of midgut epithelial cells (Gill et al., 1992; Hofmann et al., 1988; Van Rie et al., 1989). Therefore, the structure and functional properties of the PM for Bt-toxicity are of vital importance. However, the interaction of Cry toxins with the PM, a major component of the midgut lumen, is not well understood (Granados et aI.,2001).
The PM is an extracellular thin sheet composed of proteins, glycoproteins, and chitin microfibrils in a proteoglycan matrix which, in one or more stages of the insect life, envelops the midgut lumen contents and lines on the microvilli of the underlying epithelial cells (Lehane, 1997). This separates the food from the midgut epithelium in most but not all insects and plays key roles in the intestinal biology of the insect, such as regulation of insect digestion (Terra, 2001). The PM is involved in several other functions of critical importance for the insect survival; it prevents mechanical lesions of the apical cell membrane and non-specific binding to cell surface and acts as a semipermeable membrane regulating passage of nutrients from the lumen to the intestinal epithelium (Binnington et al., 1998). lt is also an important innate defense barrier against ingested parasites, pathogens and toxins
(Binnington et a|.,1998 ; Wang and Granados, 1997; Wang and Granados, 2000).
22 Toxin binding to midgut receptors, which is mediated by domains ll and lll (Ballester et al., 1999a; Lee et al., 2000b; Li et al., 1991) and is dependent on the affinity of receptors in BBMVs of the midgut in susceptible larvae (Ballester et al., 1999b; Gill et a1.,1992; Van Rie et al., 1990), is a crucial step for the toxicity of the Cry-toxins.q A major conformational change is required to transform the initially soluble Cry toxin into a structure able to insert into the membrane, where the conformational change was envisaged to expose a relatively non-polar helix hairpin from domain I to initiate membrane penetration (Li et al., 1991). The helices in domain I of Cry toxins are largely amphipathic, but the q4-loop-oS region is the most hydrophobic (Haider et al., 1986; Morse et al., 2001). The "umbrella" model for pore formation by the Cry toxins (Gazit et al., 1998; Knowles, 1994; Li et al', 1991) suggests that a pair of helices, probably q4 and o5 (Masson et al', 1999; Schwartz et al., 1997) insert into the membrane. The remaining helices will be rearranged to open on the membrane surface like the ribs of an umbrella. Some studies suggested that the toxin binding to the receptors depends on the toxin oligomeric structure (Gomez et al., 2002). While helix cr4 of domain I is involved in the toxin oligomerization process, the helix o5 plays an important role in toxin structure stabilization and pore formation (Rausell et al., 2004a; Tigue et al., 2001). However, formation of a pre-pore oligomeric structure, probably a tetramer, by intermonomeric contacts and subsequent insertion into the apical membrane to make active lytic pores (Gomez et al., 2002; Rausell et al., 2O04b) has been postulated but how a water-soluble complex penetrates the lipid bilayer is not known. The pore formation in turn rapidly immobilizes the epithelial cells of the larvae with a gradual decrease in gut pH (Aronson and Shai, 2001; Knowles and Ellar, 1987), causing the larva to stop feeding. The lower pH enables the bacterial spores to germinate, and the bacteria can then invade the host, causing its death.
Since many pore-forming toxins, such as Bt Cry-proteins, are lectins, a mechanism of pore formation involving the membrane insertion by putative leverage-mediated (LM) uptake reactions have been proposed, where oligomeric adhesion molecules bent membrane-receptors around 'hinge'-like lipophorin particles (Schmidt and
23 Theopold, 2OO4). ln addition, recent studies describing alternative experimental approaches using the model invertebrate organism Caenorhabditis elegans have revealed a novel cellular receptor for the toxin and have detailed a cellular response of the host towards intoxication (Griffitts et al., 2005). However, the knowledge of Cry toxins structures and basic celluar uptake mechanisms is required to obtain a more critical understanding of their molecular basis of mode of action and toxicity.
2.2.6 Cry toxin receptors in insect midgut
Bt toxins kill insects by binding to specific target sites, the toxin receptors and disrupting midgut membranes (Schnepf et al., 1998). lt is not surprising to have multiple receptors, as there are several different Bt toxins, but the story has become more complicated since Garner et al. (1999) suggested that the protein that binds Cry1A toxin is an aminopeptidase, while Keeton and Bulla (1997) and Gahan et al. (2001) reported that the protein that binds Cry1A toxin is a member of cadherin-like molecules. ln Manduca sexfa the receptor for Cry1Aa, Cry1Ab, and Cry1Ac toxins is 21O-kDa protein, known to be a member of the cadherin-like (BT-R1) gene family (Francis and Bulla, 1997). Cry1Aa, Cry1Ab, and Cry1Ac exhibit identical toxicities toward M. sexfa larvae and show a high degree of sequence similarities and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Midgut expressed cadherin-like cDNAs were isolated Ρom M. sexfa (Vadlamudi et al., 1995), Bombyx mori (Nagamatsu et al., 1998); Hetiothis yirescens (Gahan et al., 2001), and Pectinophora gossypiella (Morin et al., 2003). Competition assays with the cadherin-like BT-R1 protein prepared from larval M. sexta midgut and transiently expressed in cultured cells reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins (Keeton and Bulla, 1997; Keeton et al., 199S). This is an indication of a common Bt-receptor for toxins
24 of the lepidopteran-specific Cry1A family, where a 210-kDa glycoprotein BT-Rr binds all three Cry1A toxins. ln some cases, the receptor for Cry1Ab in M. sexfa is believed to be a cadherin)ike 210-kDa membrane protein (Dorsch et al., 2002). Domain ll of Cry1A binds lepidopteran midgut epithelial receptors (de Maagd et al., 2001) by interacting with two cadherin motifs (Gómez et al., 2003). Cadherin-like Bt-R1 protein has been suggested to induce a conformational change in Cry1Ab that allows the formation of a pre-pore toxin oligomer (Gomez et a1.,2002). ln B. mori, the cadherin-like protein BtR175 serves as a Cry1Aa receptor (Nagamatsu et al., 1998). lnvolvement of cadherin-superfamily gene disruption in resistance to Cry1Ac has been described for a laboratory strain ol H. virescens (Gahan et al., 2001), although no cadherin-like proteins have been purified from this insect.
A220-kDa cadherin-like peptide was identified as a midgut Cry1Ab receptor in O. nubilalis (Hua et al., 2001). Ligand blots demonstrate that Cry1Ab binds to a 220- kDa cadherin-like glycoprotein, and 145 and 154-kDa aminopeptidase (APN) isoforms in O. nubilalis (Hua et al., 2001). Candidate Cry1Ab binding receptor genes have not yet been reported in O. nubilalls. Effects of cadherin toxin binding region variation on resistance traits are unknown.
Aminopeptidase was implicated as a midgut receptor (Gill et al., 1995; Knight et al., 1994), followed by cadherin. N-terminal and internal amino acid sequences of protein from H. ylrescens show a high degree of similarity with the enzyme aminopeptidase N, an indicator for aminopeptidase N as the receptor for CrylAc toxin in the brush border membrane (Heckel et al., 1997). Cloning and expression of a cDNA that encodes BT-R1 of M. sexfa is a 210-kDa membrane glycoprotein that specifically binds the CrylAb (Vadlamudi et al., 1995). BT-R1 shares sequence similarity with the cadherin superfamily of proteins. The CrylAc binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect M. sexfa identified as aminopeptidase N (Knight et al., 1995). ln H. virescens, a 170-kDa aminopeptidase N, bound Cry1Aa, Cry1Ab, and Cry1Ac, but not Cry1C or Cry1E, whereas N-acetylgalactosamine (GalNAc) inhibited the
25 binding of Cry1Ac but not that of Cry1Aa or Cry1Ab (Luo et al., 1997). ln P. xylostella and B. mori, the Cry1Ac receptor also seems to be aminopeptidase N, while Cry1Aa and Cry1Ab bind lo a 210-kDa brush border membrane vesicle (BBMV) protein (Crickmore et al., 1998).
Membrane-bound alkaline phosphatase is also known to bind Cry1Ac receptor (Jurat-Fuentes and Adang, 2004; Ma et al., 2005). Most recently Glycolipids have been reported as CrySB receptor in nematode Caenorhabditis elegans (Griffitts et a|.,2005).
2.2.7 Pesfs reslsfanf to Cry-toxins
The success of Bt crops does not preclude resistance problems in the future, as at least seven resistant laboratory strains of three insect pests diamondback moth Ptutelta xylostella, pink bollworm Pectinophora gossypiella and cotton bollworm Helicoverpa armigera have completed development on Bt crops in laboratory and greenhouse tests (Tabashnik et al., 2003). However, using either laboratory- adapted insects or insects collected from wild populations, a number of insect populations of several insect species from different geographical areas, with different levels of resistance were obtained by laboratory selection with Cry toxins. To date, laboratory-selected Cry toxin-resistant insects are the lndian mealmoth Ptodia interpunctel/a (McGaughey, 1985), the diamondback moth P/ufella xylostella (Tabashnik et al., 1991), the Colorado potato beetle Leptinotarsa decemlineata (Whalon et al., 1993), the cabbage looper Trichoplusla ni (Estada and Ferre, 1994), the cotton leafiryorm Spodoptera littoralis (Muller-Cohn et al, 1996), the beet armyworm Spodoptera exigua (Zheng et al., 2005), the tobacco budworm Heliothis yirescens (Gould et al., 1992; Lee et al., 1995; Maclntosh et al., 1991; Forcada et al., 1996; Heckel et al., 1997), the European corn borer Ostrinia nubilalis (Huang et al., 1999), the mosquito Culex quinquefasciatus (Georghiou and Wirth, 1997), the cotton bollworm Helicoverpa armigera (Akhurst et al., 2003; Gunning et al., 2005;
26 Ma et al., 2005), flour moth Ephestia kuehnellia (Rahman et al., 2004a) and a candidate member from nematodes Caenorhabditis elegans (Griffitts et al., 2005).
2.2.8 Mechanisms of Bf Cry-toxins resisfance rn rnsecfs One or more genes may mediate lepidoteran resistance to Cry toxins. Hypothetically the mechanisms underlying Cry toxins resistance may be found in any of the steps in the proposed mode of action (Schnepf et al., 1998). Therefore, any change in insect gut physiology that affects one or more steps in this process could prevent toxicity and potentially leads to the development of resistant pest populations (Li et al., 2004). To date, most studies for Bt resistance have focused on two steps in its mode of action: proteolytic activation of protoxin and binding of active toxin to receptors.
Several mechanisms of insect resistance to Bt toxins have been proposed (Gill et al., 1992). One of the best-known mechanisms of resistance is the reduced binding of toxin to midgut receptors. Modification of midgut receptor proteins that bind Cry toxins may reduce susceptibility of insects. This mechanism has been associated with the resistance to Cry toxins in many strains of P. interpunctella (Herrero et al., 2001; Van Rie et al., 1990), P. xylostel/a (Ferre et al., 1991; Escriche et al., 1995; Tabashnik, 1994; Tang et al., 1996), H. virescens (Gahan et al., 2001; Lee et al., 1995) and S. exigua (Moar et al., 1995). However, in some laboratory-selected resistant strains of H. yftescens, reduced binding was not associated with resistance to Cry-toxins (Gould et al., 1992; Maclntosh et al., 1991).
The other mechanism of resistance may involve midgut proteinases that interact with Bt toxins. The resistant strain of P. interpunctella lacks a major gut proteinase and has low Bt protoxin-hydrolyzing activity compared to the susceptible parent strains (Oppert et al., 1997). Changes in midgut protease activity and toxin binding together result in resistance in L, decemlineafa (Loseva et al., 2002). Faster degradation of toxin by midgut proteases was associated with Bt resistance of H.
27 yirescens (Forcada et al., 1996; Forcada et al., 1999) and Spodoptera littoralis (MullerCohn et al., 1996). An increased rates of replacement or repair of damaged cells by Bt-resistant larvae compared to their susceptible counterparts is the basis of another mode of resistance in H. virescens (Martinez-Ramirez et al., 1999). However, two novel resistance mechanisms to Cry toxins have recently been identified in different species, one based on an elevated immune response in E. kuehniella (Rahman et al., 2004a) and a second mechanism was identified with glycolipids receptors in C. elegans mutants (Griffitts et al., 2005). Cry1Ac resistance was also selected in a H. armigera population by feeding the toxin with artificial diet in the laboratory, where resulting resistance was based on a single gene locus with semi-dominant trait and imposed a fitness cost, which is likely to derive from pleiotropic effects of the resistant gene rather than effects from other gene loci (Ma et al., 2005). This indicates that Cry1Ac resistance to H. armigera may not exclusively based on the inactivation of one of the major receptor genes rather than other unknown gene(s) function. Lower glycosphingolipids content in BBMV was also reported in high-level of resistance in a line of P. xylostella (Kumaraswami et a|.,2001; Maruyama, 1999). Alternative mechanisms responsible for Bt-resistance or tolerance in individual insect species will be reviewed in this section.
The stored-grain pest P. interpunctel/a was one of the first insect species to evolve resistance to a B. thuringiensis subsp. kurstaki formulation in the laboratory (McGaughey, 1985), where the resistance trait proved to be recessive (Van Rie et al., 1990). The resistance was correlated with a 5O-fold decrease in binding affinity of a receptor for the Cry1Ab protein but an increased susceptibility to Cry1Ca, a protein not present in the selective formulation, and a corresponding increase in binding sites on the midgut for the Cry1Ca protein. ln some other colonies, the level of resistance ranged lrom 24 to >2,000 fold after selection with CrylAc and Cry1Ab, but >10 for Cry1Aa, Cry1Ba, Cry1Ca, and Cry2Aa (McGaughey and Johnson, 1994). The diversity of resistance mechanisms to different toxins within species is common. For example, while resistance is associated with reduced
28 binding of Cry1Ac toxin to midgut cells, the same toxin shows unaltered binding to an BO-kDa BBMV protein (Mohammed et al., 1996). This strain has one of the two major trypsin-like proteases missing from midgut enzymes selected with Dipel or HD-133, and a reduced in vitro activation of Cry1Ac protoxin by midgut extracts when selected with HD-198, a toxin from B. thuringlensis subsp. entomocidus (Oppert et al., 1994).
Colonies of H. virescens with different levels of resistance and different resistance mechanisms have also been obtained in selection experiments with different Cry toxins (Jurat-Fuentes et al., 2003). Selected with Cry1Ab protoxin for seven generations with Dipel, showed 71-fold resistance and a lower binding affinity to a higher number of binding sites (Maclntosh et al., 1991). Larvae of H. virescens selected with Cry1Ac protoxin could not survive on transgenic tobacco plants with moderate (0.01%) levels of Cry1Ab (Schnepf et al., 1998), but survived significantly better than susceptible larvae with very high levels of resistance to Cry1Ac and to CrylAb when selection was performed with Cry1Ac (Gould et al., 1995), an indication of the existence of multiple resistance mechanisms in this insect strain. Though Cry1Ac also binds to the CrylAa binding site (Van Rie et al., 1989), it is surprising that the binding of Cry1Ac and Cry1Ab was unchanged while the binding of Cry1Aa was dramatically reduced in this strain (Lee et al., 1995). However, it was proposed that the altered Cry1Aa binding site caused resistance to all three Cry1A toxins and that the additional binding sites recognized by Cry1Ab and Cry1Ac might not be involved in toxicity (Lee et al., 1995). Cadherin was implicated as a resistance factor to Cry1Ac by transposon knockout in H. virescens YHD2 colony resulting in 40,000-fold resistance (Gahan et al., 2001), although no cadherin-like candidate proteins have been purified from this insect.
S. littoratis with more than 500-fold resistant to Cry1Ca have shown partially recessive inheritance (Schnepf et al., 1998) and cross-resistance to Cry1Da and Cry1Ea (MullerCohn et al., 1996) . But in S. exigua, only 11-fold resistance was observed after selection with Cry1C for seven generations, where the resistance is
29 not entirely based on changes in toxin binding characteristics (Moar et al., 1995). This strain shows cross- resistance to Cry1Ab, Cry2Aa, Cry9C, and a Cry1Ea- Cry1Ca hybrid protein, where resistance to a single protein is more likelyto occur first than resistance to spore-crystal mixtures (Bosch et al., 1994).
A common mechanism and a shared genetic basis of resistance to Cry1Ac have also been shown in some insects. For example, two independently-derived resistant strains of pink bollworm P. gossypiella, share a genetic locus at which three mutant alleles (r1, 12 and 13) confer resistance to Cry1Ac, where both strains are reported to show traits of "mode 1" resistance (Tabashnik et al., 2004). Cadherin was implicated as a resistance factor by phenotypic association in P. gossypiella AZP-R strain, where two copies of cadherin resistance alleles (r1, 12 or r3) were required for resistance (Morin et al., 2003).
Laboratory colonies of O. nubilalis exhibit moderate resistance to Cry1Ab toxin in artificial diet (Bolin et al., 1999). lnheritance of resistance was autosomal and incompletely dominant in an O. nubilalis strain, when selected for survival on Dipel@ Bt formulations (Huang et al., 1999). Laboratory selection with Cry1Ab produced >1000-fold resistance in two laboratory strains of O. nubilalis, which showed autosomal inheritance and a low initial frequency of major genes, which might be an important factor in delaying evolution of resistance to Bt corn in this pest (Alves et al., 2006).
It is important, however, to keep in mind that selections in the laboratory are very different from selections that occur in the field. The laboratory colonies might have a considerably lower level of genetic diversity than field populations (Schnepf et al., 1998). Resistance to Bt-toxins has been developed in the field in P. xylostella. However in this species no success in laboratory selections for Bt resistance has been achieved to date. The natural environment may contain factors affecting the viability or fecundity of resistant insects, factors excluded from the laboratory (Ferre and Van Rie, 2002). Moreover, resistance mechanisms can be associated with
30 certain fitness costs that can be deleterious under natural conditions. ln the field, natural enemies, such as predators and parasites, can influence the development of resistance to B. thuringiensis by preferring either the intoxicated, susceptible or the healthy, resistant insects populations (Schnepf et al., 1998; Schuler et al., 2003).
Despite expectations that pests would rapidly evolve resistance to Bt Cry-toxins, increases in the frequency of resistance caused by exposure to Bt formulations or to Bt crops in the field are still to be explored at a larger scale. Although many insects have shown resistance to Bt in laboratory selection, only Plutella xylostella has evolved resistance to Bt toxin in open field populations (Tabashnik et al., 2000), showing 30 fold resistance to a Bt formulation (Dipel) and rapidly increased to >1,000-fold under further laboratory selection. This resistant strain showed a reduced binding of the Cry1Ac protein to gut BBMV (Tabashnik et al., 1994), the presence of at least some receptor molecules on the midgut (Escriche et al., 1995; Masson et al., 1995) and the inheritance of resistance is conferred largely by a single autosomal recessive locus (Tabashnik et al., 1997a). This was corroborated when a major autosomal locus inherited as a recessive trait was identified (Heckel et al., 1999). Moreover, this "Hawaii" resistance allele simultaneously confers cross-resistance to Cry1Aa, CrylAb, Cry1Ac, Cry1Fa, and CrylJa but not to Cry1Ba, Cry1Bb, Cry1Ca, Cry1Da, Cry1la, or Cry2Aa (Granero et al., 1996)' The toxins in this cross-resistance group have significant amino acid sequence similarity in Domain ll, a region believed to be important for receptor binding in many species.
Binding to BBMV was reduced for CrylAb but almost unaffected for Cry1Aa, Cry1Ac, or Cry1C in a field population of diamondback moths from the Philippines, which showed partial resistance to Cry1Aa, Cry1Ab, and Cry1Ac, but sensitivity to Cry1C, Cry1F, and Cry1J, where inheritance of resistances to Cry1Aa and Cry1Ac was expressed in an autosomal dominant and semidominant fashion, respectively (Tabashnik et al., 1997b). A Cry1Ab resistance allele, associated with reduced
31 binding to BBMV receptors, was partially responsible for a high level of resistance in a field collected Malaysian strain selected with B. thuringiensls subsp. kurstaki and B. thuringiensis subsp. aizawai(Wright et al., 1997).
A field-collected colony of P. xylostella showing 31-fold resistance to Cry1C, survived and neonates completed their entire life cycle on transgenic broccoli expressing a Cry1C protein alter 24 generations of laboratory selection with Cry1C (Zhao et al., 2000). Reduced binding is not the major mechanism of high level of resistance to Cry1C in this strain. Whatever the mechanism it seems to be inherited as an autosomal and incompletely recessive factor.
The first-instar larvae of H. armigera from a population selected lor 25 generations with Cry1Ac (63 fold resistance) in laboratory conditions were able to complete their larval development on transgenic cotton expressing Cry1Ac and produced fertile adults (Akhurst et al., 2003). Resistance in this Australian strain is incompletely recessive and associated with lack of high affinity binding site. ln a laboratory selection H. armigera developed resistance to CrylAc in lndia at comparable levels with the Australian strains (Kranthi et al., 2000). Additive gene action was predicted to mediate Bt resistance in H. zea (Burd et al., 2003)
It has been suggested that an increased rates of replacement or repair of damaged cells by Bt-resistant larvae compared to their susceptible counterparts is the basis of another mechanism of resistance in H. virescens larvae (Martinez-Ramirez et al., 1999). Sublethal doses of Bt toxins AA 1-9 and HD-73 cause dose-dependent destruction of cultured midgut cells from H. virescens larvae but regenerate at higher rate in resistant larvae than susceptible (Loeb et al., 2001). This increasing rate of cell proliferation may possibly involve in Bt resistance.
Lower glycosphingolipids content in BBMV is known to associate with very high- level resistance in a line of P. xylosfe//a having 130000-fold resistant to Cry1Ac (Maruyama, 1999). Receptor binding was not the core of resistance in this strain
32 since Cry1Ac bound almost equally to a 120-kDa receptor protein of BBMVfrom both susceptible (PXS) and resistant (PXR) strains. lndeed, receptor binding must be accompanied by events such as membrane insertion and pore formation, and the evolution of resistance may be associated with appropriate changes in these post-binding events. Loss of glycolipid carbohydrates is known to involve in Bt resistance in nematodes C. elegans (Griffitts et al., 2001), where CrySB toxin, which targets nematodes directly and specifically binds glycolipids and the binding is carbohydrate {ependent and relevant for toxin action in vivo (Griffitts et al., 2005).
Bt resistance mechanisms are not unique. For example, the case history of P. xylostella (DBM) and H. yirescens resistance to Cry toxins presents a cautionary tale for the wide range of resistance mechanisms within the species. Starting in the mid 1980s, with an intensive use of B. thuringiensis subsp. kurstakiin commercial agriculture, resistant DBM insects have evolved in numerous geographically isolated regions of the world, and B. thuringiensrs subsp. aizawai resistance is beginning to appear even more rapidly. While various alleles showing cross- resistance, dominant inheritance, or stability in the absence of selection have been detected in case of very high levels of resistance in different insects, little is known about the mechanisms of low to medium level of resistance.
2.3 Inducible tolerance to Bt-toxin in insect pests 2.3.1 lntroduction
Receptor inactivation by gene mutations (mode 1) provides immediate resistance to relatively high levels of the toxins. The case history of Bt-resistance demonstrates that the emergence of resistance through gene mutations is a rare event, given that only one pest insect species have developed genetic resistance in the field. Moreover, inactivation in one gene is not necessarily expressed as a resistant phenotype because some receptor genes comprise gene families with
33 overlapping gene function. While mode 1 resistance is rare, the potential exists for a number of pest insects to become tolerant by other mechanisms.
2.3.2 lnducible tolerance mechanisms
Recent reports on inducible tolerance to low to medium levels of Bt-formulation in E. kuehniel/a (Rahman et al., 2004a) have highlighted possible alternative mode of resistance in a large number of pest insect species that have been selected in the laboratory for resistance against Bþtoxins. The observed tolerance in this strain is dependent on the induction of immune proteins, which occurs with the ingestion of sub-lethal doses of Bt-formulations and that both the immune induction and the Bt tolerance are transmitted to offspring by a maternal effect.
When larvae from a susceptible laboratory strain of E. kuehniella were selected for survival on progressively increasing levels of the Bt-formulation (Dipel) consisting of CrylAa, Cry1Ab, Cry1Ac, Cry2\a, proteins, and spores for five generations, the resulting Bt-tolerant strain displayed a constitutively elevated immune response (Rahman et al., 2004a). Treating larvae of the susceptible strain with sublethal doses of a Bt-formulation led to an elevated melanisation reaction in the hemolymph, which in turn was correlated with an increase in tolerance against the toxin at a later stage of larval development. As susceptible larvae were immune- induced and exposed to Bt-toxin in the same generation, this experiment excludes the selection of a pre-existing resistance allele as a cause of increased tolerance to the toxin in pre-treated larvae.
Reciprocal crosses of tolerant and susceptible insects revealed a transmission of both the immune induction and tolerance to the toxin from one generation to the next by a maternal effect (Rahman et al., 2004a). Offspring of tolerant females and susceptible males (TxS) were significantly more tolerant than offspring from susceptible females and tolerant males (SxT). A possible mechanism for this effect is the incorporation of an immune-elicitor into the oocyte by an immune-induced
34 female. The elicitor could interact with embryonic tissues to induce the immune system of the neonate so that by the time the neonate starts feeding; the insect is already induced thus increasing the chances of surviving the toxin.
The results from E. kuehniella were corroborated from the reports of a field- collected strain of H. armigera that has developed genetic resistance in the laboratory (Akhurst et al., 2003), comprising a complex mixture of putative receptor inactivation and elevated immune status (Ma et al., 2005) and P. xylostella (M. Sarjan, unpublished data), suggesting that the initial development of low-level Bt- tolerance through immune-related processes may be a common phenomenon. ln H. armigera, the immune-induction was associated with an increased level of the larval serum protein hexamerin, which interacts with the toxin by forming an insoluble aggregate (Ma et al., 2005).
The inducible tolerance mechanisms are quite different from other Bt-resistance mechanisms (Gonzalez-Cabrera et al., 2001), where the observed reductions in Bt- toxicity are exclusively explained in terms of alterations to receptor properties in the gut epithelium (Darboux et al., 2002; Gahan et al., 2001). While inducible tolerance mechanisms may be effective only against low to medium levels of toxin and are no immediate threat to most transgenic crops expressing the Bt Cry-proteins, it may pose long-term threat through genotype selection, given that the toxin levels in transgenic crops may not be sufficient to kill tolerant insect populations in conjunction with semi-dominant receptor mutations,
The molecular basis of inducible tolerance mechanisms in E. kuehniella is not known. However, the evidence that the magnitude of resistance is determined by more than one gene suggests that it may involve multiple metabolic and regulatory pathways. For example, mutational changes in a gene product involved in post- translational modifications, such as glycosyltransferases, may potentially interfere with pro-coagulant, cell surface receptors and elicitor functions. lf pro-coagulant molecules are transferred into the gut lumen as part of an immune response, it is
35 possible that soluble immune components in the gut lumen interact with the mature toxin causing its inactivation by a coagulation or melanization reaction. Alternatively, lectin-like immune molecules that mimic the toxin may engage the receptor in internalisation reactions without causing osmofragility. Likewise, the nature and molecular mechanism of transmission of the elicitor remains to be elucidated. lt is known that the determination of dorsal-ventral polarity in the Drosophila embryo is mediated by a group of genes that are functionally involved in immune defence reactions of the adult insect (Lemaitre et al,, 1997).
2.3.3 /nsecf immunity
Although the introduction of novel biochemical, genetic, molecular and cell biology tools to the study of insect immunity has generated an information explosion in recent years (Bariltas-Mury et al., 2000), a fundamental question in innate immunity is how microbes and other potentially damaging objects are recognised and inactivated in the absence of cells still remains to be explored (Hall et al., 1999; Kanost et al., 2004; Karlsson et al., 2004). lnsects are particularly resistant to microbial infections, although they do not have an acquired immune system that is capable of specifically recognizing and selectively eliminating foreign microorganisms and molecules (i.e., foreign antigens). Like many other multicellular organisms, insects are able to recognise and inactivate potential pathogens and toxins in the absence of cells by the rapid and transient expression of several genes encoding antibacterial peptides (Boman and Hultmark, 1987) and proteolytic coagulation cascades (Hoffmann et al., 1999).
The defense system of insects consists of different innate immune reactions. lnnate immunity is based on the recognition of microbial molecules, such as lipopolysaccharides (LPS) and peptidoglycans, by specific receptors and the subsequent activation of the cellular response, which includes phagocytosis and encapsulation, and the humoral response (Vierstraete et al., 2004). lnnate immunity, which refers to the first-line host defense against the early phase of microbial infection, is an evolutionarily ancient defense mechanism. Quite recently the areas of insect and vertebrate innate immunity received renewed attention for
36 the similarities between the human and Drosophila immune cascades and are merging as new information confirms the remarkable extent of the evolutionary conservation, at a molecular level, in the signaling pathways mediating these responses in such distant species (Barillas-Mury et al., 2000).
Melanization and other innate immune reactions, such as aggregation and coagulation are regulated by complex activating systems, involving proteolytic zymogen activation cascades (Soderhall and Cerenius, 1998; Zhang et al., 2004). This includes the assembly of a number of proteins, such as serine proteases and protease homologues (Yu and Kanost, 2003) in addition to other factors with unknown function (Lee et al., 2000a). Previous observations involving cell-free plasma proteins, such as hemolin interacting with bacteria, suggested a Lipid A- mediated binding of pattern and other recognition proteins (Daffre and Faye, 1997), followed by a Ca-dependent coagulation reaction, which is dependent on LPS- sugar moieties (Schmidt et al., 1993). Although recent experiments indicated that lipophorin and phenoloxidase are involved in coagulation (Li et a1.,2O02) and that the addition of LPS to purified lipophorin particles from insect larvae causes aggregation and inactivation of the toxin (Ma et al., 2006), the question remains: How is the binding of LPS to pattern recognition proteins translated into the inactivation of pathogens and toxins? lndirect evidence suggests that invertebrates with an open circulatory system sequester damaging microorganisms.and toxins by a combination of coagulation (Nagai and Kawabata, 2000) and melanization reactions (Kanost et al,, 2004), involving adhesive (Lee et al., 1998) and covalent cross-linking of plasma components (Jiang et al., 2003), melanin synthesis and reactive oxygen production (Nappi and Ottaviani, 2000). Since the mechanisms underlying these processes are not known, the question is whether lipophorin particles are the regulatory and effector components for cell-free immune reactions.
The induction of the immune system is dependent on elicitors and other stress- related factors (Barnes and Siva-Jothy, 2000) and increased melanization,
37 determined mainly by phenoloxidase (PO) activity, in hemolymph and cuticle has been implicated in increased resistance to pathogens (Reeson et al., 1998). While coagulation and aggregation reactions are independent of prophenoloxidase (PPO) (Rizki et al., 1985) the two are linked (Nagai and Kawabata, 2000). For example, the absence of melanization caused a reduction in the defence capacity (Charalambidis et al., 1994b), although the two are regulated at the metabolic level by independent pathways (Charalambidis et al., 1994a).
The observation that feeding of a sublethal dose of Bt-toxin to lepidopteran larvae caused immune induction in the hemolymph, probably by localised cellular damage to the gut lining, exposing elicitors from the gut lumen to the hemolymph (Ma et al., 2005; Rahman et al., 2004a). The elevated immune status, which was correlated with the degree of plasma melanization, provided larvae with a protection against subsequent application of lethal doses of the toxin (Rahman et a1.,2004a). Given that the level of melanization is correlated with immune-induction and the elevated immune status with Bt-tolerance, the question is whether melanization is responsible for the observed Bt-tolerance in immune-induced lawae?
2.3.4 Parasitoid-derived immune suppressors
Parasitoids are effective natural enemies of many pest species and are widely used in conjunction with biopesticides such as B. thuringiensis (Bt) in integrated pest management (lPM) programs. A major concern with the adoption of Bt crops worldwide is their potential impact on nontarget organisms including biological control agents like parasitoids. Laboratory and glasshouse studies have found little evidence of direct negative effects on parasitoids of Bt-formulations or of Bt- transgenic plants, nor of Bt-resistant hosts acquiring cross-resistance to parasitoids (Schuler et al., 2004). ln addition, two studies suggest that toxin uptake by parasitoid larvae can differ among species because certain larvae may avoid the gut, where most of the toxin is concentrated (Meissle et al., 2004i Vojtech et al., 2005). Field studies have confirmed that the abundance and activity of parasitoids and predators are similar in Bt and non-Bt crops (Romeis et al., 2006), indicating
38 that parasitoids can be employed in a complementary fashion with the use of Bt- formulations. However, while in previous studies host Bt-resistance has typically been due to alterations in gut protease activity or receptor insensitivity, little is known when the host larvae are immune-induced. Since the capacity of a host to overcome parasitization in large part depends on the effectiveness of its immune response, this raises the question of whether the immune induction associated with Bt-tolerance results in cross-protection against parasitism. While an elevated immune status protects against viral pathogens (Reeson et al., 1998), it does not cross-protect against insect parasitoids (Rahman et al., 2004b). This raises two questions:
Firstly, what is the effect of immune suppression by parasitoids on melanizalion levels and Bt-toxicity? Hymenopteran parasitoids that lay their eggs inside the hemocoel of another insect, suppress the host's immune system precluding immune-mediated inactivation of the egg and emerging larvae. lt is also possible that the growing larvae, which have to digest and take up hemolymph components inside the gut lumen, would be negatively affected by the melanization and coagulation of immune-active host plasma. The suppression of the host immune system, which occurs by maternal protein secretions of the female parasitoid, involving serpins (Beck et al., 2000; Nappi et al., 2005) and other immune suppressors (Asgari et al., 2003; Lavine and Beckage, 1995; Shelby et a|.,2000), is apparently strong enough to reduce the immune status to a level, where it allows the development of the parasitoid inside the host larvae (Rahman et al., 2004b). The question is, whether parasitoid survival is correlated with low levels of melanization. Since immune suppression by parasitoids can increase the virulence of viral pathogens (Washburn et al., 2000), the question whether Bt{oxicity is increased in immune-induced parasitised larvae remains to be explored.
The second question is whether melanization, which is a measure of immune induction, constitutes the cause of Bt-tolerance or represents an independent pathway that is irrelevant to Bt-toxicity and Bt-tolerance. This can be tested by an in vivo suppression of melanization involving the specific inhibition of
39 phenoloxidase by a metal ion-chelating agent tropolone (Morita et al., 2003), which in hemolymph plasma interacts mainly with copper (Nomiya et al., 2004) present in active phenoloxidase (Chase et al., 2000; Fujimoto et al., 1995; Hall et al., 1995; Kawabata et al., 1995) as an essential redox-system of the enzyme (Decker and Terwilliger, 2000).
Melanization or the activity of phenoloxidase (PO) is a good indicator for immune induction and suppression in insects. lf melanization is not involved in the reponse to the toxin what are the alternative immune pathways that protect against the toxin? One possible answer is that the mechanism of up- or down-regulation of immune reactions always include melanization in conjunction with other reactions, such as coagulation, even though these can be separated at the genetic (Asada, 1997) or metabolic level (Charalambidis et al., 1994a). lmmune suppression has been used to examine baculovirus virulence (Washburn et al., 1996). Co-infection of Manduca sexta larvae with polydnavirus from Cofesia congregafa increases susceptibility to fatal infection by Autographa californica M Nucleopolyhedrovirus (Washburn et al., 2000). Conversely, preliminary experiments in Helicoverpa armigera indicate that the elevated immune status, which provides protection against Bt-toxin (Ma et al., 2005), also protect against baculovirus infection (unpubl.). While baculoviruses and Bttoxin share similarities, there are differences, both in the route of infection and in host defence. Since there is no evidence that virions are phagocytized by tracheal cells and since insect hemocytes are unable to cross the basement membrane lining the hemolymph, the only inducible defence pathway that can prevent the spreading of virions in this tissue environment is the inactivation by aggregation and coagulation of defence molecules, although the coagulation products inside the trachea may attract hemocytes to form nodules (Washburn et al., 2000). The relevance to the protection against Bt-toxin is the fact that recognition of the pathogen and inactivation (at least at an initial stage) occurs outside the hemocoel lining. This implies that the factors involved in recognition and inactivation of pathogens and toxins are able to operate away from hemocytes, if not in their absence.
40 What are the candidates for a cell-free recognition and inactivation system? While a number of possible pro-coagulants have been identified in arthropods, the possible function and mode of action seems to differ among different species (Duvic and Brehelin, 1998; Hall et al., 1999; Korayem et al., 2004; Li et al., 2002; Theopold et al., 2002; Theopold et al., 2004). The main feature of pro-coagulants is the unique ability to recognize and form specific aggregates around pathogens and toxins, which effectively separate the damaging effects from the surrounding physiological environment of the host. Many of the pro-coagulants, such as lipophorin (Li et a|.,2002), vitellogenin (Hall et al., 1999) and hexamerin (Ma et al., 2005), serve metabolic and storage functions and in performing those functions are transferred across the hemocoel lining to accumulate in other tissues and body cavities, such as oocytes, epidermis and the gut lumen. Given their ubiquitous presence and their potential function as pro-coagulants, these structures can form a first line of defence against intruding pathogens outside the hemocoel lining. For example, lipophorin, which is secreted into the gut lumen, may be engaged in lipid shuttle between the gut nutrients and the gut cell lining. ln the immune-induced larvae these particles may be modified to attract Bt-toxin molecules, causing sequestration of toxin before it can reach the brush border membrane.
3. The aims of the study The observation, that immune induction in the embryo are caused by a maternal effect in immune-induced Bt-tolerant E kuehniella strain, raises some broad questions about the molecular and genetic basis of this Bt-tolerance mechanism. Therefore, the main aim of this study was to explore the molecular and genetic basis of this novel tolerance mechanism in insects and their long{erm management as part of IPM and IRM strategies.
Given that lipophorin and phenoloxidase are involved in coagulation and that the addition of LPS to purified lipophorin particles from insect larvae causes
41 aggregation and inactivation of the toxin, the second objective of the project was to investigate how the binding of LPS to pattern recognition proteins is translated into the inactivation of pathogens and toxins. ln particular we wanted to investigate whether lipophorin particles, which are the lipid carrier in insects, are the regulatory and effector components for cell-free immune reactions to inactivate Bt-toxins in the gut lumen.
Since the elevated immune status, which is correlated with the degree of plasma melanization, provided larvae with a protection against subsequent application of lethal doses of the toxin, the third objective of the project was to answer whether melanization, which is a measure of immune induction, constitutes the cause of Bt- tolerance or represents an independent pathway that is unrelated to Bt-toxicity and Bt-tolerance.
While an elevated immune status protects against gut-derived pathogens, it does not cross-protect against insect parasitoids in the hemocoel. The forth objective of the project was to investigate the effect of immune suppression by parasitoids on melanization levels and Bt-toxicity. Since there are no specific inhibitors of coagulation, the fifth objective of the project was to test in vivo suppression of gut melanization to determine whether PPO-inactivation affect Bt-toxicity in the gut lumen. Since melanization or the activity of phenoloxidase is a good indicator for immune induction and suppression in insects, if melanization is not involved in the reponse to the toxin, the sixth objective of the project was to investigate alternative immune pathways, for example, explore the possible role of pro-coagulants for Bt- tolerance, given that the mechanism of up- or down-regulation of immune reactions always include melanization in conjunction with other reactions, such as coagulation.
The main feature of pro-coagulants is the unique ability to recognize and form specific aggregates around pathogens and toxins, which effectively separate the damaging effects from the surrounding physiological environment of the host. Lipophorin, vitellogenin and hexamerin for their ubiquitous presence and potential
42 function as pro-coagulants can form a first line of defence against intruding pathogens in insects. Therefore the final objective of the project was to investigate the functional role of lipophorin, which is secreted into the gut lumen, where it is engaged in the lipid shuttle between gut nutrients and gut cell lining, for possible sequestration of Bt-toxin before it can reach the brush border membrane in immune-i nduced larvae.
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59 lndue;lble tolerance to BacÏllus thuringiønsís (BÍ) endotaxins M M Rahman based on cell-free immune reactions
Cell-free immune reactions in insects
M. Mahhubur Rahman, Gang lrla, Harry L. S. Robelts, Otto Schmidt
Insect Molecular biology, School of Agriculture, Food and Wine University of Adelaide, Glen Osmond, SA 5064, Australia
Journal of lnsect Physiology 2006, 522 754'762.
60 tnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions
Statement of authorshiP
The paper published in Journal of lnsect Physiology as:
M. Mahbubur Rahman, Gang Ma, Harry L. S. Roberts, Otto Schmidt (2006): Cell-free immune reactions in insects. Journal of lnsect Physiology 52:754-762.
Mohammad Mahbubur Rahman (Gandidate) Designed experiments, performed experimental work, analysed and interpreted data, and co-wrote manuscriPt.
Gang Ma Assisted in glycolipid extraction
Harry L. S. Roberts Assisted in manuscript preparation
Otto Schmidt (Principal Supervisor) Supervised work, help in analysis and interpretation of data, co'wrote manuscript and acted as communicating author.
Sþned; Date qK,/t, %' Mohammad Mahbubur Rahman
I gave consent on behalf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy.
sisned Date: z{,(/,Ob Otto Schmidt
61 Available online at www.sciencedirect.com Journal scrEN"E of d)or^=.r" Insect Physíology ELSH/IER Journal of Inscct Physiology 52 (2006) 754-762 www.el¡evier.com/locate/jinsphys
Cell-free immune reactions in insects M. Mahbubur Rahman, Gang Ma, Harry L.S. Roberts, Otto Schmidt*
Insect Moleculor Biology, (lniuersity of Adehide, GIen Osmond, SA 5064, Austrlli4
Received 3 April 2006; received in revised form 4 April 2006; accepted 4 April 2006
Abstract
Insects, like many other multicellular organisms, are able to recognise and inactivate potential pathogens and toxins in the absence of cells. Here we show that the recognition and inactivation of lipopolysaccharides (LPS) and bacteria is mediated by lipophorin particles, which are the lipid carrier in insects. In immune-induced insects sub-populations ol Iipophorin particles are associated with pattern recognition proteins and regulatory proteins that activate prophenoloxidase. Moreover, interactions with lectins result in the assembly of lipophorin particles into cage-like coagulation products, effectively protecting tbe surrounding tissues and cells from the potentially damaging effects of pathogens and phenoloxidase products. The existence of cell-free defence reactions implies that immune signals exist upstream of cell-bound receptors. O 2006 Elsevier Ltd. All rights reserved.
Keyrvords: Lipopolysaccharide; Prophenoloxidase; Lipophorin; Coagulation; Pattern recognition proteins
1. Introduction pathogens and toxins? Indirect evidence suggests that invertebrates with an open circulatory system sequester A fundamental question in innate immunity is how damaging microorganisms and toxins by a combination of microbes and other potentially damaging objects are coagulation (Nagai and Kawabata, 2000) and melanisation recognised and inactivated in the absence of cells (Hall et reactions (Kanost et al., 2()04), involving adhesive (Lee et al., 1999; Kanost et al., 2(Þ4; Karlsson et al., 2004). al., 1998) and covalent crossJinking of plasma components Invertebrates use proteolytic coagulation cascades and (Jiang et al., 2fi)3), melanin synthesis and reactive oxygen antimicrobial peptides (Boman and Hultmark, 1987) to production (Nappi and Ottaviani, 2000). Since the inactivate potentially damaging organisms and toxins mechanisms underlying these processes are not known, (Hoffmann et al., 1999). Previous observations involving we asked whether lipophorin particles are the regulatory cell-free plasma proteins, such as hemolin interacting with and effector components for cell-free immune reactions' bacteria, suggested a Lipid A-mediated binding of pattern and other recognition proteins (Daffre and Faye, 1997), 2. Material and methods followed by a Ca-dependent coagulation reaction, which is dependent on LPS-sugar moieties (Schmidt et al., 1993; 2. l. Low-density gradient centrifugation Sun et al., 1990). Although recent experiments indicated phenoloxidase are involved in coagula- that lipophorin and 50-100 fourth to fifth instar larvae frorn Ephestia 2002) and that the addition of LPS to tion (Li et al., kuehniella were washed in ethanol and dried on filter paper particles insect larvae causes purified lipophorin from before extracting hemolymph by cutting a for-leg and and inactivation of the toxin (Ma et al., 2006), aggregation bleeding into a icc-cold anticoagulant solution (30mM the question remains: How is the binding of LPS to pattern trisodium citrate, 26 mM citric acid, 20 mM EDTA, 15 mM proteins translated the inactivation of recognition into NaCl, pH 5.5) containing phenylthiourea (PTU)' Hemo- cytes were removed by centrifugation at 50009 for 3min. *Corresponding a author. Tcl.: + 618 8303 7252; fax: +618 8303 7109. Cell-free plasma (ca 1.5m1) was added to l5ml of E-ntail odth ess: [email protected] (O. Sbhmidt). solution ol 44,39 KBr in 100m1 and overlaid wilh 0.9Yo
0022-1910/S-see front matter @ 2006 Elsevier Ltd. Atl rights reserved. doi: I 0. I 0 I 6/j jinsphys.206.O{.003 M, Mahbubur Ralunan et al. / Journal of Insect Physiology 52 (2006) 754-762 755
NaCl to a final volume of 30m1. The tube was spun at which presumably prevented complete dissolution of 24,000rpm (SV/42Ti rotor) for llh at l0'C. The gradient aggregated proteins in the pellets. was eluted tn ca 7.2m1 fractions covering densities of 1.15 g/ml (top fractions) to 1.45 g/ml (bottom fractions). 2.3. Lectin bioassays The separation of lipophorin particles and lipid-free plasma components was visible after centrifugation by For the measurement of lectin-mediated aggregation, green pigment-containing proteins in the plasma fraction aliquots of gradient fractions were mixed with lectins at and melanised lipophorin sub-fractions in the low-density various concentrations in the presence and absence of area of the gradient. To reduce melanisation and lipid calcium (final concentration I mM) and incubated for I h oxidation during centrifugation p-mercaptoethanol (P-ME, at RT. Proteins were dissolved in loading buffer, heated for final concentration 2mM) and a reactive oxygen scavenger 10 min at 65 "C and separated by SDS-PAGE. To examine nitro blue trizolium (NBT, final concentration l0 pg/ml) sugar-binding properties, incubations were performed in were added to protein extracts. Under these conditions the the presence of various sugars to final concentrations of brown-coloured fraction was replaced by pink-coloured 10 mM. oxidised NBT (not shown) an indication for the production of reactive electrons in lipophorin-containing fractions. 3. Results Although attempts were made to preclude oxidisation residual pink-coloured NBT was still detectable in lipo- 3.1. Lipophorin particles contain immune proteins phorin-containing gradient fractions. To isolate less reactive particles, we used hemolymph from younger (third When lipoprotein particles from the flour moth instar) larvae of a larger moth, Galleria mellonella. E. kuehniella were isolated by low-density gradient centrifugation a brown-coloured band was clearly visible 2.2. LPS bíoassay in the low-density area separated from a blue-pigmented plasma protein complex (not shown), which could indicate We used LPS-induced melanin synthesis (Rahman et al., melanisation reactions in association with lipoprotein 2004) and lipophorin particle aggregation (Ma et al., 2006) particles. Examination of proteins from gradient-separated as an indication for elicitor-mediated cell-free immune particles revealed two major apolipoproteins (apo I/II), functions of plasma fractions obtained by low-density representing several sub-populations of lipophorin parti- gradient centrifugation. For melanin measurements plasma cles of different relative lipid content and protein associa- fractions were diluted with PBS to protein concentrations tions, such as pattern recognition proteins (Fig. la, þgbp), between 2.4 and 2.8OD280 and after addition of DOPA imaginal disc growth factors (Ma et al., 2006) and (final concentration of 10mM) melanisation was measured morphogens (Panakova et al., 2005). In addition to apo at ODae¡ in the presence of calcium (final concentration I/II, other protein bands were identified in low-density lmM) and LPS (final concentration of l0pg/ml). In fractions of the gradient as two phenoloxidase protein lipophorin-free plasma fractions measurements were per- variants (Fig. la, PPO). This indicates that lipophorin formed with the amount of proteins ranging from 2.4 up to particles are associated with immune proteins and possibly 4.3 OD2s0. Comparative measurements were always per- recognise microbes via pattern recognition proteins. formed on the same day using fraction material from the rily'e therefore incubated gradient fractions with (Sl5) and same gradient centrifugation. without (S9) lipophorin particles with bacteria and For the measurement of LPS-induced aggregation monitored aggregation. Under these conditions bacteria reactions aliquots of gradient fractions were mixed with were found in large aggregates in the presence of LPS at various concentrations in the presence and absence lipophorin particles, whereas plasma fractions from high- of calcium (final concentration 1 mM) and incubated for density areas of the gradient were not active (Fig. 2a). This I h at RT. Proteins were dissolved in loading buffer, heated suggests that lipophorin particles containing immune- for l0min at 65"C and separated by SDS-PAGE. The related proteins are able to recognise elicitors and amount of LPS-mediated covalent cross-linking in each inactivate potential pathogens by forming large aggregates. sample was estimated by the relative amount of Coomassie blue or antibody-stained apolipophorin I, using a pixel 3.2. Lipophorin particles in immune-inducecl insects estimation program. To estimate LPS-induced aggregation and distinguish adhesive linkages from covalent cross- To examine the distribution of pattern recognition linking, aliquots \vere centrifuged (10,0009 for lOmin) proteins and phenoloxidases in immune-induced larvae, after incubation and supernatants (and pellets) dissolved in we compared an E. kuehniella strain with an elevated loading buffer and after heating for lOmin at 65'C immune status (Rahman et al., 2004) with the non-induced analysed separately by SDS-PAGE. While amounts of strain. In plasma from immune-induced larvae, the relative proteins in supernatant and pellet added up in adhesive amounts of B-glucan-binding proteins in lipophorin aggregation reactions, less protein was detected in the particle lractions is higher and extends to very low-density pellets under conditions enhancing covalent cross-linking, fractions compared to plasma from non-induced larvae 7s6 M. Mahbubur Rcltman et al. I Journal of Insect Physiology 52 (2006) 754-762
plasma low-dsnslly very lo w-density s 23 R 23
lraction s7 sg s1 1 s13 M s15 s17 s1s s21 s23 BSbp non- sbp \-ç¡- induced s23 823 lraclion B7 Fì9 Hl1 M R13 R15 R17 H19 821 R23 tmmune- sbp PPO induced '¿'
vWD R R 17 Apol i-? 11 PPO Fraction Fl7 Rg M R11 R13 815 R17 819 821 823 R13 Rlg tmmune- PPO induced PAP
(a) (b)
Fig. l. Lipophorinparticlcsarctheregulatoryunitsofcell-freeimmunìty(a)Sub-populationsoflipophorinparticles(low-densityandverylow-density) are asso"iàted with proteins involved in pattern recognition and proteolytic activation of prophenoloxidase. Western blots of low-density gradient fractions from immune-induced (R) and non-induced (S) larvae (Rahman et al., 2ü)4) probed with antibodies against p-glucan binding proteins (Égbp) from Monc¡tc4 sextrt (Ma and Kanost, 2üÐ), van Willebrand factor domain (vWD) from Golleria mellonelta,which binds to apolipophorin I (Ma et al.' p-glucan 200,6), and prophenoloxidase from M. saxto (Jiang et al., t997), which binds to two protein variants (Jiang et al., lÐ7). Note that thc binding protein is a dimer, which is stable in SDS solutions (Ma et al., 2006). (b) Relativc amoünts of p-glucan binding protein (Bgbp), phenoloxidase and phenoloxidase-activating proteins (PAP2 and PAP3) from M. sexta (Jiang et a1.,2ffi3) in low-density (fraction l7) and very low-density (fraction 23) lradient fractions containing lipopholin particles derived from immune-indnced (.R) and non-induced (S) larvae (Rahman et al., 20O4). M, molecuhr weight marrkers.
(Fig. la). In contrast to pattern recognition proteins, the molecules are taken up into the lipid moiety of the lipid distribution of phenoloxidase did not show significant carrier, followed by a Ca-dependent activation of pheno- differences in gradient fractions from induced and non- loxidase. This is consistent with a small but significant induced insects (Fig. lb). Likewise, phenoloxidase-activat- delay in melanisation reactions observed after addition of ing proteases (PAP2 and PAP3) did not show significant LPS to lipophorin particles (Fig. 2b), which could be an differences in immune-induced and non-induced larvae and indication that LPS-uptake into lipophorin particles is a were not visible in lipi , s15 120 100 Apo I l : 80 c PPO .E o Apo ll E 60 o- o 40 A LPS =oo o I LPS/Ca 20 S9 ! tes/calsugar 0 0.5 1.1 2.3 4.7 9.4 18.8 37.5 75 150 LPS conc. (pg/ml) Aryl Fig 3. LPS-triggcrcd aggregation ¿rnd covalent cross-linking of purilìcd lipoporin pirrticles. LPS was added to aliquots of lipophorin lr¡ction Rl5 (x) in incrcasing ¿ìmoünts (0 5-l50pg/ml) in the prcsence and absencc of CaCl2 (lìnal conc. I mM) and the rcl¿rtive amonnt of Coommassìc blue- staincd apolipophorin I was measurcd. Similar rcsults wcrc obt¿rined when ô szpop¡,lc¿t-ps apolipophorin I w¿is measurcd on Wcstern blots alter labclling with anti- partly 0.8 E s]4/DOPA/LPS vWD antibodies (not shown). LPS-induced aggrcgation is inl.ìibitcd l^ galtrctosamine, N-acctylgalactosatninc and g S14/DOPA/CaILPS in the prcsence of nrixtnt'cs of I (lìnat conc. l0nlM each). Note thi,rt sugar alone A S14/DOPA/LPS/Ca N-arcetylglucoszrmine o.u appcars to caùsc some aggrcgation of lipophorin particlcs for unknown Ëd ! reasons. õ 3 0.4 presence and absence of calcium. When we used tetrameric 0.2 + galactose-specific peanut agglutinin (PNA), we detected a concentration-dependent aggregation of lipophorin parti- 30 60 90 cles (Fig. 4a), which could be dissolved when the reaction (b) Time (minutes) was performed in the absence of calcium, but appears to induce covalent cross-linking when performed in the Fig. 2. (a) B¿rcteria trigger aggrcgation of purilìcd lipophorin particlcs. presence of calcium. When labelled PNA was used, the grown aliquots incubated with E roll (strain JMl06) was overnight and staining was detected in the pellet (not shown), which low-dcnsity gradicnt fractions from the high-dcnsity arca (fraction 9) and the low-dcnsity arca (frtrction l5) containing similar amottnts of protcin Thc suggests that the lectin became all integral part of cxperiment wzrs performcd as describcd (Ma and Kanost, 2000)' The aggregate. This suggests that glycodeterminants from the narjor protcin bands in cach of the two fr'¿ctions ¿rre indicatcd as 230kDa lipophorin particles are cross-linked by putative particle- apolipophorin | (apo l), 70kDa apolipophorin II (apo II), 74kDa derived lectins, such as oligomerised pattern recognition proplrcnoloxidase (PPO) and 72kDa arylphorin (aryl). (b) LPS triggcrs proteins (Mellroth et al., 2005), which can be emulated by nrcl¿rnisatio¡r in purilìcd lipophorin particlcs. Protcin contcnt mcasrtring examine whether 2.4 OD (at 280 nm) fronr low-density gradicnt fraction S l4 w¡s mixcd with the addition of oligomeric lectins. To DOPA (lìnal conccntr¿rtion l0mM) and CaCl (lìnal conc lmM) and PNA interaction with lipophorin particles is based on optical dcnsity measurcd continuonsly (at 490nm). When LPS (lìnal conc. sugar-mediated binding, we incubated the mixture with l0 pg/ml) was added aftcr 40min (arrow), optical dcnsiry incrcased with a various sugars to compete with the sugar determinants of ¿rbout l0min. In contrast, when DOPA and LPS was mixcd lag-phasc of the particle. Under these conditions, the PNA-mediated with lipophorin ptrrticlcs and CaCl2 was ¿dded aftcr 40nrin (arrol), presence optical density increased immediately. No increase in optical density wirs aggregation was partially inhibited in the of obscrvcd in lipophorin-frec lractions from thc plasma (fraction S7) using galactosamine (Fig. 4b), while other sugars, such as protcin amounts up to 4.3OD (at 280nm). Similar results were obtaincd GalNAc and GlcNAc had no effect. This suggests that with corrcsponding fractions from immnnc-induccd larvac (not shown). PNA oligomers interact with galactose-containing glyco- determinants on the lipophorin particles, causing aggrega- lectins or dimerisation of irnmulectins or ø-glucan-binding tion. Tliis is consistent with previous observations proteins as described for Drosophiia peptidoglycan-binding suggesting that glycodeterminants lrom bacterial glycoli- proteins (Mellroth et al., 2005). Alternatively, LPS-derivecl picts (Schmidt et al., 1993) and cell wall components are sugar determinants may be cross-linked by the activation involved in the oligomerisation of pattern recognition of an unknown lectin. proteins (Mellroth et al., 2005). Given that PNA-mediated aggregation occurred in the absence of LPS, it is 3.5. PNA-mediated seu:q5sembly oJ' lipophorin particles conceivable that adhesive connections also include parli- cle-specific glycodeterminants. Since apolipophorin I/II Since Ca-dependent protease activation may trigger lack Gal-containing sugar modifications (Sundermeyer et adhesive and covalent cross-linking, we added oligomeric al., 1996), the sugar may reside in associated glycoproteins lectins to low-density purifled particle fractions in the or glycolipids, Conceptually the activation of lipophorin 758 M. Mahbubur Rahmun er al. / Journol of Insect Physiology 52 (2006) 754-762 100 lBo S .c 60 o E S40 ¡\ erun =ôo I eNnrca 0 0 0.5 2.3 4.6 9.31 18.7 5 (a) PNA conc. (pg/ml) 100 lBo S o =ooE erun .st40 I 5uË õ I enrucat particles form globular 0 Fig. 5. Lipophorin particle assemblies. Lipophorin 3.1 6.2 12.5 25 50 100 structures. FITC-conjugated PNA was added to lipophorin particles from G. mellonella (ftaction l5) and inspected with a confocal microscope under (b) PNA conc. (pg/ml) indirect UV-light. No labelled structures were obtained with GalNAc- Fig. 4. Aggregation of lipophorin particles by oligomeric lectins. (a) Gal- speciñc Helix po,rlatio lecaiÍ (FlTC-conjugated HPL) or GlcNAc-specific specific lectin (peanut agglutinin, PNA) can induce aggregation and wheat gcrm agglutinin (FITC-conjugated WGA). covalent crossJinking of lipophorin particle proteins in the absence of calcium. (b) PNA-induced aggregation is partly inhibited by galactosa- mine (final concentration 50mM). Note that galactosamine zrlone appears 4. Discussion to cause some aggregation of lipophorin particles for unknown reasons. Our observations suggest that lipid particles play an particles can be based on two scenarios: by elicitor- important role in the regulatory process involved in mediated enzymatic modification of particle glycodetermi- activating the LPS-mediated immune response. The pro- nants, which interact with pre-existing oligomeric lectins, cess of cell-free immune activation by LPS involves at least or by elicitor-mediated oligomerisation of monomeric two steps (Fig. 6), a Ca-independent uptake of LPS into the lectins, which connect pre-existing sugar-specific particle lipid moiety of lipophorin particles, followed by a Ca- determinants. dependent'activation' and sugar-dependent oligomerisa- tion of aclhesive molecules that cause self-assembly of 3.6. Self-assembly into cage-like aggregates lipophorin particles into globular structures. These glo- bules effectively inactivate microbes and sequester objects This suggests that lectins, interact with lipophorin with a lipid-containing coating, which may preclude particles to form a complex. It raises the question whether bacterial growth and the spread of damaging toxins as lectins simply connect the lipophorin particles to the well as reactive oxygen intermediates. bacterial surface and LPS micelles, or whether the Whether the induction of melanisation and aggregation oligomeric lectin is involved in a LPS-triggered self- of lipid particles (shown in Fig. 2) is caused by LPS or assembly of lipophorin particles. To answer this question contaminated peptidoglycan is not known from our we added FlTC-conjugated PNA to purified lipophorin experiments. We and others (Duvic and Brehelin, 1998) particles from Galleria mellonella and inspected the have observed that laminarin can induce the LPS-mediated coagulation products under the confocal microscope. aggregation but not melanization, which indicates that tlnder these conditions, we observed round or oval-shaped more than one elicitor may be involved in some of the globules less than 3 pm in size (Fig. 5). This suggests that reactions. coagulation products form regular-shaped cage-like assem- This raises the question, which type of adhesion blies in the presence of oligomeric adhesion molecules with molecules may be responsible for the transition from the potential to sequester microbes and other objects by a soluble to adhesive particles? Given that adhesiveness of lipid-containing layer of lipophorin particles. This cell-free lipid particles may emerge as an essential feature of cell-free defence reaction may form a first line of defence, such as in defence reactions, the mode of activation of adhesive the gut lumen, where inactivation of toxins can be effective proteins associated \4rith lipid particles becomes a crucial even in the absence of hemocytes (Rahman et al., 2004). step in our understanding of innate immune recognition. M. Mahbubur Rrthtntm et al. / Jownal of Insect Physiology 52 (2006) 754-762 759 LPS/1 recognition and aggregation PL Llpid canler .1 LPS + LPS uptake I cat LPS sugar - mediated cross-linking particles Fig. 6. Schematic view ol a two-step activation proccss, involving LPS uptake from lipid droplets into the lipid moiety of lipophorin (Cã-independent) and subsequent oligomerisation ol adhesion molecules (Ca-dependent) leading to the sugar-mediatcd cross-linking of particles into globularitructures. Note that lipophoiin particles are dcpicted with LPS molecules being located on one of ths two lipid bilayers only' While aggregation is possible with apolar particles, globular structures arc only expcctcd in polariscd particles for which there is no expcrimental evidence. Our analysis of lipid particles revealed several candidate R21 does (not shown). This is unexpected since particle- proteins that can potentially contribute to the adhesiveness associated PPO requires PAPs for activation. One explana- and each has a different mode of activation. tion is that PAPs are inactivated or dissociated from While it is not clear whether each adhesive protein is particles under high salt and in the presence of EDTA associated with separate particles or whether more than during centrifugation. Alternatively this observation could one adhesion nolecule is associated with a single particle, a indicate that particles are assembled in a step-wise fashion, number of potential proteins in lipophorin fractions have where PPO is recruited first but may not be inducible via the capacity to become adhesive in the presence of elicitors elicitors. Since PPO is associated with oxygen, particles (LPS) or in response to environmental changes (lipid lacking activating proteases may be involved in oxygen oxidisation etc). Firstly, the elicitor-binding protein carrier functions (Decker and Terwilliger, 2000) transport- (Bai 1996; families, immulectins and B-glucan-binding proteins con- ing oxygen in the hemocoel and into eggs et al., tain two binding domains per protein (Yu et al., 2002). Schmidt et al., 5b). This is consistent with observations Similar to the Drosophila peptidoglycan recognition describing lipophorin particles as multifunctional com- protein, these proteins become dimeric in the presence of plexes that can be associated with growth factors (Ma et elicitors (Mellroth et al., 2005), which makes them al., 2006) and morphogens (Panakova et al., 2005) to adhesive. In fact, the observed immulectin dimers lrom regulate the growth and shape of tissues. our gradient preparations are stable under denaturating Our conclusions that phenoloxidase activity is linked to conditions and visible in Western blots (Ma et al., 2006). lipid particles and regulated by particle-specific immune Secondly, we have identified an imaginal disc growth factor proteins are consistent with very early observations that to be associated with lipid particles (Ma et al., 2006), which cell-free defence reactions exist in insects (Boman and is known to have lectin properties (Kawamura et al', 1999). Hultmark, 1987). Some species, such as mosquitoes with Finally, it is known that exchangeable apolipoproteins, few or no hemocytes in the hemolymph of adult insects are such as apolipoprotein III, have LPS-binding properties nevertheless capable of defending against intruding dama- (Whitten et al., 2004), which cause the oligomeric protein ging organisms by humoral defence reactions (Goetz et al., to attach to the surface of bacteria and induce phagocy- 1987). Recent observations suggest that most of the tosis. This suggests that lipid particlcs are associated with malaria parasites encountered in the gut lumen of more than one protein with the ability to become adhesive mosquitoes are inactivated by cell-free defence reactions in response to different elicitors or under different including melanisation and other PO reactions (Kumar et conditions. al., 2003), which include reactive oxygen intermediates Another question is how lipid particles become asso- (Nappi and Ottaviani, 20 . Interestingly, specific proteins ciated with immune proteins? Fig. I shows fraction 14, involved in these reactions comprise C-type lectins, which which has PAPs both in non-induced (Sl4) and in immune- may target parasite-specific but also insect-specific deter- induced (Rl4) larvae. Preliminary experiments suggest that minants (Osta et al., 2ü)4), an observation that is S21 does not show LPS-induced melanisation, whereas consistent with a general role of particle-associated '760 M. Mahbubn' Ruhnton et al. / Journol of Insact Physiology 52 (2006) 754 762 I (a) (b) ll rlltftfttìfl.illl Cel l-free coag ulat¡on rEact¡ons Cellular clearance react¡ons Fig. 7. Schematic drawing depicting the removal lrom circulrtion ol'adhesive' lipophorin particles by cetl-free sequestration and cellular clearance with reactions. Sell-assembly into gìobular aggrcgates by adhesive lipid particlcs (a) may also occur on the cell surf¿rce il adhesion molecules interact hinge- receptors (b). Given the size dìfferences ãi lectin and lipid partictes, receptors interacting with oligomcric adhesion molccules are bend around the the like iipophorin particles, causing a curvatüre of the mcmbrane (Schmidt and Theopold, !ffi). Clustcring of lipid particlcs on the ccll surface may drivc uptaki åf the tåxin by a cellular clearance reaction based on dynamic adhesion processcs on thc cell surface (Schmidt and Schreiber, 2ü)6). adhesion molecules in lipid metabolism and cell home- keleton and round up due to lack of adhesive surface ostasis. proteins (Schmidt et al., 2005a). A protein with the In addition there is a large volume of literature capacity to inactivate hemocytes has been isolated (Asgari suggesting an involvement of lipid particles in various et al., 1997) and shown to form a complex with lipophorin defence and detoxification reactions. For example, lipo- before being taken up by hemocytes (Asgari and Schmidt, phorin has toxin-binding properties (Vilcinskas et al., 1997) 2002). Importantly, the immune suppressor alone does not and binds and detoxifies LPS (Kato et a1.,1994; Pratt and bind to surface receptors, whereas the complex is cleared 'Weers, 2004). Lipophorin is regulated by immune induc- by massive cellular uptake reactions, which remove tion (Mullen et al., 2004). Many of the effects can be traced adhesive receptors from the hemocyte surface. Interest- to apoIII, which has opsonic properties, enhancing ingly, there are no signs of coagulation in the hemocoel phagocytosis of bacteria (Whitten et al., 2004). Interest- even with the injection of large amounts of suppressor' ingly apoIII is also involved in immune activation (Kim et This raises the interesting possibility that some interactions al.,2Ul,4; rüiesner et al., 1997) and humoral defence (Park with lipophorin particles generate adhesive particles that et al., 2005) and induces the immune response in vivo engage in aggregation, whereas others lead to interactions (Niere et al., 2001). with the cell surface (Fig. 7). The implication of this model The dual role of apoIII as a lectin-like protein in cellular is that the formation of a modified (adhesive) lipophorin reactions is quite interesting, since apoIII is able to detach particle determines the interaction with other particles spread hemocytes from the substrate when applied in (coagulation or not) and with cells (lipid exchange or soluble form but enhance adhesion and phagocytosis when uptake). Self-assembly of particles into a cell-free cageJike immobilised on the surface of microbes (Whitten et al., sequestration product may resemble the assembly of 2004). Similar reports from other immune proteins, such as adhesive particles with membrane-bound receptors, which hemolin (Ladendorff and Kanost, l99l) and hemomucin- internalise the object by leverage-mediated uptake reac- binding lectins (Schmidt and Schreiber, 2006) could tions (Schmidt and Theopold, 2004) creating a dynamic indicate that lipophorin is able to interfere with cellular balance with adhesion reactions (Schmidt and Schreiber, adhesion by an unknown process. Whether this includes a 2006). role of apolipoproteins in preventing adhesion of hemo- cytes (Coodin and Caveney, 1992; Mandato et al., 1996) remains to be seen. Acknowledgements Lipophorin interacts with immune suppressors to inactivate hemocytes (Asgari and Schmidt, 2002). Im- The authors thank Mike Kanost for antisera against mune-suppressed hemocytes from larvae parasitised by pattern recognition proteins and phenoloxidase activating hymenopteran parasitoids have depolymerised actin-cytos- proteases (PAPÐ and Jana Bradley, Caspar Jonker and M. Mahbubur Ralunon et ol. I Journal of Insect Physiology 52 (2006) 754-762 761 Natasha Mclnnnes for help with the experiments. This apolipophorin-Ill and its distribution in hemocyte ftom Hyplnnu'io and Molecular Biotogy 34, l0ll 1023' work was supported by a grant from Biolnnovation SA. cLtnea.l¡sect Biochemistry Kumar, S., Christophides, G,K., Cantera, R., Charles, 8., Han, Y.S., Meister, S., Dimopoulos, G., Kafatos, F.C., Barillas-Mury, C., 2003. References The role of reactive oxygen species on PlasmodiLttn melanotic cncnpsulntion ln Anopheles gunbiae. Proceedings ol the National (USA) 100, 14139-14144. Asgari, S., Schmidt, O.,2002. A coiled-coil region of an insect immune Academy of Sciences 1991. 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Subunit Niere, M., Dcttlofl M., Maier, T.,Zieglct, M, Wicsner, A.,200l.Insect composition of pro-phcnol oxidase from Manduca sexl¿: molecular immune activation by apolipophorin III is correlated with the lipid- cloning of subunit ProPO-Pl. Insect Biochemistry and Molecular binding properties ofthis protein. Biochemistry 40, 11502-11508. Biology 27, 835-850. Osta, M.A., Christophides, G.K., Kalatos, F.C., 2004. Effects of 303, 2030 2032. Jiang, H., Wang, Y., Yu, X.-Q., Zht¡Y., Kanost, M., 2003. Propheno- mosquito genes on Plasntoclittttt development. Science 2005. loxidase-activating proteinase-3 (PAP-3) ftom Manduca se¡rí hemo- Panakova, D., Sprong, H., Marois, E., Thiele, C., Eaton, S., lymph: a clip-domain serine proteinase regulated by serpin-lJ and Lipoprotein particles are required lor Hedgehog and Wingless serine proteinase homologs. Insect Biochemistry and Molecular signalling. Nature 435, 58-65. Lee, Biology 33, 1049 1060. 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Lipopolysaccharide lipophorin complex lorma- 2004. Induction and transmission of Bocillus thuringiensis tolerance in tion in insect hemolymph-a common pathway of lipopolysaccharide the flour mo16 Ephestio kuehniello. Procecdings of the National detoxification both in insects and in mammals. Insect Biochemistry Academy of Sciences (USA) l0l, 269Ç2699. and Molecul¿rr Biology 24, 547-555. Schmidt, O., Faye, I., Lindstrom Dinnetz, I., Sun, S.C., 1993. Specific Kawamura, K., Shibata, T., Saget, O., Peel, D., Bryant, P.J., 1999. A new immune recognition olinsect hemolin. Dcvelopmental and Compara- family of growth factors produced by the fat body and active on tive Immunology 17, 195 200. Drosophila imagin.rl disc cells. Development 126, 2ll-219. Schmidt, O., Glatz, R.V., Asgari, S., Roberts, H.S.L., 2005a. Are insect Kim, H.J., Je, H.J., Park, S.Y., Lee, I.H., Jin, B.R., Yun, H K., Yun, immune suppressors driving cellular nptake reactions? Archives of C.Y., Han, Y.S., Kang, Y.J., Seo, S.J., 2004. Immune activation of Insect Biochemistry and Physiotogy 60, 153-158. 762 M. Mahbubur Rahman et ol. / Journal of Insect Physiology 52 (2006) 754-762 Schmidt, O., Li, D., Beck, M., Kinuthia, W., Bellati, J., Roberts, H'L.S., the hemolymph of the greater wax moth Galleria mellonella. 2005b. Phenoloxidase-like activities and the function of viruslike Comparative Biochemistry & Physiology-{: Comparative Pharma- particles in ovaries of the parthenogenetic parasitoid Venturiî cology & Toxicology 117, 4145. canescens. Journal of Insect Physiology 51,117-125' Whitten, M.M.A., Tew, I.F., Lee, B.L., Ratcliffe, N.A',2004. A novel role Schmidt, O., Schreiber,4., 2006. Integration of cell adhesion reactions-a for an insect apolipoprotein (apolipophorin III) in {beta}-1,3-glucan bala¡rce ol fotces? Jounral of Theoretical Biology 238, 608-615. pattern recognition and cellular encapsulation reactions. Journal of Schmidt, O., Theopold, U., 2004. An extracellular driving force of Immunology 172, 2177-2185. (Hypothesis). BioEssays 26, endocytosis and cel[-shape changes riliesner, 4., Losen, S., Kopacek, P., Weise, C', Gotz, P', 1997.Isolated 134+1350. apotipophorin III from Galleria mellonel/ø stimulates the immune Sun, S.C., Lindstrom, L, Boman, H.G., Faye, I., Schmidt, O., 1990' reactions of this insect. Journal of Insect Physiology 43,383-391. Hemolin: an insect-immune protein belonging to the immunoglobulin Yamashita, S., Sakai, N., Hirano, K., Ishigami, M., Maruyama, T., superfamily. Science 250, 1729-1732. Nakajima, N., Matsuzawa, Y., 2001. Roles of plasma lipid transfer Sundermeyer, K., Hendricks, J.K., Prasad, S.V., Wells, M.A', 1996' The cholesterol transport' Frontiers in Bioscience 6, precursor protein of the structural apolipoproteins of lipophorin: proteins in reverse cDNA and deduced amino acid sequenæ. Insect Biochemistry and D366_D387. Pattern Molecular Biology 26, 735-738. Yu, X.-Q., Zhu, Y.-F., Ma, C., Fabrick, J.4., Kanost, M.R.,2002. Vilcinskas,4., Kopacek, P., Jegorov,4., Vey,4., Matha, V., 1997. recognition proteins in Manduco sexfa plasma. Insect Biochemistry Detection of lipophorin as the major cyclosporin-binding protein in and Molecular Biology 32, 1287 1293. lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions CHAPTER 4 Mode of action of antimicrobial proteins, pore-forming toxins and biologically active pept¡des (Hypothesis) o Schmidtl, M M Rahmant, G Mal, u rheopold2, Y sun3, M Sarjana, M Fabbris and H Robertsl rlnsect Molecular biology, Faculty of Sciences (Waite Campus), University of Adelaide, Glen Osmond, SA 5064, Australia 2Department of Molecular biology and Functional Genomics, Stockholm University, S- 1 0691 Stockholm, Sweden 3The Biotechnology Research Center, Shanxi Academy of Agriculture Sciences, 64 N. Nongke, Taiyuan, Shanxi 030031 China aFaculty of Agriculture, University of Mataram, Lombok, lndonesia sDepartment of Experimental Oncology, European lnstitute of Oncology,l-2Q141 Milan, Italy lnvertebrate Survival Journal 2005, 2= 82-90. 71 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorship The paper published in lnvertebrate Survival Journal as: O Schmidt, M M Rahman, G Ma, U Theopold, Y Sun, M Sarjan, M Fabbri and H Roberts (2005): Mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides (Hypothesis). lnvertebrate Survival Journal2: 82-90. Otto Schmidt (Principal Supervisor) Supervised work, co-wrote manuscript and acted as communicating author. Mohammad Mahbubur Rahman (Gandidate) Performed experimental work, and assisted in analysis and interpretation of data. Gang Ma Assisted in Galleria mellonella colony maintenance Uli Theopold Co-wrote manuscript Yu Sun Co-wrote manuscript Muhammad Sarjan Performed experimental work. Marco Fabbri Co-wrote manuscript Harry L. S. Roberts Co-wrote manuscript Sþned Date Eg o t/' 36 Mohammad Mahbubur Rahman I gave consent on behalf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. Signed Date 28,//, Aó Otto Schmidt 72 Schmidt, O., Rahman, M. M., Ma, G., Theopold, U., Sun, Y., Sarjan. M. … Roberts, H. (2005). Mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides (hypothesis). Invertebrate Survival Journal, 2(2), 82-90. NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library. lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions CHAPTER 5 Induction and transm¡ssion ol Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella M. Mahbubur Rahman, Harry L. S. Robelts, Muhammad Sarjan*, Sassan Asgari+, and Otto Schmidt Department of Applied and Molecular Ecology, University of Adelaide, Glen Osmond, South Australia 5064, Australia * Present address: Faculty of Agriculture, University of Mataram, Lombok, lndonesia + Present address: Department of Zoology and Entomology, School of Life Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia Proceedings of the National Academy of Sciences of the United Sfafes of America 2004, 1 01 (9): 2696-2699. 82 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorship The paper published in Proceedings of the National Academy of Sciences of the United Sfafes of America as,' M. Mahbubur Rahman, Harry L. S. Roberts, Muhammad Sarjan, Sassan Asgari, and Otto Schmidt (2004): lnduction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella. Proceedings of the National Academy of Sciences of the United Sfafes of America 101(9): 2696-2699. Mohammad Mahbubur Rahman (Gandidate) Designed experiments, performed experimental work, analysed and interpreted data, and co-wrote manuscript. Harry L. S. Roberts Assisted in analysis and interpretation of data, and help in manuscript preparation. Muhammad Sarjan Help in experimental work. Sassan Asgari Help in experimental work. Otto Schmidt (Principal Supervisor) Supervised work, help in analysis and interpretation of data, co-wrote manuscript and acted as communicating author. Signed Date p¿,' Qg " //' Moh Mahbubur Rahman I gave consent on behalf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. sisned; * Daþ: 2(,t{.Ø Otto Schmidt 83 Rahman, M. M., Roberts, H. L. S., Sarjan, M., Asgari, S. & 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), 2696-2699. NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library. It is also available online to authorised users at: http://dx.doi.org/10.1073/pnas.0306669101 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions CHAPTER 6 The development of the endoparas¡to¡d Venturia canescens in Bt-tolerart, immune induced larvae of the flour moth Ephestia kuehniella M. Mahbubur Rahman, Harry L. S. Robelts, Otto Schmidt lnsect Molecular Biology Laboratory, School of Agriculture and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia Journal of lnvertebrate Pathology 87 (2-3):129-131. 8B lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorship The Published in Journal of lnvertebrate Pathology as: M. Mahbubur Rahman, Harry L. S. Roberts, Otto Schmidt (200Ð 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): 129-131. Mohammad Mahbubur Rahman (Gandidate) Designed experiments, performed experimental work, analysed and interpreted data, and co-wrote manuscript. Harry L. S. Roberts Assisted in analysis and interpretation of data, co-wrote manuscript and acted as communicating author. Otto Schmidt (Principal Supervisor) Supervised work and help in manuscript preparation Slgned Date: PS ,//' o6 Mohammad Mahbubur I gave consent on behalf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. srsned; Daþ: z<"//,0ó 89 Available online at www.sciencedirect.com Journal of "crENcE d)or^..r^ hvrrBnrpsRAIB Pannor-ocv ELSEVIER Journal of Invertebrate Patholoey 87 (2004) 129-131 www.elsevier.comlocate/yjipa Short Communication The development of the endoparasitoid Venturia canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella M. Mahbubur Rahman, Harry L.S. Roberts*, Otto Schmidt Insect Moleculat' Biology Laboratorl,, Sclnol oJ'Agriufuure and Wine, Uniuersity oJ Adelaide , Glen Osntond, SA 5064, Australia Received 13 August 2004; accepted 29 September 2004 Abstract We examined the survival and development of the endoparasitoid Venturia canescens in a Bt-tolerant laboratory strain of the flour moth Ephestia kuehniella, in which Bt-tolerance has been shown to be associated with an inducible elevation of the insect's immune ,.rpãn... The results indicate the elevated immune status associated with Bt-tolerance does not confer cross-protection against parasitism by V. canescens. No significant difference was observed in the rate ofemergent wasps from parasitised Bt-tolerant und Bt-rur..ptible hosts. In addition, wasps from Bt-tolerant hosts had longer development times and were larger than wasps from Bt-susceptible hosts. O 2004 Elsevier Inc. All rights reserved. Keyttords: Integrated pest management Venturia canescen,\: Eplrcslia løehniella; Bacillus thuringiensís; Biological control 1. Introduction fl our moth Ephestia kuelmiella (Lepidoptera: Phycitidae), a common pest of stored food products, is associated Parasitoids are effective natural enemies of many pest with an inducible increase in the rate of the melanization species and are widely used in conjunction with biopesti- reaction in haemolymph (Rahman et al., 2004), a hall- cides such as Bacillus thuringiensis (Bt) in integrated pest mark of an elevated immune response in insects (Soder- management (IPM) programmes. Previous research has hall and nius, 1998). Similar results have also been found little evidence of direct negative effects on parasi- observed for the cotton bollworm Helicouerpa armigera toids of Bt-formulations or of Bt-transgenic plants, nor (Ma et al., un iew) and the diamondbackmoth Plu- of Bt-resistant hosts acquiring cross-resistance to parasi- tella xylostella (M. Sarjan, unpublished data), suggesting toids (Glare and O'Callaghan, 2(X)0; Schuler et al., 2004), that the initial development of Bt-tolerance through indicating that parasitoids can be employed in a compli- immune-related processes may be a common phenome- mentary fashion with the use of Bt-formulations. How- non. Since the capacity of a host to overcome parasitiza- ever, while in previous studies host Bt-resistance has tion in large part depends on the effectiveness of its typically been due to alterations in gut protease activity immune response, this raises the question of whether the ol receptor insensitivity, recent research has shown that immune induction associated with Bt-tolerance results in tolerance to Bt endotoxins in a laboratory strain of the cross-protection against parasitism. To answer this ques- tion we examined the survival and development of the - Venturia canescens Grav. (Hymenoptera: Corresponding author. Fax: +61-83794095. endoparasitoid E+tail address: [email protected] (H.L.S. Roberts). Ichneumonidae) in Bt-tolerant E. kuehniella. 0022-20111$ - see lront matter @ 2004 Elsevìer Inc. All rights reserved doi: 1 0. I 0 I 6/j jip.2004.09.003 130 M.M. Ruhmnn et al. I Journal oJ InDertebrete Patltology 87 (2004) 129-l31 2. Materials and methods 24.0 day; Fig. 2A) as well as a small but significant posi- tive relationship between host mass and development time The toxin used was a commercial formulation of Bt (F:9.41, df:1,212, p:0.002). The interaction between endotoxins (DelfinV/G, Sandoz (now Syngenta), North host line and host mass was not significant, indicating that Ryde, NS'W), consisting of CrylAa, CrylAb, CrylAc, the difference in development time of wasps from the two Cry2{a, proteins, and spores. host lines was independent of host mass. The initial Bt-susceptible colony of E. kuehniella was a long established laboratory colony that had been Ë 300 maintained without selection for over 100 generations. o çct) The Bt-tolerant E. kuehniella origtnaled from the colony T T Ð zoo (2004), subsequently q) described in Rahman et al. and (J maintained on diet containing 4000ppm of the Bt-for- F 100 TT{' mulation. Both Bt-tolerant and Bt-susceptible larvae -ao o E the study were reared egg-hatch on toxin- ¡¡ used in from aaaaaa free diet. All hosts were maintained as per Rahman et al. 0 (2004). The wasps were a clonal RP line of V. canescens, 0102030 (m¡ns) maintained as per Roberts and Schmidt (2004). T¡me In the study, 150 Bt-susceptible line and 100 Bt-toler- Fig. L Melanization assays of cell-free haemolymph from Bt-tolerant ant line larvae, ranging in mass from 10 to 50mg, were (open circle) and Bt-susceptible (closed circle) E kueltniella larvae. each parasitised once by V. canescens. Following parasit- Bars represent SEM. ism each host was weighed and then maintained individ- ually in a glass vial until parasitoid emergence. Upon A 0.7 emergence parasitoids were killed by freezing, their head 0.6 capsule width measured and development time recorded, To assess whether host line differentially affected wasp 0.5 size to mass ratio a sample of 30 wasps emerging from l, c o.4 each host line were dried in an oven at 50'C lor 24h and o =C' then weighed. Procedures for obtaining single parasitised 0) 0.3 hosts and determining survivorship ancl developmental IL o.2 parameters followed the methods of Roberts et al. (2004). The immune status of eight samples E. kuehniella larvae 0.1 line was determined melanisation assays from each by 0 according to the method of Rahman et al. (20O4). 22 23 24 25 26 27 28 29 30 Data were analysed using the general linear model tlme (daYs) (GLM) platform, JMP V4.0.4 (SAS,200l), with continu- Wasp development ous factors centred by their means (Neter et al., 1990). B Analyses started with full models with all interactions, + E o interactions were dropped if p>0.25 + *o* and non-significant E + + (Winer, l97l). L Ë o '31 o f 3 + + -gl + o v, ô 3. Results o- +++ 6 ô + o â+ aa + E c Compared to Bt-susceptible larvae, cell-free haemol- ct signifi- OJ r+ o + ymph (Fig. l) flom Bt-tolerant larvae showed c, ô o increased melanization reactions (F:157.8, df : èoðaô a cantly ß8t 2 o 1,108, p<0.0001), a hallmark of an elevated immune ô ô 3 o o oÒ' response in insects (Soderhall and Cerenius, 1998). o Neither host line nor mass had a significant effect on 10 20 30 40 s0 parasitism success (rate of emergent wasps from Bt-tol- Host mass (mg) erant hosts: 87o/o,fron Bt-susceptible hosts: 88%). Analysis by GLM of wasp development time with Fig. 2. Development of Iz. canescens in E. kttehniella larvae' (A) Histo- gram of developrnent time in days ol parasitoids in Bt-susceptible revealed a significant host line and host mass as factors (black bars) hosts. (B) The relationship :7,212, (grey bars) and Bt-tolerant difference between the host lines (F:87.3, df between the mass ol the host and the head capsule width of the emer- p<0.0001; least squares mean development time: in gent parasitoid from Bt-susceptible (diamond and dashed line) and Bt- Bt-tolerant hosts:25.8 day, in Bt-susceptible hosts: tolerant (plus and solid line) hosts. Lines represent linear regression. M. M. Rahman et al. I Journal oJ'Inoertebrate Pathology 87 (2004) 129-l3l 131 A similar analysis of wasp head capsule width revealed parasitism, when the host is still able to feed and grow, is the effect of host line was significant (F:40.8, df :l,2ll, extended relative to Bt-susceptible hosts. p<0.0001), with larger wasps developing in Bt-tolerant hosts. There was also a significant positive relationship between host mass and adult wasp head capsule width Acknowledgment (F:l79.2,df :l,2ll,p<0.0001), and a significant inter- action between host mass and host line (F:5.13, This work was supported by ARC grants to HLSR df :l,2ll, p:0.025), indicative of the difference in adult and OS. wasp head capsule width between the two host lines becoming smaller as host mass increased (Fig.2B). Analysis by GLM of wasp head capsule width with References wasp dry weight and host line as factors revealed the Glare, T., O'Callaghan, M., 2000. Bacillus thuringiansls: Biology, Ecol- effects of host line were not significant, indicating that ogy and Safety. John ìüiley, Chichester. host line did not differentially affect the wasp head cap- Ma, G., Roberts, H.L.S., Sarjan, M., Featherstone, N., Lahnstein, J., sule width to mass ratio. Akhurst, R., Schmidt, O., under review. Is the mature endotoxin CrylAc from Bacillus thuringrslrsts inactivated by a coagulation reaction in the gut lumen of tolerani Helicouerpa armigera larvae2 Insect Biochem. Mol. Biol. 4. Discussion Neter, J., Wasserman, W., Kutner, M., 1990. Applied Linear Statistical Models: Regression, Analysis of Variance, and Experimental The study found no significant difference in the rate Designs. third ed. Irwin, Homewood, II. of emergent'wasps from the Bt-tolerant and Bt-suscepti- Rahman, M.M., Roberts, H.L.S., Sarjan, M., Asgari, S., Schmidt, O., ble lines of E. kuehniella, indicating that the elevated 2004. Induction and transmission of Bt-tolerance in the flour moth Ephestia kuehniella. Proc. Natl. Acad. Sci. USA l0l, 2696¿699. immune status associated with the Bt-tolerant line (Fig. Roberts, H.L.S., Schmidt, O.,2004. Lifetime egg maturation by host- l) does not provide E. kuehniella with cross-protection deprived Venluria canescens. J. Insect Physiol.50, 195-202. against parasitism by V. canescens. The study also found Roberts, H.L.S., Trüe, O., Schmidt, O., 2004. The development of the that wasps from Bt-tolerant hosts had longer develop- endoparasitoid wasp Venluria canescens in superparasitised Ephes- ment times and were larger than wasps from susceptible tiu kuehniella. J. Insect Physiol. 50, 839-846' SAS, 2001. JMP IN, Belmont, California, Duxbury Press. hosts. The increase in wasp development time was inde- Schuler, T.H., Denholm, I., Clark, S.J., Stewart, C.N., Puppy' G.M.' (as pendent of host mass, while the increase in wasp size 2004. Effects of Bt plants on the development and survival ol the measured by adult wasp head capsule width, Fig. 2B) parasitoid Cotesis plutellae(Hymenoptera: Braconidae) in suscepti- was greatest for small hosts (which themselves have the ble and Bt-resistant larvae of the diamondback moth, Plutella xylo' greatest potential for growth) and smallest for large slel/a (Lepidoptera: Plutellidae). J. Insect Physiol. 50' 435-443. K., Cerenius, L., 1998. Role of the prophenoloxidase-acti- hosts (which have the smallest potential for growth). Soderhall, vating system in invertebrate immunity. Curr. Opin. Immunol. 10, This suggests these effects represent a partial inhibition 23-28. of embryonic or early instar parasitoid development in Winer, 8.J., 1971. Statistical Principles in Experimental Design, sccontl the Bt-tolerant hosts, such that the initial stage of ed. McGraw-Hill, Tokyo. lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions CHAPTER 7 Tolerance to Bacillus thuringiensis endotox¡n in immune-suppressed larvae of the flour moth Ephestia kuehniella M. Mahbubur Rahman, Harry L, S. Robefts and Otto Schmidt lnsect Molecular Biology Laboratory, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia Manuscript under review in Applied and Environmental Microbiology 93 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorship Manuscript under review in Applied and Environmental Microbiology as: M. Mahbubur Rahman, Harry L.S. Roberts and Otto Schmidt: Tolerance to Bacittus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella. Mohammad Mahbubur Rahman (Gandidate) Designed experiments, performed experimental work, analysed and interpreted data, and co-wrote manuscript. Harry L. S. Roberts Assisted in manuscript preparation. Otto Schmidt (Principal Supervisor) Supervised work, assisted in analysis and interpretation of data, co-wrote manuscript and acted as communicating author. Sþned Date: qg ,/i, 06 Mohammad Mahbubur Rahman I gave consent on behalf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. signed: Daþ: ze"lL b{, Otto Schmidt 94 Under review in Applied and Environmental Microbiology Section : I nvertebrate Microbiology Tolerance to Bacillus thuringiensrs endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella. M. Mahbubur Rahman, Harry L.S. Roberts and Otto Schmidt. lnsect Molecular Biology Laboratory, School of Agriculture and Wine, University of Adelaide, Glen Osmond SA 5064, Australia * Corresponding author Email: [email protected] Phone: +61-83037252 Fax: +61-8303 7109 95 Abstract Tolerance to Bacittus thuringiensrs crystal endotoxins (Bt-toxins) is correlated with an elevated immune status in larvae of the flour moth Ephestia kuehniella (Rahman, M. M., H. L. S. Roberts, M. Sarjan, S. Asgari, and O. Schmidt 2OO4. PNAS 101:2696-2699). 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. To examine whether hemolymph proteins exist in the gut lumen were they can function as pro-coagulants, we compared gut and plasma proteins of immune- induced with those of non-induced larvae. Here we show that the lipid carrier lipophorin represents a major component in the gut lumen and interacts with mature Bt-toxin to form cross-linked coagulation products' Keywords: Ephestia kuehniella, crystal toxin, Bacillus thuringiensis, melanization, coagulation tropolone, parasitoid, prophenoloxidase, lipophorin' 96 1. Introduction Melanization and other defence reactions such as aggregation and coagulation are regulated by complex activating systems, involving proteolytic zymogen activation cascades (38, 47). This includes the assembly of a number of proteins, such as serine proteases and protease homologues (46), in addition to other factors with unknown function (21). The induction of the immune system is dependent on elicitors and other stress-related factors (2) and increased melanization in hemolymph and cuticle, determined mainly by phenoloxidase (PO) activity, which has been implicated in increased resistance to pathogens (32). While coagulation and aggregation reactions are independent of prophenoloxidase (PPO) (33) the two are linked (27). While the absence of melanization caused a reduction in the defence capacity (7), the two are regulated at the metabolic level by independent pathways (6), We have recently discovered that PPO and immune-related proteins are associated with lipophorin particles (Rahman et al., in press). PPO-containing lipid particles can be induced to perform melanin synthesis or aggregate into coagulation products. We previously observed that feeding of a sublethal dose of Bt-toxin to lepidopteran larvae caused immune induction in the hemolymph, probably by localised cellular damage to the gut lining, exposing hemolymph to elicitors from the gut lumen (24,30). The elevated immune status, which was correlated with the degree of plasma melanization, provided larvae with a limited but nevertheless significant protection against subsequent application of lethal doses of the toxin (30). Moreover, the elevated immune status and with it the tolerance to Bt-formulations, was transmitted to subsequent generations by a maternal effect (24, 3O). Given that the level of melanization is correlated with immune-induction and the elevated immune status with Bt-tolerance, the question is whether melanization is responsible for the observed Bt-tolerance in immune-induced larvae? Several observations suggest that this is not the case: Firstly, while Bt-tolerant Helicoverpa armigera larvae show darkened peritrophic membranes (24), we 97 did not observe any signs of melanization in the gut tissue or the gut lumen of immune-induced E. kuehniella larvae. Since PPO is involved in reactive oxygen production and protein cross-linking apart from melanin synthesis, the observed melanization in the gut of H. armigera may be one of several possible activities but not necessarily essential. Thus in immune-induced E. kuehniel/a larvae the PO activity in the gut may be restricted to other than melanization reactions. Secondly, while an elevated immune status protects against some gut-derived pathogens (32), it does not cross-protect against insect parasitoids that oviposit inside the hemocoel (31). This raises two questions: Firstly, how does immune suppression by parasitoids affect melanization levels and Bt-toxicity? Hymenopteran parasitoids that lay their eggs inside the hemocoel of another insect suppress the host's immune system, precluding immune-mediated inactivation of the egg and emerging larvae. This includes protection of the growing larvae against negative effects of melanization and coagulation of immune-active host plasma inside the gut of the parasitoid. The suppression of the host immune system, which occurs by maternal protein secretions of the female parasitoid, involving serpins (4, 28) and other immune suppressors (1, 20, 37) preclude activation of host melanization and coagulation reactions allowing the development of the parasitoid inside the host larvae (31). The question is whether immune suppression by parasitoids, which increases virulence of viral pathogens (45), can also increase Bt-toxicity in parasitised larvae. The second question is whether melanization, which is a convenient indicator of the immune status, is the cause of Bt-tolerance or represents an independent pathway that is irrelevant to Bt-toxicity and Bt-tolerance. We approached these questions by in vivo suppression of melanization involving the inhibition of phenoloxidase by a metal ion-chelating agent tropolone (25). ln hemolymph plasma tropolone interacts mainly with copper (29) present in active phenoloxidase (8, 12, 13,17) as an essential redox-system of the enzyme (9)' Here we show that, while plasma melanization is correlated with the immune status, it is not responsible for the observed tolerance to the Bt-toxin in the gut 98 lumen. lnstead, the lipid carrier lipophorin, which is a pro-coagulant in hemolymph, is present in the gut lumen and able to bind to the toxin. This suggests that Bt-toxin can potentially be inactivated in the gut lumen by an aggregation of lipophorin particles sequestering the toxin into coagulation products. 2. Materials and Method 2.1 lnsecús The Bt-susceptible colony of E. kuehniella was derived from a long established laboratory colony that had been maintained without selection for over 100 generations. The Bt-tolerant E. kuehnlel/a originated from the colony described in Rahman et al. (2004), and subsequently maintained on diet containing 4000ppm or 8000ppm of a Bt-formulation comprising a commercial formulation of Bt endotoxins (DelfinWG, Sandoz (now Syngenta, North Ryde, NSW), consisting of Cry1Aa, Cry1Ab, Cry1Ac, Cry2\a, detergents, proteins and spores. Both Bt-susceptible and a sub-population of Bt-tolerant larvae used in the study were maintained on toxin-free diet of a 10: 2: 1 mixture of oat bran: wheat germ: brewers yeast. Before each experiment the Bt-tolerant larvae were kept on toxin-free diet for a week, which removed any of the components of the Bt-formulation from the mid-gut. The wasps were a clonal RP line of a thelytokous Venturia canescens laboratory culture previously established as reported by (3). The wasps were reared in cylindrical clear plastic tubs; height 20cm and diameter 15cm. Three or four adult wasps were placed into each container with 40-50 host larvae. Upon emergence the wasps were removed from culture and were kept in gauze-covered 425 ml clear plastic cup with a 50o/o honey solution. All experiments were conducted and cultures maintained at 25 + 1oC, under a constant light-dark regime (L1 4:D10). 99 2.2 Parasitism To obtain singly parasitised E. kuehniella lawae a single V. canescens wasp was put together with 25-30 host larvae of varying sizes in a plastic container (7 cm in diameter, 8 cm high). The parasitoids were observed during oviposition and stinging attempts that resulted in a startle response from the larvae, combined with the characteristic cocking movements of the wasp's ovipositor (34) were considered as real oviposition events. Any stinging attempt that either did not evoke a startle response or was not followed by a cocking movement was regarded as uncertain and the larva was discarded. 2.3 Melanizationassays Eight to ten larvae were chilled on ice for five minutes, washed with ice-cold 70% ethanol and then ice-cold PBS. Haemolymph was extracted by cutting off a foreleg and bleeding each larva directly into 1.5 ml ice-cold PBS. The solution was centrifuged for 5 min at 30009 and the cell-free supernatant transferred to a cuvette. The absorbance was first measured at 280nm to determine the relative protein concentration, and then the absorbance at 490 nm was recorded every minute for 90 min on a Varian DMS 100s spectrophotometer' Haemolymph melanization assays of parasitised larvae were conducted 0,2, 4, 6, 12, 24, 36, 48, 72 and 96 h post-parasitism. To investigate the effects of physical insult on the melanization response, unparasitised larvae from the Bt- susceptible line were each pierced once by a sterile micropin of similar diameter to the ovipositor of V. canescens, and melanization assays conducted after 12 hours. At least 5 replicates were performed for each treatment. To investigate the effects of ingested tropolone on the melanization response, 4th instar larvae from the Bt-tolerant and susceptible lines were maintained on standard diet containing l ppm tropolone for 24h and melanization assays conducted. 2.4 Bt-toxin bioassay of parasitised larvae Three mating pairs of adult E kuehniella from both Bt-susceptible and tolerant (4000ppm F7 colony, LC50 2591ppm) colonies were allowed to lay eggs on fresh food for two days at the control temperature. After 25 days half the 100 resulting 4th instar larvae from each colony were singly parasitised by V canescens wasp (RP strain) as described above. The parasitised and unparasitised larvae from both susceptible and tolerant strains were then allotted into groups of 20 larvae and transferred to diet containing, variously: 0,100, 1000, 2000 and 4000ppm of the Bt formulation. There were five replicates at each concentration. The Bt-susceptible and Bt- tolerant larvae were maintained for 10 days, and larval survivorship was recorded. 2.5 Low-density gradient centrifugation of gut content 50-100 third to fifth instar larvae were washed in ethanol and dried on filter paper. The midgut was removed by cutting off head and terminal segments and homogenized a ice-cold anticoagulant solution (30 mM Trisodium Citrate, 26 mM Citric Acid, 20 mM EDTA, 15 mM NaCl, pH 5.5) containing phenylthiourea (PTU). The crude extractwas centrifuged at 10 0009 for 10 minutes and 1.5m1 of gut extract was added to 15 ml of a solution of 44.39 KBr in 100m1 and overlaid with 0.9% NaCl to a final volume of 30 ml. The tube was inserted into a SWT|42 rotor and spun at 24OOO rpm for 17 hours at 1OoC. The gradient was eluted in ca lml fractions covering densities of 1.15 g/ml (top fractions) to 1.45 g/ml (bottom fractions). The separation of lipophorin particles and lipid-free plasma components was visible after centrifugation by whitish protein bands in the low-density arca of the gradient. To reduce lipid oxidation during centrifugation B-mercaptoethanol (B- ME, final concentration 2 mM) and a reactive oxygen scavenger nitro blue trizolium (NBT, final concentration 1Opg/ml), were added to protein extracts. Under these conditions a pink-coloured band of oxidised NBT was visible in the low-density area of the gradient, an indication for the production of reactive electrons in lipophorin-containing fractions. 2.6 Protein separation and Western blots SDS polyacrylamide-gel electrophoresis on a Mini-Protean ll electrophoresis unit (Bio Rad) was performed as described previously (Ma et al., 2006). 101 Molecular weights were determined using prestained SeeBlueTM (lnvitrogen) molecular weight markers. The proteins were blotted onto a nitrocellulose membrane (Amersham) as described by $1). Protein amounts were measured in the spectrophotometer (280nm) and identical aliquots from each gradient fraction were used for comparison of plasma and gut extracts from immune- induced and non-induced larvae. The amount of protein loaded was ca. 5 pg/lane in Western blots or as indicated in the figure legends. The blotting efficiency was determined by staining the blot with Ponceau S. for Western-blot. Antiserum against G. mellonel/a lipophorin and vWD domain of apolipophorin I was from G. Ma (23) and antiserum against PPO from Manduca sexfa was kindly donated by M. Kanost (Kansas University, Manhattan KS, USA). Antibodies were diluted 1:10000 before incubation with Western blots and visualised with alkaline phosphatase conjugated goat anti-rabbit antibodies. 2.7 Súaúisfical analysis The rate of the melanization reaction was estimated for each replicate as the slope of the plot of absorbance against time (Rahman et a|2004). The effects of time interval following parasitism on the rate of the melanization reaction were analysed by one-way ANOVA, followed by Dunnet's test with unparasitised larvae treated as the control, using the statistical software package JMP V4.0.4 (sAS 2001\. Median lethal concentration (LCso) values were estimated by probit analysis using POLO-PC software (LeOra Software, Berkeley, CA). Samples were considered significantly different if the 95% confidence intervals (95% Cl) did not overlap. 102 3. Results 3.1 Plasma melanization in parasitised larvae Melanization assays of cell-free haemolymph from unparasitised larvae of the Blsusceptible (non-induced) E. kuehniel/a line showed that the melanization reaction proceeded at a low rate (see also (28) and Fig 1), which was almost undetectable. ln contrast, cell-free haemolymph from unparasitised E. kuehnietta larvae from the Bt-tolerant (immune-induced) line showed a high rate of melanization (F=157.8, df=1,108 p<0.0001). While the elevated rate of melanization is significantly reduced in parasitised larvae it remains at a higher rate than in susceptible parasitised larvae (Fig. 2). A one-way ANOVA revealed the melanization rates following parasitism by V. canescens showed a transient increase, which was significant (F=16.62, df=8, 54, p<0.0001), similar to the transient increase in Bt-susceptible larvae, except that the rate of melanization was higher in tolerant larvae. To examine whether the observed transient induction was based on wounding or other stress factors, melanization assays were performed after stabbing larvae with a sterile needle. The rate of plasma melanization from Bt- susceptible (non-induced) larvae ionducted 12h after the physical insult revealed that the reaction proceeded at an undetectable rate, with no significant difference from untreated controls (not shown). This suggests that parasitism causes significant reduction in hemolymph melanization, which does not affect toxicity in the gut. lf phemnoloxidase in the hemolymph and gut are regulated independently, this raises the question whether Bt-toxicity is affected in larvae with reduced phenoloxidase activity in the gut lumen? 3.2 Plasma melanization in tropolone-treated laruae Plasma from 4th instar larvae that were maintained on diet containing l ppm tropolone lor 24h showed a significantly lower rate of melanization in the Bt- tolerant (immune-induced) larvae compared to untreated controls (F=39.19, df=1, 12, p<0.0001 , Fig. 3). There was no significant difference in the melanization rate 103 between tropolone-treated Bt-tolerant (immune-induced) larvae and both tro polone-treated and untreated Bt-susceptible (non-ind uced ) larvae. 3.3 Tropolone-treatment and parasitism success Analysis of the developmental parameters wasp head capsule width, development time and survivorship revealed no significant differences between V. canescens emerging from singly parasitised tropolone-treated and non- treated E. kuehniella larvae (Table 1). This suggests that tropolone-treatment does not affect suitability of larvae as a host of the parasitoid, Alternatively any effect that tropolone may have on the physiology of non-parasitised larvae may have been abolished by the effects of epistatic immune-suppressive maternal secretions. 3.4 Bt-tolerance in parasitised laruae There were no significant differences between the LCso values for parasitised and unparasitised larvae of either the Bt-tolerant strain (parasitised: LC5s=$737, 95% Cl=1660-679412; unparasitised: LC5s=5371 ,95o/o Cl=3547-239170) or Bt- susceptible strain (parasitised: LC5s=1 147,95o/o Cl=561-1867; unparasitised: LCso=1 859, 95% Cl=1 1 97 -2526)' This suggests that the observed tolerance to the Bt-toxin in the gut is not dependent on plasma-mediated immune-induction in the hemolymph but is modified or regulated independently in the gut lumen. 3.5 Bt-tolerance in tropolone-treated laruae Since there are no specific inhibitors of coagulation, we decided to inhibit melanization in Bt-tolerant (immune-induced) larvae to determine whether tropolone-treatment of larvae, which causes specific inhibition of melanization, can affect Bt-toxicity. lf melanization is regulated independently from other humoral defence reactions, such as coagulation, reduction in melanization may or may not affect susceptibility to the toxin. Although tropolone-treatment reduced the rate of melanization in Bltolerant (immune-induced) larvae (Fig. 3), tolerance to the toxin in tropolone-treated 104 larvae was similar to non-treated larvae (Table 2). This suggests that reduction in melanization has no effect on toxicity, which implies that melanization is part of the elevated immune status in Bt-tolerant larvae, but not responsible for the protection against the toxin. 3.6 Bt-toxin interacts with lipophorin particles in the gut Western blots of Bt-tolerant (immune-induced) and susceptible (non-induced) larvae were analysed for possible differences in the amounts of prophenoloxidase (PPO) using antibodies against PPO from Manduca sexfa. ln protein extracts from hemolymph plasma the antibodies cross-reacted slightly with two or three bands around 70 kDa, which could represent isoforms and/or proteolytically cleaved phenoloxidase (Fig. 4). Bt{olerant larvae previously exposed to Bt-formulations (as indicated) have higher levels of PPO. The amount of phenoloxidase labelling is not significantly reduced in Bt-tolerant (4000ppm) larvae that have been kept in the absence of Bt for two subsequent generations (Res-Bt). To examine possible protein changes in Bttolerant (immune-induced) and susceptible (non-induced) larvae, we analysed hemolymph and gut proteins for possible differences. When hemolymph plasma proteins were examined on SDS-PAGE (Fig. 5 Hemolymph) and on Western blots (not shown), no significant protein differences were observed. Since the toxin may be inactivated in the gut lumen by a coagulation reaction, we also analysed gut proteins for possible changes in immune-induced and non-induced larvae. Proteins from the gut showed a significant amount of lipophorin, which was altered somewhat in extracts from'Bt-tolerant larvae. While non-induced larvae showed normal patterns of lipophorin proteins with a 230 kDa apolipophorin I and a 75 kDa apolipophorin ll (Fig. 5), the immune- induced larvae less 75 kDa protein and instead had a 150 kDa protein (arrow), which cross-reacted with antibodies against lipophorin. The missing band at 75 kDa and the novel band at 150 kDa suggest covalent dimerization of apolipophorin ll in tolerant strains, which could also include covalent linkages of 105 exchangeable apolipoproteins, immune proteins and lipids. Since we have recently uncovered a close association of PPO with lipid particles (Rahman et al., in press), we examined a possible cross-linking of apolipoproteins and PPO in immune-induced larvae. When Western blots were incubated with antibodies against PPO, we noticed additional bands around 150 kDa which were labelled (Figure 5). This indicates that cross-linking in lipid particles from immune- induced gut extracts comprise homo- and heterodimers including apolipoproteins and associated immune proteins such as PPO. 3.7 Separation by low-density gradient centrifugation Previous studies indicated that endotoxin interacts with lipophorin particles from hemolymph plasma lrom Gatteria mellonel/a (36) (37). To examine a possible interaction of gut lipophorin with endotoxin, we mixed gut extracts with mature Cry1Ac and separated the lipophorin particles by low-density gradient centrifugation. Under these conditions some of the toxin was found in fractions (Figure 6), which contained the major lipophorin proteins. Some of the labelling is detected in lipophorin fractions at the top of the gel having formed large protein aggregates (not shown). This is a possible indication that Cry1Ac binds to gut-derived lipophorin particles. '106 4. Discussion Taken together these results indicate that immune suppression by parasitoids does not affect susceptibility of host larvae to Bt-toxin. This implies that defence components located in the hemocoel and gut lumen are regulated independently and function as separate entities. This is an unexpected result, since PO, a major humoral defence component in lepidopteran insects is derived exclusively from hemocytes and transported to other tissues of the body, where it is involved in processes, such as cuticle hardening (39) and defence (5). This study indicates that while melanization is a good indicator for immune induction and suppression, phenoloxidase is not directly involved in the response to the toxin. This raises important questions: lf melanization is not relevant to the protection against the toxin, why is prophenoloxidase activated in Bt-tolerant larvae? More importantly, what are the alternative immune pathways that protect against the toxin? One possible answer to the first question is that the mechanism of up- or down- regulation of immune reactions may include melanization in conjunction with other reactions, such as coagulation, even though these can be separated at the genetic (33)or metabolic level (6). lf melanization is not relevant for Bt-tolerance, which defence pathways protect the larva against the toxin? lmmune suppression by parasitoids has been used to examine baculovirus virulence (45). Co-infection of Manduca sexta larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa catifornica M Nucleopolyhedrovirus (a3). Conversely, preliminary experiments in Helicoverpa armigera indicate that the elevated immune status, which provides protection against Bt-toxin (Ma et al', 2006), also protect against baculovirus infection (unpubl.). While baculoviruses and Bt- toxin share similarities, there are differences, both in the path of infection and in host defence. Firstly, while a protective line of defence inside the gut lumen is not ruled out, most of the virus-related defence reactions appear to take place after the virions are released from infected gut cells. According to one model, virions may spread through the larval body in extracellular matrix space 107 provided by trachea and other tissues attached to the gut (18). ln immune- competent larvae virions are trapped by the immune system and prevented from spreading (11, 43, 44). Since there is no evidence that virions are phagocytized by tracheal cells and since insect hemocytes are unable to cross the basement membrane lining of the hemolymph, the only inducible defence pathway that can prevent the spreading of virions in this tissue environment is the inactivation by aggregation and coagulation of defence molecules, where the virion-containing coagulation products inside the trachea attract hemocytes to form nodules (a3). The relevance to the protection against Bt-toxin is the fact that recognition of the pathogen and inactivation (at least at an initial stage) occurs outside the hemocoel lining. This implies that the factors involved in recognition and inactivation of pathogens and toxins are able to operate away from hemocytes, if not in their absence. What are the candidates for a cell-free recognition and inactivation system? While a number of possible pro-coagulants have been identified in arthropods, the possible function and mode of action seems to differ among different species (10, 14, 15, 19,22,26, 40, 42).The main feature of pro-coagulants is their unique ability to recognize and form specific aggregates around pathogens and toxins, which effectively separate the damaging effects from the surrounding physiological environment of the host. Many of the pro-coagulants, such as lipophorin (22) and hexamerin (23), serve metabolic and storage functions and in performing those functions are transferred across the hemocoel lining to accumulate in other tissues and body cavities, such as oocytes, epidermis and the gut lumen. Given their ubiquitous presence and their potential function as pro-coagulants, these structures could form a first line of defence against intruding pathogens. For example, lipophorin, which is secreted into the gut lumen may be engaged in lipid transport between the gut nutrients and the gut cell lining. ln immune-induced larvae lipophorin particles may be modified to attract Bt-toxin molecules, causing sequestration of the mature toxin before it can reach the brush border membrane. The observation that apolipophorin ll forms SDS-insoluble dimers in the gut of immune-induced larvae suggests that the protein has the capability to engage in strong protein- 108 protein linkages or to form covalent linkages, such as those mediated by transglutaminase. The observation that parasitoid-mediated suppression of the immune system in the hemocoel does not affect the defence against gut-derived Bt-toxin, suggests that the cell-free coagulation reactions in the gut lumen operate independently from the hemolymph. The fact that this is different from viruses, where immune suppression led to increased virulence, is probably due to the different line of virus defence, which is not in the gut lumen but inside the hemocoel where virions are released from infected gut cells. ln contrast, Bt-toxin may be inactivated in the gut lumen before it can reach the gut cells. The mechanism for separate regulation of pro-coagulant in the plasma and gut is not known, but if lipophorin is a major pro-coagulant in the gut lumen as indicated by the predominance of apolipophorin in gut extracts (Fig. 5), it may not be transported directly into the gut lumen, but modified by gut cells on its passage through the epithelium. The observation that mature Cry1Ac is found with lipophorin after low-density gradient centrifugation, suggest that Cry1Ac binds to lipophorin particles and can be transported into low-density areas of the gradient, where some of the proteins aggregate into coagulation products that are not resolved by SDS-PAGE. ln summary, the observations that lipophorin particles exist in the gut lumen that can act as a pro-coagulant are compatible with the existence of an immune- inducible Bttolerance mechanism based on the sequestration of the toxin by a coagulation reaction inside the gut lumen. The emergence of insect pest populations that become tolerant to Bt-toxin by transient induction mechanisms that can be transmitted to subsequent generations by a maternal effect (24,30) has possible implications for resistance management strategies in the field. While the level of tolerance is well below the toxin levels expressed in most transgenic plants, inducible tolerance mechanisms may nevertheless become relevant in situations where insects are exposed to low levels of the toxin or where semi-dominant heterozygotes of genetic mutant insects emerge with increased levels of resistance. lf the two mechanisms are additive, the 109 combined levels of resistance may become a threat to transgenic resistance strategies. Acknowledgements; We acknowledge the receipt of PhD Scholarships for MMR and HSLR from the University of Adelaide. 110 5. References 1. Asgari, S., G. Zhang, R. Zareie, and O. Schm¡dt. 2003. 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Molecular cloning of insect pro-phenol oxidase - a copper- containing protein homologous to arthropod hemocyanin. Proceedings of the National Academy of Sciences (USA,) 92:7774-7778. 18. Kirkpatrick, B.4., J. O. Washburn, E. K. Engelhard, and L. E. Volkman. 1994. Primary infection of insect tracheae by Autographa californica M nuclear polyhedrosrs virus. Virology 203:184-6' 19. Korayem, A. M., M. Fabbri, K. Takahashi, G. Scherfer, M' Lindgren' O. Schmidt, R. Ueda, M. S. Dushay, and U. Theopold. 2004. A Drosophila salivary gland mucin is also expressed in immune tissues: evidence for a function in coagulation and the entrapment of bacteria. Insect Biochemistry and Molecular Biology 34:1297-1304. 20. Lavine, M. D., and N. E. Beckage. 1995. Polydnaviruses - potent mediators of host insect immune dysfunction. Parasitology Today l1:368-378. 21. Lee, K. M., K. Y. Lee, H. W. Choi, M. Y. Gho, T. H. Kwon, S. Kawabata, and B. L. Lee. 2000. Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin- like protein in the presence of dopamine. European Journal of Bioch e mi stry 267 =3695-3703. 112 22. L¡, D., G. Scherfer, A. M. Korayem, Z. Zhao, O. Schmidt, and U. Theopold. 2002. lnsect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. lnsect Biochemistry and Molecular Biology 32:919-928. 23. Ma, G., D. Hay, D. L¡, S. Asgari, and O. Schmidt. 2006. Recognition and inactivation of LPS by lipophorin particles. Developmental and Comparative lmmunology 30, 61 9-626. 24. Ma, G., H. Roberts, M. Sarjan, N. Featherstone, J. Lahnstein, R. Akhurst, and O. Schmidt. 2005. ls the mature endotoxin Cry1Ac from Bacillus thuringiensrs inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae? Insect Biochemistry and M ole cu lar Bi ol ogy 35:7 29-7 39. 25. Morita, Y., E. Matsumura, T. Okabe, M. Shibatâ, M.Sugiura, T. Ohe, H. Tsujibo, N. lshida, and Y. Inamori. 2003. Biological activity of tropolone. Biological and Pharmaceutical Bulletin 26=1 487'1 490. 26. Muta, T., and S. lwanaga. 1996. The role of hemolymph coagulation in innate immunity. Current Opinion in lmmunology 8:41-47. 27. Nagai, T., and S. Kawabata. 2000. A link between blood coagulation and prophenol oxidase activation in arthropod host defense. Journal of Biological Chemistry 27 5:29264-7 . 28. Nappi, A. J., F. Frey, and Y. Garton. 2005. Drosophila serpin 274 is a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response. Journal of Insect Physiology 51=197-205. 29. Nomiya, K., A. Yoshizawa, N. C. Kasuga, H. Yokoyama, and S. Hirakawa . 2004. Synthesis, solid-state characterization and antimicrobial activities of three different polymorphs of a coppe(ll) complex with 4- iso pro pyltro po lo ne ( h i no kitiol ) . I n organ i ca Ch imi ca Acta 357 :1 1 68-1 17 6. 30. Rahman, M. M., H. L. S. Roberts, M. Sarjan, S. Asgari, and O. Schmidt. 2004. lnduction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella. Proceedings of the National Academy of Sciences IUSA) 101:2696-2699. 31. Rahman, M. M., H. L. S. Roberts, and O. Schmidt. 2004. The development of the endoparasitoid Venturia canescens in Bt-tolerant, immune induced larvae of the flour moth Ephestia kuehniella. Journal of I nvertebrate P athology 87 29-1 31 . =1 32. Reeson, A. F., K. Wilson, A. Gunn, R.S. Hails, and D. Goulson. 1998. Baculovirus resistance in the Noctuid Spodoptera exempta is phenotypically plastic and responds to population density. Proceedings of the Royal Society of London - Series B: Biological Sciences 265=1787- 1791. 33. Rizki, T. M., R. M. Rizki, and R. A. Bellotti. 1985. Genetics of a Drosophila phenoloxid ase. Mol Gen Genet 201:.7-13. 113 34. Rogers, D. 1972. The lchneumon wasp Venturia canescens: oviposition and avoidance of superparasitism. Entomologia Experimentalís et Applicata 15:1 90-194. 35. Sarjan, M. 2002. Resistance against endotoxin from Bacillus thuringiensrs endotoxins in lepidopteran insects. University of Adelaide, Adelaide. 36. Schmidt, O., M. M. Rahmaî, G. Ma, U. Theopold, Y. Sun, M. Sarjan, M. Fabbri, and H. S. L. Roberts. 2005. Mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides ( H ypothes is). I nve rte b rate S u rviv a I J o u rn a I 2:82-90 . 37. Shelby, K. S., O. A. Adeyeye, B. M. Okot-Kotber, and B. A. Webb. 2000. Parasitism-linked block of host plasma melanization. Journal of I nverte brate P ath ol ogy 7 5:21 8-225. 38. Soderhall, K., and L. Gerenius. 1998. Role of the prophenoloxidase- activating system in invertebrate immunity. Current Opinion in lmmunology 10:23-28. 39. Sugumaran, M. 1996. Role of insect cuticle in insect immunity., p. 355- 374. ln K. Söderhäll, S. lwanaga, and G. Vasta (ed.), New Directions in lnvertebrate lmmunology. SOS Publication., New Haven. 40. Theopold, U., D. L¡, M. Fabbri, G. Scherfer, and O. Schmidt.2002. The coagulation of insect hemolymph. Cellular and Molecular Life Sciences 59:363-372. 41. Theopold, U., C. Samakovlis, H. Erdjument-Bromagê, N. Dillon, B. Axelsson, O. Schmidt, P. Tempst, and D. Hultmark. 1996. Helix pomatia lectin, an lnducer of Drosophila immune response, binds to hemomucin, a novel surface mucin. Journal of Biological Chemistry 271=12708-12715. 42. Theopold, U., O. Schmidt, K. Soderhall, and M. S. Dushay. 2004. Coagulation in arthropods: defence, wound closure and healing. Trends in lmmunology 25=289-294. 43. Washburn, J. O., E. J. Haas-Stapleton, F. F. Tan, N. E. Beckage, and L. E. Volkman. 2000. Co-infection of Manduca sexfa larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa californica M Nucleopolyhedrovirus. Journal of lnsect Physiology 46: 1 79-1 90. 44. Washburn, J. O., B. A. Kirkpatrick, and L. E. Volkman. 1995. Comparative pathogenesis of Autographa californica M nuclear polyhedrosis yftus in larvae of Trichoplusia ni and Heliofhis virescens. Virology 209:561-8. 45. Washburn, J. O., B. A. Kirkpatrick, and L. E. Volkman. 1996. lnsect protection against viruses. Nature 383:767. 114 46 Yu, X.-Q., and M. R. Kanost. 2003. Manduca sexfa lipopolysaccharide- specific immulectin-2 protects larvae from bacterial infection. Developmental and Comparative lmmunology 27=189-1 96. 47. Zhang, G., Z.-Q. Lu, H. Jiang, and S. Asgari. 2004. Negative regulation of prophenoloxidase (proPO) activation by a clip'domain serine proteinase homolog (SPH) from endoparasitoid venom. /nsecf Bioch e mi stry a nd Molecu I ar Biology 34=477 -483. 115 6. List of tables Table l. Developmental parametersfor V.canescens emerging from singly parasitised tropolone-treated and non-treated E. kuehniella larvae. Least squares Mean Percentage N mean head development parasitoid capsule width (mm) time (d) survival Treatment 25 1.260 24.18 80.0 Control 24 1.263 24.24 79.2 Table 2. Exposure of Bt-tolerant strain with two different doses of the Bt- formulation after treatments with tropolone". Tropolone Bt-toxin No of Percentage Percentage Mean (ppm) formulation larvae larval adult develop- survival (ppm) exposed emergence mentaltime after 7 (days) days 0 5000 51 82 47 28.5 1 000 5000 50 76 44 27.59 0 10000 50 54 34 27.47 1 000 1 0000 51 75 33 28.53 "The experiment was performed with 4th instar larvae of an E kuehniella strain, which has been kept for seven generations on food with 4000ppm of the Bt-formulation under laboratory conditions. Exposure to Bt-formulation above 5000ppm kills all non-induced (susceptible) larvae. 116 Figure Legends Figure 1. Melanization assays of cell-free haemolymph from immune-induced Bt-tolerant (open circle) and non-induced Bt-susceptible (closed circle) E. kuehniella larvae (see also 31). Bars represent SEM. Figure 2. Mean rates of the melanization reaction [as the slope of the plot of absorbance against time (arbitrary units)l of cell-free haemolymph from E. kuehnietta larvae at different time intervals following parasitism by V. canescens. Rates of melanization from Bt-susceptible (grey bars) and the Bt- tolerant (black bars) larvae are shown for comparison at each time interval. Bars represent SEM. Figure 3. Mean rates of the melanization reaction [as the slope of the plot of absorbance against time (arbitrary units)l of cell-free haemolymph from Bt-tolerant and Bt- susceptible line E. kuehniella larvae maintained on diet containing 1 ppm tropolone for 24h. Bars represent SEM. Figure 4. Prophenoloxidase-detection of Western blots containing hemolymph plasma proteins from susceptible and Bt-tolerant larvae kept on various concentrations (11Oppm, SOOppm, 2000ppm, 4000ppm and 8000ppm) of Bt- formulation (Dipel). Antibodies against PPO from M. sexta were visualised with alkaline phosphatase-conjugated secondary antibodies. The level of phenoloxidase labelling in Bt-tolerant larvae is maintained for several generations in the absence of Bt (Res-Bt). Figure 5. Gut protein extracts separated by SDS-PAGE and analysed on Western blots using anti-lipophorin and anti-PPO antibodies. Equal amounts of proteins from susceptible (non-induced) and Bt-tolerant (immune-induced) larvae were applied. The protein bands of ca 230 and 75 kDa cross-react with antibodies against lipophorin and represent apol and apoll respectively. These antibodies also cross-react to a 150 kDa protein in immune-induced larvae (arrow). A similar band is detected in Western blots incubated with antibodies 117 against PPO, which is associated with lipophorin particles (Rahman et al., in press). The appearance of a 150 kDa band in immune-induced larvae, coincides with a ladder of thin bands leading to the 150 kDa band, which could be covalently attached lipid molecules in addition to covalent dimerization of the apolipophorin ll or hetero-dimerization with PPO by cross-linking agents, such as transglutaminase. Figure 6. Low-density gradient centrifugation of gut extracts from immune- induced larvae mixed with trypsin-activated Cry1Ac. Aliquots from the gradient were analysed on Western blots using antibodies against Cry1Ac, van Willebrand Factor D domain of apolipophorin I from Galleria mellonella (23) and prophenoloxidase from Manduca sexfa (16).Trypsin-digested protoxin comprised a mixture of protein bands with a 69 kDa band as a major protein in addition to smaller bands down to 60 kDa, which are over-digested mature toxin and high molecular weight proteins which could be toxin oligomers (26). Below is a Coomassie blue staining of hemolymph plasma separated in the same centrifugation run showing arylphorin found only in high-density fractions (7-13), whereas apolipophorin I and ll are only found in low-density fractions (13-23) with a peak in fraction 17. Cry1Ac and PPO are found across the gradient and have to be associated with lipophorin particles to be distributed over low-density fractions. Note that Cry1Ac coincides with the major peak of apolipophorin at fraction 17, whereas PPO is also found in lipophorin sub-populations with very high lipid contents, which is consistent with a dual function of PPO as a melanization enzyme and oxygen carrier (Rahman et al., in press). 118 Figures Figure I 350 E q 300 -a o \fot 250 È, IE 200 -¿ o o tr 150 IE .ct ¡- 100 ,/ o 3n ¡t 50 ./ 0 0 20 40 60 80 100 Time (mins) 119 Figure 2 1 8 tr o I 6 I Susceptible I Tolerant (E 1 4 .N gc 1 2 o 1 0 = Ë I .z 6 o IE 4 o o- 2 È 0 UP 2 6122436487296 Hours after parasitism 120 Figure 3 4 c 3.5 o (úrO") É o E 2-5 (u th) gc q 1.5 t rÈo1 o (ú T É o.s I 0 Gontrol Tropolone Control Tropolone Bt-tolerent Bt-susceptible 121 Figure 4 Troo Tuoo Trooo Toooo Trooo T-"* Sus M -25OKD " - 98KDa - 641(Da ' - 50KDa Figure 5 Gut extract Sus Tzooo Toooo M Sus Trooo T¿ooo M Sus Trooo Toooo 254 k ( Ë{ ,ü - ïl Coomassie blue anti-lipophorin anti-PPO 122 Figure 6 high density low density M 7 9 11 13 15 17 19 21 23 64 \ às Glll Æ {;.IÞ-' o-Cryl Ac rl q-PPO 64 a* rr,ül q'a¡,i {rt"a I ,,u*tÜ. 7M911131517192123 98 Coomassie blue 64 123 tnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions 3 Ò Sequestration of mature Bacillus thuringiensis endotoxin by liPoPhorin Pafticles M. Mahbubur Rahman and Otto Schmidt lnsect Molecular Biology Laboratory, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia Manuscript under review in Applied and Environmental Microbiology 124 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorshiP Manuscript under review in Applied and Environmental Microbiology as: M. Mahbubur Rahman, Harry L. S. Roberts and Otto Schmidt: Sequestration of mature Bacillus thuringiensis endotoxin by lipophorin particles Mohammad Mahbubur Rahman (Candidate) Designed experiments, performed experimental work, analysed and interpreted data, and co-wrote manuscriPt. Otto Schmidt (Principal Supervisor) Supervised work, assisted in analysis and interpretation of data, co-wrote manuscript and acted as communicating author. Signed: Date ,1/, "2& 06 Moh am m ad M ah bu bu r Rah man I gave consent on behatf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. Signed Date: ?g(('cþ Otto Schmidt 125 lJnder review in Applied and Environmental Microbiology Secfion; I nvertebrate Microbiology Sequestration of mature Bacillus thuringiensts endotoxin by lipophorin particles. M. Mahbubur Rahman and Otto Schmidt* lnsect Molecular Biology Laboratory, School of Agriculture and Wine, University of Adelaide, Glen Osmond SA 5064, Australia * Corresponding author Email: [email protected] Phone: +61-83037252 Fax: +61-8303 7109 126 Abstract Tolerance lo Baciltus thuringiensis crystal endotoxins (Bt-toxins) is correlated with an elevated immune status in larvae of the flour moth Ephestia kuehniella (Rahman, M. M., H. L. S. Roberts, M. Sarjan, S. Asgari, and O. Schmidt2004. PNAS 101:2696-2699). These studies imply that insect larvae with an elevated immune status can potentially engage the mature toxin in a cell-free coagulation reaction, inactivating the toxin in the gut lumen before it can reach the brush border membrane. This suggests that immune components exist in the gut lumen that recognise and inactivate the toxin in the absence of cells. Here we show that lipophorin particles in the gut interact with the mature toxin like lectin that inactivates the toxin by a coagulation reaction. Keywords: Bacillus thuringiensls, crystal toxin, tolerance, immune-induction, lipophorin, coagulation. 127 1. Introduction Endotoxins from the soil bacterium Bacillus thuringiensis (Bt-toxins) are among the most effective biopesticides due to a number of unique properties, such as high lethality in many invertebrate pest species and virtual absence of toxicity in mammals. Moreover, even with the extensive use of transgenic plants expressing Bt-toxins (15) and traditional sprays of Bt-formulations to control insect pests, only few cases of genetic resistance against Bt-toxins have been reported from field populations (3). This could indicate that mutations inactivating crucial Bt-receptors (4), are extremely rare and remain low in insect populations (14). Alternatively, mutant receptors may not always produce resistance phenotypes in some insect species due to the presence of multiple Bt-receptor gene families (2) and post-translational receptor modifications (8) with overlapping binding properties. While known cases of receptor inactivation generate high levels of resistance, lethality is often restricted to neonates or early larval stages (5). One explanation is that older larvae stop feeding after encountering contaminated food and with enough fat reserves available may survive the bioassay or pupate prematurely. However, some insects are known to resume feeding after several days overcoming othen¡uise lethal doses of the toxin. This could suggest that other modes of resistance exist, some based on inducible mechanisms (6). We have recently identified novel tolerance mechanisms against low to medium levels of toxin in immune-induced insects (12) (10). The observed tolerance is dependent on the induction of immune proteins, which occurs with the ingestion of sub-lethal doses of Bt-formulations (12). The conclusions from these studies imply that larvae with an elevated immune status can potentially engage the mature toxin in a cell-free coagulation reaction, inactivating the toxin in the gut lumen before it can reach the brush border membrane. This suggests that immune components exist in the gut lumen that recognise and inactivate the toxin in the absence of cells. 128 Cell-free defence reactions have recently been discovered in Ephestia kuehnielta, where lipophorin particles are found to be associated with immune proteins, such as pattern recognition proteins, prophenoloxidase and its activating proteases (11). lsolation of particles by low-density gradient centrifugation revealed that these particles possess the ability to interact with lipopolysaccharide (LPS), forming aggregates that prevent LPS from interacting with active lipophorin particles (9). The aggregation appears to require at least two steps, one where the LPS molecule is incorporated into the lipid moiety of the lipophorin particle and a subsequent activation of putative lectin-like adhesion molecules that cross-link sugar determinants across lipid particles. While the first step occurs in the absence of calcium, the second step requires calcium but can be simulated with the addition of oligomeric adhesion molecules (11). For example, when Gal-specific peanut agglutinin (PNA) was added to isolated lipophorin particles in the absence of calcium, the outcome was aggregation of lipid particles into globular structures with F|TC-conjugated PNA trapped inside a lipid layer effectively shielding the lectin from further interactions (9,11). Since many pore-forming toxins have lectin-like properties, we asked whether the B. thuringiensis crystal toxin can interact with lipophorin particles in the gut lumen and become inactivated by aggregation reactions. Here we show that isolated lipid particles from hemolymph plasma and the gut lumen are able to interact with mature Cry1Ac like a lectin. This suggests that inducible tolerance is mediated by the secretion of immune components into the gut, which bind and sequester damaging toxins into coagulation products before they can reach the gut lining. 129 2. Materials and Methods 2.1 Bt-toxin The Cry1Ac protoxin used in the coagulation assays was purified from B. thuringiensrs subsp. kurstaki HD73 by sucrose gradients (M. Sarjan and N. Featherstone, unpubl. results). Purified crystals were suspended in a solution containing 30 mM NazCOg and 1% mercaptoethanol at pH 9.6 and digested with trypsin or gut juice extracts from Piers rapae. Aliquots of the digestion mixture were analysed on SDS-PAGE after different time periods. Incubation conditions were chosen, were the protoxin has completely disappeared and the mature toxin at 69kDa and a tetrameric complex were the only toxin-staining proteins (Fig. 1). 2,2 Low-density gradient centrifugation 50-100 third to fifth instar larvae were washed in ethanol and dried on filter paper before extracting hemolymph by cutting a for-leg and bleeding into a ice- cold anticoagulant solution (30 mM trisodium citrate, 26 mM citric acid, 20 mM EDTA, 15 mM NaCl, pH 5.5) containing phenylthiourea (PTU). Hemocytes were removed by centrifugation at 50009 for 3 minutes. 1.5m1 of plasma was added to 15 ml of a solution of 44.39 KBr in 100m1 and overlaid with 0.9% NaCl to a final volume of 30 ml. The tube was spun at 24000 rpm (SWT!42 rotor) for 17 hours at 1OoC. The gradient was eluted in ca 1ml fractions covering densities of 1.15 g/ml (top fractions) to 1.45 g/ml (bottom fractions). Midgut preparations were obtained by cutting head and abdomen segments with a microscissor and removing midgut section with a pair of forceps. The section (from about fifty larvae) were immersed in ice-cold anti-coagulation buffer and homogenised. After centrifugation at 50009 for 10 minutes the supernatant was separated by low-density gradient centrifugation as described above. 2.3 Western þloús Aliquots of dialysed low-density gradient fractions were mixed with loading buffer, heated at 65oC and analysed by SDS-PAGE and Western blots using 130 antisera against recombinant vWD from Gatteria mellonella, which is part of apolipoprotein I (Ma, et al., 2006) and Cry1Ac (kind gift from Sarjeet Gill). After staining gels with Coomassie blue or Western blots with phosphatase- conjugated secondary antibodies, the apolipoprotein I band at 230 kDa was used as an indication for the presence of soluble lipid particles and quantified using a pixel estimation program (11). 2.4 lnsecú gut staining Gut tissues were dissected from caterpillars and midgut sections were separated with microscissors leaving the peritrophic membrane intact. The gut was fixed in paraformaldehyde (4%), containing 0.5% Tween 20 in the presence or absence of phenylthiourea (PTU) for several hours. After extensive washing overnight, gut tissues were treated with antibodies against recombinant vWD from Galteria mellonella, which is part of apolipoprotein I (Ma, et al', 2006)' lncubation of primary antibody (1:1000 dilution), and FITC- or TRITC- conjugated secondary antibodies (1:5000 dilution) were done for at least four hours, followed by four washing steps each. To keep non-specific staining to a minimum, fixation and incubations were performed in the presence of PTU. 2.5 Bt-toxin aggregation assay For the measurement of toxin-induced aggregation aliquots of gradient fractions were mixed with mature crystal toxin (see Figure 1) at various concentrations in the absence and presence of calcium (final concentration 1mM) and incubated for t hour at RT. Proteins were dissolved in loading buffer, heated for 10 min at 65oC and separated by SDS-PAGE. To examine sugar-binding properties, incubations were performed in the presence of various sugars to final concentrations of 1 OmM. 131 3. Results 3.1" CrylAc causes aggregation of lipophorin particles Since lectin-like pore-forming toxins from B. thuringiensis have been shown to bind glycoproteins (1) and glycolipids (7), we explored the possibility that crystal endotoxins interact with lipophorin particles like lectins. When mature toxin was added to plasma or LGC-isolated lipophorin fractions, apolipophorin I and ll proteins where selectively removed from the supernatant forming SDS-insoluble aggregates. The observed covalent cross-linking was probably based on transglutaminase activities or prophenoloxidase activation, producing cross- linking intermediates. To test whether aggregation involved lectin-like adhesion by mature toxins as an initial step, we attempted to inhibit the reaction by competing sugar moieties. To demonstrate a reversible sugar competition, the covalent cross-linking of lipid particles had to be precluded. We noticed previously that Ca-free or aged lipophorin fractions fail to aggregate in the presence of LPS but retain the ability to aggregate with oligomeric lectins (11)' We therefore asked whether Cry1Ac interaction with aged lipophorin particles resembled lectin interactions and could be reduced by competition with lectin- binding sugars. When activated Cry1Ac, which forms oligomeric complexes (Figure 1), was added to aged lipophorin particles in the absence of calcium, we observed aggregation illustrated by the absence of apolipophorin I in the supernatant (Fig. 2a). These aggregates were at least partly dissolved in detergent (not shown) and aggregation was reduced in the presence of galactosamine (Fig. 2a). To test whether this reaction was due to bacterial elicitor contaminations in the toxin fraction, we examined the reactivity of aged lipophorin fractions in the presence of LPS. When the reaction was performed in the presence of LPS extracts in the presence and absence of calcium, no aggregation occurred even in the presence of calcium (Fig. 2b). This suggests that the Cry1Ac-mediated aggregation of aged particles was due to its lectin properties rather than other cross-linking activities triggered by bacterial contaminants acting as elicitors in the mature toxin solution. Likewise, residual trypsin in the toxin fraction could have triggered aggregation. When trypsin was used in these assays, aggregation was observed only in the 132 presence of calcium (Figure 2c), whereas no aggregation was observed under the Ca-free conditions used in the toxin experiment (Figure 2d). Again this eliminates possible contaminations of proteases in the aggregation assay by activating particle-specific adhesion molecules. Together, these experiments suggests that the bacterial endotoxin Cry1Ac interacted with lipophorin particles like oligomeric lectins causing adhesive aggregation through cross-linking of lipid particle-specific glycodeterminants. 3.2 Lipophorin-staining in the gut The question is therefore whether Bt-toxin can bind to lipophorin particles from the gut. Previous experiments showed that mature Bt-toxin co-located with lipophorin particles as oligomeric toxin complexes when incubated with plasma and separated by low-density gradient centrifugation (13). To investigate whether Cry1Ac-binding lipophorin particles existed in the gut, we mixed midgut extracts with mature Cry1Ac and separated lipophorin particles by low-density gradient centrifugation. Under these conditions monomeric (Figure 3) and high molecular weight aggregates (not shown) are carried with particles in the low- density area of the gradient (Figure 4, asterix). This suggests that lectins, including pore-forming toxins interact with lipophorin particles to form a complex. 3,3 Lipophorin particles form aggregates in the gut Cell-free defence reaction may form a first line of defence in the gut lumen, which can be effective even in the absence of hemocytes. Given that lipophorin particles involved in extracting lipids from food in the gut lumen are likely candidates for sensing and inactivating damaging organisms and toxins, we examined the gut content from Bt-tolerant and susceptible larvae (12) using confocal microscopy on whole mid-guts. Overall, the gut content in the Bt- tolerant larvae contained more unprocessed food compared to susceptible larvae. When lipophorin-staining was performed on whole mounts, the antibodies stained some of the food pieces in addition to small globular structures (Figure 4), whereas the staining was much more homogeneous and without globular structures in the gut lumen of susceptible larvae. This could suggest that resistant larvae are less effective in digesting food and secrete 133 more and/or different lipophorin particles into the gut lumen. The observed co- location of lipophorin to globular structures in the gut of Bt-tolerant but not in susceptible E. kuehnietta (Figure 4) suggests that lipophorin particles are probably instrumental in immune-related tolerance against Bt-toxin in some insects. 134 4. Discussion The observed aggregation of lipophorin particles by the lectin-like Bt-toxin has profound implications for our understanding of inducible tolerance mechanisms based on cell-free defence reactions. However, our abilities to detect and analyse these r."".tion, are limited due to the highly fragile and reactive nature of immune-activated lipid particles. For example, the isolation of lipid particles by LGC in the absence of EDTA causes spontaneous and rapid aggregation due to activation of proteolytic regulatory cascades that render the particles adhesive. Conversely, particles isolated in the presence of EDTA are stable but quickly lose their ability to respond to elicitors. Under these conditions, the most effective way to use particles for experiments was to dialyse aliquots immediately after LGC fractionation and restore calcium before or during experimentation. Nevertheless, after several days of storage elicitor responses fade away, but lipid particles can still aggregate in the presence of oligomeric lectins. Another problem is the possible biophysical changes to lipophorin particles imposed by LGC. While LGC constitutes a gentle and effective method of separating lipid particles, it inevitably occurs under high salt and changing redox conditions, which may cause changes to the particles. lf these particles represent indeed immune and other sensors, many conditions that differ from the physiological status in the gut lumen or in the hemolymph plasma may 'activate' particles and generate adhesive particles that spontaneously aggregate. Likewise, the oxidization of lipid carbohydrates during centrifugation may have precluded the isolation of glycolipids from LGC-isolated lipophorin particles. When membrane preparations from whole gut are used, Gal- containing glycolipids are detected, which bind to the toxin (7). The presence of lipophorin-containing globules in the gut lumen of Bt-tolerant larvae (Figure 4) suggests a possible inactivation of the toxin by cell-free defence reactions, which can explain the observed tolerance to the toxin by an inducible mechanism. However, it is not clear from our experiments what molecular status lipophorin particles acquire with immune induction. One 135 possibility is that certain immune molecules, such as pattern recognition proteins, are recruited to the particles and in the process acquire the ability to recognize microbial patterns of potentially damaging organisms. Another possibility is that particle-specific glycoproteins or glycolipids are modified in immune-induced larvae to attract pathogens or toxins in the gut lumen. This is particularly interesting, since many pathogenic bacteria and pore-forming toxins are known to use glycodeterminants on the brush border membrane to gain entry into the gut cells. While toxin-binding glycolipids in lipid particles remain to be identified, the presence of lipophorin particles in the gut lumen with glycodeterminants that are similar to the brush border membrane could constitute a first line of defence, where lipid particles interact with damaging organisms and toxins before they reach membrane-bound receptors' A mechanism where membrane-like lipid particles form the first line of defence in the gut lumen raises a number of questions. lf lipid particles are indeed protective, why are immune-active lipophorin particles reduced in the gut lumen of susceptible larvae? One possibility is that immune-activation and secretion of lipid particles into the gut lumen reduces lipid carrier and other metabolic functions of lipophorin particles in the hemolymph, which imposes fitness penalties on the insect. ln fact, exposure to increasing concentrations of Bt- formulations in the food increases developmental time in a direct correlation to the toxin concentration (Rahman, unpubl. data). Thus the insect has to return to non-induced status as soon as the exposure to the toxin is over to compete with other larvae for growth. Another question is how the elevated immune status and with it tolerance to Bt- toxin is transmitted to offspring? Since immune activation is based on inducible regulatory processes rather than mutations, the next generation is expected to start like a non-induced insect. However, when subsequent generations of insects where exposed to increasing levels of toxicity, the tolerance to the toxin (and with ¡t the fitness penalty) increased (M. Rahman, unpubl' data). Subsequent analysis of reciprocal crosses suggested that the mode of transmission is based on a maternal effect (12). Given that lipophorin particles are incorporated into the ooplasma of developing oocytes, it is conceivable that 136 'activated' particles in the circulation of immune-induced females are stored in the egg and become involved in the induction of the immune system of the developing embryo. Experiments are under way to test this assumption. Acknowledgement: The authors thank Nicki Featherstone for the preparation of Cry1Ac crystals, Sarjeet Gill and Mike Kanost for antibodies and Jana Bradley and Natasha Mclnnnes for help with the experiments. This work was supported by a grant from Biolnnovation SA. 5. References 1. Burton, S. L., D. J. Ellar, J. Li, and D. J. Derbyshire. 1999. N- acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain lll lectin-like fold of a Bacillus thuringiensrs insecticidal toxin. Journal of Molecular Biology 287l. 1011- 1022. 2. Chang, W. X. 2., L. J. Gahan, B. E. Tabashnik, and D. G. Heckel. 1ggg. A new aminopeptidase from diamondback moth provides evidence for a gene duplication event in Lepidoptera. /nsect Molecular Biology. 8:171-177. 3. Ferre, J., and J. Van Rie. 2002. Biochemistry and genetics of insect resistance to Bacitlus thuringiensrs. Annual Reviews of Entomology 47= 501-533. 4. Gahan, L. J., F. Gould, and D. G. Heckel.2001. ldentification of a gene associated with Bt-resistance in Heliothis vftescens. Sclence 293l. 857- 860. 5. Gilliland, 4., C. E. Chambers, E. J. Bone, and D. J. Ellar.2002. Role of Bacittus thuringiensrs Cry1 õ-endotoxin binding in determining potency during Lepidopteran larval development. Applied and Environmental Microbiology 68= 1 509-1 51 5. 6. Griffitts, J. S., and R. V. Aroian. 2005. Many roads to resistance: how invertebrates adapt to Bt toxins. BloEssays 27= 614-624. 7. Griffitts, J. s., s. M. Haslam, T. Yang, S. F. Garczynski, B. Mulloy, H. Morris, P. S. Gremer, A. Dell, M. J. Adang, and R. V. Aroian. 2005. Glycolipids as receptors for Bacillus thuringiensls crystal toxin. Science 307:922-925. 8. Knight, P. J. K., J. Garroll, and D. J. Ellar. 2003. Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacittus thuringiensis Cry1Ac toxin. /nsecf Biochemistry and Molecular Biology SU -. 137 I Ma, G., D. Hay, D. L¡, S. Asgari, and O. Schmidt. 2006' Recognition and inactivation of LPS by lipophorin particles. Developmental and Comparative lmmunology 30: 61 9-626 10. Ma, G., H. RobertS, M. Sarjan, N. Featherstone, J. Lahnstein, R. Akhurst, and O. Schmidt. 2005. ls the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae? lnsect Biochemistry and M ol e cu I ar Biology 35=7 29-7 39. 11 Rahman, M. M., G. Ma, H. L. S. Roberts, and O. Schmidt. Cell-free immune reactions in insects. Journal of Insect Physiology 52:.754'762. 12 Rahman, M. M., H. L. S. Roberts, M. Sarian, S. Asgari, and O. Schmidt. 2004. lnduction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella. Proceedings of the National Academy of Sciences (USA) 101:2696-2699' 13 Schmidt, O., M. M. Rahmaî, G. Ma, U. Theopold, Y' Sun, M. Sarjan' M. Fabbri, and H. S. L. Roberts. 2005. Mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides ( H ypoth es is). I nve rte brate S u rv iva I J o u rn a I 2:82-90 . 14 Tabashnik, B. E., A. L. Patin, T. J. Dennehy, Y. B. Liu, Y. Garriere, M. A. Sims, and L. Antilla. 2000. Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm. Proceedings of the Nationat Academy of Sciences of the United Sfafes of America 97=12980-12984. 15 Van Rie, J. 2000. Bacillus thuringiensis and its use in transgenic insect control technologies. lnternational Journal of Medical Microbiology 290:463-469. 138 6. Figure legends Figure 1 Processing of pro-toxin (Cry1Ac) into mature Bt{oxin. Crystals of the pro-toxin are solubilized under reducing conditions at high pH revealing a ca 130 kDa protein (-GJ), which is processed in the presence of trypsin (1pg/ml final concentration) or an extract from the gut of the lepidopteran larvae Pieris rapae (1¡ll/ml) to form mature toxin (arrowhead) of ca 69 kDa (+GJ). In addition to the monomeric toxin an oligomeric SDS-stable complex of the toxin (arrow) is found (10), that could be trimers or tetramers. Less oligomers are detected in trypsin- digested protoxin. lt is not known whether monomeric toxin becomes oligomeric in association with lipophorin particles or whether oligomeric toxin interacts directly with particles. Figure 2 a) Pore-forming lectin-like toxins, such as crystal toxin (Cry1Ac) from Bacillus thuringiensrs (Bt-toxin), mediate aggregation of aged lipophorin particles in the absence of calcium. Trypsin-activated Cry1Ac was added to lipophorin particles in the presence and absence of galactosamine (final conc. 30mM) and in the absence of calcium. b) Under the conditions of aged fractions, LPS did not elicit an aggregation reaction even in the presence of calcium. c) Trypsin did not have an effect on aged lipophorin particles in the absence of calcium, but showed some aggregation in the presence of calcium. d) Diagram of three independent Bt-toxin-mediated aggregation experiments (separate gradients) and relative significance of Bt-treatments in the presence and absence of galactosamine. Figure 3 Pore-forming toxins with lectin properties interact with lipophorin particles from the gut in the absence of calcium. Gut extracts were mixed with gut juice- activated Cry'lAc and separated by low-density gradient centrifugation. Upper panel shows Coomassie blue-stained aliquots of gradient fractions from gut extracts. lt shows the separation of proteins that stay with plasma proteins (arylphorin) and those that are enriched in low-density areas of the gradient 139 (such as apolipophorin ll, which has a peak at fraction 17, asterix) and prophenoloxidase, which is carried by lipophorin particles. Lower panels show corresponding Western blots incubated with antibodies against apolipophorin I (which also jhas a peak in fraction 17, asterix) and antibodies against Cry1Ac, visualised with alkaline phosphatase-conjugated secondary antibodies. Anti- Cry1Ac-labelled bands at 60 kDa and 250 kDa (not shown), which represent monomeriç and multimeric toxin proteins, were visible in low-density areas (fraction 1 7, asterix). Figure 4 Detection of lipophorin in the gut lumen of immune-induced (Bt-tolerant) and non-induced (susceptible) E. kueniella (12) using antibodies against the vWD domain of apolipophorin l[Ma, in press #10945]. Note the homogeneous distribution of staining in the non-induced gut (upper panel), in contrast to the gut of immune-induced laryae, which contained non-digested food and dot-like stained coagulation products (lower panel). These dot-like coagulation products appears to form in the absence of melanin synthesis. This is in contrast to other lepidopteran species, such as Helicoverpa armigera, where melanization reactions are observed inside the gut lumen (10) 140 Figure 1 GrylAc .GJ +GJ M -> - -250 98 64 50 +¡* - 141 Figure 2 a +Bt d -Bt +Bt +Gal f apol * ** +LpS b -¡p5 +LPS +Ca G apol ?] c +Tryp +Bt +Bt -Tryp +Tryp +Ga -Bt +Gal ffi Frt T G aPol Figure 3 lipophorin plasma R7 M R9 R11 R13 R15 Rl7 R19 R2l R23 98 bw.- :'-:*ru¡ Goomassie 64 M R7 R9 Rll R13 R15 R17 R19 R21 R23 2so ru1tllf apor ' F¡" ,,F t*'-,.'*i i$cvrrc 142 Figure 4 vWD gut 143 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Summary and Conclusion 144 Summary and Conclusion By exposing larvae from a laboratory culture of E. kuehniella to an increasing concentration of a complex formula of endotoxins and spores from B. thuringiensis, a tolerant population emerged within a few generations, which survived on levels of the toxin that was lethal to the starting population (Rahman et al., 2OO4). The observations that the tolerant strain displayed an elevated immune response compared to the susceptible strain that is directly correlated to the observed Bt-tolerance suggested that the basis of tolerance is an elevated immune response (Rahman et al., 2004). As susceptible larvae were immune-induced and subsequently exposed to othenrvise lethal Bt-toxin concentrations in the same generation, observed survival in induced larvae excludes the selection of a pre-existing resistance allele as a cause of tolerance development. Reciprocal crosses of tolerant (T) and susceptible (S) insects suggested a maternal transmission of the immune-elicitor and Bt-tolerance (Rahman et al., 2004). The level of the immune response in the TXS larvae, as measured by the scale of melanization reactions, was variable among single pair offspring, but directly correlated with the extent of Bt-tolerance, suggesting that the observed variation in the magnitude of the potential immune response is determined by more than one gene. Moreover, when offspring from the backcross were immune-induced by a sub-lethal dose of the toxin, the observed melanization reaction was significantly greater than that detected in immune- induced susceptible larvae, indicating that the genetic disposition to respond to an elicitor was genetically determined by multiple alleles that were different in the tolerant compared to the susceptible population (Rahman et al., 2004). This suggests that with the exposure of insect populations to increasing concentrations of the toxin, genotype selection processes could produce tolerant insects with particular allele combinations. Genotype selection is also indicated by a reduction in fitness penalty imposed by to the toxin. This fitness penalty can be visible by a delay in development, which is roughly proportional to the toxin level to which the larvae were 145 exposed. At BOOOppm larval development measured in months rather than weeks as in larvae exposed to lower toxin concentrations. However, when some of the few larvae survived on BOOOppm, the offspring were able to finish development much faster than the previous generation. This could suggests that the combination of favorable alleles in some offspring allows to reduce the fitness penalty. Bacterial endotoxins, such as Cry-toxins inactivate eukaryotic cells by creating pores, but the mechanisms of how water-soluble toxins are inserted into the cellular membrane are not known (Lacy and Raymond, 1998). Current models imply îhat monomeric toxins interact with a receptor and are inserted into the membrane bilayer forming an oligomeric pore-forming complex (Bhakdi et al., 1996). Our preliminary observations suggest that lipophorin components may be involved in Bt-toxicity by a lectin-mediated endocytosis process where binding of the toxin to lipid-particles may be required to transport the toxin to the gut lining but an alternative reaction may also aggregate the toxin as part of an immune reaction (Sarjan, 2OO2). Since the functional role of lipophorin particles in cell-free immunity and their potential involvement in recognition and inactivation of pathogens and toxins are not known, we investigated whether lipophorin particles are the regulatory and effector components in immune- mediated inactivation of Bt-toxins, in particular when the larvae are exposed to low to medium levels of the toxin. The observation that lipophorin particles containing phenoloxidase from insect larvae when mixed with LPS causes aggregation and inactivation of the LPS (Ma et al., 2006) are consistent with our observations that lipid particles play an important role in the regulatory process involved in activating the adhesive properties of lipid particles (Rahman et ql., 2006). The lipophorin particles described multifunctional complexes associated with growth factors (Ma et al., 2006) and morphogens (Panakova et al., 2005) are also linked to phenoloxidase activity to regulate particle-specific immune proteins, which are consistent with very early observations that cell-free defence reactions exist in insects (Boman and Hultmark, 1987). 146 Although it is not known from our experiments whether the induction of melanisation and aggregation of lipid particles is caused either by LPS or contaminated peptidoglycan. The observed round or oval-shaped coagulation products forming regular-shaped cage-like assemblies in the presence of oligomeric adhesion molecules (Rahman et al., 2006) has the potential to inactivate microbes and sequester objects with a lipid-containing layer of lipophorin particles. These may also preclude bacterial growth and the spread of damaging toxins by forming a first line of defence in the gut lumen by a cell- free immune reaction, where the inactivation of toxins can be effective even in the absence of hemocytes. Given that lipophorin interacts with immune suppressors to inactivate hemocytes (Asgari and Schmidt, 2002), ¡t is surprising that the immune suppression by parasitoids does not affect susceptibility of host larvae to Bt- toxin (Rahman et al., under review-l). lt implies that defence components located in the hemocoel and gut lumen are regulated independently and function as separate entities. This is an unexpected result, since PO, a major humoral defence component in lepidopteran insects is derived exclusively from hemocytes and transported to other tissues of the larval body, where it is involved in processes, such as cuticle hardening (Ashida and Brey, 1995) and defence (Brey et al., 1993). Our observations suggest that LPS mediated phenoloxidase activation or melanization is a good indicator for immune induction and suppression but not directly involved in the response to the detoxification of toxin (Rahman et al., under review-l). Since melanization is not relevant to the protection against the toxin, why is prophenoloxidase activated in Bt-tolerant lawae? More importantly, what are the alternative immune pathways that protect against the toxin? One possible answer to the first question is that the mechanism of up- or down- regulation of immune reactions may include melanization in conjunction with other reactions, such as coagulation, even though these can be separated at the genetic (Rizki et al., 1985) or metabolic level (Charalambidis et al., 1994). 147 The observation that melanization is not relevant for Bt-tolerance indicates that some alternative defence pathways may exist to protect the larva against the toxin? Parasitoids mediated immune suppression has been studied to examine baculovirus virulence (Washburn et al., 1996). M. sexta larvae co-infected with polydnavirus from C. congregafa increases susceptibility to fatal infection by Autographa californica M Nucteopotyhedrovirus (Washburn et al., 2000)' Conversely, preliminary experiments in H. armigera indicate that the elevated immune status, which provides protection against Bt-toxin (Ma et al', 2005), also protects against baculovirus infection (Ma, 2005). While baculoviruses and Bt-toxin share similarities, there are differences, both in the path of infection and in host defence. Firstly, while a protective line of defence inside the gut lumen is not ruled out, most of the virus-related defence reactions appear to take place after the virions are released from infected gut cells. ln immune-competent larvae virions are trapped by the immune system and prevented from spreading (Engelhard et al., 1gg4; Washburn et al., 1995). Since there is no evidence that virions are phagocytized by tracheal cells and since insect hemocytes are unable to cross the basement membrane lining of the hemolymph, the only inducible defence pathway that can prevent the spreading of virions in this tissue environment is the inactivation by aggregation and coagulation of defence molecules, where the virion-containing coagulation products inside the trachea attract hemocytes to form nodules (Washburn et al., 2000). The relevance to the protection against Bt-toxin is the fact that recognition of the pathogen and inactivation (at least at an initial stage) occurs outside the hemocoel lining. This implies that the factors involved in recognition and inactivation of pathogens and toxins are able to operate away from hemocytes, if not in their absence. The results in E. kuehnietta are consistent with results from H. armigera, where elevated levels of melanotic immune activity are associated with Bt-resistance (Ma et al., 2005) and tolerance to baculovirus infection (Sarjan, 2002). The findings in the two lepidopteran laboratory strains of an elevated melanotic immune status in Bt-tolerant insects providing protection against other pathogens are also consistent with recent observations of malaria-refractory 148 Anophetes gambiae strains, which have elevated oxidative activities compared to susceptible insects (Dimopoulos et al., 2000). What are the candidates for a cell-free recognition and inactivation system? While a number of possible pro-coagulants have been identified in arthropods, the possible function and mode of action seems to differ among different species (Duvic and Brehelin, 1998; Hall et al., 1999; Korayem et al., 2004; Li et al.,2OO2; Muta and lwanaga, 1996; Theopold et al., 2002; Theopold et al., 2OO4). The main feature of pro-coagulants is their unique ability to recognize and form specific aggregates around pathogens and toxins, which effectively separate the damaging effects from the surrounding physiological environment of the host. Many of the pro-coagulants, such as lipophorin (Li et al', 2002), vitellogenin (Hall et al., 1999) and hexamerin (Ma et al., 2006), serye metabolic and storage functions and in performing those functions are transferred across the hemocoel lining to accumulate in other tissues and body cavities, such as oocytes, epidermis and the gut lumen. Given their ubiquitous presence and their potential function as pro-coagulants, these structures could form a first line of defence against intruding pathogens. For example, lipophorin, which is secreted into the gut lumen may be engaged in lipid transport between the gut food and lipid droplets and the gut cell lining. ln immune-induced larvae lipophorin particles may be modified to attract Bt-toxin molecules, causing sequestration of the mature toxin before it can reach the brush border membrane. The observation that apolipophorin ll forms SDS-insoluble dimers in the gut of immune-induced larvae (Rahman et al., 2006) suggests that the protein has the capability to engage in strong protein-protein linkages or to form covalent linkages, such as those mediated by transglutaminase or PO' The observed aggregation of lipophorin particles by the lectin-like Bt-toxin (Rahman et al., under review-l) has profound implications for our understanding of inducible tolerance mechanisms based on cell-free defence reactions. The presence of lipophorin-containing globules in the gut lumen of Bt-tolerant larvae (Rahman and Schmidt, under review) suggests a possible inactivation of the toxin by cell-free defence reactions, which can explain the observed tolerance to the toxin by an inducible mechanism (Rahman et al., 2004). However, it is not 149 clear from our experiments what molecular status lipophorin particles acquire with immune induction. One possibility is that certain immune molecules, such as pattern recognition proteins, are recruited to the particles and in the process acquire the ability to recognize microbial patterns of potentially damaging organisms. Another possibility is that particle-specific glycoproteins or glycolipids are modified in immune-induced larvae to attract pathogens or toxins in the gut lumen. This is particularly interesting, since many pathogenic bacteria and pore-forming toxins are known to use glycodeterminants on the brush border membrane to gain entry into the gut cells. lf lipophorin particles with similar glycodeterminants are present in the gut lumen it is possible that particles interact with damaging organisms and toxins before they reach membrane-bound receptors. The inducible tolerance mechanisms are quite different from other Bt-resistance mechanisms (Gonzalez-Cabrera et al., 2001), where the observed reductions in Bt-toxicity are exclusively explained in terms of alterations to receptor properties in the gut epithelium (Darboux et al., 2002; Gahan et al., 2001). While inducible tolerance mechanisms may be effective only against low to medium levels of toxin and are no immediate threat to most transgenic crops expressing the Bt Cry-proteins, it may pose long-term threat through genotype selection, given that the toxin levels in transgenic crops may not be sufficient to kill tolerant insect populations in conjunction with semi-dominant receptor mutations. An immune-related Bt-tolerance mechanism, which can be transmitted by a maternal effect (Ma, 2005; Rahman et al., 2004), may have practical implications in the field. Although the levels of immune-related tolerance may be low and gut-associated immune-elicitors may not induce tolerance in all insects, moderately resistant insect populations can emerge in the field and survive in sufficient numbers under continued selection pressure to genetically fix the elevated immune status (Rahman et al, unpublished data) or develop rare mutations in receptor genes (Gahan et al., 2001). 150 A number of strategies have been developed to prevent the emergence of insect resistance in field population (Bates et al., 2005) given that receptor inactivation by gene mutations (mode 1) provides resistance to relatively high levels of the toxins (Baxter et al., 2005; Tabashnik et al., 2002). The most effective is the so-called 'refuge strategy', where susceptible insects are kept at relatively high level in transgenic crops to prevent offspring of rare heterozygotic mutants from becoming homozygotic (Tabashnik et al., 2003). While mode 1 resistance is rare, the potential exists for a number of pest insects to become tolerant by other mechanisms. The observations that immune-induced Bt- tolerant insects undergo dramatic changes in gut physiology (Ma, 2005), with induced insects switching from food-digestion to pathogen-defence (Rahman et al., under review-ll) suggests that the elevated immune status includes a number of physiological alterations, such as changes in the types and quantities of gut proteases (Li et al., 2005), increased secretion of lipid particles (Rahman et al., 2006), increased coagulation and melanization reactions (Rahman et al., under review-l). The significance of systemic immune-induction is that it provides the physiological conditions and the immune components for cell-free coagulation reactions in the gut lumen (Rahman et al., under review-l), causing the sequestration of Bt-toxin (Rahman et al., under review-ll) before it can reach the brush border membrane of the gut lining. Since the coagulation reaction is probably based on lectin-properties of the Bt-toxin (Rahman et al., under review-ll), causing the cross-linking of putative lipid-particle-specific glycolipids, it may be less selective than toxin interactions with membrane-bound glycoprotein receptors, lf this proves to be correct, the inducible Bt-tolerance mechanism may not discriminate among different Cry-toxins and therefore make pyramiding of Cry-toxin genes in transgenic plants less effective if not obsolete. Although the toxin levels in transgenic crops may be sufficient to kill tolerant insect populations in conjunction with semi-dominant receptor mutations, the observed inducible tolerance mechanisms based on the sequestration of the toxin in the gut lumen by a transient immune induction may pose a risk for the long-term use of Bt-toxin in integrated pest management strategies. Given that the effects of inducible tolerance are likely to be additive to resistance (mode 1) 151 in semi-dominant heterozygotes, the chances of receptor mutations emerging within tolerant insect populations are becoming increasingly higher the longer insect populations are exposed to the toxin. Under these conditions it is possible that semi-dominant heterozygotes, in conjunction with induced tolerance, will be resistant to high levels of the toxin, posing a possible threat to the long-term use of toxin as a biopesticide. Since the refuge strategy relies on resistance levels of heterozygotes being well below the levels of toxin expression in transgenic crops, it is important to understand how Bt-toxicity in the gut lumen is related to the sequestration mechanism and to develop management strategies to overcome the emergence of tolerance in insect pests. Attempts to use other elicitors, such as LPS or zymosan, to induce the immune system of E. kuehnielta were not successful. Given that larvae feed on substrate, which is frequently contaminated by bacteria or fungi, it is possible that some insects, including E. kuehniella, G. mellonella and D. melanogaster, require direct elicitor contact with the haemolymph to be responsive. ln this context it is possible that sub-lethal concentrations of the toxin may damage gut epithelium enough to allow elicitors to reach haemolymph, but retain enough integrity of the gut epithelium to allow transport or secretion of soluble immune components into the gut lumen. Further experiments using different elicitors, such as baculoviruses, may shed light on the induction process. Moreover, further studies are required to identify the exact mechanism of cell- free defence reactions. Our abilities to detect and analyse immune reactions are limited due to the highly fragile and reactive nature of immune-activated lipid particles. For example, the isolation of lipid particles by LGC in the absence of EDTA causes spontaneous and rapid aggregation due to activation of proteolytic regulatory cascades that render the particles adhesive. Conversely, particles isolated in the presence of EDTA are stable in solution but quickly lose their ability to respond to elicitors. Under these conditions, the most effective way to use particles for experiments was to dialyse aliquots immediately after LGC fractionation and restore calcium before or during experimentation' Nevertheless, after several days of storage elicitor responses fade away, 152 although aggregation of particles can still be mediated by the addition of oligomeric adhesion molecules. This requires that all experiments have to be performed with controls that assess the immune status of each LGC fraction. Another problem is the possible biophysical changes to lipophorin particles imposed by LGC. While LGC constitutes a gentle and effective method of isolating lipid particles, it inevitably occurs under high salt and changing redox conditions, which may cause changes to the particles. lf these particles represent indeed immune and other sensors (Rahman et al., under review-ll), many conditions that differ from the physiological status in the gut lumen or in the hemolymph plasma may'activate' particles and therefore generate adhesive particles that can aggregate. Another problem is oxidation of lipids during LGC. For example, attempts to isolate glycolipids from LGC-isolated lipophorin particles failed, probably due to the oxidization of lipid carbohydrates during centrifugation. The nature and molecular mechanism of transmission of the elicitor are not known from our experiments. lnterestingly, it is known that the determination of dorsal-ventral polarity in the Drosophila embryo is mediated by a group of genes that are functionally involved in immune defence reactions of the adult insect (Lemaitre et al., 1997). Our observations, indicating that immune induction processes in the embryo are caused by a maternal effect, raises the question of how the developmental and immune functions are separated in immune-induced Bt-tolerant insects. One possibility is that components involved in dorsal-ventral polarity are deposited in the perivitellogenic egg space, whereas immune elicitors may be stored in the yolk interacting with primordial mesodermal tissues after the dorsal-ventral axis has been established. A mechanism where membrane-like lipid particles form the first line of defence in the gut lumen raises a number of questions. lf lipid particles are indeed protective, why are immune-active lipophorin particles absent or reduced in the gut lumen of susceptible larvae? One possibility is that immune-activation and secretion of lipid particles into the gut lumen reduces lipid carrier and other metabolic functions of lipophorin particles in the hemolymph, which imposes 153 fitness penalties on the insect. ln fact, exposure to increasing concentrations of Bt-formulations in the food increases developmental time in a direct correlation to the toxin concentration (Rahman, unpubl. data). Thus the insect has to return to non-induced status as soon as the exposure to the toxin is over to compete with other larvae for growth. Another question is how the elevated immune status and with it tolerance to Bt- toxin is transmitted to offspring? Since immune activation is based on inducible regulatory processes rather than mutations, the next generation is expected to start from a tolerance baseline that is comparable to non-induced insects. However, When subsequent generations of insects where exposed to increasing levels of toxicity, the tolerance to the toxin (and with it the fitness penalty) increased (Rahman, unpubl. data). Subsequent analysis of reciprocal crosses suggested that the mode of transmission is based on a maternal effect (Rahman et al., 2OO4). Given that lipophorin particles are incorporated into the ooplasma of developing oocytes, it is conceivable that 'activated' particles in the circulation of immune-induced females are stored in the egg and become involved in the induction of the immune system of the developing embryo. Experiments are under way to test this assumption. 154 Reference Asgari, S., and Schmidt, O. 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(2002): lnsect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. lnsect Biochemistry and Molecular Biology 32, 919-928. Ma, G. (2005): The molecular biology of tolerance to Bacillus thuringiensis endotoxin in Helicoverpa armigera: a novel mechanism and its genetic transmission., pp. 167l: Schoot of Agriculture, Food and Wine, University of Adelaide, Adelaide (PhD Thesis). Ma, G., Hay, D., Li, D., Asgari, S., and Schmidt, O. (2006): Recognition and inactivation of LPS by lipophorin particles." Developmental and Comparative I mmu nology 30(7): 61 9-626. Ma, G., Roberts, H., Sarjan, M., Featherstone, N., Lahnstein, J', Akhurst, R., and Schmidt, O. (2005): ls the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae? lnsect Biochemistry and Molecular Biology 35,729-739. 156 Muta, T., and lwanaga, S. (1996): The roleof hemolymph coagulation in innate immunity. Current Opinion in lmmunology 8,41-47. Panakova, D., Sprong, H., Marois, E., Thiele, C., and Eaton, S. (2005): Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435,58-65. Rahman, M. M., Akter, K. F., Naidu, R., and Schmidt, O. (under review): Arsenic interactions with lipid particles containing iron. Journal Environmental Pollution. 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. Rahman, M. M., Roberts, H. L. S., Sarjan, M., Asgari, S., and Schmidt, O. (2004): lnduction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella. Proceedings of the National Academy of Sciences of the United Sfafes of America 101, 2696-2699. Rahman, M. M., Roberts, H. L. S. and Schmidt, O. (under review-l): Tolerance lo Bacittus thuringiensrs endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella. Applied and Environmental Microbiology. Rahman, M. M. and Schmidt, O. (under review-ll): Cell-free sequestration of mature Bacittus thuringiensrs endotoxin by lipophorin particles. Applied and Environmental M icrobiology. Rizki, T. M., Rizki, R. M., and Bellotti, R. A. (19S5): Genetics of a Drosophila phenoloxid ase. Mol Gen Genet 201, 7-13. Sarjan, M. (2002): Resistance against endotoxin from Bacillus thuringiensis in Lepidopteran insects, pp. 129 Department of Applied and Molecular Ecology, UNiversity of Adelaide, Adelaide (PhD thesis). Tabashnik, B. E., Carrière, Y., Dennehy, T. J., Morin, S., Sisterson, M. S', Roush, R. T., Shelton, A. M., and Zhao, J.-2. (2003): lnsect resistance to transgenic Bt crops: Lessons from the laboratory and field. Journal of Economic Entomology 96, 1031 - 1038. Tabashnik, B.-E., Dennehy, T.-J., Sims, M.-A., Larkin, K., Head, G.-P., Moar, W.-J., and Carriere, Y. (2002): Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacil/us thuringiensls toxin Cry2\b. Applied and Environmental Microbiology. Theopold, U., L¡, D., Fabbri, M., Scherfer, C., and Schmidt, O' (2002): The coagulation of insect hemolymph. Cellular and Molecular Life Sciences 59, 363-372. Theopold, U., Schmidt, O., Soderhall, K., and Dushay, M' S. (2004): Coagulation in arthropods: defence, wound closure and healing. Trends in I mmu nology 25, 289-294. Washburn, J. O., Haas-Stapleton, E. J., Tan, F. F., Beckage, N. E', and Volkman, L. E. (2000): Co-infection of Manduca sexfa larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa californica M Nucleopolyhedrovirus. Journal of Insect Physiology 46, 179-190. 157 Washburn, J.O., Kirkpatrick,8.4., and Volkman, L. E. (1995): Comparative pathogenesis of Autographa californica M Nuclear polyhedrosrsvfius in larvae of Trichoplusia Ni and Heliothis virescens. Virology 209, 561-568. Washburn, J. O., Kirkpatrick, 8.4., and Volkman, L. E. (1996): Insect protection against viruses. Nature 383, 767 -7 67 . 158 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions APPENDIX A) Arsenic interactions w¡th lipid particles containing iron (Manuscr¡pt under rev¡ew in Environmental Pollutionl B) Factors affecting growth in the koinobiont endoparasitoid Venturia canescens in the flour moth Ephestia kuehniella (Manuscr¡pt under rev¡ew in Journal of lnsect Physiologyl C) Gell-free immune react¡ons in insect (Poster presented in School Research Day,2006) D) Bt tolerance by an elevated immune response in the flour moth Ephestia kuehniella (Poster presented in IGE 20041 E) Gell-free sequestration of mature Bt-toxin Gry1Ac in the gut lumen of Ephestia kuehnielta larvae (Poster presented in School Research Day,2006) F) lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins based on cell-free immune reactions (PhD final presentation) 159 tnducibte tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Appendix A Arsenic interactions w¡th lipid part¡cles containing iron M Mahbubur Rahmanl, K. Farzana Akter2, Ravi Naidu2'3 and Otto Schmidtl llnsect Molecular Biology, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, 545064, Australia. 2Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lake, SA 5095, Australia 3CRC for Contamination Assessment and Remediation of the Environment, SPRI Building, Mawson Lake, SA 5095, Australia. Manuscript under review in Environmental Pollution 160 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorshiP Manuscript under review in Environmental Pollution as: M Mahbubur Rahmanl, K. FarzanaAkteÉ, Ravi Naidu2'3 and Otto Schmidtl : Arsenic interactions with lipid particles containing iron. Mohammad Mahbubur Rahman (Gandidate) Designed experiments, performed experimental work, analysed and interpreted data, co-wrote manuscript and acted as communicating author' Kazi Farzana Akter Co-designed experiments, assisted in experimental work for the determination of total arsenic and iron by ICP-MS. Ravi Naidu Providing pig blood samples and instrumental support for ICP-MS analysis. Otto Schmidt (Principal Supervisor) Supervised work, assisted in analysis and interpretation of data, and co-wrote manuscript. Signed: Date ¡î8,it' Ø;" Mohammad Mahbubur Rahman I gave consent on behalf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. Signed Date: 2{,(1,ô6 Otto Schmidt 161 lJnder review in Journal of Environmental Pollution Arsenic interactions with lipid particles containing ¡ron. M Mahbubur Rahman1., K. FarzanaAkteÉ, Ravi Naidu2'3 and Otto Schmidtl llnsect Molecular Biology, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, 545064, Australia. 2Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lake, SA 5095, Australia 'CRC for Contamination Assessment and Remediation of the Environment, SPRI Building, Mawson Lake, SA 5095, Australia. * Corresponding author: Email: [email protected] Phone: +61 8 8303 7274 Fax: +61 I 8303 7109 162 Abstract While arsenic is toxic to all multicellular organisms, some organisms become tolerant by an unknown mechanism. We have recently uncovered an inducible tolerance mechanism in insects, which is based on a sequestration of toxins and pathogens by lipid particles. To examine whether arsenic interacts with lipid particles from mammals we compared binding of arsenic to lipid particles from insect and pig plasma after separation of lipid particles by low-density gradient centrifugation (LGC). Arsenic was found in both organisms in an area of the gradient, which corresponds to lipid rich lipid particles. Since iron is known to affect arsenic toxicity in some organisms, we asked whether iron may be present in lipid particles. When LDC gradient fractions were analysed for the presence of iron we detected a peak in very low-density fractions similar to those that carried arsenic. This could indicate that arsenic interacts with lipid particles that contain iron and if arsenic is removed from the plasma by lipid particles that would also reduce iron-containing lipid particles at the time of arsenic emergence in the plasma. To test this assumption we measured the iron content in plasma at various time periods after the toxin ingestion. This time course revealed that iron is depleted in plasma fractions when arsenic shows a peak. Our data suggest that arsenic interacts with invertebrate and vertebrate lipid particles that are associated with proteins that may lead to detoxification by cell-free or cellular sequestration mechanisms. Keywords; blood plasma, arsenic, inducible tolerance, lipid particles, sequestration mechanisms. 163 1. lntroduction Since arsenic (As) naturally occurs in soil, water and food, all organisms are exposed to variable levels of this substance although the exposure is likely to depend on the bioavailable fraction. At higher doses of As uptake or chronic contact due to occupational or environmental exposure or to cases of accidental intoxication, As poisoning may occur, which is associated in humans with cancer, gastrointestinal and respiratory diseases and neurotoxic symptoms (Yoshida et al., 2OO4).lt is apparent that the mode of action is based on multiple and overlapping effects on cellular processes including oxidative stress, signal transduction pathways and indirectly by damaging DNA (Schoen et al., 2004). It also appears that the chemical forms and oxidation states of As as well as the form of uptake by the organisms is crucial for the response and the development of short and long-term adverse effects (Langdon et al., 2003). While there is no clear support of genetic resistance to As, there is anecdotal evidence in earthworms suggesting that tolerance to the toxin can be induced relatively quickly and exists in local earthworm populations at mine sites and other contaminated areas (Langdon et al., 2003). Since there are no hints of a mode of action in this system, we asked whether tolerance to As can be studied using an insect model system. We have recently uncovered a transient tolerance mechanism in insects to formulations of endotoxins from the soil bacterium Bacillus thuringiensis (Rahman et al., 2OO4). The mechanism is based on a transient induction of the immune system, which leads to dramatic changes in the gut lumen and sequestration of toxins and pathogens by a cell-free aggregation reaction (Rahman and Schmidt, under review). The major components involved in cell- free sequestration of toxin are lipid carriers, which in insects are lipophorin particles (Rahman et al., in press). These particles are involved in shuttling lipids inside the hemocoel (Canavoso et al., 2001) and inside the gut lumen, where they extract lipids from food droplets and transport to the brush border membrane. Thus lipid particles can act as sensors for damaging objects in the food as well as effector components by inactivating the objects by a cell-free 164 coagulation reaction (Rahman et al., in press) (Ma et al., 2005). The binding of Bt-toxin to lipophorin particles in the gut lumen inactivates the toxin by an aggregation reaction before it can reach the brush border membrane to form a pore (Rahman et al., under review). This raises the question whether the insect model can be used to study the interaction of lipid particles with other toxins, such as As. lf As-binding proteins are associated with lipid particles, the presence of As may alter particle properties causing aggregation and sequestration of the toxin. To determine whether As interacts with lipid particles we separated lipid particles by low- density gradient centrifugation (LGC) from other plasma proteins. Here we show that As binds to lipid-rich lipophorin particles in insects and to LDL-like particles in vertebrates. The particles that interact with As are associated with iron. 2. Materials and Methods 2.1 Low-density gradient centrifugation of plasma A solution containing 250 pg/ml As in water was mixed with Galleria standard diet (Ref) and dried for six hours at room temperature. Third to fourth instar larvae were exposed to arsenic-containing diet for 48 hr when about half of the larvae showed signs of reduced turgor and darkening of hemolymph colour. Only larvae that did not show any sign of toxicity after 48 hours of exposure were used for plasma islolation and lipid particle separation. Hemolymph plasma was collected from fifty larvae on ice-cold 4 ml anti-coagulation buffer following the methods of Rahman et al (2004). Cell-free plasma was isolated by centrifugation at 50009 for 5 minutes. The plasma solution was then subjected to low-density gradient centrifugation with minor modifications according to Rahman et al (2006). ln a separate experiment, arsenic was added to cell-free hemolymph plasma from arsenic-free Galleria larvae (adjusted to 2 pg/ml in 30ml) and fractionated by LGC following the same procedure. 165 2.2 Plasma from arsenic-fed pigs A single dose of 99.8 pg/kg of body weight (BW) of As was orally administered to each pig under fasting conditions and serial blood was collected after 0, 1,2, 3, 4,5, 6, 7,8, 10, 24 and 26 hours. Blood cells were removed by centrifugation at 50009 for 5 minutes. Blood plasma from the three-hour time period was used for separation of lipid particles by LGC as described above. 2.3 Determination of total arsenic and iron The total blood plasma from different time periods was analysed for total As and iron content using lnductively Coupled Plasma Mass Spectrometry (|CP-MS). Plasma aliquots (500U1) were diluted in 4.5m1 in milliQ water, mixed with HNOg to a final concentration of 2o/o and left for 24 hours at room temperature. Preparations were filtered with 0.45pm membrane filters and arsenic and iron measured directly using high-performance liquid chromatography (HPLC) ICP- MS. 2.4 Protein analysis by SDS-PAGE Gomassie blue staining An aliquot (2 pl) from each gradient fraction was first diluted with 18 pl milli Q water and then with 5 pl loading buffer and incubated for 15 minutes at 650C. Protein samples were separated by SDS-PAGE according to (Ma et al., 2005). 3. Results 3.1 Arsenic-binding to lipophorin particles To examine whether As interacts with lipophorin particles from insects, we added arsenate (Asu) to cell-free hemolymph plasma from two insect species, the flour moth Eph estia kuehniella and the wax moth Galleric mellonella and separated lipophorin particles by low-density gradient centrifugation (LGC). Under these conditions a peak of As can be detected in a very low-density area of the gradient, which contains lipid-rich lipophorin particles (Fig. 1A). A similar peak of As was observed when As was fed to insect larvae and plasma was separated by LGC (Fig. 1). While the majority of lipid particles is found in fractions between 12 and 16 as indicated by the peak of apolipoprotein I and ll 166 (Fig. 1B) a sub-population of lipid-rich particles is found in areas corresponding to fractions 17 to 23. Previous observations indicated that lipid-rich particles are associated with immune proteins, such as prophenoloxidase (Rahman et al., in press). This suggests that As binds to lipid particles and is carried into the low- density areas of the gradient. 3.2 Arsenic-binding to lipid particles from pig plasma To examine whether As binds to lipid particles in mammals, As was fed to pigs and plasma taken and analysed at various time points after ingestion for the presence of As. Within a few hours As was detected in plasma with a peak of As between 3 and 5 hours after ingestion (Figure 2). To determine whether As was bound to lipid particles, cell-free plasma obtained 3 hours after ingestion of the toxin was separated by LGC and As was measured in each gradient fraction. Under these conditions a high amounts of As were detected at very low-density areas of the gradient (Fig. 3A) similar to insect lipid-rich particles and close to a peak of low-density lipid (LDL) particles containing apolipoprotein B1 00. 3.3 Arsenic and iron interaction with lipid particles Since iron (Fe) is known to affect As toxicity in plants (Warren et al., 2003), we asked whether As may interact with iron in lipid particles. When LDC gradient fractions were analysed for the presence of iron we detected a peak in very low- density fractions similar to those that carried arsenic (Fig. 3B). This could indicate that arsenic and iron interact with the same lipid particles and if arsenic is removed from the plasma by lipid particles that would also reduce iron- containing lipid particles at the time of As emergence in the plasma. To test this assumption we measured the iron content in plasma at various time periods after the toxin ingestion. This time course revealed that iron is depleted in plasma fractions when arsenic peaks and reappears in the plasma when arsenic is removed (Fig. a). 167 4. Discussion Our data suggest that invertebrate and vertebrate lipid particles interact with As, which may lead to detoxification by cell-free or cellular sequestration mechanisms. The dynamics of As and iron in the plasma of As-fed pigs suggests that as the amount of As increases in the plasma the amount of iron is reduced and only increases when As decreases. This and the observation that As and iron are found in a similar fraction in a very low-density area of the gradient suggests that As interacts with lipid particles that contain iron, probably in the form of ferritin and transferrin (Georgieva et al., 2002) (Nappi and Vass, 2000), but hemoglobin is also known to carry iron. How is As removed from plasma by lipid particles? The insect model describing inactivation of toxins from hemolymph provides two possible avenues: One is the cell-free sequestration by an aggregation of adhesive lipid particles into globules (Rahman et al., in press), the other is an interaction of adhesive lipid particles with cellular receptors leading to internalisation by a leverage- mediated uptake reaction. ln the insect model both, the cellular and the acellular reactions are driven by adhesion molecules that are associated with lipid particles and become adhesive in the process of the toxin interacting with the particles. The nature of the adhesion molecules and the mechanisms of becoming adhesive are not known. The observation that iron is associated with lipid particles could indicate a functional role in the process. lron-poor apoferritin is produced in the fat body of insects (Nichol and Locke, 1999) and probably associated with lipid particles. Arsenic could bind with ferritin lacking iron or interact indirectly by altering the lipid particle, such as by oxidisation of lipids. Since it is known that ferritin forms multimers, which store iron inside cells, one possible scenario is that ferritin forms oligomers in the presence of arsenic. lnteraction of oligomeric proteins across lipid particles may lead to aggregation and coagulation (Karlsson et al., 2004), which is actually observed in arsenic containing lipid particles from insects, where high molecular weight proteins appear above apolipoprotein bands (Fig. 1A). ln addition, large particle aggregates are formed in As-treated preparations, which are found in the plasma fractions after low-density gradient centrifugation ((Fig. 1A). This 168 suggest that As may trigger the self-assembly of lipid particles into large aggregates, which sequester the toxin in the absence of cells. While toxin- mediated aggregation was easily detected in insects, such reactions were much less obvious in pig plasma. Given the closed circulatory system in vertebrates, where large protein aggregates are a threat, it is possible that the major detoxification reactions in mammals are based on cellular clearance rather than cell-free aggregation reactions. For example, it is known that transferrin is internalised by cells in an actin-independent process (Conner and Schmid, 2003) (Fujimoto et al., 2000) where ferritin or transferrin interact with corresponding receptors on the cell surface (Ramalingam et al., 2000) and plays an important role in immune clearance (Ponka and Lok, 1999) and phagocytosis, such as the removal of apoptotic cells (Yuan, 1999). Thus As- containing lipid particles may become adhesive by direct or indirect (Wang et al., 2006) effects of arsenic and subsequently interact with cell-surface receptors causing the uptake of arsenic-containing complex by a leverage- mediated endocytosis reaction. Whatever the mode of As clearance from the plasma, the involvement of iron- containing lipid particles allows us to focus on possible detoxification reactions by using the insect models as a paradigm. 169 5. References Canavoso, L.E., Jouni,2.8., Karnas, K. J., Pennington, J. E', and Wells, M' A' (2001). Fat metabolism in insects. Annual Review of Nutrition 21,23'46. Conner, S. D., and Schmid, S. L. (2003). Differential requirements for AP-2 in clathrin-mediated endocytosis. J Cell Biol 162,773-780' Fujimoto, L. M., Roth, R., Heuser, J. E., and Schmid, S' L. (2000). Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis. Traffic 1, 161'171. Georgieva, T., Dunkov, B. C., Dimov, S., Ralchev, K., and Law, J' H' (2002). Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types. Insect Biochemistry and Molecular Biology 32, 295-302' Karlsson, C., Korayem, A. M., Scherfer, C., Loseva, O', Dushay, M. S., and Theopold, U. (2004). Proteomic analysis of the Drosophila larval hemolymph clot. Journal of Biological chemistry 279, 52033-52041. Langdon, C. J., Piearce, T. G., Meharg, A. 4., and Semple, K' T' (2003). lnteractions between earthworms and arsenic in the soil environment: a review. Environmental Pollution 1 24, 361 -373. Ma, G., Roberts, H., Sarjan, M., Featherstone, N., Lahnstein, J', Akhurst, R., and Schmidt, O. (2005). ls the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae? lnsect Biochemistry and Molecular Biology 35,729-739. Nappi, A. J., and Vass, E. (2000). lron, metalloenzymes and cytotoxic reactions. Cellular & Molecular Biology 46,637-647. Nichol, H., and Locke, M.(1999). Secreted ferritin subunits are of two kinds in insects - Molecular cloning of cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius. lnsect Biochemistry & Molecular Biology 29,999-1013. Ponka, P., and Lok, C. N. (1999). The transferrin receptor: role in health and disease. lnternational Journal of Biochemistry & Cell Biology 31, 1111' 1137. Rahman, M. M., Ma, G., Roberts, H. S. L., and Schm¡dt, O. (in press). Cell-free immune reactions in insects. Journal of lnsect Physiology. 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 (USA) 101,2696-2699. Rahman, M. M., Roberts, H. L. S., and Schmidt, O. (under review). Tolerance to Bacillus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella. Applied and Molecular Microbiology. 170 Rahman, M. M., and Schmidt, O. (under review). Cell-free sequestration of mature Bacillus thuringiensis endotoxin by lipophorin particles. Applied and Environmental Microbiology. Ramalingam, T. S., West, A. P., Lebron, J. 4., Nangiana, J' S', Hogan, T. H., Enns, C. A., and Bjorkman, P. J. (2000). Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis. Nature Cell Biology 2,953-957. Schoen, A., Beck, 8., Sharmâ, R., and Dube, E. (2004). Arsenic toxicity at low doses: epidemiological and mode of action considerations. Toxicology and Applied Pharmacology Arsenic in Biology and Medicine 198, 253-267. Wang, L., Xu, Z. R., Jia, X. Y., Jiang, J. F., and Han, X' Y. (2006). Effects of arsenic (As-lll) on lipid peroxidation, glutathione content and antioxidant enzymes in growing pigs. Asian-Australasian Journal of Animal Sciences 19,727-733. Yoshida, T., Yamauchi, H., and Fan Sun, G. (2004). Chronic health effects in people exposed to arsenic via the drinking water: dose-response relationships in review. Toxicology and Applied Pharmacology Arsenic in Biology and Medicine 198, 243-252. Yuan, X. M. (1999). Apoptotic macrophage-derived foam cells of human atheromas are rich in iron and ferritin, suggesting iron-catalysed reactions to be involved in apoptosis. Free Radical Research 30,221-+' Warren, G.P., Alloway, 8.J., Lepp, N.W., Singh, 8., Bochereau, F.J.M. and Penny, C. (2003) Field trials to assess the uptake of arsenic by vegetables from contaminated soils and soil remediation with iron oxides, The Science of The Total Environment, 31 1, 19-33. 171 6. Figure Legends Figure 1. A) Separation of gradient fractions by SDS-PAGE. Cell-free plasma from Gatteria larvae fed with arsenic was separated by low-density gradient centrifugation and aliquots of each fraction (ca 1i100) stained with Coomassie blue after electrophoretic separation. Molecular markers are Seeblue@ prestained standard, myosin 250 kDa, BSA 98 kDa, glutamic dehydrogenase 64 kDa, and alcohol dehydrogenase 50 kDa. The location of apolipoproteins I and ll, which are 230 and 72 kDa in size are indicated on the right site of the gel. The proteins bands between 65 and 75 kDa in size consist of multiple bands comprising arylphorin, which is found in high-density plasma fractions, at least two prophenoloxidase bands, which are found in high- and low-density areas of the gradient and apolipoprotein ll, which is only visible in low-density areas of the gradient. Additional protein bands at the top of the gel are protein aggregates, which form in arsenic-containing plasma fractions. B) Arsenic content of gradient fractions from arsenic-fed insects (grey bars) and from gradient fractions where arsenic has been added to plasma from arsenic- free insects (black bars). Although there is no visible Coomassie blue-staining in fractions 19-24, previous analysis of gradient proteins using Western blots, suggests the presence of lipid-rich lipophorin particles which are associated with immune proteins, such as prophenoloxidase and proteases (Rahman et al., in press). Figure2. Time course of arsenic content in the plasma of pigs. After administering a single dose of 99.8 ug/kg of body weight (BW) of arsenic serial blood was collected and the amount of arsenic measured using HPLC/ ICP-MS. Figure 3. Comparison of arsenic and iron distribution in low-density gradients of pig plasma. Fractions 21 to 23 contain significant amounts of arsenic and iron. 172 Figure 4. Time course of arsenic and iron contents in the plasma of pigs. Note the inverse dynamics of the two substances indicating a reduction of iron-containing lipid particles in the presence of arsenic. 173 Figure I 12345678M9 10 11 12 13 14 15 16 M 1718',19 20 21 2223 2425 M FI Itt Þ. Asg 250 ú ff ur Apo 9B Apo il 64 L. ¡<' ;ilsü¡i E"E 50 plasma low-density very low-density I 7 6 5 o tr 4 o o 3 ø 2 I 0 (\eeÓ1e($êtê6($<'9..ûrtrf Fraction 174 Figure 2 t20 O 1 00 e g0 80 É60a c) 840 at) o 20 O 0 0 5 10 15 20 25 30 Time(h) 175 Figure 3 0 07 0 .06 E 0 .05 CL CL 0.04 o c 0 03 o (J 0 02 ID 0 0 1 0 (\ e 'io IT (\€rþÓ1e ('$ (^P (^þ ($ ôq ..û a*' 176 Figure 4 Nudey-As VS time Hairy-As VS time t20 t20 00 o 100 o B80 è80 ã = .E 60 É60 ãoo 340 o 4zo -20 o a 0 0 2s 30 0 5 l0 15 20 25 30 0 5 l0 l5 20 Tine(h) Time(h) Nudey-Fe vs time Hairy-Fe vs time 3.5 25 3 a a 2.5 l5 2 1.5 o l 0.5 0.5 0 0 25 30 0 l0 l5 20 25 30 0 l0 l5 20 177 tnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Appendix B Factors affecting growth in the koinobiont endoparasito¡d Venturia canescens in the flour moth EPhestia kuehniella M. Mahbubur Rahman, Harry L. S. Robelts, Otto Schmidt lnsect Molecular Biology Laboratory, School of Agriculture and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia Manuscript under review in Journal of lnsect Physiology 178 lnducibte tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Statement of authorshiP Manuscript under review in Journal of Insect Physiology as: M. Mahbubur Rahman, Harry L. S. Roberts, Otto Schmidt: Factors affecting growth in the koinobiont endoparasitoid Venfuria canescens in the flour moth Ephestia kuehniella Mohammad Mahbubur Rahman (Candidate) Designed experiments, performed experimental work, analysed and interpreted data, co-wrote manuscript and acting aS communicating author. Harry L. S. Roberts Assisted in analysis and interpretation of data, and co-wrote manuscript. Otto Schmidt (Principal Supervisor) Supervised work and help in manuscript preparation. Signed Date Ce'//-06 Mohammad Mahbubur Rahman I gave consent on behatf of co-authors for M. M. Rahman to present this paper for examination towards the Doctor of Philosophy. signed: Date: 2€^(1,oÓ Otto Schmidt 179 Manuscript under review in Journal of lnsect Physiology Factors affecting growth in the koinobiont endoparasitoid Venturia canescens in the flour moth Ephestia kuehniella M. Mahbubur Rahman*, Harry L.S. Roberts and Otto Schmidt lnsect Molecular Biology Laboratory, School of Agriculture and Wine, University of Adelaide, Glen Osmond SA 5064, Australia * Corresponding author: Email: [email protected] Phone: +61 8 8303 7274 Fax: +61 8 8303 7109 180 Abstract With resistance of insect pests to synthetic pesticides on the increase, the role of parasitoid wasps as biological control agents is expanding in pest and resistance management strategies. One of the predictors of reproductive success of endoparasitoids is the relative size of the wasp at host emergence. While in idiobiont parasitoids, where the host stops feeding after parasitism, the wasp size is determined by the host size at the time of parasitism, the size of koinobiont wasps, where the host continues to feed after parasitism, is dependent on additional factors. Here we show that the host mass and temperature are important factors that determine survival and development of the koinobiont endoparasitoid Venturia canescens in late instar larvae of the flour moth Ephestia kuehniella. Keywords: E. kuehniella, biological control, endoparasitoid, V. canescens, parasitism success, host mass, temperature. 181 1. lntroduction Endoparasitic wasps deposit their eggs inside the body of their host, typically an immature stage of another arthropod, and after hatching the developing parasitoid feeds exclusively on the tissues of the host. Hosts parasitised by idiobiont wasps cease to grow or develop following parasitism so that the resources available to the developing wasp are fixed at the time of parasitism. ln contrast, hosts parasitised by koinobiont wasps continue to feed and grow until the functional integrity of the host is destroyed by the developing parasitoid, allowing the resources available to the parasitoid to increase past the time of attack. Being ectotherms, one of the key determinants of the development (and hence growth) rate of an insect is temperature. Between upper and lower thermal developmental thresholds an insect's rate of development increases approximately linearly with temperature (Blunck 1914, 1924), and can be described by the equation: DR=a+bT equationl where DR is development rate, equal to the inverse of the total time required for the insect to complete its development, a and b are constants and I is the environmental temperature. The total "degreedays" required for the insect to complete development is the inverse of the slope b, called the "thermal constant" K. lnterestingly, the developmental threshold of the host is typically lower than that of the parasitoid, while the thermal constant is typically higher. This may result in significant developmental consequences for the development of koinobiont parasitoids at different temperatures. 182 The time (t) required for the development of the parasitoid to reach a point where the host is incapacitated will be some fixed fraction of the development time (DT) of the parasitoid such that: t= kt*DTeeRAstrotD)= h/ DR¡ennestoro¡ equation 2 ln that time the host grows an additional amount equal to the product of its growth rate (GR) during that stage and the time f in equationl. To a first approximation, this growth rate will be a proportional to its development rate, so that: Additionat growth = f*GR =t*kz*DRHosD equation 3 Substituting equation 2 into equation 3: Additionat growth = k1*kz* DRHosr) / DR¡pnaesroro¡ equation 4 It has been shown in single temperature studies that the size of an emergent parasitoid varies with the size of the host (eg Harvey et al 1994 Roberts et al 2OO4). Thus, it is plausible that any additional growth by the host will translate directly into additional growth by the parasitoid and, unless the relationships between development rate and temperature for the parasitoid and its host are the same, the relations between host mass at oviposition and adult wasp size will vary with rearing temperature. Using developmental data for the solitary koinobiont endoparasitoid Venturia canescens Grav. (Hymenoptera: lchneumonidae) (Eliopoulos and Stathas 2003) and its host the flour moth Ephestia kuehniella (Lepidoptera: Phycitidae) (Jacob and Cox 1977)to solve for DR¡rosr,¡ and DR¡enaesrorol (Fig. 1a) and then solving for equation four at different temperatures (Fig. 1b), it is predicted that the size of adult V. canescens from equivalent size E. kuehniel/a will decrease with increasing rearing temperature. To investigate this we examined the development of V. canescens in late instars of E. kuehniella at different constant temperatures. 183 2. Materials and Method 2,1. lnsects The wasps were a clonal RP line of V. canescens, founded from wasps collected near Mt. Boron in Southern France and maintained in the laboratory for two years. The wasps were reared in cylindrical clear plastic tubs (height 20cm and diameter 15cm). Three or four adult wasps were placed into each container with 40-50 hosts. Upon emergence the wasps were removed from culture and were kept in gauze-covered 425 ml clear plastic cups (Party Rite Jumbo TumblersrM, Harris Paper Pty. Ltd,, West Heidelberg, Australia) with a 50% honey solution. Wasps used in the experiments were 5-10 days old. Hosts were E. kuehniella, reared on a 10:2:1 mixture of oat bran, wheat germ, and dried brewers yeast. All cultures maintained at 25 ! 1oC, under a constant light dark regime (114:D10). 2.2. Parasitism conditions ln the study,4th and Sth instar E. kuehniellalawae ranging in mass from 15 to 50 mg, were each parasitised once by V. canescens. Parasitised hosts were assigned to one of five constant temperature conditions, 15, 20, 24, 28 and 32C. To obtain singly parasitised hosts a single wasp was put together with 25-30 host larvae of varying sizes in a plastic container (7 cm in diameter, I cm high). The parasitoids were observed during oviposition and stinging attempts that resulted in a startle response from the larvae, combined with the characteristic cocking movements of the wasp's ovipositor (Rogers 1972) were considered as real oviposition events. Any stinging attempt that either did not evoke a startle response or was not followed by a cocking movement was regarded as uncertain and the larva was discarded. After parasitisation the hosts were weighed and maintained individually in glass vials with excess food until parasitoid eclosion. The cohort size was 50 parasitised larvae at 15, 20 and 32.C and 80 parasitised larvae at 24 and 2B'C. All experiments were conducted synchronously. 184 2.3. Developmental parameters Emergent wasps were killed by freezing and the time and date of emergence recorded, which allowed development time to be calculated. Head capsule width was measured to the nearest 0.0125 mm with an optical micrometer. Hosts from which a wasp had not emerged 20 days after the last wasp had emerged were dissected to determine whether developmental failure had occurred as an early instar (dry host mummy), a late instar or pupa (dead larva/pupa identifiable in host) or a pharate (some part of the developing adult cuticle visible). 2.4. Statistical analysis Data were analysed using the generalised linear model (GLM) platform, JMP V4.0.4 (SAS 2OO1), with continuous factors centred by their means (Neter et al 1990). Analyses started with full models with all interactions, and non-significant interactions were progressively dropped. The lower developmental threshold (To) was estimated by fitting the experimental data to a linear regression equation of the form: y=a+bT where y is the rate of development (the inverse of the mean development time) at temperature f, and a and b are constants. Following Eliopoulos and Stathas (2003), data for T=32C were excluded from this calculation as the assumed linear relationship ceases to be valid at temperatures close to the upper thermal threshold (Campbell et al. 1974), with the relationship becoming non-linear (Mills 1981). The thermal constant K was estimated as the slope of the regression line (1/b) (Campbell et al 1974). 185 3. Results 3.1 Parasitism success Parasitism success was higher for hosts maintained at temperatures in the range 2O-28C compared to those maintained at the extreme temperatures of 15 and 32C (Table 1). Analysis by logistic regression of the stage at which developmental failure occurred revealed that as temperature increased there was a significant decrease in the fraction of parasitoids failing as pharates and an associated increase in the fraction failing as late instar larvae (X=7.95, df=1,5g, p=0.0048) while there was no significant change in the fraction that failed as early instars (Fig. 2). The effects of host mass on parasitism success and stage of parasitism failure were not significant. 3.2. Wasp size Analysis of wasp head capsule width with temperature and host mass as factors revealed a significant negative relationship (F=54.1, df=1,194, p<0.0001) between temperature and wasp size and a significant positive relationship (F=251.1, df=1,194, p<0.0001) between host mass and wasp size (Fig. 3). 3. 3. Te m pe ratu re etïecfs A similar analysis on wasp development time showed the time required lor V. canescens to complete its development decreased significantly with temperature (F=1999, df=4,206, p<0.0001, Table 2) while the effects of host mass were not significant. The rate of development increased linearly with temperature within the temperature range 15-28'C (R2=0.993, Fig. 4)' Linear regression of development rate (D) of V. canescens on temperature (T) returned the equation: D = 0.00247T- 0.0239 indicating a lower developmental threshold of To = 9'68 + o'77"C and a thermal constant of K = 404.3 + 24.0 degree-days (mean t SE). 186 4. Discussion Our observations on the developmental outcomes of koinobiont endoparasitoids have basic and applied implications relating to wasp development inside another insect and mass culturing of biological control agents. ln a physiological context it appears that temperature thresholds that impose developmental barriers affect the growth of the endoparasitoid. While the rates of early larval death were not changed significantly at different temperature regimes, the rate of pharate pupal death decreased with increasing temperatures, whereas the rate of death at late instar larvae increased. The simplest explanation for this observation is a temperature-dependent developmental barrier that affects late instar larvae and becomes more pronounced at higher temperatures. At lower temperatures larvae may be able to enter pupariation but die as late larvae at higher temperatures. Since prepupal and pharate pupal mortality has been associated with high melanization levels (Roberts et al 2004\ it will be interesting to explore phenoloxidase levels in parasitoid larvae at different temperatures. Determination of wasp size at four temperature regimes within upper and lower thermal development thresholds suggest that while the size of adult parasitoids correlated with the size of the host as expected from idiobiont systems, the size of a koinobiont wasp emerging from equivalent host sizes decreased with increasing rearing temperatures. This suggests that the additional growth ensuing from the continuous feeding of the host is dependent on the rearing temperature. This suggests that the assumptions leading to the mathematical description of developmental thresholds and thermal constants are correct. The resulting equations predicting a decrease of adult wasps emerging from equivalent sized hosts at increased rearing temperatures and its confirmation by experimental observations indicate a complex interaction of two organisms where temperature may have different effects on metabolic and immune functions and where the endoparasitoid has to manipulate host physiology while suppressing the latter but maintaining the former host functions. Acknowledgments: This work was supported by an ARC grant to OS. 187 References Blunck, H. (1914). Development in Dytiscus, Coleoptera: embryo. Zeitschrift fuer Wissenshaftliche Zoologie 111: 76-157 . Blunck, H. (1924). Development in Dytiscus, Coleoptera: larva and pupa. Zeitschrift fuer Wissenschaftliche Zoologie 121: 171-391. Campbell,4., B. D.Frazer, N. Gilbert, A. P. Gutierre and M. MacKauer. (1974). Temperature requirements of some aphids and their parasites Journal of Applied Ecology 11 (2): 431-438. Eliopoulos, P. A. and G. J. Stathas (2003). Temperature-dependent development of the koinobiont endoparasitoid Venturia canescens (Gravenhorst) (Hymenoptera: lchneumonidae): Effect of host instar. Environmental Entomology 32(5): 1 049-1 055. Harvey, J.4., l. F. Harvey and D. J. Thompson (1994). Flexible larval growth allows use of a range of host sizes by a parasitoid wasp. Ecology 75(5): 1420-1428. Jacob, T. A. and P. D. Cox (1977). The influence of temperature and humidity on the life-cycle of Ephestia kuehnietla Zeller (Lepidoptera: Pyralidae). Journal of Stored Products Research 13:107-118. Mills, N. J. (19S1). Some aspects of the rate of increase of a Coccinellid. Ecological Entomology 6:(3): 293-299. Neter, J., W. Wasserman and M. Kutner (1990). Applied linear statistical models: regression, analysis of variance, and experimental designs. Homewood, ll., lnruin. Roberts, H. L. S., O. Trüe and O. Schmidt (2004). The development of the endoparasitoid wasp Venturia canescens in superparasitised Ephestia kuehniella. Journal of lnsect Physiology 50(9): 839-846. Roberts, H. L. S., M. Keller and O. Schmidt (2006). An empirical model of the sympatric coexistence of two strains of the endoparasitoid wasp Venturia canescens . Achives of lnsect Biochemistry and Physiology 61 (3): 184- 194. Rogers, D. (1972). The lchneumon wasp Venturia canescens: oviposition and avoidance of superparasitism. Entomologia Experimentalis et Applicata 15: 1 90-1 94. SAS (2001). JMP V4.0.4. Belmont, California, Duxbury Press. 188 Tables Table 1. Survival of V. canescens larvae developing in singly parasitised larvae of E. kuehniella at different constant temperatures. Temperature ('C) 15 20 24 28 32 Survival (%) 40 92 83.75 80 28 Table 2. Development time in days of V. canescens in singly parasitised larvae of E. kuehniella at different constant temperatures. Temperature ("G) N Mean SE Range 15 19 82.1 1.3 74-95 20 46 37.6 0.2 34-41 24 67 27.6 0.4 19-37 28 64 22.6 0.2 19-28 32 14 20.9 0.2 1s-23 189 Figure Legends Figure 1. The influence of temperature on development. a) Linear regression of development rate against temperature of V. canescens (open circle; from Eliopoulos 2003) and E. kuehniella (closed circle; from Jacob 1979)' b) Predicted relationship between gain in mass by E. kuehniella following parasitization by V. canescens and rearing temperature, as the ratio of their development rates as described in equation 3. Figure 2. Logistic regression of the developmental stage at which parasitism failed on temperature, for V. canescens developing in singly parasitised E. kuehniella larvae. Lines represent the cumulative probability plots. Figure 3. Relationship between the mass of the host E. kuehniella at parasitism and the head capsule width of the emergent V. canescens, for parasitised hosts maintained at different constant temperatures. Lines represent linear regression. Open Square and solid line, T=15'C; closed square and dashed line, T=2O.C; closed triangle and dotted line,T=24oC; open diamond and dash- dot line, T=28'C; closed diamond and dash-dot-dot line, T=32oC. Figure 4. Relationship between the rate of development time of V. canescens in singly parasitised larvae of E. kuehniella and rearing temperature. Line represents linear regression 190 Figure 1a 0.05 U' 0.04 o (It E (¡) 0.03 g o c 0) 0.02 E o. o o t¡, 0.01 oct 0 10 15 20 25 30 Temperature ('C) Figure 1b 0.7 0.ô5 Èø J ¿' (ú 0.55 E (u 0.5 tt v, o 0.45 0.4 15 20 25 30 Temperature ('C) 191 Figure 2 1.00 I Pharate I ¡ I E: 075 I o a -o Late lnstar o T o- I q) ¡ 0.50 I (ú T I E I f ¡ O 0.25 ! I I I Early lnstar a I T 0.00 15 20 25 30 Temperature ('C) 192 Figure 3. 1.5 I 20"c 15"C 1.45 / a / ts 2g"c 1.4 E 32"C tt , t 1^a '= o , 1.35 -9 a o 11, oLa CL t! a o 1 .3 t,t! tr o t¿ A , CL Ø IE 1 25 E, o ëco-a = 1) ,lo 1.2 a a o o a a o 1.15 10 15 20 25 30 35 40 45 50 55 Host mass (mg) 193 Figure 4. 0.05 o) 0.04 (ú (¡) E 0.03 o o) (¡) ô o.02 0.01 10 15 20 25 30 Temperature (oC) 194 lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Appendix C Bt tolerance by an elevated immune response in the flour moth Ephestia kuehniella M Mahbubur Rahman, Harry L. H. RobeÊs, M. Sarjan, S. Asgari and O. Schmidt lnsect Molecular Biology Laboratory, Discipline of Plant and Pest, School of Agriculture and Wine, University of Adelaide, Glen Osmond, 545064, Australia. Poster presented in the XXII lnternational Gongress of Entomology, 15-21August 2004, Brisbane, Queensland, Australia (Poster lD PS2S240). 195 Bt tolerance by an elevated immune respgn_se th Ephestia kuehniella Roberts, M. Sarjan, S. Asgari & O" S ci"t ine of Plant & Pest, Schooi of Agricu & '¡:; ,:e [email protected] m0 lntroduction: The use of Bacillus thuringiensis endotoxins to control insect 350 E vectors of human dlseases and agricultural pests is under threat from the 2m0 300 Tolerant evolution of resistance in major pest species. ln addition to high levels of Bioassav of rec¡orocal 250 crosses-shows fiìalernal by several ance 1500 200 re recept Y, c transm¡ss¡on of tolerance rePorled aìns of 150 aga toxin leve in 't000 100 es. Becau ular basis o of o 50 resistance to the toxin is not known, we explored alternative mechanisms. 500 0 Here, we describe an investigation of a laboratory culture of the flour moth 0 20 ¡10 60 80 for a possible correlation between systemic immune Tim (m¡ns) Ephestia kuehniella 0 induction and Bt tolerance. s@püô sxR ÐG R6isttl lmmum ln Irvæ 350 I Reclorocal show È 3oo crossas Pre-treated matdrnal tansmlsslon of s 250 6 ¡mmune lnducdon ! zoo ./I -{ E tso 4 € I o 100 Control <50 2 o 0 6 '--_i_--- lo 20 30 4o 50 60 70 90 É. 0 Time (m¡ns) R?XSé S? XRó CROSS wlür to ¡ncrea¡cd Tre atm e nt 95%Cl Slope RR Lrwl of toþdcs cmspdds to 3Ea ollîmum ßspoßc Control '1940 1252-4128 0.94 ftm 11000 ke-exposed 32610 ' 8404-1x106 0.52 16.8 1m m 6@0 l o ,0x, 2æ0 0 3 1 6 ln: PNAS (2004) 101 (9), 2696-2699. Funded by ARC grants to OS. I ndueible tolerance to Êacil[us thwingtensls (Bt) endotoxÍns M M Rahman based on cetþfree immune rèactlons Cell-free immune reactions in insect M Mahbubur Rahman, Harry L. H. RobeÊs and O. Schmidt lnsect Molecular Biology, Discipline of Plant and Food Science School of Agriculture, Food and \iline, University of Adelaide Glen Osmond, 545064, Australia Poster presented in the School Research Day at the Sunnybrae Funetion Centre, 31 October 2004 School of Agriculture, Food and Wine 197 ìi a :: . 1: M. Mahbubur Rahman, Harry L. S. Robefts and Otto Schmidt lnsect Molecular Biology, Plant and Food Science :t¡( a School of Agriculture, Food and Wine, Waite Gampus like many other multicellular organisms, are able to recognise and inactivate potential pathogens and toxins in the absence of cells. Here we show that the : Abstnct lnsects, i ì I of pathogens and phenoloxidase products. The existence of cell-free defence reactions implies thal immune signals exist upstream of cell-bound receptors. r7 T9 M T11 T13 T15 117 119 f21 r23 I a-5 ¿! lc $ lL¡ ¡zJ 15 tÐ 3t 62 125 25 50 100 Fig. 1. Lipophorin particles are the regulatory T13 T19 Þ¡À 6ìq units of cell-free immunity l^9hì PNA conc- llps/n¡) F¡g.4. Aggregation of lipophorin particles by oligomer¡c lectins sl5 trsr!DoP!Fs play in the regulatory l I '; '1r}ü'ì*, Summary: Lipid particles an important role -:ij .),-'" -.. process involved in activating the LPS-mediated immune response '::: I ,iåi . .':':.:'"Í PPO ¡l € causing self-assembly of lipophorin particles into globular structures. Apo 04 + These globules effectively inactivate microbes and sequester objects S9 o2 tf with a lipid-containing coating, which may preclude bacterial growth and 90 the spread of damaging toxins as well as reactive oxygen intermediates. 30 60 $i,i Tlms (mlnut€s) The observations that PPO activity is linked to lipid particles and Fig. 3. LPS-mediated phenoxidase # 5u¡ regulated by particle-specific immune proteins are consistent with very riC I Lipophorìn pildrcl$¡rcablclo rccosnisc activation requires an association with F¡9.5. L¡pophor¡n padicles form globular ( licrrú¡s rnJ il' !ri\Jrc put(nUrl frrho;(n{ lipophorin particles structures w¡th F ITC-conjugated PNA, early observations that cell-free defence reactions exist in insects. Some species, such as mosquitoes with few or no hemocytes in the (1 LPS recognition and aggregation hemolymph of adult insects are nevertheless capable of defending .4 against intruding damaging organisms by humoral defence reactions. oililto suggest that rnost of the malaria parasites ._$L, PL oi¡ti¡9 Lipid carrier Recent observations -/z¡¡1:- u lh¡ encountered in the gut Iumen of mosquitoes are inactivated by cell-free defence reactions including melanisation and other PO reactions, which include reactive oxygen intermediates. lnterestingly, specific proteins a Pp PoP involved in these reactions comprise C{ype lectins, which may target + LPS uptake LPS OlltllO óÍirfo t parasite-specific but also insect-specific determinants, an observation d{'u oe that is consistent with a general role of parlicle-associated adhesion 0 Cell-fræ coagulation Gactions pÞ pOQ Ð o"4m."o molecules in Iipid metabolism and cell homeostasis. d,,iu5c&, i,cèr u rló -co* '?#m,t$ i-'21;::::î. E;-,:;-:.'. ä. ;;1.-â ?:rr,ìis.'tri,?lï,1: . ,, 'e LqI_rygaj:medlqlqq qross-llnking ceilutar ctearance Þact¡ons, '.'' " J '..;'a.þ" :.q'. ... 2.'-:'.. ''9 iB+¡'îr,.ÐãiG- 6.ri' ,ôSE ä¡çr 3,ëiË, : -tilli,ll- , - ?:3 ., ãtl ¡:;t¡ ;-a: 6 Fig. 6" Schematic view of a two-step activation process of lipophorin particles I --7.ñ TiTr-f--f---ryt-!T-ElFq';7::iroF .r:îE lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Appendix E Cell-free sequestration of mature Bt-toxin CryLAc in the gut lumen of EphestÍa kuehniella larvae M Mahbubur Rahman and O. Schmidt lnsect Molecular Biology, Discipline of Plant and Food Science School of Agriculture, Food and Wine, University of Adelaide Glen Osmond, 545064, Australia Poster presented in the School Research Day at the Sunnybrae Function Gentre, 31 October 2004 School of Agriculture, Food and Wine 199 s. €ú Foo¿ wa$F a NBT 1b lowd.n¡lty v.dlow-d.n.lty 1c s23 T23 F¡IE Fsbp Very ld¡Y+nsty lBcüon S9 s11 100 [ptþrtr Psbp EO Loü.dcßity Pink t'¡Sf 17 T9 117 T19 121 f23 fraction ¡phorfi PPo !-¡ 60 \ Psbp trl Apu prdsll a0 T PNA Plasm Erc p¡rucar csrtår Tt7 20 E { Apol Ttl PPO 0 T7 T9 M T11 T13 115 117 f19 T21 123 --._ Fmction ¡suL$lal¡tltsis 3.1 62 12.5 100 Fig. l. Lipophorin particles are the regulatory T13 -T19 PPO uÉcb¡d¡ units of cell-free ¡mmunity F¡9. ¡1. Aggregat¡on of by ol¡gomer¡c lectins. sl5 tr $do"rr3 0,8 Apo I I 0.8 PPO ^ A ll Þo 0.4 + S9 0.2 30 80 90 aty Tlh.lmlnul.sl Fig. 3. LPS-mediated phenoxidase 5!F Fic 2 Lipophorin padicles are ablc to recogtrise act¡vat¡on requires an assoc¡ation with F¡g 5. Lipophorin particles form globular el¡ciloß ard ¡traclivâlc potm¡¡al pathogms lipo nn articles structures w¡th F ITC-conjugated PNA LPS rscognltion and aggregalion Lip¡d carr¡er (¡) (b) ll tlr tillillrtlilr lfLilf Eætions 0 coagddion g+ a år.mðdl.t€d oroBs-llnkln Cdlular cleüarc€ Fig. 6. Schematic view of a two-step activation process of lipophorin particles lnducible tolerance to Bacillus thuringiensis (Bt) endotoxins M M Rahman based on cell-free immune reactions Appendix F Inducible tolerance to Bacillus thuringiensis (Bt) endotox¡ns based on cell-free immune reactions M Mahbubur Rahman lnsect Molecular Biology, Discipline of Plant and Food Science School of Agriculture, Food and Wine, University of Adelaide Glen Osmond, 5A5064, Australia PhD Final Seminar at the Charles Hawker Conference Centre 24 July 2OO4 201 Inducible tolerance to Bacillus Overview th u n g ( ri iensis Bt) endotoxins 1). lnnate immunity and its reactíons based on cell-free immune in the gut lumen in insects. reactions 21. lmmune inductíon in the gut lumen r!.. IHEUiJVTlSft and inducible tolerance to Bt-toxin. .::- ArooFADET¡|0E h#\ltHHflt 3). fmmune suppressíon and Bt-toxicity. 4). lnactivation of mature Bt'toxin CrylAc by cell-free coagulatíon reactions in the gut lumen. Innate immune react¡ons in insects Are lipophorin part¡cles the O Recognítion and inactivation of regulatory un¡ts and effector lipopolysaccharides (LPS) and bacteria by recognition complexes. components for innate immune react¡ons in insects? O RecognítÍon compfexes consist of pattern recognition proteins, phenoloxidase (PPO) and proteolytic activation proteins. 1 particles Lipophorin particles separated Lipophorin are assoc¡ated with immune by LGC of hemolymph plasma Prote¡ns from Ephestia kuehniella larvae 9 tl '13 l5 17 19 21 23 25 27 11 13 15 17 19 21 23 25 27 a ..Ë-t'r - j - d".- +Apol PPO a--*..- I Apo i + i i Èfihi¡rib-tnducod F) l¡oÞlnduood (S) .- i ì5-.; - - [10 Íi I fl3 T15 Tl7 Tto 121 T23 P"+\ç- FEcüÞñ t Ì9 T11 T13 115 ll7 119 f2l f23 PPO particles Lipophorin can Lipophorin particles in immune- recognise elicitor"s and inactivate potential pathogens induced E, kuehniella larvae a b h¡.iiÙ vqr*lsdg PPô\é 1 Fcdbn r ru M lll ll3 r15 r11 f19 v1 n3 induod PAP '3 2 LPS mediated phenoloxidase LPS can trigger aggregation and activation requires an association covalent cross-linking of purified with lipophorin particles lipophorin particles 120 El slmoPflca 100 08 80 06 ö0 04 40 LPs I LPs/cå 20 02 t I rrstc.lsusu' 0 0 5 1 1 23 4,7 5.4 18.8 37.5 75 150 30 60 PNA oligomers interact with galactose Oligomeric lectin interacts w¡th containing glycodeterminants on the lipophorin particles to form cage lipophorin particles like coagulation products a ^ 100 ; 80 :.0 60 È60 Ä p¡¡ å I PNA prrc¡ &o D E PNrc.r X o^ zo 0 0 05 23 40 s3 107175 75 3t 62 125 25 I 1oo PNAcono {Æ/ml) PNA conc ll¡s/ml) 5!h J Cell-free sequestration of elicitors Summary L LPS recogn¡tion and aggregation Lipophorin particles are the p Lipid carr¡€r o regulatory units of cell-free immunity. o This cell-free immune reactions mediated by lipophorin particles may I potentially involve in detoxification of c!) pore-forming toxins (Bt-toxins) in the gut lumen. LPS .med¡ãted Overview Immune induction in the gut lumen and inducible 1). f nnate immunity and its reactions ín the gut fumen in insects. tolerance to Bt-toxins o Most Bt-resistance mechanisms are based on the selection of pre-existing receptor alleles in the population. lmmune and Bt-toxicity. 3). suppression o However, we explored a novel 4). lnactivation of mature Bt-toxin CrylAc mechanism where tolerance to the Bt-toxin be by cell-free coagulation reactíons in (low to medium level of resistance) can increased by immune induction. the gut lumen. 4 The immune response Bt-tolerance is increased by is increased by gut-derived gut-derived im mune induction elicitors (Bt pre-exposure) (Bt pre-exposure) s0 Ê 300 Treâtment I LC."(PPm) 95%Cl RR Pree¡PoW /- 8 250 : 1940 1250-4120 e 200 Control t 150 Pre-exoosed ì 3261 0 8400-1xE6 16.8 100 Contml 50 o . Tolerance correlates with an elevated 20 40 60 æ rmmune response. The rate of melanization Summary 2 correlates w¡th LCuo values for 3'd instar TÇxScY larvae 1) Bt-tolerance is induced in insects as part of an active immune response. Ê e 3 2) The strength of the expressed immune response can be increased by elicitors. 3) The magnitude of the immune response and Bt-tolerance are determined by more than i lmmune-induction and Bt-tolerance are due to one gene. more than one gene 5 Overview Immune-suppression 11. lnnate immuníty and its reactions and Bt-toxicity ín the gut lumen in insects. 2). lmmune induction in the gut lumen and inducible tolerance to Bt-toxin 1). lmmune suppression by endoparasitoid wasp Venfuria canescens, which affects hemolymph immunity. 4). lnactivation of mature Bt-toxin CrylAc 2). lnhibition of phenoloxidase (PPO) activity coagulation reactions in by cell-free by tropolone, which affects gut immunity. the gut lumen. Parasitoids suppress Bt-tolerant E, kuehníella are not protected against ¡n the immune response paras¡t¡sm by V. canescens host insects Ê '*'= rotsr¿nt E Does an elevated immune status in ! ' + a Bt-tolerant E. kuehniella larvae .25 provide cross-protection against 'a' = SuscsPllblo parasitism Tol.r.nt súc.pllbl. - by V. canescens? E. kuehnlella s!ilalî 6 Development time of parasitoids in immune-induced Parasitoid-mediated (Bt-tolerant) and non-induced immune-suppression and ( Bt-suscepti ble) hosts Bt-toxicity 1) How does parasitoid-mediated - imm une-s uppression affect melanization (PPO activity)? 2) What is the effect of parasitoíd- mediated W.!Þ d.v.loÞn.nl lln. (d.y.) imm une-suppression on Bt-toxicity? Melanization of plasma correlates The amount of PPO in with an elevated immune status hemolymph is correlated with in tolerant host Bt-exposure (in ppm) Troo T$o Tmoo T.ooo T*oo T-sr Sus M -250KDa Tá .4 t9 4 ùUscepuo¡e -98KDa Ð -64KDa llm (hln.) -5()KDa 7 Melanization in hemolymph is Immune suppression by V, significantly reduced after canescens does not affect Bt- parasitlsm by V. canescens toxicity in E. kuehniella larvae i'¡L.l,ilirlnil i il , v¿llll.:ll l;or íilJ(lll(ìu l llrìll (ppr ì ìì u )5 S usccpttt;lc IJ 't.\i l .ilr 1 r' 2111i I /(l f oir:rant 5/i! lii(ii) (i/11 I l.l Summary 3 Is melanization in the gut required for Bt- o Tolerance to Bt-toxin in the gut is not affected by parasitoid-mediated to lera nce? immune suppression in the hemolymph This can be tested by the suppression The defence reactions in the gut a of melanízation in the gut. and in the hemolymph are probably regulated separately. 8 Defence reactions in the gut Tropolone can be used in conjunction w¡th Bt-toxin on o lnactivation of the Bt-toxin in the E. kuehniella larvae gut may involve cell-free defence reactions: PeÍ(icnlaue M()arl L()a:il squaro:l .lutvtvitl developrrrcrìL¡l rncJrl ht)od 1) Melanization (PPO activity) lrûìo (d) crDsulc wtdth (rtr0) 2) Coagulation (Pro-coagulant ?) Conlnrl ll) 24 24 1 ;ì6:'l I re¡ lrnc r ìl BO 24 18 1 260 o Lipophorin (involved in lipid extraction and lipid shuttle in the gut lumen) Melanization in Bt-tolerant Tropolone does not affect larvae was s¡gnificantly reduced Bt-efficacy in Bt-tolerant after tropolone treatment E. kuehniella larvae [roDolorìo Bl loxrns P(ìrc(]nl¿ee Mcan Pclccnl¡ge (pprn) (pprn) f¿rval Devuloptrrcnlal 3(lult survlval ( l0(lays) tllne (d) elrlergerlce c 5000 32 28 50 4/ 1 000 5000 ;'6 27 5S 4,1 l t0000 54 27 4t 34 1 00t) t0000 t4 28 53 33 9 SummarV 4 Overview 1). lnnate immuníty and its reactíons Reduction in melanization has no o in the gut lumen ín insects. effect on Bt-toxicity. 2). lmmune induction in the gut lumen is with o Melanization correlated and inducible tolerance to Bt-toxin. immune-induction, but not causing Bt-tolerance. 3). lmmune suppression and Bt-toxÍcity. t This leaves coagulation as a likely mechanism for Bt-tolerance in the gut lumen. Tolerant larvae secrete more and Lipophorin is changed in the I or dtfferent lipophorin particles gut of tolerant larvae into the gut lumen 10 Lipophorin particles CryTAc interacts with lipophorin interact w¡th Bt-toxin CrylAc particles like oligomeric lectins in the gut lumen causing adhesive aggregation a 1Bl +st +&l d . lipophor¡n -Bt plasma .OJ +OJ T + tpd 17 M T0 Tll Tl3 T15 Tl7 ttgt2tt2l + -*fi --2& L+Þs5.m tes *. ' *c. s <- apd u. tl o& H t t0 Ttt 113 Ti5 fi7 ll9l2ll¡3 OE c +fry llnù+Bt 9," _Ìry ¡lryP *c. -8 +8 ll t u .- +âpol +Gå *' ,i siL Conclusion Acknowledgement Lipophorin particles interact with mature lectin-like Bt-toxin or llirrry Robcrtlì Wof k oxpcrrtrttcc strldont:i inactivating the toxin by cell-free Mr:tnLre rg ìtr OS l¿tb School & t)iscipltnc stilff nlc¡tlller¡; coagulation reactions in the gut I lumen before it can reach the brush r ' r:r ill t,,,,,,',,,,,1, border membrane. 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