Onderzoeken van de cellulaire en moleculaire aspecten in het immuunsysteem van Locusta migratoria

The locusta species - Locusta migratoria and Schistocerca gregaria - are responsible for the cyclic devastating hopper bands and swarm in many parts of Africa up to the Eurasian region. Present day locust swarms can be predicted but still most accurate control is based on post outbreak insecticide treatment resulting in high risks for resistancedevelopment and major pollution concerns. Hitherto the need for sustainable alternative bio-control agents, though more expensive, become realistic. Such biological control agent most probably will be entomopathogen based. However, for optimal efficacy of such approach, the innateimmune system of the locust needs to be turned down or at least be compromised. We believe that cytokines are important physiological player inthe locust cellular immune system. It is because the cellular immune system of the locust needs cytokines to keep up with high speed multiplication of hemocytes that are needed either for phagocytosis, encapsulationor nodula formation. Therefore, we will carry out the following research activities in the next four years. i) Since no locust cell line is available today, we start the development of a locust derived cell line from hemocyte progenitor cells (blood stem cells). This will result in a cell line that is most representative for the identification of cytokines involved in the innate immune response. As a suitable alternative approach, we will also consider developing an embryo derived stable cell line. ii) We will do in vivo and in vitro identification of importantcytokines in the locust innate immune system, the cellular immune system in particular. iii) We will target encoding mRNA of the identified cytokines for degradation by RNA interference and study the locust response towards immunological challenges with locust specific pathogens, i.e. bacteria (gram positive and negative), fungi and nematods.TABLE OF CONTENTS LIST OF ABBREVIATIONS ix SUMMARY xi SAMENVATTING xiv INTRODUCTION: Purposes and Scope of the study 1 General introduction 2 Purposes of the study 3 Scope of the study 4 CHAPTER 1: Mechanisms of the insect immune system 5 1.1. Introduction 6 1.2. What is insect immunity 7 1.3. Sources of the insect immune effectors 7 1.4. Mechanisms of the insect systemic immune response 10 1.4.1. Pathogen recognition 10 1.4.2. Humoral immune system 11 1.4.3. Cellular immune system 13 1.4.4. Anti-viral immune system 14 1.4.5. Insect cytokines and immune signaling 16 1.4.5.1. Insect immune signaling 16 1.4.5.2. Cytokine-based insect immune signaling 19 1.4.5.3. Direct immune signals in insect cells 22 1.4.5.4. Interaction in insect immune signaling 23 1.5. Immune system in insect epithelial tissues 24 1.6. Reinfection and adaptability in insects 25 CHAPTER 2: Current understanding of locust immunity 29 2.1. Cellular and molecular mechanisms of the locust immune system 30 2.2. Antimicrobial peptides and humoral immune response in locusts 30 2.3. Hematopoietic tissue and cellular immune response in locusts 32 2.4. Pathogen attack and locust prophylactic strategies 35 CHAPTER 3: Locust cellular defense against infections: Sites of pathogen clearance and hemocyte proliferation 37 3.1. INTRODUCTION 38 3.2. MATERIALS AND METHODS 40 3.2.1. Locust stocks 40 3.2.2. Injection and total hemocyte count 40 3.2.3. Hemocyte harvesting and fixation 41 3.2.4. Hematopoietic tissue preparation 41 3.2.5. Sarcina lutea and Candida albicans labeling 42 3.2.6. Phagocytosis assay 43 3.2.7. Determining total pathogen clearance 43 3.2.8. BrdU treatment techniques 44 3.2.9. BrdU detection and mitosis studies 44 3.2.10. BrdU clearance 44 3.2.11. Cell death detection 45 3.2.12. Microscopy and data analysis 45 3.3. RESULTS 45 3.3.1. Pathogen clearance by locust hemocytes and hematopoietic tissue 45 3.3.2. Primary response of adult locusts to infection includes release of newly synthesized hemocytes into the circulation 46 3.3.3. Infection induces substantial cell death but no cell division in the hematopoietic tissue of adult locusts 48 3.3.4. Circulating hemocytes are the primary sources for new hemocyte production in adult locusts 51 3.3.5. The hematopoietic tissue grows continuously in cell number and tissue size despite infection 52 3.4. DISCUSSION 52 CHAPTER 4: Hemocytes or phagocytic tissue cells for a locust cell line development? Possibilities and challenges 59 4.1. INTRODUCTION 60 4.2. METHODOLOGY 62 4.2.1. Animals, media and chemicals 62 4.2.2. Cytological staining 62 4.2.3. Collection and in vitro culture of locust hemocytes 63 4.2.4. Collection and in vitro culture of locust phagocytic tissue cells 63 4.2.5. Primary cell culture validation tests 64 4.3. RESULTS & DISCUSSION 65 4.3.1. Development of primary cell culture using locust hemocytes and phagocytic tissue cells 65 4.3.2. Possibilities and challenges in using locust hemocytes and phagocytic tissue cells for cell line development 70 CHAPTER 5: Identification and functional characterization of a novel locust peptide belonging to the family of insect growth blocking peptides 74 5.1. INTRODUCTION 75 5.2. MATERIALS AND METHODS 77 5.2.1. Locust breeding and synchronization 77 5.2.2. Hemocyte in vitro culture and cell spreading assay 78 5.2.3. Locust serum preparation 78 5.2.4. HPLC and MALDI MS 79 5.2.5. Trypsinization and LC-MS/MS 79 5.2.6. LC-MS/MS and sequence data analysis 80 5.2.7. Peptide synthesis and preparation 81 5.2.8. RNA extraction, cDNA synthesis and QRT-PCR analysis 81 5.2.9. Total hemocyte count and molting experiments 82 5.3. RESULTS 83 5.3.1. Response of hemocytes to different fractions of locust serum 83 5.3.2. Purification and validation of locust growth blocking peptide 84 5.3.3. Functional characterization of locust growth blocking peptide 87 5.3.4. GBP homologue in Schistocerca gregaria 88 5.4. DISCUSSION 89 CHAPTER 6: A quantitative peptidomics approach to unravel immunological functions of angiotensin converting enzyme in Locusta migratoria 94 6.1. INTRODUCTION 95 6.2. MATERIALS AND METHODS 97 6.2.1. Locust breeding and injection materials 97 6.2.2. RNA extraction and cDNA synthesis 97 6.2.3. Quantitative real-time PCR (QRT-PCR) 98 6.2.4. LmACE gene mapping 99 6.2.5. dsRNA synthesis and knockdown of LmACE gene 99 6.2.6. Loss-of-function studies 100 6.2.7. Nanoscale reversed phase LC-MS/MS 101 6.2.8. LC-MS/MS data analysis 102 6.3. RESULTS 103 6.3.1. LmACE is an extracellular protein transcribed from an at least 14 exon gene in the locust Genome 103 6.3.2. Following LPS or PGN challenge, LmACE transcript was upregulated in hemocytes but not in fat body tissue 105 6.3.3. Immune challenge rescued partially the knockdown of LmACE gene 106 6.3.4. Differential peptidomics unraveled putative enzymatic roles of LmACE in the locust hemolymph 107 6.3.5. Some LmACE regulated peptides are encoded in large protein precursors adjacent to unique protein domains 113 6.4. DISCUSSION 115 CHAPTER 7: General discussion and future prospects 119 7.1. Cellular characterization of the locust immunity 120 7.2. Molecular characterization of the locust immunity 122 7.3. Future prospects 125 7.3.1. In vivo characterization of locust hemocytes and phagocytic organ 125 7.3.2. Continuous cell line development 126 7.3.3. Functional characterization of the locust growth blocking peptide 126 7.3.4. Functional characterization of the locust angiotensin converting enzyme 127 ADDENDUM CHAPTER 6 129 REFERENCES 131 LIST OF PUBLICATIONS 147nrpages: 164status: publishe