by Wynstan, H.K. Cheuk. / Thesis submitted in: Dec., 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 57-65). / Abstract also in Chinese. / Chapter I. --- ABSTRACT --- p.i / Chapter II. --- ACKNOWLEDGMENTS --- p.iii / Chapter III. --- TABLE OF CONTENTS --- p.iv / Chapter IV. --- LIST OF TABLES --- p.viii / Chapter V. --- LIST OF FIGURES --- p.ix / Chapter VI. --- INTRODUCTION --- p.1 / Chapter VII. --- LITERATURE REVIEW --- p.3 / Chapter A. --- Cordyceps militaris infection --- p.3 / Chapter B. --- The diagnostic criteria for C. militaris infection --- p.3 / Chapter 1. --- Telemorphic stage of C. militaris --- p.4 / Chapter 2. --- Anamorphic stage of C. militaris --- p.4 / Chapter C. --- The natural occurrence of C. militaris infection --- p.5 / Chapter D. --- The epizootiology of C. militaris infection --- p.5 / Chapter E. --- The values of studying C. militaris infection --- p.6 / Chapter 1. --- Potential insect biocontrol agent --- p.6 / Chapter 2. --- Exploitation of medicinal values --- p.8 / Chapter (i) --- Chemical constituents --- p.8 / Chapter (ii) --- Pharmacological action --- p.9 / Chapter VIII. --- MATERIALS AND METHODS --- p.11 / Chapter A. --- Pathogen culture establishment --- p.11 / Chapter 1. --- Source of pathogen and strain maintenance --- p.11 / Chapter 2. --- Selection of the artificial medium for C. militaris --- p.11 / Chapter (i) --- Colony diameter --- p.11 / Chapter (ii) --- Enumeration of conidia --- p.12 / Chapter 3. --- Examination of the morphological features of C. militaris in selected agar medium of RF Agar --- p.13 / Chapter B. --- Host species establishment --- p.13 / Chapter 1. --- Source of host species and laboratory rearing --- p.13 / Chapter 2. --- Assessment of viable insect population --- p.13 / Chapter C. --- The Biological characteristics of C. militaris in RF broth --- p.14 / Chapter 1. --- Methods for the cultivation of C. militaris in RF broth --- p.14 / Chapter (i) --- Pathogen inoculum and culture medium --- p.14 / Chapter (ii) --- Culture conditions --- p.15 / Chapter 2. --- Techniques for harvesting --- p.15 / Chapter 3. --- Methodologies of various assessments --- p.15 / Chapter (i) --- Germination --- p.15 / Chapter (ii) --- Morphological developments --- p.16 / Chapter (iii) --- Biomass increase --- p.16 / Chapter (iv) --- Key enzymes profiles --- p.16 / Chapter a. --- Inocula preparation --- p.16 / Chapter 1. --- Extracellular enzymes --- p.16 / Chapter 2. --- Intracellular enzymes --- p.16 / Chapter b. --- Preliminary screening --- p.17 / Chapter c. --- Procedures for quantitative measure of selected enzymes --- p.17 / Chapter d. --- Eight selected key enzymes --- p.18 / Chapter 1. --- Esterase --- p.18 / Chapter 2. --- Lipase --- p.18 / Chapter 3. --- Peroxidase --- p.18 / Chapter 4. --- Acid phosphatase --- p.18 / Chapter 5. --- β- galactosidase --- p.19 / Chapter 6. --- N-acetyl-β-glucosaminidase --- p.19 / Chapter 7. --- Trypsin --- p.19 / Chapter 8. --- β-glucosidase --- p.20 / Chapter e. --- Total protein analysis --- p.20 / Chapter (v) --- Quantation of cordycepin --- p.20 / Chapter a. --- "Standard, mobile phase and reagents" --- p.20 / Chapter b. --- High Performance Liquid Chromatography (HPLC) apparatus --- p.20 / Chapter c. --- Assay procedures --- p.21 / Chapter D. --- C. militaris infection process --- p.22 / Chapter 1. --- "Sterilization of insect host, Antheraea pernyi" --- p.22 / Chapter 2. --- Criteria for eliminating unfit pupae for the infection experiments --- p.22 / Chapter 3 --- Techniques for inducing infection --- p.22 / Chapter (i) --- Cuticular contact --- p.22 / Chapter (ii) --- Injection --- p.23 / Chapter (iii) --- Dipping --- p.23 / Chapter 4. --- Assessment of gross infection and the percentage of parasitization --- p.24 / Chapter 5. --- Verification of Cordyceps infection --- p.24 / Chapter 6. --- Methodologies of histological and ultrastructural examinations --- p.24 / Chapter (i) --- Scanning Electron Microscopy (SEM) --- p.24 / Chapter a. --- "Sample preparation, fixation and dehydration" --- p.24 / Chapter b. --- Critical point drying --- p.25 / Chapter c. --- Mounting --- p.25 / Chapter d. --- SEM examination --- p.25 / Chapter (ii) --- Transmission Electron Microscopy (TEM) --- p.25 / Chapter a. --- "Sample preparation, fixation and dehydration" --- p.25 / Chapter b. --- Infiltration --- p.26 / Chapter c. --- "Semi-thin sectioning, ultra-thin sectioning and post-staining" --- p.26 / Chapter d. --- TEM examination --- p.27 / Chapter (iii) --- Light Microscopy (LM) --- p.27 / Chapter a. --- Sample preparation --- p.27 / Chapter b. --- Fixation and dehydration --- p.27 / Chapter c. --- Embedding --- p.28 / Chapter d. --- Sectioning and staining --- p.28 / Chapter e. --- LM examination --- p.28 / Chapter 7. --- Methodology of HPLC assay for cordycepin in infected host --- p.28 / Chapter 8. --- In vitro susceptibility testing of the microbes isolated from A. pernyi against cordycepin --- p.29 / Chapter (i) --- Isolation of microbes from the cuticle surface of the healthy A. pernyi --- p.29 / Chapter (ii) --- Isolation of microbes from the non-cuticle surface of the healthy A. pernyi --- p.29 / Chapter (iii) --- Isolation of microbes from the necrotic tissue of A pernyi --- p.29 / Chapter (iv) --- Conditions for incubation --- p.29 / Chapter (v) --- Identification of microbial isolates of A. pernyi --- p.30 / Chapter a. --- Fungal isolates --- p.30 / Chapter b. --- Bacterial isolates --- p.30 / Chapter (vi) --- Assay procedures of cordycepin susceptibility testing of isolated microbes --- p.31 / Chapter IX. --- RESULTS --- p.32 / Chapter A. --- Biological characteristics of C. militaris grown in vitro --- p.32 / Chapter 1. --- Selection of the artificial medium for C. militaris --- p.32 / Chapter 2. --- Morphological examinations of C. militaris in RF agar --- p.32 / Chapter (i) --- Appearance of C. militaris --- p.32 / Chapter (ii) --- Conidiogenesis --- p.33 / Chapter (iii) --- Morphological developments --- p.33 / Chapter 3. --- Physiological examinations of C. militaris in RF Broth --- p.34 / Chapter (i) --- Germination --- p.34 / Chapter (ii) --- Biomass and pH of the culture filtrate --- p.34 / Chapter 4. --- Biochemical examinations of C. militaris in RF Broth --- p.34 / Chapter (i) --- Key enzyme profiles --- p.34 / Chapter (ii) --- Quantitation of cordycepin --- p.35 / Chapter B. --- C. militaris infection of A pernyi --- p.35 / Chapter 1. --- Percentage of parasitization by cuticular contact methods --- p.35 / Chapter 2. --- Morphological examinations of C. militaris infection --- p.36 / Chapter (i) --- Gross infection of C. militaris --- p.36 / Chapter (ii) --- Site susceptibility of A.pernyi to C. militaris infection --- p.38 / Chapter (iii) --- Attachment of C. militaris --- p.38 / Chapter (iv) --- Penetration of A. pernyi by C. militaris hyphae --- p.38 / Chapter (v) --- Utilization of A. pernyi by C. militaris hyphae --- p.39 / Chapter 3. --- Biochemical examinations of C. militaris infection --- p.39 / Chapter (i) --- Cordycepin --- p.39 / Chapter 4. --- In vitro susceptibility testing of the microflora isolated from A. pernyi to cordycepin --- p.39 / Chapter (i) --- Isolation and identification of microflora associated with A. pernyi --- p.39 / Chapter (ii) --- Cordycepin susceptibility testing to microbes isolated from A. pernyi --- p.40 / Chapter X. --- DISCUSSION --- p.41 / Chapter A. --- Biological characteristics of C. militaris grown in vitro --- p.41 / Chapter 1. --- Medium selection --- p.42 / Chapter 2. --- Morphological characteristics of C. militaris in RF medium --- p.42 / Chapter 3. --- Biochemical characteristics of C. militaris in RF broth -enzymes production --- p.43 / Chapter 4. --- Biochemical characteristics of C. militaris in RF broth -cordycepin production --- p.45 / Chapter B. --- C. militaris infection of A. pernyi --- p.45 / Chapter 1. --- Induction method --- p.46 / Chapter 2. --- Host selection --- p.47 / Chapter 3. --- Site susceptibility --- p.48 / Chapter 4. --- Attachment --- p.48 / Chapter 5. --- Germination --- p.49 / Chapter 6. --- Penetration --- p.50 / Chapter 7. --- Tissue utilization by C. militaris --- p.52 / Chapter 8. --- Sexual reproduction --- p.53 / Chapter 9. --- Cordycepin --- p.54 / Chapter 10. --- Host susceptibility --- p.55 / Chapter XI. --- CONCLUSION --- p.56 / Chapter XII. --- LITERATURE CITED --- p.57 / Chapter XIII. --- TABLES --- p.66 / Chapter XIV. --- FIGURES --- p.74