A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/129924 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Elucidating the natural function of cordycepin, a secondary metabolite of the fungus Cordyceps militaris, and its potential as a novel biopesticide in Integrated Pest Management By Victoria Clare Woolley A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Life Sciences University of Warwick, School of Life Sciences September 2018 Table of Contents List of Figures ......................................................................................................... 1 List of Tables ........................................................................................................... 3 Abbreviations .......................................................................................................... 4 Acknowledgements .................................................................................................. 6 Declarations ............................................................................................................ 7 Abstract ................................................................................................................... 8 Chapter 1-Introduction ........................................................................................... 9 1. 1 The importance of effective crop protection for global food production ............... 9 1.2 Crop protection and Integrated Pest Management .................................................. 12 1.3 The use of entomopathogens as biopesticides in IPM ............................................... 13 1.4 Entomopathogenic fungi and their use as biopesticides ........................................... 16 1.4.1 Use of anamorphic hypocrealean EPF as biopesticides in IPM ....................................... 19 1.5 Hypocrealean Entomopathogenic fungi; fungal life cycle and mechanisms of infection of arthropod hosts ................................................................................................ 22 1.5.1 Attachment of conidia to the host integument and penetration of the cuticle ................... 23 1.5.2 Growth of EPF within the host insect ............................................................................ 23 1.5.3 Host death and EPF transmission .................................................................................. 24 1.5.4 Endophytic interactions of EPF ..................................................................................... 25 1.6 Biology of Cordyceps militaris ....................................................................................... 26 1.7 The role of the insect immune system in defence against microbial infection ...... 28 1.7.1 The cellular immune response of insects ....................................................................... 29 1.7.2 The humoral immune response in insects ...................................................................... 29 1.7.2.1 The Toll pathway ....................................................................................................... 31 1.7.2.2 The IMD pathway ...................................................................................................... 32 1.7.2.3 Cross-talk between immune pathways ........................................................................ 32 1.7.2.4 The role of AMPs in insect immunity ......................................................................... 33 1.7.2.5 The Jak-Stat pathway ................................................................................................. 35 1.7.2.6 The prophenol oxidase cascade .................................................................................. 35 1.7.3 The role of lysozyme in insect immunity ....................................................................... 36 1.7.4 The wounding response in insects ................................................................................. 37 1.7.5 The immune response to EPF in insects......................................................................... 38 1.8 Metabolites of entomopathogenic fungi: role in insect infection and use as novel natural products.................................................................................................................... 39 1.8.1 Classes of EPF secondary metabolites ........................................................................... 39 1.8.2 The role of EPF metabolites in insect infection .............................................................. 40 1.8.3 Medicinal uses of EPF metabolites ................................................................................ 42 1.8.4 Cordycepin: A secondary metabolite of C. militaris....................................................... 42 1.8.5 The potential of cordycepin as a novel pest control agent ............................................... 46 1.9 Aims and Objectives ...................................................................................................... 47 ii Chapter 2 - Determining the effect of entomopathogenic fungi and cordycepin on the survival of Galleria mellonella ........................................................................ 48 2.1 Background ..................................................................................................................... 48 2.1.1 The model insect G. mellonella ..................................................................................... 48 2.1.2 Laboratory bioassays investigating the infection of insects by EPF ................................ 49 2.1.3 Galleria mellonella as a tool for studying the effect of fungal secondary metabolites on insects ................................................................................................................................... 50 2.1.4 The insect gut as a barrier to infection ........................................................................... 50 2.1.5 Aims ............................................................................................................................ 51 2.2 Materials and methods .................................................................................................. 52 2.2.1 Insect material .............................................................................................................. 52 2.2.2 Fungal material ............................................................................................................. 52 2.2.3 Preparing conidial suspensions...................................................................................... 53 2.2.4 Confirmation of fungal species identity using nucleotide sequences ............................... 54 2.2.5 Galleria mellonella bioassay- fungal injection ............................................................... 55 2.2.6 Galleria mellonella bioassay - fungal topical application ............................................... 56 2.2.7 Galleria mellonella bioassay– cordycepin injection ....................................................... 57 2.2.8 Identification of bacteria isolated from G. mellonella cadavers ...................................... 57 2.3 Results .............................................................................................................................. 59 2.3.1 Confirmation of fungal species identity ......................................................................... 59 2.3.2 Galleria mellonella bioassay techniques........................................................................ 62 2.3.3 Galleria mellonella bioassay- fungal injection ............................................................... 62 2.3.4 Topical application of EPF conidia onto G. mellonella .................................................. 64 2.3.5 Effect of cordycepin on survival of G. mellonella .......................................................... 65 2.2.6 Identification of bacteria isolated from G. mellonella .................................................... 67 2.4 Discussion......................................................................................................................... 70 2.5 Conclusions ..................................................................................................................... 73 Chapter 3- Determining the effect of entomopathogenic fungi on the expression of genes associated with immune reactions in Galleria mellonella ........................... 74 3.1 Background ..................................................................................................................... 74 3.1.1 The G. mellonella humoral immune response ................................................................ 74 3.1.2 The immune response of G. mellonella to fungal infection ............................................ 76 3.1.3 Monitoring the progression on EPF infection ................................................................ 77 3.1.4 Aims and objectives...................................................................................................... 78 3.2