Bombus Terrestris) Fat Body and Haemolymph -An Immune Perspective
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A proteomic and cytological characterisation of the buff-tailed bumblebee (Bombus terrestris) fat body and haemolymph -An immune perspective A thesis submitted to the Maynooth University, for the degree of Doctor of Philosophy by Dearbhlaith Ellen Larkin B. Sc. October 2018 Supervisor Head of Department Dr Jim Carolan, Prof Paul Moynagh, Applied Proteomics Lab, Dept. of Biology, Dept. of Biology, Maynooth University, Maynooth University, Co. Kildare. Co. Kildare Table of contents ii List of figures ix List of tables xiii Dissemination of research xvi Acknowledgments xviii Declaration xix Abbreviations xx Abstract xxiii Table of contents Chapter 1 General introduction 1.1 Bumblebees ............................................................................................................................. 2 1.1.1 Bumblebee anatomy ............................................................................................................. 4 1.2 Bombus terrestris .................................................................................................................... 5 1.3 Global distribution and habitat ................................................................................................ 8 1.4 Pollination ............................................................................................................................... 8 1.5 Bumblebee declines, cause and effect ................................................................................... 10 1.5.1 Environmental stressors and reduced genetic diversity ..................................................... 11 1.5.2 Pathogens and parasites...................................................................................................... 12 1.5.3 Pesticides ............................................................................................................................ 13 1.6 Social immunity .................................................................................................................... 15 1.7 Innate immunity in insects .................................................................................................... 15 1.7.1 Pathogen recognition .......................................................................................................... 17 1.7.2 Haemocytes and cellular immunity .................................................................................... 18 1.7.2.1 Encapsulation .................................................................................................................. 20 1.7.2.2 Phagocytosis .................................................................................................................... 20 1.7.2.3 Nodulation ....................................................................................................................... 21 1.7.3 Humoral responses ............................................................................................................. 21 1.7.3.1 Signalling pathways ........................................................................................................ 21 1.7.3.2 Antimicrobial peptides .................................................................................................... 23 1.7.3.3 Melanisation, coagulation and the phenoloxidase cascade ............................................. 23 1.8 Haemolymph ......................................................................................................................... 26 ii 1.9 Fat body................................................................................................................................. 26 1.9.1 Fat body development and maturation ............................................................................... 27 1.9.2 Fat body structure ............................................................................................................... 28 1.9.3 Cell types and structural composition ................................................................................ 29 1.9.4 Storage and utilisation of energy and nutrients .................................................................. 31 1.9.5 Immune mediation ............................................................................................................. 32 1.10 Application of molecular techniques to investigate the B. terrestris immune response ..... 34 1.11 Overview of thesis objectives ............................................................................................. 36 1.12 Thesis hypotheses ............................................................................................................... 37 Chapter 2 Materials and methods 2.0 General chemicals, reagents and sterilisation procedures ..................................................... 40 2.1 Maintenance and preparation of bacterial and fungal strains ................................................ 40 2.1.1 Bacterial culture mediums.................................................................................................. 40 2.1.2 Maintenance of bacterial strains ......................................................................................... 40 2.1.3 Bacterial cell culturing and enumeration ........................................................................... 41 2.1.4 Fungal culture mediums ..................................................................................................... 41 2.1.5 Culture and maintenance of fungal strains ......................................................................... 41 2.1.6 Fungal cell preparation and enumeration ........................................................................... 42 2.2 Insects, treatments and dissections ........................................................................................ 42 2.2.1 Bumblebee maintenance .................................................................................................... 42 2.2.2 Bumblebee infection procedure ......................................................................................... 42 2.2.3 Haemolymph collection ..................................................................................................... 43 2.2.4 Fat body dissection and collection ..................................................................................... 43 2.2.5 Pesticide preparation and exposure .................................................................................... 44 2.3 Bioassays ............................................................................................................................... 44 2.3.1 Encapsulation/melanisation assay ...................................................................................... 44 2.3.1.1 Filament preparation and inoculation .............................................................................. 44 2.3.1.2 Dissection and fixing....................................................................................................... 45 2.3.1.3 Image analysis ................................................................................................................. 45 2.3.2 Haemocyte counts .............................................................................................................. 46 2.3.3 Statistical analysis .............................................................................................................. 46 2.4 Imaging and microscopy ....................................................................................................... 46 2.4.1 Confocal fluorescent microscopy ....................................................................................... 46 iii 2.4.1.1 Fluorescent microscopy of fat cells ................................................................................. 46 2.4.1.3 Fluorescent microscopy of haemocytes .......................................................................... 47 2.4.2 Histology and cytology ...................................................................................................... 47 2.4.2.1 Haemocyte cytology........................................................................................................ 47 2.4.2.2 Fat body histology ........................................................................................................... 48 2.4.2.3 Imaging and statistical analysis ....................................................................................... 49 2.5 Mass spectrometry preparation ............................................................................................. 49 2.5.1 Haemolymph collection for proteomic analysis ................................................................. 49 2.5.2 Fat body collection and preparation for proteomic analysis .............................................. 49 2.5.3 Protein quantification ......................................................................................................... 50 2.5.4 One dimensional gel electrophoresis and protein staining ................................................. 50 2.5.5 2D Sample clean-up and purification ................................................................................. 50 2.5.6 Protein sample digestion .................................................................................................... 51 2.5.7 Peptide clean-up and purification for LC-MS\MS ............................................................. 51 2.5.8 Mass spectrometric analysis ..............................................................................................