Competition and co-existence of Photorhabdus temperata subspecies temperata and Photorhabdus temperata subspecies cinerea, symbionts of Heterorhabditis downesi Mohamed A M Asaiyah, M.Sc A thesis submitted to the National University of Ireland, Maynooth, for the degree of Doctor of Philosophy Department of Biology October 2017 Head of Department: Prof. Paul Moynagh Supervisors: Prof. Christine T. Griffin and Dr Abigail Maher Declaration This thesis has not been submitted in whole or in part to any university for any degree, and is except where is stated, the original work of the author. Signed --------------------------- i Contents Acknowledgement ................................................................................................................... vii Publications ............................................................................................................................... ix Abbreviations ........................................................................................................................... vii Abstract ...................................................................................................................................... x Chapter 1 Introduction .............................................................................................................. 1 1.1 Bacterial symbiosis in entomopathogenic nematodes ..................................................... 1 1.2 Entomopathogenic Nematodes ........................................................................................ 2 1.2.1 Entomopathogenic nematode lifecycle ..................................................................... 3 1.3 Infective juvenile behaviour in the entomopathogenic nematodes .................................. 4 1.4 Photorhabdus taxonomy .................................................................................................. 5 1.4.1 Association of different Photorhabdus subspecies with different Heterorhabditis . 9 1.4.2 Photorhabdus - Pathogenicity................................................................................. 11 1.5 Production of secondary metabolites ............................................................................. 13 1.6 Competition and Co-existence ....................................................................................... 14 1.7 Aims and objectives of this project. ............................................................................... 15 Chapter 2 General Methods and materials .............................................................................. 17 2.1 Source of Heterorhabditis downesi isolates and Photorhabdus temperata ................... 17 2.1.1 Culturing of nematodes ........................................................................................... 18 2.1.2 Culturing nematodes from liquid nitrogen stocks ................................................... 19 ii 2.2 Source of symbiotic bacteria .......................................................................................... 19 2.2.1 Isolation of Bacteria from infective juveniles ......................................................... 19 2.2.2 Isolation of Bacteria from haemolymph of infected insects (G. mellonella) .......... 19 2.2.3 Culturing bacteria.................................................................................................... 20 2.3 Media preparation .......................................................................................................... 20 2.3.1 MacConkey agar ..................................................................................................... 20 2.3.2 Nutrient agar ........................................................................................................... 20 2.3.3 LB agar, Miller/ Pyruvate: ...................................................................................... 20 2.3.4 Lipid Agar ............................................................................................................... 21 2.3.5 Phosphate Buffered Saline (PBS): .......................................................................... 21 2.4 Identification of bacterial symbionts ............................................................................. 21 2.4.1 Extraction of DNA .................................................................................................. 21 2.4.2 PCR amplification ................................................................................................... 22 2.4.3 DNA sequencing ..................................................................................................... 24 Chapter 3 Effects of P. temperata subspecies on H. downesi Fitness ................................... 25 3.1 Introduction .................................................................................................................... 25 3.2 Methods.......................................................................................................................... 26 3.2.1 Production Capacity of Heterorhabditis downesi Bull Island isolates (experiment 1) .......................................................................................................................................... 26 3.2.2 Production Capacity of Heterorhabditis downesi Bull Island isolates (experiment 2) .......................................................................................................................................... 29 iii 3.2.3 Effect of P. temperata subspecies on production of IJs from cadavers under dry conditions in sand ............................................................................................................ 30 3.2.4 Desiccation of cadavers infected with H. downesi - P. temperata hybrids at 0% relative humidity .............................................................................................................. 32 3.2.5 Effect of salt concentration on P. temperata growth .............................................. 33 3.3 Results ........................................................................................................................... 34 3.3.1 Production capacity of Heterorhabditis downesi Bull Island isolates (experiment 1) .......................................................................................................................................... 34 3.3.2 Production Capacity of Heterorhabditis downesi Bull Island isolates (experiment 2) .......................................................................................................................................... 42 3.3.3 Effect of P. temperata subspecies on production of IJs from cadavers under dry conditions in sand ............................................................................................................ 43 3.3.4 Desiccation of cadavers infected with H. downesi- P. temperata hybrids at 0% relative humidity .............................................................................................................. 50 3.3.5 Effect of salt concentration on P. temperata growth .............................................. 52 3.4 Discussion ...................................................................................................................... 54 Chapter 4 Competition and coexistence of P. temperata subspecies in vivo and in vitro ..... 59 4.1 Introduction .................................................................................................................... 59 4.2 Materials and methods ................................................................................................... 62 4.2.1 Effect of environmental conditions on competition between subspecies of Photorhabdus temperata co-infecting the same insects .................................................. 62 4.2.2 Colonisation of IJs in cadavers with mixed bacterial infection (in vivo) ............... 63 4.2.3 Preference of P. temperata subspecies by H. downesi in vitro .............................. 64 iv 4.2.4 Competition in liquid culture (co-culture) .............................................................. 66 4.2.5 Developing molecular method to distinguish between P. temperata subspecies Photorhabdus Genome sequencing (pur1 and yel3) ........................................................ 67 4.3 Results ............................................................................................................................ 75 4.3.1 Effect of environmental conditions on competition between subspecies of Photorhabdus temperata co-infecting the same insects .................................................. 75 4.3.2 Colonisation of IJs in cadavers with mixed bacterial infection (in vivo) ............... 78 4.3.3 Preference of H. downesi for P. temperata subspecies on agar plates ................... 80 4.3.4 Competition in liquid culture (co-culture) .............................................................. 81 4.3.5 Developing molecular method to distinguish between P. temperata subspecies Photorhabdus Genome sequencing (pur1 and yel3) ........................................................ 82 4.4 Discussion ...................................................................................................................... 94 Chapter 5 Cadaver Defence ................................................................................................. 100 5.1 Introduction .................................................................................................................