A Molecular and Phylogenetic Analysis of Cryobiosis in Nematodes of the Genus Panagrolaimus Lorraine Marie McGill, B. Sc. Thesis submitted to the National University of Ireland Maynooth in fulfilment of the requirements for the Degree of Doctor of Philosophy Department of Biology National University of Ireland Maynooth Co. Kildare Supervisor: Prof. Ann Burnell Head of Department: Prof. Kay Ohlendieck October 2011 Table of Contents Abbreviations ........................................................................................................... i! List of Figures......................................................................................................... iv! List of Tables ........................................................................................................ viii! Acknowledgements.................................................................................................. x! Declaration............................................................................................................. xii! Abstract................................................................................................................. xiii! Chapter I General Introduction .............................................................1! 1.1 Nematodes.......................................................................................................... 1! 1.1.1 Habitat and ecology ..................................................................................... 1! 1.1.2 Nematode morphology................................................................................. 1! 1.1.3 Molecular taxonomy of the Phylum Nematoda ........................................... 2! 1.1.3.1 Ribosomal small subunit gene .............................................................. 2! 1.1.3.2 Ribosomal large subunit gene ............................................................... 4! 1.1.3.3 rRNA internal transcribed spacer.......................................................... 4! 1.1.3.4 Mitochondrial cytochrome c oxidase subunit 1 .................................... 5! 1.1.4 Panagrolaimus sp. ....................................................................................... 5! 1.2 Phylogenetics .................................................................................................. 7! 1.2.1 Methods used in phylogenetic studies ......................................................... 7! 1.2.1.1 Maximum likelihood methods .............................................................. 8! 1.2.1.2 Bayesian methods ................................................................................. 9! 1.2.2 The fossil record and evolutionary timescales............................................. 9! 1.2.2.1 Molecular clock models ...................................................................... 10! 1.2.2.2 Molecular clock calibration ................................................................ 12! 1.2.3 Nematode evolution ................................................................................... 13! 1.3 Freezing survival ............................................................................................. 15! 1.3.1 Definition and occurrence of freezing-adapted organisms ........................ 17! 1.3.1.1 Microorganisms .................................................................................. 17! 1.3.1.2 Plants................................................................................................... 17! 1.3.1.3 Animals ............................................................................................... 19! 1.3.2 Why study freeze tolerance? ...................................................................... 20! 1.4 Freezing injury ................................................................................................ 20! 1.5 Mechanisms of freezing tolerance ................................................................. 21! 1.5.1 Accumulation of colligative cryoprotectants ............................................. 22! 1.5.1.1 Colligative cryoprotectants ................................................................. 22! 1.5.1.2 Glycerol............................................................................................... 22! 1.5.1.3 Sorbitol................................................................................................ 24! 1.5.1.4 Glucose................................................................................................ 24! 1.5.2 Accumulation of low-molecular weight cryoprotectants........................... 25! 1.5.2.1 Trehalose............................................................................................. 25! 1.5.2.2 Proline ................................................................................................. 28! 1.5.2.3 Glycine betaine ................................................................................... 30! 1.5.3 Antifreeze proteins..................................................................................... 30! 1.5.3.1 Mechanism that Antifreeze proteins bind to the ice............................ 31! 1.5.3.2 Fish antifreeze proteins ....................................................................... 36! 1.5.3.3 Fish Type I antifreeze proteins............................................................ 36! 1.5.3.4 Fish Type II antifreeze proteins .......................................................... 38! 1.5.3.5 Fish Type III antifreeze proteins......................................................... 38! 1.5.3.6 Fish Type IV antifreeze proteins......................................................... 39! 1.5.3.7 Fish Type V antifreeze proteins .......................................................... 40! 1.5.3.8 Insect and arthropod antifreeze proteins ............................................. 41! 1.5.3.9 Plant antifreeze proteins...................................................................... 44! 1.5.3.10 Algal AFPs........................................................................................ 48! 1.5.3.11 Fungal AFPs...................................................................................... 49! 1.5.3.12 Yeast AFPs........................................................................................ 49! 1.5.3.13 Bacterial AFPs .................................................................................. 50! 1.5.4 Ice nucleator proteins ................................................................................. 50! 1.5.5 Gene and protein induction in response to low temperature stress............ 53! 1.5.5.1 Cold-shock proteins ............................................................................ 55! 1.5.5.2 Cold-regulated genes........................................................................... 55! 1.5.5.3 Heat shock proteins............................................................................. 58! 1.5.5.4 Alteration of cell membrane lipid composition .................................. 59! 1.6 Relationship between freezing and desiccation survival ............................. 62! 1.7 Freezing survival in nematodes ..................................................................... 62! 1.8 Objectives of this project................................................................................ 65! Chapter II Materials and Methods ......................................................66! 2.1 Materials .......................................................................................................... 66! 2.1.1 Chemicals................................................................................................... 66! 2.1.2 Bacterial strains and plasmids.................................................................... 66! 2.1.3 Source of Panagrolaimus species and strains............................................ 66! 2.2 Methods............................................................................................................ 68! 2.2.1 Nematode culturing methods ..................................................................... 68! 2.2.1.1 Culturing of Panagrolaimus species and strains................................. 68! 2.2.1.2 Mass culturing nematodes................................................................... 68! 2.2.1.3 Harvesting nematodes ......................................................................... 68! 2.2.1.4 Cleaning nematode stocks................................................................... 69! 2.2.1.5 Freezing nematode stocks ................................................................... 69! 2.2.1.6 Recovery of frozen nematode stocks .................................................. 70! 2.2.1.7 Desiccation stress experiments ........................................................... 70! 2.2.1.8 Freezing stress experiments ................................................................ 70! 2.2.2 Molecular methods..................................................................................... 71! 2.2.2.1 Genomic DNA extraction ................................................................... 71! 2.2.2.2 RNA extraction ................................................................................... 72! 2.2.2.3 cDNA synthesis................................................................................... 72! 2.2.2.4 Determination of RNA/DNA concentration and quality .................... 73! 2.2.2.5 Agrose gel electrophoresis .................................................................