A Molecular and Phylogenetic Analysis of Cryobiosis in Nematodes of the Genus Panagrolaimus

Many organisms are able to survive freezing temperatures through the development of biochemical and physiological adaptations. These biochemical adaptations may include the synthesis of proteins such as antifreeze proteins or cryoprotectants such as trehalose, the elimination of ice nucleators, and the expression of stress associated proteins (such as molecular chaperones, antioxidants, late embryogenesis abundant (LEA) proteins). Physiological adaptations include the ability to undergo cryoprotective dehydration. The molecular mechanisms underlying freezing stress tolerance are poorly understood. One of the main aims of this project was to employ phylogenetic, proteomic, and transcriptomic approaches to gain insights into the adaptations that aid the survival of the free- living cryotolerant nematode Panagrolaimus superbus. Panagrolaimus sp. from temperate, subpolar, polar and continental geographic regions show a range of freezing ability from strains that show high survival upon direct exposure to -80 oC to those that are freezing sensitive. Acclimation significantly improves the freezing survival of temperate, subpolar, polar and continental Panagrolaimus strains and species. They also undergo anhydrobiosis and a correlation exists between desiccation tolerance and freezing tolerance. A phylogenetic study did not find a relationship between freezing phenotype, biogeography and phylogeny. The freezing and desiccation tolerance of ten new tropical Panagrolaimus strains was investigated. These strains are desiccation tolerant but not freezing tolerant, suggesting that freezing survival requires some specialised adaptations. A phylogenetic study of all the Panagrolaimus strains used in this study showed that the desiccation tolerant tropical Panagrolaimus strains are more divergent from the other strains and species in this study. Protein extracts from freezing tolerant Panagrolaimus sp. can inhibit the growth of ice along specific planes of an ice crystal, resulting in hexagonal bipyrimidal ice crystals. This ice faceting capacity was considered most likely to be due to the presence of ice binding proteins. An ice affinity purification method was implemented to purify ice-binding proteins from P. superbus. Several proteins found to be enriched in the ice fraction were identified by mass spectrometry. As none of the identified proteins was an obvious ice binding protein, it was not possible to determine whether these proteins had ice-binding protein activity. The divergence times for five Panagrolaimus strains and species were estimated using the relaxed molecular clock approach. The Panagrolaimus sp. were found to have diverged from other nematodes 70.12 million years ago. The Antarctic nematode P. davidi diverged from its Californian sister species PS1579 approximately 17.18 million years ago and the Arctic nematode P. superbus diverged from its Pennsylvanian sister species Panagrolaimus sp. AF36 9.97 million years ago. The genes that are differentially expressed in response to a period of cold acclimation were determined using the next generation sequencing method RNA-seq. A large number of novel genes were significantly up-regulated (P-value