© COPYRIGHT by Deborah Jane Weinstein 2019 ALL RIGHTS RESERVED GENOME-WIDE ANALYSIS OF GENE EXPRESSION IN HALICEPHALOBUS MEPHISTO (THE DEVIL WORM) BY Deborah Jane Weinstein ABSTRACT The nematode Halicephalobus mephisto was discovered in an isolated aquifer, 1.3km below ground. H. mephisto thrives under extreme conditions including elevated heat (37.2°C) and minimal oxygen, classifying it as an extremophile. H. mephisto is a vital discovery for evolution and adaptation, with particular interest in its thermophilic abilities. Here we report the full transcriptome and genome of H. mephisto. In the process we identified a unique adaptation: over amplification of AIG1 and Hsp70 genes, with 168 and 142 domains respectively. Hsp70 was over-expressed under elevated heat conditions, along with ARMET and Bax inhibitor-1, suggesting these genes help H. mephisto to survive elevated heat. AIG1 was not upregulated in elevated heat suggesting its use for non-heat abiotic stressors such as hypoxia. This paper sheds light on the genomic adaptations that have evolved in H. mephisto to survive its challenging environment. ii TABLE OF CONTENTS ABSTRACT .................................................................................................................. ii LIST OF TABLES ............................................................................................................. iv LIST OF ILLUSTRATIONS .............................................................................................. v LIST OF ABBREVIATIONS ............................................................................................ vi INTRODUCTION ........................................................................................................ 1 OVERVIEW OF METHODS UTILIZING GENOMIC PROGRAMS ....................... 6 INPUT DATA ............................................................................................................... 8 MAKER 2 ................................................................................................................... 10 DOMAIN ANALYSIS ............................................................................................... 11 GENE EXPRESSION ANALYSIS BY TOPHAT2-STRINGTIE- BALLGOWN .............................................................................................................. 13 GENE ONTOLOGY AND MANUAL INSPECTION OF TOP 20 UP- AND DOWN-REGULATED ............................................................................................... 17 TREE BUIDLING ...................................................................................................... 19 CROSS-SPECIES HSP70 COMPARISON AND VENN DIAGRAM ..................... 22 CONCLUSION ........................................................................................................... 24 APPENDIX ....................................................................................................................... 30 REFERENCES ................................................................................................................. 35 iii LIST OF TABLES Table 1: Preliminary RNA samples from H. mephisto ................................................................... 8 Table 2: Transcripts Generated by Trinity ...................................................................................... 9 Table 3: Final Genome (Omega) Assembly ................................................................................... 9 Table 4: Gene Predictions from Maker2 ....................................................................................... 10 Table 5: GO Terms for H. mephisto ............................................................................................. 18 iv LIST OF ILLUSTRATIONS Figure 1: Phylogenetic Tree of Nematode Phylum Containing P. redivivus .................................. 2 Figure 2: Phylogenetic Tree of H. mephisto ................................................................................... 3 Figure 3: The Computational Programs Utilized During this Research Proposal. ......................... 6 Figure 4: Protein Domain Analysis. .............................................................................................. 12 Figure 5: Transcriptome Analysis of Gene Expression in H. mephisto. ...................................... 15 Figure 6: RNA Expression Data Analysis of H. mephisto. .......................................................... 16 Figure 7: Bayesian Phylogenetic Tree of Hsp70. ......................................................................... 20 Figure 8: RAxML Phylogenetic Tree of AIG1. ............................................................................ 21 Figure 9: Graph Displaying the Number of Hsp70 Genes Across Nematode Species. ................ 22 Figure 10: Venn Diagram Comparing Orthologous Genes Clusters between H. mephisto, P. redivivus, C. elegans and D. malangogaster. .................................................................... 23 v LIST OF ABBREVIATIONS H. mephisto Halicephalobus mephisto C.elegans Caenorhabditis elegans P. redivivus Panagrellus redivivus HGT Horizontal gene transfer HSP Heat-shock proteins ARMET Arginine-rich, mutated in early-stage tumors BI-1 Bax Inhibitor-1 S. maltophila Stenotrophomonas maltophila ER Endoplasmic reticulum UPR Unfolded protein response PERK PKR-like ER kinase RIDD Regulated IRE1a Dependent Decay AIG1 avrRpt2-induced gene NGM Nematode Growth Media BAC Bacterial Artificial Chromosome vi INTRODUCTION Extremophiles are classified as organisms that inhabit physically or geochemically extreme environments and are of significant interest to science (Rothschild 2001). These organisms extend the boundaries of survival for parameters such as temperature, pH, salinity, water and pressure). Most extremophiles belong to Archaea and Bacteria (Rothschild 2001), yet they expand into Eukaryota as well. Currently eukaryote extremophiles have been the focus of fewer research studies compared to the other two domains within the three-domain system (Rothschild 2001). One newly discovered nematode is Halicephalobus mephisto (The Devil Worm), which was discovered in 2011 in the Beatrix Gold Mine in South Africa, 1.3km below the surface. H. mephisto of Phylum Nematoda, is the first multicellular animal discovered at such sub-terrestrial depths. The environment it inhabits provides multiple survival challenges, leading to it being classified as an extremophilic nematode (Borgonie et al., 2011). Environmental temperature was recorded at 37.2°C, which is the highest temperature recorded for a free-living nematode, and the dissolved oxygen was recorded at 13-72 μM (0.42-2.3 mg/L); the minimum level required for fish to survive is 4-6mg/L (Utah State University, 2018). Put another way, H. mephisto can survive with ~10-40 fold less than standard surface water oxygen levels (Borgonie et al., 2011). The combined effect of low oxygen levels in the water and an elevated temperature create extremely harsh survival conditions for H. mephisto. The water containing the H. mephisto was carbon-dated to be 3,000-12,000 years old and contained minimal tritium (Borgonie et al., 2011). This leads to the conclusion that the water was not mixing with any surface water and is an isolated environment. Therefore, the evolution of the organism H. mephisto occurred within an 1 ‘underground Galapagos’ akin to a subsurface island. The existence of H. mephisto proves that not only bacteria or fungi can live in such harsh oxygen deficient conditions but also multicellular animals, thereby making its genome highly interesting to science. Due to their adaptive ability nematodes, also known as roundworms, are a highly diverse phylum and one of the most abundant, with organisms in nearly every type of habitat (Corsi et al., 2015). Figure 1: Phylogenetic Tree of Nematode Phylum Containing P. redivivus 99 protein tree including C. elegans and P. redivivus, and position to H. mephisto. Image credit: Maggie Lau from Princeton The genomes of over one hundred nematodes have been sequenced, including Trichinella spirilus (Mitreva and Jasmer, 2008), Caenorhabditis briggsae and Caenorhabditis elegans (Martin et al., 2014). C. elegans was the first multicellular animal ever sequenced and is the most widely used model for nematodes (Corsi et al., 2015). However, C. elegans is not the closest relative to H. mephisto since it belongs to the family Rhabditidae, (Corsi et al., 2015) while H. mephisto belongs to family Panagrolaimidae (Borgonie et al., 2011). A closer relative to H. 2 mephisto was recently sequenced, Panagrellus redivivus, also known as “the microworm” providing a more adjacent species for evolutionary comparison (Srinivasan et al., 2013). This is displayed in Figure 2, which shows the placement of P. redivivus within the nematode phylum (Srinivasan et al., 2013). Figure 1, shows the current phylogeny of H. mephisto based on rDNA data from Bayesian inference (Borgonie et al., 2011). Figure 2: Phylogenetic Tree of H. mephisto Recent phylogenetic tree is based on small-subunit rDNA data using Bayesian inference (50% majority rule). Image credit: G Borgonie et al. Nature. 474, 79-82 (2011) doi:10.1038/nature09974 With the availability of sequence technology in the growing field there is the ability to sequence various classes of organisms. Micro-aerobes, like H. mephisto, have various medical and agricultural applications and could lead to possible solutions for environmental damage especially pollution (Urbieta et al. 2015). Nematodes