METAGENOMICS AND METATRANSCRIPTOMICS OF LAKE ERIE ICE Opeoluwa F. Iwaloye A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2021 Committee: Scott Rogers, Advisor Paul Morris Vipaporn Phuntumart © 2021 Opeoluwa Iwaloye All Rights Reserved iii ABSTRACT Scott Rogers, Lake Erie is one of the five Laurentian Great Lakes, that includes three basins. The central basin is the largest, with a mean volume of 305 km2, covering an area of 16,138 km2. The ice used for this research was collected from the central basin in the winter of 2010. DNA and RNA were extracted from this ice. cDNA was synthesized from the extracted RNA, followed by the ligation of EcoRI (NotI) adapters onto the ends of the nucleic acids. These were subjected to fractionation, and the resulting nucleic acids were amplified by PCR with EcoRI (NotI) primers. The resulting amplified nucleic acids were subject to PCR amplification using 454 primers, and then were sequenced. The sequences were analyzed using BLAST, and taxonomic affiliations were determined. Information about the taxonomic affiliations, important metabolic capabilities, habitat, and special functions were compiled. With a watershed of 78,000 km2, Lake Erie is used for agricultural, forest, recreational, transportation, and industrial purposes. Among the five great lakes, it has the largest input from human activities, has a long history of eutrophication, and serves as a water source for millions of people. These anthropogenic activities have significant influences on the biological community. Multiple studies have found diverse microbial communities in Lake Erie water and sediments, including large numbers of species from the Verrucomicrobia, Proteobacteria, Bacteroidetes, and Cyanobacteria, as well as a diverse set of eukaryotic taxa. Sequences obtained from the metagenomic, and transcriptomic analyses match diverse organisms from thirty-two bacterial, two archaeal, and eight eukaryotic phyla. Some of the organisms found were capable of nitrogen, carbon, iron, sulfur, and hydrocarbon metabolism. Sequences from pathogenic and toxin-producing organisms were found. Organisms associated with several iv human activities, including pollution, agriculture, cultivation, manufacturing, shipping, and other activities were found. These results suggest that the ice contains a large diversity of organisms that are indicative of the diverse biological and anthropogenic influences in and around Lake Erie. The results also showed that the lake ice contains a considerable number of cyanobacterial sequences, which often are in low concentrations in the lake water during winter. v ACKNOWLEDGMENTS I would like to express my heartfelt gratitude to my advisor, Dr. Scott Rogers, for his support, mentorship, and guidance through the course of this research. Also, I am grateful for his consistent input, suggestions, time, and responsiveness to my questions while writing this thesis. I am thankful to my thesis committee members, Dr. Paul Morris and Dr. Vipaporn Phuntumart for their time and advise that greatly contributed to the success of my thesis project. I thank the previous members of the Rogers lab who have worked assiduously on this project. Their contribution to this project is greatly appreciated, they include Dr. Zeynep Kocer, Dr. Yury Shtarkman and Brenna Michaud. I would like to the current members of the Rogers lab, they include Tessa Alloy and Sri Devan Appasamy who have contributed to this project in multiple ways. I am grateful to Dr. George Bullerjahn, Dr. Nigel D’Souza and Dr. Robert McKay for their help in the collection of the sample used in this project. Finally, I am grateful to the Department of Biological Sciences, Bowling Green State University for their support throughout my graduate education. vi TABLE OF CONTENTS Page CHAPTER I. INTRODUCTORY REVIEW ........................................................................... 1 Lake Erie ....................................................................................................................... 1 Life in Lake Erie under ice ........................................................................................... 2 Microbial metabolism under ice conditions .................................................................. 2 Human activities around Lake Erie ............................................................................... 5 Ice drilling/sample collection ........................................................................................ 6 Ultracentrifugation ........................................................................................................ 6 RNA and DNA extraction ............................................................................................. 7 cDNA synthesis ............................................................................................................ 7 Polymerase chain reaction ............................................................................................ 9 454 pyrosequencing ...................................................................................................... 10 Basic local alignment search tool ................................................................................. 12 Curation of data (Python and Biopython) ..................................................................... 13 Chapter i references ...................................................................................................... 14 CHAPTER II. ORGANISMS DIVERSITY IN LAKE ERIE ................................................. 24 Lake Erie ....................................................................................................................... 24 Metabolic activities in lake ice ..................................................................................... 24 Previous research .......................................................................................................... 25 Purpose of research ....................................................................................................... 26 Materials and methods .................................................................................................. 27 Sample preparation ........................................................................................... 27 DNA/RNA extraction ....................................................................................... 28 vii cDNA synthesis ................................................................................................ 28 Sequence analysis ............................................................................................. 29 Taxonomic proportions ..................................................................................... 29 Metabolic classifications ................................................................................... 29 Comparison with previous Lake Erie water study ............................................ 30 Results and discussion .................................................................................................. 30 Result summary ................................................................................................ 30 Summary of organisms ..................................................................................... 39 Archaea ................................................................................................. 39 Bacteria ................................................................................................. 39 Extremophiles ................................................................................................... 41 Cyanobacteria ................................................................................................... 43 Cellulolytic bacteria .......................................................................................... 44 Oil/hydrocarbon degrader ................................................................................. 45 Pathogens .......................................................................................................... 45 Human pathogens.................................................................................. 46 Pathogenic in animals ........................................................................... 46 Pathogenic in plants .............................................................................. 47 Comparison of the bacterial community with Lake Erie water study .............. 48 Eukaryotes......................................................................................................... 48 SAR (stramenopiles, alveolates and rhizaria) ....................................... 49 Fungi ..................................................................................................... 54 viii Arthropods ............................................................................................ 56 Chordata ................................................................................................ 57 Human activities associated with organisms found in Lake Erie ..................... 58 Metabolic analysis ............................................................................................ 58 Methanogenesis..................................................................................... 59 Methylotrophy......................................................................................